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Rapid and Sensitive Detection of Salmonella in Chickens Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

  • Liu, Zhi-Ke;Zhang, Qiu-Yu;Yang, Ning-Ning;Xu, Ming-Guo;Xu, Jin-Feng;Jing, Ming-Long;Wu, Wen-Xing;Lu, Ya-Dong;Shi, Feng;Chen, Chuang-Fu
    • Journal of Microbiology and Biotechnology
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    • v.29 no.3
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    • pp.454-464
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    • 2019
  • Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, $MgSO_4$ concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as $89fg/{\mu}l$, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.

Detection of Geosmin Production Capability Using geoA Gene in Filamentous Cyanobacteria (Nostocales, Oscillatoriales) Strains (geoA 유전자를 이용한 사상형 남조류(Nostocales, Oscillatoriales)의 Geosmin 생성능 검출)

  • Ryu, Hui-Seong;Shin, Ra-Young;Seo, Kyung-Ae;Lee, Jung-Ho;Kim, Kyunghyun
    • Journal of Korean Society on Water Environment
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    • v.34 no.6
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    • pp.661-668
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    • 2018
  • Geosmin is volatile metabolites produced by a range of filamentous cyanobacteria which causes taste and odor problems in drinking water. Molecular ecological methods which target biosynthetic genes (geoA) are widely adopted to detect geosmin-producing cyanobacteria. The aim of this study was to investigate the potential production capability of 8 strains isolated from the Nakdong River. Ultimately, a suggestion for a genetical monitoring tool for the identification of geosmin producers in domestic waters was to be made. Geosmin was detected using solid phase microextraction gas chromatography mass spectrometry (SPME GC-MS) in two strains of Dolichospermum plactonicum (DGUC006, DGUC012) that were cultured for 28 day. The highest concentrations during the experiment period was $17,535ngL^{-1}$ and $14,311ngL^{-1}$ respectively. Additionally, geoA genes were amplified using two primers (geo78F/971R and geo78F/982R) from strains shown to produce geosmin, while amplification products were not detected in any of non-producing strains. PCR product (766 bp) was slightly shorter than the expected size for geosmin producers. According to the BLAST analysis, amplified genes were at nucleotide level with Anabaena ucrainica (HQ404996, HQ404997), Dolichospermum planctonicum (KM13400) and Dolichospermum ucrainicum (MF996872) between 99 ~ 100 %. Both strains were thus confirmed as potential geosmin-producing species. We concluded that the molecular method of analysis was a useful tool for monitoring potential cyanobacterial producers of geosmin.

Role of Interleukin-4 (IL-4) in Respiratory Infection and Allergy Caused by Early-Life Chlamydia Infection

  • Li, Shujun;Wang, Lijuan;Zhang, Yulong;Ma, Long;Zhang, Jing;Zu, Jianbing;Wu, Xuecheng
    • Journal of Microbiology and Biotechnology
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    • v.31 no.8
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    • pp.1109-1114
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    • 2021
  • Chlamydia pneumoniae is a type of pathogenic gram-negative bacteria that causes various respiratory tract infections including asthma. Chlamydia species infect humans and cause respiratory infection by rupturing the lining of the respiratory which includes the throat, lungs and windpipe. Meanwhile, the function of interleukin-4 (IL-4) in Ch. pneumoniae respiratory infection and its association with the development of airway hyperresponsiveness (AHR) in adulthood and causing allergic airway disease (AAD) are not understood properly. We therefore investigated the role of IL-4 in respiratory infection and allergy caused by early life Chlamydia infection. In this study, Ch. pneumonia strain was propagated and cultured in HEp-2 cells according to standard protocol and infant C57BL/6 mice around 3-4 weeks old were infected to study the role of IL-4 in respiratory infection and allergy caused by early life Chlamydia infection. We observed that IL-4 is linked with Chlamydia respiratory infection and its absence lowers respiratory infection. IL-4R α2 is also responsible for controlling the IL-4 signaling pathway and averts the progression of infection and inflammation. Furthermore, the IL-4 signaling pathway also influences infection-induced AHR and aids in increasing AAD severity. STAT6 also promotes respiratory infection caused by Ch. pneumoniae and further enhanced its downstream process. Our study concluded that IL-4 is a potential target for preventing infection-induced AHR and severe asthma.

