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1.  Patterning human neuronal networks on photolithographically engineered silicon dioxide substrates functionalized with glial analogues 
Interfacing neurons with silicon semiconductors is a challenge being tackled through various bioengineering approaches. Such constructs inform our understanding of neuronal coding and learning and ultimately guide us toward creating intelligent neuroprostheses. A fundamental prerequisite is to dictate the spatial organization of neuronal cells. We sought to pattern neurons using photolithographically defined arrays of polymer parylene-C, activated with fetal calf serum. We used a purified human neuronal cell line [Lund human mesencephalic (LUHMES)] to establish whether neurons remain viable when isolated on-chip or whether they require a supporting cell substrate. When cultured in isolation, LUHMES neurons failed to pattern and did not show any morphological signs of differentiation. We therefore sought a cell type with which to prepattern parylene regions, hypothesizing that this cellular template would enable secondary neuronal adhesion and network formation. From a range of cell lines tested, human embryonal kidney (HEK) 293 cells patterned with highest accuracy. LUHMES neurons adhered to pre-established HEK 293 cell clusters and this coculture environment promoted morphological differentiation of neurons. Neurites extended between islands of adherent cell somata, creating an orthogonally arranged neuronal network. HEK 293 cells appear to fulfill a role analogous to glia, dictating cell adhesion, and generating an environment conducive to neuronal survival. We next replaced HEK 293 cells with slower growing glioma-derived precursors. These primary human cells patterned accurately on parylene and provided a similarly effective scaffold for neuronal adhesion. These findings advance the use of this microfabrication-compatible platform for neuronal patterning.
PMCID: PMC4243028  PMID: 23733444
parylene; photolithography; cell patterning; neuron; silicon dioxide
2.  Live birth following serial vitrification of embryos and PGD for fragile X syndrome in a patient with the premutation and decreased ovarian reserve 
To present a live birth resulting from serial vitrification of embryos and pre-implantation genetic diagnosis (PGD).
A 31-year-old with primary infertility, fragile-X premutation, and decreased ovarian reserve (DOR) (baseline FSH level 33 IU/L), presented after failing to stimulate to follicle diameters >10 mm with three cycles of invitro fertilization (IVF). After counseling, the couple opted for serial in-vitro maturation (IVM), embryo vitrification, and genetic testing using array comparative genomic hybridization (aCGH) and PGD. Embryos were vitrified 2 days after intra-cytoplasmic sperm injection (ICSI). Thawed embryos were biopsied on day-three and transferred on day-five.
The couple underwent 20 cycles of assisted reproductive technology. A total of 23 in-vivo mature and five immature oocytes were retrieved, of which one matured in-vitro. Of 24 embryos, 17/24 (71 %) developed to day two and 11/24 (46 %) survived to blastocyst stage with a biopsy result available. Four blastocysts had normal PGD and aCGH results. Both single embryo transfers resulted in a successful implantation, one a blighted ovum and the other in a live birth.
Young patients with DOR have potential for live birth as long as oocytes can be obtained and embryos created. Serial vitrification may be the mechanism of choice in these patients when PGD is needed.
PMCID: PMC3879937  PMID: 24062195
In-vitro maturation (IVM); Embryo vitrification; Diminished Ovarian Reserve (DOR); Fragile X Syndrome (FXS); Preimplantation Genetic Diagnosis (PGD); Array Comparative Genomic Hybridization (aCGH)
3.  Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates 
Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2 wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.
PMCID: PMC4143168  PMID: 24637580
Bioengineering; Issue 85; Receptors; Cell Surface; Polymers; Cell Adhesion; Biomedical and Dental Materials; parylene-C; silicon dioxide; photolithography; cell adhesion; Cell Patterning
4.  Development and validation of a high throughput intrinsic ATPase activity assay for the discovery of MEKK2 inhibitors 
Journal of biomolecular screening  2012;18(4):388-399.
The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. In order to identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high throughput screening and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small molecule inhibitors of MEKK2 activity.
