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No pharmacy program, however well-resourced, has sufficient time or resources to teach students all current, practice-relevant knowledge. And while the volume of potential pharmacy education curriculum content increases exponentially each year, available time for direct instruction continues to decline. Given these constraints, pharmacy curricula must focus on promoting deep learning of the most critical, fundamental, broadly applicable, and lasting knowledge. Yet, in terms of didactic knowledge, pharmacy education currently has no agreed upon, evidence-based criteria for determining which foundational concepts are most important to teach nor any research-based assessment tools to demonstrate how well students have learned those core concepts.This lack of consensus regarding core conceptual knowledge makes disparities in learning outcomes both more likely to occur and less likely to be detected or addressed. Over the past 30 years, several scientific disciplines undergirding pharmacy have developed research-based lists of core concepts and related concept inventories, demonstrating their transformative educational potential. Core concepts are big, fundamental ideas that experts agree are critical for all students in their discipline to learn, remember, understand, and apply. Concept inventories are research-based, psychometrically validated, multiple-choice tests designed to uncover learners' prior knowledge and potential misconceptions and determine their depth of understanding of disciplinary core concepts.This commentary proposes adapting and applying this evidence-based core concepts approach to enhance pharmacy education's overall effectiveness and efficiency and outlines an ongoing, multinational research initiative to identify and define essential pharmacy concepts to be taught, learned, and assessed.

The adenosine A

Pharmacology education currently lacks a research‐based consensus on which core concepts all graduates should know and understand, as well as a valid and reliable means to assess core conceptual learning. The Core Concepts in Pharmacology Expert Group (CC‐PEG) from Australia and New Zealand recently identified a set of core concepts of pharmacology education as a first step toward developing a concept inventory—a valid and reliable tool to assess learner attainment of concepts. In the current study, CC‐PEG used established methodologies to define each concept and then unpack its key components. Expert working groups of three to seven educators were formed to unpack concepts within specific conceptual groupings: what the body does to the drug (pharmacokinetics); what the drug does to the body (pharmacodynamics); and system integration and modification of drug–response. First, a one‐sentence definition was developed for each core concept. Next, sub‐concepts were established for each core concept. These twenty core concepts, along with their respective definitions and sub‐concepts, can provide pharmacology educators with a resource to guide the development of new curricula and the evaluation of existing curricula. The unpacking and articulation of these core concepts will also inform the development of a pharmacology concept inventory. We anticipate that these resources will advance further collaboration across the international pharmacology education community to improve curricula, teaching, assessment, and learning., The second stage of development of core concepts of pharmacology education; defining each concept, and unpacking the concept by articulation of sub‐concepts.

Pharmacology education currently lacks an agreed knowledge curriculum. Evidence from physics and biology education indicates that core concepts are useful and effective structures around which such a curriculum can be designed to facilitate student learning. Building on previous work, we developed a novel, criterion‐based method to identify the core concepts of pharmacology education. Five novel criteria were developed, based on a literature search, to separate core concepts in pharmacology from topics and facts. Core concepts were agreed to be big ideas, enduring, difficult, applicable across contexts, and useful to solve problems. An exploratory survey of 33 pharmacology educators from Australia and New Zealand produced 109 terms, which were reduced to a working list of 26 concepts during an online workshop. Next, an expert group of 12 educators refined the working list to 19 concepts, by applying the five criteria and consolidating synonyms, and added three additional concepts that emerged during discussions. A confirmatory survey of a larger group resulted in 17 core concepts of pharmacology education. This list may be useful for educators to evaluate existing curricula, design new curricula, and to inform the development of a concept inventory to test attainment of the core concepts in pharmacology., Over 50 pharmacology educators from Australia and New Zealand worked together to identify the core concepts of pharmacology education. The figure shows the 19 concepts identified and the process taken to produce this list

