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Introduction: The aim of this study was to perform a clinicopathologic analysis of PHLPP1 expression in gastric cancer patients and analyze AKT activity with chemotherapy drug treatment in cancer subtypes. Materials and Methods: Surgically resected gastric cancer tissue specimens were obtained from 309 patients who underwent gastrectomy, and PHLPP1 expression was validated by tissue microarray analysis with immunohistochemistry. We assessed whether PHLPP1 selectively dephosphorylates Ser473 of AKT in an in-vitro study. Results: We found that the PHLPP1 overexpression (OE) group showed significantly greater proportions of differentiated subtype samples and early T stage samples, lower lymph node metastasis, and lower TNM stage than the PHLPP1 underexpression (UE) group. The overall survival of the PHLPP1-OE group was significantly higher (53.39 ± 0.96 months) than that of the PHLPP1-UE group (47.82 ± 2.57 months) ( P  = .01). In vitro analysis, we found that the PHLPP1-OE group showed a significant decrease in relative AKT S-473 levels in both cell lines (MKN-74 and KATO-III). We found that treatment with chemotherapy drugs decreased the activity of Ser473 in the MKN-74 cell line with PHLPP1 OE, but it did not affect the activity of Ser473 in KATO-III cells. Conclusion: We found that patients who overexpressed PHLPP1 showed low recurrence and good prognosis. PHLPP1 was found to work by lowering the activity of AKT Ser473 in gastric cancer. Additionally, we found a clue regarding the mechanism of chemotherapeutic drug resistance in a cell line of signet ring cell origin and will uncover this mechanism in the future.

Background: Sulforaphane (SFN) is an isothiocyanate compound present in cruciferous vegetables. Although the anti-inflammatory effects of SFN have been reported, the precise mechanism related to the inflammatory genes is poorly understood., Objectives: This study examined the relationship between the anti-inflammatory effects of SFN and the differential gene expression pattern in SFN treated ob/ob mice., Methods: Nitric oxide (NO) level was measured using a Griess assay. The inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression levels were analyzed by Western blot analysis. Pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). RNA sequencing analysis was performed to evaluate the differential gene expression in the liver of ob/ob mice., Results: The SFN treatment significantly attenuated the iNOS and COX-2 expression levels and inhibited NO, TNF-α, IL-1β, and IL-6 production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA sequencing analysis showed that the expression levels of 28 genes related to inflammation were up-regulated (> 2-fold), and six genes were down-regulated (< 0.6-fold) in the control ob/ob mice compared to normal mice. In contrast, the gene expression levels were restored to the normal level by SFN. The protein-protein interaction (PPI) network showed that chemokine ligand (Cxcl14, Ccl1, Ccl3, Ccl4, Ccl17) and chemokine receptor (Ccr3, Cxcr1, Ccr10) were located in close proximity and formed a "functional cluster" in the middle of the network., Conclusions: The overall results suggest that SFN has a potent anti-inflammatory effect by normalizing the expression levels of the genes related to inflammation that were perturbed in ob/ob mice., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Korean Society of Veterinary Science.)

The endoplasmic reticulum (ER) is the main site of protein synthesis, folding, and secretion to other organelles. The capacity of the ER to process proteins is limited, and excessive accumulation of unfolded and misfolded proteins can induce ER stress, which is associated with plant diseases. Here, a transgenic Arabidopsis system was established to express anti-cancer monoclonal antibodies (mAbs) that recognize the tumor-associated antigen GA733-2. Monoclonal antibody (mAb) CO17-1A recognize a tumor-associated epitope expressed on the colorectal cancer cell surface. The ER retention Lys-Asp-Glu-Leu (KDEL) motif sequence was added to the C-terminus of the heavy chain to retain anti-colorectal cancer mAbs in the ER, consequently boosting mAb production. Agrobacterium-mediated floral dip transformation was used to generate T1 transformants, and homozygous T4 seeds obtained from transgenic Arabidopsis plants expressing anti-colorectal cancer mAbs were used to confirm the physiological effects of KDEL tagging. Germination rates were not significantly different between both plants expressing mAb CO without KDEL mAb CO (CO plant) and mAb CO with KDEL mAb COK (COK plant). However, COK plants primary root lengths were shorter than those of CO plants and non-transgenic Arabidopsis plants in in vitro media. Most ER stress-related genes, with the exception of bZIP28 and IRE1a, were upregulated in COK plants compared to CO plants. Western blot and SDS-PAGE analyses showed that COK plants exhibited up to five times higher expression and mAb amounts than plants. Enhanced expression in mAb COK plants was confirmed by immunohistochemical analyses. mAb COK was distributed across most of the area of leaf tissues, whereas mAb CO was mainly distributed in extracellular areas. Surface plasmon resonance analyses revealed that mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. N-glycosylation analysis showed that mAb CO had plant specific residues whereas mAb COK mainly showed an oligo-mannose N-glycan structure without the plant specific glycan residues. In this study, the reduction of plant growth and biomass induced by ER retention signal peptide might be only in in vitro conditions, and thus should be carefully considered for the initial screening for transgenic lines on culture media. Taken together, nevertheless the fusion of ER retention signal peptide is an effective approach for enhancing the yields of recombinant proteins in vivo., Competing Interests: The authors have declared that no competing interests exist.

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

PMA1 encodes a transmembrane polypeptide that functions to pump protons out of the cell. Ectopic PMA1 overexpression in Saccharomyces cerevisiae enhances tolerance to weak acids, reactive oxygen species (ROS) and ethanol, and changes the following physiological properties: better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide production. The enhanced stress tolerance was dependent on the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway, but not the MAPK Slt2 of the cell wall integrity (CWI) pathway; however, a PMA1 overexpression constitutively activated both Hog1 and Slt2. The constitutive Hog1 activation required the MAPK kinase kinase (MAP3K) Ssk2 of the HOG pathway, but not Ste11 and Ssk22, two other MAP3Ks of the same pathway. The constitutive Slt2 activation did not require Rom2 and the membrane sensors of the CWI pathway, whereas Bck1 was indispensable. The PMA1 overexpression activated the stress response element but not the cyclic AMP response element and the Rlm1 transcription factor. PMA1 overexpression may facilitate the construction of industrial strains with simultaneous tolerance to weak acids, ROS, and ethanol.

Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines, which are important for innate immunity. NLRs, i.e., nucleotide-binding oligomerization domain (NOD)-like receptors, play a crucial role as innate immune sensors and form multiprotein complexes called inflammasomes, which mediate caspase-1-dependent processing of pro-IL-1β. To elucidate the role of inflammasome components in T. gondii -infected THP-1 macrophages, we examined inflammasome-related gene expression and mechanisms of inflammasome-regulated cytokine IL-1β secretion. The results revealed a significant upregulation of IL-1β after T. gondii infection. T. gondii infection also upregulated the expression of inflammasome sensors, including NLRP1, NLRP3, NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and NAIP, in a time-dependent manner. The infection also upregulated inflammasome adaptor protein ASC and caspase-1 mRNA levels. From this study, we newly found that T. gondii infection regulates NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and neuronal apoptosis inhibitor protein (NAIP) gene expressions in THP-1 macrophages and that the role of the inflammasome-related genes may be critical for mediating the innate immune responses to T. gondii infection.