PMCID: PMC3723327  PMID: 23134735
MEKK2; MAP3K2; ATPase; crizotinib; bosutinib
5.  Family Understanding of Seriously-ill Patient Preferences for Family Involvement in Healthcare Decision Making 
Surrogate accuracy in predicting patient treatment preferences (i.e., what patients want) has been studied extensively, but it is not known whether surrogates can predict how patients want loved ones to make end-of-life decisions on their behalf.
To evaluate the ability of family members to correctly identify the preferences of seriously-ill patients regarding family involvement in decision making.
Cross-sectional survey.
Twenty-five pancreatic cancer and 27 amyotrophic lateral sclerosis (ALS) patients and their family members (52 dyads total).
Main Measures
Patients and family members completed the Decision Control Preferences (DCP) scale regarding patient preferences for family involvement in health care decisions using conscious and unconscious scenarios.
Key Results
Patient and family member agreement was 56% (29/52 dyads) for the conscious scenario (kappa 0.29) and 46% (24/52 dyads) for the unconscious scenario (kappa 0.15). Twenty-four family members identified the patient’s preference as independent in the unconscious scenario, but six of these patients actually preferred shared decision making and six preferred reliant decision making. In the conscious scenario, preference for independent decision making was associated with higher odds of patient–family agreement (AOR 5.28, 1.07–26.06). In the unconscious scenario, cancer patients had a higher odds of agreement than ALS patients (AOR 3.86; 95% CI 1.02–14.54).
Family members were often unable to correctly identify patient preferences for family involvement in end-of-life decision making, especially when patients desired that decisions be made using the best-interest standard. Clinicians and family members should consider explicitly eliciting patient preferences for family involvement in decision making. Additional research is still needed to identify interventions to improve family member understanding of patient preferences regarding the decision-making process itself.
Electronic supplementary material
The online version of this article (doi:10.1007/s11606-011-1717-6) contains supplementary material, which is available to authorized users.
PMCID: PMC3138973  PMID: 21499822
decision making; patient preference; advance care planning; terminal care
6.  Learner feedback and educational outcomes with an internet-based ambulatory curriculum: a qualitative and quantitative analysis 
BMC Medical Education  2012;12:55.
Online medical education curricula offer new tools to teach and evaluate learners. The effect on educational outcomes of using learner feedback to guide curricular revision for online learning is unknown.
In this study, qualitative analysis of learner feedback gathered from an online curriculum was used to identify themes of learner feedback, and changes to the online curriculum in response to this feedback were tracked. Learner satisfaction and knowledge gains were then compared from before and after implementation of learner feedback.
37,755 learners from 122 internal medicine residency training programs were studied, including 9437 postgraduate year (PGY)1 residents (24.4 % of learners), 9864 PGY2 residents (25.5 %), 9653 PGY3 residents (25.0 %), and 6605 attending physicians (17.0 %). Qualitative analysis of learner feedback on how to improve the curriculum showed that learners commented most on the overall quality of the educational content, followed by specific comments on the content. When learner feedback was incorporated into curricular revision, learner satisfaction with the instructive value of the curriculum (1 = not instructive; 5 = highly instructive) increased from 3.8 to 4.1 (p < 0.001), and knowledge gains (i.e., post test scores minus pretest scores) increased from 17.0 % to 20.2 % (p < 0.001).
Learners give more feedback on the factual content of a curriculum than on other areas such as interactivity or website design. Incorporating learner feedback into curricular revision was associated with improved educational outcomes. Online curricula should be designed to include a mechanism for learner feedback and that feedback should be used for future curricular revision.
PMCID: PMC3418189  PMID: 22788677
Online education; Curriculum development; Feedback; Learner satisfaction
7.  Everyday ethics in internal medicine resident clinic: an opportunity to teach 
Medical Education  2011;45(7):712-721.
Being a good doctor requires competency in ethics. Accordingly, ethics education during residency training is important. We studied the everyday ethics-related issues (i.e. ordinary ethics issues commonly faced) that internal medical residents encounter in their out-patient clinic and determined whether teaching about these issues occurred during faculty preceptor–resident interactions.
