The Impact of Web-Based Continuing Medical Education Using Patient Simulation on Real-World Treatment Selection in Type 2 Diabetes: Retrospective Case-Control Analysis.

BACKGROUND: Despite guidelines recommending the use of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) in certain patients with type 2 diabetes (T2D), they are not being prescribed for many of these patients. Web-based continuing medical education (CME) patient simulations have been used to identify clinicians' practice gaps and improve clinical decision-making as measured within a simulation, but the impact of this format on real-world treatment has not been researched. OBJECTIVE: This study aimed to evaluate the effect of a simulation-based CME intervention on real-world use of GLP-1 RAs by endocrinologists and primary care physicians. METHODS: Two evaluation phases of the CME simulation were conducted: phase I, the CME simulation phase, was a paired, pre-post study of 435 physician learners in the United States; and phase II, the real-world phase, was a retrospective, matched case-control study of 157 of the 435 physicians who had claims data available for the study period. RESULTS: Phase I CME results showed a 29 percentage point increase in correct decisions from pre- to postfeedback (178/435, 40.9% to 304/435, 69.9%; P<.001) in selecting treatment that addresses both glycemic control and cardiovascular event protection. Phase II results showed that 39 of 157 (24.8%) physicians in the intervention group increased use of GLP-1 RAs, compared to 20 of 157 (12.7%) in the comparison group. Being in the intervention group predicted GLP-1 RA use after education (odds ratio 4.49; 95% CI 1.45-13.97; P=.001). CONCLUSIONS: A web-based CME simulation focused on secondary prevention of cardiovascular events in a patient with T2D was associated with increased use of evidence-based treatment selection in the real world.

Viral Shedding in Mice following Intravenous Adenovirus Injection: Impact on Biosafety Classification.

There have been numerous advances in gene therapy and oncolytic virotherapy in recent years, especially with respect to cutting-edge animal models to test these novel therapeutics. With all of these advances, it is important to understand the biosafety risks of testing these vectors in animals. We performed adenovirus-based viral shedding studies in murine models to ascertain when it is appropriate to downgrade the animals from Biosafety Level (BSL) 2 to BSL 1 for experimental handling and transport. We utilized intravenous injections of a replication-competent adenovirus and analyzed viral shedding via the collection of buccal and dermal swabs from each animal, in addition to obtaining urine and stool samples. The adenovirus hexon copy number was determined by qPCR, and plaque formation was analyzed to assess the biologic activity of viral particles. Our results demonstrate that after 72 h following viral inoculation, there is no significant quantity of biologically active virus shedding from the animals. This observation suggests that on day 4 following adenovirus injection, mice can be safely downgraded to BSL 1 for the remainder of the experiment with no concern for hazardous exposure to laboratory personnel.

Behaviors Related to Medication Safety and Use During Pregnancy.

Background: Most women take medication during pregnancy despite limited scientific evidence on safety. We investigated medication use, including changes in and reasons for changes in use during pregnancy, with attention to medication use in pregnant women with chronic conditions. Materials and Methods: We conducted an online survey of pregnant women aged ≥18 years (n = 1,226). We calculated descriptive statistics for aspects of medication use and performed multivariable logistic regression to examine associations between change in use and chronic conditions. Results: Seventy-nine percent of women took at least one medication during pregnancy. Among those, 63.2% made at least one medication change: 42.0% started, 34.9% stopped, 30.0% missed dose(s), and 18.1% lowered dose(s) from that originally prescribed or recommended. More than a third (36.5%) of women who stopped, lowered, or missed medication did so independent of health care provider advice; 54.0% cited concern about birth or developmental defects as reasons for change. Odds of medication change were higher for women with chronic conditions: digestive conditions-starting (adjusted odds ratio [AOR] = 1.8, 95% confidence interval [CI] = 1.1-2.7), stopping (AOR = 2.1, 95% CI = 1.4-3.3), and lowering (AOR = 2.4, 95% CI = 1.7-3.3) medication; mental health conditions-starting (AOR = 1.6, 95% CI = 1.2-2.2), stopping (AOR = 3.0, 95% CI = 2.3-4.0), or missing (AOR = 2.1, 95% CI = 1.6-2.8) medication; pain conditions-stopping (AOR = 2.9, 95% CI = 2.0-4.2); and respiratory conditions-starting (AOR = 2.0, 95% CI = 1.3-3.1), stopping (AOR = 1.7, 95% CI = 1.1-2.6), and missing (AOR = 2.2, 95% CI = 1.4-3.4) medication. Conclusions: Most pregnant women take medication and many, including those with chronic conditions, change their medication use during pregnancy. Medication change may occur independent of health care provider advice and due to women's safety concerns.