In consensus, myocardial infarction (MI) is defined as irreversible cell death secondary to prolonged ischemia in heart. The aim of our study was to evaluate the therapeutic potential of anti-fibrotic human Relaxin-expressing plasmid DNA with hypoxia response element (HRE) 12 copies (HR1) delivered by a dendrimer type PAM-ABP polymer G0 (HR1/G0) after MI on functional, hemodynamic, geometric, and cardiac extracellular matrix (ECM) remodeling in rats. HR1/G0 demonstrated significantly improved LV systolic function, hemodynamic parameters, and geometry on 1 wk and 4 wks after MI in rats, compared with I/R group. The resolution of regional wall motional abnormalities and the increased blood flow of infarct-related coronary artery supported functional improvements of HR1/G0. Furthermore, HR1/G0 polyplex showed favorable post-infarct cardiac ECM remodeling reflected on the favorable cardiac ECM compositions. Overall, this is the first study, which presented an advanced platform for the gene therapy that reverses adverse cardiac remodeling after MI with a HR1 gene delivered by a bioreducible dendrimer polymer in the cardiac ECM., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes., (Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme with potent anti-oxidant and anti-inflammatory activities. Previous studies have shown that systemic induction of HO-1 by chemical inducers reduces adiposity and improves insulin sensitivity. To dissect the specific function of HO-1 in adipose tissue, we generated transgenic mice with adipose HO-1 overexpression using the adipocyte-specific aP2 promoter. The transgenic (Tg) mice exhibit similar metabolic phenotype as wild type (WT) control under chow-fed condition. High fat diet (HFD) challenge significantly increased the body weights of WT and Tg mice to a similar extent. Likewise, HFD-induced glucose intolerance and insulin resistance were not much different between WT and Tg mice. Analysis of the adipose tissue gene expression revealed that the mRNA levels of adiponectin and interleukin-10 were significantly higher in chow diet-fed Tg mice as compared to WT counterparts, whereas HFD induced downregulation of adiponectin gene expression in both Tg and WT mice to a similar level. HFD-induced proinflammatory cytokine expression in adipose tissues were comparable between WT and transgenic mice. Nevertheless, immunohistochemistry and gene expression analysis showed that the number of infiltrating macrophages with preferential expression of M2 markers was significantly higher in the adipose tissue of obese Tg mice than WT mice. Further experiment demonstrated that myeloid cells from Tg mice expressed higher level of HO-1 and exhibited greater migration response toward chemoattractant in vitro. Collectively, these data indicate that HO-1 overexpression in adipocytes does not protect against HFD-induced obesity and the development of insulin resistance in mice.

Follicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte.

Objective: The associations between arrest-defective protein 1 (ARD1) gene expression and the clinicopathological characteristics and clinical outcomes of 94 patients undergoing hepatectomy for hepatocellular carcinoma (HCC) were investigated., Methods: ARD1 mRNA levels in HCC and corresponding non-cancerous tissues were quantified by real-time PCR. The gene expression of the tumor relative to that in the non-tumor tissues was calculated using the 2(-)(ΔΔ)(CT) method. The subjects were classified into high expression (2(-)(ΔΔ)(CT) > 1, n = 38) and low expression (2(-)(ΔΔ)(CT) ≤ 1, n = 56) groups., Results: The HCCs did not differ from matched liver tissues in terms of ARD1 mRNA levels. The high expression group had more often microvascular invasion than the low expression group (32 vs. 14%; p = 0.045). The two groups did not differ significantly in terms of other patient or tumor variables. The median follow-up period was 92.1 months. The 5-year recurrence-free and overall survival rates were 34 and 76% for the high expression group, respectively, which were similar to the rates of the low expression group (46 vs. 73%, p = 0.98 and p = 0.52, respectively)., Conclusions: Intratumoral ARD1 mRNA overexpression was involved in the microvascular invasion process in patients with HCC, although it did not associate strongly with postresectional outcomes., (Copyright © 2012 S. Karger AG, Basel.)

Our previous study has demonstrated that the methanol extract of Hyul-Tong-Ryung (HM) specifically suppresses the phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) production through the inhibition of MMP-9 mRNA expression in MCF-7 human breast carcinoma cells. However, the molecular mechanisms involved in transcriptional suppression of MMP-9 by HM in PMA-induced MCF-7 cells are not known. In this study, we aimed to elucidate the molecular mechanisms involved in the inhibition of MMP-9 expression by HM in PMA-induced MCF-7 cells. The results of promoter assay and EMSA showed that HM specifically inhibits MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, PMA-stimulated phosphorylation of extracellular signal regulated kinase 1/2 (ERK 1/2) was suppressed by HM treatment, whereas the phosphorylation of either c-Jun N-terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK) was not affected. HM could inhibit the PMA-induced MMP-9 expression through suppression of the transcriptional activity of MMP-9 gene in MCF-7 cells. These results indicate that HM inhibits PMA-induced MMP-9 expression by blocking the activation of activator protein-1 (AP-1) via extracellular signal regulated kinase 1/2 (ERK 1/2) signaling pathway.

Reactive oxygen species (ROS) are produced during seed desiccation, germination, and ageing, leading to cellular damage and seed deterioration and, therefore, decreased seed longevity. The effects of simultaneous over-expression of two antioxidant enzymes on seed longevity and seed germination under stressful conditions were investigated. Transgenic tobacco simultaneously over-expressing the Cu/Zn-superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes in plastids showed normal growth and seed development. Furthermore, the transgenic seeds displayed increased CuZnSOD and APX enzymatic activities during seed development and maintained antioxidant enzymatic activity after two years of dried storage at room temperature. The two-year stored non-transgenic seeds (aged NT seeds) had higher levels of ion leakage than the two-year stored transgenic seeds (aged CA seeds), indicating membrane damage caused by ROS was more severe in the aged NT seeds than the aged CA seeds. The aged CA seeds decreased germination rates as compared to newly harvested transgenic and non-transgenic seeds. The aged CA seeds, however, significantly increased germination rates under various abiotic stress conditions as compared to aged NT seeds. These data strongly suggest that simultaneous over-expression of the CuZnSOD and APX genes in plastids improves seed longevity and germination under various environmental stress conditions by attenuating the effects of oxidative stress produced by elongated storage conditions and harsh environmental stresses.

In vitro oocyte maturation (IVM) holds great promise as a tool for enhancing clinical treatment of infertility, enhancing availability of nonhuman primates for development of disease models, and facilitating endangered species preservation. However, IVM outcomes have remained significantly below the success rates obtained with in vivo matured (VVM) oocytes from humans and nonhuman primates. A cDNA array-based analysis is presented, comparing the transcriptomes of VVM oocytes with IVM oocytes. We observe a small set of just 59 mRNAs that are differentially expressed between the two cell types. These mRNAs are related to cellular homeostasis, cell-cell interactions including growth factor and hormone stimulation and cell adhesion, and other functions such as mRNA stability and translation. Additionally, we observe in IVM oocytes overexpression of PLAGL1 and MEST, two maternally imprinted genes, indicating a possible interruption or loss of correct epigenetic programming. These results indicate that, under certain IVM conditions, oocytes that are molecularly highly similar to VVM oocytes can be obtained; however, the interruption of normal oocyte-somatic cell interactions during the final hours of oocyte maturation may preclude the establishment of full developmental competence.

