Difluoromethylornithine (DFMO) and AMXT 1501 inhibit capsule biosynthesis in pneumococci.

Polyamines are small cationic molecules that have been linked to various cellular processes including replication, translation, stress response and recently, capsule regulation in Streptococcus pneumoniae (Spn, pneumococcus). Pneumococcal-associated diseases such as pneumonia, meningitis, and sepsis are some of the leading causes of death worldwide and capsule remains the principal virulence factor of this versatile pathogen. α-Difluoromethyl-ornithine (DFMO) is an irreversible inhibitor of the polyamine biosynthesis pathway catalyzed by ornithine decarboxylase and has a long history in modulating cell growth, polyamine levels, and disease outcomes in eukaryotic systems. Recent evidence shows that DFMO can also target arginine decarboxylation. Interestingly, DFMO-treated cells often escape polyamine depletion via increased polyamine uptake from extracellular sources. Here, we examined the potential capsule-crippling ability of DFMO and the possible synergistic effects of the polyamine transport inhibitor, AMXT 1501, on pneumococci. We characterized the changes in pneumococcal metabolites in response to DFMO and AMXT 1501, and also measured the impact of DFMO on amino acid decarboxylase activities. Our findings show that DFMO inhibited pneumococcal polyamine and capsule biosynthesis as well as decarboxylase activities, albeit, at a high concentration. AMXT 1501 at physiologically relevant concentration could inhibit both polyamine and capsule biosynthesis, however, in a serotype-dependent manner. In summary, this study demonstrates the utility of targeting polyamine biosynthesis and transport for pneumococcal capsule inhibition. Since targeting capsule biosynthesis is a promising way for the eradication of the diverse and pathogenic pneumococcal strains, future work will identify small molecules similar to DFMO/AMXT 1501, which act in a serotype-independent manner.

Efficacy and safety of allogenic canine adipose tissue-derived mesenchymal stem cell therapy for insulin-dependent diabetes mellitus in four dogs: A pilot study.

Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and can control the immune dysregulation that leads to ß-cell destruction. Stem-cell transplantation could thus manage insulin-dependent diabetes mellitus (IDDM) in dogs. In this pilot study, we aimed to assess canine adipose tissue-derived MSCs (cAT-MSCs) transplantation as a treatment for canine diabetes mellitus. This study included four dogs with over a year of insulin treatment for IDDM, following diagnosis at the Veterinary Medicine Teaching Hospital of Seoul National University. Allogenic cAT-MSCs were infused intravenously three or five times monthly to dogs with IDDM. Blood and urine samples were obtained monthly. General clinical symptoms, including changes in body weight, vitality, appetite, and water intake were assessed. Three of the four owners observed improvement of vitality after stem cell treatment. Two of the four dogs showed improvement in appetite and body weight, polyuria, and polydipsia. C-peptide has increased by about 5-15% in three of the cases, and fructosamine and HbA1c levels have improved in two of the cases. Hyperlipidemia was resolved in two of the dogs, and there was no concurrent bacterial cystitis in any of the dogs. C-peptide secretion and lipid metabolism are associated with diabetic complications. Improvement in these parameters following the treatment suggests that cAT-MSC transplantation in dogs with IDDM might help to improve their insulin secretory capacity and prevent diabetic complications.

A mitochondrial proteome profile indicative of type 2 diabetes mellitus in skeletal muscles.