Nrf2 induces Ucp1 expression in adipocytes in response to β3-AR stimulation and enhances oxygen consumption in high-fat diet-fed obese mice

  • Chang, Seo-Hyuk;Jang, Jaeyool;Oh, Seungjun;Yoon, Jung-Hoon;Jo, Dong-Gyu;Yun, Ui Jeong;Park, Kye Won
    • BMB Reports
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    • v.54 no.8
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    • pp.419-424
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    • 2021
  • Cold-induced norepinephrine activates β3-adrenergic receptors (β3-AR) to stimulate the kinase cascade and cAMP-response element-binding protein, leading to the induction of thermogenic gene expression including uncoupling protein 1 (Ucp1). Here, we showed that stimulation of the β3-AR by its agonists isoproterenol and CL316,243 in adipocytes increased the expression of Ucp1 and Heme Oxygenase 1 (Hmox1), the principal Nrf2 target gene, suggesting the functional interaction of Nrf2 with β3-AR signaling. The activation of Nrf2 by tert-butylhydroquinone and reactive oxygen species (ROS) production by glucose oxidase induced both Ucp1 and Hmox1 expression. The increased expression of Ucp1 and Hmox1 was significantly reduced in the presence of a Nrf2 chemical inhibitor or in Nrf2-deleted (knockout) adipocytes. Furthermore, Nrf2 directly activated the Ucp1 promoter, and this required DNA regions located at -3.7 and -2.0 kb of the transcription start site. The CL316,243-induced Ucp1 expression in adipocytes and oxygen consumption in obese mice were partly compromised in the absence of Nrf2 expression. These data provide additional insight into the role of Nrf2 in β3-AR-mediated Ucp1 expression and energy expenditure, further highlighting the utility of Nrf2-mediated thermogenic stimulation as a therapeutic approach to diet-induced obesity.

Investigating the value optimized forest carbon offset projects based on forest management scenarios in South Korea

  • Woo, Heesung;Park, Joowon;Park, Soo-Kyoo
    • Korean Journal of Agricultural Science
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    • v.47 no.4
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    • pp.951-962
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    • 2020
  • One hundred ninety-five countries reached agreement on a new climate treaty in Paris, France to reduce the carbon emissions. South Korea has been selected as a target country for reducing greenhouse gas (GHG) obligations since 2020. In this context, the Korean government developed several GHG emissions reduction programs using forests called the "Forest carbon offset scheme (FCOS)." The forest management method is one of the tools to implement FCOS. Most of the participants registered forest management as the preferred methodology to participate in the FCOS. For a successful implementation of the FCOS, it is necessary to explore the optimal methods by considering the cost-effective aspect of conducting the forest management as a tool to increase carbon absorption. In this context, this study investigated the value optimized FCOS projects based on the forest management methodology in South Korea. Three forest management scenarios, 1) extending the final age of maturity of Pinus densiflora stands (S1), 2) extending the final age of maturity of Quercus acutissima stands (S2), and 3) reforestation with new species (Pinus densiflora to Quercus acutissima) (S3), were examined and evaluated to identify the optimal carbon absorption and value optimized economic perspective. The results of the scenario-based modelling indicated that S3 showed value optimized from an economical perspective, and S2 was the most effective method to absorb carbon among the scenarios. It is anticipated that this paper will contribute to provide valuable information by presenting innovative approaches as a value optimized FCOS implementing tool in a GHG reduction program in South Korea.

Chikungunya Virus nsP2 Impairs MDA5/RIG-I-Mediated Induction of NF-κB Promoter Activation: A Potential Target for Virus-Specific Therapeutics

