Sexual Dimorphism in Balance and Coordination in p75NTRexonIII Knock-Out Mice.

The p75 neurotrophin receptor (p75NTR) is implicated in various biological functions during development and adulthood. Several animal models have been developed to identify the roles of p75NTR in vivo and in vitro. P75NTRExonIII knock-out mice are widely used to study the neurotrophin receptor and its signaling pathways. Similar to other models of p75NTR knock-out (p75NTRExon IV KO) or conditional knock-out (p75NTRfl/fl) mice, p75NTRExonIII knock-out mice present severe abnormalities in walking, gait, balance and strength. The present study identifies a sexual dimorphism in the p75NTRExonIII knock-out strain regarding balance and coordination. Using Kondziela's inverted grid test, we observed that p75NTRExonIII knock-out males performed poorly at the task, whereas p75NTRExonIII knock-out females did not exhibit any defects. We also observed that female p75NTRExonIII knock-out mice performed significantly better than male p75NTRExonIII knock-out mice at the beam balance test. There were no differences in strength, skin innervation, or the number of ulcers on the toes between p75NTRExonIII knock-out males and females. The literature regarding the role of p75NTR in behavior is controversial; our results suggest that studies investigating the role of p75NTR in vivo using p75NTR knock-out mice should systematically report data from males and females.

Efficient co-cultivation of human fibroblast cells (HFCs) and adipose-derived stem cells (ADSs) on gelatin/PLCL nanofiber.

In this study, we investigated whether the nanofibers produced by natural-synthetic polymers can probably promote the proliferation of co-cultured adipose-derived stem cells/human fibroblast cells (ADSs/HFCs) and synthesis of collagen. Nanofiber was fabricated by blending gelatin and poly (L-lactide co-ɛ-caprolactone) (PLCL) polymer nanofiber (Gel/PLCL). Cell morphology and the interaction between cells and Gel/PLCL nanofiber were evaluated by FESEM and fluorescent microscopy. MTS assay and quantitative real-time polymerase chain reaction were applied to assess the proliferation of co-cultured ADSs/HFCs and the collagen type I and III synthesis, respectively. The concentrations of two cytokines including fibroblast growth factor-basic and transforming growth factor-ß1 were also measured in culture medium of co-cultured ADSs/HDCs using enzyme-linked immunosorbent assay assay. Actually, nanofibers exhibited proper structural properties in terms of stability in cell proliferation and toxicity analysis processes. Gel/PLCL nanofiber promoted the growth and the adhesion of HFCs. Our results showed in contact co-culture of ADSs/HFCs on the Gel/PLCL nanofiber increased cellular adhesion and proliferation synergistically compared to non-coated plate. Also, synthesis of collagen and cytokines secretion of co-cultured ADSs/HFCs on Gel/PLCL scaffolds is significantly higher than non-coated plates. To conclude, the results suggest that Gel/PLCL nanofiber can imitate physiological characteristics in vivo and enhance the efficacy of co-cultured ADSs/HFCs in wound healing process.

Combination of gold nanoparticles with low-LET irradiation: an approach to enhance DNA DSB induction in HT29 colorectal cancer stem-like cells.

PURPOSE: High-linear energy transfer (high LET) irradiation has significant cytotoxic effects on different cancerous stem-like cells (CSLCs) such as colorectal CSLCs. A review of the literature has indicated that the presence of gold nanoparticles (GNPs) enables low-LET irradiation to produce highly non-homogeneous dose distributions like high-LET irradiation. The purpose of this study was to evaluate the radioresponsiveness of HT29 colorectal CSLCs under low-LET irradiation (X-ray) and in the presence of GNPs. METHODS: Radioresponsiveness was evaluated using the ϒ-H2AX foci formation assay, the clonogenic assay, the cell cycle progression assay and analyses of radiobiological parameters. RESULTS: In the presence of GNPs, the survival fraction of HT29 CSLCs was significantly reduced and caused significant changes in the radiobiological parameters after irradiation. In addition, ϒ-H2AX assay showed that in the presence of GNPs, the persistent DNA double-strand breaks were significantly increased in irradiated HT29 CSLCs. The relative biological effectiveness value of GNPs with X-rays was about 1.6 for HT-29 CSLCs at the 10% of cell survival fraction (D10 level) when compared to X-rays alone. CONCLUSION: Therefore, the combination of GNPs with X-ray irradiation has the potential to kill HT29 CSLCs greater than the X-ray alone, and may be considered as an alternative for high-LET irradiation.

