A Unique Partnership Between the University of Hawai'i Cancer Center and the University of Guam: Fifteen Years of Addressing Cancer Health Disparities in Pacific Islanders in Hawai'i and Guam.

This manuscript describes the efforts in research, education, and outreach of a unique partnership between the University of Hawai'i Cancer Center and the University of Guam in addressing cancer health disparities faced by Pacific Islanders in Hawai'i, Guam, and other parts of Micronesia. Significant accomplishments of this 15-year collaboration in research, training Micronesian students, and impact on the local communities are highlighted.

Identification of a new inhibitor of KRAS-PDEδ interaction targeting KRAS mutant nonsmall cell lung cancer.

Oncogenic KRAS is considered a promising target for anti-cancer therapy. However, direct pharmacological strategies targeting KRAS-driven cancers remained unavailable. The prenyl-binding protein PDEδ, a transporter of KRAS, has been identified as a potential target for pharmacological inhibitor by selectively binding to its prenyl-binding pocket, impairing oncogenic KRAS signaling pathway. Here, we discovered a novel PDEδ inhibitor (E)-N'-((3-(tert-butyl)-2-hydroxy-6,7,8,9-tetrahydrodibenzo[b,dfuran-1-yl)methylene)-2,4-dihydroxybenzohydrazide(NHTD) by using a high-throughput docking-based virtual screening approach. In vitro and in vivo studies demonstrated that NHTD suppressed proliferation, induced apoptosis and inhibited oncogenic K-RAS signaling pathways by disrupting KRAS-PDEδ interaction in nonsmall cell lung cancer (NSCLC) harboring KRAS mutations. NHTD redistributed the localization of KRAS to endomembranes by targeting the prenyl-binding pocket of PDEδ and exhibited the suppression of abnormal KRAS function. Importantly, NHTD prevented tumor growth in xenograft and KRAS mutant mouse model, which presents an effective strategy targeting KRAS-driven cancer.

Inhibition of KRAS-dependent lung cancer cell growth by deltarasin: blockage of autophagy increases its cytotoxicity.

Deltarasin is a recently identified small molecule that can inhibit KRAS-PDEδ interactions by binding to a hydrophobic pocket on PDEδ, resulting in the impairment of cell growth, KRAS activity, and RAS/RAF signaling in human pancreatic ductal adenocarcinoma cell lines. Since KRAS mutations are the most common oncogene mutations in lung adenocarcinomas, implicated in over 30% of all lung cancer cases, we examined the ability of deltarasin to inhibit KRAS-dependent lung cancer cell growth. Here, for the first time, we document that deltarasin produces both apoptosis and autophagy in KRAS-dependent lung cancer cells in vitro and inhibits lung tumor growth in vivo. Deltarasin induces apoptosis by inhibiting the interaction of with PDEδ and its downstream signaling pathways, while it induces autophagy through the AMPK-mTOR signaling pathway. Importantly, the autophagy inhibitor, 3-methyl adenine (3-MA) markedly enhances deltarasin-induced apoptosis via elevation of reactive oxygen species (ROS). In contrast, inhibition of ROS by N-acetylcysteine (NAC) significantly attenuated deltarasin-induced cell death. Collectively, these observations suggest that the anti-cancer cell activity of deltarasin can be enhanced by simultaneously blocking "tumor protective" autophagy, but inhibited if combined with an anti-oxidant.

Common Genetic Variants Found in HLA and KIR Immune Genes in Autism Spectrum Disorder.