In 2017, a revamped bachelor of pharmacy program was introduced at Monash University and incorporated a predominantly flipped classroom-based pedagogy. The attitudes and preferences of students towards this program had yet to be assessed using a reliable instrument. Since no instrument was readily available, the objective of this study was to identify, contextualize, and validate a suitable instrument.We conducted a literature search to identify and adapt a validated instrument. Cognitive interviews were conducted to examine students' understanding of scales and definitions of items. The instrument was then evaluated by education experts for further refinement. The reliability of the final instrument was assessed in a cohort of students, and unsuitable items were removed.Students had issues understanding the scales and specific terms used in the original instrument, potentially due to differences in terminologies used in the university's context and variance in English proficiency levels and exposure. In the preference domain, wording of the instrument to present exclusively traditional classroom or exclusively flipped classroom statements greatly influenced its reliability. This could be due to exposure of students to a predominantly flipped classroom environment since inception. The final instrument optimized in this study had α = 0.85, 0.86, and 0.9 for the pre-activities, in-class lectures, and in-class workshops attitude domains, respectively, and α = 0.73 for the preference domain.Our study highlights the necessity of contextualizing instruments to fit the local context in which they are administered and provides key recommendations when conducting such adaptations.

Adenosine modulates many aspects of human physiology and pathophysiology through binding to the adenosine family of G protein-coupled receptors, which are comprised of four subtypes, the A1R, A2AR, A2BR and A3R. Modulation of adenosine receptor function by exogenous agonists, antagonists and allosteric modulators can be beneficial for a number of conditions including cardiovascular disease, Parkinson's disease, and cancer. Unfortunately, many preclinical drug candidates targeting adenosine receptors have failed in clinical trials due to limited efficacy and/or severe on-target undesired effects. To overcome the key barriers typically encountered when transitioning adenosine receptor ligands into the clinic, research efforts have focussed on exploiting the phenomenon of biased agonism. Biased agonism provides the opportunity to develop ligands that favour therapeutic signalling pathways, whilst avoiding signalling associated with on-target undesired effects. Recent studies have begun to define the structure-function relationships that underpin adenosine receptor biased agonism and establish how this phenomenon can be harnessed therapeutically. In this review we describe the recent advancements made towards achieving therapeutically relevant biased agonism at adenosine receptors.

The adenosine A

The adenosine A1 G protein-coupled receptor (A1AR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the A1AR orthosteric site is located within the receptor's transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein-coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of A1AR-ECL2 to explore the role of this domain on A1AR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5'-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in A1AR orthosteric ligand binding and receptor activation.

Aberrant ligand-independent G protein-coupled receptor constitutive activity has been implicated in the pathophysiology of a number of cancers. The adenosine A2B receptor (A2BAR) is dynamically upregulated under pathologic conditions associated with a hypoxic microenvironment, including solid tumors. This, in turn, may amplify ligand-independent A2BAR signal transduction. The contribution of A2BAR constitutive activity to disease progression is currently unknown yet of fundamental importance, as the preferred therapeutic modality for drugs designed to reduce A2BAR constitutive activity would be inverse agonism as opposed to neutral antagonism. The current study investigated A2BAR constitutive activity in a heterologous expression system and a native 22Rv1 human prostate cancer cell line exposed to hypoxic conditions (2% O2). The A2BAR inverse agonists, ZM241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol] or PSB-603 (8-(4-(4-(4-chlorophenyl)piperazide-1-sulfonyl)phenyl)-1-propylxanthine), mediated a concentration-dependent decrease in baseline cAMP levels in both cellular systems. Proliferation of multiple prostate cancer cell lines was also attenuated in the presence of PSB-603. Importantly, both the decrease in baseline cAMP accumulation and the reduction of proliferation were not influenced by the addition of adenosine deaminase, demonstrating that these effects are not dependent on stimulation of A2BARs by the endogenous agonist adenosine. Our study is the first to reveal that wild-type human A2BARs have high constitutive activity in both model and native cells. Furthermore, our findings demonstrate that this ligand-independent A2BAR constitutive activity is sufficient to promote prostate cancer cell proliferation in vitro. More broadly, A2BAR constitutive activity may have wider, currently unappreciated implications in pathologic conditions associated with a hypoxic microenvironment.