Kryptolebias marmoratus is the only known internally self-fertilizing vertebrate. It shows high susceptibility to many chemical carcinogens and has been proposed as a potential cancer model species alternative to mammals. Since use of this fish species is expected to rise in cancer research, regulation of oncogenes from K. marmoratus needs proper understanding. We cloned and deduced full-length sequence of cDNA of N-ras oncogene from K. marmoratus. Study of expression profile of N-ras by using quantitative real-time RT-PCR revealed that brain had the highest level of expression compared to other tissues. Some embryonic stages showed more N-ras expression than juveniles and adults. Exposure to two environmental endocrine disrupting chemicals (EDCs), bisphenol A (BPA) and 4-nonylphenyl (NP) caused up-regulation of N-ras in gonad, intestine and liver of hermaphrodite K. marmoratus. It is suggested that K. marmoratus may be a suitable model species for oncogene expression studies. The observed EDC-induced expression of N-ras supports the assumption that EDC exposure may predispose the host to the risk of environmental carcinogenesis.

We focused on the transcriptional responses induced by low and very low doses of ionizing radiation with time effect. Regardless of their importance only a few limited studies have been done. Here we applied a large-scale gene transcript profile to elucidate the genes and biological pathways. Immortalized human mesenchymal stem cells were irradiated with 0.01, 0.05, 0.2 and 1 Gy of gamma radiation and total RNA was extracted from each cell line at 1, 4, 12 and 48 h after exposure. The essential transcriptional responses were identified according to dose and time. A total of 6,016 genes showed altered expression patterns at more than one time point or dose level among the investigated 10,800 genes. Genes that showed dose-dependent expression responses were involved in signal transduction, regulation of transcription, proteolysis, peptidolysis and metabolism. Those that showed time-dependent responses were divided into two distinct groups: the up-and-down group was associated with 'cellular defense mechanisms' such as apoptosis, cell adhesion, stress response and immune response and the down-and-up group with 'fundamental cellular processes' such as DNA replication, mitosis, RNA splicing, DNA repair and translation initiation. Genes showing both dose-and time-dependent responses exhibited a mixture of both features. A highly non-linear relationship between the IR dose and the transcriptional relative response was obtained from the dose-dependent group. The time-dependent group also exhibited a non-linear relationship as the complex effect group did. Some of the early-reactive-phase (1-4 h) genes showed a differential expression response to 0.01, 0.05 and 0.2 Gy but were unresponsive to 1 Gy. Some of the late-recovery-phase (12-48 h) genes showed a differential expression to 1 Gy but were relatively unresponsive to other doses. We further characterized the gene expression patterns that could be implicated in the molecular mechanism of the cellular responses to low and very low-dose irradiation.

Background: IL-31 is a novel cytokine that, when overexpressed in transgenic mice, induces severe itching dermatitis resembling human eczema., Objective: We aimed to evaluate the importance of polymorphisms in the human IL-31 gene (IL31) in the genetic susceptibility to eczema., Methods: We sequenced the entire IL-31 gene, including the promoter region, and determined the haplotype structure. Single nucleotide polymorphisms tagging the main haplotypes were genotyped in 3 independent European populations comprising 690 affected families. An association analysis of IL31 gene variants with atopic and nonatopic eczema was performed., Results: We found significant association of a common IL31 haplotype with the nonatopic type of eczema in all 3 study populations (combined P = 4.5 x 10(-5)). Analysis of PBMCs in healthy individuals revealed a strong induction IL31 mRNA expression on stimulation with anti-CD3 and anti-CD28 that was 3.8-fold higher in individuals homozygous for the risk haplotype (AA) in contrast to non-A haplotype carriers, suggesting that altered regulation of IL-31 gene expression is the disease-causing factor., Conclusion: Our results lend strong support to an important role of IL-31 in the pathogenesis of nonatopic eczema., Clinical Implications: This study presents the first genetic risk factor for the nonatopic type of eczema and indicates a primary role of IL-31-induced pruritus in the initiation of this disease, thus proposing a new target for the prevention and therapy of eczema.

The purpose of this study was to examine the changes in gastric ghrelin and leptin with respect to Helicobacter pylori infection and whether such changes affect the plasma levels of leptin and ghrelin. In addition, we examined the relationship between changes in gastric mucosal ghrelin and leptin levels and gastrointestinal symptoms. Sixty-three patients diagnosed with chronic gastritis were enrolled in the study. Twenty-nine patients were Helicobacter pylori negative and 34 were Helicobacter pylori positive. Expression of ghrelin and leptin mRNA in the gastric mucosa was measured using endoscopic biopsies from the fundus. Plasma levels of ghrelin and leptin were measured by radioimmunoassay. Expression of leptin mRNA in the gastric mucosa was significantly higher in Helicobacter pylori-positive patients than in negative patients (0.38 +/- 0.17 vs. 0.24 +/- 0.12, p = 0.039). The expression of ghrelin was lower in positive patients than in the negative group, although this difference was not significant (p = 0.07). However, there was no significant difference in plasma leptin and ghrelin levels. Gastric mucosal ghrelin mRNA expression was significantly lower in patients with dyspepsia than in those without (0.15 +/- 0.11 vs. 0.23 +/- 0.20, p = 0.05). Helicobacter pylori infection and gastrointestinal symptoms could be associated with leptin and ghrelin expression in the gastric mucosa.

Type 1 diabetes results from insulin deficiency caused by destruction of pancreatic beta cells. Glucagon-like peptide (GLP)-1 stimulates beta cell growth and differentiation. To determine whether continuous expression of GLP-1 in vivo can regenerate beta cells and remit type 1 diabetes in mice for a prolonged time, we constructed an adenoviral vector containing the cytomegalovirus promoter/enhancer and albumin leader sequence followed by GLP-1 cDNA (rAd-GLP-1). A single administration of rAd-GLP-1 via the tail vein into streptozotocin (STZ)-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in remission of diabetes within 10 days; normoglycemia remained until the experiment was terminated. The number of insulin-positive cells in the pancreas and insulin secretion significantly increased in rAd-GLP-1-treated mice compared with STZ-induced diabetic mice treated with rAd-beta-galactosidase. Glucose tolerance tests in mice that achieved normoglycemia after treatment with rAd-GLP-1 showed that the kinetics of glucose clearance was similar to normal NOD/SCID mice. Treatment of autoimmune diabetic mice with rAd-GLP-1 restored normoglycemia, which was maintained for 1 year when mice were also treated with an immunoregulator to halt the autoimmune response to beta cells. We suggest that regeneration of insulin-producing cells by GLP-1 gene therapy may be a potential method for prolonged control of type 1 diabetes in humans.

Changes in gene expression in liver of medaka in response to 4-nonylphenol (4-NP) were investigated with a microarray consisting of 3.4 K medaka-specific cDNA probes. Upon exposure to 4-NP at two different concentrations (20 and 100 microg/L) for 24 h, we found 44 different genes that were up- or downregulated with statistically significant changes. Digestive enzyme genes were downregulated but vitellogenin, choriogenin and novel biomarker genes were upregulated. Additionally an analysis of the changes of gene expression with respect to time was conducted at 100 microg/L of 4-NP with samples at 0, 24, 48 and 96 h. The expression of 75 genes was altered and these were categorized into four different clusters. The validity of the results was checked by semi-quantitative PCR with 12 representative genes and this confirmed the microarray data. Therefore, we suggest that this medaka 3.4 K chip would be useful in generating gene expression profiles for studies of ecotoxicogenomics in medaka.

