New machine learning-based automatic high-throughput video tracking system for assessing water toxicity using Daphnia Magna locomotory responses.

Daphnia magna is an important organism in ecotoxicity studies because it is sensitive to toxic substances and easy to culture in laboratory conditions. Its locomotory responses as a biomarker are highlighted in many studies. Over the last several years, multiple high-throughput video tracking systems have been developed to measure the locomotory responses of Daphnia magna. These high-throughput systems, used for high-speed analysis of multiple organisms, are essential for efficiently testing ecotoxicity. However, existing systems are lacking in speed and accuracy. Specifically, speed is affected in the biomarker detection stage. This study aimed to develop a faster and better high-throughput video tracking system using machine learning methods. The video tracking system consisted of a constant temperature module, natural pseudo-light, multi-flow cell, and an imaging camera for recording videos. To measure Daphnia magna movements, we developed a tracking algorithm for automatic background subtraction using k-means clustering, Daphnia classification using machine learning methods (random forest and support vector machine), and tracking each Daphnia magna location using the simple online real-time tracking algorithm. The proposed tracking system with random forest performed the best in terms of identification (ID) precision, ID recall, ID F1 measure, and ID switches, with scores of 79.64%, 80.63%, 78.73%, and 16, respectively. Moreover, it was faster than existing tracking systems such as Lolitrack and Ctrax. We conducted an experiment to observe the impact of toxicants on behavioral responses. Toxicity was measured manually in the laboratory and automatically using the high-throughput video tracking system. The median effective concentration of Potassium dichromate measured in the laboratory and using the device was 1.519 and 1.414, respectively. Both measurements conformed to the guideline provided by the Environmental Protection Agency of the United States; therefore, our method can be used for water quality monitoring. Finally, we observed Daphnia magna behavioral responses in different concentrations after 0, 12, 18, and 24 h and found that there was a difference in movement according to the concentration at all hours.

Insulin resistance mediates high-fat diet-induced pulmonary fibrosis and airway hyperresponsiveness through the TGF-ß1 pathway.

Prior studies have reported the presence of lung fibrosis and enhanced airway hyperresponsiveness (AHR) in mice with high-fat-diet (HFD)-induced obesity. This study evaluated the role of TGF-ß1 in HFD-induced AHR and lung fibrosis in a murine model. We generated HFD-induced obesity mice and performed glucose and insulin tolerance tests. HFD mice with or without ovalbumin sensitization and challenge were also treated with an anti-TGF-ß1 neutralizing antibody. AHR to methacholine, inflammatory cells in the bronchoalveolar lavage fluid (BALF), and histological features were evaluated. Insulin was intranasally administered to normal diet (ND) mice, and in vitro insulin stimulation of BEAS-2b cells was performed. HFD-induced obesity mice had increased insulin resistance, enhanced AHR, peribronchial and perivascular fibrosis, and increased numbers of macrophages in the BALF. However, they did not have meaningful eosinophilic or neutrophilic inflammation in the lungs compared with ND mice. The HFD enhanced TGF-ß1 expression in the bronchial epithelium, but we found no differences in the expression of interleukin (IL)-4 or IL-5 in lung homogenates. Administration of the anti-TGF-ß1 antibody attenuated HFD-induced AHR and lung fibrosis. It also attenuated goblet cell hyperplasia, but did not affect the AHR and inflammatory cell infiltration induced by OVA challenge. The intranasal administration of insulin enhanced TGF-ß1 expression in the bronchial epithelium and lung fibrosis. Stimulating BEAS-2b cells with insulin also increased TGF-ß1 production by 24 h. We concluded that HFD-induced obesity-associated insulin resistance enhances TGF-ß1 expression in the bronchial epithelium, which may play an important role in the development of lung fibrosis and AHR in obesity.

Potential of serum soluble CD93 as a biomarker for asthma in an ovalbumin-induced asthma murine model.

BACKGROUND: CD93 is a membrane-associated glycoprotein, which can be released in a soluble form (sCD93) into the serum. CD93 has received renewed attention as a candidate biomarker of inflammation in various inflammatory and immune-mediated diseases, including asthma. OBJECTIVE: We aimed to evaluate the effects of airway inflammation on CD93 levels in murine models. METHODS: We established an ovalbumin (OVA)-induced acute asthma murine model (OVA model) and a lipopolysaccharide (LPS)-induced airway inflammation murine model (LPS model). Dexamethasone was administered by gavage to attenuate the airway inflammation. RESULTS: The OVA model demonstrated typical allergic asthma features with increased airway hyper-responsiveness, inflammatory cell infiltration, increased Th2 cytokine levels, compared to the control group. CD93 levels were decreased in lung homogenates and, respiratory epithelial cells, whereas serum sCD93 levels were increased in the OVA model, as compared to the control group. Dexamethasone reversed these effects of OVA. In contrast, in the LPS model, CD93 levels were not affected in neither respiratory epithelial cells nor serum. CONCLUSIONS: Our findings demonstrate the potential of using sCD93 as a biomarker for allergic asthma.

