A Retro-Aldol Reaction Prompted the Evolvability of a Phosphotransferase System for the Utilization of a Rare Sugar.

The evolution of the bacterial phosphotransferase system (PTS) linked to glycolysis is dependent on the availability of naturally occurring sugars. Although bacteria exhibit sugar specificities based on carbon catabolite repression, the acquisition and evolvability of the cellular sugar preference under conditions that are suboptimal for growth (e.g., environments rich in a rare sugar) are poorly understood. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. We detected a minimal set of adaptive mutations in the d-fructose-specific PTS to render E. coli capable of d-tagatose utilization. These E. coli mutant strains lost the tight regulation of both the d-fructose and N-acetyl-galactosamine PTS following deletions in the binding site of the catabolite repressor/activator protein (Cra) upstream from the fruBKA operon and in the agaR gene, encoding the N-acetylgalactosamine (GalNAc) repressor, respectively. Acquired d-tagatose catabolic pathways then underwent fine-tuned adaptation via an additional mutation in 1-phosphofructose kinase to adjust metabolic fluxes. We determined the evolutionary trajectory at the molecular level, providing insights into the mechanism by which enteric bacteria evolved a substrate preference for the rare sugar d-tagatose. Furthermore, the engineered E. coli mutant strain could serve as an in vivo high-throughput screening platform for engineering non-phosphosugar isomerases to produce rare sugars. IMPORTANCE Microorganisms generate energy through glycolysis, which might have preceded a rapid burst of evolution, including the evolution of cellular respiration in the primordial biosphere. However, little is known about the evolvability of cellular sugar preferences. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. Consequently, we identified mutational hot spots and determined the evolutionary trajectory at the molecular level. This provided insights into the mechanism by which enteric bacteria evolved substrate preferences for various sugars, accounting for the widespread occurrence of these taxa. Furthermore, the adaptive laboratory evolution-induced cellular chassis could serve as an in vivo high-throughput screening platform for engineering tailor-made non-phosphorylated sugar isomerases to produce low-calorigenic rare sugars showing antidiabetic, antihyperglycemic, and antitumor activities.

Comparison of out-of-pocket expenditure and catastrophic health expenditure for severe disease by the health security system: based on end-stage renal disease in South Korea.

BACKGROUND: Korea's health security system named the National Health Insurance and Medical Aid has revolutionized the nation's mandatory health insurance and continues to reduce excessive copayments. However, few studies have examined healthcare utilization and expenditure by the health security system for severe diseases. This study looked at reverse discrimination regarding end-stage renal disease by the National Health Insurance and Medical Aid. METHODS: A total of 305 subjects were diagnosed with end-stage renal disease in the Korea Health Panel from 2008 to 2013. Chi-square, t-test, and ANCOVA were conducted to identify the healthcare utilization rate, out-of-pocket expenditure, and the prevalence of catastrophic expenditure. Mixed effect panel analysis was used to evaluate total out-of-pocket expenditure by the National Health Insurance and Medical Aid over a 6-year period. RESULTS: There were no significant differences in the healthcare utilization rate for emergency room visits, admissions, or outpatient department visits between the National Health Insurance and Medical Aid because these healthcare services were essential for individuals with serious diseases, such as end-stage renal disease. Meanwhile, each out-of-pocket expenditure for an admission and the outpatient department by the National Health Insurance was 2.6 and 3.1 times higher than that of Medical Aid (P < 0.05). The total out-of-pocket expenditure, including that for emergency room visits, admission, outpatient department visits, and prescribed drugs, was 2.9 times higher for the National Health Insurance than Medical Aid (P < 0.001). Over a 6-year period, in terms of total of out-of-pocket expenditure, subjects with the National Health Insurance spent more than those with Medical Aid (P < 0.01). If the total household income decile was less than the median and subjects were covered by the National Health Insurance, the catastrophic health expenditure rate was 92.2%, but it was only 58.8% for Medical Aid (P < 0.001). CONCLUSION: Individuals with serious diseases, such as end-stage renal disease, can be faced with reverse discrimination depending on the type of insurance that is provided by the health security system. It is necessary to consider individuals who have National Health Insurance but are still poor.

Association between domperidone use and adverse cardiovascular events: A nested case-control and case-time-control study.

