Selective, non-covalent conjugation of synthetic peptides with recombinant proteins mediated by host-guest chemistry.

The combination of potent chemical moieties with biologically active proteins is key to some of today's most innovative therapeutic drugs. In order to obviate any chemical modification of the proteins, we present a novel and powerful strategy for the selective conjugation of recombinant protein domains with synthetically derived peptides via a cucurbit[8]uril host-guest chemistry approach.We thank Alexandra Rieck, Isabelle Sermadiras, Benjamin Kemp and Jennifer Spooner for help with the expression and purification of the Fc and Tn3 domains, Jefferson Revell and Jacky Metcalfe for help with the synthesis of modified peptides and Christel Veyssier for help with the light scattering experiments. RJG was supported by the MedImmune postdoctoral program. SS was supported by ERC Starting Investigator grant ASPiRe (No. 240629).This is the final version of the article. It first appeared from Royal Society of Chemistry via http://dx.doi.org/10.1039/C6CC00405

Practical consensus guidelines for the management of enuresis

Despite the high prevalence of enuresis, the professional training of doctors in the evaluation and management of this condition is often minimal and/or inconsistent. Therefore, patient care is neither optimal nor efficient, which can have a profound impact on affected children and their families. Once comprehensive history taking and evaluation has eliminated daytime symptoms or comorbidities, monosymptomatic enuresis can be managed efficaciously in the majority of patients. Non-monosymptomatic enuresis is often a more complex condition; these patients may benefit from referral to specialty care centers. We outline two alternative strategies to determine the most appropriate course of care. The first is a basic assessment covering only the essential components of diagnostic investigation which can be carried out in one office visit. The second strategy includes several additional evaluations including completion of a voiding diary, which requires extra time during the initial consultation and two office visits before treatment or specialty referral is provided. This should yield greater success than first-line treatment. Conclusion: This guideline, endorsed by major international pediatric urology and nephrology societies, aims to equip a general pediatric practice in both primary and secondary care with simple yet comprehensive guidelines and practical tools (i.e., checklists, diary templates, and quick-reference flowcharts) for complete evaluation and successful treatment of enuresis

Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases

<p>Abstract</p> <p>Background</p> <p>Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH₃/B₆/F₁₀, originally subcloned from GH₃cells based on its ability to express high levels of the membrane estrogen receptor-α.</p> <p>Methods</p> <p>We measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities.</p> <p>Results</p> <p>Four phytoestrogens (coumestrol, daidzein, genistein, and <it>trans</it>-resveratrol) were studied over wide concentration ranges. Except <it>trans</it>-resveratrol, all phytoestrogens increased GH₃/B₆/F₁₀cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for <it>trans</it>-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens.</p> <p>Conclusion</p> <p>Phytoestrogens can block physiological estrogen-induced tumor cell growth <it>in vitro </it>and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary.</p

Curcumin and resveratrol inhibit nuclear factor-kappaB-mediated cytokine expression in adipocytes

