Multiple variants of African swine fever virus circulating in Vietnam.

African swine fever (ASF) is a contagious and deadly viral disease affecting swine of all ages. ASF was first reported in Vietnam in February 2019, and it is now considered endemic in Vietnam. In this study, 122 ASF-positive samples collected from domestic pigs in 28 different provinces of northern, central, and southern Vietnam during outbreaks in 2019-2021 were genetically characterized. The findings confirmed that all ASF virus (ASFV) strains circulating in Vietnam belonged to p72 genotype II, p54 genotype II, CD2v serogroup 8, and CVR gene variant type I. However, further analysis based on the tandem repeat sequences located between I73R and I329L genes revealed that there were three different variants of ASFV, IGR I, II, and III, circulating in the domestic pig population in Vietnam. The IGR II variants were the most prevalent (117/122 strains) and were detected in pigs in all of the provinces tested, followed by IGR III (4/122 strains) and IGR I (1/122 strains). This study confirms for the first time the presence of IGR III variants in Vietnam.

Protective Effect of Gomisin N against Endoplasmic Reticulum Stress-Induced Hepatic Steatosis.

Gomisin N is a physiological substance derived from Schisandra chinensis. In the present study, the in vitro and in vivo effects of gomisin N on endoplasmic reticulum (ER) stress and hepatic steatosis were investigated. We quantified the expression of markers of ER stress, including glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homolog protein (CHOP), and X-box-binding protein-1 (XBP-1), and triglyceride (TG) accumulation, in HepG2 cells treated with tunicamycin or palmitate. Tunicamycin treatment in HepG2 cells induced expression of markers of ER stress and increased TG levels; Gomisin N reversed these effects, reducing the expression of markers of ER stress and TG levels. Similar effects were seen following palmitate pretreatment of HepG2 cells. The inhibitory effects of gomisin N were further confirmed in mice injected with tunicamycin. Gomisin N reduced expression of markers of ER stress and decreased TG levels in mouse liver after tunicamycin injection. Furthermore, gomisin N decreased expression of inflammatory and lipogenic genes in palmitate-incubated HepG2 cells. These results suggest that gomisin N inhibits ER stress and ameliorates hepatic steatosis induced by ER stress.

ATF3 inhibits PPARγ-stimulated transactivation in adipocyte cells.

Previously, we reported that activating transcription factor 3 (ATF3) downregulates peroxisome proliferator activated receptor (PPARγ) gene expression and inhibits adipocyte differentiation in 3T3-L1 cells. Here, we investigated another role of ATF3 on the regulation of PPARγ activity. ATF3 inhibited PPARγ-stimulated transactivation of PPARγ responsive element (PPRE)-containing reporter or GAL4/PPARγ chimeric reporter. Thus, ATF3 effectively repressed rosiglitazone-stimulated expression of adipocyte fatty acid binding protein (aP2), PPARγ target gene, in 3T3-L1 cells. Coimmunoprecipitation and GST pulldown assay demonstrated that ATF3 interacted with PPARγ. Accordingly, ATF3 prevented PPARγ from binding to PPRE on the aP2 promoter. Furthermore, ATF3 suppressed p300-mediated transcriptional coactivation of PPRE-containing reporter. Chromatin immunoprecipitation assay showed that overexpression of ATF3 blocked both binding of PPARγ and recruitment of p300 to PPRE on aP2 promoter induced by rosiglitazone treatment in 3T3-L1 cells. Taken together, these results suggest that ATF3 interacts with PPARγ and represses PPARγ-mediated transactivation through suppression of p300-stimulated coactivation in 3T3-L1 cells, which may play a role in inhibition of adipocyte differentiation.

Protective Effects of Alisma orientale Extract against Hepatic Steatosis via Inhibition of Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.

ATF3 represses PPARγ expression and inhibits adipocyte differentiation.

Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3 in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of -2.6Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5' deleted-reporters showed that ATF3 repressed the activity of -2037bp promoter, whereas it did not affect the activity of -1458bp promoter, suggesting that ATF3 responsive element is located between the -2037 and -1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5'-TGACGTTT-3') between -1537 and -1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated inhibition of PPARγ expression may contribute to inhibition of adipocyte differentiation during cellular stress including ER stress.

ATF3 plays a role in adipocyte hypoxia-mediated mitochondria dysfunction in obesity.

Obesity-associated adipose tissue hypoxia plays a pivotal role in insulin resistance via impaired adipocyte dysfunction including mitochondria dysfunction. In this study, we investigated the involvement of hypoxia-inducible ATF3 in adipocyte hypoxia-mediated mitochondrial dysfunction. While HIF-1α and ATF3 were increased in white adipose tissue of high fat diet (HFD) obese mice compared with control lean mice, mitochondria-related genes were significantly reduced. Treatment with hypoxia mimetics CoCl(2) or incubation with 2% O(2) impaired mitochondria function as demonstrated by decreases in ATP production, NADH dehydrogenase activity, mitochondrial membrane potential, and reduced expression of mitochondria-related genes including NRF-1, PGC-1α, COX1 and SOD in 3T3-L1 adipocyte cells. Furthermore, overexpression of ATF3 in 3T3-L1 cells also decreased mitochondria function as well as expression of mitochondria-related genes. ATF3 knockdown in 3T3-L1 cells partly prevented the hypoxia-mediated decrease in mitochondria function and expression of mitochondria-related genes. The mitochondria-related genes were decreased in white adipose tissue of ATF3-overexpressing mice compared with wild-type mice. These results suggest that ATF3 may play a role in adipocyte hypoxia-mediated mitochondrial dysfunction in obesity.

Antiobesity activity of Vigna nakashimae extract in high-fat diet-induced obesity.

In this study, we evaluated the antiobesity effects of Vigna nakashimae (VN) extract and elucidated the underlying mechanisms. VN extract suppressed adipocyte differentiation and significantly attenuated the expression of adipogenic genes in 3T3-L1 cells. It decreased the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes in fully differentiated 3T3-L1 cells. Moreover, it enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase (ACC), and increased the expression of fatty acid oxidation genes. In high-fat diet (HFD) fed mice, VN extract suppressed HFD-induced increases in body weight, epididymal fat tissue weight, and hepatic lipid levels, and decreased the plasma levels of triacylglycerols, fatty acid, total cholesterol, and inflammatory cytokines. Consistently with in vitro study results, VN extract prevented HFD-induced increases in the expression of PPARγ and its target genes, and restored the decrease in the phosphorylation of AMPK and ACC in epididymal fat and liver tissues. These findings suggest that Vigna nakashimae prevents obesity through suppression of PPARγ expression and activation of AMPK, and that it might be a useful dietary supplement for the prevention of obesity.

Predictors of early hospital readmission in patients receiving home mechanical ventilation.

BACKGROUND: Although the proportion of patients with chronic respiratory failure requiring home mechanical ventilation (HMV) is increasing, hospital readmissions in these patients are also increasing. OBJECTIVE: We investigated the factors for early readmission in patients receiving HMV. METHODS: We retrospectively analyzed the data of adult patients readmitted to the hospital within a year who first received HMV and were discharged from the Asan Medical Center between March 2014 and February 2019. We compared the clinical characteristics at discharge before readmission between the early (readmission within 30 days) and late readmission groups (readmission between day 31 and 1 year) and investigated the clinical characteristics and outcomes at readmission. RESULTS: Of the 116 patients identified, 36.2% had been readmitted early. The patients who received invasive HMV had a higher rate of early readmission than those who received non-invasive HMV. Pneumonia was the most common reason of readmission in the two groups. The rate of aspiration was significantly higher in the early readmission group (28.6% vs. 8.1%; P = .003). In multivariate logistic regression analysis, nasogastric tube feeding, sequelae of pneumonia or acute respiratory distress syndrome, and central nervous system disorders as causes for HMV were significantly associated with early readmission. CONCLUSION: Feeding methods and causes for HMV were associated with early readmission. Educating caregivers on respiratory care (suction and feeding methods) is important for preventing early readmission.