Determinants of Hormone Replacement Therapy Knowledge and Current Hormone Replacement Therapy Use.

Background: The use of hormone replacement therapy (HRT) to treat menopausal symptoms has declined since the early 2000s, and little is known about the contemporary determinants of use in the United States. We aim to understand women's knowledge of HRT as a treatment of menopausal symptoms and to assess the factors associated with HRT use. Materials and Methods: Weighted multivariate logistic regression models evaluated the correlates of high HRT knowledge and current HRT use among a sample of 2,548 women aged ≥45 years who participated in an online survey between August 2019 and May 2020. Results: In total, 82% of the women surveyed reported experiencing one or more menopausal symptoms, yet only 10.5% reported using HRT. Only 33% reported high HRT knowledge. The odds of reporting high HRT knowledge increased with increasing age. Racial, ethnic minority women were less likely to report high HRT knowledge (adjusted odds ratio [AOR] = 0.69; 95% confidence interval [CI] = 0.5-0.9). Hispanic and non-Hispanic women of other racial and ethnic groups were less likely to use HRT compared with non-Hispanic White women (AOR = 0.3; 95% CI = 0.1-0.6) (AOR = 0.4; CI = 0.2-0.9), respectively. Women experiencing irregular periods were less likely to report current HRT use (AOR = 0.1, 95% CI = 0.4-0.7). Compared with past users, never users appeared to be more risk averse, and reported concern over HRT risks and side effects as reasons for nonuse. Conclusions: Many factors impact women's perceived HRT knowledge level and to a lesser extent HRT use. Future research should better define the most important factors influencing decisions to use HRT for symptom relief.

Clinician knowledge and behaviors related to the 4Ms framework of Age-Friendly Health Systems.

BACKGROUND: The Age-Friendly Health Systems (AFHS) aims to improve the experience of care for adults aged 65 years and older through the 4Ms framework, an evidence-based approach to care planning that emphasizes what matters most to the older person, mentation, mobility, and medication. The aim of this study was to examine clinicians' attitudes, knowledge, and practices concerning AFHS and the 4Ms. METHODS: We surveyed U.S.-based health care providers randomly identified from the Medscape database. The sample was weighted based on sex, U.S. Census region, and ethnic diversity of health occupations. We examined the differences between cohorts using proportions tests and logistic regression models. RESULTS: More than 90% of clinicians (n = 1684) agreed that "older patients require a different approach to care than younger patients." Fifty percent of clinicians "always" take the age of their patient into consideration when determining care. A majority of clinicians said they discuss each of the 4Ms with older patients and/or their family caregivers. Screening for depression and review of high-risk medication use are among the leading types of age-friendly care that clinicians provide to older patients. A minority of clinicians are asking older adults about and aligning the care plan with What Matters. CONCLUSIONS: A majority of clinicians acknowledged the benefits of providing care via AFHS but reported limited knowledge of the specificities of the 4Ms framework and are not necessarily taking the age of their patients into consideration when determining the best form of care. Health care settings that have implemented the 4Ms framework appear to be doing so in an incomplete way. Our study reinforces the case for training primary care providers on how to adopt the evidence-based 4Ms framework in clinical practice effectively and consistently.

Cancer imaging and therapy utilizing a novel NIS-expressing adenovirus: The role of adenovirus death protein deletion.

Encoding the sodium iodide symporter (NIS) by an adenovirus (Ad) is a promising strategy to facilitate non-invasive imaging and radiotherapy of pancreatic cancer. However, insufficient levels of NIS expression in tumor cells have limited its clinical translation. To optimize Ad-based radiotherapy and imaging, we investigated the effect of Ad death protein (ADP) deletion on NIS expression. We cloned two sets of oncolytic NIS-expressing Ads that differed only in the presence or absence of ADP. We found that ADP expression negatively affected NIS membrane localization and inhibited radiotracer uptake. ADP deletion significantly improved NIS-based imaging in pancreatic cancer models including patient-derived xenografts, where effective imaging was possible for up to 6 weeks after a single virus injection. This study demonstrates that improved oncolysis may hinder the therapeutic effect of oncolytic viruses designed to express NIS. In vivo studies in combination with 131I showed potential for effective radiotherapy. This also highlights the need for further investigation into optimal timing of 131I administration and suggests that repeated doses of 131I should be considered to improve efficacy in clinical trials. We conclude that ADP deletion is essential for effective NIS-based theranostics in cancer.

