Cell physiology and antibiotic production of Streptomyces coelicolor grown on solid medium

Summary In order to examine the physiology ofStreptomyces coelicolor when growing on solid media, we have employed a membrane overlay technique and used a new approach to extract substrate and product compounds from the agar. Comparisons made with liquid grown cultures indicate a change from non-growth associated productivity of actinorhodin in liquid culture, to growth associated production on agar plates. In contrast, the temporal control of methylenomycin production was virtually identical under both culture conditions. Considerable extracellular protein production was observed during growth on agar.     

Fast classification and compositional analysis of cornstover fractions using Fourier transform near-infrared techniques

The objectives of this research were to determine the variation of chemical composition across botanical fractions of cornstover, and to probe the potential of Fourier transform near-infrared (FT-NIR) techniques in qualitatively classifying separated cornstover fractions and in quantitatively analyzing chemical compositions of cornstover by developing calibration models to predict chemical compositions of cornstover based on FT-NIR spectra. Large variations of cornstover chemical composition for wide calibration ranges, which is required by a reliable calibration model, were achieved by manually separating the cornstover samples into six botanical fractions, and their chemical compositions were determined by conventional wet chemical analyses, which proved that chemical composition varies significantly among different botanical fractions of cornstover. Different botanic fractions, having total saccharide content in descending order, are husk, sheath, pith, rind, leaf, and node. Based on FT-NIR spectra acquired on the biomass, classification by Soft Independent Modeling of Class Analogy (SIMCA) was employed to conduct qualitative classification of cornstover fractions, and partial least square (PLS) regression was used for quantitative chemical composition analysis. SIMCA was successfully demonstrated in classifying botanical fractions of cornstover. The developed PLS model yielded root mean square error of prediction (RMSEP %w/w) of 0.92, 1.03, 0.17, 0.27, 0.21, 1.12, and 0.57 for glucan, xylan, galactan, arabinan, mannan, lignin, and ash, respectively. The results showed the potential of FT-NIR techniques in combination with multivariate analysis to be utilized by biomass feedstock suppliers, bioethanol manufacturers, and bio-power producers in order to better manage bioenergy feedstocks and enhance bioconversion.     

Environmental footprints of British Columbia wood pellets from a simplified life cycle analysis

Purpose  

Environmental footprints of wood pellets produced in British Columbia (BC) of Canada are to be estimated based on industry surveys and published emission factor data.     

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C‐zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ‐related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid‐binding ability, and PLCζ level along with its distribution were evaluated using computer‐assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid‐binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.     

Lenalidomide overcomes suppression of human natural killer cell anti-tumor functions by neuroblastoma microenvironment-associated IL-6 and TGFβ1

Background

Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression.

Methods

Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma.

Results

CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide.

Conclusion

Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1.     

Ectomycorrhizal fungi of exotic pine plantations in relation to native host trees in Iran: evidence of host range expansion by local symbionts to distantly related host taxa

Introduction of exotic plants change soil microbial communities which may have detrimental ecological consequences for ecosystems. In this study, we examined the community structure and species richness of ectomycorrhizal (EcM) fungi associated with exotic pine plantations in relation to adjacent native ectomycorrhizal trees in Iran to elucidate the symbiont exchange between distantly related hosts, i.e. Fagales (Fagaceae and Betulaceae) and Pinaceae. The combination of morphological and molecular identification approaches revealed that 84.6 % of species with more than one occurrence (at least once on pines) were shared with native trees and only 5.9 % were found exclusively on pine root tips. The community diversity of ectomycorrhizal fungi in the pine plantations adjacent to native EcM trees was comparable to their adjacent native trees, but the isolated plantations hosted relatively a species-poor community. Specific mycobionts of conifers were dominant in the isolated plantation while rarely found in the plantations adjacent to native EcM trees. These data demonstrate the importance of habitat isolation and dispersal limitation of EcM fungi in their potential of host range expansion. The great number of shared and possibly compatible symbiotic species between exotic Pinaceae and local Fagales (Fagaceae and Betulaceae) may reflect their evolutionary adaptations and/or ancestral compatibility with one another.     

Copper Oxide Nanomaterials Derived from Zanthoxylum armatum DC. and Berberis lycium Royle Plant Species: Characterization,Assessment of Free Radical Scavenging and Antibacterial Activity

Copper oxide nanomaterials were synthesized by a facile sustainable biological method using two plant species (Zanthoxylum armatum DC. and Berberis lycium Royle ). The formation of materials was confirmed by FT‐IR, ATR, UV‐visible, XRD, TEM, SEM, EDX, TGA and PL. The antibacterial activity was evaluated by agar well diffusion method to ascertain the efficacy of plant species extract and extract derived copper oxide nanomaterials against six Gram‐positive bacteria namely Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes, Corynebacterium diphtheriae, Corynebacterium xerosis, Bacillus cereus and four Gram‐negative bacteria such as Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris against the standard drug, Ciprofloxacin for Gram‐positive and Gentamicin for Gram‐negative bacteria, respectively. In both cases, copper oxide nanomaterials were found to be sensitive in all the bacterial species. Sensitivity of copper oxide nanomaterials shows an be higher as compared to plant species extract against different bacteria. Scavenging activity of plant extracts along with nanomaterials have been accessed using previously reported protocols employing ascorbic acid as standard. Scavenging activity of copper oxide nanomaterials shows an increase with increase in concentration. The biological activity (bactericidal and scavenging efficiency) of plant derived copper oxide nanomaterials revealed that these materials can be used as potent antimicrobial agent and DPPH scavengers in industrial as well as pharmacological fields.     

Calcitonin administration improves endometrial receptivity via regulation of LIF,Muc-1 and microRNA Let-7a in mice

Calcitonin (CT) is one of the factors affecting the embryo implantation, but its effects on the implantation window have not been fully investigated. The current study investigated the effects of CT on the endometrium receptivity by morphological study and evaluation of leukemia inhibitory factor (LIF), mucin 1 (Muc-1), and microRNA (miRNA) Let-7a in the ovarian stimulation and the normal ovarian cycle. Then the mechanism of the CT effects through the mammalian target of rapamycin (mTOR) signaling pathway was studied by using PP242. A total of 64 BALB/c mice were divided into the normal ovarian cycle and ovarian stimulation groups. Each group consisted of four subgroups: control, calcitonin, PP242, and calcitonin+PP242. CT and PP242 were injected on the fourth of pregnancy into the mice and 24 hr later all the mice were killed. The uterine tissue samples were used for morphological analysis, and endometrial cells were mechanically isolated for evaluation of gene and protein expression. The results showed that ovarian stimulation induced mTOR phosphorylation as well as increased expression of the Let-7a miRNA. In addition, CT injection increased the expression of LIF and miRNA Let-7a in ovarian stimulation similar to that in normal ovarian cycles. However, injection of PP242 reduced expression of miRNA Let-7a and increased Muc-1 expression in ovarian stimulation group. In conclusion, the administration of CT improved endometrial receptivity in mice. This phenomenon occurred by upregulation of LIF, miRNA Let-7a and downregulation of Muc-1 via mTOR signaling pathway.     

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K _d) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.     

Membrane-protein stability in a phospholipid-based crystallization medium

Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.