Previous studies on ras proto-oncogene genes in fish have been focused on chemical-associated carcinogenesis, and the expression of fish ras genes was not well-characterized. We investigated Ki- and Ha-ras genes from the hermaphroditic fish Rivulus marmoratus to understand better their expression patterns in specific tissues, as well as their responses to endocrine-disrupting chemicals such as 4-nonylphenol (4-NP). By investigating expression patterns, we found that the R. marmoratus Ki-ras (Rm Ki-ras) gene showed an alternative splicing event between exons 4A and 4B according to tissue types, which is different from the expression pattern of mammalian Ki-ras genes. In the Rm Ki-ras gene, there were two different expressed types, with exons 1-2-3-4A-4B (long form) and with exons 1-2-3-4B (short form). In the Rm Ki-ras gene, the long form was expressed strongly in the gonad and intestine, and the short form was expressed ubiquitously, except for a low level of expression in the liver. Following 4-NP exposure (300 microg/L), the Rm Ki-ras long form in the liver was significantly expressed, while it was expressed moderately in the ovaries. However, the Rm Ha-ras gene was significantly over-expressed in the brain, while its expression in the gonad was down-regulated. In relation to these modulations after 4-NP exposure, we searched the Rm Ha- and Ki-ras promoter regions and found several ERE-half sites, that may be involved in the modulation of ras gene expression following 4-NP exposure. These genes could be applicable as new biomarker genes for assessing exposure to endocrine-disrupting chemicals (EDCs). Further, this implies the disturbance of ras-dependent signal transduction following EDC exposure.

Sonic hedgehog functions to induce floor plate in early stages, and spinal motor neurons and midbrain dopaminergic neurons in later stages of development. Here, we investigated the effects of sonic hedgehog on tyrosine hydroxylase expression in three cell lines that correspond to different stages of neural development. Sonic hedgehog increased the tyrosine hydroxylase gene expression in pluripotent P19 cells but repressed it in tyrosine hydroxylase-producing PC12 cells. Promoter analysis in mouse neural stem cells indicated that the N-terminal of sonic hedgehog repressed both the basal and cAMP-dependent protein kinase A-mediated tyrosine hydroxylase activity. These results suggest that the N-terminal of sonic hedgehog increases tyrosine hydroxylase gene expression in cells to acquire dopaminergic phenotypes, but decreases expression in late born neurons by antagonizing the protein kinase A cAMP-responsive element binding protein pathway.

This study determined whether exercise training prevents pathological hypertrophy in the left ventricle by modulation of myocardial and apoptosis-associated genes. We used spontaneously hypertensive rats (n=15, non-exercise SHR), exercise-trained SHR (n=15, treadmill exercise for 12 weeks), and sedentary Wistar-Kyoto (WKY) rats (n=15). Exercise-trained SHR expressed adaptive changes such as reduced body weight, heart rate, blood pressures, left ventricle wall thickness, lipid profiles, and homocysteine level. The mRNA expression of angiotensin converting enzyme, endothelin-1, and brain natriuretic peptides in the heart was lower in the exercise-trained SHR and in the WKY than in the non-exercise SHR, whereas mRNA expression of caveolin-3 and eNOS in the heart was higher. Bcl-2 protein was higher in the exercise-trained SHR than in the WKY and the non-exercise SHR. In contrast, Bax protein levels were lower in the exercise-trained SHR and in the WKY than in the non-exercise SHR. Furthermore, the levels of the active forms of caspase-3 (20 kDa) were lower in the exercise-trained SHR and in the WKY than in the non-exercise SHR. These findings suggest that exercise training prevents pathological hypertrophy in the left ventricle by modulation of myocardial genes and that it interferes with a signal transduction pathway of apoptosis secondary to the pathological cardiac hypertrophy.

Astragali radix, which has tonifying and circulatory effect as well as immune response, is one of the oldest and most frequently used crude drug for oriental medicine in many Asian countries. The present study was conducted to evaluate the effect of aqueous extract of Astragali radix (ARE) on the functions of murine macrophage cell line, RAW 264.7 macrophage cells. In the cell proliferation assay, methotrexate (MTX), an agent of immune suppression, decreased the cell proliferation of RAW 264.7 macrophage cells (IC(50): 100 microg/ml), but the suppression of cell proliferation was significantly protected by ARE treatment in RAW 264.7 macrophage cells. The expressions of cytokine gene by ARE were investigated using reverse transcription polymerase chain reaction (RT-PCR). In RT-PCR, IL-1alpha, IL-1beta and IL-6 mRNA expressions was induced in ARE-treated RAW 264.7 macrophage cells. We also investigated the effect of the nitric oxide (NO) synthesis and inducible nitric oxide synthase (iNOS) mRNA expression by ARE. ARE alone had no effect on NO synthesis and iNOS mRNA expression in RAW 264.7 cells. In the case of lipopolysaccharide (LPS) stimulation, NO production and iNOS mRNA expression were detected in RAW 264.7 cells. However, NO production and iNOS mRNA expression which is induced by LPS decreased after treatment of ARE. These data demonstrate that ARE can reduce the suppression of macrophage cell proliferation induced by MTX, and induce IL-1alpha, IL-1beta and IL-6 mRNA expressions in RAW 264.7 macrophage cells. Also, ARE inhibit NO production in LPS-stimulated RAW 264.7 macrophage cells, and the inhibition of NO production may be associated with the inhibition of iNOS mRNA expression.

The intertidal harpacticoid copepod Tigriopus japonicus is an important species in the study of marine pollution. To facilitate molecular biomonitoring using T. japonicus, we constructed a T. japonicus unidirectional cDNA library using lambdaZAP expression vector, excised to pBluescript vector with the aid of helper phage, and analyzed 686 randomly picked expressed sequence tags (ESTs) from this species. From the 686 ESTs sequenced, we found several functional genes such as vitellin, kinases and potential detoxification-related genes. We are now preparing a T. japonicus cDNA chip for molecular ecotoxicological studies. In this paper, we discuss the potential use of T. japonicus ESTs and their importance in ecotoxicology.

In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTalpha gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTalpha gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTalpha gene as rm-GSTalpha. The cDNA of the rm-GSTalpha gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTalpha was subcloned into a 6 x His-tagged pCRT7 TOPO TA expression vector to produce the recombinant 6 x His-tagged rm-GST protein. This will be used in future to raise an rm-GSTalpha antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 microg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTalpha mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTalpha gene as well as its potential use in relation to environmental pollution.

