Invasion of Caco-2 Cells by Salmonella typhimurium (Copenhagen) Isolates from Healthy and Sick Chickens
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In a previous study, Salmonella isolates of sick birds were distinguished from those of apparently healthy birds by their high degree of invasion of tissue culture cells. In this study, a single pair of Salmonella isolates was examined to determine the source of this observed difference in invasion. When isolates were allowed to invade Caco-2 cells for 8 hours, the isolate from the sick bird (S) appeared to invade in greater numbers than did the isolate from the healthy bird (H). However, when invasion was distinguished from intracellular growth/survival, it was found that H invaded in greater numbers than S, but once inside the cell, H declined in number, and S increased. Inhibition of RNA, protein, and DNA syntheses lessened the degree to which both invaded. The presence of mannose inhibited invasion by S but did not appear to inhibit invasion by H. Trypsin treatment of monolayers affected invasion of S and H, whereas neuraminidase treatment did not. There was no significant difference noted between S and H in ability to adhere to fixed monolayers. Therefore, the two isolates tested differ in their mechanisms of entry into Caco-2 cells, the efficiency with which they invade, and their ability to survive within Caco-2 cells.Keywords:
Gentamicin protection assay
Caco-2
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Patterns of invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin in Caco-2 cells (without centrifugation) were compared with previously published studies of the rabbit ileal invasion assay (RIIA) and (where relevant) a HEp-2 cell invasion assay. Optimal conditions for the use of Caco-2 cell monolayers in bacterial invasion assays were defined. Centrifuge-assisted attachment of bacteria to cells was not used routinely as this increased the invasiveness of known hypo-invasive strains and detachment of Caco-2 cells. Inocula with too high bacterial numbers resulted in rapid acidification of media and detachment of the monolayers. The invasiveness of Typhimurium strains TML, WAKE, WII8, LT7, SL1027 and M206 in Caco-2 cells reflected that seen in the RIIA. The invasiveness of Choleraesuis strain A50 was similar to that in the RIIA except that bacteria grown at 37 degrees C and used without storage at 4 degrees C were slightly more invasive than those grown at 37 degrees C and stored at 4 degrees C before use. Dublin strain 3246 showed no apparent temperature-regulated invasiveness in Caco-2 cells, in contrast to the results observed in the RIIA. Dublin strain 3246 did not cleave tight junctions in the Caco-2 cell monolayer as it did in rabbit ileal epithelia both in vitro and in vivo. Three TnphoA insertion LPS mutants of Typhimurium TML were uniformly hypo-invasive in both Caco-2 cells and the RIIA; in contrast, they were differentially invasive in HEp-2 cells. Three smooth TnphoA insertion mutants of Typhimurium TML (invH, invG and pagC) were hypo-invasive in both the Caco-2 and HEp-2 cell invasion assays but not in the RIIA.
Caco-2
Gentamicin protection assay
Tryptic soy broth
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Objective To study the effect and molecular mechanism of EGFR pathway mediating proliferation and invasion of Caco-2 cell line.Methods The tetrazolium-based colorimetric assay was used to evaluate the cell growth treated by EGF,AG1478 or PD98059.The in vitro invasion assay was used to examine the invasiveness of Caco-2 cells treated by EGF, AG1478 or PD98059.MMP-2,MMP-9,TIMP-1 and TIMP-2 mRNA of Caco-2 cells were measured by RT-PCR.Expressions of P-EGFR and P-ERK protein of Caco-2 cells were determined by Western blotting.Results Exogenous EGF enhanced significantly the growth and proliferation of Caco-2 cells.The growth ratio increased 23.35%(P0.01) at 24 h.AG1478(20 μmol/L) as EGFR inhibitor and PD98059(40 μmol/L) as ERK inhibitor inhibited the growth and proliferation of Caco-2 cells in a time-independent manner(P0.01).In vitro invasion assay showed that 10 μg/L EGF increased the invasion(P0.01) of Caco-2 cells,while both AG1478 and PD9805 inhibited EGF-induced invasion of Caco-2 cells(P0.01).RT-PCR assays revealed that exogenous EGF up-regulated mRNA levels of MMP-2 and MMP-9 and down-regulated mRNA levels of TIMP-1 and TIMP-2,and AG1478 and PD98059 decreased the levels of MMP-2,MMP-9 mRNA and increased the levels of TIMP-1 and TIMP-2 mRNA.The MMP-2 to TIMP-2 ratio and the MMP-9 to TIMP-1 ratio were decreased significantly by AG1478 or PD98059(P0.01).Conclusion EGFR signal pathway adjusts the growth,invasion and metastasis of human colon carcinoma cells.The mechanisms may be that EGFR changes matrix metalloproteinases and its inhibitors by ERK/MAPK signal pathway.
