Designing an Inclusive Learning Training Series for Pharmacy Educators.

This article describes the design, implementation, and evaluation of five faculty development sessions focused on inclusive teaching strategies in pharmacy education. Inclusive strategies ensure that every student can clearly understand and engage in meaningful learning opportunities. Three sessions were implemented in fall 2020 and two in spring 2021. Sessions focused on experiential, didactic, and graduate education. A convergent parallel mixed methods evaluation was conducted using descriptive statistics and thematic analysis. Sessions were highly rated, and participants provided suggestions for curriculum improvement (e.g., creating resources, surveying students, and peer auditing syllabi for aspects of inclusiveness). Given the increasing emphasis on inclusion in pharmacy education, this work is timely for sharing strategies aimed at faculty development and teaching practices.

Effect of oxidative stress on telomere maintenance in aortic smooth muscle cells.

Reactive oxygen species (ROS) and telomere dysfunction are both associated with aging and the development of age-related diseases. Although there is evidence for a direct relationship between ROS and telomere dysfunction as well as an independent association of oxidative stress and telomere attrition with age-related disorders, there has not been sufficient exploration of how the interaction between oxidative stress and telomere function may contribute to the pathophysiology of cardiovascular diseases (CVD). To better understand the complex relationships between oxidative stress, telomerase biology and pathophysiology, we examined the telomere biology of aortic smooth muscle cells (ASMCs) isolated from mutant mouse models of oxidative stress. We discovered that telomere lengths were significantly shorter in ASMCs isolated from superoxide dismutase 2 heterozygous (Sod2+/-) mice, which exhibit increased arterial stiffness with aging, and the observed telomere attrition occurred over time. Furthermore, the telomere erosion occurred even though telomerase activity increased. In contrast, telomeres remained stable in wild-type and superoxide dismutase 1 heterozygous (Sod1+/-) mice, which do not exhibit CVD phenotypes. The data indicate that mitochondrial oxidative stress, in particular elevated superoxide levels and decreased hydrogen peroxide levels, induces telomere erosion in the ASMCs of the Sod2+/- mice. This reduction in telomere length occurs despite an increase in telomerase activity and correlates with the onset of disease phenotype. Our results suggest that the oxidative stress caused by imbalance in mitochondrial ROS, from deficient SOD2 activity as a model for mitochondrial dysfunction results in telomere dysfunction, which may contribute to pathogenesis of CVD.

Exploring the Future of Graduate Education in Pharmaceutical Fields.

To ensure graduate students remain at the forefront of healthcare, curricula must be aligned with current and emerging innovations likely to influence students' ability to be successful. In this study, a modified Delphi technique was utilized to determine and prioritize the innovations and professional skills needed. For innovations, the top three areas experts identified were (1) personalized medicine, (2) big data, and (3) cell and gene therapy. For professional skills, the top three areas were (1) creative problem solving, (2) communication, and (3) data literacy. These results can be used to inform graduate curriculum development within various pharmaceutical fields.

Prosocial effects of an oxytocin metabolite, but not synthetic oxytocin receptor agonists, in a mouse model of autism.

Currently, there are no established pharmaceutical strategies that effectively treat social deficits in autism spectrum disorder (ASD). Oxytocin, a neurohormone that plays a role in multiple types of social behaviors, has been proposed as a possible therapeutic against social impairment and other symptoms in ASD. However, from the standpoint of pharmacotherapy, oxytocin has several liabilities as a standard clinical treatment, including rapid metabolism, low brain penetrance, and activity at the vasopressin (antidiuretic hormone) receptors. The present studies describe findings from a preclinical screening program to evaluate oxytocin receptor (OXTR) agonists and oxytocin metabolites for potential clinical use as more optimal treatments. We first investigated two synthetic oxytocin analogs, TC-OT-39 and carbetocin, using in vitro cell-based assays for pharmacological characterization and behavioral tests in the BALB/cByJ mouse model of ASD-like social deficits. Although both TC-OT-39 and carbetocin selectively activate the OXTR, neither synthetic agonist had prosocial efficacy in the BALB/cByJ model. We next evaluated two oxytocin metabolites: OT(4-9) and OT(5-9). While OT(5-9) failed to affect social deficits, the metabolite OT(4-9) led to significant social preference in the BALB/cByJ model, in a dose-dependent manner. The increased sociability was observed at both 24 h and 12 days following the end of a subchronic regimen with OT(4-9) (2.0 mg/kg). Overall, these results suggest that the prosocial effects of oxytocin could be mediated by downstream activity of oxytocin metabolites, raising the possibility of new pathways to target for drug discovery relevant to ASD.

