Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions.

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short ß-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREBTAD) construct. The CREBTAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

Establishing an Australian pediatric spinal cord disorder register using consumer, health professional, and researcher perspectives.

CONTEXT/OBJECTIVE: Pediatric spinal cord disorders (SCD) are rare, and epidemiological data available to support treatment are lacking. The implementation of a national data register tailored to this population would greatly assist clinicians and therapists in guiding clinical practice. This study gathered perspectives surrounding a prospective national pediatric spinal cord disorder register. DESIGN: Survey and modified Delphi technique. SETTING: Australia. PARTICIPANTS: SCD consumers, health professionals, and researchers. INTERVENTIONS: None. OUTCOME MEASURES: None. RESULTS: Purposive sampling recruited 6 consumers and 52 health professionals and researchers working in the field of SCD to participate. The consumer survey contained items including demographic information, general and pediatric-specific SCD health issues, and questions regarding activity and participation. The modified Delphi survey required health professionals and researchers to identify which "collection items" and "administrative features" should be included in a national SCD register for both clinical and research purposes. Seventeen essential and nine optional items, two outcome measures, data collection methods, consumer access, definition of "pediatric," and use of International Data Standards were included in the consensus for a minimum dataset. CONCLUSION: This study developed a minimum dataset that could inform an Australian register for pediatric SCD. A register linking to an adult database is recommended to ensure coverage across the lifespan. While items for a minimum dataset have been recommended, this dataset is large. Review and refinement of this list are recommended to ensure the register is not overly time-consuming for practical use.

Benralizumab for PDGFRA-Negative Hypereosinophilic Syndrome.

BACKGROUND: Hypereosinophilic syndrome is a group of diseases defined by marked eosinophilia in blood or tissue and eosinophil-related clinical manifestations. Benralizumab is a monoclonal antibody against interleukin-5 receptor α, which is expressed on human eosinophils. METHODS: In this randomized, double-blind, placebo-controlled, phase 2 trial, we administered a series of three monthly subcutaneous injections of either benralizumab (at a dose of 30 mg) or placebo in 20 symptomatic patients who had PDGFRA-negative hypereosinophilic syndrome and an absolute eosinophil count of at least 1000 cells per cubic millimeter; all the patients were receiving stable therapy (drugs or dietary changes) for this disease. This regimen was followed by an open-label phase, during which the patient's background therapy could be tapered as tolerated, and an extension phase. The primary end point of the randomized phase was a reduction of at least 50% in the absolute eosinophil count at week 12. RESULTS: During the randomized phase, the primary end point occurred in more patients in the benralizumab group than in the placebo group (9 of 10 patients [90%] vs. 3 of 10 patients [30%], P = 0.02). During the open-label phase, clinical and hematologic responses were observed in 17 of 19 patients (89%) and were sustained for 48 weeks in 14 of 19 patients (74%); in the latter group, in 9 of 14 patients (64%), background therapies could be tapered. Bone marrow and tissue eosinophilia were also suppressed with benralizumab therapy. The most common drug-related adverse events, headache and an elevated lactate dehydrogenase level, occurred in 32% of the patients after the first dose of benralizumab and resolved within 48 hours in all patients. Other adverse events occurred with similar frequency in the two groups. Of the many potential predictors of response that were examined, only clinical disease subtype appeared to be associated with the initial response or relapse. CONCLUSIONS: In this small phase 2 trial, patients with PDGFRA-negative hypereosinophilic syndrome who received benralizumab for 12 weeks had lower absolute eosinophil counts than those who received placebo. During the open-label phase, clinical and hematologic responses were sustained for 48 weeks in 74% of the patients. Adverse events did not limit treatment. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov numbers, NCT00001406 and NCT02130882.).

Native wildflower plantings support wild bee abundance and diversity in agricultural landscapes across the United States.