Objective. To examine the effects of student demographics, prior academic performance, course engagement, and time management on pharmacy students’ performance on course examinations and objective structured clinical examinations (OSCEs). Methods. Study participants were one cohort of pharmacy students enrolled in a five-year combined Bachelor and Master of Pharmacy degree program at one institution. Variables included student demographics, baseline factors (language assessment and situational judgement test scores), prior academic performance (high school admission rank), course engagement, and student time management of pre-class online activities. Data were collected from course, learning management system, and institutional databases. Data were analyzed for univariate, bivariate, and multivariate associations (four linear regression models) between explanatory factors and outcome variables. Results. Three years of data on 159 pharmacy students were obtained and entered in the dataset. Significant positive predictors of OSCE communication performance included domestic (ie, Australian) student designation, higher baseline written English proficiency, and pre-class online activity completion. Positive predictors of OSCE problem-solving included workshop attendance and low empathy as measured by a baseline situational judgment test (SJT). Positive predictors of performance on year 2 end-of-course examinations included the Australian Tertiary Academic Rank, completing pre-class online activities prior to lectures, and high integrity as measured by an SJT. Conclusion. Several explanatory factors predicted pharmacy students’ examination and OSCE performance in the regression models. Future research should continue to study additional contexts, explanatory factors, and outcome variables.

Clinical guidelines increasingly emphasise the importance of comprehensive and holistic care for older people. The objective of this education brief is to describe a workshop designed to improve first year pharmacy students' empathy and attitudes towards older people.A two-hour, interactive, university-based workshop was developed and evaluated. Small groups of first year pharmacy students (approximately five per group) worked with an older person to complete a number of scaffolded activities focused on the older person's life experiences with pharmacy and medication usage. The effectiveness of this intervention was measured using an eight-item, pre- and post-workshop survey adapted from published scales.Engaging older people as university-based instructors for first year pharmacy students was associated with significant improvements in three of the eight attitudinal items. Following the workshop, students were more likely to report that older people are: pleasant to be with (p 0.001), more likely to understand what it feels like to have problems with aging (p 0.005) and less likely to believe older people become less organised and more confused as they age (p 0.001).Engaging older people as university-based instructors for first year pharmacy students may be a useful strategy to develop empathy and positive attitudes towards older people. Further research is needed to determine if the attitudinal improvements are sustained over time.

Student engagement during classes includes behavioural, cognitive and emotional components, and is a pre-requisite for successful active learning environments. A novel approach to measuring student engagement was developed, involving triangulation of real-time student-self report, observation by trained observers and heart rate measurement. The self-report instrument was evaluated in four separate cohorts (n = 123) at Monash University and the University of North Carolina. The six item self-report demonstrated good reliability (Cronbach’s alpha values ranged from 0.7–0.81). The self-report showed predictive validity in that small group activities were rated as significantly more engaging than didactic lecturing. Additionally, there was significant inter-instructor variability and within-class variability, indicating good discrimination between classroom activities. This self-report may prove useful to academic teaching staff in evaluating and refining their active learning activities. Independent observation was not found to correlate with student self-report, due in part to students who were pretending to engage being rated as engaged by an observer. Strikingly, students reported that they were pretending to engage for 23% of class time, even for highly regarded instructors. Individual participants were rated as engaged for 42 of the 46 intervals for which they reported that they had “pretended to engage”, indicating that the two observers were unable to detect disengagement during periods in which students pretended to engage. Instructors should be aware that student cues such as eye contact and nodding may indicate pretending to engage. One particular self-report item; “I tried a new approach or way of thinking about the content”, correlated positively with heart rates, and a controlled study reproduced this finding during two activities that required students to try a new approach to understanding a concept. Agreement with this item also correlated with superior performance on two in-class written assessment tasks (n = 101, p