Student Learning with Generated and Answered Peer-written Questions.

Objective. To investigate the degree to which student-generated questions or answering student-generated multiple-choice questions predicts course performance in medicinal chemistry. Methods. Students enrolled in Medicinal Chemistry III over a 3-year period were asked to create at least one question per exam period using PeerWise; within the software, they were also asked to answer and rate one peer question per class session. Students' total reputation scores and its components (question authoring, answering, and rating) and total answer scores (correctness of answers submitted indicating agreement with the author's chosen answer) were analyzed relative to final course grades. Results. Students at the non-satellite campus and those who generated more highly rated questions performed better overall in the course accounting for 12% of the variability in course grades. The most notable differences were between the top third and bottom third performing students within the course. The number of questions answered by students was not a significant predictor of course performance. Conclusion. Student generation of more highly rated questions (referred to as more thoughtful in nature by the software program) is predictive of course performance but it only explained a small variability in course grades. The correctness of answers submitted, however, did not relate to student performance.

Reversal of social deficits by subchronic oxytocin in two autism mouse models.

Social deficits are a hallmark feature of autism spectrum disorder (ASD) and related developmental syndromes. Although there is no standard treatment for social dysfunction, clinical studies have identified oxytocin as a potential therapeutic with prosocial efficacy. We have previously reported that peripheral oxytocin treatment can increase sociability and ameliorate repetitive stereotypy in adolescent mice from the C58/J model of ASD-like behavior. In the present study, we determined that prosocial oxytocin effects were not limited to the adolescent period, since C58/J mice, tested in adulthood, demonstrated significant social preference up to 2 weeks following subchronic oxytocin treatment. Oxytocin was also evaluated in adult mice with underexpression of the N-methyl-d-aspartate receptor NR1 subunit (encoded by Grin1), a genetic model of autism- and schizophrenia-like behavior. Subchronic oxytocin had striking prosocial efficacy in male Grin1 knockdown mice; in contrast, chronic regimens with clozapine (66 mg/kg/day) or risperidone (2 mg/kg/day) failed to reverse deficits in sociability. Neither the subchronic oxytocin regimen, nor chronic treatment with clozapine or risperidone, reversed impaired prepulse inhibition in the Grin1 knockdown mice. Overall, these studies demonstrate oxytocin can enhance sociability in mouse models with divergent genotypes and behavioral profiles, adding to the evidence that this neurohormone could have therapeutic prosocial efficacy across a spectrum of developmental disorders.

Telomere maintenance as a target for drug discovery.

The observation that the enzyme telomerase is up-regulated in 80-90% of cancer cells isolated from primary human tumors but is absent in neighboring cells of healthy tissue has resulted in significant efforts to validate telomerase as an anticancer drug target and to develop effective approaches toward its inhibition. In addition to inhibitors that target the enzymatic function of telomerase, efforts toward immunotherapy using peptides derived from its catalytic subunit hTERT and hTERT-promoter driven gene therapy have made significant advances. The increased level of telomerase in cancer cells also provides a potential platform for cancer diagnostics. Telomerase inhibition leads to disruption of a cell's ability to maintain the very ends of the chromosomes, which are called telomeres. Thus, the telomere itself has also attracted attention as an anticancer drug target. In this Perspective, interdisciplinary efforts to realize the therapeutic potential of targeting telomere maintenance with a focus on telomerase are discussed.

Prosocial effects of oxytocin in two mouse models of autism spectrum disorders.

Clinical evidence suggests that oxytocin treatment improves social deficits and repetitive behavior in autism spectrum disorders (ASDs). However, the neuropeptide has a short plasma half-life and poor ability to penetrate the blood-brain barrier. In order to facilitate the development of more bioavailable oxytocinergic compounds as therapeutics to treat core ASD symptoms, small animal models must be validated for preclinical screens. This study examined the preclinical utility of two inbred mouse strains, BALB/cByJ and C58/J, that exhibit phenotypes relevant to core ASD symptoms. Mice from both strains were intraperitoneally administered oxytocin, using either acute or sub-chronic regimens. Acute oxytocin did not increase sociability in BALB/cByJ; however, sub-chronic oxytocin had significant prosocial effects in both BALB/cByJ and C58/J. Increased sociability was observed 24 h following the final oxytocin dose in BALB/cByJ, while prosocial effects of oxytocin emerged 1-2 weeks post-treatment in C58/J. Furthermore, acute oxytocin decreased motor stereotypy in C58/J and did not induce hypoactivity or anxiolytic-like effects in an open field test. This study demonstrates that oxytocin administration can attenuate social deficits and repetitive behavior in mouse models of ASD, dependent on dose regimen and genotype. These findings provide validation of the BALB/cByJ and C58/J models as useful platforms for screening novel drugs for intervention in ASDs and for elucidating the mechanisms contributing to the prosocial effects of oxytocin.