The pathogenesis of type 2 diabetes mellitus (T2DM) is closely associated with mitochondrial functions in insulin-responsive tissues. The mitochondrial proteome, compared with the mitochondrial genome, which only contains 37 genes in humans, can provide more comprehensive information for thousands of mitochondrial proteins regarding T2DM-associated mitochondrial functions. However, T2DM-associated protein signatures in insulin-responsive tissues are still unclear. Here, we performed extensive proteome profiling of mitochondria from skeletal muscles in nine T2DM patients and nine nondiabetic controls. A comparison of the mitochondrial proteomes identified 335 differentially expressed proteins (DEPs) between T2DM and nondiabetic samples. Functional and network analyses of the DEPs showed that mitochondrial metabolic processes were downregulated and mitochondria-associated ER membrane (MAM) processes were upregulated. Of the DEPs, we selected two (NDUFS3 and COX2) for downregulated oxidative phosphorylation and three (CALR, SORT, and RAB1A) for upregulated calcium and protein transport as representative mitochondrial and MAM processes, respectively, and then confirmed their differential expression in independent mouse and human samples. Therefore, we propose that these five proteins be used as a potential protein profile that is indicative of the dysregulation of mitochondrial functions in T2DM, representing downregulated oxidative phosphorylation and upregulated MAM functions.

Clinical and laboratory profiles of hospitalized children with acute respiratory virus infection.

PURPOSE: Despite the availability of molecular methods, identification of the causative virus in children with acute respiratory infections (ARIs) has proven difficult as the same viruses are often detected in asymptomatic children. METHODS: Multiplex reverse transcription polymerase chain reaction assays were performed to detect 15 common respiratory viruses in children under 15 years of age who were hospitalized with ARI between January 2013 and December 2015. Viral epidemiology and clinical profiles of single virus infections were evaluated. RESULTS: Of 3,505 patients, viruses were identified in 2,424 (69.1%), with the assay revealing a single virus in 1,747 cases (49.8%). While major pathogens in single virus-positive cases differed according to age, human rhinovirus (hRV) was common in patients of all ages. Respiratory syncytial virus (RSV), influenza virus (IF), and human metapneumovirus (hMPV) were found to be seasonal pathogens, appearing from fall through winter and spring, whereas hRV and adenovirus (AdV) were detected in every season. Patients with ARIs caused by RSV and hRV were frequently afebrile and more commonly had wheezing compared with patients with other viral ARIs. Neutrophil-dominant inflammation was observed in ARIs caused by IF, AdV, and hRV, whereas lymphocyte-dominant inflammation was observed with RSV A, parainfluenza virus, and hMPV. Monocytosis was common with RSV and AdV, whereas eosinophilia was observed with hRV. CONCLUSION: In combination with viral identification, recognition of virus-specific clinical and laboratory patterns will expand our understanding of the epidemiology of viral ARIs and help us to establish more efficient therapeutic and preventive strategies.

The relationship between age of onset and risk factors including family history and life style in Korean population with type 2 diabetes mellitus.

[Purpose] The purpose of the present study was to assess the relationship between age of onset and risk factors including family history and life style in Korean population with type 2 diabetes mellitus (T2D). [Subjects and Methods] Subjects with T2D patients who received outpatient care for blood sugar control were randomly sampled at 13 general hospitals and 969 subjects were included. Cox proportional hazard models were used to confirm associations between age of onset and risk factors including family history and life style in Korean population with T2D. [Results] Parent history of T2D was significantly associated with age of onset. Compared to none of family members with T2D, those whose both father and mother had a history showed the highest the risk of early-onset (HR=2.36; 95% CI=1.45-3.85). Mother and father's history of T2D (HR=1.73; 95% CI=1.46-2.05; HR=1.83; 95% CI=1.40-2.37) were associated with the risk of early-onset. Moreover, exercise (HR=1.23, CI=1.08-1.40) smoking status (HR=1.62, CI=1.32-1.99), and drinking (HR=1.32, CI=1.13-1.54) were associated with a higher risk for the early-onset. [Conclusion] Family history as well as life style including exercise, smoking, and drinking are the risk factors for early-onset factor in Korean population with T2D.

Silencing carboxylesterase 1 in human THP-1 macrophages perturbs genes regulated by PPARγ/RXR and RAR/RXR: down-regulation of CYP27A1-LXRα signaling.