Global trends in pollinator-dependent crops have raised awareness of the need to support managed and wild bee populations to ensure sustainable crop production. Provision of sufficient forage resources is a key element for promoting bee populations within human impacted landscapes, particularly those in agricultural lands where demand for pollination service is high and land use and management practices have reduced available flowering resources. Recent government incentives in North America and Europe support the planting of wildflowers to benefit pollinators; surprisingly, in North America there has been almost no rigorous testing of the performance of wildflower mixes, or their ability to support wild bee abundance and diversity. We tested different wildflower mixes in a spatially replicated, multiyear study in three regions of North America where production of pollinator-dependent crops is high: Florida, Michigan, and California. In each region, we quantified flowering among wildflower mixes composed of annual and perennial species, and with high and low relative diversity. We measured the abundance and species richness of wild bees, honey bees, and syrphid flies at each mix over two seasons. In each region, some but not all wildflower mixes provided significantly greater floral display area than unmanaged weedy control plots. Mixes also attracted greater abundance and richness of wild bees, although the identity of best mixes varied among regions. By partitioning floral display size from mix identity we show the importance of display size for attracting abundant and diverse wild bees. Season-long monitoring also revealed that designing mixes to provide continuous bloom throughout the growing season is critical to supporting the greatest pollinator species richness. Contrary to expectation, perennials bloomed in their first season, and complementarity in attraction of pollinators among annuals and perennials suggests that inclusion of functionally diverse species may provide the greatest benefit. Wildflower mixes may be particularly important for providing resources for some taxa, such as bumble bees, which are known to be in decline in several regions of North America. No mix consistently attained the full diversity that was planted. Further study is needed on how to achieve the desired floral display and diversity from seed mixes.

Thermochemical studies of epoxides and related compounds.

Gas-phase heats of formation for the four butene oxide isomers are reported. They were obtained by measuring the condensed-phase heat of reduction to the corresponding alcohol using reaction calorimetry. Heats of vaporization were determined and allow gas-phase heats of formation to be obtained. The experimental measurements are compared to calculations obtained using a variety of computational methods. Overall, the G3 and CBS-APNO methods agree quite well with the experimental data. The influence of alkyl substituents on epoxide stability is discussed. Comparisons to alkenes, cyclopropanes, aziridines, thiiranes, and phosphiranes are also made. Isodesmic-type reactions were used to determine strain energies of the epoxides and related compounds with various substituents.

Development of flavone propargyl ethers as potent and selective inhibitors of cytochrome P450 enzymes 1A1 and 1A2.

Naturally occurring flavonoids are known to be metabolized by several cytochrome P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, 3A4, and 3A5. In general flavonoids can act as substrates, inducers, and/or inhibitors of P450 enzymes. The position of the substituents on the flavone backbone has been shown to impact the biological activity against P450 enzymes. To explore the effect of a propargyl ether substitution on flavones and flavanones, 2´-flavone propargyl ether (2´-PF), 3´-flavone propargyl ether (3´-PF), 4´-flavone propargyl ether (4´-PF), 5-flavone propargyl ether (5-PF), 6-flavone propargyl ether (6-PF), 7-flavone propargyl ether (7-PF), 6-flavanone propargyl ether (6-PFN), and 7- flavanone propargyl ether (7-PFN) were synthesized. All of the newly synthesized compounds and the parent hydroxy flavones were tested for both direct inhibition and mechanism-based inhibition of cytochrome P450 enzymes 1A1, 1A2, 2A6, and 2B1. The flavone propargyl ether derivatives were found to be more potent inhibitors of P450s 1A1 and 1A2. None of the flavones and flavanones in our study showed any inhibition of P450 2A6. Only 2´-PF and 6-PFN inhibited P450 2B1. 3´-PF showed direct inhibition of P450 1A1 with the highest observed potency of 0.02 µM, in addition to its ability to cause mechanism-based inhibition with KI and kinactivation values of 0.24 µM and 0.09 min-1 for this enzyme. 7- Hydroxy flavone also exhibited mechanism-based inhibition of P450 1A1 with KI and kinactivation values of 2.43 µM and 0.115 min-1. Docking studies and QSAR studies on P450 enzymes 1A1 and 1A2 were performed which revealed important insights into the nature of binding of these molecules and provided us with good QSAR models that can be used to design new flavone derivatives.

Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome.

Protein folding is a fundamental biological process of great significance for cell function and life-related processes. Surprisingly, very little is presently known about how proteins fold in vivo. The influence of the cellular environment is of paramount importance, as molecular chaperones, the ribosome, and the crowded medium affect both folding pathways and potentially even equilibrium structures. Studying protein folding in physiologically relevant environments, however, poses a number of technical challenges due to slow tumbling rates, low concentrations and potentially non-homogenous populations. Early work in this area relied on biological assays based on antibody recognition, proteolysis, and activity studies. More recently, it has been possible to directly observe the structure and dynamics of nascent polypeptides at high resolution by spectroscopic and microscopic techniques. The fluorescence depolarization decay of nascent polypeptides labeled with a small extrinsic fluorophore is a particularly powerful tool to gain insights into the dynamics of newly synthesized proteins. The fluorophore label senses both its own local mobility and the motions of the macromolecule to which it is attached. Fluorescence anisotropy decays can be measured both in the time and frequency domains. The latter mode of data collection is extremely convenient to capture the nanosecond motions in ribosome-bound nascent proteins, indicative of the development of independent structure and folding on the ribosome. In this review, we discuss the theory of fluorescence depolarization and its exciting applications to the study of the dynamics of nascent proteins in the cellular environment.

Production of ribosome-released nascent proteins with optimal physical properties.

The growing interest in protein folding under physiologically relevant conditions has prompted investigations requiring direct comparisons between ribosome-bound and ribosome-released nascent proteins. Such studies, involving the ad hoc release of newly synthesized proteins from stalled ribosomes, demand a release agent able to produce nonaggregated native proteins and preserve the overall nature of the medium. Here, we explore hydroxylamine, a reactant rarely used to release nascent chains, and compare it to other ribosome-release agents: puromycin, RNase A/EDTA, and sodium hydroxide. Ribosome-bound nascent chains corresponding to the sequence of apoHmpH, the Escherichia coli N-terminal domain of Hmp, were used as a model system. Fluorescence anisotropy decays were employed to probe the self-association and overall physical properties of nascent proteins. Gel electrophoresis and RNA chip microfluidic capillary electrophoresis yielded information on the integrity of nascent peptidyl-tRNAs and ribosomes, respectively. Of the four reagents examined, only hydroxylamine releases nascent apoHmpH without causing extensive aggregation or degradation of the ribosome. Hydroxylamine does not introduce large hydrophobic C-terminal modifications and functions at nearly physiological pH. It is therefore a suitable reagent for the ad hoc release of nascent proteins from the ribosome.

Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions.

We still know very little about how proteins achieve their native three-dimensional structure in vitro and in the cell. Folding studies as proteins emerge from the mega Dalton-sized ribosome pose special challenges due to the large size and complicated nature of the ribosome-nascent chain complex. This work introduces a combination of three-component analysis of fluorescence depolarization decays (including the presence of two local motions) and in-cone analysis of diffusive local dynamics to investigate the spatial constraints experienced by a protein emerging from the ribosomal tunnel. We focus on E. coli ribosomes and an all-alpha-helical nascent globin in the presence and absence of the cotranslationally active chaperones DnaK and trigger factor. The data provide insights on the dynamic nature and structural plasticity of ribosome-nascent chain complexes. We find that the sub-ns motions of the N-terminal fluorophore, reporting on the globin dynamics in the vicinity of the N terminus, are highly constrained both inside and outside the ribosomal tunnel, resulting in high-order parameters (>0.85) and small cone semiangles (<30 degrees ). The shorter globin chains buried inside the tunnel are less spatially constrained than those of a reference sequence from a natively unfolded protein, suggesting either that the two nascent chain sequences have a different secondary structure and therefore sample different regions of the tunnel or that the tunnel undergoes local structural adjustments to accommodate the globin sequence. Longer globins emerging out of the ribosomal tunnel are also found to have highly spatially constrained slow (ns) motions. There are no observable spectroscopic changes in the absence of bound chaperones.

Chain dynamics of nascent polypeptides emerging from the ribosome.

Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.