The "common variant-common disease" hypothesis was proposed to explain diseases with strong inheritance. This model suggests that a genetic disease is the result of the combination of several common genetic variants. Common genetic variants are described as a 5% frequency differential between diseased vs. matched control populations. This theory was recently supported by an epidemiology paper stating that about 50% of genetic risk for autism resides in common variants. However, rare variants, rather than common variants, have been found in numerous genome wide genetic studies and many have concluded that the "common variant-common disease" hypothesis is incorrect. One interpretation is that rare variants are major contributors to genetic diseases and autism involves the interaction of many rare variants, especially in the brain. It is obvious there is much yet to be learned about autism genetics. Evidence has been mounting over the years indicating immune involvement in autism, particularly the HLA genes on chromosome 6 and KIR genes on chromosome 19. These two large multigene complexes have important immune functions and have been shown to interact to eliminate unwanted virally infected and malignant cells. HLA proteins have important functions in antigen presentation in adaptive immunity and specific epitopes on HLA class I proteins act as cognate ligands for KIR receptors in innate immunity. Data suggests that HLA alleles and KIR activating genes/haplotypes are common variants in different autism populations. For example, class I allele (HLA-A2 and HLA-G 14 bp-indel) frequencies are significantly increased by more than 5% over control populations (Table 2). The HLA-DR4 Class II and shared epitope frequencies are significantly above the control populations (Table 2). Three activating KIR genes: 3DS1, 2DS1, and 2DS2 have increased frequencies of 15, 22, and 14% in autism populations, respectively. There is a 6% increase in total activating KIR genes in autism over control subjects. And, more importantly there is a 12% increase in activating KIR genes and their cognate HLA alleles over control populations (Torres et al., 2012a). These data suggest the interaction of HLA ligand/KIR receptor pairs encoded on two different chromosomes is more significant as a ligand/receptor complex than separately in autism.

Preparing for events for physically challenged athletes.

The participation in sports for physically challenged athletes continues to expand in multiple domains from recreational, novice, and competitive to elite competitions such as the Paralympics. Physically challenged athletes have various disabilities such as visual impairments, spinal cord injuries, amputations, cerebral palsy, or other neuromuscular impairments and have different levels of functional ability within these broad categories. The spectrum of medical illnesses and musculoskeletal injuries seen with sports is similar to that of able-bodied athletes; however medical teams caring for athletes with disabilities need to be familiar with medical risks such as skin breakdown, thermoregulation problems, dehydration, autonomic dysreflexia, infections, orthotic and prosthetic issues, and psychiatric comorbidities that may be encountered. The medical team preparation for events involving physically challenged athletes should include obtaining appropriate medical supplies, ensuring disability-compatible access to medical areas, and preparing for emergency extraction from adaptive equipment.

Activating killer-cell immunoglobulin-like receptors (KIR) and their cognate HLA ligands are significantly increased in autism.

Killer-cell immunoglobulin-like receptor (KIR) proteins are expressed on natural killer (NK) cells and appear important in innate and adaptive immunity. There are about 14 KIR genes on chromosome 19q13.4, composed of those that inhibit and those that activate NK cell killing. Haplotypes have different combinations of these genes meaning that not all genes are present in a subject. There are two main classes of cognate human leukocyte antigen (HLA) ligands (HLA-Bw4 and HLA-C1/C2) that bind to the inhibitory/activating receptors. As a general rule, the inhibitory state is maintained except when virally infected or tumor cells are encountered; however, both increased activation and inhibition states have been associated with susceptibility and protection against numerous disease states including cancer, arthritis, and psoriasis. Utilizing DNA from 158 Caucasian subjects with autism and 176 KIR control subjects we show for the first time a highly significant increase in four activating KIR genes (2DS5, 3DS1, 2DS1 and 2DS4) as measured by chi square values and odds ratios. In addition, our data suggests a highly significant increase in the activating KIR gene 2DS1 and its cognate HLA-C2 ligand (2DS1+C2; p = 0.00003 [Odds ratio = 2.87]). This information ties together two major immune gene complexes, the human leukocyte complex and the leukocyte receptor complex, and may partially explain immune abnormalities observed in many subjects with autism.

Isolation, characterization, and in vitro propagation of infantile hemangioma stem cells and an in vivo mouse model.

BACKGROUND: Infantile hemangiomas (IH) are the most common benign tumors of infancy. The typical clinical course consists of rapid growth during the first year of life, followed by natural and gradual involution over a multi-year time span through unknown cellular mechanisms. Some tumors respond to medical treatment with corticosteroids or beta-blockers, however, when this therapy fails or is incomplete, surgical extirpation may be necessary. Noninvasive therapies to debulk or eliminate these tumors would be an important advance. The development of an in vitro cell culture system and an animal model would allow new insights into the biological processes involved in the development and pathogenesis of IH. RESULTS: We observed that proliferative stage IH specimens contain significantly more SALL4+ and CD133+ cells than involuting tumors, suggesting a possible stem cell origin. A tumor sphere formation assay was adapted to culture IH cells in vitro. Cells in IH tumor spheres express GLUT1, indicative of an IH cell of origin, elevated levels of VEGF, and various stem/progenitor cell markers such as SALL4, KDR, Oct4, Nanog and CD133. These cells were able to self-renew and differentiate to endothelial lineages, both hallmarks of tumor stem cells. Treatment with Rapamycin, a potent mTOR/VEGF inhibitor, dramatically suppressed IH cell growth in vitro. Subcutaneous injection of cells from IH tumor spheres into immunodeficient NOD-SCID mice produced GLUT1 and CD31 positive tumors with the same cellular proliferation, differentiation and involution patterns as human hemangiomas. CONCLUSIONS: The ability to propagate large numbers of IH stem cells in vitro and the generation of an in vivo mouse model provides novel avenues for testing IH therapeutic agents in the future.

