Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO^M) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO^P) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO^P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO^P (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO^M (21%). The cytoprotective effect of the asialo-rhuEPO^P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.     

Comparison of analytical methods for profiling <Emphasis Type="Italic">N-</Emphasis> and <Emphasis Type="Italic">O-</Emphasis>linked glycans from cultured cell lines

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.     

Fabrication of Au/Fe3O4/RGO based aptasensor for measurement of miRNA‐128, a biomarker for acute lymphoblastic leukemia (ALL)

Due to their high sensitivity, simplicity, portability, self‐contained, and low cost, the development of electrochemical biosensors is a beneficial way to diagnose and anticipate many types of cancers. An electrochemical nanocomposite‐based aptasensor is fabricated for the determination of miRNA‐128 concentration as the acute lymphoblastic leukemia (ALL) biomarker for the first time. The aptamer chains were immobilized on the surface of the glassy carbon electrode (GCE) through gold nanoparticles/magnetite/reduced graphene oxide (AuNPs/Fe₃O₄/RGO). Fast Fourier transform infrared (FTIR), X‐ray diffraction (XRD), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM) were used to characterize synthesized nanomaterials. Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were used to characterize the modified GCE in both label‐free and labeled methods. The results indicate that the modified working electrode has high selectivity and for miRNA‐128 over other biomolecules. The hexacyanoferrate redox system typically operated at around 0.3 V (vs. Ag/AgCl), and the methylene blue redox system ran at about 0 V, were used as an electrochemical probe. The detection limit and linear detection range for hexacyanoferrate and methylene blue are 0.05346 fM, 0.1–0.9 fM, and 0.005483 fM, 0.01–0.09 fM, respectively. The stability and diffusion control analyses were performed as well. In both label‐free and labeled methods, the modified electron showed high selectivity for miRNA‐128. The use of methylene blue as a safer redox mediator caused miRNA‐128 to be detected with greater accuracy at low potentials in PBS media. The findings also show the substantial improvement in detection limit and linearity by using reduced graphene oxide‐magnetite‐gold nanoparticles that can be verified by comparing with previous studies on the detection of other miRNAs.     

Curcumin as an anti-inflammatory agent: Implications to radiotherapy and chemotherapy

Cancer is the second cause of death worldwide. Chemotherapy and radiotherapy are the most common modalities for the treatment of cancer. Experimental studies have shown that inflammation plays a central role in tumor resistance and the incidence of several side effects following both chemotherapy and radiotherapy. Inflammation resulting from radiotherapy and chemotherapy is responsible for adverse events such as dermatitis, mucositis, pneumonitis, fibrosis, and bone marrow toxicity. Chronic inflammation may also lead to the development of second cancer during years after treatment. A number of anti-inflammatory drugs such as nonsteroidal anti-inflammatory agents have been proposed to alleviate chronic inflammatory reactions after radiotherapy or chemotherapy. Curcumin is a well-documented herbal anti-inflammatory agents. Studies have proposed that curcumin can help management of inflammation during and after radiotherapy and chemotherapy. Curcumin targets various inflammatory mediators such as cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor κB (NF-κB), thereby attenuating the release of proinflammatory and profibrotic cytokines, and suppressing chronic production of free radicals, which culminates in the amelioration of tissue toxicity. Through modulation of NF-κB and its downstream signaling cascade, curcumin can also reduce angiogenesis, tumor growth, and metastasis. Low toxicity of curcumin is linked to its cytoprotective effects in normal tissues. This protective action along with the capacity of this phytochemical to sensitize tumor cells to radiotherapy and chemotherapy makes it a potential candidate for use as an adjuvant in cancer therapy. There is also evidence from clinical trials suggesting the potential utility of curcumin for acute inflammatory reactions during radiotherapy such as dermatitis and mucositis.     

Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

The cryptococcal capsule is a critical virulence factor of an important pathogen, but little is known about how it is associated with the cell or released into the environment. Two mutants lacking PBX1 and PBX2 were found to shed reduced amounts of the capsule polysaccharide glucuronoxylomannan (GXM). Nuclear magnetic resonance, composition, and physical analyses showed that the shed material was of normal mass but was slightly enriched in xylose. In contrast to previous reports, this material contained no glucose. Notably, the capsule fibers of pbxΔ mutant cells grown under capsule-inducing conditions were present at a lower than usual density and were loosely attached to the cell wall. Mutant cell walls were also defective, as indicated by phenotypes including abnormal cell morphology, reduced mating filamentation, and altered cell integrity. All observed phenotypes were shared between the two mutants and exacerbated in a double mutant. Consistent with a role in surface glycan synthesis, the Pbx proteins localized to detergent-resistant membrane domains. These results, together with the sequence motifs in the Pbx proteins, suggest that Pbx1 and Pbx2 are redundant proteins that act in remodeling the cell wall to maintain normal cell morphology and precursor availability for other glycan synthetic processes. Their absence results in aberrant cell wall growth and metabolic imbalance, which together impact cell wall and capsule synthesis, cell morphology, and capsule association. The surface changes also lead to increased engulfment by host phagocytes, consistent with the lack of virulence of pbx mutants in animal models.     

