Identification of domains within megalomicin and erythromycin polyketide synthase modules responsible for differences in polyketide production levels in Escherichia coli

The megalomicin and erythromycin polyketide synthases (PKSs) produce the same aglycon product, 6-deoxyerythronolide B (6-dEB). Both PKSs were examined in an Escherichia coli strain metabolically engineered to support complex polyketide biosynthesis. Production of 6-dEB in shake flask fermentations was undetectable by mass spectrometry in the strain expressing the megalomicin (Meg) PKS genes, whereas 31 mg/L 6-dEB was produced by the strain with the erythromycin (DEBS) PKS. The genes for each of the three subunits comprising the PKSs were expressed in different combinations from three compatible expression vectors (e.g., DEBS1, DEBS2, and MegA3) to identify two Meg PKS subunits, MegA1 and MegA3, which conferred lower 6-dEB titers than their DEBS counterparts. Comparison of protein expression levels and 6-dEB titers by engineered hybrid DEBS/Meg PKS genes further defined regions within modules 2 and 6 of MegA1 and MegA3, respectively, which limit protein expression and 6-dEB production in E. coli. Meg module 2 + TE (M2 + TE) and a hybrid DEBS M2/Meg M2 + TE protein were engineered and purified for in vitro comparisons with DEBS M2 + TE. The specific activity of the hybrid M2 + TE was approximately 16-fold lower than DEBS M2 + TE and only twice as high as the Meg M2 + TE enzyme in diketide elongation assays. Since the hybrid M2 worked comparably to DEBS M2 in vivo, this suggests that boosting subunit concentration could serve as a useful approach to overcome enzyme deficiencies in heterologous polyketide production.     

Combination unilateral amygdaloid and ventromedial hypothalamic lesions: evidence for a feeding pathway

Previous studies have reported hyperphagia and obesity in female rats with bilateral lesions of the most posterodorsal part of the amygdala. In rats with unilateral posterodorsal amygdaloid lesions, a dense pattern of anterograde degeneration appears in the ipsilateral ventromedial hypothalamus, but not the contralateral nucleus. In the present study, female rats with unilateral ventromedial hypothalamic lesions or sham lesions were given either sham lesions or unilateral lesions of the posterodorsal amygdala (PDA) 20 days later. Unilateral lesions of the ventromedial hypothalamus resulted in hyperphagia and excessive weight gain. Subsequent amygdaloid lesions that were contralateral to the initial hypothalamic lesions resulted in hyperphagia and additional excessive weight gains, but amygdaloid lesions ipsilateral to the initial hypothalamic lesions did not. It is concluded that the effects of the two lesions on body weight are not additive and that the PDA and ventromedial hypothalamus are part of the same ipsilateral pathway regulating feeding behavior and body weight regulation.     

A synthesis of two decades of research documenting the effects of noise on wildlife

Global increases in environmental noise levels – arising from expansion of human populations, transportation networks, and resource extraction – have catalysed a recent surge of research into the effects of noise on wildlife. Synthesising a coherent understanding of the biological consequences of noise from this literature is challenging. Taxonomic groups vary in auditory capabilities. A wide range of noise sources and exposure levels occur, and many kinds of biological responses have been observed, ranging from individual behaviours to changes in ecological communities. Also, noise is one of several environmental effects generated by human activities, so researchers must contend with potentially confounding explanations for biological responses. Nonetheless, it is clear that noise presents diverse threats to species and ecosystems and salient patterns are emerging to help inform future natural resource‐management decisions. We conducted a systematic and standardised review of the scientific literature published from 1990 to 2013 on the effects of anthropogenic noise on wildlife, including both terrestrial and aquatic studies. Research to date has concentrated predominantly on European and North American species that rely on vocal communication, with approximately two‐thirds of the data set focussing on songbirds and marine mammals. The majority of studies documented effects from noise, including altered vocal behaviour to mitigate masking, reduced abundance in noisy habitats, changes in vigilance and foraging behaviour, and impacts on individual fitness and the structure of ecological communities. This literature survey shows that terrestrial wildlife responses begin at noise levels of approximately 40 dBA, and 20% of papers documented impacts below 50 dBA. Our analysis highlights the utility of existing scientific information concerning the effects of anthropogenic noise on wildlife for predicting potential outcomes of noise exposure and implementing meaningful mitigation measures. Future research directions that would support more comprehensive predictions regarding the magnitude and severity of noise impacts include: broadening taxonomic and geographical scope, exploring interacting stressors, conducting larger‐scale studies, testing mitigation approaches, standardising reporting of acoustic metrics, and assessing the biological response to noise‐source removal or mitigation. The broad volume of existing information concerning the effects of anthropogenic noise on wildlife offers a valuable resource to assist scientists, industry, and natural‐resource managers in predicting potential outcomes of noise exposure.     