The incidence of aggressive behavior in patients with schizophrenia is higher than in the general population. Among particular gene polymorphisms posited to be involved in psychiatric disorders, the catecholamine-O-methyltransferase (COMT) and serotonin transporter (5-HTTPR) genes have been the focus of recent research on aggression. In this study, we hypothesized that both the COMT and the 5-HTTPR genotypes may be dependent on and related to aggression in Korean patients with schizophrenia. The subjects were 168 unrelated male schizophrenic patients diagnosed according to DSM-IV. Among two psychiatric hospital staff and medical university students, 158 unrelated male subjects with no lifetime history of psychiatric disorders were recruited to establish the COMT and 5-HTTPR genotype distribution in the general population. All episodes of aggression from the last discharge to readmission were rated. The Total Overt Aggression Scale (OAS) score (sum of the scores of all episodes of aggression), highest OAS score (highest individual episode score, 0-16), OAS category, and OAS category score (mean score within each category) were recorded. There were statistically significant effects of COMT genotype on the mean OAS 4 (physical aggression against other people) score and the highest OAS score. The most predictive was the OAS 4 score. There was a statistically significant effect of 5-HTTPR genotype on mean total score. Thus, the COMT gene is associated with the severity of aggression and with physical aggression against other people, whereas the 5-HTTPR gene is associated with the summary score of all episodes of aggression.

Prostaglandin E2 (PGE2), an abundant eicosanoid in bone, has been implicated in a number of pathological states associated with bone loss and is also known to stimulate matrix metalloproteinase-1 synthesis and secretion in rat and human osteoblast cells, although the intracellular reactions responsible for this remain unclear. Interleukin-1beta (IL-1beta) is a cytokine that plays a critical role in bone remodeling and appears to act as a downstream effector of most bone-resorbing agents. However, the issue of whether PGE2 regulates the expression of IL-1beta in mouse osteoblasts has not been resolved. In this work, we demonstrate that PGE2 is a potent inducer of IL-1beta production by fetal osteoblasts and show that PGE2 stimulates the activity of the IL-1beta promoter in osteoblasts, suggesting that PGE2 controls IL-1beta gene expression at least at the transcriptional level. PGE2 was found to induce IL-1beta mRNA expression in the cells within 4 h and the level of expression was maintained for 36 h. A dose-related increase in IL-1beta production was found with 0.1-2.0 microM PGE2. The induction of IL-1beta protein in the medium paralleled the induction of IL-1beta mRNA levels. The role of cAMP activation in PGE2-mediated IL-1beta production was examined by the effects of forskolin, an adenylyl cyclase (AC) activator and dideoxyadenosine (DDA), an AC inhibitor. Forskolin enhanced and DDA blocked the production of IL-1beta by PGE2. In addition, PGE2-mediated IL-1beta induction was completely inhibited by the cAMP antagonist, Rp-cAMP, and protein kinase A (PKA) inhibitors of KT5720 and H89. The PGE2-induced production of IL-1beta was also blocked by the PKA inhibitor PKI14-22. However, a specific inhibitor of protein kinase C, calphostin C, had no affect on PGE2-induced IL-1beta gene expression. Among the potential agonists, forskolin was a potent inducer of IL-1beta expression, while phorbol myristate acetate and serum had little effect. These findings indicate that PGE2 involves the cAMP-PKA signaling pathway in regulating IL-1beta gene expression in osteoblasts., (Copyright 2004 Elsevier B.V.)

The effects of THI 52 (1-naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) on (a) inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) expression in RAW 264.7 cells stimulated by lipopolysaccharide (LPS)/interferon gamma (IFN-gamma), (b) plasma nitrate concentration as well as iNOS protein expression (lung) in vivo in LPS-treated rats, and (c) the restoration of vascular contractility to vasoconstrictor agents in LPS-treated vessels in vitro were investigated. THI 52 concentration-dependently reduced not only nitric oxide (NO) production (IC(50) value, 12.5 microM) but also the expression of TNF-alpha and iNOS mRNA in RAW 264.7 cells. Incubation of rat endothelium-denuded thoracic aorta with LPS (300 ng/mL) in vitro for 8 hr resulted in the suppression of vasoconstrictor effects to phenylephrine (PE), effects that were restored by co-incubation with THI 52. Administration of THI 52 (10 and 20mg/kg, i.p.) 30 min before injection of LPS (10mg/kg, i.p.) resulted in a significant reduction of the expression of iNOS protein in rat lung tissue and in the plasma nitrite/nitrate (NOx) level. Addition of THI 52-treated macrophage-conditioned medium to a TNF-sensitive L929 fibroblast cell line (CCL1) increased cell viability, depending on the concentration of THI 52. Finally, THI 52 inhibited the activation of nuclear factor kappaB (NF-kappaB) by inhibition of IkappaB degradation through the prevention of IkappaB phosphorylation. Collectively, these results strongly suggest that THI 52 suppresses both TNF-alpha and iNOS gene expression by inhibiting NF-kappaB. Thus, THI 52, a new synthetic isoquinoline alkaloid, may be beneficial in inflammatory disorders where the overproduction of NO and TNF-alpha is a matter of concern.

Background: Androgenetic alopecia (AGA) is a common and chronic problem characterized by hair follicle miniaturization. Aims: In this study, heat‐treated Limosilactobacillus fermentum LM1020 (HT‐LM1020) was investigated in human follicle dermal papilla cell (HFDPC), scalp tissue, and clinical trials for patients with AGA. Patients/Methods: Cell proliferation and the expression of cyclins and cyclin‐dependent kinases (CDKs) were measured in HFDPC. The relative gene expression of 5α‐reductase and growth factors were investigated in hair scalp. This double‐blind, randomized, placebo‐controlled clinical trial was conducted over 24 weeks. Primary efficacy was evaluated by measuring hair density, and secondary efficacy was assessed by experts and self‐assessment. Changes in the microbiota of the hair scalps were analyzed using 16S metagenome amplicon sequencing. Results: HT‐LM1020 promoted cell growth (p < 0.001) and cyclin B1 expression, and it reduced 5α‐reductase and induced fibroblast growth factor 7 (FGF7), FGF10, and epithelial growth factor7 (EGF7) (p < 0.001). In the clinical trial, the experimental group demonstrated an increase in hair density from 133.70 to 148.87 n/cm2 at Week 24 (p < 0.001), while also expressing satisfaction with their hair density, reduced hair loss, and hairline. At Week 24, the total ratio of lactic acid bacteria operational taxonomic unit (OTU) in the scalp increased from 6.65% to 26.19%. At the same period, placebo‐controlled group decreased Staphylococcus caprae OTU from 77.95% to 14.57% while experimental group decreased from 65.80% to 41.02%. Conclusions: These present results showed that HT‐LM1020 was a co‐effector of ingredients for anti‐hair loss contributing to cell proliferation and the expression of CDKs. [ABSTRACT FROM AUTHOR]