This study involved a multi-method qualitative research design combining observation of preceptor-resident discussions with preceptor interviews. The study was conducted in two different internal medicine training programme clinics over a 2-week period in June 2007. Fifty-three residents and 19 preceptors were observed, and 10 preceptors were interviewed. Transcripts of observer field notes and faculty interviews were carefully analysed. The analysis identified several themes of everyday ethics issues and determined whether preceptors identified and taught about these issues.
Everyday ethics content was considered present in 109 (81%) of the 135 observed case presentations. Three major thematic domains and associated sub-themes related to everyday ethics issues were identified, concerning: (i) the Doctor–Patient Interaction (relationships; communication; shared decision making); (ii) the Resident as Learner (developmental issues; challenges and conflicts associated with training; relationships with colleagues and mentors; interactions with the preceptor), and; (iii) the Doctor–System Interaction (financial issues; doctor–system issues; external influences; doctor frustration related to system issues). Everyday ethics issues were explicitly identified by preceptors (without teaching) in 18 of 109 cases (17%); explicit identification and teaching occurred in only 13 cases (12%).
In this study a variety of everyday ethics issues were frequently encountered as residents cared for patients. Yet, faculty preceptors infrequently explicitly identified or taught these issues during their interactions with residents. Ethics education is important and residents may regard teaching about the ethics-related issues they actually encounter to be highly relevant. A better understanding of the barriers to teaching is needed in order to promote education about everyday ethics in the out-patient setting.
PMCID: PMC3233355  PMID: 21649704
8.  A Video-Intervention to Improve Clinician Attitudes Toward Patients with Sickle Cell Disease: The Results of a Randomized Experiment 
Clinician attitudes toward patients are associated with variability in the quality of health care. Attitudes are typically considered difficult to change, and few interventions have attempted to do so. Negative attitudes toward adults with sickle cell disease have been identified as an important barrier to the receipt of appropriate pain management for this patient population.
To test the effect of a video-intervention designed to improve clinician attitudes toward adults with sickle cell disease.
An 8-minute video depicting a clinician expert and patients discussing challenges in seeking treatment for sickle cell pain.
A randomized post-test only control group design was used to assess the impact of the intervention on the attitudes of 276 nurses and housestaff working at a large, urban, academic medical center.
Attitudes toward adult sickle cell patients assessed using 5- and 6-point Likert-scale items. Exploratory factor analysis was used to identify underlying attitudinal domains and develop scales. Examples of the negative and positive attitudes assessed include clinician estimates of the percentage of SCD patients that exaggerate pain (negative) or make clinicians glad they went into medicine (positive).
Compared to the control group, the intervention group exhibited decreased negative attitudes (Difference in means = -8.9, 95%CI [-14.2, -3.6]; Cohen’s d = 0.41), decreased endorsement of certain patient behaviors as “concern-raising” (Difference in means = -7.8, 95%CI [-13.1, -2.5]; Cohen’s d = 0.36), and increased positive attitudes toward sickle cell patients (Difference in means = 6.6, 95% CI [0.6, 12.6]; Cohen’s d = 0.27).
Our results suggest that the attitudes of clinicians toward sickle cell patients may be improved through a short and relatively easy to implement intervention. Whether the attitudinal differences associated with our intervention are sustainable or are linked to clinical outcomes remains to be seen.
Electronic supplementary material
The online version of this article (doi:10.1007/s11606-010-1605-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3077483  PMID: 21181560
healthcare provider attitudes; sickle cell disease; randomized experiment; video interventions
9.  Expectant Management of Vestibular Schwannoma: A Retrospective Multivariate Analysis of Tumor Growth and Outcome 
Skull Base  2011;21(5):295-302.