PURPOSE: To assess the association between domperidone and adverse cardiovascular events. METHODS: We conducted nested case-control and case-time-control studies using Korea's healthcare database (2002-2015). We identified patients without history of hospitalization, cancer, or cardiovascular diseases in 2002. From our cohort, those diagnosed with an adverse cardiovascular event (case), composite of arrhythmia, hypertension, or acute myocardial infarction were matched to two controls using risk-set sampling on various sociodemographic variables. Exposure was assessed in the 1 to 7 days, or in the 1 to 7 days (hazard period) and 91 to 97 days (control period) prior to index date, in nested case-control and case-time control studies, respectively. We compared domperidone to metoclopramide or non-use and estimated odds ratios (OR) with 95% confidence intervals (CI) using conditional logistic regression. RESULTS: From 627 799 patients, we identified 71 555 cases and 141 833 controls. In the nested case-control study, while the risk of cardiovascular events was increased with domperidone (OR 1.38, 95% CI 1.28-1.47) compared to non-use, the risk was reduced (0.64, 0.57-0.72) compared to metoclopramide. In the case-time-control study, similar increased risk was found when compared to non-use (1.40, 1.29-1.52) but a reduced risk as compared with metoclopramide (0.63, 0.54-0.72). Risk of myocardial infarction associated with domperidone was highest (nested case-control: 1.94, 1.33-2.83; case-time-control: 1.91, 1.01-3.62) when compared to non-use but did not indicate an increased risk when compared to metoclopramide (nested case-control: 0.60, 0.32-1.13; case-time-control: 0.70, 0.25-1.98). CONCLUSION: Our findings support a positive association between domperidone and adverse cardiovascular events. However, domperidone may have a safer cardiovascular profile than metoclopramide.

Associations of Food-Chewing Discomfort with Health Behaviors and Cognitive and Physical Health Using Pooled Data from the Korean Health Panel (2010-2013).

Using 4 years of pooled data from the Korean Health Panel (2010-2013), the prevalence of food-chewing discomfort in adults over the age of 19 was investigated and the cross-sectional relationship between food-chewing discomfort and health behaviors and cognitive and physical health was identified. The prevalence of food-chewing discomfort was 31%: young adults (<40 years), 17.9%; middle-aged adults (40-64 years), 28.9%; and older adults (≥65 years), 57.1% (p < 0.0001). When food-chewing discomfort was sometimes, often, or always rather than never, odds ratios (ORs) were analyzed after controlling for sociodemographic characteristics. Significant OR results of target variables were smoking (OR 1.15, 1.37, 1.50), drinking (1.08, 0.87, 0.73), problem drinking (1.87, 1.67, 1.34), abstinence from drinking (1.23, 1.34, 1.42), nonphysical activity (OR 0.87 only significant, 0.94 nonsignificant, 1.10 nonsignificant), memory decline (2.07, 2.56, 3.31), decision-making difficulty (1.76, 2.78, 4.37), limitation of daily life due to illness (2.29, 3.60, 3.92), and the presence of a chronic disease (1.28, 1.62, 1.73), respectively. In conclusion, there were associations of food-chewing discomfort with increased smoking and decreased alcohol consumption, with increased difficulty in decision-making and memory decline, limitations in daily life due to disease, and the presence of chronic diseases. Therefore, it is necessary to investigate the causal relationship between chewing and health behaviors and cognitive and physical health through longitudinal studies.

Mitochondrial Mutations in Cholestatic Liver Disease with Biliary Atresia.

Biliary atresia (BA) results in severe bile blockage and is caused by the absence of extrahepatic ducts. Even after successful hepatic portoenterostomy, a considerable number of patients are likely to show progressive deterioration in liver function. Recent studies show that mutations in protein-coding mitochondrial DNA (mtDNA) genes and/or mitochondrial genes in nuclear DNA (nDNA) are associated with hepatocellular dysfunction. This observation led us to investigate whether hepatic dysfunctions in BA is genetically associated with mtDNA mutations. We sequenced the mtDNA protein-coding genes in 14 liver specimens from 14 patients with BA and 5 liver specimens from 5 patients with choledochal cyst using next-generation sequencing. We found 34 common non-synonymous variations in mtDNA protein-coding genes in all patients examined. A systematic 3D structural analysis revealed the presence of several single nucleotide polymorphism-like mutations in critical regions of complexes I to V, that are involved in subunit assembly, proton-pumping activity, and/or supercomplex formation. The parameters of chronic hepatic injury and liver dysfunction in BA patients were also significantly correlated with the extent of hepatic failure, suggesting that the mtDNA mutations may aggravate hepatopathy. Therefore, mitochondrial mutations may underlie the pathological mechanisms associated with BA.

