Background: Human islet amyloid polypeptide (hIAPP) fibrils of unknown structure are formed in type 2 diabetes. Results: A hIAPP fibril structure was derived from EPR data, electron microscopy, and computer modeling. Conclusion:The fibril is a left-handed helix that contains hIAPP monomers in a staggered conformation. Significance: The results provide the basis for therapeutic prevention of fibril formation and growth.

NADPH is an important cofactor in many biosynthesis pathways that control fundamental cellular processes. We recently determined the crystal structure of HSCARG, with functions previously unknown, and demonstrated it is an NADPH sensor, which undergoes restructuring and redistribution in response to changes of intracellular NADPH/NADP levels. In this study, we identified argininosuccinate synthetase (AS), a rate-limiting enzyme in nitric oxide synthesis, as capable of associating with HSCARG and demonstrated further that HSCARG decreased nitric oxide synthesis by down-regulating AS activity, whereas AS overexpression up-regulated hscarg mRNA transcription, suggesting a negative feedback mechanism. A decrease in the NADPH/NADP ؉ ratio, induced by dehydroepiandrosterone treatment, enhanced the interaction between HSCARG and AS, which resulted in stronger inhibition of AS activity and nitric oxide production. The dimerization region of HSCARG, amino acids 153-189, was identified to undergo critical interactions with AS. Furthermore, the viability of HSCARG RNA interference-treated epithelial cells decreased significantly, accompanied by an increase of the activity of caspase-3, which suggested that the loss of viability was because of apoptosis. These results indicate that HSCARG regulation of AS activity is crucial for maintaining the intracellular balance between redox state and nitric oxide levels.Nitric oxide (NO), 2 a cellular signaling molecule, has been shown to be involved in vascular regulation, autoimmunity, and neurotransmission and impacts diverse biological processes, including cell survival (1-7). The impaired production of NO can result in the vascular dysfunction, whereas overproduction of NO will induce some diseases such as the cerebral infarction, diabetes mellitus, and neurodegenerative disorders (7-11). Arginine is the sole amino acid substrate that is required for the production of NO (12, 13), and its regeneration from citrulline, the co-product of NO synthesis, is rate-limited by argininosuccinate synthetase (AS) (13-16). In addition, the reducing reagent donor, NADPH, and oxygen are necessary for NO production (13, 17). For this reason, NO production is not only limited by the regeneration of arginine but is also affected by the intracellular NADPH concentration, which requires cross-talk between the signaling pathways of NADPH and NO.In addition to its well known function in energy metabolism, NADH, along with its phosphorylated relative NADPH, has been recognized as an important regulatory molecule. Together, their roles are crucial in signaling pathways that control fundamental cellular processes, such as transcription, regulation of calcium homeostasis, and apoptosis (18 -20). NAD mainly exists in its oxidized state (NAD ϩ ), whereas NADP is largely found in its reduced form, NADPH (21, 22). The predominant function of NADP is to maintain a pool in its reduced form to ensure a rapid regeneration of the defense systems to protect cells from oxidative damage. NADPH holds a key position in...

Electron paramagnetic resonance (EPR) using site-directed spin-labeling (SDSL) can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of inter-label distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of inter-label distances. We describe an algorithm, PRONOX, for rapid computation of inter-label distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12, and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein; a total of 44 inter-label distances. The program reproduced these distances accurately (r2=0.94, slope=0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggests that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure.

The aim of this study was to investigate the effects of Clostridium butyricum (C. butyricum) on the performance, serum lipid metabolism, muscle morphology, meat quality, and fatty acid profiles of Peking ducks. A total of 1,500 Peking ducks were randomly divided into five groups with five replicates and were fed a non-antibiotic basal diet (Control) or a basal diet supplemented with either 200, 400, or 600 mg/kg of C. butyricum (2.0 × 109 CFU/g) or 150 mg of aureomycin/kg for 42 d. Compared with the control group, supplementation with C. butyricum increased the average daily weight gain but reduced the feed/gain ratio from 1 to 42 d of age. Similarly, dietary C. butyricum increased the activities of antioxidant enzymes but decreased the malondialdehyde (MDA) and lipid metabolites concentration. C. butyricum supplementation increased the muscle pH value at 45 min postmortem, the redness of the meat, and the contents of inosine acid (IMP) and intramuscular fat (IMF) in Peking ducks. By contrast, C. butyricum supplementation lowered the lightness, drip loss, and the shear force of breast meat. Supplementation with C. butyricum increased the concentrations of essential amino acids and flavor amino acids, as well as arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and total polyunsaturated fatty acids (PUFA) in breast muscle. Dietary C. butyricum could positively improve performance, lipid metabolism, meat quality, and the amino acid and fatty acid composition in a dose-dependent manner. Therefore, C. butyricum is proposed as a feasible alternative feed additive for the production of healthier Peking duck meat with favorable properties.