Macrophage foam cells store excess cholesterol as cholesteryl esters, which need to be hydrolyzed for cholesterol efflux. We recently reported that silencing expression of carboxylesterase 1 (CES1) in human THP-1 macrophages [CES1KD (THP-1 cells with CES1 expression knocked down) macrophages] reduced cholesterol uptake and decreased expression of CD36 and scavenger receptor-A in cells loaded with acetylated low-density lipoprotein (acLDL). Here, we report that CES1KD macrophages exhibit reduced transcription of cytochrome P45027A1 (CYP27A1) in nonloaded and acLDL-loaded cells. Moreover, levels of CYP27A1 protein and its enzymatic product, 27-hydroxycholesterol, were markedly reduced in CES1KD macrophages. Transcription of LXRα (liver X receptor α) and ABCA1 (ATP-binding cassette transporter A1) was also decreased in acLDL-loaded CES1KD macrophages, suggesting reduced signaling through PPARγ-CYP27A1-LXRα. Consistent with this, treatment of CES1KD macrophages with agonists for PPARγ, RAR, and/or RAR/RXR partially restored transcription of CYP27A1 and LXRα, and repaired cholesterol influx. Conversely, treatment of control macrophages with antagonists for PPARγ and/or RXR decreased transcription of CYP27A1 and LXRα Pharmacologic inhibition of CES1 in both wild-type THP-1 cells and primary human macrophages also decreased CYP27A1 transcription. CES1 silencing did not affect transcript levels of PPARγ and RXR in acLDL-loaded macrophages, whereas it did reduce the catabolism of the endocannabinoid 2-arachidonoylglycerol. Finally, the gene expression profile of CES1KD macrophages was similar to that of PPARγ knockdown cells following acLDL exposures, further suggesting a mechanistic link between CES1 and PPARγ. These results are consistent with a model in which abrogation of CES1 function attenuates the CYP27A1-LXRα-ABCA1 signaling axis by depleting endogenous ligands for the nuclear receptors PPARγ, RAR, and/or RXR that regulate cholesterol homeostasis.

Skin Regeneration with Self-Assembled Peptide Hydrogels Conjugated with Substance P in a Diabetic Rat Model.

The wound healing process requires enough blood to supply nutrients and various growth factors to the wound area. However, chronic wounds such as diabetic skin ulcers have limited regeneration due to a lack of cellular and molecular signals because of a deficient blood flow. Mesenchymal stem cells (MSCs) are known to provide various factors, including growth factors, cytokines, and angiogenic mediators. Although MSCs have great therapeutic potential, their transplantation has many obstacles, including the time required to culture the cells, the invasiveness of the procedure, and limited stem cell sources. In this study, we induced a diabetic 1 model in rats aged 7 weeks by injecting streptozotocin and citrate buffer solution. After confirming that diabetes was induced in the rats, we created critical sized wounds on the dorsal area of the rats and then injected hydrogels. We performed the experiments with four groups (defect model for the control, self-assembled peptides (SAPs), SAP with soluble substance P, and SAP conjugated with substance P) to treat the wound defect. Tissues were harvested at 1, 2, and 3 weeks after injection and examined for the wound closure, histological analysis, quantitative real-time polymerase chain reaction analysis, and quantification of collagen deposits to investigate stem cell recruitment and full recovery of wounds at an accelerated time period. As our results show, the wounds treated with SAP and substance P exhibited significantly accelerated wound closure, enhanced collagen deposition, and increased angiogenesis. Furthermore, we confirmed the ability of SAP with substance P to promote the recruitment and homing of cells by immunofluorescence staining of a MSC marker. In addition, it was observed that substance P remained in the wound area up to 3 weeks after the injection of SAP with substance P. It is believed that the endogenous MSCs mobilized by substance P had therapeutic effects through their proper differentiation and release of paracrine factors into the wound sites. In conclusion, this study shows that SAP with substance P can promote wound healing to enhance skin regeneration without cell transplantation in a diabetic model.