Simple Summary: This study explores SMARCD3's role in gastric cancer, focusing on its elevated expression in signet ring cell (SRC) versus well-differentiated (WD) groups. Elevated SMARCD3 levels in SRC correlated with poorer survival outcomes (HR 2.16, p < 0.001), as shown by Kaplan–Meier analysis. Functional assays involving SMARCD3 knock-in and knock-out highlighted that its depletion reduces cell proliferation, migration, invasion, and EMT marker expression, while overexpression increases cell irregularity and area (p < 0.001). Further investigations into signaling pathways revealed that SMARCD3 overexpression boosts p-AKT-S473 and p-ERK levels in MKN-74 cells and β-catenin and PI3Kp85 activities in KATO III cells. Conversely, its knock-out decreases these activities in SNU 601 cells. These results suggest that SMARCD3′s overexpression could serve as a negative prognostic marker and a potential target for gastric cancer therapy. This study investigates the role of SMARCD3 in gastric cancer by comparing its expression in signet ring cell (SRC) and well-differentiated (WD) groups within gastric cancer cell lines and tissues. We observed elevated SMARCD3 levels in the SRC group compared to the WD group. Functional analysis was conducted through both SMARCD3 knock-in and knock-out methods. Kaplan–Meier survival analysis indicated that higher SMARCD3 expression correlates with poorer overall survival in gastric cancer patients (HR 2.16, p < 0.001). SMARCD3 knock-out cells showed decreased proliferation, migration, invasion, and expression of epithelial–mesenchymal transition (EMT) markers, contrasting with results from temporary and stable SMARCD3 overexpression experiments, which demonstrated increased cell area and irregularity (p < 0.001). Further analysis revealed that SMARCD3 overexpression in MKN-74 cells significantly enhanced p-AKT-S473 and p-ERK levels (p < 0.05), and in KATO III cells, it increased β-catenin and PI3Kp85 activities (p < 0.05). Conversely, these activities decreased in SNU 601 cells following SMARCD3 depletion. The study concludes that SMARCD3 overexpression may serve as a negative prognostic marker and a potential therapeutic target in gastric cancer treatment due to its role in promoting EMT. [ABSTRACT FROM AUTHOR]

Energy deprivation triggers various physiological, biochemical and molecular changes in plants under abiotic stress. We investigated the oxidative damages in the high altitude grown conifer Korean fir (Abies koreana) exposed to waterlogging stress. Our experimental results showed that waterlogging stress led to leaf chlorosis, 35 days after treatment. A significant decrease in leaf fresh weight, chlorophyll and sugar content supported this phenotypic change. Biochemical analysis showed a significant increase in leaf proline, lipid peroxidase and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical content of waterlogged plants. To elucidate the molecular mechanisms, we conducted RNA-sequencing (RNA-seq) and de novo assembly. Using RNA-seq analysis approach and filtering (P  < 0.05 and false discovery rate <0.001), we obtained 134 unigenes upregulated and 574 unigenes downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis placed the obtained differentially expressed unigenes in α-linoleic pathway, fatty acid degradation, glycosis, glycolipid metabolism and oligosaccharide biosynthesis process. Mapping of unigenes with Arabidopsis using basic local alignment search tool for nucleotides showed several critical genes in photosynthesis and carbon metabolism downregulated. Following this, we found the repression of multiple nitrogen (N) assimilation and nucleotide biosynthesis genes including purine metabolism. In addition, waterlogging stress reduced the levels of polyunsaturated fatty acids with a concomitant increase only in myristic acid. Together, our results indicate that the prolonged snowmelt may cause inability of A. koreana seedlings to lead the photosynthesis normally due to the lack of root intercellular oxygen and emphasizes a detrimental effect on the N metabolic pathway, compromising this endangered tree's ability to be fully functional under waterlogging stress. [ABSTRACT FROM AUTHOR]

Chlorpyrifos (CPF), an organophosphorus pesticide (OP), is one of the most widely used pesticides in the world. Subchronic exposures to CPF that do not cause cholinergic crisis are associated with problems in cognitive function (i.e., learning and memory deficits), but the biological mechanism(s) underlying this association remain speculative. To identify potential mechanisms of subchronic CPF neurotoxicity, adult male Long Evans (LE) rats were administered CPF at 3 or 10mg/kg/d (s.c.) for 21 days. We quantified mRNA and non-coding RNA (ncRNA) expression profiles by RNA-seq, microarray analysis and small ncRNA sequencing technology in the CA1 region of the hippocampus. Hippocampal slice immunohistochemistry was used to determine CPF-induced changes in protein expression and localization patterns. Neither dose of CPF caused overt clinical signs of cholinergic toxicity, although after 21 days of exposure, cholinesterase activity was decreased to 58% or 13% of control levels in the hippocampus of rats in the 3 or 10mg/kg/d groups, respectively. Differential gene expression in the CA1 region of the hippocampus was observed only in the 10mg/kg/d dose group relative to controls. Of the 1382 differentially expressed genes identified by RNA-seq and microarray analysis, 67 were common to both approaches. Differential expression of six of these genes (Bdnf, Cort, Crhbp, Nptx2, Npy and Pnoc) was verified in an independent CPF exposure study; immunohistochemistry demonstrated that CRHBP and NPY were elevated in the CA1 region of the hippocampus at 10mg/kg/d CPF. Gene ontology enrichment analysis suggested association of these genes with receptor-mediated cell survival signaling pathways. miR132/212 was also elevated in the CA1 hippocampal region, which may play a role in the disruption of neurotrophin-mediated cognitive processes after CPF administration. These findings identify potential mediators of CPF-induced neurobehavioral deficits following subchronic exposure to CPF at a level that inhibits hippocampal cholinesterase to less than 20% of control. An equally significant finding is that subchronic exposure to CPF at a level that produces more moderate inhibition of hippocampal cholinesterase (approximately 50% of control) does not produce a discernable change in gene expression.

PURPOSE: High-grade neuroendocrine carcinoma (HGNEC) of the lung is an aggressive cancer with a complex biology. We aimed to explore the prognostic value of genetic aberrations and poly(ADP-ribose) polymerase-1 (PARP1) expression in HGNEC and to establish a novel prognostic model. MATERIALS AND METHODS: We retrospectively enrolled 191 patients with histologically confirmed HGNEC of the lung. Tumor tissues were analyzed using PARP1 immunohistochemistry (IHC; N = 191) and comprehensive cancer panel sequencing (n = 102). Clinical and genetic data were used to develop an integrated Cox hazards model. RESULTS: Strong PARP1 IHC expression (intensity 3) was observed in 153 of 191 (80.1%) patients, and the mean PARP1 H-score was 285 (range, 5-300). To develop an integrated Cox hazard model, our data set included information from 357 gene mutations and 19 clinical profiles. When the targeted mutation profiles were combined with clinical profiles, 12 genes (ATRX , CCND2 , EXT2 , FGFR2 , FOXO1 , IL21R , MAF , TGM7 , TNFAIP3 , TP53 , TSHR , and DDR2) were identified as prognostic factors for survival. The integrated Cox hazard model, which combines mutation profiles with a baseline model, outperformed the baseline model (incremental area under the curve 0.84 v 0.78; P = 8.79e-12). The integrated model stratified patients into high- and low-risk groups with significantly different disease-free and overall survival (integrated model: hazard ratio, 7.14 [95% CI, 4.07 to 12.54]; P <.01; baseline model: 4.38 [2.56 to 7.51]; P <.01). CONCLUSION: We introduced a new prognostic model for HGNEC that combines genetic and clinical data. The integrated Cox hazard model outperformed the baseline model in predicting the survival of patients with HGNEC. [ABSTRACT FROM AUTHOR]