Genetic Profile of African Swine Fever Viruses Circulating at Pig Farms in South Korea during the Outbreaks between 2022 and April 2023.

Fifteen pig farms were affected by African swine fever (ASF) in South Korea during the outbreaks between 2022 and April 2023. The ASF virus (ASFV) genome was directly extracted from the blood and tissue samples of 15 ASFV-positive pig farms to analyze the genetic characteristics. Phylogenetic analysis revealed that the 15 strains belonged to p72 genotype II and CD2v serogroup 8, which were the central variable region (CVR) I variants of the B602L gene. Fourteen strains were intergenic region (IGR) II variants, containing an additional tandem repeat sequence (TRS), between I73L and I329R, with the exception of one strain from an ASFV-infected pig farm reported on 22 January 2023, which was an IGR I variant. In addition, a single-nucleotide polymorphism (SNP) was detected at position 107 from the start of the IGR between A179L and A137R in six isolates. The findings of this study suggest that the sources of the virus at the pig farms from which these variants originated differed from those of other pig farms.

Pathogenicity and Pathological Characteristics of African Swine Fever Virus Strains from Pig Farms in South Korea from 2022 to January 2023.

Since the first African swine fever (ASF) outbreak occurred at a pig farm in South Korea in September 2019, as of 31 January 2023, 31 ASF cases have occurred at pig farms, while 2799 ASF virus (ASFV)-infected wild boars have been identified. The circulation of ASFV in wild boar populations poses a high risk of spillover to pig farms in the country. However, information on the changes in the pathogenicity of Korean ASFV strains from wild boars is not available. Investigating the pathogenicity of ASFV strains from pig farms is the only way to predict their alterations. In a previous study, no changes in the pathogenicity of ASFV strains circulating during 2019-2021 were identified through animal experiments. In this study, we chose two ASFV strains with potentially reduced pathogenicity among ten viruses obtained from pig premises from 2022 to January 2023 and estimated their pathogenicities and pathological characteristics. All the inoculated pigs died 8-10 days post-inoculation after showing pyrexia, depression, anorexia, and recumbency together with the common pathological lesions of enlarged hemorrhagic lymph nodes and splenomegaly with infarction. These results support that the pathogenicity among ASFV isolates in South Korea still remained unchanged during the study period.

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells.

ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBPα transcript and repressed the activity of the 3.6-kb mouse C/EBPα promoter, demonstrating that ATF3 downregulates C/EBPα expression. Transfection studies using mutant constructs containing 5'-deletions in the C/EBPα promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBPα promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBPα mRNA and repress the promoter activity of the C/EBPα gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBPα expression. Collectively, these results demonstrate that ATF3 represses the C/EBPα gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation.

Detection of African Swine Fever at an Abattoir in South Korea, 2020.

In October 2020, a suspect case of African swine fever (ASF) was detected at an abattoir located in the north-central border region of South Korea. The farm of origin was traced and confirmed positive for ASF. This recurrence was following a period of absence of outbreaks in domestic pigs after the first incursion in 2019, during which a total of 14 domestic pig farms were confirmed between September and October 2019. In 2020, a total of two farms were confirmed, and the molecular characterization of key regions of the genome showed that the two isolates from 2020 were identical with the previous isolates from South Korea in 2019. The continued spread and circulation of ASF in the wild boar population represents an increased risk of spill-over outbreaks in domestic pigs, and, therefore, additional control measures should be implemented for farms in these regions, including a heightened level of surveillance. This was the case for the index farm, which was required to send pigs only to the designated abattoir at which the suspect case was quickly detected. The improvement of biosecurity in pig farms, particularly at the wild boar-domestic pig interface, will be key to the successful control of ASF in the region.

Genetic Characterization of African Swine Fever Virus from Pig Farms in South Korea during Outbreaks in 2019-2021.