  • Bae, Sojung;Lee, Jeong Yoon;Myoung, Jinjong
    • Journal of Microbiology and Biotechnology
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    • v.30 no.12
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    • pp.1801-1809
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    • 2020
  • Chikungunya virus (CHIKV) was first identified in 1952 as a causative agent of outbreaks. CHIKV is transmitted by two mosquito species, Aedes aegypti and A. albopictus. Symptoms after CHIKV infection in human are typically fever and joint pain, but can also include headache, muscle pain, joint swelling, polyarthralgia, and rash. CHIKV is an enveloped single-stranded, positive-sense RNA virus with a diameter of approximately 70 nm. The pathogenesis of CHIKV infection and the mechanism by which the virus evades the innate immune system remain poorly understood. Moreover, little is known about the roles of CHIKV-encoded genes in the viral evasion of host immune responses, especially type I interferon (IFN) responses. Therefore, in the present study, we screened CHIKV-encoded genes for their regulatory effect on the activation of nuclear factor kappa B (NF-κB), a critical transcription factor for the optimal activation of IFN-β. Among others, non-structural protein 2 (nsP2) strongly inhibited melanoma differentiation-associated protein 5 (MDA5)-mediated induction of the NF-κB pathway in a dose-dependent manner. Elucidation of the detailed mechanisms of nsP2-mediated inhibition of the MDA5/RIG-I signaling pathway is anticipated to contribute to the development of virus-specific therapeutics against CHIKV infection.

Anti-inflammatory mechanisms of suppressors of cytokine signaling target ROS via NRF-2/thioredoxin induction and inflammasome activation in macrophages

  • Kim, Ga-Young;Jeong, Hana;Yoon, Hye-Young;Yoo, Hye-Min;Lee, Jae Young;Park, Seok Hee;Lee, Choong-Eun
    • BMB Reports
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    • v.53 no.12
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    • pp.640-645
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    • 2020
  • Suppressors of cytokine signaling (SOCS) exhibit diverse anti-inflammatory effects. Since ROS acts as a critical mediator of inflammation, we have investigated the anti-inflammatory mechanisms of SOCS via ROS regulation in monocytic/macrophagic cells. Using PMA-differentiated monocytic cell lines and primary BMDMs transduced with SOCS1 or shSOCS1, the LPS/TLR4-induced inflammatory signaling was investigated by analyzing the levels of intracellular ROS, antioxidant factors, inflammasome activation, and pro-inflammatory cytokines. The levels of LPS-induced ROS and the production of pro-inflammatory cytokines were notably down-regulated by SOCS1 and up-regulated by shSOCS1 in an NAC-sensitive manner. SOCS1 up-regulated an ROS-scavenging protein, thioredoxin, via enhanced expression and binding of NRF-2 to the thioredoxin promoter. SOCS3 exhibited similar effects on NRF-2/thioredoxin induction, and ROS downregulation, resulting in the suppression of inflammatory cytokines. Notably thioredoxin ablation promoted NLRP3 inflammasome activation and restored the SOCS1-mediated inhibition of ROS and cytokine synthesis induced by LPS. The results demonstrate that the anti-inflammatory mechanisms of SOCS1 and SOCS3 in macrophages are mediated via NRF-2-mediated thioredoxin upregulation resulting in the downregulation of ROS signal. Thus, our study supports the anti-oxidant role of SOCS1 and SOCS3 in the exquisite regulation of macrophage activation under oxidative stress.

Qualitative Analysis for Metabolites of Pharmaceuticals Formed in Daphnia magna and Gammarus pulex Using Liquid Chromatogram-High Resolution Mass Spectrometry (LC-HRMS) (LC-HRMS를 이용한 Daphnia magna 및 Gammarus pulex 생체내 의약품 대사체 정성분석)

  • Jeon, Junho
    • Journal of Environmental Analysis, Health and Toxicology
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    • v.21 no.4
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    • pp.243-251
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    • 2018
  • Pharmaceuticals in wastewater effluents have been recognized as emerging pollutants threatening freshwater organisms. To extend understanding for bioaccumulation and toxicity in those organisms, information on biotransformation products (or metabolites) and their metabolic pathway are crucial. The aim of the present study is to identify and elucidate metabolites of pharmaceuticals formed in exposed organisms using suspect and nontarget screening approach using LC-HRMS. As the target pharmaceuticals, carbamazepine, ketoprofen, metoprolol, propranolol, and verapamil were selected whereas Daphnia magna and Gammarus pulex were used as test organisms. After 24h exposure, metabolites formed in the organisms were identified using LC-HRMS. The structures of metabolites were elucidated via analysis of MS/MS fragment pattern and the comparison with fragment database. As the results, a total of 10 metabolites were identified for 5 parent compounds (C253/C356 for carbamazepine, K211 for ketoprofen, M256 for metoprolol, P218/P276/P306 for propranolol, V196/V291/V441 for verapamil). Among them, the presence of C253 and V291 was confirmed using standard materials. Most of the identified metabolites were formed through oxidative reactions such as hydroxylation, N-demethylation, and dealkylation. Cysteine conjugation (phase II reaction) metabolite (C356) for carbamazepine was found in daphnia. The metabolic pathway of verapamil showed similar metabolic pathways and metabolic pathways for both species. Although the toxicological information on the identified metabolites could not be confirmed, the molecular structure information of the proposed metabolites can be used for future evaluation and prediction of toxicity.

