Demineralization enables reeling of wild silkmoth cocoons

Wild Silkmoth cocoons are difficult or impossible to reel under conditions that work well for cocoons of the Mulberry silkmoth, Bombyx mori . Here we report evidence that this is caused by mineral reinforcement of Wild Silkmoth cocoons and that washing these minerals out allows for the reeling of commercial lengths of good quality fibers with implications for the development of the "Wild Silk" industry. We show that in the Lasiocampid silkmoth Gonometa postica , the mineral is whewellite (calcium oxalate monohydrate). Evidence is presented that its selective removal by ethylenediaminetetraacetic acid (EDTA) leaves the gum substantially intact, preventing collapse and entanglement of the network of fibroin brins, enabling wet reeling. Therefore, this method clearly differs from the standard "degumming" and should be referred to as "demineralizing". Mechanical testing shows that such preparation results in reeled silks with markedly improved breaking load and extension to break by avoiding the damage produced by the rather harsh degumming, carding, or dry reeling methods currently in use, what may be important for the development of the silk industries not only in Asia but also in Africa and South America.     

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli

Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.     

Hepatocyte CYP2E1 overexpression and steatohepatitis lead to impaired hepatic insulin signaling

Insulin resistance and increased cytochrome P450 2E1 (CYP2E1) expression are both associated with and mechanistically implicated in the development of nonalcoholic fatty liver disease. Although currently viewed as distinct factors, insulin resistance and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signaling. To test this possibility, the effects of in vitro and in vivo CYP2E1 overexpression on hepatocyte insulin signaling were examined. CYP2E1 overexpression in a hepatocyte cell line decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in response to insulin. CYP2E1 overexpression was also associated with increased inhibitory serine 307 and 636/639 IRS-1 phosphorylation. In parallel, the effects of insulin on Akt activation, glycogen synthase kinase 3, and FoxO1a phosphorylation, and glucose secretion were all significantly decreased in CYP2E1 overexpressing cells. This inhibition of insulin signaling by CYP2E1 overexpression was partially c-Jun N-terminal kinase dependent. In the methionine- and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression, insulin-induced IRS-1, IRS-2, and Akt phosphorylation were similarly decreased. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signaling, potentially contributing to the insulin resistance associated with nonalcoholic fatty liver disease.     

Restrained molecular dynamics studies on complex of adriamycin with DNA hexamer sequence d-CGATCG

Adriamycin is an anthracycline anticancer drug used widely for solid tumors in spite of its adverse side effects. The solution structure of 2:1 adriamycin-d-(CGATCG)(2) complex has been studied by restrained molecular dynamics simulations. The restraint data set consists of several intramolecular and intermolecular nuclear Overhauser enhancement cross-peaks obtained from two-dimensional nuclear magnetic resonance spectroscopy data. The drug is found to intercalate between CG and GC base pairs at two d-CpG sites. The drug-DNA complex is stabilized via specific hydrogen bonding and van der Waal's interactions involving 4OCH(3), O5, 6OH, and NH(3)(+) moiety of daunosamine sugar, and rings A protons. The O-glycosidic bond C7-O7-C1'-C2' lies in the range 138 degrees -160 degrees during the course of simulations. The O6-H6...O5 hydrogen bond is stable while O11-H11...O12 hydrogen bond is not favored. The intercalating base pairs are buckled and minor groove is wider in the complex. The phosphate on one strand at intercalation site C1pG2 is in B(I) conformation and the phosphates directly lying on opposite strand is in B(II) conformation. The phosphorus on adjacent site G2pA3 is in B(II) conformation and hence a distinct pattern of B(I) and B(II) conformations is induced and stabilized. The role of various functional groups by which the molecular action is mediated has been discussed and correlated to the available biochemical evidence.     

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).     

Signaling through CD14 attenuates the inflammatory response to Borrelia burgdorferi, the agent of Lyme disease

Lyme disease is a chronic inflammatory disorder caused by the spirochetal bacterium, Borrelia burgdorferi. In vitro evidence suggests that binding of spirochetal lipoproteins to CD14, a pattern recognition receptor expressed on monocytes/macrophages and polymorphonuclear cells, is a critical requirement for cellular activation and the subsequent release of proinflammatory cytokines that most likely contribute to symptomatology and clinical manifestations. To test the validity of this notion, we assessed the impact of CD14 deficiency on Lyme disease in C3H/HeN mice. Contrary to an anticipated diminution in pathology, CD14(-/-) mice exhibited more severe and persistent inflammation than did CD14(+/+) mice. This disparity reflects altered gene regulation within immune cells that may engender the higher bacterial burden and serum cytokine levels observed in CD14(-/-) mice. Comparing their in vitro stimulatory activity, live spirochetes, but not lysed organisms, were a potent CD14-independent stimulus of cytokine production, triggering an exaggerated response by CD14(-/-) macrophages. Collectively, our in vivo and in vitro findings support the provocative notion that: 1) pattern recognition by CD14 is entirely dispensable for elaboration of an inflammatory response to B. burgdorferi, and 2) CD14-independent signaling pathways are inherently more destructive than CD14-dependent pathways. Continued study of CD14-independent signaling pathways may provide mechanistic insight into the inflammatory processes that underlie development of chronic inflammation.     

Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site

IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.     

Molecular docking analysis of azithromycin and hydroxychloroquine with spike surface glycoprotein of SARS-CoV-2

Millions of people are affected by COVID-19 since the last quarter of 2019. Treatment using hydroxychloroquine (HCQ) as monotherapy in combination with azithromycin (HCQ-AZ) were administered at several clinical centres to patients tested positive to the virus across continents. Therefore, it is of interest to document the molecular docking analysis data of azithromycin and hydroxychloroquine drug with the spike surface glycoprotein of novel COVID-19. Thus, we report the molecular modelling docking based structural binding features of HCQ-AZ with the spike surface glycoprotein of COVID-19 for further evaluation in this regard.     

gamma-cleavage-independent functions of presenilin, nicastrin, and Aph-1 regulate cell-junction organization and prevent tau toxicity in vivo

Genetic analysis of familial Alzheimer's disease has revealed that mutations in the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however, presenilin mutations might also influence tau hyperphosphorylation and neurodegeneration through gamma-secretase-independent mechanisms. To address this possibility and determine whether other components of the gamma-secretase complex possess similar regulatory functions, we analyzed the roles of presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced neurodegeneration. Here, we show that presenilin and nicastrin prevent tau toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these transmembrane proteins differentially regulate the intracellular localization of GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity neither interfered with these kinase pathways nor induced aberrant tau phosphorylation. These results establish new in vivo molecular functions for the three components of the gamma-secretase complex and reveal a different mechanism that might contribute to neuronal degeneration in Alzheimer's disease.