Table of Contents | Genet. Mol. Res. 2016 (3)
Enterococcus faecalis is the major pathogen of post-endodontic disease and refractory periapical periodontitis, and recent research on this species has focused on its pathogenicity. E. faecalis most often causes disease in the form of a biofilm, and total protein expression shows a strong association with its virulence. Therefore, the purpose of our study was to explore different methods of extracting the total proteins of the E. faecalis (ATCC 33186 standard strain) biofilm. The total proteins in the biofilm were extracted using an ultrasonication method with varied parameters, including duration, amplitude setting, period, and duty cycle. After the optimal conditions of ultrasonication were determined based on the protein profile from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the total protein content in the biofilm was detected using the bicinchoninic acid assay, Bradford Coomassie brilliant blue assay, and Lowry assay, and the results were compared and analyzed. The parameters for the optimal conditions of ultrasonication were as follows: a processing duration of 2 min, amplitude setting of 20%, and ultrasonication period of 4 s at a 50% duty cycle. The total protein content was 2299.1 mg/dish when measured by the bicinchoninic assay, 3793.8 mg/dish when measured by the Bradford Coomassie brilliant blue assay, and 1858.0 mg/dish when measured by the Lowry assay. These results demonstrate that the Bradford Coomassie brilliant blue assay is a simple and feasible method for use in detecting the total protein content in a bacterial biofilm.
Cancer subtype recognition and feature selection are important problems in the diagnosis and treatment of tumors. Here, we propose a novel gene selection approach applied to gene expression data classification. First, two classical feature reduction methods including locally linear embedding (LLE) and rough set (RS) are summarized. The advantages and disadvantages of these algorithms were analyzed and an optimized model for tumor gene selection was developed based on LLE and neighborhood RS (NRS). Bhattacharyya distance was introduced to delete irrelevant genes, pair-wise redundant analysis was performed to remove strongly correlated genes, and the wavelet soft threshold was determined to eliminate noise in the gene datasets. Next, prior optimized search processing was carried out. A new approach combining dimension reduction of LLE and feature reduction of NRS (LLE-NRS) was developed for selecting gene subsets, and then an open source software Weka was applied to distinguish different tumor types and verify the cross-validation classification accuracy of our proposed method. The experimental results demonstrated that the classification performance of the proposed LLE-NRS for selecting gene subset outperforms those of other related models in terms of accuracy, and our proposed approach is feasible and effective in the field of high-dimensional tumor classification.
Here, we applied a two-stage clonal expansion model of morphological (cell-size) evolution to a long-term evolution experiment with Escherichia coli. Using this model, we derived the incidence function of the appearance of cell-size stability, the waiting time until this morphological stability, and the conditional and unconditional probabilities of morphological stability. After assessing the parameter values, we verified that the calculated waiting time was consistent with the experimental results, demonstrating the effectiveness of the two-stage model. According to the relative contributions of parameters to the incidence function and the waiting time, cell-size evolution is largely determined by the promotion rate, i.e., the clonal expansion rate of selectively advantageous organisms. This rate plays a prominent role in the evolution of cell size in experimental populations, whereas all other evolutionary forces were found to be less influential.
We analyzed the publicly available ChromHMM BED files of the ENCODE project and tested the Markov properties of the different chromatin states in the human genome. Nucleotide frequency profiles of regional chromatin segmentations were analyzed, and Markov chains were built to detect Markov properties in the chromatin states of different ChromHMM regions. By estimating the transition probabilities of 200-base pair nucleotide sequences of the human genome, we constructed a nucleotide-sequence-based Markovian chromatin map called SeqChromMM.
