Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinctive mechanoresponses during the early stages of hPSC differentiation. Taken together, these results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs and integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.

Crossover of surface waves and capillary-viscous-elastic transition in soft biomaterials detected by resonant acoustic rheometry.

Viscoelastic properties of hydrogels are important for their application in science and industry. However, rheological assessment of soft hydrogel biomaterials is challenging due to their complex, rapid, and often time-dependent behaviors. Resonant acoustic rheometry (RAR) is a newly developed technique capable of inducing and measuring resonant surface waves in samples in a non-contact fashion. By applying RAR at high temporal resolution during thrombin-induced fibrin gelation and ultraviolet-initiated polyethylene glycol (PEG) polymerization, we observed distinct changes in both frequency and amplitude of the resonant surface waves as the materials changed over time. RAR detected a series of capillary-elastic, capillary-viscous, and visco-elastic transitions that are uniquely manifested as crossover of different types of surface waves in the temporally evolving materials. These results reveal the dynamic interplay of surface tension, viscosity, and elasticity that is controlled by the kinetics of polymerization and crosslinking during hydrogel formation. RAR overcomes many limitations of conventional rheological approaches by offering a new way to comprehensively and longitudinally characterize soft materials during dynamic processes.

Resonant Acoustic Rheometry to Measure Coagulation Kinetics in Hemophilia A and Healthy Plasma: A Novel Viscoelastic Method.

Compared with conventional coagulation tests and factor-specific assays, viscoelastic hemostatic assays (VHAs) can provide a more thorough evaluation of clot formation and lysis but have several limitations including clot deformation. In this proof-of-concept study, we test a noncontact technique, termed resonant acoustic rheometry (RAR), for measuring the kinetics of human plasma coagulation. Specifically, RAR utilizes a dual-mode ultrasound technique to induce and detect surface oscillation of blood samples without direct physical contact and measures the resonant frequency of the surface oscillation over time, which is reflective of the viscoelasticity of the sample. Analysis of RAR results of normal plasma allowed defining a set of parameters for quantifying coagulation. RAR detected a flat-line tracing of resonant frequency in hemophilia A plasma that was corrected with the addition of tissue factor. Our RAR results captured the kinetics of plasma coagulation and the newly defined RAR parameters correlated with increasing tissue factor concentration in both healthy and hemophilia A plasma. These findings demonstrate the feasibility of RAR as a novel approach for VHA, providing the foundation for future studies to compare RAR parameters to conventional coagulation tests, factor-specific assays, and VHA parameters.

High Frequency Spectral Ultrasound Imaging Detects Early Heterotopic Ossification in Rodents.

Heterotopic ossification (HO) is a devastating condition in which ectopic bone forms inappropriately in soft tissues following traumatic injuries and orthopedic surgeries as a result of aberrant mesenchymal progenitor cell (MPC) differentiation. HO leads to chronic pain, decreased range of motion, and an overall decrease in quality of life. While several treatments have shown promise in animal models, all must be given during early stages of formation. Methods for early determination of whether and where endochondral ossification/soft tissue mineralization (HO anlagen) develop are lacking. At-risk patients are not identified sufficiently early in the process of MPC differentiation and soft tissue endochondral ossification for potential treatments to be effective. Hence, a critical need exists to develop technologies capable of detecting HO anlagen soon after trauma, when treatments are most effective. In this study, we investigate high frequency spectral ultrasound imaging (SUSI) as a noninvasive strategy to identify HO anlagen at early time points after injury. We show that by determining quantitative parameters based on tissue organization and structure, SUSI identifies HO anlagen as early as 1-week postinjury in a mouse model of burn/tenotomy and 3 days postinjury in a rat model of blast/amputation. We analyze single cell RNA sequencing profiles of the MPCs responsible for HO formation and show that the early tissue changes detected by SUSI match chondrogenic and osteogenic gene expression in this population. SUSI identifies sites of soft tissue endochondral ossification at early stages of HO formation so that effective intervention can be targeted when and where it is needed following trauma-induced injury. Furthermore, we characterize the chondrogenic to osteogenic transition that occurs in the MPCs during HO formation and correlate gene expression to SUSI detection of the HO anlagen.

Resonant acoustic rheometry for non-contact characterization of viscoelastic biomaterials.