Adenosine receptors are a family of GPCRs containing four subtypes (A(1), A(2A), A(2B) and A(3) receptors), all of which bind the ubiquitous nucleoside adenosine. These receptors play an important role in physiology and pathophysiology and therefore represent attractive drug targets for a range of conditions. The theoretical framework surrounding drug action at adenosine receptors now extends beyond the notion of prototypical agonism and antagonism to encompass more complex pharmacological concepts. New paradigms include allostery, in which ligands bind a topographically distinct receptor site from that of the endogenous agonist, homomeric or heteromeric interactions across receptor oligomers and biased agonism, that is, ligand‐dependent differential intracellular signalling. This review provides a concise overview of allostery, oligomerization and biased agonism at adenosine receptors and outlines how these paradigms may enhance future drug discovery endeavours focussed on the development of novel therapeutic agents acting at adenosine receptors. LINKED ARTICLES: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc

The adenosine A

The A

We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology.

Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites.

A concise synthesis of a series of N(6)-substituted adenosines with bicyclo[3.2.1]octan-6-yl and polycyclic N(6)-substituents has been developed. The adenosine A(1) receptor (A(1)R) affinity and potency of these compounds was initially assessed using competitive binding assays and cyclic adenosine monophosphate (cAMP) accumulation assays in DDT(1) MF-2 cells. The potency and receptor subtype selectivity of selected examples was further evaluated by measuring their effects on cAMP accumulation at all human adenosine receptor subtypes expressed in CHO cells. The results of these assays indicated that all of the synthesised N(6)-substituted adenosines are full agonists at A(1) R and activate this receptor selectively over the other adenosine receptor subtypes. The two standout compounds in terms of potency were N(6)-(3-thiabicyclo[3.2.1]octan-6-yl)adenosine and N(6)-(cubanylmethyl)adenosine with EC(50) values at human A(1)R of 2.3 nM and 1.1 nM, respectively. The cubanylmethyl derivative in particular proved to be highly receptor subtype selective. These two compounds were further evaluated in a simulated ischaemia model in cultured cardiomyoblasts, where they were found to impart protective effects under hypoxic conditions that resulted in a significant reduction in cell death.

Item does not contain fulltext BACKGROUND: Despite policy and financial support for patient-experience surveys as feedback to modify clinical practice, their benefits and other effects remain unclear. AIM: This study aimed to examine the perspective of GPs who have engaged with such tools. METHOD: Qualitative semi-structured interviews with 30 GPs across south Wales, UK. The transcripts were analysed using a constant comparative method. RESULTS: The participants regarded patient feedback as highly important but felt that patient-experience surveys, as currently implemented, were hard to interpret. Reflecting on the doctor-patient interaction, participants perceived ambiguity about whether results were evaluating the individual or the GP practice, and whether they were directed towards the 'practitioner-as-person' or towards specific behaviours. A potential threat was posed in both interpretations, with the risk of damaging consequences for the practitioner and the nature of general practice. The tension between satisfying patients and performing good health care was a clearly voiced dilemma. Doctors did not feel confident in their ability to change in response to feedback, particularly outside a formal training environment. CONCLUSION: Patient evaluation is seen as a key part of multisource feedback for practising doctors. Clarity is needed about the purpose of evaluation - whether for organisational or personal 'feedback', and whether formative or summative - and there is a need for attention to the process, with trained facilitators, to ensure that doctors gain from structured reflection, and can minimise the potential negative effects of evaluation.