We conducted a retrospective observational study to assess the consequences of conservative management of vestibular schwannoma (VS). Data were collected from tertiary neuro-otological referral units in United Kingdom. The study included 59 patients who were managed conservatively with radiological diagnosis of VS. The main outcome measures were growth rate and rate of failure of conservative management. Multivariate analysis sought correlation between tumor growth and (i) demographic features, (ii) tumor characteristics. The mean tumor growth was 0.66 mm/y. 11 patients (19%) required intervention. Mean time to intervention was 37 months with two notable late “failures” occurring at 75 and 84 months. Tumors extending into the cerebellopontine angle (CPA) grew significantly faster than intracanalicular tumors (p = 0.0045). No association was found between growth rate and age, sex, tumor laterality, facial nerve function, and grade of hearing loss. Conservative management is acceptable for a subset of patients. Tumors extending into the CPA at diagnosis grow significantly faster than intracanalicular tumors. No growth within 5 years of surveillance does not guarantee a continued indolent growth pattern; surveillance must therefore continue.
PMCID: PMC3312137  PMID: 22451829
Vestibular schwannoma; conservative; surveillance
10.  Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS) 
Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents.
PMCID: PMC3134944  PMID: 21760877
Glycolysis; GAPDHS; high throughput screening; sperm; contraceptive.
11.  A High Throughput Assay for Discovery of Bacterial β-Glucuronidase Inhibitors 
CPT-11 is a widely-used anti-cancer drug that is converted in vivo to its active metabolite, SN-38. In the liver, enzymes detoxify SN-38 by coupling it to a glucuronidate moiety and this inactive compound (SN-38G) is excreted into the gastrointestinal tract. In the intestine, commensal bacteria convert the SN-38G back to the active and toxic SN-38 using bacterial β-glucuronidase enzyme (GUS). This intestinal SN-38 causes debilitating diarrhea that prevents dose-intensification and efficacy in a significant fraction of patients undergoing CPT-11 treatment for cancer. This CPT-11 metabolic pathway suggests that small molecule inhibitors of GUS may have utility as novel therapeutics for prevention of dose-limiting diarrhea resulting from CPT-11 therapy. To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library. Novel inhibitors of GUS were identified with IC50 values ranging from 50 nM to 4.8 µM. These compounds may be useful as chemical probes for use in proof-of-concept experiments designed to determine the efficacy of GUS inhibitors in altering the intestinal metabolism of drugs. Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.
PMCID: PMC3106358  PMID: 21643506
β-glucuronidase; CPT-11; screen; inhibitor.
12.  The Business of Self-Monitoring of Blood Glucose: A Market Profile 
The market for self-monitoring of blood glucose (SMBG) approached $8.8 billion worldwide in 2008. Yet despite dramatic double-digit growth in sales of SMBG products since 1980, the business is now facing declining prices and slower dollar growth. Given that SMBG meters and test strips are viewed by consumers and insurers as essentially generic products, it will be extremely challenging for new market entrants to displace well-entrenched existing competitors without a truly innovative technology. Also, in the face of declining glucose test strip prices, market expansion can only occur through identification of more of the undiagnosed diabetes population and convincing existing diabetes patients to adopt glucose testing or to test more frequently. Ultimately, a combination of technology innovations, patient education, and economic incentives may be needed to significantly expand the SMBG market and build sustainable long-term dollar growth for SMBG vendors.
PMCID: PMC2769893  PMID: 20144440
blood glucose; continuous blood glucose monitoring; glucose monitoring; market growth; market size; noninvasive; self-monitoring of blood glucose
13.  Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli 
Journal of biomolecular screening  2009;14(9):1092-1101.
The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with non-classical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by HTS, e.g., RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Towards the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, we have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in six distinct chemotype clades. The most potent class of hits was further examined, and one member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures.
PMCID: PMC2877376  PMID: 19675313
RecA; antibiotic resistance; ATPase inhibition; ciprofloxacin; SOS response
14.  Novel Inhibitors of E. coli RecA ATPase Activity 
The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents.
PMCID: PMC2905775  PMID: 20648224
Antibiotic resistance; ATPase inhibition; high throughput screening; microbial drug resistance; RecA; SOS response.
15.  Development and validation of the Family Decision-Making Self-Efficacy Scale 
Several studies have reported high levels of distress in family members who have made health care decisions for loved ones at the end of life. A method is needed to assess the readiness of family members to take on this important role. Therefore, the purpose of this study was to develop and validate a scale to measure family member confidence in making decisions with (conscious patient scenario) and for (unconscious patient scenario) a terminally ill loved one.