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Gentamicin protection assay
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This chapter contains sections titled: Introduction Influenza Neuraminidase as a Drug Target Neuraminidase Active Site and Inhibitor Binding Small-Molecule Inhibitors of Influenza Neuraminidase Mechanism of Resistance Influenza Neuraminidase Inhibitors Based on Other Scaffolds Clinical Use of Neuraminidase Inhibitors Concluding Remarks References
Neuraminidase inhibitor
Oseltamivir
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Salmonella is capable of harming human and animal health, and its multidrug resistance (MDR) has always been a public health problem. In addition, antibiotic-free or antibiotic-reduced policies have been implemented in poultry production. Therefore, the search for antibiotic alternatives is more urgent than ever before. The aim of this study was to assess the antibacterial activity of star anise-cinnamon essential oil (SCEO) in vitro and its prophylactic effect against the infections of Salmonella pullorum, Salmonella give, and Salmonella kentucky in vivo. The results demonstrated that SCEO is effective against Salmonella pullorum, Salmonella give, and Salmonella kentucky in vitro. Supplementation with SCEO could significantly decrease the infections of Salmonella pullorum and Salmonella give, whereas it could slightly but not significantly decrease the infection of Salmonella kentucky, while also significantly alleviating the body weight (BW) loss caused by the infections of Salmonella pullorum, Salmonella give, and Salmonella kentucky in Yellow chickens. The SCEO had the best prophylactic effect against the infection of Salmonella give in Yellow chickens, followed by the infection of Salmonella pullorum and the infection of Salmonella kentucky. The SCEO, used as an antibiotic alternative, could be an effective prevention strategy against the infections of Salmonella pullorum, Salmonella give, and Salmonella kentucky in Yellow chickens.
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Neuraminidase inhibitor
High-Throughput Screening
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Objective:To determine the inhibitory effect of compounds L20051117 and AMMS607 on influenza virus neuraminidase at molecular level.Methods:The anti-influenza virus neuraminidase activity of L20051117 and AMMS607 was examined by the fluorimetric assay.Results:The inhibitory effect of L20051117 on influenza virus neuraminidase was higher than that of AMMS607,the inhibition of influenza A virus neuraminidase by L20051117 and AMMS607 was higher than that of influenza B virus neuraminidase.Conclusion:L20051117 is a potent inhibitor of the neuraminidase activity of both influenza A and B virus.Its in vitro activity is higher than AMMS607.
Neuraminidase inhibitor
Influenzavirus B
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본 연구에서는 인플루엔자 바이러스 표면에 존재하는 neuraminidase 효소의 활성을 평가 할 수있는 종이칩 기반의 분석 시스템을 구축하였다. 종이칩의 장점을 살려 분석 전문가와 장비 없이 현장 진단(Point-of-care)이 가능하도록 X-Neu5Ac 기질을 이용한 비색분석법을 통해 시료 내 neuraminidase 효소의 존재를 정량적으로 확인 할 수 있도록 설계 및 제작하였다. Neuraminidase 효소의 활성을 확인할 수 있는 종이칩 센서(Paper-based neuraminidase assay sensor; PNAS) 성능 실험 결과 neuraminidase를 0.004 U/mL 농도부터 검출 가능하였으며, 인간 혈청에 각기 다른 농도로 존재하는 neuraminidase 효소의 양을 활성 평가를 통해 정량적으로 검출할 수 있음을 입증하였다($R^2$ > 0.99). 또한, 보관기간에 따른 종이칩의 안정성 평가 결과 빛이 차단 된 $4^{\circ}C$ 환경에서 보관 시 70일까지 초기 성능이 안정하게 유지됨을 확인하였다. 마지막으로, PNAS 상에서 효소 반응의 신뢰성 평가를 위해 미카엘리스-멘텐 동역학 (Michaelis-Menten kinetics)을 적용하여 X-Neu5Ac 기질에 대한 neuraminidase의 동역학 분석 결과 $K_m$ 값은 $8.327{\times}10^{-3}M$ 으로 확인되었으며, 이 값은 용액상에서의 효소 반응 속도 계산으로부터 산출된 값과($K_m=8.327{\times}10^{-3}M$ ) 근사한 수치임을 확인하였다. 본 연구로부터 개발된 종이칩 기반의 neuraminidase 효소 활성 평가 시스템은 인플루엔자 바이러스의 신속하고 안전한 검출에 다양하게 응용 될 수 있을 것으로 생각된다. In this study, we described a paper-based neuraminidase assay sensor (PNAS) which can be applied to detect the infection by influenza viruses. The PNAS was designed and manufactured to quantitatively identify the levels of neuraminidase in the sample, which is based on colorimetric analysis using the X-Neu5Ac substrate. The limit of detection of the PNAS was determined as 0.004 U/mL of neuraminidase. According to the amount of neuraminidase in human serum, the PNAS could monitor the enzyme activity with a good linearity ($R^2$ > 0.99). In addition, the initial performance of the PNAS has been maintained up to 70 days in the $4^{\circ}C$ . Finally, we demonstrated whether the Michaelis-Menten kinetics is applied to the PNAS, which can show the reliability of the enzyme reactions. The kinetic studies indicated that the PNAS provides the good condition for enzyme reactions ($K_m=8.327{\times}10^{-3}M$ ), but they were performed on paper chip nonetheless. The paper-based neuraminidase assay sensor may be useful in a wide range of rapid and safe detection of influenza virus.