Factors associated with intention to be tested for HIV among men who have sex with men in a country with a very low HIV prevalence.

This study examined factors associated with the intention to take an HIV test among men who have sex with men (MSM) in South Korea. An internet website-based survey was conducted among users of the only and largest online MSM website between 20 July 2016, and 20 August 2016. A total of 2915 participants completed the survey and answered questions related to sociodemographic information, health behaviors, sexual behaviors, and HIV testing history. Of these, 2587 (88.7%) participants responded as having an intention to take an HIV test. A multivariable logistic regression analysis revealed the following as having reduced the intention to undergo HIV testing: very good subjective health status and no sexual interactions during the last 6 months (Adjusted odds ratios [AOR] 0.45 and 0.54, respectively). In contrast, increased intention to take an HIV test was associated with being 20-29 years old, 30-39 years old, not paying or receiving money for sex, having a history of HIV testing, and taking an HIV test once per 12 months (AOR 2.64, 2.13, 1.54, 1.81, and 2.17, respectively). In conclusion, HIV testing among MSM in this study was associated with age, subjective health status, sex(es) of one's sexual partner(s) during the last 6 months, sexual risk behaviors, HIV testing history, and undergoing regular HIV testing.

Unusual bilateral vulvar liposarcoma.

Liposarcoma of vulva is a rare malignant tumor. Only fifteen cases of vulvar liposarcoma have been reported in English literature. Because of extreme rarity of vulvar liposarcoma, it is initially misdiagnosed as benign tumor such as lipoma. This paper aims to present a unique case of liposarcoma of bilateral vulvae with a review of the literature of previously reported cases.

ATP induced microglial cell migration through non-transcriptional activation of matrix metalloproteinase-9.

In response to brain insults, microglia, the resident inflammatory cells in CNS, migrate into injured sites to initiate inflammatory responses in brain. ATP, released from apoptotic or necrotic cells induce chemoattractive responses but the mechanism is not clear yet. In this study, we investigated whether ATP modulates microglial migration by regulating the activity of matrix metalloproteinases (MMPs). ATP induced rapid microglial migration and increased the activity of MMP-9 in the culture supernatants (secreted compartments) in a concentration-dependent manner. The increased activity of secreted MMP-9 is due to the increased protein secretion, but not by the increased MMP-9 mRNA and protein expression. Inhibition of MMP-9 activity by treatment with specific inhibitors including GM6001 and SB-3CT prevented ATP-induced microglial migration. ATP-induced microglial migration was also inhibited by P2Y receptor antagonists including clopidogrel as well as PI3K inhibitor such as wortmanin. Taken together, ATP non-transcriptionally increased MMP-9 activity by activation of P2Y and PI3K. The results from the present investigation may provide further insights into the regulation of the activity of MMP-9 during microglial migration, which may play essential role in the regulation of inflammatory responses in pathological situations such as neurodegenerative disorders.

Enhanced angiogenesis mediated by vascular endothelial growth factor plasmid-loaded thermo-responsive amphiphilic polymer in a rat myocardial infarction model.

Thermo-responsive hydrogel-mediated gene transfer may be preferred for the muscle, because the release of DNA into the surrounding tissue can be controlled by the 3-dimensional structure of the hydrogel. Such a system for the controlled release of a therapeutic gene may extend the duration of gene expression. Here, a thermo-responsive, biodegradable polymeric hydrogel was synthesized for local gene transfer in the heart. Initially, the luciferase gene was delivered into mouse heart. The intensity of gene expression assessed by optical imaging was closely correlated with the expressed protein concentration measured by luciferase assay in homogenized heart. Polymeric hydrogel-based gene transfer enhanced gene expression up to 4 fold, compared with naked plasmid, and displayed 2 bi-modal expression profiles with peaks at 2 days and around 25 days after local injection. Histological analyses showed that gene expression was initially highest in the myocardium, whereas lower and longer expression was seen mainly in fibrotic or inflammatory cells that infiltrated the injury site during injection. Next, a rat myocardial infarction model was made for 1 week, and human vascular endothelial growth factor (hVEGF) plasmid was injected into the infarct area with an amphiphilic thermo-responsive polymer. Enhanced and sustained hVEGF expression in the infarct region mediated by amphiphilic thermo-responsive polymer increased capillary density and larger vessel formation, thus enabling effective angiogenesis.