Resonant Acoustic Rheometry (RAR) is a new, non-contact technique to characterize the mechanical properties of soft and viscoelastic biomaterials, such as hydrogels, that are used to mimic the extracellular matrix in tissue engineering. RAR uses a focused ultrasound pulse to generate a microscale perturbation at the sample surface and tracks the ensuing surface wave using pulse-echo ultrasound. The frequency spectrum of the resonant surface waves is analyzed to extract viscoelastic material properties. In this study, RAR was used to characterize fibrin, gelatin, and agarose hydrogels. Single time point measurements of gelled samples with static mechanical properties showed that RAR provided consistent quantitative data and measured intrinsic material characteristics independent of ultrasound parameters. RAR was also used to longitudinally track dynamic changes in viscoelastic properties over the course of fibrin gelation, revealing distinct phase and material property transitions. Application of RAR was verified using finite element modeling and the results were validated against rotational shear rheometry. Importantly, RAR circumvents some limitations of conventional rheology methods and can be performed in a high-throughput manner using conventional labware. Overall, these studies demonstrate that RAR can be a valuable tool to noninvasively quantify the viscoelastic mechanical properties of soft hydrogel biomaterials.

Modes of Action of a Bi-domain Plant Defensin MtDef5 Against a Bacterial Pathogen Xanthomonas campestris.

Defensins are small cysteine-rich endogenous host defense peptides expressed in all higher plants. They are thought to be important players in the defense arsenal of plants against fungal and oomycete pathogens. However, little is known regarding the antibacterial activity of these peptides. The genome of the model legume Medicago truncatula contains 63 genes each encoding a defensin with a tetradisulfide array. A unique bi-domain defensin, designated MtDef5, was recently characterized for its potent broad-spectrum antifungal activity. This 107-amino acid defensin contains two domains, 50 amino acids each, linked by a short peptide APKKVEP. Here, we characterize antibacterial activity of this defensin and its two domains, MtDef5A and MtDef5B, against two economically important plant bacterial pathogens, Gram-negative Xanthomonas campestris and Gram-positive Clavibacter michiganensis. MtDef5 inhibits the growth of X. campestris, but not C. michiganensis, at micromolar concentrations. MtDef5B, but not MtDef5A, exhibits more potent antibacterial activity than its parent MtDef5. MtDef5 and each of its two domains induce distinct morphological changes and cell death in X. campestris. They permeabilize the bacterial plasma membrane and translocate across membranes to the cytoplasm. They bind to negatively charged DNA indicating these peptides may kill bacterial cells by inhibiting DNA synthesis and/or transcription. The cationic amino acids present in the two γ-core motifs of MtDef5 that were previously shown to be important for its antifungal activity are also important for its antibacterial activity. MtDef5 and its more potent single domain MtDef5B have the potential to be deployed as antibacterial agents for control of a Xanthomonas wilt disease in transgenic crops.

Systems analysis of dynamic transcription factor activity identifies targets for treatment in Olaparib resistant cancer cells.

The development of resistance to targeted therapeutics is a challenging issue for the treatment of cancer. Cancers that have mutations in BRCA, a DNA repair protein, have been treated with poly(ADP-ribose) polymerase (PARP) inhibitors, which target a second DNA repair mechanism with the aim of inducing synthetic lethality. While these inhibitors have shown promise clinically, the development of resistance can limit their effectiveness as a therapy. This study investigated mechanisms of resistance in BRCA-mutated cancer cells (HCC1937) to Olaparib (AZD2281) using TRACER, a technique for measuring dynamics of transcription factor (TF) activity in living cells. TF activity was monitored in the parental HCC1937 cell line and two distinct resistant cell lines, one with restored wild-type BRCA1 and one with acquired resistance independent of BRCA1 for 48 h during treatment with Olaparib. Partial least squares discriminant analysis (PLSDA) was used to categorize the three cell types based on TF activity, and network analysis was used to investigate the mechanism of early response to Olaparib in the study cells. NOTCH signaling was identified as a common pathway linked to resistance in both Olaparib-resistant cell types. Western blotting confirmed upregulation of NOTCH protein, and sensitivity to Olaparib was restored through co-treatment with a gamma secretase inhibitor. The identification of NOTCH signaling as a common pathway contributing to PARP inhibitor resistance by TRACER indicates the efficacy of transcription factor dynamics in identifying targets for intervention in treatment-resistant cancer and provides a new method for determining effective strategies for directed chemotherapy. Biotechnol. Bioeng. 2017;114: 2085-2095. © 2017 Wiley Periodicals, Inc.

