Gene regulatory networks are based on simple building blocks such as promoters, transcription factors (TFs) and their binding sites on DNA. But how diverse are the functions that can be obtained by different arrangements of promoters and TF binding sites? In this work we constructed synthetic regulatory regions using promoter elements and binding sites of two noninteracting TFs, each sensing a single environmental input signal. We show that simply by combining these three kinds of elements, we can obtain 11 of the 16 Boolean logic gates that integrate two environmental signals in vivo. Further, we demonstrate how combination of logic gates can result in new logic functions. Our results suggest that simple elements of transcription regulation form a highly flexible toolbox that can generate diverse functions under natural selection.evolution | gene regulation | regulatory logic | signal integration

The expression of inducible nitric oxide synthase (NOS2) in glial cells is inhibited by neurotransmitters such as norepinephrine (NE) which elevate cAMP levels. We examined the molecular basis for this effect using a 2.2-kb fragment of the rat NOS2 promoter transfected into rat C6 glioma cells. Promoter activation (up to six-fold) by lipopolysaccharide (LPS) and interferon-g (IFNg) was reduced by NE, which alone had no effect. However, a promoter construct extending to bp 2130 and containing the proximal nuclear factor-kappa B (NF-kB) binding site was minimally activated by LPS and cytokines, but activated up to three-fold by NE. Deletion analysis identi®ed a 27-bp region (bp 2187 to 2160) as critical for mediating this suppressive effect. This region also enhanced promoter activation by LPS and cytokines, and prevented activation by NE alone. Gel shift analysis revealed constitutive binding to this region, and induction by NE of additional complexes which could be blocked by an antibody against CREB. NE also increased levels of the IkBa protein which could contribute to its suppressive effects. These results identify a critical role for this 27-bp region in regulation of NOS2 promoter activation and suppression by cAMP.

Background and Objective There is general agreement that certain fatty acids and lipopolysaccharides (LPS) promote inflammation through toll-like receptor 4 (TLR4), and that inflammation promotes insulin resistance. We therefore hypothesized that mice with periodontitis and a TLR4 loss-of-function (LOF) mutation fed a high-fat (HF) diet would develop improved glucose homeostasis compared with wild-type (WT) animals with periodontitis fed a HF diet. Material and Methods Wild-type and TLR4 mutant mice fed a HF diet were divided into four groups (n = 6/group): WT; WT with periodontitis (WT/P); mutant (Mut); and mutant with periodontitis (Mut/P). Periodontitis was induced by placing LPS soaked ligatures around maxillary second molars. Fasting insulin and glucose levels were measured weekly for 10 wk. Glucose tolerance was evaluated at baseline (week 1) and at 9 wk. Insulin signaling (phosphorylation of Akt) and tumor necrosis factor-α (TNF-α) mRNA levels in liver were determined when the mice were killed at week 10. Results Mut/P mice developed less alveolar bone loss compared with WT/P mice (p < 0.05). Fasting glucose levels were improved after 8 wk of feeding a HF diet (weeks 9 and 10) in Mut/P mice compared with Mut, WT and WT/P mice (p < 0.05). Glucose tolerance was impaired in all groups compared with baseline (p < 0.05), except for the Mut/P group. Insulin signaling was improved (p < 0.05), and expression of TNF-α was decreased (p < 0.05) in the liver of Mut/P mice compared with the liver of WT/P mice. Conclusion The TLR4 LOF mutation partially protects against alveolar bone loss and improves glucose homeostasis in mice with periodontitis fed a HF diet.

Clinical trials evaluating the management of acute exacerbations of COPD assess heterogeneous outcomes, often omitting those that are clinically relevant or more important to patients. We have developed a core outcome set, a consensus-based minimum set of important outcomes that we recommend are evaluated in all future clinical trials on exacerbations management, to improve their quality and comparability. COPD exacerbations outcomes were identified through methodological systematic reviews and qualitative interviews with 86 patients from 11 countries globally. The most critical outcomes were prioritized for inclusion in the core outcome set through a two-round Delphi survey that was completed by 1,063 participants (256 patients, 488 health professionals and 319 clinical academics) from 88 countries in 5 continents. Two global, multi-stakeholder, virtual consensus meetings were conducted to (i) finalize the core outcome set and (ii) prioritize a single measurement instrument to be used for evaluating each of the prioritized outcomes. Consensus was informed by rigorous methodological systematic reviews. The views of patients with COPD were accounted for in all stages of the project. Survival, treatment success, breathlessness, quality of life, activities of daily living, need for higher level of care, arterial blood gases, disease progression, future exacerbations and hospital admissions, treatment safety and adherence were all included in the core outcome set. Focused methodological research was recommended to further validate and optimize some of the selected measurement instruments. The panel did not consider the prioritized set of outcomes and associated measurement instruments burdensome for patients and health professionals to use.

Expression of inflammatory nitric oxide synthase (NOS2) is mediated by transcription factor NFB. By using the specific proteasome inhibitor lactacystin to examine IB degradation, we observed a paradoxical increase in lipopolysaccharide-and cytokine-dependent NOS2 expression at low concentrations or when lactacystin was added subsequent to cytokines. Lactacystin reduced the initial accumulation of NOS2 mRNA but reduced its subsequent decrease. Lactacystin increased NOS2 promoter activation after 24 h, but not after 4 h, and similarly prevented initial NFB activation and at later times caused NFB reactivation. Lactacystin reduced initial degradation of IB-␣ and IB-␤, however, at later times selectively increased IB-␤, which was predominantly non-phosphorylated. Expression of fulllength rat IB-␤, but not a carboxyl-terminal truncated form, inhibited NOS2 induction and potentiation by lactacystin. Lactacystin increased IB-␤ expression in the absence of NOS2 inducers, as well as expression of heat shock protein 70, and the heat shock response due to hyperthermia increased IB-␤ expression. These results suggest that IB-␤ contributes to persistent NFB activation and NOS2 expression in glial cells, that IB-␤ is a stress protein inducible by hyperthermia or proteasome inhibitors, and that delayed addition of proteasome inhibitors can have stimulatory rather than inhibitory actions.In brain, inflammatory responses of astroglial cells occur during disease, infection, trauma, and ischemia. These responses include release of pro-inflammatory cytokines as well as synthesis and release of nitric oxide (NO).1 In astrocytes, NO is primarily biosynthesized by the calcium-independent isoform of NO synthase (NOS2) which is normally not present but whose expression is induced by a variety of inflammatory stimuli. Increasing evidence points to a role for NOS2-derived NO in the pathogenesis of a variety of human neurological diseases and brain trauma (1). In human brain, astrocytes express NOS2 during demyelinating diseases (2-4), cerebral ischemia (5, 6), viral infection (7), traumatic brain injury (8), Alzheimer's disease (9), and possibly during AID's dementia (10). Beneficial aspects of preventing or reducing NOS2 expression have been demonstrated in animal studies of experimental autoimmune encephalomyelitis (EAE) (11) and cerebral ischemia (12) demonstrating that suppression of astroglial NOS2 can be of therapeutic value in the prevention of neurological damage. Primary cultures of astrocytes from rat (13, 14), mouse (15), and human (16) have been used extensively to characterize the inductive process of astroglial NOS2 expression. In vitro studies have demonstrated that stimulation of cells with bacterial endotoxin lipopolysaccharide (LPS) or with a combination of cytokines which nominally includes IL1-␤ leads to de novo expression of NOS2. Extensive characterization of glial NOS2 expression and regulation has also been carried out with the rat C6 glioma cell line (17, 18), which shares many properties with primary astrocyte cu...