Background: Familial chylomicronemia syndrome (FCS) is a rare monogenic form of severe hypertriglyceridemia, caused by mutations in genes involved in triglyceride metabolism. Herein, we report the case of a Korean family with familial chylomicronemia syndrome caused by compound heterozygous deletions of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1). Case presentation: A 4-year-old boy was referred for the evaluation of severe hypertriglyceridemia (3734 mg/dL) that was incidentally detected 4 months prior. His elder brother also demonstrated an elevated triglyceride level of 2133 mg/dL at the age of 9. Lipoprotein electrophoresis revealed the presence of chylomicrons, an increase in the proportion of pre-beta lipoproteins, and low serum lipoprotein lipase levels. The patient's parents and first elder brother had stable lipid profiles. For suspected FCS, genetic testing was performed using the next-generation sequencing-based analysis of 31 lipid metabolism-associated genes, which revealed no pathogenic variants. However, copy number variant screening using sequencing depth information suggested large heterozygous deletion encompassing all the coding exons of GPIHBP1. A real-time quantitative polymerase chain reaction was performed to validate the deletion site. The results showed that the siblings had two heterozygous copy number variants consisting of the whole gene and an exon 4 deletion, each inherited from their parents. During the follow-up period of 17 months, the patient did not develop pancreatitis, following dietary intervention. Conclusion: These siblings' case of familial chylomicronemia syndrome caused by rare GPIHBP1 deletions highlight the implementation of copy number variants—beyond next-generation sequencing—as an important consideration in diagnosis. Accurate genetic diagnosis is necessary to establish the etiology of severe hypertriglyceridemia, which increases the risk of pancreatitis. [ABSTRACT FROM AUTHOR]

BackgroundAtopic dermatitis (AD) is a major inflammatory condition of the skin caused by inherited skin barrier deficiency, with mutations in the filaggrin gene predisposing to development of AD. Support for barrier deficiency initiating AD came from flaky tail mice, which have a frameshift mutation in Flg and also carry an unknown gene, matted, causing a matted hair phenotype.ObjectiveWe sought to identify the matted mutant gene in mice and further define whether mutations in the human gene were associated with AD.MethodsA mouse genetics approach was used to separate the matted and Flg mutations to produce congenic single-mutant strains for genetic and immunologic analysis. Next-generation sequencing was used to identify the matted gene. Five independently recruited AD case collections were analyzed to define associations between single nucleotide polymorphisms (SNPs) in the human gene and AD.ResultsThe matted phenotype in flaky tail mice is due to a mutation in the Tmem79/Matt gene, with no expression of the encoded protein mattrin in the skin of mutant mice. Matt(ft) mice spontaneously have dermatitis and atopy caused by a defective skin barrier, with mutant mice having systemic sensitization after cutaneous challenge with house dust mite allergens. Meta-analysis of 4,245 AD cases and 10,558 population-matched control subjects showed that a missense SNP, rs6684514, [corrected] in the human MATT gene has a small but significant association with AD.ConclusionIn mice mutations in Matt cause a defective skin barrier and spontaneous dermatitis and atopy. A common SNP in MATT has an association with AD in human subjects.

Summary: Numerous staple crops exhibit polyploidy and are difficult to genetically modify. However, recent advances in genome sequencing and editing have enabled polyploid genome engineering. The hexaploid black nightshade species Solanum nigrum has immense potential as a beneficial food supplement. We assembled its genome at the scaffold level. After functional annotations, we identified homoeologous gene sets, with similar sequence and expression profiles, based on comparative analyses of orthologous genes with close diploid relatives Solanum americanum and S. lycopersicum. Using CRISPR‐Cas9‐mediated mutagenesis, we generated various mutation combinations in homoeologous genes. Multiple mutants showed quantitative phenotypic changes based on the genotype, resulting in a broad‐spectrum effect on the quantitative traits of hexaploid S. nigrum. Furthermore, we successfully improved the fruit productivity of Boranong, an orphan cultivar of S. nigrum suggesting that engineering homoeologous genes could be useful for agricultural improvement of polyploid crops. [ABSTRACT FROM AUTHOR]

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway plays a central role in the development of various organisms. However, dysregulation of microRNAs (miRNAs), which are crucial regulators of gene expression, can disrupt signaling pathways at all stages of development. Although Notch signaling is involved in wing development in Drosophila, the mechanism underlying miRNA‐based regulation of the Notch signaling pathway is unclear. Here, we report that loss of Drosophila miR‐252 increases the size of adult wings, whereas the overexpression of miR‐252 in specific compartments of larval wing discs leads to patterning defects in the adult wings. The miR‐252 overexpression‐induced wing phenotypes were caused by aberrant Notch signaling with intracellular accumulation of the full‐length Notch receptor during development, which could be due to defects in intracellular Notch trafficking associated with its recycling to the plasma membrane and autophagy‐mediated degradation. Moreover, we identified Rab6 as a direct target of miR‐252‐5p; Rab6 encodes a small Ras‐like GTPase that regulates endosomal trafficking pathways. Consistent with this finding, RNAi‐mediated downregulation of Rab6 led to similar defects in both wing patterning and Notch signaling. Notably, co‐overexpression of Rab6 completely rescued the wing phenotype associated with miR‐252 overexpression, further supporting that Rab6 is a biologically relevant target of miR‐252‐5p in the context of wing development. Thus, our data indicate that the miR‐252‐5p‐Rab6 regulatory axis is involved in Drosophila wing development by controlling the Notch signaling pathway. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the correlation between melatonin and the reproductive mechanism of farmed female red‐spotted groupers, Epinephelus akaara. Through the reproductive cycle experiment, the sexual maturity period was determined to be from June to July, following which the spawning season occurred in August. In addition, kiss1, kiss2, and GPR54 mRNA expression levels peaked in January, whereas that of sbGnRH mRNA remained at high levels from January to May and decreased during the sexual maturity and spawning seasons. During the day–night cycle, a nocturnal rhythm of AANAT2 mRNA expression was observed in the brain, and AANAT2 mRNA expression showed a seasonal pattern with increased expression during summer compared with that in winter. A correlation between melatonin and reproduction‐related genes could not be observed via melatonin administration, and sexual maturity was observed to be more induced under long photoperiod conditions compared with short photoperiod conditions. These results indicate that the reproductive mechanism of the farmed female red‐spotted grouper is related to changes in photoperiod and melatonin, and further research is needed to clarify the reproductive mechanism. Our findings provide basic information for identifying the reproductive mechanisms of farmed E. akaara. [ABSTRACT FROM AUTHOR]

Tagatose is a rarely occurring sugar found in food and sweet fruits. It is a potential sweetener with low calories but a sweetness similar to that of sucrose. In this study, we developed a whole-cell biocatalyst using Corynebacterium glutamicum for direct bioconversion of fructose to tagatose. First, we constructed a biological conversion pathway for the conversion of fructose to tagatose by expressing tagatose 4-epimerase (TN) from Thermotoga neapolitana in C. glutamicum. Next, to increase the expression level of the enzyme, we engineered copy number of plasmid. Plasmid library was constructed by randomizing the cgrI antisense RNA region in the plasmid, and the plasmid with high-copy number was isolated using fluorescence-activated single cell sorting (FACS)-based high-throughput screening. The isolated plasmid, pHCP7, had 2-fold higher copy numbers than the original plasmid. A higher expression level and conversion yield could be achieved using pHCP7. Finally, we examined a novel tagatose 4-epimerase TN(KNF4E) isolated from Kosmotoga olearia. The gene expression level was further increased (33.9% of total fraction) through codon optimization and expression in pHCP7, and a conversion yield as high as 21.7% was achieved. [ABSTRACT FROM AUTHOR]