In South Korea, a total of 21 African swine fever (ASF) infected farms were confirmed during 2019-2021. ASF viruses (ASFVs) were isolated from the blood and spleen samples of the 21 affected farms and their genetic characteristics were analyzed. Phylogenetic analysis indicated that the 21 Korean ASFV strains belonged to p72 genotype II and serogroup 8. All isolates were of the intergenic region (IGR) II variant with 10 tandem repeat sequences between I73R and I329L and the central variable region (CVR) 1 variant of the B602L gene. There were no IGR variations between the A179L and A137R and between the MGF 505 9R and10R nor mutations in the O174L, K145R, MGF 505-5R, CP204L, and Bt/Sj regions. The genes of the 21 ASFV strains were identical to those of Georgia 2007/1 and Chinese and Vietnamese strains (Pig/HLJ/2018, China/2018/AnhuiXCGQ, and ASFV_NgheAn_2019); however, X69R of the J268L region of the 18th isolate (Korea/Pig/Goseong/2021) had three nucleotide (CTA) insertions at the 209th position, which led to the addition of one tyrosine (Y) at the C-terminal. This suggests that there are variations among ASFVs circulating in South Korea and the 18th ASFV-infected farm was due to a variant different from those of the other 20 pig farms.

Comparison of the Virulence of Korean African Swine Fever Isolates from Pig Farms during 2019-2021.

African swine fever (ASF) was first reported in South Korea in September 2019, and as of 31 December 2021, a total of 21 cases in domestic pig farms and 1875 ASFV-infected wild boars have been confirmed in the country. With the continued circulation of ASF in wild boars, and subsequent outbreaks in domestic pigs, concerns were raised about the possible changes in virulence occurring among African swine fever viruses (ASFV) circulating in South Korea. In this study, four Korean ASFV strains isolated from domestic pig farms at different time points between 2019 and 2021 were chosen, and used to experimentally infect domestic pigs by intramuscular inoculation to compare their virulence. All challenged pigs died at 4-9 days post-inoculation, with many showing clinical symptoms of fever, depression, loss of appetite, and recumbency. Gross lesions observed at necropsy included enlargement and hemorrhage of the lymph nodes and hydropericardium. The study showed that all four Korean ASFV isolates caused acute forms of illness, which supports the view that virulence among the circulating ASFV isolates in South Korea remained unchanged and highly virulent during this period.

ATF3 represses PDX-1 expression in pancreatic ß-cells.

The downregulation of PDX-1 expression plays an important role in development of type 2 diabetes. However, the negative regulator of PDX-1 expression is not well known. In this study, we analyzed the mouse PDX-1 promoter to characterize the effects of ATF3 on PDX-1 expression in pancreatic ß-cells. Both thapsigargin treatment, an inducer of ER stress, and ATF3 expression decreased PDX-1 expression in pancreatic ß-cells, MIN6N8. Furthermore, they also repressed the activity of -4.5 Kb promoter of mouse PDX-1 gene. Transfection studies with 5' deleted-reporters showed that ATF3 repressed the activity of 0.9Kb PDX-1 promoter, whereas it did not affect the activity of 0.7 Kb PDX-1 promoter, suggesting that ATF3 responsive element is located between the -903 and -702. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds directly to the promoter region spanning from -759 to -738. Moreover, mutation of the putative ATF/CRE site between -752 and -745 abrogated ATF3-mediated transrepression of the PDX-1 promoter. PDX-1 was decreased in MIN6N8 cells treated with high glucose or high palmitate, whereas ATF3 was increased, indicating that ATF3 plays a role in hyperglycemia or hyperlipidemia-mediated downregulation of PDX-1 expression. Collectively, these results demonstrate that ATF3 represses PDX-1 expression via binding to an ATF3-responsive element in its promoter, which plays an important role in suppression of pancreatic ß-cells function.

ATF3 inhibits PDX-1-stimulated transactivation.