<p>Abstract</p> <p>Background</p> <p>Adipocytes express inflammatory mediators that contribute to the low-level, chronic inflammation found in obese subjects and have been linked to the onset of cardiovascular disorders and insulin resistance associated with type 2 diabetes mellitus. A reduction in inflammatory gene expression in adipocytes would be expected to reverse this low-level, inflammatory state and improve cardiovascular function and insulin sensitivity. The natural products, curcumin and resveratrol, are established anti-inflammatory compounds that mediate their effects by inhibiting activation of NF-κB signaling. In the present study, we examined if these natural products can inhibit NF-κB activation in adipocytes and in doing so reduce cytokine expression.</p> <p>Methods</p> <p>Cytokine (TNF-α, IL-1β, IL-6) and COX-2 gene expression in 3T3-L1-derived adipocytes was measured by quantitative real-time PCR (qRT-PCR) with or without TNFα-stimulation. Cytokine protein and prostaglandin E₂(PGE₂) expression were measured by ELISA. Effects of curcumin and resveratrol were evaluated by treating TNFα-stimulated adipocytes with each compound and 1) assessing the activation state of the NF-κB signaling pathway and 2) measuring inflammatory gene expression by qRT-PCR and ELISA.</p> <p>Results</p> <p>Both preadipocytes and differentiated adipocytes express the genes for TNF-α, IL-6, and COX-2, key mediators of the inflammatory response. Preadipocytes were also found to express IL-1β; however, IL-1β expression was absent in differentiated adipocytes. TNF-α treatment activated NF-κB signaling in differentiated adipocytes by inducing IκB degradation and NF-κB translocation to the nucleus, and as a result increased IL-6 (6-fold) and COX-2 (2.5-fold) mRNA levels. TNF-α also activated IL-1β gene expression in differentiated adipocytes, but had no effect on endogenous TNF-α mRNA levels. No detectable TNFα or IL-1β was secreted by adipocytes. Curcumin and resveratrol treatment inhibited NF-κB activation and resulted in a reduction of TNF-α, IL-1β, IL-6, and COX-2 gene expression (IC₅₀= 2 μM) and a reduction of secreted IL-6 and PGE₂(IC₅₀~ 20 μM).</p> <p>Conclusion</p> <p>Curcumin and resveratrol are able to inhibit TNFα-activated NF-κB signaling in adipocytes and as a result significantly reduce cytokine expression. These data suggest that curcumin and resveratrol may provide a novel and safe approach to reduce or inhibit the chronic inflammatory properties of adipose tissue.</p

Modulation of Cytochrome P450 Metabolism and Transport across Intestinal Epithelial Barrier by Ginger Biophenolics

Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE) confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural ‘‘milieu’’ confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G) and 6-shogaol (6S), through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP) enzyme activity in human liver microsomes by monitoring metabolites of CYPspecific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE’s inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp). Intriguingly, however, 10G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an indepth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens

Windbreaks in North American Agricultural Systems

Windbreaks are a major component of successful agricultural systems throughout the world. The focus of this chapter is on temperate-zone, commercial, agricultural systems in North America, where windbreaks contribute to both producer profitability and environmental quality by increasing crop production while simultaneously reducing the level of off-farm inputs. They help control erosion and blowing snow, improve animal health and survival under winter conditions, reduce energy consumption of the farmstead unit, and enhance habitat diversity, providing refuges for predatory birds and insects. On a larger landscape scale windbreaks provide habitat for various types of wildlife and have the potential to contribute significant benefits to the carbon balance equation, easing the economic burdens associated with climate change. For a windbreak to function properly, it must be designed with the needs of the landowner in mind. The ability of a windbreak to meet a specific need is determined by its structure: both external structure, width, height, shape, and orientation as well as the internal structure; the amount and arrangement of the branches, leaves, and stems of the trees or shrubs in the windbreak. In response to windbreak structure, wind flow in the vicinity of a windbreak is altered and the microclimate in sheltered areas is changed; temperatures tend to be slightly higher and evaporation is reduced. These types of changes in microclimate can be utilized to enhance agricultural sustainability and profitability. While specific mechanisms of the shelter response remain unclear and are topics for further research, the two biggest challenges we face are: developing a better understanding of why producers are reluctant to adopt windbreak technology and defining the role of woody plants in the agricultural landscape

Working passionately does not always pay off : the negative moderating role of passion on the relationship between deliberate practice and venture performance

Deliberate practice, an iterative process that leads to expertise, is found to be positively associated with superior performance in domains such as sports, education, and entrepreneurship. At the same time, deliberate practice is also seen as being less than enjoyable and difficult to pursue consistently. As such, passion is considered to be a vital motivator of engagement in and maintenance of deliberate practice. Despite the evident importance of passion, the relationship between passion and deliberate practice in entrepreneurship has not been subject to sufficient empirical evaluation. Therefore, in this study, we consider the way in which passion moderates the relationship between deliberate practice and venture performance. We hypothesize that deliberate practice is positively related to venture performance and that passion positively moderates this relationship. We find support for our first hypothesis, in line with previous studies. However, contrary to our second hypothesis, we find that entrepreneurial passion negatively moderates the deliberate practice-venture performance relationship. In response to this finding, we provide possible explanations as to why this negative moderation effect was observed by drawing on Kolb’s experiential learning cycle