Endocannabinoid hydrolases in avian HD11 macrophages identified by chemoproteomics: inactivation by small-molecule inhibitors and pathogen-induced downregulation of their activity.

The endocannabinoids (eCBs) are endogenous arachidonoyl-containing lipid mediators with important roles in host defense. Macrophages are first-line defenders of the innate immune system and biosynthesize large amounts of eCBs when activated. The cellular levels of eCBs are controlled by the activities of their biosynthetic enzymes and catabolic enzymes, which include members of the serine hydrolase (SH) superfamily. The physiologic activity of SHs can be assessed in a class-specific way using chemoproteomic activity-based protein profiling (ABPP) methods. Here, we have examined avian (chicken) HD11 macrophages, a widely used cell line in host-pathogen research, using gel-based ABPP and ABPP-multidimensional protein identification technology (MudPIT) to profile the changes in SH activities under baseline, chemical-inhibitor-treated, and pathogen-challenged conditions. We identified α/ß-hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH) as the principal SHs responsible for 2-arachidonoylglycerol (2AG) hydrolysis, thereby regulating the concentration of this lipid in HD11 cells. We further discovered that infection of HD11 macrophages by Salmonella Typhimurium caused the activities of these 2AG hydrolases to be downregulated in the host cells. ABHD6 and FAAH were potently inhibited by a variety of small-molecule inhibitors in intact live cells, and thus these compounds might be useful host-directed adjuvants to combat antimicrobial resistance in agriculture. 2AG was further shown to augment the phagocytic function of HD11 macrophages, which suggests that pathogen-induced downregulation of enzymes controlling 2AG hydrolytic activity might be a physiological mechanism to increase 2AG levels, thus enhancing phagocytosis. Together these results define ABHD6 and FAAH as 2AG hydrolases in avian macrophages that can be inactivated pharmacologically and decreased in activity during Salmonella Typhimurium infection.

Prevalence of Malnutrition in Hospitalized Patients: a Multicenter Cross-sectional Study.

BACKGROUND: Malnutrition is associated with many adverse clinical outcomes. The present study aimed to identify the prevalence of malnutrition in hospitalized patients in Korea, evaluate the association between malnutrition and clinical outcomes, and ascertain the risk factors of malnutrition. METHODS: A multicenter cross-sectional study was performed with 300 patients recruited from among the patients admitted in 25 hospitals on January 6, 2014. Nutritional status was assessed by using the Subjective Global Assessment (SGA). Demographic characteristics and underlying diseases were compared according to nutritional status. Logistic regression analysis was performed to identify the risk factors of malnutrition. Clinical outcomes such as rate of admission in intensive care units, length of hospital stay, and survival rate were evaluated. RESULTS: The prevalence of malnutrition in the hospitalized patients was 22.0%. Old age (≥ 70 years), admission for medical treatment or diagnostic work-up, and underlying pulmonary or oncological disease were associated with malnutrition. Old age and admission for medical treatment or diagnostic work-up were identified to be risk factors of malnutrition in the multivariate analysis. Patients with malnutrition had longer hospital stay (SGA A = 7.63 ± 6.03 days, B = 9.02 ± 9.96 days, and C = 12.18 ± 7.24 days, P = 0.018) and lower 90-day survival rate (SGA A = 97.9%, B = 90.7%, and C = 58.3%, P < 0.001). CONCLUSION: Malnutrition was common in hospitalized patients, and resulted in longer hospitalization and associated lower survival rate. The rate of malnutrition tended to be higher when the patient was older than 70 years old or hospitalized for medical treatment or diagnostic work-up compared to elective surgery.

MALAT1 promoted invasiveness of gastric adenocarcinoma.