Unmasking Arrhythmogenic Hubs of Reentry Driving Persistent Atrial Fibrillation for Patient-Specific Treatment.

Background Atrial fibrillation (AF) driver mechanisms are obscured to clinical multielectrode mapping approaches that provide partial, surface-only visualization of unstable 3-dimensional atrial conduction. We hypothesized that transient modulation of refractoriness by pharmacologic challenge during multielectrode mapping improves visualization of hidden paths of reentrant AF drivers for targeted ablation. Methods and Results Pharmacologic challenge with adenosine was tested in ex vivo human hearts with a history of AF and cardiac diseases by multielectrode and high-resolution subsurface near-infrared optical mapping, integrated with 3-dimensional structural imaging and heart-specific computational simulations. Adenosine challenge was also studied on acutely terminated AF drivers in 10 patients with persistent AF. Ex vivo, adenosine stabilized reentrant driver paths within arrhythmogenic fibrotic hubs and improved visualization of reentrant paths, previously seen as focal or unstable breakthrough activation pattern, for targeted AF ablation. Computational simulations suggested that shortening of atrial refractoriness by adenosine may (1) improve driver stability by annihilating spatially unstable functional blocks and tightening reentrant circuits around fibrotic substrates, thus unmasking the common reentrant path; and (2) destabilize already stable reentrant drivers along fibrotic substrates by accelerating competing fibrillatory wavelets or secondary drivers. In patients with persistent AF, adenosine challenge unmasked hidden common reentry paths (9/15 AF drivers, 41±26% to 68±25% visualization), but worsened visualization of previously visible reentry paths (6/15, 74±14% to 34±12%). AF driver ablation led to acute termination of AF. Conclusions Our ex vivo to in vivo human translational study suggests that transiently altering atrial refractoriness can stabilize reentrant paths and unmask arrhythmogenic hubs to guide targeted AF driver ablation treatment.

Silencing miR-370-3p rescues funny current and sinus node function in heart failure.

Bradyarrhythmias are an important cause of mortality in heart failure and previous studies indicate a mechanistic role for electrical remodelling of the key pacemaking ion channel HCN4 in this process. Here we show that, in a mouse model of heart failure in which there is sinus bradycardia, there is upregulation of a microRNA (miR-370-3p), downregulation of the pacemaker ion channel, HCN4, and downregulation of the corresponding ionic current, If, in the sinus node. In vitro, exogenous miR-370-3p inhibits HCN4 mRNA and causes downregulation of HCN4 protein, downregulation of If, and bradycardia in the isolated sinus node. In vivo, intraperitoneal injection of an antimiR to miR-370-3p into heart failure mice silences miR-370-3p and restores HCN4 mRNA and protein and If in the sinus node and blunts the sinus bradycardia. In addition, it partially restores ventricular function and reduces mortality. This represents a novel approach to heart failure treatment.

Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node.

Mechanisms for human sinoatrial node (SAN) dysfunction are poorly understood and whether human SAN excitability requires voltage-gated sodium channels (Nav) remains controversial. Here, we report that neuronal (n)Nav blockade and selective nNav1.6 blockade during high-resolution optical mapping in explanted human hearts depress intranodal SAN conduction, which worsens during autonomic stimulation and overdrive suppression to conduction failure. Partial cardiac (c)Nav blockade further impairs automaticity and intranodal conduction, leading to beat-to-beat variability and reentry. Multiple nNav transcripts are higher in SAN vs atria; heterogeneous alterations of several isoforms, specifically nNav1.6, are associated with heart failure and chronic alcohol consumption. In silico simulations of Nav distributions suggest that INa is essential for SAN conduction, especially in fibrotic failing hearts. Our results reveal that not only cNav but nNav are also integral for preventing disease-induced failure in human SAN intranodal conduction. Disease-impaired nNav may underlie patient-specific SAN dysfunctions and should be considered to treat arrhythmias.

Human Atrial Fibrillation Drivers Resolved With Integrated Functional and Structural Imaging to Benefit Clinical Mapping.