Identification of Major Flavone C-Glycosides and Their Optimized Extraction from Cymbidium kanran Using Deep Eutectic Solvents.

Cymbidium kanran, an orchid exclusively distributed in Northeast Asia, has been highly valued as a decorative plant and traditional herbal medicine. Here, C. kanran extracts were prepared in 70% aqueous methanol using ultrasound-assisted extraction (UAE) and subjected to liquid chromatography-photodiode array detection and ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry analysis, which were used for quantitative and qualitative analysis, respectively. It was found that the extracts were rich in flavone C-glycosides including vicenin-2, vicenin-3, schaftoside, vitexin, and isovitexin. Ten deep eutectic solvents (DESs) were synthesized by combining choline chloride (hydrogen bond acceptor) with various polyols and diols (hydrogen bond donors) and were tested as a medium for the efficient production of extracts enriched with potentially bioactive flavone C-glycosides from C. kanran. A DES named ChCl:DPG, composed of choline chloride and dipropylene glycol at a 1:4 molar ratio, exhibited the best extraction yields. Then, the effects of extraction conditions on the extraction efficiency were investigated by response surface methodology. Lower water content in the extraction solvent and longer extraction time during UAE were desirable for higher extraction yields. Under the statistically optimized conditions, in which 100 mg of C. kanran powder were extracted in 0.53 mL of a mixture of ChCl:DPG and water (74:26, w/w) for 86 min, a total of 3.441 mg g-1 flavone C-glycosides including 1.933 mg g-1 vicenin-2 was obtained. This total yield was 196%, 131%, and 71% more than those obtained using 100% methanol, water, and 70% methanol, respectively.

Characterization of a Major Allergen from Mongolian Oak, Quercus mongolica, a Dominant Species of Oak in Korea.

BACKGROUND: Oaks are the most common trees in Korean forests, and Mongolian oak, Quercus mongolica, is the dominant species. However, no allergen has been characterized from Mongolian oak. In this study, we tried to characterize a major allergen from Mongolian oak. METHODS: A molecule homologous to pathogenesis-related 10 (PR-10)-like protein, Que m 1, was cloned by RT-PCR. Its recombinant protein, along with Que a 1, an allergen from white oak (Q. alba), was produced. The allergenicity and diagnostic value of recombinant Que m 1, Que a 1, and Bet v 1 proteins were compared by ELISA using sera from oak-sensitized subjects. A basophil activation test was also performed using CD63 expression as an activation marker. RESULTS: Que m 1 sequence shares 57.5-96.2% amino acid sequence identity with PR-10-like allergens from various plants. Specific IgE to recombinant Que m 1, Que a 1, and Bet v 1 were detected in 92.0, 74.0, and 38.0% of 50 serum samples from Korean tree pollinosis patients. Recombinant Que m 1 was able to inhibit IgE reactivity to Que a 1 and Bet v 1, indicating its strong cross-reactivity. The activation patterns of basophils from 5 patients were similar in terms of the CD63 expression and protein concentration of challenged Bet v 1 and Que m 1. CONCLUSIONS: A major allergen, Que m 1, was cloned, and its recombinant protein was produced from Mongolian oak, a dominant species in Korea. Recombinant Que m 1 is potentially useful for the diagnosis and treatment of tree pollinosis in Korea.

The complete chloroplast genome sequence of the Japanese Camellia (Camellia japonica L.).

The complete chloroplast genome sequence of valuable ornamental tree, Camellia japonica L. (Theaceae), was determined. The genome size was 156,971 bp in length, containing a pair of 25,798 bp inverted repeat (IR) regions, which were separated by small and large single copy regions (SSC and LSC) of 18,394 and 86,673 bp, respectively. The cp genome contained 134 genes, including 91 coding genes, six rRNA genes, and 37 tRNA genes. The overall GC content of the chloroplast genome was 37.3%. Phylogenetic analysis revealed the position of C. japonica being sister to the clade containing C. crapnelliana and C. oleifera (subgenus Camellia).

Improvement Characteristics of Bio-active Materials Coated Fabric on Rat Muscular Mitochondria.