On the basis of a survey of family members of patients with amyotrophic lateral sclerosis (ALS) enriched by in-depth interviews guided by Self-Efficacy Theory, we developed six themes within family decision making self-efficacy. We then created items reflecting these themes that were refined by a panel of end-of-life research experts. With 30 family members of patients in an outpatient ALS and a pancreatic cancer clinic, we tested the tool for internal consistency using Cronbach’s alpha and for consistency from one administration to another using the test–retest reliability assessment in a subset of 10 family members. Items with item to total scale score correlations of less than .40 were eliminated.
A 26-item scale with two 13-item scenarios resulted, measuring family self-efficacy in decision making for a conscious or unconscious patient with a Cronbach’s alphas of .91 and .95, respectively. Test–retest reliability was r = .96, p = .002 in the conscious senario and r = .92, p = .009 in the unconscious scenario.
Significance of results
The Family Decision-Making Self-Efficacy Scale is valid, reliable, and easily completed in the clinic setting. It may be used in research and clinical care to assess the confidence of family members in their ability to make decisions with or for a terminally ill loved one.
PMCID: PMC2814174  PMID: 19788773
Decision making; Self-efficacy; End of life; Family; Scale
16.  Temporal and geographic evidence for evolution of Sin Nombre virus using molecular analyses of viral RNA from Colorado, New Mexico and Montana 
Virology Journal  2009;6:102.
All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.
Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.
Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (πs) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (πa) was 7.0 × 10-4 in the M segment and 17.3 × 10-4 in the S segment sequences. The low πa relative to πs suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.
Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.
PMCID: PMC2716327  PMID: 19602267
17.  A High-Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction 
The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high-throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gαi1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z′-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z′-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
PMCID: PMC2440576  PMID: 18537560
Fluorescence anisotropy; fluorescence polarization; GoLoco motif; heterotrimeric G-proteins; high-throughput screening
18.  When Patients Lack Capacity 
Current approaches to end-of-life decision making are widely considered inadequate. We explored these complexities by examining how patients with terminal diagnoses would choose to involve their physicians and loved ones in making medical decisions, assuming they were able and unable to participate. Cross-sectional interviews of 130 patients recently diagnosed with fatal conditions were conducted. Patients were recruited from two academic medical centers using a modification of the Decision Control Preferences Scale, ranging from independent decision making to decision making that relies upon others. Patients were asked how they would balance their own wishes relative to the input of physician and loved ones in making medical decisions, and to weigh the input of loved ones relative to physician. Most patients (52%), assuming they had the capacity, would opt to share decision making with their physicians, but 15% would defer to their physicians and 34% would make decisions independently. Similarly, 44% would share decision making with their loved ones, but fewer (6%) would defer to their loved ones. Thirty-nine percent would rely upon their physicians’ judgments about what would be best for them rather than their own wishes if they became unconscious, compared with 15% who would do so if they were conscious (P < 0.001). Nonetheless, patients were more likely to weigh their loved ones’ input more heavily than their physicians’ input if they were unconscious (33%) than if they were conscious (7%, P = 0.05). Race, religion, gender, diagnosis, and health status were largely unassociated with patients’ decision control preferences. Patients with terminal diagnoses report a wide diversity of decision control preferences, but most would opt to share decision making with their physicians and loved ones. If unable to decide for themselves, they shift toward greater reliance on physician input relative to their own wishes but would weigh loved ones’ input more heavily than physician input. Deciding for patients who cannot speak for themselves may be more complex than has previously been reflected in law, policy, or clinical ethics.
PMCID: PMC2604910  PMID: 16256898
Ethics; end of life; decision making; advance care planning
19.  How Would Terminally Ill Patients Have Others Make Decisions for Them in the Event of Decisional Incapacity? A Longitudinal Study 
To determine the role terminally ill patients would opt to have their loved ones and physicians play in healthcare decisions should they lose decision-making capacity and how this changes over time.
Serial interviews.
The study institutions were The Johns Hopkins Medical Institutions in Baltimore, Maryland, and St. Vincent’s Hospital, in New York.
One hundred forty-seven patients with cancer, amyotrophic lateral sclerosis, or heart failure, at baseline and 3 and 6 months.
Patients’ baseline decision control preferences varied widely, but most opted for shared decision-making, leaning slightly toward independence from their loved ones. This did not change significantly at 3 or 6 months. Fiftyseven percent opted for the same degree of decision control at 3 months as at baseline. In a generalized estimating equation model adjusted for time, more-independent decision-making was associated with college education (P =.046) and being female (P =.01), whereas more-reliant decision-making was associated with age (P<.001). Patients leaned toward more reliance upon physicians to make best-interest determinations at diagnosis but opted for physicians to decide based upon their own independent wishes (substituted judgment) over time, especially if college educated.
Terminally ill patients vary in how much they wish their own preferences to control decisions made on their behalf, but most would opt for shared decisionmaking with loved ones and physicians. Control preferences are stable over time with respect to loved ones, but as they live longer with their illnesses, patients prefer somewhat less reliance upon physicians.
PMCID: PMC2583169  PMID: 18031490
decision-making; end of life; decisional incapacity; surrogates; ethics
20.  Family health care decision making and self-efficacy with patients with ALS at the end of life 
Palliative & supportive care  2008;6(3):273-280.
Persons with ALS differ from those with other terminal illnesses in that they commonly retain capacity for decision making close to death. The role patients would opt to have their families play in decision making at the end of life may therefore be unique. This study compared the preferences of patients with ALS for involving family in health care decisions at the end of life with the actual involvement reported by the family after death.
A descriptive correlational design with 16 patient–family member dyads was used. Quantitative findings were enriched with in-depth interviews of a subset of five family members following the patient's death.
Eighty-seven percent of patients had issued an advance directive. Patients who would opt to make health care decisions independently (i.e., according to the patient's preferences alone) were most likely to have their families report that decisions were made in the style that the patient preferred. Those who preferred shared decision making with family or decision making that relied upon the family were more likely to have their families report that decisions were made in a style that was more independent than preferred. When interviewed in depth, some family members described shared decision making although they had reported on the survey that the patient made independent decisions.
Significance of results:
The structure of advance directives may suggest to families that independent decision making is the ideal, causing them to avoid or underreport shared decision making. Fear of family recriminations may also cause family members to avoid or underreport shared decision making. Findings from this study might be used to guide clinicians in their discussions of treatments and health care decision making with persons with ALS and their families.
PMCID: PMC2572768  PMID: 18662421
Decision making; End of life; Family; Ethics
21.  Rapid Field Immunoassay for Detecting Antibody to Sin Nombre Virus in Deer Mice 
Emerging Infectious Diseases  2007;13(10):1604-1607.
We developed a 1-hour field enzyme immunoassay (EIA) for detecting antibody to Sin Nombre virus in deer mice (Peromyscus maniculatus). The assay specificity and sensitivity were comparable to those of a standard EIA. This test will permit identification of rodents with antibody to this and perhaps other hantaviruses.
PMCID: PMC2851528  PMID: 18258020
Sin Nombre virus; deer mouse; hantavirus; rapid diagnostic field test; EIA; rodent; PAGEIA; dispatch
22.  Effect of an Internet-based Curriculum on Postgraduate Education 
Journal of General Internal Medicine  2004;19(5 Pt 2):505-509.
We hypothesized that the Internet could be used to disseminate and evaluate a curriculum in ambulatory care, and that internal medicine residency program directors would value features made possible by online dissemination. An Internet-based ambulatory care curriculum was developed and marketed to internal medicine residency program directors. Utilization and knowledge outcomes were tracked by the website; opinions of program directors were measured by paper surveys. Twenty-four programs enrolled with the online curriculum. The curriculum was rated favorably by all programs, test scores on curricular content improved significantly, and program directors rated highly features made possible by an Internet-based curriculum.