New models of Tetrahymena telomerase RNA from experimentally derived constraints and modeling.

The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA (TER) provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment toward understanding its nontemplating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of the Tetrahymena telomerase reverse transcriptase (tTERT) to characterize the conformational changes of tTER telomerase assembly. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footprinting allow us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major groove of the helix-capping loop.

Identification of G-Quadruplex Inducers Usinga Simple, Inexpensiveand Rapid High Throughput Assay, and TheirInhibition of Human Telomerase.

Telomeres are protein and DNA complexes located atchromosome ends. Telomeric DNA is composed of a double stranded region of repetitive DNA followed by single-stranded 3' extension of aG-rich sequence. Single-stranded G-rich sequencescan fold into G-quadruplex structures,and molecules that stabilize G-quadruplexes are known to inhibit the enzyme telomerase and disrupt telomere maintenance. Because telomere maintenance is required for proliferation of cancer cells, G-quadruplex stabilizers have become attractive prospects for anticancer drug discovery.However, telomere-targeting G-quadruplex ligands have yet to enter the clinic owing in part to poor pharmacokinetics and target selectivity. Increasing the pharmacophore diversity of G-quadruplex and specifically telomeric-DNA targeting agents should assist in overcoming these shortcomings. In this work, we report the identification and validation ofligands that bind telomeric DNA and induce G-quadruplex formationusing the NCI Diversity Set I, providing validation of anextremely simple, rapid and high-throughput screen using FRET technology. Hits from the screen were validated by examining telomerase inhibition and G-quadruplex inductionusing CD spectroscopy and DNA polymerase stop assays. We show that two known DNA binding molecules, ellipticine derivativeNSC 176327 (apyridocarbazole) and NSC 305831 (an antiparasitic hetero-cyclediamidine referred to as furamidine and DB75),are selective induceG-quadruplex formation in the human telomeric sequence and bind telomeric DNA quadruplexes in the absence of stabilizing monovalent cations with molar ratios(molecule: DNA)of 4:1and 1.5:1, respectively.

ortho-Quinone tanshinones directly inhibit telomerase through an oxidative mechanism mediated by hydrogen peroxide.

The tanshinone natural products possess a variety of pharmacological properties including anti-bacterial, anti-inflammatory, anti-oxidant, and anti-neoplastic activity. The molecular basis of these effects, however, remains largely unknown. In the present study, we explored the direct effect of tanshinones on the enzyme telomerase. Telomerase is up-regulated in the majority of cancer cells and is essential for their survival, making it a potential anti-cancer drug target. We found that the ortho-quinone tanshinone II-A inhibits telomerase in a time- and DTT-dependent fashion, and the hydrogen peroxide scavenger catalase protected telomerase from inactivation. These findings demonstrate that ortho-quinone containing tanshinones can inhibit telomerase owing to their ability to generate reactive oxygen species. The results also provide evidence that telomerase is directly and negatively regulated by reactive oxygen species.

The catalytic subunit of human telomerase is a unique caspase-6 and caspase-7 substrate.

Telomerase is a ribonucleoprotein complex that is essential for persistent cellular proliferation. The catalytic subunit of human telomerase, hTERT, functions as a reverse transcriptase and promotes vitality by maintaining telomeric DNA length. hTERT is tightly regulated with complex but poorly understood positive and negative regulation at several levels including transcription, protein-protein interactions, and post-translation modifications. Because evidence implicates hTERT as an apoptosis inhibitor and because telomerase activity tends to decrease during apoptosis, we hypothesized that hTERT is a caspase substrate leading to down regulation during apoptosis. Caspases are proteases that initiate and execute apoptosis by cleaving target proteins. Indeed, we found that caspases-6 and -7 cleave hTERT during apoptosis in cultured cells. Caspase-6 cleaves at residues D129 and D637, and caspase-7 cleaves at E286 and D628. Three of the caspase cleavage sites are unique motifs. All four caspase motifs appear conserved in TERTs from Old World monkeys and apes, and the caspase-6 sites appear conserved in all primates. The caspase site that cleaves at D129 appears conserved in amniotes. hTERT fragments generated by cleavage were remarkably persistent, lasting hours after caspase activation. These results reveal a new biologically relevant mechanism for telomerase down regulation through caspase-mediated cleavage of hTERT and expand the list of known caspase motifs.

MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA) expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ) fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT) that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

Structures of telomerase subunits provide functional insights.