Technical Efficiency of the Shrimp Trawl Fishery in Aru and the Arafura Sea, the Eeastern Part of Indonesia

  • MUAWANAH, Umi;KASIM, Kamaluddin;ENDROYONO, Semm;ROSYIDI, Imron
    • Asian Journal of Business Environment
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    • v.11 no.2
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    • pp.5-13
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    • 2021
  • Purpose: This paper aims to study the fishing capacity of the shrimp trawlers in the Aru and Arafura Sea. Research design, data and Methodology: The abundant shrimp resources call for its sustainable use to advance economic activity. Time-series data of effort and catch from shrimp trawlers from 1980-2015 were utilized. The technical efficiencies of the vessels and the target capacity of the shrimp fishery were estimated using Peak to Peak Method. Results: Arafura's shrimp fishery presents high technical efficiency, which is suggested by the high scores of its fishing capacity utilization. The issuance of a ministerial decree on the prohibition of the use of trawls has impacted the plummeting of Arafura's shrimp trawler production right after the regulation implemented in 2015, which is far below the total production of the Arafura's shrimp trawler in 1980. Today, the Aru and Arafura Sea has different sets of fishing gears that are dominated by Squid Jigging and gillnet vessels. Conclusions: Arafura shrimp fishery performs high technical efficiency, which is indicated by the high utilization value of its catching capacity ranging from 71% to 100% annually from 1980 to 2015 This has shown that the resource-utilization has shifted from shrimps to other economically important species during the post trawl ban such as squid and pelagic. Any future intention to utilize shrimp resources, interaction of gears harvesting shrimp with current gears should be well considered in the policy making process.

Heat stress on microbiota composition, barrier integrity, and nutrient transport in gut, production performance, and its amelioration in farm animals

  • Patra, Amlan Kumar;Kar, Indrajit
    • Journal of Animal Science and Technology
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    • v.63 no.2
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    • pp.211-247
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    • 2021
  • Livestock species experience several stresses, particularly weaning, transportation, overproduction, crowding, temperature, and diseases in their life. Heat stress (HS) is one of the most stressors, which is encountered in livestock production systems throughout the world, especially in the tropical regions and is likely to be intensified due to global rise in environmental temperature. The gut has emerged as one of the major target organs affected by HS. The alpha- and beta-diversity of gut microbiota composition are altered due to heat exposure to animals with greater colonization of pathogenic microbiota groups. HS also induces several changes in the gut including damages of microstructures of the mucosal epithelia, increased oxidative insults, reduced immunity, and increased permeability of the gut to toxins and pathogens. Vulnerability of the intestinal barrier integrity leads to invasion of pathogenic microbes and translocation of antigens to the blood circulations, which ultimately may cause systematic inflammations and immune responses. Moreover, digestion of nutrients in the guts may be impaired due to reduced enzymatic activity in the digesta, reduced surface areas for absorption and injury to the mucosal structure and altered expressions of the nutrient transport proteins and genes. The systematic hormonal changes due to HS along with alterations in immune and inflammatory responses often cause reduced feed intake and production performance in livestock and poultry. The altered microbiome likely orchestrates to the hosts for various relevant biological phenomena occurring in the body, but the exact mechanisms how functional communications occur between the microbiota and HS responses are yet to be elucidated. This review aims to discuss the effects of HS on microbiota composition, mucosal structure, oxidant-antioxidant balance mechanism, immunity, and barrier integrity in the gut, and production performance of farm animals along with the dietary ameliorations of HS. Also, this review attempts to explain the mechanisms how these biological responses are affected by HS.