This study surveys the improvement characteristics in old-aged muscular mitochondria by bio-active materials coated fabric (BMCF). To observe the effects, the fabric (10 and 30%) was worn to old-aged rat then the oxygen consumption efficiency and copy numbers of mitochondria, and mRNA expression of apoptosis- and mitophagy-related genes were verified. By wearing the BMCF, the oxidative respiration significantly increased when using the 30% materials coated fabric. The mitochondrial DNA copy number significantly decreased and subsequently recovered in a dose-dependent manner. The respiratory control ratio to mitochondrial DNA copy number showed a dose-dependent increment. As times passed, Bax, caspase 9, PGC-1α and ß-actin increased, and Bcl-2 decreased in a dose-dependent manner. However, the BMCF can be seen to have had no effect on Fas receptor. PINK1 expression did not change considerably and was inclined to decrease in control group, but the expression was down-regulated then subsequently increased with the use of the BMCF in a dose-dependent manner. Caspase 3 increased and subsequently decreased in a dose-dependent manner. These results suggest that the BMCF invigorates mitophagy and improves mitochondrial oxidative respiration in skeletal muscle, and in early stage of apoptosis induced by the BMCF is not related to extrinsic death-receptor mediated but mitochondria-mediated signaling pathway.

Different methylation profiles between intestinal and diffuse sporadic gastric carcinogenesis.

OBJECTIVE: Gastric cancer (GC) is histologically classified into intestinal type and diffuse type, and diffuse type cancer can be further subdivided into poorly differentiated carcinoma (PDC) and signet ring cell carcinoma (SRCC). Recent evidence suggests that early SRCC is an initial, differentiated form of diffuse GC that may evolve into PDC. This study aimed at identifying the molecular features of epigenetic methylation changes in histologic differentiation status of GC. METHODS: Included in this study are 149 samples of paraffin-embedded tissues and 115 fresh endoscopically biopsied tissues. Multiple paraffin tissues involving normal (n=22), dysplasias (GDs, n=39), differentiated cancers (DCs, n=35), PDCs (n=33) and SRCCs (n=20) were included as an experimental group. For the validation group, endoscopically biopsied tissues of DCs (n=50), PDCs (n=31), and SRCs (n=34) were analyzed. DNAs, isolated from each group were analyzed to determine the methylation status of 6 genes (GDNF, RORA, MINT25, KLF7, CDH1, LINE-1) using pyrosequencing. RESULTS: LINE-1 was hypomethylated in GCs compared to normal and GD. GDNF, RORA and MINT25 were more hypermethylated in intestinal type GCs than those of diffuse type GCs, whereas CDH1 showed opposite patterns of methylation. Among diffuse type GCs, SRCCs showed lower level of methylation for GDNF, RORA, MINT25 and KLF7, and higher level for CDH1 compared to PDCs. CONCLUSIONS: In conclusion, intestinal type of GCs shows different epigenetic methylation profiles compared to the diffuse one. Moreover, SRCCs have different methylation profiles compared with PDCs, suggesting a unique molecular pathway in the gastric carcinogenesis.

TP53BP2 locus is associated with gastric cancer susceptibility.

We investigated the association of the TP53BP2 locus with gastric cancer susceptibility in a Korean population. We assayed 9 single nucleotide polymorphisms (SNP) in an 82.5 kb region that included the TP53BP2 locus in 233 male gastric cancer patients and 390 unaffected healthy male controls. The allelic frequencies of 4 SNP within TP53BP2, g.206692C>T, g.198267A>T, g.164895G>A and g.152389A>T, differed significantly between cases and controls (p < or = 0.0376). When compared to carriers of non-risk alleles, individuals homozygotic for each of the risk alleles had a 50% increase in risk of gastric cancer (age-adjusted odds ratio [OR] > or = 1.48; p < or = 0.0371). Furthermore, these 4 significantly associated SNP were in strong linkage disequilibrium (r2 > or = 0.51). Haplotype analysis showed that individuals with the CAGA haplotype, consisting of the risk alleles at each SNP, had a 1.55-fold higher risk for gastric cancer than individuals with the haplotype TTAT, consisting of the non-risk alleles at each SNP (OR = 1.55; 95% confidence interval [CI] = 1.13-2.14; p = 0.00705). Two other SNP were not polymorphic in the study subjects, whereas the other 3 SNP, located toward the outside of the TP53BP2 locus, were not associated with gastric cancer susceptibility. Although the location of the pathogenic variant is not yet known, our results suggest that the TP53BP2 locus is associated with susceptibility to gastric cancer in the Korean population.