Background: The pathogenesis of diabetic kidney disease (DKD) is complex, involving metabolic and hemodynamic factors. Although DKD has been established as a heritable disorder and several genetic studies have been conducted, the identification of unique genetic variants for DKD is limited by its multiplex classification based on the phenotypes of diabetes mellitus (DM) and chronic kidney disease (CKD). Thus, we aimed to identify the genetic variants related to DKD that differentiate it from type 2 DM and CKD. Methods: We conducted a large-scale genome-wide association study mega-analysis, combining Korean multi-cohorts using multinomial logistic regression. A total of 33,879 patients were classified into four groups—normal, DM without CKD, CKD without DM, and DKD—and were further analyzed to identify novel single-nucleotide polymorphisms (SNPs) associated with DKD. Additionally, fine-mapping analysis was conducted to investigate whether the variants of interest contribute to a trait. Conditional analyses adjusting for the effect of type 1 DM (T1D)-associated HLA variants were also performed to remove confounding factors of genetic association with T1D. Moreover, analysis of expression quantitative trait loci (eQTL) was performed using the Genotype-Tissue Expression project. Differentially expressed genes (DEGs) were analyzed using the Gene Expression Omnibus database (GSE30529). The significant eQTL DEGs were used to explore the predicted interaction networks using search tools for the retrieval of interacting genes and proteins. Results: We identified three novel SNPs [rs3128852 (P = 8.21×10−25), rs117744700 (P = 8.28×10−10), and rs28366355 (P = 2.04×10−8)] associated with DKD. Moreover, the fine-mapping study validated the causal relationship between rs3128852 and DKD. rs3128852 is an eQTL for TRIM27 in whole blood tissues and HLA-A in adipose-subcutaneous tissues. rs28366355 is an eQTL for HLA-group genes present in most tissues. Conclusions: We successfully identified SNPs (rs3128852, rs117744700, and rs28366355) associated with DKD and verified the causal association between rs3128852 and DKD. According to the in silico analysis, TRIM27 and HLA-A can define DKD pathophysiology and are associated with immune response and autophagy. However, further research is necessary to understand the mechanism of immunity and autophagy in the pathophysiology of DKD and to prevent and treat DKD. [ABSTRACT FROM AUTHOR]

Background: Curcuma longa has been used as spices, food preservative, coloring material, and traditional medicine. This plant also has long been used for a variety of diseases including dyslipidemia, stomach disorders, arthritis, and hepatic diseases. The aim of the present investigation was to examine the anti-neuroinflammatory effects of the 50% ethanolic extract of C. longa in lipopolysaccharide (LPS)-induced BV2 microglial cells. Methods: Griess reaction was employed to measure the production of nitric oxide (NO), and the levels of prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin 1-beta (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were determined by using profit ELISA kits. Western blotting was used to determine the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), mitogen activated protein kinases (MAPKs), heme oxygenase-1 (HO-1) and nuclear factor erythroid-2-related factor 2 (Nrf2). Results: Pre-treatment with CLE inhibited the overproduction and overexpression of pro-inflammatory mediators including NO, PGE2, iNOS, COX-2, and pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α in LPS-induced BV2 cells. In addition, CLE suppressed the activation of the NF-κB and three MAPK signaling pathways. Treatment with CLE induced HO-1 protein expression by activating Nrf2 pathway, and inhibiting the HO-1 expression reversed the anti-inflammatory effect of CLE. Conclusion: CLE showed anti-neuroinflammatory effects against LPS-induced microglial cells activation through the inhibition of production and expression of pro-inflammatory mediators by negative regulation of the NF-κB and MAPK signaling pathways. These anti-neuroinflammatory effects of CLE were mediated by HO-1/Nrf2 signaling pathway. Taken together, the present study suggests a potent effect of CLE to prevent neuroinflammatory diseases. It is necessary to perform additional efficacy evaluation through in vivo experiments. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a self-regulating gaseous molecule as well as an interacting partner with different reactive oxygen species (ROS). Endogenous NO increased, but hydrogen peroxide (H2O2) and superoxide radical anion ( O 2 ⋅ − ) decreased in Chinese cabbage leaves treated with sodium nitroprusside (SNP) (0.5 mM). Antioxidant enzyme genes BrCat1, BrCat2, BrAPX, BrGR1, and BrGST1 expressions were transiently induced by the SNP (0.5 mM). Pretreated SNP (0.5 mM) enhanced tolerance to subsequent challenges by phytotoxic SNP (5 mM) and methyl viologen (MV) (50 μM), but accelerated cellular damage by H2O2 (4 M) shown by electrolyte leakage and lipid peroxidation. The SNP (0.5 mM) differentially modulated endogenous NO and ROS levels as well as antioxidant enzyme gene expressions by excess SNP, MV, and H2O2 stresses. These results suggest that NO as a signal molecule differentially interacts with challenging stresses in Chinese cabbage leaves, followed by differential cellular damage and antioxidant responses. [ABSTRACT FROM AUTHOR]

Simple Summary: Desmoid tumors are rare and aggressive tumors that currently do not receive satisfactory systemic treatment. Thus, an in-depth study into desmoid tumors was needed. In this study, we performed the comprehensive molecular profiling for desmoid tumor samples from patients and found the druggable biomarker. Notably, the TGF-β signaling pathway was consistently identified as enriched in desmoid tumors using comprehensive molecular profiling. It suggested that therapeutic interventions targeting TGF-β in fibroblasts using TGF-β receptor inhibitors may be clinically beneficial in patients with desmoid tumors. After that, we validated it by using a desmoid patient-derived primary cell model that displayed high concordance with desmoid primary tissues. Finally, we proved that that the inhibition of the TGF-β pathway is useful as a potential treatment for patients with desmoid tumors. (1) Background: Desmoid tumors have a relatively high local failure rate after primary treatment using surgery and/or radiotherapy. Moreover, desmoid tumors recur at the primary site for many patients. An effective therapeutic strategy for the desmoid tumor is needed to maintain quality of life and prolong survival. (2) Method: First of all, we collected desmoid tumor tissues and investigated the status of protein expression for beta-catenin and alpha-SMA through immunohistochemistry. Then, we performed targeted sequencing and whole RNA sequencing. To compare the data with other cancer types, we used NGS data from sarcoma patients at Yonsei Cancer Center (YCC-sarcoma cohort, n = 48) and The Cancer Genome Atlas (TCGA, n = 9235). Secondly, we established the novel patient-derived preclinical models (n = 2) for the validation of treatment strategy. The same gene alteration of primary tissue was demonstrated. (3) Results: We discovered specific gene sets related to the TGF-β signaling pathway. Moreover, we selected the combination treatment comprising TGF-β inhibitor, vactosertib, and imatinib. In screening for the anti-proliferation effect, the combination treatment of TGF-β inhibitor was more effective for tumor suppression than monotherapy. (4) Conclusion: We found preclinical indications that TGF-β inhibitors could prove useful as a potential treatment for patients with desmoid tumors. Moreover, we could find some examples in clinical trials. [ABSTRACT FROM AUTHOR]

Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species. [ABSTRACT FROM AUTHOR]