Human red blood cells express the RNA sensor TLR7 and bind viral RNA

Red blood cells (RBCs) express the nucleic acid-sensing toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-sensing TLRs and bind RNA is unknown. Here we show that human RBCs express the RNA sensor, TLR7. TLR7 is present on the red cell membrane and associates with the RBC membrane protein Band 3. RBCs bind synthetic single-stranded RNA and RNA from pathogenic single-stranded RNA viruses. RNA acquisition by RBCs is attenuated by recombinant TLR7 and inhibitory oligonucleotides. Thus, RBCs may represent a previously unrecognized reservoir for RNA, although how RNA-binding by RBCs modulates the immune response has yet to be elucidated. These findings add to the growing list of non-gas exchanging RBC immune functions.

The Effect of Salt on the Gelling Properties and Protein Phosphorylation of Surimi-Crabmeat Mixed Gels

The effects of different salt additions (1.0%, 1.5%, 2.0%, 2.5%, 3.0%, and 3.5%) on the gelling properties and protein phosphorylation of the mixed gels (MG) formed by silver carp (Hypophthalmichthys molitrix) surimi with 10% crabmeat were investigated. The MG’s breaking force, deformation, gel strength, and water-holding capacity (WHC) increased as the salt concentration increased. The intrinsic fluorescence intensity of the samples initially decreased and then increased, reaching the lowest when the NaCl concentration was 2.5%. The result of SDS–polyacrylamide gel electrophoresis indicated that large aggregates were formed by protein–protein interaction in the MG containing 2.5% or 3.0% NaCl, decreasing the protein band intensity. It was also found that with the addition of NaCl, the phosphorus content initially increased and then decreased, reaching the maximum when the NaCl concentration was 2% or 2.5%, which was similar to the changing trend of actin band intensity reported in the results of Western blot. These results revealed that the amount of salt used had a significant effect on the degree of phosphorylation of the MG protein. The increase in phosphorylation was linked to improved gelling properties, which could lead to new ideas for manufacturing low-salt surimi products in the future.

Differential expression of erythrocyte membrane protein band 4.2 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding erythrocyte membrane protein band 4.2, EPB42, when comparing primary tumors of the breast to the tissue of origin, the normal breast. EPB42 mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of EPB42 in primary tumors of the breast was correlated with overall survival in patients with normal-like subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. EPB42 may be of relevance to initiation, maintenance or progression of cancers of the female breast.

EPB41L4B expression associates with survival in triple negative breast cancer.

We mined published microarray data (1) to understand the most significant gene expression differences in the tumors of triple negative breast cancer patients based on survival following treatment: dead or alive. We observed significant transcriptome-wide differential expression of erythrocyte membrane protein band 4.1-like 4B, encoded by EPB41L4B when comparing the primary tumors of triple negative breast cancer patients dead or alive. Importantly, EPB41L4B expression was correlated with distant metastasis-free survival in basal subtype breast cancer, a molecular subtype sharing significant overlap with triple negative breast cancer. EPB41L4B may be of relevance as a biomarker or as a molecule of interest in understanding the etiology or progression of triple negative breast cancer.

Cloning and secretory expression of functional diisopropyl-fluorophosphatase (DFPase) in Bacillus subtilis

Background and aims: Synthetic organophosphates (OPs) inhibit acetylcholinesterase resulting in the accumulation of acetylcholine, failure of organs, and eventually death. Diisopropyl-fluorophosphatase (DFPase) is one of the OPs degrading enzymes that has broad substrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order to accelerate the biodegradation rate of OPs. Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET28-inaV/N-dfpase plasmid. The PCR product was subcloned in the pWB980 plasmid. Competent B. subtilis WB600 were transformed with recombinant plasmid. SDS PAGE technique was used to study the expression of protein secreted in superrich medium. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Approximately, 35 kDa protein band was shown in culture medium after incubating at 35°C for 72 hours and 150 rpm. Measurement of enzyme’s activity was done by monitoring the release of fluoride from diisopropyl fluorophosphate (DFP), using ion-meter. Results showed that enzyme’s activity was 3333 U/L. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.