Objective Enhanced de novo lipogenesis (DNL) in hepatocytes is a major contributor to nonalcoholic fatty liver disease (NAFLD). Fatty acid translocase (FAT/CD36) is involved in the pathogenesis of NAFLD through facilitating free fatty acids uptake. Here, we explored the effects of CD36 on DNL and elucidated the underlying mechanisms. Methods We generated hepatocyte-specific CD36 knockout (CD36LKO) mice to study in vivo effects of CD36 on DNL under high-fat diet (HFD). Lipid deposition and DNL were analyzed in primary hepatocytes isolated from CD36LKO mice or HepG2 cells with CD36 overexpression. RNA sequence, co-immunoprecipitation, and proximity ligation assay were carried out to determine its role in regulating DNL. Results Hepatic CD36 expression was upregulated in NAFLD mice and patients, and CD36LKO mice exhibited attenuated HFD-induced hepatic steatosis and insulin resistance. We identified hepatocyte CD36 as a key regulator for DNL in the liver. Sterol regulatory element-binding protein 1 (SREBP1) and its downstream lipogenic enzymes such as FASN, ACCα, and ACLY were significantly downregulated in the liver of HFD-fed CD36LKO mice, whereas overexpression CD36 stimulated insulin-mediated DNL and lipid droplet formation in vitro. Mechanistically, CD36 was activated by insulin and formed a complex with insulin-induced gene-2 (INSIG2) that disrupts the interaction between SREBP cleavage-activating protein (SCAP) and INSIG2, thereby leading to the translocation of SREBP1 from ER to Golgi for processing. Furthermore, treatment with 25-hydroxycholesterol or betulin molecules shown to enhance SCAP–INSIG interaction, reversed the effects of CD36 on SREBP1 cleavage. Conclusions Our findings identify a previously unsuspected role of CD36 in the regulation of hepatic lipogenic program through mediating SREBP1 processing by INSIG2, providing additional evidence for targeting CD36 in NAFLD.

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BackgroundThis retrospective study was designed to investigate the sole influence of orthokeratology (OK) lens fitting decentration on the Zernike coefficients of the reshaped anterior corneal surface.MethodsThis study comprised a review of 106 right eyes and measurements of corneal topography both before OK and at 1-month follow-up visit. A routine was designed to calculate local corneal surface astigmatism and assist the determination of OK lens fitting decentration from pupil center. The pupil-centered corneal Zernike coefficients of baseline (PCCB) and post-treatment (PCCP) were calculated. Meanwhile, the OK-lens-centered corneal Zernike coefficients of post-treatment (OCCP) were also calculated and considered as the presumptive ideal fitting group without decentration. Relationships between lens fitting decentration and the change of Zernike coefficients including (PCCP − PCCB) and (PCCP − OCCP) were analyzed.ResultsPatients with a mean age of 11 ± 2.36 years old had an average spherical equivalent refractive error of −3.52 ± 1.06 D before OK. One month after treatment, OK lens fitting decentration from pupil center was 0.68 ± 0.35 mm. RMS of 3rd-order (P < 0.05), RMS of 4th-order (P < 0.001) and RMS of total high order (P < 0.001) corneal Zernike coefficients were increased in PCCP by comparing with OCCP, which was solely caused by lens fitting decentration. Nevertheless, no significant difference was observed in (P > 0.05). For the high order corneal Zernike coefficients in (PCCP – OCCP), radial distance of decentration was correlated with (r = −0.296, P < 0.05), (r = −0.396, P < 0.001), and (r = 0.449, P < 0.001), horizontal decentration was significantly correlated with (r = 0.901, P < 0.001) and (r = 0.340, P < 0.001), and vertical decentration was significantly correlated with (r = 0.904, P < 0.001).ConclusionsOK lens fitting decentration within 1.5 mm hardly influenced the change of corneal spherical power for myopia correction, but significantly induced additional corneal high order Zernike coefficients including , , , and .

Water plays an important role in the mediation of biomolecular interactions. Thus, accurate prediction and evaluation of water-mediated interactions is an important element in the computational design of interfaces involving proteins, RNA, and DNA. Here, we use an algorithm (WATGEN) to predict the locations of interfacial water molecules for a data set of 224 protein-RNA interfaces. The accuracy of the prediction is validated against water molecules present in the X-ray structures of 105 of these complexes. The complexity of the water networks is deconvoluted through definition of the characteristics of each water molecule based on its bridging properties between the protein and RNA and on its depth in the interface with respect to the bulk solvent. This approach has the potential for scoring the water network for incorporation into the computational design of protein-RNA complexes.