Cyclic analogs of galanin and neuropeptide Y by hydrocarbon stapling

Hydrocarbon stapling is an effective strategy to stabilize the helical conformation of bioactive peptides. Here we describe application of stapling to anticonvulsant neuropeptides, galanin (GAL) and neuropeptide Y (NPY), that are implicated in modulating seizures in the brain. Dicarba bridges were rationally introduced into minimized analogs of GAL and NPY resulting in increased α-helical content, in vitro metabolic stability and n-octanol/water partitioning coefficient (log D). The stapled analogs retained agonist activities towards their respective receptors and suppressed seizures in a mouse model of epilepsy.     

The unique nucleotide specificity of the sucrose synthase from Thermosynechococcus elongatus

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. In filamentous cyanobacteria, the sucrose cleavage direction plays a key physiological function in carbon metabolism, nitrogen fixation, and stress tolerance. In unicellular strains, the function of sucrose synthase has not been elucidated. We report a detailed biochemical characterization of sucrose synthase from Thermosynechococcus elongatus after the gene was artificially synthesized for optimal expression in Escherichia coli. The homogeneous recombinant sucrose synthase was highly specific for ADP as substrate, constituting the first one with this unique characteristic, and strongly suggesting an interaction between sucrose and glycogen metabolism.     

Molecular Mechanisms of EAST/SeSAME Syndrome Mutations in Kir4.1 (KCNJ10)

Inwardly rectifying potassium channel Kir4.1 is critical for glial function, control of neuronal excitability, and systemic K⁺ homeostasis. Novel mutations in Kir4.1 have been associated with EAST/SeSAME syndrome, characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt waste. Patients are homozygous for R65P, G77R, C140R or T164I; or compound heterozygous for A167V/R297C or R65P/R199Stop, a deletion of the C-terminal half of the protein. We investigated the functional significance of these mutations by radiotracer efflux and inside-out membrane patch clamping in COSm6 cells expressing homomeric Kir4.1 or heteromeric Kir4.1/Kir5.1 channels. All of the mutations compromised channel function, but the underlying mechanisms were different. R65P, T164I, and R297C caused an alkaline shift in pH sensitivity, indicating that these positions are crucial for pH sensing and pore gating. In R297C, this was due to disruption of intersubunit salt bridge Glu²⁸⁸–Arg²⁹⁷. C140R breaks the Cys¹⁰⁸–Cys¹⁴⁰ disulfide bond essential for protein folding and function. A167V did not affect channel properties but may contribute to decreased surface expression in A167V/R297C. In G77R, introduction of a positive charge within the bilayer may affect channel structure or gating. R199Stop led to a dramatic decrease in surface expression, but channel activity was restored by co-expression with intact subunits, suggesting remarkable tolerance for truncation of the cytoplasmic domain. These results provide an explanation for the molecular defects that underlie the EAST/SeSAME syndrome.     

Chromatin insulators specifically associate with different levels of higher-order chromatin organization in Drosophila

Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Here, we have analyzed the distribution of insulator proteins on the 56F–58A region of chromosome 2R in Drosophila polytene chromosomes to assess the role of chromatin insulators in shaping genome architecture. Data show that the suppressor of Hairy-wing protein [Su(Hw)] is found in three structures differentially associated with insulator proteins: bands, interbands, and multi-gene domains of coexpressed genes. Results show that bands are generally formed by condensation of chromatin that belongs to genes containing one or more Su(Hw) binding sites, whereas, in interbands, Su(Hw) sites appear associated with open chromatin. In addition, clusters of coexpressed genes in this region form bands characterized by the lack of CP190 and BEAF-32 insulator proteins. This pattern correlates with the distribution of specific chromatin marks and is conserved in nurse cells, suggesting that this organization may not be limited to one cell type but represents the basic organization of interphasic chromosomes.     

Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C₁₇H₂₇NO_9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.