OBJECTIVES: This study sought to improve atrial fibrillation (AF) driver identification by integrating clinical multielectrode mapping with driver fingerprints defined by high-resolution ex vivo 3-dimensional (3D) functional and structural imaging. BACKGROUND: Clinical multielectrode mapping of AF drivers suffers from variable contact, signal processing, and structural complexity within the 3D human atrial wall, raising questions on the validity of such drivers. METHODS: Sustained AF was mapped in coronary-perfused explanted human hearts (n = 11) with transmural near-infrared optical mapping (∼0.3 mm2 resolution). Simultaneously, custom FIRMap catheters (∼9 × 9 mm2 resolution) mapped endocardial and epicardial surfaces, which were analyzed by Focal Impulse and Rotor Mapping activation and Rotational Activity Profile (Abbott Labs, Chicago, Illinois). Functional maps were integrated with contrast-enhanced cardiac magnetic resonance imaging (∼0.1 mm3 resolution) analysis of 3D fibrosis architecture. RESULTS: During sustained AF, near-infrared optical mapping identified 1 to 2 intramural, spatially stable re-entrant AF drivers per heart. Driver targeted ablation affecting 2.2 ± 1.1% of the atrial surface terminated and prevented AF. Driver regions had significantly higher phase singularity density and dominant frequency than neighboring nondriver regions. Focal Impulse and Rotor Mapping had 80% sensitivity to near-infrared optical mapping-defined driver locations (16 of 20), and matched 14 of 20 driver visualizations: 10 of 14 re-entries seen with Rotational Activity Profile; and 4 of 6 breakthrough/focal patterns. Focal Impulse and Rotor Mapping detected 1.1 ± 0.9 false-positive rotational activity profiles per recording, but these regions had lower intramural contrast-enhanced cardiac magnetic resonance imaging fibrosis than did driver regions (14.9 ± 7.9% vs. 23.2 ± 10.5%; p < 0.005). CONCLUSIONS: The study revealed that both re-entrant and breakthrough/focal AF driver patterns visualized by surface-only clinical multielectrodes can represent projections of 3D intramural microanatomic re-entries. Integration of multielectrode mapping and 3D fibrosis analysis may enhance AF driver detection, thereby improving the efficacy of driver-targeted ablation.

Redundant and diverse intranodal pacemakers and conduction pathways protect the human sinoatrial node from failure.

The human sinoatrial node (SAN) efficiently maintains heart rhythm even under adverse conditions. However, the specific mechanisms involved in the human SAN's ability to prevent rhythm failure, also referred to as its robustness, are unknown. Challenges exist because the three-dimensional (3D) intramural structure of the human SAN differs from well-studied animal models, and clinical electrode recordings are limited to only surface atrial activation. Hence, to innovate the translational study of human SAN structural and functional robustness, we integrated intramural optical mapping, 3D histology reconstruction, and molecular mapping of the ex vivo human heart. When challenged with adenosine or atrial pacing, redundant intranodal pacemakers within the human SAN maintained automaticity and delivered electrical impulses to the atria through sinoatrial conduction pathways (SACPs), thereby ensuring a fail-safe mechanism for robust maintenance of sinus rhythm. During adenosine perturbation, the primary central SAN pacemaker was suppressed, whereas previously inactive superior or inferior intranodal pacemakers took over automaticity maintenance. Sinus rhythm was also rescued by activation of another SACP when the preferential SACP was suppressed, suggesting two independent fail-safe mechanisms for automaticity and conduction. The fail-safe mechanism in response to adenosine challenge is orchestrated by heterogeneous differences in adenosine A1 receptors and downstream GIRK4 channel protein expressions across the SAN complex. Only failure of all pacemakers and/or SACPs resulted in SAN arrest or conduction block. Our results unmasked reserve mechanisms that protect the human SAN pacemaker and conduction complex from rhythm failure, which may contribute to treatment of SAN arrhythmias.

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

BACKGROUND: Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). METHODS: We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). RESULTS: Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10-100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10-100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. CONCLUSIONS: This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine.

BMP-2 expression correlates with local failure in head and neck squamous cell carcinoma.