BACKGROUND: Gastric cancer is the second leading cause of cancer globally, and the mechanism of its pathogenesis is still largely unknown. Recently, non-coding RNAs have been recognized to promote metastasis in various cancers, including gastric cancer. METHODS: We found that metastasis associated lung adenocarcinoma transcript-1 (MALAT1) is upregulated in gastric cancer tissue compared to adjacent normal tissue, as determined by microarray and subsequent qRT-PCR, then investigated the impact of MALAT1 on apoptosis, cell proliferation, and the cell cycle to dissect the carcinogenesis of gastric cancer, and examined mechanisms of invasion and metastasis. Expression of MALAT1 and U6 was determined by SYBR qRT-PCR in nine-teen gastric cancer cell lines and fifty fresh samples of cancer tissue and adjacent tissues. Downregulation of MALAT1 was accomplished with two different siRNAs. Cell proliferation was determined after treatment with these siRNAs. FACS using PI/Annexin-V staining was carried out. To analyze the invasiveness, a scratch wound-healing assay and a Matrigel invasion assay were performed. Cancer related gene expression assay was done after transfection of siR- MALAT1. RESULTS: The expression of MALAT1 was significantly elevated in various gastric cancer cell lines and gastric cancer tissues compared to normal cell lines and tissues (p < 0.01). siR-MALAT1 significantly reduced viable AGS cell numbers and induced apoptosis (p < 0.05). Deep invasion of tumor (advanced T stages) was more common in the high MALAT1-level group (p = 0.039). siR-MALAT1 significantly decreased AGS cell invasiveness and migration. siR-MALAT1 reduced expression of snail and N-cadherin, and elevated E-cadherin. The Wnt/ß-catenin related genes were significantly decreased by transfection of siRNA MALAT1. MALAT1 is involved in gastric carcinogenesis via inhibition of apoptosis and promotes invasiveness via the epithelial-to-mesenchymal transition. CONCLUSIONS: In our study, we found that deregulation of MALAT1 could be involved in both tumorigenesis and invasiveness in gastric cancer cells.

Association of Some Vitamins and Minerals with Periodontitis in a Nationally Representative Sample of Korean Young Adults.

The purpose of this study was to investigate whether the intakes of some kinds of vitamins and minerals are associated with periodontitis in a nationally representative sample of young adults. This study comprised 2049 young adults aged 19-39 years who took both periodontal examination and nutrition survey. The vitamin and mineral intakes were calculated from dietary intake data gained by complete one-day 24-h recall interviews, and the intake levels for each nutrient were classified by the Recommended Nutrient Intake (RNI) in Dietary Reference Intakes for Koreans and median values. Periodontitis was assessed using Community Periodontal Index (CPI). Multivariate logistic regression analyses were performed in a whole sample and subgroups with the strata of gender or smoking, following a complex sampling design. In analyses according to RNI, a lower intake of niacin was significantly associated with periodontitis in young adults (odd ratio [OR] 1.47, 95% confidential interval [CI] 1.09-2.00) and in its subgroup of women (OR 1.70; 95% CI 1.10-2.64) and current non-smokers (OR 1.75; 95% CI 1.22-2.51). Whereas, in analyses according to median intake values, there were significant associations of periodontitis with a lower intake of niacin in women (OR 1.58; 95% CI 1.02-2.46) and current non-smokers (OR 1.50; 95% CI 1.01-2.22), with lower intake of vitamin C in women (OR 1.66; 95% CI 1.04-2.64) and in current non-smokers (OR 1.49; 95% CI 1.04-2.14), with lower intake of iron in women (OR 1.85; 95% CI 1.11-3.07), and with lower intake of vitamin A marginally in women (OR 1.56; 95% CI 1.00-2.44). In young adults, periodonitis is significantly associated with the lower intakes of niacin, vitamin C, and iron, especially in women and current non-smokers.

Delayed treatment with fenofibrate protects against high-fat diet-induced kidney injury in mice: the possible role of AMPK autophagy.