Antisense oligonucleotides are emerging as an exciting therapeutic strategy for treating skin diseases such as psoriasis. Potential antisense targets are proteins upregulated in psoriatic skin, in particular those associated with inflammation (intercellular adhesion molecule [ICAM]-1, IL-2 and -8), proliferation (insulin-like growth factor type I receptor [IGF-IR], epidermal growth factor) and hyperangiogenesis (vascular endothelial growth factor [VEGF]). Whereas topical application and subsequent penetration of large oligonucleotides into normal skin is problematic, the impaired barrier function of psoriatic lesions permits the uptake of antisense drugs. Studies to date indicate that topically applied antisense molecules can be delivered to target cells in the epidermis and dermis of psoriatic skin. Antisense-mediated suppression of target mRNA and protein has been demonstrated in models of human skin grafted to immunosuppressed mice and in hairless mouse models of skin inflammation. In a xenograft model of human psoriasis, treatment with repeated intradermal injections of IGF-IR antisense caused a normalisation of the epidermal hyperproliferation. This class of drug, therefore, holds much potential for the successful treatment of psoriasis in the clinical setting.

We have previously shown that antisense oligonucleotides effectively reduced insulin-like growth factor I receptor expression in human psoriatic skin grafted on to nude mice when injected intradermally. We therefore investigated the penetration of C-5 propyne modified antisense oligonucleotides into human normal and psoriatic skin after topical administration. Oligonucleotide (37.5 microg; 250 microM) was applied in aqueous solution or 5% methylcellulose gel for 24 h, prior to live confocal microscopy and fluorescence microscopy of fixed sections. We found that oligonucleotide could penetrate through the stratum corneum of psoriatic but not normal human skin over large regions of the epidermis. The oligonucleotide was localized to the nucleus of large parakeratotic cells in the psoriatic skin as well as smaller basal and suprabasal keratinocytes. In normal human skin, oligonucleotide was confined to the stratum corneum, with little or no oligonucleotide apparent in the viable epidermis. Electrophoresis of oligonucleotide recovered from treated psoriatic and normal skin revealed that the oligonucleotide remained intact over the 24 h period. In summary, we found that C-5 propyne modified antisense oligonucleotides could reach the target cells (in this case basal keratinocytes) after topical administration to psoriatic but not normal skin.

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.

Anti-sense oligonucleotide uptake by keratinocytes in human skin grafts on athymic mice was examined using live confocal microscopy. Fluorescein isothiocyanate-labeled 15-mer C-5 propyne modified phosphorothioate anti-sense oligonucleotide (10-50 microM) was intradermally injected into normal human skin grafts on athymic mice, and the localization of the anti-sense oligonucleotide was assessed after 1-24 h postinjection. Anti-sense oligonucleotide was found to localize in the nuclei of basal and suprabasal keratinocytes after 1-2 h, and this localization was still observed after 24 h. This live in vivo observation of anti-sense oligonucleotide uptake in basal keratinocytes was confirmed using conventional fluorescence microscopy of fixed sections of skin grafts. Neither single nucleotides which were fluorescein isothiocyanate-labeled nor fluorescein isothiocyanate alone was able to penetrate into the nuclei of human skin graft keratinocytes after intradermal injection, and hence it is likely that the anti-sense oligonucleotide was not degraded prior to intracellular localization. Topical administration of anti-sense oligonucleotide and anti-sense oligonucleotide-liposome complexes resulted primarily in localization in the stratum corneum of human skin grafts. When grafts were tape stripped prior to anti-sense oligonucleotide administration, however, as little as 5 microM anti-sense oligonucleotide was required to observe nuclear anti-sense oligonucleotide accumulation. These results suggest that cutaneous anti-sense strategies can be tested using delivery via intradermal anti-sense oligonucleotide injection in human skin grafts on athymic mice, and that agents providing penetration of anti-sense oligonucleotide across the stratum corneum are likely to be required for successful topical therapies.

