Structural,Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare

Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 Å resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the −7 to −4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.     

Iraqi propolis increases degradation of IL-1β and NLRC4 by autophagy following Pseudomonas aeruginosa infection

Autophagy is a cellular process for maintaining cellular homeostasis. This process can be induced by different factors, such as immune stimuli and pathogen-associated molecules. Autophagy has an important role in the control of IL-1β secretion by macrophages and other cell types. In present study, we describe a novel role for Iraqi propolis affecting autophagy in controlling the secretion of IL-1β in bone-marrow macrophages (BMDMs). After infection with Pseudomonas aeruginosa in the presence of propolis, the degradation of IL-1β was induced, and the activity of inflammasome was reduced. Iraqi propolis-induced autophagy in in vitro and in vivo models decreased the levels of IL-1β and caspase-1. Results indicated that IL-1β pathway production is regulated by autophagy via two different novel mechanisms, namely, regulation of the activation of NLRC4 inflammasome and IL-1β targeting for lysosomal degradation.     

Failure of neutralizing gp120 monoclonal antibodies to prevent HIV infection of choriocarcinoma-derived trophoblasts

Although placental trophoblasts, the only fetal cells in direct contact with infectious maternal blood, can be infected with HIV, the precise cause for the low transmission rate of virus across the placental barrier is unknown. One of the most common conjectures is that maternal anti-HIV antibodies (Abs) contribute to the protection of the fetus. This hypothesis has been tested in vitro by infecting the CD4-negative placental trophoblast line, BeWo, with HIV-1_IIIB in the presence of serial dilutions of neutralizing monoclonal Abs against the V3 loop (No. 694) or CD4-binding conformational domain (No. 588). The results, based on measurement of p24 production from virus-exposed cells, reveal that the titers of Abs, adequate in preventing the infection of control MT-4 T lymphocytes, were less effective in protecting trophoblasts. Furthermore, PCR analysis of HIV DNA formed after a single round of infection has shown no significant decrease in the number of viral copies in Ab-protected BeWo cells. An anti-HIV serum from a pregnant woman did also have no effect. Although our in vitro observations do not necessarily apply to the in vivo situation, the results suggest that the humoral immune response sustained by neutralizing Abs may be able to protect T lymphocytes, but not placental trophoblasts. The findings are consistent with recent clinical studies demonstrating a lack of correlation between the presence of neutralizing anti-HIV Abs in pregnant women and HIV transmission in utero.     

Use of SRAP markers to assess genetic diversity and population structure of wild, cultivated, and ornamental pomegranates (Punica granatum L.) in different regions of Iran

Sequence-related amplified polymorphism (SRAP) was used to assess the genetic diversity of 63 cultivated, wild, and ornamental pomegranate genotypes from five different geographical regions of Iran. A total of 250 fragments were amplified using 13 primer combinations; among these, 133 bands (53?%) were polymorphic. The average PIC value was 0.28 over all PCs. The genetic distance among genotypes ranged from 0.10 to 0.37 with an average of 0.24. Cluster analysis using the neighbor-joining (NJ) method suggested there are close relationships between ornamental and some wild genotypes. Although AMOVA results revealed significant differences in the genetic diversity among the regions (P?=?0.0048), the genetic variation was mainly caused by variation of intra regions. The results indicated low genetic differentiation (Fst?=?0.025) and high gene flow (Nm?=?2.28) among regions. These results confirmed that SRAP markers could be powerful tools and an effective marker system for determining the genetic diversity and population genetic structure of the pomegranate.     

Binding of divalent metal ions to calcium-free peroxidase: thermodynamic and kinetic studies

Thermodynamics of binding of divalent metal ions including Ca(2+) , Mg(2+) , Ba(2+) , and Cd(2+) to Ca-free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular-mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca(2+) >Cd(2+) >Mg(2+) >Ba(2+) . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation-time method) for binding of Ca(2+) (as the typical metal ion) to Ca-free HRP, which was found a second-order type having a two-step mechanism involving fast formation of Ca-free HRP/1?Ca(2+) as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M(2+) metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca(2+) ion was occurred on distal and proximal binding sites of Ca-free HRP associated with higher stability energies (E(total) ). Indeed, among the divalent metal ions, Ca(2+) with the highest binding affinity (maximum value of K(bin) and minimum value of ΔG$\rm{{_{bin}^{0}}}$), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.     

Community impacts of anthropogenic disturbance: natural enemies exploit multiple routes in pursuit of invading herbivore hosts

Background  

Biological invasions provide a window on the process of community assembly. In particular, tracking natural enemy recruitment to invading hosts can reveal the relative roles of co-evolution (including local adaptation) and ecological sorting. We use molecular data to examine colonisation of northern Europe by the parasitoid Megastigmus stigmatizans following invasions of its herbivorous oak gallwasp hosts from the Balkans. Local host adaptation predicts that invading gallwasp populations will have been tracked primarily by sympatric Balkan populations of M. stigmatizans (Host Pursuit Hypothesis). Alternatively, ecological sorting allows parasitoid recruitment from geographically distinct populations with no recent experience of the invading hosts (Host Shift Hypothesis). Finally, we test for long-term persistence of parasitoids introduced via human trade of their hosts' galls (Introduction Hypothesis).     

Machine learning reveals mesenchymal breast carcinoma cell adaptation in response to matrix stiffness

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.     

Nano silver entrapped in phospholipids membrane: synthesis, characteristics and antibacterial kinetics

The antimicrobial property of stabilized silver nanoparticles (AgNPs) with phospholipid membrane was investigated on both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial strains. The influence of phospholipid concentrations on antibacterial kinetics actions of AgNPs was studied with two different methodologies in order to understand the bactericidal and bacteriostatic effects. The bacterial inactivation of synthesized AgNPs fitted well to the Chick-Watson model with a high regression coefficient, R(2) > 0.91. The antibacterial properties of AgNPs depend on the particle size, stabilizer and lecithin concentrations. Only the stabilized AgNPs that have the K(lec/Ag) values of 1 and 2 presented the inhabitation zone, while unstabilized AgNPs agglomerated quickly, settled on the wells and did not diffuse in agar. In addition, the specific coefficient of lethality depends on the lecithin concentration. An increase in lecithin concentration caused multilayer creation on the AgNPs' surface and reduced the release of AgNPs which led to low bacterial killing rate.