Abnormal Expression of EPB41L1 in Colon Adenocarcinoma and Effect on Prognosis

Background: Colon adenocarcinoma (COAD) is the most common pathological type of colorectal cancer (CRC) and further study of the molecular mechanism will help to improve the quality of life of patients with COAD. Erythrocyte membrane protein band 4.1 like 1(EPB41L1), a gene encoding protein 4.1N, has been reported to be closely associated with tumorigenesis and progression. However, the role of EPB41L1 in COAD is largely unknown and remains to be fully studied.Methods: In this study, we analyzed the mRNA expression of EPB41L1 in COAD through the Oncomine and GEO databases. Then, the relative expressions of EPB41L1 across the sub-groups of COAD were performed by the UALCAN data portal. Next, we investigated the prognostic value of EPB41L1 in COAD patients by using the UALCAN and HPA online databases. Further, the mutation of EPB41L1 in COAD was analyzed by c-Bioportal. The co-expression genes of EPB41L1 in COAD were displayed from the LinkedOmics database, and function enrichment analysis was analyzed by DAVID. The co-expression gene network was constructed through the STRING database, and the MCODE plug-in of which was used to build the gene modules, both of them were visualized by Cytoscape software. Finally, the pathway enrichment of the top modular genes in the co-expression gene network was analyzed by DAVID.Results: The results revealed that EPB41L1 is significantly upregulated with the development of COAD, leading to a poor prognosis. There are mutations in the EPB41L1 gene, but it has no significant effect on the prognosis of COAD. Moreover, the genes correlated with EPB41L1 in COAD, identified in the most highly connected sub-network, were enriched in the cell cycle. Conclusions: In summary, the above results suggest that EPB41L1 was significantly upregulated in the COAD tissues and the high expression level of EPB41L1 predicts a poor prognosis of COAD patients. Therefore, we suggest that EPB41L1 can be a potential candidate biomarker for the diagnosis and prognosis of COAD.

Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

Abstractω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.

Cytotoxic Potential from the Extracellular Filtrate from Aspergillus niveus Cultured in YPD Medium against Human Tumor Cells

Different fungal species, especially from the genus Aspergillus, have been reported as producers of small molecules, including proteins, with biological activity and a better understanding of their sources, structure, function and toxicity is essential for their biotechnological applications. According to this, our aim was to evaluate the cytotoxic activity of the extracellular filtrate produced by A. niveus. The crude filtrate obtained in YPD medium containing 18 kDa protein, after cultivation for 120 h, was selected for cytotoxic assay, assessed by Giemsa staining, against different human tumor cell lines. Crude filtrate inhibited (from 27% to 50%) the ONS-76 (medulloblastoma), HT144T (melanoma), HOS (osteosarcoma), T98G (glioblastoma) human tumor cell lines and MRC-5 (fibroblasts) human normal cells, at 20 µg/mL for 72 h treatment. According to this, the 18 kDa protein band and the fractions obtained after DEAE-Cellulose procedure were evaluated through mass spectrometry (MS/MS) analysis, revealing the presence of peptides with similarity to the alpha-sarcin, mitogillin and Aspf1 ribotoxins described for other Aspergillus species. In conclusion, the A. niveus extracellular filtrate containing ribotoxin-like proteins reduced, in vitro, the growth of human tumor cell lines indicating their biotechnological potential, indicating a possible future application in the elaboration of immunotoxins.

The Purification Of Rennin-Like Protease From Lactobacillus Paracasei Isolated From Ettawa Goat Milk

There is a protease produced by bateria that has characteristics similar to rennin from a calf. Rennin has the ability to clot casein in milk. Rennin-like protease (RLP) is produced by bacteria extracellularly. Lactic Acid Bacteria (LAB) have the potential to be developed for RLP production because they are safe and non-pathogenic bacteria. Rennin is needed in the process of milk coagulation to subsequently obtain a curd in the process of making cheese. In this study, the LAB isolated from Ettawa goat milk (isolate 2.12) which produced RLP was 99% identical to Lactobacillus paracasei based on 16S rRNA gene sequence analysis. The purification of the RLP L. paracasei 2.12 with 60% ammonium sulfate deposition, dialysis, and filtration gel chromatography Sephadex G-50 showed a single 38 kDa protein band with SMCA/SPA was 4.48 higher than that of the calf rennet with a ratio value of 1, therefore in this study, RLP L. paracasei 2.12 was developed as an alternative to renin in cheese making.