Fenofibrate activates not only peroxisome proliferator-activated receptor-α (PPARα) but also adenosine monophosphate-activated protein kinase (AMPK). AMPK-mediated cellular responses protect kidney from high-fat diet (HFD)-induced injury, and autophagy resulting from AMPK activation has been regarded as a stress-response mechanism. Thus the present study examined the role of AMPK and autophagy in the renotherapeutic effects of fenofibrate. C57BL/6J mice were divided into three groups: normal diet (ND), HFD, and HFD + fenofibrate (HFD + FF). Fenofibrate was administered 4 wk after the initiation of the HFD when renal injury was initiated. Mouse proximal tubule cells (mProx24) were used to clarify the role of AMPK. Feeding mice with HFD for 12 wk induced insulin resistance and kidney injury such as albuminuria, glomerulosclerosis, tubular injury, and inflammation, which were effectively inhibited by fenofibrate. In addition, fenofibrate treatment resulted in the activation of renal AMPK, upregulation of fatty acid oxidation (FAO) enzymes and antioxidants, and induction of autophagy in the HFD mice. In mProx24 cells, fenofibrate activated AMPK in a concentration-dependent manner, upregulated FAO enzymes and antioxidants, and induced autophagy, all of which were inhibited by treatment of compound C, an AMPK inhibitor. Fenofibrate-induced autophagy was also significantly blocked by AMPKα1 siRNA but not by PPARα siRNA. Collectively, these results demonstrate that delayed treatment with fenofibrate has a therapeutic effect on HFD-induced kidney injury, at least in part, through the activation of AMPK and induction of subsequent downstream effectors: autophagy, FAO enzymes, and antioxidants.

Oxyradical stress increases the biosynthesis of 2-arachidonoylglycerol: involvement of NADPH oxidase.

NADPH oxidase (Nox)-derived oxyradicals contribute to atherosclerosis by oxidizing low-density lipoproteins (LDL), leading to their phagocytosis by vascular macrophages. Endocannabinoids, such as 2-arachidonoylglycerol (2-AG), might be an important link between oxidative stress and atherosclerosis. We hypothesized that 2-AG biosynthesis in macrophages is enhanced following ligation of oxidized LDL by scavenger receptors via a signal transduction pathway involving Nox-derived ROS that activates diacylglycerol lipase-ß (DAGL-ß), the 2-AG biosynthetic enzyme. To test this idea, we challenged macrophage cell lines and murine primary macrophages with a xanthine oxidase system or with nonphysiological and physiological Nox stimulants [phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA)]. Each stressor increased cellular superoxide levels and enhanced 2-AG biosynthetic activity in a Nox-dependent manner. Levels of cytosolic phospholipase A2-dependent AA metabolites (eicosanoids) in primary macrophages were also dependent on Nox-mediated ROS. In addition, 2-AG levels in DAGL-ß-overexpressing COS7 cells were attenuated by inhibitors of Nox and DAGL-ß. Furthermore, ROS induced by menadione (a redox cycling agent) or PMA could be partially attenuated by the cannabinoid 1/2 receptor agonist (WIN 55,212-2). Finally, cells that overexpress Nox2 components (Phox-COS7) synthesized larger amounts of 2-AG compared with the parental COS7 cells. Together, the results suggest a positive correlation between heightened oxygen radical flux and 2-AG biosynthesis in macrophage cell lines and primary macrophages. Because of the antioxidant and anti-inflammatory effects associated with 2-AG, the increased levels of this bioactive lipid might be an adaptive response to oxidative stress. Thus oxyradical stress may be counteracted by the enhanced endocannabinoid tone.

Promoter methylation of PCDH10 by HOTAIR regulates the progression of gastrointestinal stromal tumors.