OBJECTIVE: Preclinical data show that exogenous administration of recombinant human bone morphogenetic protein-2 (rhBMP-2) to human oral carcinoma cell lines increases pathogenicity using a nude mouse model. The objectives of this study are to (1) describe the characteristics of baseline protein expression of BMP-2 in head and neck squamous cell carcinomas (HNSCC) and (2) determine if BMP-2 expression level correlates with worse oncologic outcomes. STUDY DESIGN: Retrospective analysis of previously harvested patient samples. SETTING: Academic medical center. SUBJECTS: In total, 149 patients with oral cavity, oropharynx, larynx, and hypopharynx HNSCC treated between January 1, 1997, and December 31, 2004. METHODS: A tissue microarray of HNSCC was assembled and immunohistochemistry for BMP-2 performed. Staining was quantified using a standardized scoring system. Specimens were dichotomized into high or low expression level. Statistical analyses using log-rank, Wilcoxon, and Fisher exact test were performed for associations between BMP-2 protein level and clinicopathologic features and patient survival. RESULTS: BMP-2 expression at any level was noted in 146 of 149 (98%) of samples. Tumors with high BMP-2 expression had higher rates of local failure compared with low-expressing tumors (17.3% vs 6.3%; P = .04). There was no significant association for BMP-2 expression level with tumor location, T stage, N stage, overall survival, regional failure, or distant failure. CONCLUSION: Head and neck squamous cell carcinomas with high baseline BMP-2 protein level are associated with higher rates of local recurrence. These data have important implications for using rhBMP-2 in tissue engineering reconstructive approaches in the setting of cancer-related defects.

PDCD2 knockdown inhibits erythroid but not megakaryocytic lineage differentiation of human hematopoietic stem/progenitor cells.

Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and γ-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.

rhBMP-2 has adverse effects on human oral carcinoma cell lines in vivo.

OBJECTIVES/HYPOTHESIS: To establish the relevance of the bone morphogenetic protein (BMP) signaling pathway in human oral squamous cell carcinoma (OSCCA) cell lines and determine if there is a biologic impact of stimulating this pathway with recombinant human (rh) BMP-2. STUDY DESIGN: In vitro laboratory investigations and in vivo analysis using an orthotopic animal model for oral cancer. METHODS: Gene expression profiles for BMP-2 and components of the BMP-signaling pathway were determined using reverse transcriptase-polymerase chain reaction. In vivo effects were evaluated using Kaplan-Meier survival analysis and studying histopathologic changes in established tumor xenografts with or without rhBMP-2 pretreatment. A phosphokinase array was used to detect levels of activation in signaling kinases. RESULTS: The BMP-2 gene was expressed in 90% of the 30 OSCCA cell lines tested. Gene expression of all components of the BMP-signaling pathway was highly conserved. Tumor xenografts established with rhBMP-2-treated cells showed more rapid local growth that resulted in worse animal survival as compared to the control group. These tumors had a more poorly differentiated morphology. Changes in protein kinases suggested interactions of BMP-2 signaling with the Wnt-ß-catenin, and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways. CONCLUSIONS: Human OSCCA cell lines frequently express BMP-2 and all necessary components of the BMP-signaling pathway. Exogenous treatment of human OSCCA cell lines with rhBMP-2 prior to engraftment in an orthotopic animal model caused the subsequent tumors to be more locally aggressive with worse survival. Continued caution should be used for considering rhBMP-2 for reconstruction of bone defects in oral cancer patients.

Treatment effects of rhBMP-2 on invasiveness of oral carcinoma cell lines.

OBJECTIVE: To determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) has biological effects on the invasiveness of human oral squamous cell carcinoma (OSCCA) cell lines. STUDY DESIGN: Laboratory investigation using six human OSCCA cell lines, with three cell lines having baseline gene expression of BMP-2 and three cell lines without baseline gene expression of BMP-2. METHODS: The invasiveness of each cell line was measured using a matrigel invasion assay with or without stimulation by rhBMP-2. A tumor metastasis quantitative PCR array was used to establish whether observed findings from the invasion assay correlated to changes in gene expression. RESULTS: There was a significant increase in tumor cell invasion in response to rhBMP-2 in all BMP-2 positive cell lines but no change in the cell lines that did not express the BMP-2 gene. Quantitative PCR revealed that changes in gene expression were distinctly different based on the baseline gene expression of BMP-2 and favored a more metastatic genotype in the BMP-2-positive cells. CONCLUSIONS: Recombinant human BMP-2 has an adverse biological effect on invasiveness of human OSCCA cell lines in vitro. This adverse effect is dependent on the baseline gene expression of BMP-2. Changes in expression of genes involved with tumor metastasis correlated to the invasion assay findings. These data raise concern for the safe application of rhBMP-2 for reconstruction of bone defects in oral cancer patients.

Expectations, validity, and reality in gene expression profiling.