The success of anti-sense strategies has been limited, at least in part, by the poor uptake of these agents into the target cells. In keratinocytes, there is conflicting evidence as to the amount and location of oligonucleotide uptake into these cells, with variable proportions of cells reported to take up oligodeoxynucleotide, and also cytoplasmic and nuclear localization reported. In this study, the uptake of oligodeoxynucleotides in cultured normal human keratinocytes and the HaCaT cell line was quantitated in the presence of various lipids designed to enhance uptake and in varying culture conditions. About 12% of cells in a confluent normal human keratinocyte culture showed nuclear uptake, with a small and variable proportion showing cytoplasmic localization after 24 h incubation with 1 microM oligodeoxynucleotide. Uptake of oligodeoxynucleotide was found to be increased by liposome encapsulation (to a maximum of 28.1% +/- 2.1% of cells), low confluence (39.5% +/- 2.5%), and further increased by a combination of the two conditions (55.4% +/- 4.3%). HaCaT cell populations showed sparse but consistent uptake of oligodeoxynucleotide, with about 1% of cells showing nuclear localization in the presence of 1 microM oligodeoxynucleotide, increasing to 13.5% +/- 4.9% in the presence of cationic lipid (Tfx-50) in low confluence HaCaT monolayers. We conclude that normal keratinocytes exhibit reliable, substantial uptake of oligonucleotides in conditions controlled for confluence and aided by liposome encapsulation.

A series of adenosine-5'-N-alkylcarboxamides and N(6)-(2,2-diphenylethyl)adenosine-5'-N-alkylcarboxamides bearing antioxidant moieties in the 2-position were synthesized from the versatile intermediate, O(6)-(benzotriazol-1-yl)-2-fluoro-2',3'-O-isopropylideneinosine-5'-N-alkylcarboxamide (1). These compounds were evaluated as A(2A) adenosine receptor (A(2A)R) agonists in a cAMP accumulation assay, and a number of potent and selective agonists were identified. Three of these compounds were evaluated further in an ischemic injury cell survival assay and a reactive oxygen species (ROS) production assay whereby 15b and 15c were shown to reduce ROS activity and cell death due to ischemia.

Recent results using animal models of inflammatory skin conditions have shown that blockers of the voltage-gated potassium channel, Kv1.3 hold great promise for clinical utility. Kv1.3 blockers act as immunosuppressants by modulating the various subsets of inflammatory T and B cells involved in autoimmune disorders. While peptidic inhibitors based on naturally occurring venoms demonstrate potent and selective Kv1.3 blockade, these require parenteral administration and may face potential immunogenicity problems. Small molecule blockers show considerable diversity, however selectivity over other Kv1- family channels has been difficult to achieve. More recent advances have added to the evidence that Kv1.3 channels are a suitable therapeutic target and that the development of novel and selective agents will herald new drugs for inflammatory skin disorders.

Antisense oligonucleotides as a therapeutic platform have been slow to progress since the approval of the first antisense drug in 1998. Recently, there have been several examples of convincing antisense interventions in animal models and promising clinical trial data. This review considers the factors determining the success of antisense oligonucleotides as therapeutic agents. In order to produce target knockdown after systemic delivery, antisense oligonucleotides must avoid nuclease degradation, reticuloendothelial-system uptake and rapid renal excretion, and extravasate to the target cell type outside the vasculature. They then must enter the target cell, and escape the endosome–lysosome pathway so as to be free to interact with the target mRNA. We consider the significance of these limiting factors based on the literature and our own experience using systemic administration of antisense oligonucleotides.

1. RNA interference in vivo has tremendous potential, both with respect to the elucidation of protein function in animals and as a therapeutic platform in humans. In vitro, short interfering RNA (siRNA) has been shown to completely silence gene expression in mammalian cells at low picomolar concentrations. 2. Although many good publications have shown specific silencing to occur in vivo, there are few that have transferred the combination of maximal efficacy and high potency to this setting. The present review considers the biological barriers that limit the movement of siRNA from vascular lumen to target cell cytoplasm and the strategies that have been used to overcome them. 3. Intravenous administration of siRNA results in rapid, extensive removal of siRNA from the blood via renal excretion, tissue distribution and nuclease degradation. Movement across vascular capillaries appears to be a limiting factor in some cases; few examples of silencing have been reported in organs with a conventional capillary endothelium. 4. Cellular uptake and endosomal trapping are significant barriers, but can be overcome using strategies such as antibody mediated cellular uptake or polyethyleneimine-mediated endosomal escape.