Colonization and Biodegradation of Photo-Oxidized Low-Density Polyethylene (LDPE) by New Strains of Aspergillus sp. and Lysinibacillus sp.

The primary objective of this study was the isolation of low-density polyethylene (LDPE)-degrading microorganisms. Soil samples were obtained from an aged municipal landfill in Tehran, Iran, and enrichment culture procedures were performed using LDPE films and powder. Screening steps were conducted using linear paraffin, liquid ethylene oligomer, and LDPE powder as the sole source of carbon. Two landfill-source isolates, identified as Lysinibacillus xylanilyticus XDB9 (T) strain S7-10F and Aspergillus niger strain F1-16S, were selected as super strains. Photo-oxidation (25 days under ultraviolet [UV] irradiation) was used as a pretreatment of the LDPE samples without pro-oxidant additives. The PE biodegradation process was performed for 56 days in a liquid mineral medium using UV-irradiated pure LDPE films without pro-oxidant additives in the presence of the bacterial isolate, the fungal isolate, and the mixture of the two isolates. The process was monitored by measuring the fungal biomass, the bacterial growth, and the pH of the medium. During the process, the fungal biomass and the bacterial growth increased, and the pH of the medium decreased, which suggests the utilization of the preoxidized PE by the selected isolates as the sole source of carbon. Carbonyl and double bond indices exhibited the highest amount of decrement and increment, respectively, in the presence of the fungal isolate, and the lowest indices were obtained from the treatment of a mixture of both fungal and bacterial isolates. Fourier transform infrared (FT-IR), x-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses showed that the selected isolates modified and colonized preoxidized pure LDPE films without pro-oxidant additives.     

Structure of a capsular polysaccharide isolated from Salmonella enteritidis

Salmonella enteritidis is a food-borne enteric human pathogen that can form a complex protective extracellular matrix. We describe here a component of this matrix which is distinct from other known salmonella extracellular polysaccharides such as cellulose and colanic acid. We have used glycosyl composition and linkage analysis, as well as 1D and 2D NMR spectroscopy to determine the structure of this polysaccharide. We propose that the primary saccharide in the S. enteritidis capsule has a branched tetrasaccharide repeating unit having the following structure: -->3)-alpha-D-Galp-(1-->2)-[alpha-Tyvp-(1-->3)]-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->. This structure is partially substituted on both tyvelose and galactose with a glucose-containing side chain. It further bears considerable similarity to the O antigen from this organism, a feature found in a number of other capsules from Gram-negative bacteria. In addition, we have detected fatty acids at levels that indicate the presence of a lipid anchor.     

Evidence of inhibin/activin subunit betaC and betaE synthesis in normal human endometrial tissue

Background  

Inhibins are important regulators of the female reproductive system. Recently, two new inhibin subunits betaC and betaE have been described, although it is unclear if they are synthesized in normal human endometrium.     

Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

Integrin-growth factor receptor cross-talk plays a role in growth factor signaling, but the specifics are unclear. In a current model, integrins and growth factor receptors independently bind to their ligands (extracellular matrix and growth factors, respectively). We discovered that neuregulin-1 (NRG1), either as an isolated EGF-like domain or as a native multi-domain form, binds to integrins αvβ3 (with a K_D of 1.36 × 10⁻⁷ m) and α6β4. Docking simulation predicted that three Lys residues at positions 180, 184, and 186 of the EGF-like domain are involved in integrin binding. Mutating these residues to Glu individually or in combination markedly suppressed integrin binding and ErbB3 phosphorylation. Mutating all three Lys residues to Glu (the 3KE mutation) did not affect the ability of NRG1 to bind to ErbB3 but markedly reduced the ability of NRG1 to induce ErbB3 phosphorylation and AKT and Erk1/2 activation in MCF-7 and T47D human breast cancer cells. This suggests that direct integrin binding to NRG1 is critical for NRG1/ErbB signaling. Notably, stimulation of cells with WT NRG1 induced co-precipitation of ErbB3 with α6β4 and with αvβ3 to a much lower extent. This suggests that WT NRG1 induces integrin-NRG1-ErbB3 ternary complex formation. In contrast, the 3KE mutant was much less effective in inducing ternary complex formation than WT NRG1, suggesting that this process depends on the ability of NRG1 to bind to integrins. These results suggest that direct NRG1-integrin interaction mediates integrin-ErbB cross-talk and that α6β4 plays a major role in NRG-ErbB signaling in these cancer cells.