Transcriptional regulation of the mouse calbindin-D9k gene by the ovarian sex hormone.

Calbindin-D9k levels in the rat uterus are under the control of estrogen. We found that the putative estrogen response element (ERE) failed to bind to the estrogen receptor from the mouse uterus. We therefore isolated mouse genomic clones of the calbindin-D9K gene and analyzed their expression in the mouse uterus. The promoter region of the gene contained several putative steroid hormone receptor binding sites. To characterize these elements, we constructed several promoter-reporter plasmids, and transiently transfected them into T47D breast cancer cells that express both estrogen and progesterone receptors. Luciferase activity was expressed from a promoter region containing the putative progesterone response element (PRE) and expression was stimulated by progesterone. In the uterus of oophorectomized mice, the calbindin-D9k gene was up-regulated by progesterone, but not by estrogen. These results suggest that the mouse uterine calbindin-D9k gene is expressed under the control of a PRE.

Macrophage inhibitory cytokine-1 induces the invasiveness of gastric cancer cells by up-regulating the urokinase-type plasminogen activator system.

In our search for genes associated with gastric cancer progression, we identified macrophage inhibitory cytokine-1 (MIC-1), a member of the transforming growth factor beta superfamily, as an overexpressed gene in gastric tumor tissues. Expression analysis of MIC-1 in gastric tumor tissues revealed a specific expression in gastric cancer cells, and this expression level was well correlated with invasive potential in various human gastric cancer cell lines. Stable transfection of MIC-1 into SNU-216, a human gastric cancer cell line, significantly increased its invasiveness. The overexpression of MIC-1 into SNU-216 cells significantly increased the activity of urokinase-type plasminogen activator (uPA), and the expressions of uPA and urokinase-type plasminogen activator receptor (uPAR). Similarly, the stimulation of gastric cancer cell lines with purified recombinant MIC-1 dose-dependently increased cell invasiveness, uPA activity, and uPA and uPAR expression. However, MIC-1 did not significantly suppress the proliferation of gastric cancer cell lines. We also found that the stimulation of human gastric cell lines with recombinant MIC-1 strongly induced activation of mitogen-activated protein kinase kinase-1/2 and extracellular signal-regulated kinase-1/2. Additional analysis revealed that PD98059, a selective inhibitor of mitogen-activated protein kinase kinase-1/2, suppressed not only gastric cancer cell invasiveness and uPA activity, but also the mRNA expressions of uPA and uPAR, as induced by recombinant MIC-1. Our results indicate that MIC-1 may contribute to the malignant progression of gastric cancer cells by inducing tumor cell invasion through the up-regulation of the uPA activation system via extracellular signal-regulated kinase-1/2-dependent pathway.

Interleukin-12 p40 gene expression is induced in lipopolysaccharide-activated pituitary glands in vivo.

Proinflammatory cytokines have several functions including activation of the hypothalamo-pituitary-adrenal (HPA) axis and regulation of the immune system. The present study focuses on the regulation of interleukin 12 (IL-12) and its receptor gene expression in the HPA axis under artificially induced immune stress, brought on by administration of lipopolysaccharide (LPS) to Sprague-Dawley (SD) rats. RT-PCR analyses showed that expression of the IL-12 p40 gene was significantly increased and peaked at 2 h in the pituitary gland, but not in the hypothalamus. LPS-induced IL-12 p40 gene induction in the pituitary gland was suppressed after beta-adrenoceptor agonist pretreatment in vivo. Both IL-12 p40 gene induction and IL-12 production were also observed when freshly isolated pituitary glands from non-treated SD rats were incubated with LPS in vitro. Furthermore, CD14, which is known as a LPS receptor, was found to be expressed in the pituitary gland. Gel mobility shift assays using nuclear extracts prepared from the pituitary glands of rats administered LPS showed induction of NF-kappaB and AP-1 DNA-binding activity. These results suggest that LPS stimulates the pituitary gland directly in vivo to increase IL-12 p40 gene expression and IL-12 protein production.