The most reliable surface marker for the identification of colorectal cancer stem-like cells: A systematic review and meta-analysis.

Several surface markers have been proposed for the identification and characterization of colorectal cancer stem-like cells (CR-CSLCs). However, their reliability in CR-CSLCs identification remains controversial. This study evaluated the correlation between all candidate surface marker's expression and CSLCs properties (tumorigenicity) through monitoring in vivo tumor incidence and final tumor volume. PubMed, Web of Science, and Scopus databases were systematically searched until November 2017. A total of 27 studies were found that met the inclusion criteria for cluster of differentiation 133 (CD133) and CD44 markers. Results indicated that either CD133 or CD44 positive cells caused about twofold increase in tumor volume compared with the negative cells (p < 0.05). In two groups of cells derived from primary tumors and cell lines, CD133 + cells had 25 and 1.45 times higher tumor incidence potential than CD133 - cells, respectively ( p < 0.05). Also, cohort evaluation showed that CD133 overexpression at protein level is a marker of poor overall survival in colorectal cancer (CRC) patients. While CD44 + cells displayed twofold tumorigenicity compared with the negative cells ( p < 0.05), combination of CD44 and CD133 showed about sevenfold tumorigenicity potential ( p < 0.05). In conclusion, the present meta-analysis suggests that CD133 is a robust biomarker to identify primary tumor CSLCs and can be proposed as a prognostic marker of CRC patient whereas it should be used with caution in cell lines. It seems to be more reliable to use CD133 in combination with CD44 as target biomarkers for the isolation of CR-CSLCs in both cell line and primary tumor cells populations.

Using of keratin substrate for enrichment of HT29 colorectal cancer stem-like cells.

Eradication of cancer stem-like cells (CSLCs) are becoming increasingly an important target for new cancer therapies. The ability to study their behavior in vitro will provide the opportunity for high-throughput testing of more effective treatments. In this study, spheroid-like structures' formation and enrichment of HT29 CSLCs were evaluated on a wool keratin-based substrate as a bio-mimic of natural extracellular matrix (ECM) proteins. The results indicated that culturing on keratin substrate increased spheroid formation ability and radio-/chemoresistance of HT29 cells. Moreover, cell surface expression of CD133 CSLCs' marker and the mRNA level of stemness genes such as Nanog, Oct4, and c-MYC were increased. These data suggest that keratin can potentially be used for spheroid-like structure formation and enrichment of HT29 CSLCs. In addition, it seems that the induction of stemness characteristics on keratin substrate is probably because of the activation of α2 ß1 integrin signaling pathway. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1264-1271, 2019.

Gold nanoparticles in combination with megavoltage radiation energy increased radiosensitization and apoptosis in colon cancer HT-29 cells.

PURPOSE: Gold nanoparticles (GNP) act as a radiosensitizer in radiation therapy. However, recent studies have shown contradictory evidence in terms of radiosensitization in the presence of GNP combined with X-ray megavoltage energy (MV) on different cell types. In this study, the effect of GNP on radiosensitization enhancement of HT-29 human colorectal cancer cells at MV X-ray energy was evaluated. MATERIALS AND METHODS: The cytotoxicity and radiosensitization of GNP were evaluated in HT-29 human colorectal cancer cells by MTS-assay and multiple MTS-assay, respectively. Cellular uptake was assayed using graphite furnace atomic absorption spectrometry (GFAAS). Apoptosis and cell cycle progression were determined by an Annexin V-FITC/propidium iodide (PI) kit and PI/RNase solution with flow cytometry, respectively. RESULTS: Results showed that the cell viability of the HT-29 cells was not influenced by exposure to different concentrations of GNP (10-100 µM). GNP alone did not affect the cell cycle progression and apoptosis. In contrast, GNP, in combination with radiation (9 MV), induced more apoptosis. The interaction of GNP with MV energy resulted in a significant radiosensitization enhancement compared with irradiation alone. CONCLUSION: It was concluded that GNP may work as bio-inert material on HT-29 cancer cells and their enhancement of radiosensitization may be due to increase in the absorbed irradiation dose.