Comparison of circulating MicroRNA 141 to circulating tumor cells, lactate dehydrogenase, and prostate-specific antigen for determining treatment response in patients with metastatic prostate cancer.

Our aim was to determine the utility of circulating micro RNA miR-141 as a potential biomarker of therapeutic response in prostate cancer (CaP) patients. We compared the values of miR-141 in plasma of 21 CaP patients to the levels of prostate specific antigen (PSA), circulating tumor cells (CTC) and lactate dehydrogenase (LDH). Data suggest a strong correlation of miR-141 values and clinical course. For prostate cancer (CaP), the measurement of prostate-specific antigen (PSA) and radiographic studies do not adequately predict response to therapy and survival, and, therefore, new relevant biomarkers are needed. We and other researchers have shown that longitudinal measurements of PSA, circulating tumor cells (CTC), and lactate dehydrogenase (LDH) may aid in predicting response to therapy. Results of recent studies have determined that circulating microRNA (miRNA) miR-141 is detected in plasma of patients with CaP. We, therefore, compared the temporal changes of miR-141 with the levels of CTC, LDH, and PSA in 21 patients with CaP, and longitudinally examined these markers alone or in combinations to determine the utility of miR-141 in the predicting a patient's clinical course and response to therapy. Levels of miR-141 in plasma of 21 patients with CaP were measured by using quantitative reverse transcription-polymerase chain reaction. A total of 35 intervals were assessed. Directional changes (increasing or decreasing) in PSA, CTC, and miR-141 had sensitivity in predicting clinical outcome (progression vs. nonprogressing) of 78.9%. Logistic regression modeling of the probability of clinical progression demonstrates that miR-141 levels predicted clinical outcomes with an odds ratio of at least 8.3. miR-141 also had the highest correlation with temporal changes of PSA with a correlation of R = 0.77 (P < .001). In this retrospective study, miR-141 demonstrated a similar ability to predict clinical progression when compared with other clinically validated biomarkers. Furthermore, miR-141 demonstrated high correlation with changes of the other biomarkers.

Circulating tumor cells as a predictive biomarker in patients with hormone-sensitive prostate cancer.

UNLABELLED: Little information exists regarding the utility circulating tumor cell (CTC) enumeration in hormone sensitive prostate cancer. We enumerated CTC in 33 consecutive patients undergoing androgren deprivation therapy (ADT) at our institution. Multivariate analysis revealed baseline CTC as the only independent predictor of progression to CRPC. These data suggest that baseline CTC may identify those unlikely to benefit from ADT. INTRODUCTION: Circulating tumor cell (CTC) enumeration by using the Cellsearch platform has established prognostic and predictive value in patients with metastatic castration-resistant prostate cancer (mCRPC). Limited information exists regarding the clinical utility of CTC enumeration in metastatic hormone-sensitive prostate cancer (mHSPC). The goal of this study was to prospectively determine the relative clinical utility of CTCs in mHSPC. PATIENTS AND METHODS: We analyzed serial CTC in conjunction with other classic biomarkers in 33 consecutive patients treated at the Nevada Cancer Institute with HSPC initiating androgen deprivation therapy and correlated these patients with prognostic prostate-specific antigen (PSA) endpoints and onset of CRPC. RESULTS: Initial CTC correlated positively with lactate dehydrogenase and alkaline phosphatase, and were unrelated to PSA and testosterone. In univariate analysis, baseline CTC, alkaline phosphatase, lactate dehydrogenase, testosterone, and follow-up CTC were individual predictors of progression to CRPC. In a multivariate Cox regression, only baseline CTC retained independent predictive value. Threshold analysis revealed the cutpoint that optimized specificity and sensitivity of the test to be 3 cells per 7.5 mL whole blood. Baseline CTC also correlated well with PSA nadir benchmarks. CONCLUSIONS: Initial CTC values predict the duration and magnitude of response to hormonal therapy. CTC enumeration may identify patients at risk of progression to CRPC before initiation of androgen deprivation therapy.

Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFG(b1)/EGF in murine induced pluripotent stem cell-derived alveolar Type II-like cells.

Induced pluripotent stem (iPS) cells are derived from reprogrammed somatic cells and are similar to embryonic stem (ES) cells in morphology, gene/protein expression, and pluripotency. In this study, we explored the potential of iPS cells to differentiate into alveolar Type II (ATII)-like epithelial cells. Analysis using quantitative real time polymerase chain reaction and immunofluorescence staining showed that pulmonary surfactant proteins commonly expressed by ATII cells such as surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC) were upregulated in the differentiated cells. Microphilopodia characteristics and lamellar bodies were observed by transmission electron microscopy and lipid deposits were verified by Nile Red and Periodic Acid Schiff staining. C3 complement protein, a specific feature of ATII cells, was present at high levels in culture supernatants demonstrating functionality of these cells in culture. These data show that the differentiated cells generated from iPS cells using a culture method developed previously (Rippon et al., 2006) are ATII-like cells. To further characterize these ATII-like cells, we tested whether they could undergo epithelial to mesenchymal transition (EMT) by exposure to drugs that induce lung fibrosis in mice, such as bleomycin, and the combination of transforming growth factor beta1 (TGF(b1)) and epidermal growth factor (EGF). When the ATII-like cells were exposed to either bleomycin or a TGF(b1)-EGF cocktail, they underwent phenotypic changes including acquisition of a mesenchymal/fibroblastic morphology, upregulation of mesenchymal markers (Col1, Vim, a-Sma, and S100A4), and downregulation of surfactant proteins and E-cadherin. We have shown that ATII-like cells can be derived from skin fibroblasts and that they respond to fibrotic stimuli. These cells provide a valuable tool for screening of agents that can potentially ameliorate or prevent diseases involving lung fibrosis.

Ovarian cancer biomarker performance in prostate, lung, colorectal, and ovarian cancer screening trial specimens.

Establishing a cancer screening biomarker's intended performance requires "phase III" specimens obtained in asymptomatic individuals before clinical diagnosis rather than "phase II" specimens obtained from symptomatic individuals at diagnosis. We used specimens from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial to evaluate ovarian cancer biomarkers previously assessed in phase II sets. Phase II specimens from 180 ovarian cancer cases and 660 benign disease or general population controls were assembled from four Early Detection Research Network or Ovarian Cancer Specialized Program of Research Excellence sites and used to rank 49 biomarkers. Thirty-five markers, including 6 additional markers from a fifth site, were then evaluated in PLCO proximate specimens from 118 women with ovarian cancer and 474 matched controls. Top markers in phase II specimens included CA125, HE4, transthyretin, CA15.3, and CA72.4 with sensitivity at 95% specificity ranging from 0.73 to 0.40. Except for transthyretin, these markers had similar or better sensitivity when moving to phase III specimens that had been drawn within 6 months of the clinical diagnosis. Performance of all markers declined in phase III specimens more remote than 6 months from diagnosis. Despite many promising new markers for ovarian cancer, CA125 remains the single-best biomarker in the phase II and phase III specimens tested in this study.

A framework for evaluating biomarkers for early detection: validation of biomarker panels for ovarian cancer.

A panel of biomarkers may improve predictive performance over individual markers. Although many biomarker panels have been described for ovarian cancer, few studies used prediagnostic samples to assess the potential of the panels for early detection. We conducted a multisite systematic evaluation of biomarker panels using prediagnostic serum samples from the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) screening trial. Using a nested case-control design, levels of 28 biomarkers were measured laboratory-blinded in 118 serum samples obtained before cancer diagnosis and 951 serum samples from matched controls. Five predictive models, each containing 6 to 8 biomarkers, were evaluated according to a predetermined analysis plan. Three sequential analyses were conducted: blinded validation of previously established models (step 1); simultaneous split-sample discovery and validation of models (step 2); and exploratory discovery of new models (step 3). Sensitivity, specificity, sensitivity at 98% specificity, and AUC were computed for the models and CA125 alone among 67 cases diagnosed within one year of blood draw and 476 matched controls. In step 1, one model showed comparable performance to CA125, with sensitivity, specificity, and AUC at 69.2%, 96.6%, and 0.892, respectively. Remaining models had poorer performance than CA125 alone. In step 2, we observed a similar pattern. In step 3, a model derived from all 28 markers failed to show improvement over CA125. Thus, biomarker panels discovered in diagnostic samples may not validate in prediagnostic samples; utilizing prediagnostic samples for discovery may be helpful in developing validated early detection panels.

Increases in quantitative D-dimer levels correlate with progressive disease better than circulating tumor cell counts in patients with refractory prostate cancer.

Changes in quantitative D-dimer levels, circulating tumor cell (CTC) counts, and prostate-specific antigen (PSA) levels were measured in 28 patients with refractory castration-resistant prostate cancer to assess their concordance during the course of therapy and their relationship with risk of progressive disease. A significant correlation was identified between changes in PSA and both CTC counts and D-dimer levels (r = 0.67 and 0.58, respectively; P < .001). In addition, there was a significant correlation between changes in CTC count and D-dimer level (r = 0.62; P < .001). A significantly stronger concordance between these biomarkers was noted for increasing values (sensitivity, 72%-77.8%) compared with decreasing values (specificity, 43.8%-71.4%). Notably, increases in PSA and D-dimer levels, not CTC counts, were associated with increased risks for progressive disease (P < .024). Increases in quantitative D-dimer levels correlate with progressive disease better than CTC counts in patients with refractory prostate cancer.

Tumor growth factor expression in obesity and changes in expression with weight loss: another cause of increased virulence and incidence of cancer in obesity.

BACKGROUND: Obesity is associated with increased tumorigenesis. Previously, we demonstrated that inflammation in obesity caused cancer fighting cells to display greater surface receptor levels, predisposing them to early cell death. We measured the inflammatory tumor growth factor levels to determine whether inflammation in obesity increases expression of these factors, potentially predisposing these patients to greater rates of neoplasia. METHODS: A total of 24 patients undergoing weight loss surgery had samples collected preoperatively and at 6 and 12 months after surgery. The growth factors analyzed included tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor, interferon-γ, interleukin (IL)-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, vascular endothelial growth factor, hepatocyte growth factor, TNF-receptor I (TNF-RI), TNF-RII, death receptor 5, leptin, and adiponectin. Control samples were obtained from 10 healthy, normal weight volunteers. RESULTS: The tumor growth factors TNF-α, TNF-RI, TNF-RII, vascular endothelial growth factor, hepatocyte growth factor, interferon-γ, IL-2, IL-5, and IL-6 all decreased significantly (P <.05) compared with the preoperative values. The IL-4, IL-8, leptin, death receptor 5, adiponectin, and granulocyte-macrophage colony-stimulating factor levels did not change significantly over time. The IL-1b and IL-10 levels were less than the detection limit at all points. When obese patient serum was compared with healthy volunteer pooled serum, we found that the leptin, death receptor 5, hepatocyte growth factor, vascular endothelial growth factor, TNF-RI, TNF-RII, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, IL-4, IL-5, IL-6, and IL-8 levels were all 2-37 times greater than the levels in the controls at baseline. The concentrations of these same growth factors had decreased levels only 1-3.5 times greater than those of the controls at 12 months postoperatively. CONCLUSION: Many inflammatory tumor growth factors are present in greater concentrations in obese individuals. This could explain the greater prevalence of neoplasia in the morbidly obese population.

Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic beta-like cells.

Diabetes mellitus is characterized by either the inability to produce insulin (type 1 diabetes) or as insensitivity to insulin secreted by the body (type 2 diabetes). In either case, the body is unable to move blood glucose efficiently across cell membranes to be used. This leads to a variety of local and systemic detrimental effects. Current treatments for diabetes focus on exogenous insulin administration and dietary control. Here, we describe a potential cure for diabetes using a cellular therapy to ameliorate symptoms associated with both reduced insulin secretion and insulin sensitivity. Using induced pluripotent stem (iPS) cells, we were able to derive beta-like cells similar to the endogenous insulin-secreting cells in mice. These beta-like cells secreted insulin in response to glucose and corrected a hyperglycemic phenotype in two mouse models of type 1 and 2 diabetes via an iPS cell transplant. Long-term correction of hyperglycemia was achieved, as determined by blood glucose and hemoglobin A1c levels. These data provide an initial proof of principle for potential clinical applications of reprogrammed somatic cells in the treatment of diabetes type 1 or 2.