BACKGROUND: Telomerase continues to generate substantial attention both because of its pivotal roles in cellular proliferation and aging and because of its unusual structure and mechanism. By replenishing telomeric DNA lost during the cell cycle, telomerase overcomes one of the many hurdles facing cellular immortalization. Functionally, telomerase is a reverse transcriptase, and it shares structural and mechanistic features with this class of nucleotide polymerases. Telomerase is a very unusual reverse transcriptase because it remains stably associated with its template and because it reverse transcribes multiple copies of its template onto a single primer in one reaction cycle. SCOPE OF REVIEW: Here, we review recent findings that illuminate our understanding of telomerase. Even though the specific emphasis is on structure and mechanism, we also highlight new insights into the roles of telomerase in human biology. GENERAL SIGNIFICANCE: Recent advances in the structural biology of telomerase, including high resolution structures of the catalytic subunit of a beetle telomerase and two domains of a ciliate telomerase catalytic subunit, provide new perspectives into telomerase biochemistry and reveal new puzzles.

Preparation of G-quartet structures and detection by native gel electrophoresis.

Mounting evidence supporting the existence of DNA structures containing G-quartets in vivo makes these unique and diverse nucleic acid structures an important research subject, and future investigations aimed at elucidating their biological significance are expected. The purification and characterization of G-quartet structures can be challenging because their inherent structural diversity, complexity, and stability are sensitive to an array of variables. The stability of G-quartet structures depends on many factors including number of DNA strands involved in G-quartet formation, the identity of the stabilizing cation(s), the number and sequence context of the guanosines involved in stacking, the presence of single-stranded overhangs, the intervening loop size, and the identity of nucleosides in the loop. Here we detail current methods used in G-quartet preparation and their purification and characterization by native gel electrophoresis.

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1.

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC(50) values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.

Effect of locked-nucleic acid on a biologically active g-quadruplex. A structure-activity relationship of the thrombin aptamer.

Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG), by using locked nucleic acid (LNA) to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity - the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion.

Interrogation of G-quadruplex-protein interactions.

The ability to accurately examine the interaction of G-quadruplex DNA with proteins is essential for revealing the biological roles of these unusual DNA structures. In this regard, there are four primary G-quadruplex-related activities of proteins that have been studied including simple equilibrium binding, promotion or catalysis of G-quadruplex formation, dissociation of G-quadruplex structures, and covalent modification of G-quadruplexes, which includes both nucleolytic cleavage and nucleotide addition. Here, assays used to examine the interactions of G-quadruplexes with proteins will be reviewed and specific methods to study the interactions of G-quadruplexes from telomeric DNA sequences with a variety of proteins will be described. Importantly, this review emphasizes the importance of evaluating the integrity of the G-quadruplex being studied as single sequences can often form a variety of folded structures.

The human telomere and its relationship to human disease, therapy, and tissue engineering.

The chromosomes of eukaryotes end in a specialized complex of proteins and repetitive DNA called the telomere. Telomeres form a protective cap that prevents chromosome fusions, protects chromosome ends from degradation, and assists in positioning chromosomes in the nucleus. In the absence of replenishing mechanisms, telomeric DNA is lost during each cell cycle owing to incomplete replication, oxidative damage, and nucleolytic degradation. The ribonucleoprotein complex telomerase offsets this loss of telomeric DNA, but its activity is absent in most differentiated human cells. Thus, the aging process results in ever shortening lengths of telomeric DNA. Related to this is the requirement for a mechanism of telomeric DNA maintenance in tumors, leading to telomerase expression in >85% of all cancers cells. The integral roles of telomere biology in these pathophysiological states have substantially motivated its investigation. Here, the literature on the human telomere will be reviewed with an emphasis on the relationship to human health.

The unmasking of telomerase.

Telomerase is a ribonucleoprotein complex that reverse transcribes a portion of its RNA subunit during the synthesis of G-rich DNA at the 3' end of each chromosome in most eukaryotes. This activity compensates for the inability of the normal DNA replication machinery to fully replicate chromosome termini. The roles of telomerase in cellular immortality and tumor biology have catalyzed a significant interest in this unusual polymerase. Recently the first structures of two domains, the CR4/CR5 and pseudoknot, of human telomerase RNA (hTR) were reported, offering a structural basis for interpreting biochemical studies and possible roles of hTR mutations in human diseases. Structures of the stem II and stem IV domains of Tetrahymena thermophila TR as well as the N-terminal domain of the T. thermophila telomerase reverse transcriptase have also been determined. These studies complement previous biochemical studies, providing rich insight into the structural basis for telomerase activity.