Recombinant production, purification and characterization of vessel dilator in E. coli.

Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.

Immune related transcriptional responses and performance of Litopenaeus vannamei post-larvae fed on dietary probiotic PrimaLac(®).

The present study investigated the effects of various levels of multi-strain probiotic on the immune related gene expression, digestive enzyme activity, growth performance, body chemical composition and survival of Litopenaeus vannamei post-larvae. After transferring post-larvae of L. vannamei to indoor conditions and subsequent acclimation to laboratory condition for 14 days, the shrimp were fed multi-strain probiotic at four different doses of 0, 0.25, 0.5 and 1.0 g kg(-1) for eight weeks. Shrimp fed 0.5 and 1.0 g kg(-1) probiotic PrimaLac(®) exhibited significantly (p < 0.05) higher weight gain, specific growth rate, body crude protein as well as lower FCR compared to other groups. Feeding on 0.5 and 1 g kg(-1) dietary multi-strain probiotic significantly (p < 0.05) increased the level of body crude protein. Oral administration of 0.5 and 1.0 g kg(-1) multi-strain probiotic significantly (p < 0.05) decreased body crude lipid and body moisture respectively. 30 days after feeding, protease, amylase and lipase activity increased in groups fed 0.5 and 1.0 g kg(-1) probiotic PrimaLac(®). However, on the 60th day, specific protease and amylase activity in all treatment groups were significantly higher than control group (p < 0.05) but lipase activity was higher (p < 0.05) in groups fed 0.5 and 1.0 g kg(-1) multi-strain probiotic. Oral administration of 1.0 g kg(-1) probiotic increased (p < 0.05) the level of prophenoloxidase and g-type lysozyme gene on day 30th and 60th after treatment. On day 30th and 60th, penaeidin gene expression was significantly higher in all treatment groups compared to the control group (p < 0.05). In general, findings of this study demonstrated that oral administration of 0.5 and 1.0 g kg(-1) multi-strain probiotic improved the performance of the fish and increased the expression of immune related genes.

Multiple MTS Assay as the Alternative Method to Determine Survival Fraction of the Irradiated HT-29 Colon Cancer Cells.

A multiple colorimetric assay has been introduced to evaluate the proliferation and determination of survival fraction (SF) of irradiated cells. The estimation of SF based on the cell-growth curve information is the major advantage of this assay. In this study, the utility of multiple-MTS assay for the SF estimation of irradiated HT-29 colon cancer cells, which were plated before irradiation, was evaluated. The SF of HT-29 colon cancer cells under irradiation with 9 MV photon was estimated using multiple-MTS assay and colony assay. Finally, the correlation between two assays was evaluated. Results showed that there are no significant differences between the SF obtained by two assays at different radiation doses (P > 0.05), and the survival curves have quite similar trends. In conclusion, multiple MTS-assay can be a reliable method to determine the SF of irradiated colon cancer cells that plated before irradiation.

Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli.

BACKGROUND: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host. MATERIALS AND METHODS: First the sequence encoding of IGFBP3 was designed based on the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis. CONCLUSION: DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3) expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa) are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein.

Easy method for production of a home-made DNA ladder in every laboratory.

BACKGROUND: Molecular DNA markers are one of the essential tools in molecular biology labs with varied applications. In the present study, we suggest an efficient and available strategy to produce molecular size marker in routine laboratories. MATERIALS AND METHODS: To achieve the desired sizes of DNA fragments, we recruited PCR and bioinformatics techniques to synthesize 14 DNA fragments ranging from 100 to 3000 bp. RESULTS: Holistic analysis of different parameters in primers design resulted in amplification of fragments in just one PCR program without any by-product and purification step. Our applied method enables researchers to modify amplified DNA fragments by wide range of chemical modifications toward varied applications. CONCLUSION: Method of home-made DNA ladder production by available ingredients and routine techniques reported in this study can be used in common laboratories for different applications.