Effects of chromosomal deletion of the operon encoding the multiple resistance and pH-related antiporter in Vibrio cholerae.

To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.

Staging a reflective capstone course to transition PharmD graduates to professional life.

OBJECTIVE: To develop and implement a capstone course that would allow students to reflect on their development as a professional, assess and share their achievement of the college's outcomes, complete a professional portfolio, establish a continuing professional development plan, and prepare to enter the pharmacy profession. DESIGN: Students were required to complete a hybrid course built around 4 online and inclass projects during the final semester of the curriculum. ASSESSMENT: Faculty used direct measures of learning, such as reading student portfolios and program outcome reflections, evaluating professional development plans, and directly observing each student in a video presentation. All projects were evaluated using standardized rubrics. Since 2012, all graduating students met the course's minimum performance requirements. CONCLUSION: The course provided an opportunity for student-based summative evaluation, direct observation of student skills, and documentation of outcome completion as a means of evaluating readiness to enter the profession.

Meeting the challenge of public health information delivery in the digital age.

OBJECTIVE: To discuss the current status of public health messaging and how pharmacists can become more active participants. SUMMARY: Public health needs can be addressed by using Internet videos as a model to disseminate medical information. Introducing student pharmacists to new ways of developing and delivering targeted public health messages can increase their awareness that public health intervention is part of the emerging scope of practice. CONCLUSION: For pharmacy to affect change in public health at the local and national level, pharmacists should consider providing public health advocacy messages through virtual platforms.

Using focus groups to inform pharmacy research.

BACKGROUND: Focus groups are a powerful research tool for collecting qualitative information across many contexts. The focus group offers pharmacy researchers benefits that support many of the important lines of investigation at the forefront of contemporary pharmacy-based research, particularly within the areas of patient compliance/concordance, customer behavior, patient-provider collaboration, health literacy research, and disease management. This article introduces the focus group as a research method that offers powerful investigative potential to researchers who are attempting to understand human-based phenomena. OBJECTIVES: To provide sufficient background, examples, and how to information to enable a pharmacy researcher to include focus group methodologies in their initial design decisions, and provide guidance to additional resources necessary for successful implementation of this powerful qualitative approach. METHODS: The article is organized into sections describing what a focus group is and what it can be used for; the unique benefits and drawbacks of using focus group methodology; organization and planning considerations including participant and recruitment considerations; and sampling strategies, session and question development, practical details of session management, and follow-up activities, including data analysis. RESULTS/CONCLUSION: Although often considered quick and easy focus groups require thoughtful consideration of need and purpose, considerable planning, and effort to succeed. Because of the unique insight that can be gained, their flexibility, and their ability to mesh with other methods, focus group is gaining currency as an important research tool within health care.

Pharmacist-facilitated enrollment in medication assistance programs in a private ambulatory care clinic.

PURPOSE: Efforts to reduce patients' out-of-pocket expenses for prescription drugs by incorporating pharmaceutical manufacturer assistance programs (PMAPs) into a private ambulatory care facility's pharmaceutical services are described. SUMMARY: From March 2001 through March 2002, the clinic's pharmacist prospectively evaluated patients for PMAP enrollment. The pharmacist completed the pharmaceutical manufacturer's application forms for patients identified as candidates for PMAP assistance and helped them obtain documentation. Some companies sent medications directly to patients, and others sent them to the clinic, where they were stored until the patients picked them up. The patients informed the pharmacist when refills were needed, which allowed the pharmacist to review patient records, verify compliance, and ensure proper follow-up. Forty-four patients were enrolled in 22 PMAPs during the year. The mean +/- S.D. annual household income for all patients was 15,631 dollars +/- 7,845 dollars. A total of 115 medication orders were processed for the patients. The mean +/- S.D. number of medications per patient obtained through PMAPs was 2.5 +/- 1.8, and the mean +/- S.D. number of refills was 1.8 +/- 1.0. The total cost of the medications was 48,143 dollars. The pharmacist needed an average of 15-20 minutes to perform the initial patient interview, 10-15 minutes to complete each initial application, and 5-15 minutes to complete a renewal application. Total pharmacist time needed to process each medication order was estimated at about one hour per year. CONCLUSION: A pharmacist in a private ambulatory care facility helped patients enroll in PMAPs and reduced the patients' expenses for prescription drugs.