HOTAIR, a long non-coding RNA (lncRNA), plays a crucial role in tumor initiation and metastasis by interacting with the PRC2 complex and the modulation of its target genes. The role of HOTAIR in gastrointestinal stromal tumors (GISTs) is remains unclear. Herein we investigate the mechanism of HOTAIR in the genesis and promotion of GISTs. The expression of HOTAIR was found to be higher in surgically resected high-risk GISTs than that in low- and intermediate-risk GISTs. Using GIST-T1 and GIST882 cells, we demonstrated that HOTAIR repressed apoptosis, was associated with cell cycle progression, and controlled the invasion and migration of GIST cells. Using a gene expression microarray and lists of HOTAIR-associated candidate genes, we suggested that protocadherin 10 (PCDH10) is a key molecule. PCDH10 expression was significantly decreased in GIST-T1 and GIST882 cells, possibly as a consequence of hypermethylation. We observed that HOTAIR induced PCDH10 methylation in a SUZ12-dependent manner. In this study, we found that the malignant character of GISTs was initiated and amplified by PCDH10 in a process regulated by HOTAIR. In summary, our findings imply that PCDH10 and HOTAIR may be useful markers of disease progression and therapeutic targets.

Novel Role of Endogenous Catalase in Macrophage Polarization in Adipose Tissue.

Macrophages are important components of adipose tissue inflammation, which results in metabolic diseases such as insulin resistance. Notably, obesity induces a proinflammatory phenotypic switch in adipose tissue macrophages, and oxidative stress facilitates this switch. Thus, we examined the role of endogenous catalase, a key regulator of oxidative stress, in the activity of adipose tissue macrophages in obese mice. Catalase knockout (CKO) exacerbated insulin resistance, amplified oxidative stress, and accelerated macrophage infiltration into epididymal white adipose tissue in mice on normal or high-fat diet. Interestingly, catalase deficiency also enhanced classical macrophage activation (M1) and inflammation but suppressed alternative activation (M2) regardless of diet. Similarly, pharmacological inhibition of catalase activity using 3-aminotriazole induced the same phenotypic switch and inflammatory response in RAW264.7 macrophages. Finally, the same phenotypic switch and inflammatory responses were observed in primary bone marrow-derived macrophages from CKO mice. Taken together, the data indicate that endogenous catalase regulates the polarization of adipose tissue macrophages and thereby inhibits inflammation and insulin resistance.

Clinicopathological Characteristics of Patients with Gastric Cancer according to the Expression of LIN28A.

BACKGROUND/AIMS: Although LIN28A is known to potentially play a role in the oncogenesis of various cancers, whether LIN28A expression is a predictor of poor prognosis in patients with gastric cancer has not been fully explored. We sought to evaluate clinicopathological characteristics according to the expression of LIN28A in numerous gastric cancer tissue samples. METHODS: LIN28A expression was evaluated by immunohistochemical (IHC) analysis of a tissue microarray comprising 288 gastric cancer tissues and 288 adjacent normal tissues. Clinicopathological characteristics, including overall survival, were compared according to LIN28A expression. RESULTS: The IHC staining score was lower for the cancer tissues than the normal tissues (p<0.001). However, no significant differences were observed in the clinicopathological characteristics between the low and high LIN28A expression groups. In addition, the 5-year overall survival rate did not differ between the two groups: 75.3% (95% confidence interval [CI], 69.3% to 81.7%) versus 71.6% (95% CI, 63.3% to 80.9%) for low versus high expression, respectively. CONCLUSIONS: The expression of LIN28A did not appear to play a distinct role in predicting the clinicopathological characteristics of patients with gastric cancer. In addition, LIN28A expression was not an independently associated factor for overall survival in patients with gastric cancer.

The relationship between synovial fluid VEGF and serum leptin with ultrasonographic findings in knee osteoarthritis.