Generation of murine hepatic lineage cells from induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can be generated from somatic cells of individuals by retrodifferentiation using defined transcription factors. Similar to embryonic stem (ES) cells, iPS cells can be differentiated into a variety of specific cell types. However, to date, no detailed hepatic differentiation of mouse iPS cells has been reported. In this study, we successfully developed a stepwise protocol to induce hepatic differentiation of iPS cells reprogrammed from mouse tail tip fibroblasts. At day 25 of differentiation, the iPS cell-derived hepatocytes morphologically resemble mouse primary hepatocytes with a distinct polygonal shape. Immunostaining and reverse transcription-polymerase chain reaction analysis revealed expression of specific hepatic markers including alpha-fetoprotein, albumin and alpha-1-anti-trypsin. In addition, these iPS cell-derived hepatocytes successfully demonstrated mature liver cell functions in vitro. Furthermore, in vivo assays revealed that the mouse iPS cell-derived hepatocytes successfully engrafted into the recipient livers with typical hepatic morphology. Thus, iPS cell-derived hepatocytes may hold great promise as a unique system for basic liver research and liver regeneration in the near future.

Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies.

OBJECTIVE: To study the potential relationship between two sperm nuclear attributes: persistence of histones and occurrence of chromosomal aneuploidies. DESIGN: The two variables were examined by double probing of the same spermatozoa. SETTING: Academic Andrology Laboratory. PATIENT(S): Semen samples subjected for analyses were examined. INTERVENTION(S): We studied >58,000 spermatozoa, in seven men, first with aniline blue histone staining, graded as light (mature sperm), intermediate (moderately immature), and dark (severely arrested maturation). After recording the staining patterns and destaining, the same spermatozoa were subjected to fluorescence in situ hybridization (FISH), using centrometric X, Y, and 17 chromosome probes. MAIN OUTCOME MEASURE(S): Proportions of sperm with light, intermediate, and dark staining were assessed, and ploidy of these sperm was evaluated. RESULT(S): The aneuploidy frequencies in intermediate versus light (mature) spermatozoa were increased four- to sixfold. In addition, aneuploidy frequencies and proportions of intermediate sperm were related. There was no FISH signal detectable in the darkly stained, severely arrested mature sperm. CONCLUSION(S): The data suggest that in sperm with arrested maturity and DNA fragmentation, the binding of FISH probes is diminished. DNA damage is further aggravated by the decondensation and denaturation steps of FISH. Thus, there is a strong likelihood that in oligozoospermic men, with a higher proportion of sperm with arrested maturation, the sperm disomy frequencies are historically underestimated.

Enhanced detection of staphylococcal genomes in positive blood cultures using a polymeric enzyme complex.

This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin-horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1 fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35 min using either a thin film biosensor chip or a microtiter plate-based assay.

Generation and characterization of functional cardiomyocytes using induced pluripotent stem cells derived from human fibroblasts.

We have successfully developed both spontaneous and inductive cardiomyocyte differentiation of iPS cells reprogrammed from human foreskin fibroblasts. The reprogrammed iPS cells morphologically resemble human cardiomyocytes which can beat. RT-PCR and immunostaining show that cardiac markers are expressed that are comparable to the differentiation pattern of authentic human embryonic stem cells, indicating the existence of both immature and mature differentiated cardiomyocytes. 5-Azacytidine greatly enhanced the efficiency of cardiomyocyte differentiation, whereas dimethylsulfoxide had no effect. Low serum and bone morphogenetic protein-2 marginally improved differentiation efficiency. iPS cell-derived cardiomyocytes changed their beat frequency in response to cardiac drugs, which included ion channel blockers and alpha/beta adrenergic stimulators. Derived cardiomyocytes look promising as an in vitro system for potential drug screen and/or toxicity, making this system closer to practical use in the near future.