A versatile oscillating-flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis

We report the development of a versatile system based on the oscillating-flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181-bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating-flow system and a commercial real-time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample-to-result time by eliminating an additional 30 min melt curve step required in commercial PCR systems.     

Myxobacteria versus sponge-derived alkaloids: the bengamide family identified as potent immune modulating agents by scrutiny of LC-MS/ELSD libraries

A nuclear factor-κB (NF-κB) luciferase assay has been employed to identify the bengamides, previously known for their anti-tumor activity, as a new class of immune modulators. A unique element of this study was that the bengamide analogs were isolated from two disparate sources, Myxococcus virescens (bacterium) and Jaspis coriacea (sponge). Comparative LC-MS/ELSD and NMR analysis facilitated the isolation of M. viriscens derived samples of bengamide E (8) and two congeners, bengamide E' (13) and F' (14) each isolated as an insperable mixture of diastereomers. Additional compounds drawn from the UC, Santa Cruz repository allowed expansion of the structure activity relationship (SAR) studies. The activity patterns observed for bengamide A (6), B (7), E (8), F (9), LAF 389 (12) and 13-14 gave rise to the following observations and conclusions. Compounds 6 and 7 display potent inhibition of NF-κB (at 80 and 90 nM, respectively) without cytotoxicity to RAW264.7 macrophage immune cells. Western blot and qPCR analysis indicated that 6 and 7 reduce the phosphorylation of IκBα and the LPS-induced expression of the pro-inflammatory cytokines/chemokines TNFα, IL-6 and MCP-1 but do not effect NO production or the expression of iNOS. These results suggest that the bengamides may serve as therapeutic leads for the treatment of diseases involving inflammation, that their anti-tumor activity can in part be attributed to their ability to serve as immune modulating agents, and that their therapeutic potential against cancer merits further consideration.     

Hormonal state influences aspects of female mate choice in the Túngara Frog (Physalaemus pustulosus)

Females alter their mate choices as they transition through different reproductive stages; however, the proximal mechanisms for such behavioral fluctuation are unclear. In many taxa, as females transition through different reproductive stages, there is an associated change in hormone levels; therefore, we examined whether fluctuation in hormone levels serves as a proximal mechanism for within-individual variation in mate choice in female túngara frogs (Physalaemus pustulosus). We manipulated hormone levels of females by administering 0, 10, 100, 500 or 1,000 IU of human chorionic gonadotropin (HCG), which is a ligand for luteinizing hormone (LH) receptors and will therefore cause increased gonadal hormone production. Phonotaxis assays were conducted to measure three aspects of mate choice behavior before and after HCG administration; receptivity (response to a conspecific mate signal), permissiveness (response to a signal that is less attractive than conspecific signals) and discrimination (ability to discern signal differences). The probability of response to a conspecific and an artificial hybrid signal significantly increased at the highest HCG doses. The difference in mean response time between pre- and post-HCG tests was significantly different for both the receptivity and permissiveness tests among the five doses. Increased permissiveness, however, was not due to decreased discrimination because females could discriminate between calls even at the highest HCG doses. These hormonal manipulations caused the same behavioral pattern we reported in females as they transitioned through different reproductive stages (Lynch, K.S., Rand, A.S., Ryan, M.J., Wilczynski, W., 2005. Plasticity in female mate choice associated with changing reproductive states. Anim. Behav. 69, 689-699), suggesting that changes in hormone levels can influence the female's mate choice behavior.     

Synthesis and SAR of pyrrolotriazine-4-one based Eg5 inhibitors

Synthesis and SAR of substituted pyrrolotriazine-4-one analogues as Eg5 inhibitors are described. Many of these analogues displayed potent inhibitory activities in the Eg5 ATPase and A2780 cell proliferation assays. In addition, pyrrolotriazine-4-one analogue 26 demonstrated in vivo efficacy in an iv P388 murine leukemia model. Both NMR and X-ray crystallographic studies revealed that these analogues bind to an allosteric site on the Eg5 protein.     

Assessing compound binding to the Eg5 motor domain using a thermal shift assay

Eg5 is a kinesin whose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetry of our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5′-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP-Eg5 complexes using a thermal shift assay. We measured up to a 7 °C increase in the thermal melting (T_m) of Eg5 for an inhibitor that produced IC₅₀ values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase (ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assays correlated with the magnitude of the thermal stability enhancement of ADP-Eg5. The thermal shift assay also confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assay is applicable to a range of chemotypes and can be useful in evaluating both potent (nM) and relatively weakly binding (μM) leads. Overall, the thermal shift assay was found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assay screens by providing an alternative determination of inhibitor potency.     

Analyzing the coordinated gene network underlying temperature-dependent sex determination in reptiles

Although gonadogenesis has been extensively studied in vertebrates with genetic sex determination, investigations at the molecular level in nontraditional model organisms with temperature-dependent sex determination are relatively new areas of research. Results show that while the key players of the molecular network underlying gonad development appear to be retained, their functions range from conserved to novel roles. In this review, we summarize experiments investigating candidate molecular players underlying temperature-dependent sex determination. We discuss some of the problems encountered unraveling this network, pose potential solutions, and suggest rewarding future directions of research.     

Testosterone induction of male‐typical sexual behavior is associated with increased preoptic NADPH diaphorase and citrulline production in female whiptail lizards

In rodents, male‐typical copulatory behavior is generally dependent on gonadal sex steroids such as testosterone, and it is thought that the mechanism by which the hormone gates the behavior involves the gaseous neurotransmitter nitric oxide. According to one model, testosterone induces an up‐regulation of nitric oxide synthase (NOS) in the preoptic area, increasing nitric oxide synthesis following exposure to a sexual stimulus. Nitric oxide in turn, possibly through its effect on catecholamine turnover, influences the way the stimulus is processed and enables the appropriate copulatory behavioral response. In whiptail lizards (genus Cnemidophorus), administration of male‐typical levels of testosterone to females induces the display of male‐like copulatory responses to receptive females, and we hypothesized that this radical change in behavioral phenotype would be accompanied by a large change in the expression of NOS in the preoptic area. As well as comparing NOS expression using NADPH diaphorase histochemistry between testosterone‐treated females and controls, we examined citrulline immunoreactivity (a marker of recent nitric oxide production) in the two groups, following a sexual stimulus and following a nonsexual stimulus. Substantially more NADPH diaphorase‐stained cells were observed in the testosterone‐treated animals. Citrulline immunoreactivity was greater in testosterone‐implanted animals than in blank‐implanted animals, but only following exposure to a sexual stimulus. This is the first demonstration that not only is NOS up‐regulated by testosterone, but NOS thus up‐regulated is activated during male‐typical copulatory behavior. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006