OBJECTIVE: To provide a critical overview of gene expression profiling methodology and discuss areas of future development. RESULTS: Gene expression profiling has been used extensively in biological research and has resulted in significant advances in the understanding of the molecular mechanisms of complex disorders, including cancer, heart disease, and metabolic disorders. However, translating this technology into genomic medicine for use in diagnosis and prognosis faces many challenges. In addition, gene expression profile analysis is frequently controversial, because its conclusions often lack reproducibility and claims of effective dissemination into translational medicine have, in some cases, been remarkably unjustified. In the last decade, a large number of methodological and technical solutions have been offered to overcome the challenges. STUDY DESIGN AND SETTING: We consider the strengths, limitations, and appropriate applications of gene expression profiling techniques, with particular reference to the clinical relevance. CONCLUSION: Some studies have demonstrated the ability and clinical utility of gene expression profiling for use as diagnostic, prognostic, and predictive molecular markers. The challenges of gene expression profiling lie with the standardization of analytic approaches and the evaluation of the clinical merit in broader heterogeneous populations by prospective clinical trials.

Urine metabolomics analysis for kidney cancer detection and biomarker discovery.

Renal cell carcinoma (RCC) accounts for 11,000 deaths per year in the United States. When detected early, generally serendipitously by imaging conducted for other reasons, long term survival is generally excellent. When detected with symptoms, prognosis is poor. Under these circumstances, a screening biomarker has the potential for substantial public health benefit. The purpose of this study was to evaluate the utility of urine metabolomics analysis for metabolomic profiling, identification of biomarkers, and ultimately for devising a urine screening test for RCC. Fifty urine samples were obtained from RCC and control patients from two institutions, and in a separate study, urine samples were taken from 13 normal individuals. Hydrophilic interaction chromatography-mass spectrometry was performed to identify small molecule metabolites present in each sample. Cluster analysis, principal components analysis, linear discriminant analysis, differential analysis, and variance component analysis were used to analyze the data. Previous work is extended to confirm the effectiveness of urine metabolomics analysis using a larger and more diverse patient cohort. It is now shown that the utility of this technique is dependent on the site of urine collection and that there exist substantial sources of variation of the urinary metabolomic profile, although group variation is sufficient to yield viable biomarkers. Surprisingly there is a small degree of variation in the urinary metabolomic profile in normal patients due to time since the last meal, and there is little difference in the urinary metabolomic profile in a cohort of pre- and postnephrectomy (partial or radical) renal cell carcinoma patients, suggesting that metabolic changes associated with RCC persist after removal of the primary tumor. After further investigations relating to the discovery and identity of individual biomarkers and attenuation of residual sources of variation, our work shows that urine metabolomics analysis has potential to lead to a diagnostic assay for RCC.

Microarray analysis.

Microarrays and related technologies have allowed investigators to ask biological questions in far greater detail than has previously been possible. Microarrays had a troubled beginning, but most of these problems resulted from the growing pains of this technology, which, like many new things, was initially more promise than delivery. Nevertheless, over the past few years, investigators have learned how to achieve optimal performance of technology, and now exciting discoveries are made using microarray-based research. Many of the advances have come from the realization that microarrays are not a magic tool but rather are like any other measurement device. Unless microarray experimentation is coupled with good experimental practices, it will not yield valid results or, worse yet, may lead to misleading results. In this chapter, we highlight some of the important steps that should be taken to successfully conduct a microarray study. These steps include a clearly stated biological question, experimental design, careful experimental conduct, complete statistical analysis, validation/verification of results, and dissemination of the data.

Optimal allocation of replicates for measurement evaluation studies.

Optimal experimental design is important for the efficient use of modern high-throughput technologies such as microarrays and proteomics. Multiple factors including the reliability of measurement system, which itself must be estimated from prior experimental work, could influence design decisions. In this study, we describe how the optimal number of replicate measures (technical replicates) for each biological sample (biological replicate) can be determined. Different allocations of biological and technical replicates were evaluated by minimizing the variance of the ratio of technical variance (measurement error) to the total variance (sum of sampling error and measurement error). We demonstrate that if the number of biological replicates and the number of technical replicates per biological sample are variable, while the total number of available measures is fixed, then the optimal allocation of replicates for measurement evaluation experiments requires two technical replicates for each biological replicate. Therefore, it is recommended to use two technical replicates for each biological replicate if the goal is to evaluate the reproducibility of measurements.