PMCID: PMC1492333  PMID: 15109313
curriculum; computer-assisted instruction
23.  Increased bone mass is an unexpected phenotype associated with deletion of the calcitonin gene 
The Journal of Clinical Investigation  2002;110(12):1849-1857.
Calcitonin (CT) is a known inhibitor of bone resorption. Calcitonin gene–related peptide-α (CGRPα), produced by alternative RNA processing of the CT/CGRP gene, has no clearly defined role in bone. To better understand the physiologic role of the CT/CGRP gene we created a mouse in which the coding sequences for both CT and CGRPα were deleted by homologous recombination. The CT/CGRP–/– knockout (KO) mice procreated normally, there were no identifiable developmental defects at birth, and they had normal baseline calcium-related chemistry values. However, KO animals were more responsive to exogenous human parathyroid hormone as evidenced by a greater increase of the serum calcium concentration and urine deoxypyridinoline crosslinks, an effect reversed by CT and mediated by a greater increase in bone resorption than in controls. Surprisingly, KO mice have significantly greater trabecular bone volume and a 1.5- to 2-fold increase in bone formation at 1 and 3 months of age. This effect appears to be mediated by increased bone formation. In addition, KO mice maintain bone mass following ovariectomy, whereas wild-type mice lose approximately one-third of their bone mass over 2 months. These findings argue for dual roles for CT/CGRP gene products: prevention of bone resorption in hypercalcemic states and a regulatory role in bone formation.
PMCID: PMC151647  PMID: 12488435
24.  Adaptation of Influenza A Viruses to Cells Expressing Low Levels of Sialic Acid Leads to Loss of Neuraminidase Activity 
Journal of Virology  2001;75(8):3766-3770.
Influenza A viruses possess two virion surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA binds to sialyloligosaccharide viral receptors, while the NA removes sialic acids from the host cell and viral sialyloligosaccarides. Alterations of the HA occur during adaptation of influenza viruses to new host species, as in the 1957 and 1968 influenza pandemics. To gain a better understanding of the contributions of the HA and possibly the NA to this process, we generated cell lines expressing reduced levels of the influenza virus receptor determinant, sialic acid, by selecting Madin-Darby canine kidney cells resistant to a lectin specific for sialic acid linked to galactose by α(2-3) or α(2-6) linkages. One of these cell lines had less than 1/10 as much N-acetylneuraminic acid as its parent cell line. When serially passaged in this cell line, human H3N2 viruses lost sialidase activity due to a large internal deletion in the NA gene, without alteration of the HA gene. These findings indicate that NA mutations can contribute to the adaptation of influenza A virus to new host environments and hence may play a role in the transmission of virus across species.
PMCID: PMC114867  PMID: 11264365
25.  Development of a 950-gene DNA array for examining gene expression patterns in mouse testis 
Genome Biology  2001;2(4):research0014.1-research0014.9.
Over the past five years, interest in and use of DNA array technology has increased dramatically, and there has been a surge in demand for different types of arrays. Although manufacturers offer a number of pre-made arrays, these are generally of utilitarian design and often cannot accommodate the specific requirements of focused research, such as a particular set of genes from a particular tissue. We found that suppliers did not provide an array to suit our particular interest in testicular toxicology, and therefore elected to design and produce our own.
We describe the procedures used by members of the US Environmental Protection Agency MicroArray Consortium (EPAMAC) to produce a mouse testis expression array on both filter and glass-slide formats. The approaches used in the selection and assembly of a pertinent, nonredundant list of testis-expressed genes are detailed. Hybridization of the filter arrays with normal and bromochloroacetic acid-treated mouse testicular RNAs demonstrated that all the selected genes on the array were expressed in mouse testes.
We have assembled two lists of mouse (950) and human (960) genes expressed in the mouse and/or human adult testis, essentially all of which are available as sequence-verified clones from public sources. Of these, 764 are homologous and will therefore enable close comparison of gene expression between murine models and human clinical testicular samples.
PMCID: PMC31483  PMID: 11305942

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