miR-223-RhoB signaling pathway regulates the proliferation and apoptosis of colon adenocarcinoma.

MicroRNAs (miRNAs) can function as tumor suppressor or oncogenic genes. The putative targets of miR-223 include tumor suppressor gene, RhoB. Here we sought to investigate the role of miR-223-RhoB signaling pathway in proliferation of colon cancer. We used Western blot, immunofluorescence staining, or RT-PCR to detect expression levels of miR-223 and RhoB in colon adenocarcinoma and adjacent non-cancerous tissue samples, or in human colon adenocarcinoma cell lines. MTT assay was used to determine proliferation and apoptosis in cell lines. We further used Western blot to determine levels of cell cycle regulators CDK1 and Cyclin B1 with anti-miR-223 or apoptosis with overexpression of RhoB. The expression level of miR-223 was significantly upregulated in clinical samples and cell lines of colon adenocarcinoma, in contrast to down-regulation of RhoB. In addition, we showed that inhibition of miR-223 led to upregulation of RhoB and in turn suppression of proliferation of colon adenocarcinoma. Moreover, inhibition of miR-223 or overexpression of RhoB induced cell arrest or apoptosis in colon adenocarcinoma. These results suggest that miR-223-RhoB signaling pathway plays an important role in modulation of proliferation, cell arrest, and apoptosis in colon cancer.

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

The influence of mtDNA deletion on lung cancer cells under the conditions of hypoxia and irradiation.

PURPOSE: This study was to evaluate the influence of mtDNA deletion on the lung cancer cells under the conditions of hypoxia or irradiation. METHOD: The treatment conditions of lung cancer cell lines with (A549) and without mtDNA (ρ0A549: obtained by inducing from A549) included 2 h of hypoxia and 4 Gy irradiation (group 1: without treatment; group 2: 2 h of hypoxia; group 3: 4 Gy irradiation; group 4: 2 h of hypoxia plus 4 Gy irradiation). The Human OneArray™ microarray was used to hybridize with the Cy5-labeled aRNA in microarray sample preparation. Differentially expressed genes (DEGs) between the lung cancer cells with and without mtDNA were identified using NOISeq package in R. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the online tool of DAVID. RESULT: In the KEGG pathway analysis of down-regulated DEGs, nineteen pathways were simultaneously enriched in the four groups, which were mainly metabolism- and biosynthesis-related pathways. Nine lung cancer-related pathways were enriched in group 4, and more cancer-associated DEGs, such as MYC, MAX, and E2F1 were found in group 4 than in the other groups. CONCLUSION: The mtDNA deletion could inhibit the biosynthesis and metabolism of lung cancer cells and promote the effect of hypoxia and radiation on lung cancer cells. MYC might be the key gene of the cooperation of hypoxia and radiation and MYC, MAX, and E2F1 might play roles in hypoxia- and radiation-induced cell death in lung cancer cells without mtDNA.

Enhanced L-(+)-lactic acid production by an adapted strain of Rhizopus oryzae using corncob hydrolysate.

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the L-isomer and a simple nutrition requirement by the fungus. Production of L-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of L-(+)-lactic acid from corncob hydrolysate. It increased L-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production.

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).

Fed-batch fermentation of Lactobacillus lactis for hyper-production of L-lactic acid.

A fed-batch fermentation of Lactobacillus lactis to produce L-lactic acid was developed in which the residual glucose concentration in the culture was used to control a continuous feeding strategy. Up to 210 g L-lactic acid l(-1) (97% yield) was obtained. The maximal dry cell was 2.7 g l(-1) and the average L-lactic acid productivity was 2 g l(-1) h(-1).