Chronic endoplasmic reticulum (ER) stress leads to ß-cell failure via reduction of pancreatic and duodenal homeobox-1 (PDX-1) activity, which contributes to the pathogenesis of type 2 diabetes. However, the exact mechanisms by which ER stress reduces PDX-1 activity in pancreatic ß-cells are unclear. Previously, we showed that ATF3 downregulates PDX-1 gene expression in MIN6N8 pancreatic ß-cells. Here, we investigated another role of ATF3 on the regulation of PDX-1 activity. ATF3 significantly inhibited PDX-1-stimulated transactivation of reporter plasmid containing promoters for PDX-1 binding element and the PDX-1 target gene glucokinase, which is dependent on C-terminal domain of ATF3. ATF3 interacted with PDX-1, and effectively inhibited p300-mediated transcriptional coactivation of the PBE-containing promoter, whereas C-terminal domain-deleted ATF3 did not inhibit the transcoactivation of p300. ATF3 decreased the interaction of p300 with PDX-1 in MIN6N8 cells coexpressing PDX-1 and ATF3. In addition, chromatin immunoprecipitation analysis demonstrated that both tunicamycin treatment and ATF3 overexpression inhibited the recruitment of p300 to PDX-1 on the insulin promoter in MIN6N8 cells. Taken together, these results suggest that ATF3 inhibits PDX-1-mediated transactivation through the inhibition of p300-stimulated coactivation, which may lead to ß-cell dysfunction by ER stress.

Enhancement of the thermostability of a recombinant beta-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis.

Random mutagenesis was performed on beta-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K-T307I were approx. 140, 190 and 200%, respectively, of wild type beta-agarase (661 U/mg) at 40 degrees C. All three mutant enzymes were stable up to 50 degrees C and E99K-T307I had the highest thermostability. The melting temperature (Tm) of E99K-T307I, determined by CD spectra, was increased by 5.2 degrees C over that of the wild-type enzyme (54.6 degrees C). Activities of both the wild-type and E99K-T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl2. The E99K-T307I enzyme was stable at 55 degrees C with 1 mM CaCl2, reaching 260% of the activity the wild-type enzyme held at 40 degrees C without CaCl2.

Retraction: Sasa borealis extract exerts an antidiabetic effect via activation of the AMP-activated protein kinase.

[This retracts the article on p. 15 in vol. 7, PMID: 23423690.].

Isolation and Genetic Characterization of African Swine Fever Virus from Domestic Pig Farms in South Korea, 2019.

On 17 September 2019, the first outbreak of African swine fever in a pig farm was confirmed in South Korea. By 9 October, 14 outbreaks of ASF in domestic pigs had been diagnosed in 4 cities/counties. We isolated viruses from all infected farms and performed genetic characterization. The phylogenetic analysis showed that all of fourteen ASFV isolates in South Korea belong to genotype II and serogroup 8. Additionally, all isolates had an intergenic region (IGR) II variant with additional tandem repeat sequences (TRSs) between the I73R and I329L genes and showed characteristics of central variable region (CVR) 1 of the B602L gene and IGR 1 of MGF 505 9R/10R genes. These are identical to the genetic characteristics of some European isolates and Chinese isolates.

Transcriptional auto-regulation of the dopamine receptor regulating factor (DRRF) gene.

The murine dopamine receptor regulating factor (DRRF) gene is transcribed from a TATA-less promoter that has several putative Sp1 binding sites. The present investigation identifies functional transcription factors that modulate the expression of this gene. In the D2-dopamine receptor expressing NB41A3 cells, Sp1 potently activates transcription from the DRRF promoter in pCAT-DRRF-1153/+17, while DRRF itself effectively inhibits it. Sp1 also activates this promoter in pCAT-DRRF-310/+17. In competitive co-transfection experiments, DRRF represses the transcriptional activation induced by Sp1, while Sp1 de-represses the inhibitory effect of DRRF. Deletion of the 31 bp fragment between -1153 and -1122 decreased basal transcription down to about 60%. This fragment contains a functional AP1 binding site. In addition, deletion of the 129 bp region between -901 and -772 further decreased transcription. The latter region has a functional AP2 binding site. The present observations suggest that DRRF auto-regulates its own promoter by competing with Sp1 and that both AP1 and AP2 modulate expression of this gene.