AIM: This study aimed to determine synovial fluid and serum biomarkers which could accord with radiological and ultrasonographic findings in knee osteoarthritis. METHODS: Thirty-four patients with knee osteoarthritis were detected with joint effusion by clinical examination. Both knee joints were examined using plain radiographs and ultrasonography. Questions were obtained for visual analog scale (VAS), Western Ontario McMaster Universities Osteoarthritis Index and Health Assessment Questionnaire (HAQ). Synovial fluid (SF) and serum levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-13, leptin, resistin and cartilage oligomeric matrix protein (COMP) were measured using enzyme-linked immunosorbent assay. RESULTS: Synovial fluid VEGF level was positively correlated with Kellgren-Lawrence (KL) grades and it was higher in patients with KL grade 4 than those with KL grade 2. SF VEGF correlated with ultrasonographic findings, such as the length of medial osteophytes. The amount of effusion was positively correlated with SF resistin. Serum leptin level had positive correlation with HAQ and the length of medial osteophytes. MMP-13 or COMP levels were not correlated with radiographic or ultrasonographic findings. CONCLUSION: Synovial fluid VEGF level was correlated with radiographic grading, ultrasonographic findings and functional statues in knee osteoarthritis, and serum leptin level also correlated with the ultrasonographic findings and functional status of knee osteoarthritis.

Lipopolysaccharide suppresses carboxylesterase 2g activity and 2-arachidonoylglycerol hydrolysis: A possible mechanism to regulate inflammation.

Inflammation is an important part of the innate immune response and is involved in the healing of many disease processes; however, chronic inflammation is a harmful component of many diseases. The regulatory mechanisms of inflammation are incompletely understood. One possible regulatory mechanism is the endocannabinoid system. Endocannabinoids such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA) are generally anti-inflammatory via engagement of the cannabinoid receptor 2 (CB2) on innate cells; therefore, preventing the degradation of endocannabinoids by specific serine hydrolases such as fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), and carboxylesterases (CES) might decrease inflammation. We hypothesized that the activities of these catabolic enzymes would decrease with a subsequent increase in 2-AG and AEA in a model of inflammation. Mice were injected with lipopolysaccharide (LPS) for 6 or 24h, and inflammation was confirmed by an increase in interleukin-6 (il6) and il17 gene expression. Activity-based protein profiling (ABPP) of serine hydrolases showed no significant difference in various serine hydrolase activities in brain or liver, whereas a modest decrease in Ces activity in spleen after LPS administration was noted. 2-AG hydrolase activity in the spleen was also decreased at 6h post LPS, which was corroborated by LPS treatment of splenocytes ex vivo. ABPP-MudPIT proteomic analysis suggested that the decreased 2-AG hydrolysis in spleen was due to a reduction in Ces2g activity. These studies suggest that the endocannabinoid system could be activated via suppression of a 2-AG catabolic enzyme in response to inflammatory stimuli as one mechanism to limit inflammation.

Morphological changes in human gastric epithelial cells induced by nuclear targeting of Helicobacter pylori urease subunit A.

Nuclear targeting of bacterial proteins and their pathological effects on host cells are an emerging pathogenic mechanism in bacteria. We have previously reported that urease subunit A (UreA) of Helicobacter pylori targets the nuclei of COS-7 cells through nuclear localization signals (NLSs). This study further investigated whether UreA of H. pylori targets the nuclei of gastric epithelial cells and then induces molecular and cellular changes in the host cells. H. pylori 26695 strain produced and secreted outer membrane vesicles (OMVs). UreA was translocated into gastric epithelial AGS cells through outer membrane vesicles (OMVs) and then targeted the nuclei of AGS cells. Nuclear targeting of rUreA did not induce host cell death, but resulted in morphological changes, such as cellular elongation, in AGS cells. In contrast, AGS cells treated with rUreA?NLS proteins did not show this morphological change. Next generation sequencing revealed that nuclear targeting of UreA differentially regulated 102 morphogenesis- related genes, of which 67 and 35 were up-regulated and down-regulated, respectively. Our results suggest that nuclear targeting of H. pylori UreA induces both molecular and cellular changes in gastric epithelial cells.

Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.

Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 µM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox) levels and enhanced p47(phox) membrane localization compared to that in vehicle-treated cells. p47(phox) is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47(phox). In addition, ATK also blocked OC-induced p47(phox) serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation.