Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research.

Hominids adapted to metabolize ethanol long before human-directed fermentation.

Paleogenetics is an emerging field that resurrects ancestral proteins from now-extinct organisms to test, in the laboratory, models of protein function based on natural history and Darwinian evolution. Here, we resurrect digestive alcohol dehydrogenases (ADH4) from our primate ancestors to explore the history of primate-ethanol interactions. The evolving catalytic properties of these resurrected enzymes show that our ape ancestors gained a digestive dehydrogenase enzyme capable of metabolizing ethanol near the time that they began using the forest floor, about 10 million y ago. The ADH4 enzyme in our more ancient and arboreal ancestors did not efficiently oxidize ethanol. This change suggests that exposure to dietary sources of ethanol increased in hominids during the early stages of our adaptation to a terrestrial lifestyle. Because fruit collected from the forest floor is expected to contain higher concentrations of fermenting yeast and ethanol than similar fruits hanging on trees, this transition may also be the first time our ancestors were exposed to (and adapted to) substantial amounts of dietary ethanol.

Expression of pluripotent stem cell reprogramming factors by prostate tumor initiating cells.

PURPOSE: We identified a discrete population of stem cell-like tumor cells expressing 5 essential transcription factors required to reprogram pluripotency in prostate tumor cell lines and primary prostate cancer tissue. MATERIALS AND METHODS: DU145 and PC3 human prostate cancer cell lines (ATCC), tumor tissue from patients with prostate cancer and normal prostate tissue were evaluated for the reprogramming factors OCT3/4 (Cell Signaling Technology), SOX2, Klf4 (Santa Cruz Biotechnology, Santa Cruz, California), Nanog (BioLegend) and c-Myc (Cell Signaling) by semiquantitative reverse transcriptase-polymerase chain reaction, histological and immunohistochemical analysis. Stem cell-like tumor cells were enriched by flow cytometric cell sorting using E-cadherin (R&D Systems) as a surface marker, and soft agar, spheroid and tumorigenicity assays to confirm cancer stem cell-like characteristics. RESULTS: mRNA expression of transcription factors OCT3/4 and SOX2 highly correlated in primary prostate tumor tissue samples. The number of OCT3/4 or SOX2 expressing cells was significantly increased in prostate cancer tissue compared to that in normal prostate or benign prostate hyperplasia tissue (p <0.05). When isolated from the DU145 and PC3 prostate cancer cell lines by flow cytometry, stem cell-like tumor cells expressing high OCT3/4 and SOX2 levels showed high tumorigenicity in immunodeficient mice. In vivo growth of the parental DU145 and PC3 prostate cancer cell lines was inhibited by short hairpin RNA knockdown of OCT3/4 or SOX2. CONCLUSIONS: Data suggest that prostate tumor cells expressing pluripotent stem cell transcription factors are highly tumorigenic. Identifying such cells and their importance in prostate cancer growth could provide opportunities for novel targeting strategies for prostate cancer therapy.

Differentiation of human embryonic stem cells into immunostimulatory dendritic cells under feeder-free culture conditions.

PURPOSE: The objective of this study was to develop a scalable and broadly applicable active immunotherapy approach against cancer, circumventing the limitations typically encountered with autologous vaccination strategies. We hypothesized that human embryonic stem cells (hESC) can serve as a virtually unlimited source for generating dendritic cells (DC) with potent antigen-presenting function. Here, we investigated the developmental processes and requirements for generating large numbers of mature, antigen-presenting DC from pluripotent hESC. EXPERIMENTAL DESIGN: A feeder cell-free culture system was developed to differentiate hESC into mature DC sequentially through hematopoietic and myeloid precursor stages. RESULTS: Using this method, we were able to yield large numbers of mature immunostimulatory DC from hESC to enable clinical investigation. Upon activation, the hESC-derived DC secreted interleukin-12p70, migrated in response to MIP-3beta, and exhibited allostimulatory capacity. Most importantly, antigen-loaded, hESC-derived DC were capable of stimulating potent antigen-specific CD8(+) T-cell responses in an HLA class I-matched semiallogeneic assay system. Moreover, HLA class II-mismatched hESC-derived DC induced a potent Th1-type cytokine response without expanding FOXP3(+) regulatory T cells in vitro. CONCLUSIONS: These data suggest the development of a novel active immunotherapy platform to stimulate potent T-cell immunity in patients with intractable diseases, such as cancer or viral infection.

Flavopiridol displays preclinical activity in acute lymphoblastic leukemia.

BACKGROUND: New agents are needed for treatment of children with relapsed acute lymphoblastic leukemia (ALL). Based on altered expression of cell cycle regulatory proteins, including frequent p16 (INK4A) and p15 (INK4B) deletions, flavopiridol (FP; Alvocidib) is an attractive agent for relapsed ALL. PROCEDURE: We evaluated the efficacy of FP in ALL cell lines using cell proliferation assays, determined the effects of FP treatment on cell growth and viability in cell lines and patient samples, examined cell cycle kinetics, and evaluated the effect of FP on endogenous cyclin-dependent kinase (CDK) activity, Mcl-1 expression, and RNA polymerase II expression and phosphorylation. RESULTS: ALL cell lines are sensitive to FP. At lower concentrations, FP induces transient G(1)-S cell cycle arrest and modest levels of apoptosis in cell lines. In contrast, a sustained G(1)-S and G(2)-M arrest and substantial apoptosis are observed following exposure to higher FP concentrations. After treatment with FP, ALL cell lines have decreased expression of retinoblastoma protein phosphorylated at serines 795 and 807/811, indicating reduced CDK activity. We also show that ALL cell lines are sensitive to clinically achievable concentrations of FP in medium supplemented with human serum and that FP reduces the expression of Mcl-1 and phosphorylated forms of the C-terminal domain of RNA polymerase II. FP also increases cell death by approximately twofold over baseline in primary ALL blasts. CONCLUSIONS: These data provide a biological rationale for testing FP in relapsed ALL.

17 Beta-estradiol improves survival in male mice with cardiomyopathy induced by cardiac-specific tumor necrosis factor-alpha overexpression.

A transgenic mouse model of congestive heart failure (CHF) consequent to cardiac-specific overexpression of tumor necrosis factor-alpha (TNF-alpha) (TNF1.6) displays marked sex-related phenotypic differences. To clarify the potential contributions of estrogen to these sex-specific differences, male TNF1.6 mice were treated with 17beta- estradiol (E2). E2 treatment started at 25 +/- 1 days old (group A), but not at 36 +/- 2 days old (group B), significantly improved survival rate (p < 0.05). Furthermore, ventricular weight/body weight ratio was significantly decreased by E2 treatment in group A (p < 0.05). Echocardiography revealed that E2-treated hearts in group A exhibited less left ventricular dilatation (p < 0.05) relative to untreated male TNF1.6 mice (control). Moreover, in group A, E2 treatment partially reversed basal and isoproterenol-stimulated fractional shortening in TNF1.6 mice (p < 0.05). The cardiac content of TNF-alpha and interleukin-1beta (IL-1beta) was not changed by E2 treatment regardless of the timing of treatment. Thus, E2 exposure prior to puberty can limit the severity of cardiomyopathy in male TNF1.6 mice.

Tumor necrosis factor receptors 1 and 2 differentially regulate survival, cardiac dysfunction, and remodeling in transgenic mice with tumor necrosis factor-alpha-induced cardiomyopathy.

BACKGROUND: Tumor necrosis factor (TNF)-alpha plays a pathophysiological role in heart failure. Although both TNF receptor 1 (TNFR1) and 2 (TNFR2) are present in the heart, comparatively little is known about the role of TNFR2. METHODS AND RESULTS: We bred TNFR1-knockout (KO) or TNFR2KO mice to transgenic (TG) mice with cardiac-specific overexpression of TNF-alpha and analyzed resultant progeny. Six groups of male and female mice were studied: wild type (WT) with wild receptors (WT/W), TG with wild receptors (TG/W), TG with heterozygous receptor KO (TG/R1+/- or TG/R2+/-), and TG with homozygous receptor KO (TG/R1-/- or TG/R2-/-). Both male and female TG mice displayed cardiac hypertrophy, dilation, and reduced cardiac function. Male TG mice were more severely affected than genotypically matched females and died of heart failure at a younger age. Survival, cardiac function, and remodeling of TG/R1+/- and TG/R1-/- mice were improved relative to TG/W mice in both males and females. However, the survival of female TG/R2+/- and TG/R2-/- mice was worse than that of TG/W mice, with increased left ventricular dimension and left ventricular weight/body weight ratios. The cardiac TNF-alpha protein level was upregulated in TG/R1-/- and TG/R2-/- compared with TG/W mice, whereas the level of TNF receptors was not downregulated in TG/W relative to WT/W mice. CONCLUSIONS: Ablation of the TNFR2 gene exacerbates heart failure and reduces survival, whereas ablation of TNFR1 blunts heart failure and improves survival. Signaling via TNFR2 may play a cardioprotective role in the pathogenesis of cytokine-mediated heart failure.

Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-alpha.

Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-alpha (TNF-alpha), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca(2+) (Ca(i)(2+)) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of beta-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by approximately 20%). The amplitude of Ca(i)(2+) transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD(50%)) of the Ca(i)(2+) transient, an index of the rate of sarcoplasmic reticulum Ca(2+) uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca(i)(2+) transients and contractions were reduced, TD(50%) of the Ca(i)(2+) transient was prolonged, and the inotropic effect of Iso on Ca(i)(2+) transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca(2+) handling in the former. These results indicate that cardiac-specific overexpression of TNF-alpha induces myocyte hypertrophy and gender-dependent alterations in Ca(i)(2+) handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.

Plasma levels of TNF-alpha and IL-6 are inversely related to cytochrome P450-dependent drug metabolism in patients with congestive heart failure.

BACKGROUND: Cytochrome P450 (CYP) enzymes are important mediators of drug metabolism, and activity of these enzymes is a major determinant of the duration and intensity of drug effect. Circulating plasma concentrations of pro-inflammatory cytokines (e.g., tumor necrosis factor [TNF]-alpha and interleukin [IL]-6) are elevated in patients with heart failure and these cytokines have been shown to down-regulate CYP enzyme activity. The purpose of this study was to evaluate the relationship between plasma cytokine concentrations and CYP enzyme activities in patients with heart failure. METHODS AND RESULTS: Sixteen patients with congestive heart failure (New York Heart Association classes II-IV) received a metabolic probe cocktail consisting of caffeine, mephenytoin, dextromethorphan, and chlorzoxazone to assess the activities of the CYP enzymes 1A2, 2C19, 2D6, and 2E1. Blood and urine samples were collected for drug and metabolite determinations by high-performance liquid chromatography (HPLC); cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). We found a striking inverse relationship between both TNF-alpha and IL-6 plasma concentrations and the activity of CYP2C19; metabolism of caffeine (CYP1A2) also had a negative association with IL-6 plasma concentrations. CONCLUSIONS: Cytokine-mediated decreases in drug metabolism may contribute to observed variability in drug response and augment the risk of adverse drug effects in CHF patients.

Chronic exposure to interleukin 1beta induces a delayed and reversible alteration in excitation-contraction coupling of cultured cardiomyocytes.

While proinflammatory cytokines can depress cardiac contractility, the mechanism by which this occurs remains unclear. To clarify the cellular effects of interleukin (IL)-1beta, we assessed contractility, calcium homeostasis, and gene expression in cardiomyocytes exposed to this proinflammatory cytokine. Neonatal rat cardiomyocytes were exposed to IL-1beta in the presence or absence of an inhibitor of nitric oxide (NO) synthase. Videomicroscopy was used to follow calcium transients (Fura-2 fluorescence) and amplitude of contraction, both unstimulated and after isoproterenol challenge. Gene expression was assessed by Northern and Western blot analyses. Both basal contractility (amplitude of contraction, maximum speed of contraction and relaxation) and amplitude of calcium transients were decreased, respectively, ca. 60% ( P< or =0.05) and ca. 40% ( P< or =0.05) after 3 days of IL-1beta exposure. Contractile function and amplitude of calcium transients returned to control values when cells where cultured an additional 3 days in the absence of IL-1beta. IL-1beta-treated cells had reduced responses to isoproterenol as evidenced by a lack of enhanced amplitude of contraction and a reduction in cAMP production. IL-1beta decreased the expression of genes important to the regulation of calcium homeostasis (phospholamban, sarcoplasmic reticulum calcium ATPase) at both the transcript and protein level. Alterations in contractile function did not occur through NO-mediated pathways. These results support the hypothesis that IL-1beta may play an important role in contractile dysfunction through alterations in calcium homeostasis.