Purpose: P-glycoprotein (Pgp) is a major prognostic factor for chemotherapy failure in acute myeloid leukemia (AML). This study compared the influence of genetic and leukemia-specific factors on Pgp. Experimental Design: Eight hundred and seventeen samples were studied prospectively for Pgp protein expression and function and G1199A, G2677T, and C3435T polymorphisms in the encoding gene ABCB1. Results: Age, low WBC count, high bcl-2, secondary AML and myelodysplastic syndrome, and adverse cytogenetics all correlated strongly with high Pgp (MRK16) protein expression. However, ABCB1 3435TT homozygosity was negatively correlated with Pgp. Pgp protein is only expressed in 41% of samples such that the negative effect of the polymorphism was not seen at baseline Pgp levels but was marked in the upper 41% of samples (MRK16 Δmean fluorescence intensity of 75th centile sample = 9 units for TT variant samples and 26 units for CC/CT; P = 0.003). However, no association was found between genetic factors and Pgp function using rhodamine 123 accumulation. Conclusions: The genetic polymorphism 3435TT (which results in unstable mRNA) has a significant effect on Pgp expression, but this is only seen in ∼40% of cases in which mRNA and protein are detectable. Moreover, leukemia-specific factors, such as low WBC count and poor risk cytogenetics, have a much greater effect than genetic polymorphisms on Pgp expression in AML blasts.

Human keratinocytes are highly responsive to mitogenic and antiapoptotic signaling by the insulin-like growth factor-I receptor (IGF-IR). IGF-IR hyperstimulation is a feature of hyperplastic skin conditions, making the IGF-IR an appealing target for antisense therapeutic intervention. In this study, we used a C5-propynyl-dU,dC-phosphorothioate oligo-2'-deoxyribonucleotide antisense 15-mer to the human IGF-IR mRNA, along with liposome transfection, to inhibit IGF-IR activity in a human keratinocyte cell line and demonstrated potent inhibition of cell growth despite the presence of serum. To investigate the sequence specificity of these effects and to establish the concentration range over which a purely antisense effect could be demonstrated, we introduced 1, 2, 4, 8, and 15 base mismatches into the oligonucleotide and analyzed changes in inhibitory efficacy. In the 10-30 nM concentration range, the introduction of 1 and 2 mismatches into the middle of the 15-mer only modestly affected inhibitory efficacy, whereas >4 mismatches profoundly reduced mRNA, protein, and growth-inhibitory effects. From these results, we conclude that (1) sequence-specific antisense inhibition of IGF-IR activity in keratinocytes is achievable, (2) potent anti-IGF-IR antisense inhibition can be achieved in vitro at concentrations as low as 10 nM, and (3) a sequence-dependent mechanism is likely to underpin the observed in vivo therapeutic effects (Wraight et al. Nat. Biotechnol. 2000;18:521) of these antisense oligonucleotides (AS-ODN) in cutaneous hyperplastic disorders, such as psoriasis.

Antisense oligonucleotides have been the subject of intense interest as research tools to elucidate the functions of gene products and as therapeutic agents. Initially, their mode of action was poorly understood and the biological effects of oligonucleotides were often misinterpreted. However, research into these gene-based inhibitors of cellular action recently has succeeded in realising their exciting potential, particularly as novel therapeutic agents. An emerging application of this technology is in cutaneous therapy. The demand for more effective dermatological drugs will ensure further development of antisense strategies in skin, with key issues being drug delivery, therapeutic target selection, and clinical applicability.