Neuraminidase inhibitor
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To determine whether the requirements for sialic acid varies and whether several types of silaic acid independent receptors utilized for invasion mechanisms of fresh filed isolates collected around Nanay river basin, Iquitos. The field isolates were cultured as described previously by Jensen and Trager and MR4 protocol with little modifications. The erythrocytes preparation and subsequent enzyme treatment was done as described previously by Sharma. with little modification. Invasion assay was performed as described previously by Sharma et al with little modification. The Nanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions. Here we observed that an equal numbers of neuraminidase sensitive and resistant invasion receptor-ligand interaction profiles as the most common receptor-ligand invasion profiles. Neuraminidase resistance trypsin sensitive chymotrypsin sensitive (NMRTSCTS) invasion of receptor-ligand interaction profile was found in seven isolates, Five field isolates and one reference strain showed neuraminidase sensitive, trypsin sensitive and chymotrypsin resistant (NMSTSCTR) invasion of receptor-ligand interactions, six isolates including one reference strains dd2 showed neuraminidase sensitive, trypsin and chymotrypsin resistance (NMSTRCTR) indicating its dependence on sialic acids and independence of trypsin and chymotrypsin sensitive proteins. Four isolates showed neuraminidase sensitive, trypsin sensitive and chymotrypsin sensitive (NMSTSCTS) invasion of receptor-ligand interactions, seven isolates were neuraminidase resistant, trypsin sensitive and chymotrypsin resistance (NMRTSCTR) invasion of receptor-ligand interactions, indicating its dependence on trypsin sensitive proteins. The Nanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions. A full understanding of theses invasion mechanisms may allow the development of novel prophylactic and therapeutic strategies that block erythrocyte receptor-ligand invasion mechanisms.
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Objective:To determine whether the requirements for sialic acid varies and whether several types of silaic acid independent receptors utilized for invasion mechanisms of fresh filed isolates collected around Nanay river basin,Iquitos.Methods:The field isolates were cultured as described previously by Jensen and Trager and MR4 protocol with little modifications.The erythrocytes preparation and subsequent enzyme treatment was done as described previously by Sharma.with little modification.Invasion assay was performed as described previously by Sharma et al with little modification.Results:The Nanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions.Here we observed that an equal numbers of neuraminidase sensitive and resistant invasion receptor-ligand interaction profiles as the most common receptor-ligand invasion profiles.Neuraminidase resistance trypsin sensitive chymotrypsin sensitive(NM_RT_SCT_S) invasion of receptor-ligand interaction profile was found in seven isolates,Five field isolates and one reference strain showed neuraminidase sensitive, trypsin sensitive and chymotrypsin resistant(NM_RT_SCT_R) invasion of receptor-ligand interactions, six isolates including one reference strains dd2 showed neuraminidase sensitive,trypsin and chymotrypsin resistance(NM_ST_RCT_R) indicating its dependence on sialic acids and independence of trypsin and chymotrypsin sensitive proteins.Four isolates showed neuraminidase sensitive, trypsin sensitive and chymotrypsin sensitive(NM_ST_SCT_S) invasion of receptor-ligand interactions, seven isolates were neuraminidase resistant,trypsin sensitive and chymotrypsin resistance (NM_RT_SCT_R) invasion of receptor-ligand interactions,indicating its dependence on trypsin sensitive proteins.Conclusions:The Nanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions.A full understanding of theses invasion mechanisms may allow the development of novel prophylactic and therap
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