Improving aspirin prophylaxis in a primary care diabetic population.

STUDY OBJECTIVE: To evaluate and improve adherence to American Diabetes Association guidelines for prophylactic aspirin therapy in ambulatory patients with diabetes using a pharmacy-directed intervention. DESIGN: Unblinded, single intervention. SETTING: Rural, primary care clinic. SUBJECTS: Eighty-five patients with a diagnosis of diabetes mellitus. INTERVENTION: Patients with diabetes were identified from database searches and routine clinic visits. Medical records were screened for aspirin use, allergies, adverse events, and contraindications. During routine clinic visits or structured telephone interviews, patients with indications for aspirin therapy were advised to begin enteric-coated aspirin 81 mg/day A follow-up survey assessed adherence. MEASUREMENTS AND MAIN RESULTS: At baseline, 28 (33%) of 85 patients were receiving aspirin therapy An additional 8 patients had contraindications to aspirin, and 2 patients had no indications for aspirin therapy Aspirin was recommended to 27 patients during clinic interventions and to 15 patients during telephone interventions. Two patients declined the recommendation. At the completion of this intervention, 70 (82%) of 85 patients were receiving daily aspirin or had accepted the recommendation to begin therapy. CONCLUSIONS: A pharmacy-directed intervention increased prophylactic aspirin therapy in patients with diabetes from 33% of patients at baseline to 82% at the end of the study The intervention, which has a simple, patient-focused design, serves as a template for improving aspirin prophylaxis among patients with diabetes in other ambulatory settings.

Writing tasks performed by doctor of pharmacy students during clerkship rotations.

The range of writing tasks undertaken by students during doctor of pharmacy clerkship rotations was studied. Data collection involved a review between August and November 1998 of writing samples selected by postbaccalaureate Pharm. D. students at Albany College of Pharmacy for inclusion in their required writing portfolios. The first 200 samples (accounted for by 35 students each submitting two documents for each of three clerkship rotations) were reviewed. Of these, 198 were coded to identify the four rhetorical components of clerkship location, document type, intended audience, and rhetorical purpose. Institutional sites served as the clerkship location for 164 (82.8%) of the 198 documents analyzed. The documents were placed in 28 categories; 5 of these accounted for 126 (63.6%) of the documents: 45 inservice presentations, 32 summaries, 18 patient case write-ups, 16 formulary reviews, and 15 newsletters. Students wrote most frequently to health care providers (34.8%), other pharmacists (32.3%), and teachers (16.7%), with the most frequent rhetorical purposes being informing (73.2%) and demonstrating (14.6%). Analysis of writing samples prepared by pharmacy students during clerkship rotations revealed a variety of clerkship sites, document types, audiences, and rhetorical purposes.

On-the-job writing tasks of clerkship preceptors.

The frequency with which various types of documents were written on-the-job by Pharm. D. clerkship preceptors was studied, along with the value that these documents added to their professional practice. A survey was mailed in April 1999 to 129 practicing pharmacists serving as preceptors for Albany College of Pharmacy Pharm. D. clerkship rotations. The survey asked recipients to indicate the frequency with which they wrote each of 23 types of documents and how valuable it was to their practice. In addition, participants were invited to identify documents they wrote that were not on the list. Sixty-six preceptors returned usable surveys (response rate, 51%). Sixty-four (97%) had either direct or indirect patient care responsibilities. Four types of documents (memorandum or letter, pharmacy care plan, progress notes, and patient consultation notes) were written daily, weekly, or monthly. Sixteen of the 23 document types were rated as highly valuable; of these, most were written at least quarterly and 1 was written daily. The respondents indicated 15 additional types of documents they generated in their practice; 11 of these were rated as being of high or highest value. Clerkship preceptors reported writing numerous types of documents. Document types that were written most often were generally considered valuable to the respondents' practice.