Antisense strategies targeting skin conditions are attractive in concept, with a number of possible pathologic conditions, such a psoriasis, apparently suitable for such an approach. Because in vitro screening of candidate sequences is usually desirable, we have attempted to use a range of new generation cationic lipids to produce significant antisense oligodeoxynucleotide (ODN) uptake in an immortalized keratinocyte cell line (HaCaT). A large number of commercially available lipids were screened for the ability to induce nuclear ODN localization: Tfx-50, Tfx-20, Tfx-10, Superfect, Cytofectin GSV, Perfect lipids 1-8, Lipofectin, and Lipofectamine. All lipids were used at a range of concentrations (1-20 microg/ml) and with a range of ODN concentrations (1-1000) nM). Of all lipids used, only Cytofectin GSV and Superfect produced significant (>30% of cells) levels of nuclear positive cells, with Superfect also producing significant toxicity at the effective concentration used. Only two treatments produced a significant reduction in target mRNA: insulin-like growth factor-1 receptor (IGF-1R)-ODN 64 complexed with Cytofectin GSV (27.1% +/- 3.5% of IGF-1R mRNA in untreated cells,p < 0.01) and ODN 64 complexed with 10 microg/ml Lipofectin (62.2% +/- 3.4% of IGF-1R mRNA in untreated cells, p < 0.05). Only one treatment, ODN 64 complexed with Cytofectin GSV, produced a reduction in cell growth and survival as assessed by amido black assay. These results demonstrate that in HaCaT keratinocytes, Cytofectin GSV alone of all commercially available cationic lipids was effective in delivering antisense ODN into cell nuclei such that a profound antisense effect could be demonstrated.

Insulin-like growth factor-I (IGF-I) stimulation of basal keratinocytes is an essential component of normal epidermal homeostasis. In addition to the IGF receptor, basal keratinocytes synthesize insulin-like growth factor binding protein-3 (IGFBP-3). The HaCaT keratinocyte cell line, which has many characteristics of basal keratinocytes, synthesizes IGFBP-3 that in vitro reduces its IGF-I responsiveness. IGFBP-3 has attracted interest as a potential growth arrest protein, both via its ability to modulate IGF-I responsiveness, and more controversially via IGF-I-independent mechanisms. Intracellular modes of action have been proposed, and a nuclear localization consensus sequence has previously been identified within IGFBP-3. Using immunocytochemistry with a biotinylated antibody specific for IGFBP-3, we investigated the intracellular localization of IGFBP-3 in subconfluent monolayer cultures of HaCaT cells. Diffuse cellular staining was visible, potentially corresponding to cell surface and nascent cytoplasmic IGFBP-3. Of particular interest however, was the localization of staining over the nuclei of a large proportion of cells that were undergoing cell division. Antibody staining was specific for IGFBP-3 because addition of recombinant human IGFBP-3 to the antibody prior to incubation with the cells inhibited these staining patterns. Optical sections obtained using a confocal laser scanning microscope showed that in keratinocytes undergoing cell division, IGFBP-3 was localized inside the nucleus. These results show that intracellular IGFBP-3 localization is altered during the cell cycle and suggest a possible nuclear role for IGFBP-3 during cell division.

Objective: To investigate the relationship between student engagement with the key elements of a flipped classroom approach (preparation and attendance), their attitudes to learning, including strategy development, and their performance on two types of examination questions (knowledge recall and providing rational predictions when faced with novel scenarios). Methods. This study correlated student engagement with the flipped classroom and student disposition to learning with student ability to solve novel scenarios in examinations. Results. Students who both prepared for and attended classes performed significantly better on examination questions that required analysis of novel scenarios compared to students who did not prepare and missed classes. However, there was no difference for both groups of students on examination questions that required knowledge and comprehension. Student motivation and use of strategies correlated with higher examination scores on questions requiring novel scenario analysis. Conclusion. There is a synergistic relationship between class preparation and attendance. The combination of preparation and attendance was positively correlated to assessment type; the relationship was apparent for questions requiring students to solve novel problems but not for questions requiring knowledge or comprehension.