Formation of hepatocyte spheroids is a necessary strategy for increasing liver-specific function in vitro. In this study, HepG2 cells showed good viability when grown on a polylactic acid-chitosan (PLA-CS) nanofiber and aggregated to form multicellular spheroids on the PLA-CS nanofibers with a diameter of approximately 100-200 mm in 5 days of culture, whereas no such aggregation was observed in cells cultured on 24-well plates. Hepatocyte spheroids formed on the PLA-CS nanofibers displayed excellent hepatic-related protein expression, such as albumin and urea, compared to HepG2 cells cultured on the 24-well plates. These results indicated that formation of the hepatocyte spheroids in nanofibers can increase and maintain hepatocyte functions for a longer time, supporting a new strategy for bioartificial liver development.
The role of estrogen in inducing chemoresistance is not yet fully understood. The objective of this study was to observe the relationship between estrogen levels and cellular response to chemotherapeutic drugs in non-small cell lung cancer (NSCLC) and to reveal the potential mechanisms involved. Cell viability was analyzed after pre-treating NSCLC cells with different levels of estrogen (E2), followed by treatment with an anti-tumor drug for 48 h. The roles of various estrogen receptors (ERs) were examined in vitro by blocking the activity of each ER individually. The ER pathway was further confirmed in NSCLC tissues. It was found that 10-1000 nM E2 resulted in a decreased cellular response to DDP in H1650 cells compared to the use of cisplatin alone (P
Paecilomyces hepiali (PH), a well-known medicinal fungus, has various pharmacological efficacies. In our study, the antinociceptive effects of PH and underlying mechanisms were evaluated using various mouse models. An acetic acid-induced writhing test, hot plate test, and formalin test were employed to evaluate the antinociceptive activities of PH. The levels of neuronal nitric oxide synthase (nNOS) in the hypothalamus and monoamine neurotransmitters in the serum and hypothalamus of experimental mice were examined. Additionally, hot plate tests using mice pretreated with various antagonists were used to determine the mechanisms of PH-mediated antinociception. The PH-enhanced latency period of mice in the hot plate test was significantly blocked by pretreatment with atropine and glibenclamide. PH shortened the phase I and phase II reaction times of formalin-treated mice. Strongly reduced writhing and stretching induced by acetic acid were observed in PH-treated mice, indicating that PH mainly exerts antinociceptive activity on neurogenic pain. After thermal pain stimulation for 30 s, compared to control mice, 7-day PH-treated mice had lower nNOS and dopamine levels, and increased levels of serotonin in both the serum and hypothalamus. Collectively, our data showed that PH mediated antinociceptive activities via multiple pathways, including monoamines, nNOS/ATP-sensitive K+ channels, and M-type acetylcholine receptors.
Neurokinin-1 receptor (NK1R) is a high affinity Substance P (SP) receptor and plays a key role in visceral hypersensitivity in irritable bowel syndrome (IBS). Early life stress is a significant risk factor in IBS. The aim of the present study was to investigate the influence of neonatal maternal separation on the expression and distribution of SP and its receptor along the brain-gut axis in a neonatal maternally separated rat model with visceral hypersensitivity. Male neonatal Sprague-Dawley rats, 2-21-day old, were randomly distributed into maternal separation groups of 3 h daily maternal separation (MS) or non-handling (NH). These rats underwent colorectal balloon distention (CRD) upon reaching adulthood. Immunofluorescence was used to examine the distal colon, lumbosacral spinal cord, and the brainstem to semi-quantitatively determine SP and NK1R expression before and after CRD. The following features were assessed: percentage SP-positive area in colonic muscle layer, the number of NK1R-positive myenteric plexus, SP-positive area and NK1-positivity score in the dorsal horn and the brainstem. Neither of these was altered in the MS and NH groups before or after CRD. These results suggest that the SP system might play little role in the development of visceral hyperalgesia in the neonatal maternal separation rat model.
To determine the cytotoxic effect of lymphocytes activated by melanoma-associated antigen 3 (MAGE-3)-sensitized dendritic cells (DCs) on BIU-87 tumor cells, and to evaluate the possibility of MAGE-3-peptide-pulsed DCs as a vaccine in bladder cancer immunotherapy, the proliferation of T cells and the activity of cytotoxic T lymphocytes (CTLs) were examined by the MTT method. CTLs were induced by MAGE-3-sensitized DCs, or by ovalbumin (OVA) peptide and non-sensitized DCs as controls, respectively. The results indicated that MAGE-3-sensitized DCs have the ability to promote the proliferation of T cells as well as the cytotoxic activity of CTLs on bladder cancer cells in comparison with OVA peptide and non-sensitized DCs. In other words, DCs sensitized by the MAGE-3 antigen peptide could obviously upregulate the proliferation of T cells, which resulted in the growth inhibition of bladder cancer BIU-87 cells. In addition, MAGE-3-sensitized DCs played an important role in inhibiting the growth of human BIU-87 tumor xenografts in nude mice.
Many studies exist for reconstructing gene regulatory networks (GRNs). In this paper, we propose a method based on an advanced neuro-fuzzy system, for gene regulatory network reconstruction from microarray time-series data. This approach uses a neural network with a weighted fuzzy function to model the relationships between genes. Fuzzy rules, which determine the regulators of genes, are very simplified through this method. Additionally, a regulator selection procedure is proposed, which extracts the exact dynamic relationship between genes, using the information obtained from the weighted fuzzy function. Time-series related features are extracted from the original data to employ the characteristics of temporal data that are useful for accurate GRN reconstruction. The microarray dataset of the yeast cell cycle was used for our study. We measured the mean squared prediction error for the efficiency of the proposed approach and evaluated the accuracy in terms of precision, sensitivity, and F-score. The proposed method outperformed the other existing approaches.
Recent advances in computational epigenetics have provided new opportunities to evaluate n-gram probabilistic language models. In this paper, we describe a systematic genome-wide approach for predicting functional roles in inactive chromatin regions by using a sequence-based Markovian chromatin map of the human genome. We demonstrate that Markov chains of sequences can be used as a precursor to predict functional roles in heterochromatin regions and provide an example comparing two publicly available chromatin annotations of large-scale epigenomics projects: ENCODE project consortium and Roadmap Epigenomics consortium.
Silica (SiO2) nanoparticles are being progressively applied in various applications, including cosmetics, food technology, and medical diagnostics. Although crystalline SiO2 is a known carcinogen, the carcinogenicity of SiO2 nanoparticles remains unclear. Here, we assessed the cytotoxic effects and DNA injury induced by exposure to various dosages of SiO2 nanoparticles at 0-2400 mg/mL (0-3200 mg/mL microscale SiO2 as positive control) for 24 h using RAW264.7 cells, followed by methyl tetrazolium (MTT) assay. Cells were also treated by 31.25, 125, and 500 mg/mL SiO2 nanoparticles (500 mg/mL microscale SiO2 as positive control) for 24 h and examined by single cell gel electrophoresis assay (SCEG) and flow cytometry. Outstanding dose-related decline in cell viability was observed with enhancing dosages of SiO2 nanoparticles by MTT assay. The inhibitory concentration 50% of SiO2 nanoparticles and microscale SiO2 was 16690 and 5080 mg/mL, respectively. The comet rate (comet%), length of tail, the percentage in DNA tail (TDNA%) and olive tail moment (OTM) induced by SiO2 nanoparticles were significantly increased in comparison with control and microscale SiO2 at 500 mg/mL. 500 mg/mL SiO2 nanoparticles and microscale SiO2 caused a significant increase in apoptosis rate, decreased proliferation index and increased cell proportions in G0/G1 phases by contrast to the negative control (P 2 nanoparticles are more cytotoxic than microscale SiO2 particles; they induce DNA injury, increase apoptosis, and decrease the proliferation index in RAW264.7 cells. DNA injury and apoptosis may be involved in reducing cell proliferation.
The peel of mango (Mangifera indica L.) is a special plant tissue that contains many compounds that interfere with protein extraction. A successful separation with Two-dimensional electrophoresis (2-DE) is the key step for proteomic analysis. To evaluate the efficiencies of mango peel protein extraction for 2-DE, four extraction methods were tested: 1) 2-D clean-up kit, 2) trichloroacetic acid/acetone precipitation, 3) phenol extraction, 4) phenol with methanol/ammonium acetate precipitation. The results showed that the phenol with methanol/ammonium acetate precipitation produced the best quality protein extraction and separation. Proteins were separated in 30-70 and >70 kDa ranges better than with the other methods. Acidic proteins had better resolution with fewer horizontal and vertical streaks. Sixteen proteins were identified by maxtrix-assisted laser desorption/ ionisation time-of-flight tandem mass spectrometry (MALDI-TOF/ TOF-MS/MS). The result demonstrated that each of these four methods can be used to prepare mango peel proteins. The phenol with methanol/ ammonium acetate precipitation was the best choice for proteomic analysis of mango peel.
Stearoyl-coenzyme A desaturase 1 (SCD1) is the key limiting enzyme in the synthesis of monounsaturated fatty acids, and plays a crucial role in the regulation of oleic acid. In this study, 165 ten-week-old Cherry Valley ducks were used to investigate single nucleotide polymorphisms (SNPs) in the 5' regulatory region of the SCD1 gene, and their associations with duck serum biochemical levels and fatty acid composition. Two novel SNPs, g.936516 C > G and g.936551 T > C, were found by polymerase chain reaction-single-strand conformation polymorphism analysis and DNA sequencing methods, exhibiting six genotypes (AA, BB, CC, AB, AC, and BC). The frequency of the dominant genotype BB and allele B was 0.321 and 0.403, respectively. The polymorphism information content value was 0.617, indicating high polymorphism. The chi-square test indicated that the genotype distribution deviated markedly from Hardy-Weinberg equilibrium (P SCD1 gene had significant effects on the serum albumin, total protein, globulin, triglyceride, total cholesterol, and cholinesterase levels, as well as on 16 kinds of fatty acids except for C14:1 and C20:0 (P SCD1-gene-specific SNPs in the 5’ regulatory region may be a useful marker for serum lipid, serum protein, and fatty acid composition in future marker-assisted selection for duck breeding.
Thyroid orbitopathy (TO) is an autoimmune disease that is complicated by ocular surface disorders, leading to discomfort. Dry eye is very prevalent in patients with TO. Recent studies on the pathogenesis of dry eye have focused on the inflammatory process, and some supporting evidence has been discovered. Because TO is a disorder of autoimmune origin, we assumed that the association between TO and dry eye is related to inflammation. Inflammation of the ocular surface in TO-related dry eye has not been well studied. In this study, we assessed cellular inflammation of the ocular surface and the cytokine profiles in patients with TO-related dry eye. Conjunctival impression cytology (CIC) was assessed with an immunofluorescent assay. TO-related dry eye was diagnosed by using the Schirmer test, tear break-up time, thyroid function, and clinical signs. CIC was combined with immunological staining of interleukin-1a (IL-1a), IL-1b, and IL- 6. The immunological impression cytology (IC) grade was compared to the clinical activity score of TO. All TO patients with dry eye were positive for IL-1a, IL-1b, and IL-6. However, the normal controls were also positive for IL-1a. A trend was observed between the clinical inflammatory score and immunological IC grade. This study was the first to delineate the immunological IC of TO-related dry eye. Our study aimed to investigate the pathogenesis of dry eye in TO. Our findings suggest that the conjunctival cytokines IL-1a, IL-1b, and IL-6 may play a role. The results of this study will be useful for future studies of additional inflammatory cytokines, and the levels of these cytokines could be used as an outcome to assess the efficacy of treatment, such as anti-cytokine or immunosuppression therapy, in patients with TO-related dry eye or other ocular surface inflammatory disorders.