scholarly journals miR-215 Targeting Novel Genes EREG, NIPAL1 and PTPRU Regulates the Resistance to E.coli F18 in Piglets

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1053
Author(s):  
Chao-Hui Dai ◽  
Fang Wang ◽  
Shi-Qin Wang ◽  
Zheng-Chang Wu ◽  
Sheng-Long Wu ◽  
...  
Keyword(s):  
Target Genes ◽  
Core Promoter ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nucleotide Polymorphisms ◽  
Weaned Piglets ◽  
Promoter Regions ◽  
E Coli ◽  
Rnai Technology ◽  
Genes Expression

Previous research has revealed that miR-215 might be an important miRNA regulating weaned piglets’ resistance to Escherichia coli (E. coli) F18. In this study, target genes of miR-215 were identified by RNA-seq, bioinformatics analysis and dual luciferase detection. The relationship between target genes and E. coli infection was explored by RNAi technology, combined with E. coli stimulation and enzyme linked immunosorbent assay (ELISA) detection. Molecular regulating mechanisms of target genes expression were analyzed by methylation detection of promoter regions and dual luciferase activity assay of single nucleotide polymorphisms (SNPs) in core promoter regions. The results showed that miR-215 could target EREG, NIPAL1 and PTPRU genes. Expression levels of three genes in porcine intestinal epithelial cells (IPEC-J2) in the RNAi group were significantly lower than those in the negative control pGMLV vector (pGMLV-NC) group after E. coli F18 stimulation, while cytokines levels of TNF-α and IL-1β in the RNAi group were significantly higher than in the pGMLV-NC group. Variant sites in the promoter region of three genes could affect their promoter activities. These results suggested that miR-215 could regulate weaned piglets’ resistance to E. coli F18 by targeting EREG, NIPAL1 and PTPRU genes. This study is the first to annotate new biological functions of EREG, NIPAL1 and PTPRU genes in pigs, and provides a new experimental basis and reference for the research of piglets disease-resistance breeding.

scholarly journals Elisa preliminary studies of immobilization and specific detection of bacterial strains

2020 ◽  
Vol 2 (2) ◽  
pp. 64-69
Author(s):  
Madalina Mihalache ◽  
◽  
Alina Banciu ◽  
Lucian Ionescu ◽  
Mihai Nita-Lazar
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescence Intensity ◽  
Polyclonal Antibody ◽  
Specific Binding ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Strains ◽  
Specific Detection ◽  
E Coli ◽  
Alexa Fluor

The paper aims to emphasize the specific detection of bacterial strains using enzyme-linked immunosorbent assay. The assay is based on the specific binding of polyclonal antibody anti-E. coli tagged with FITC to E.coli and monoclonal antibody anti-Ps. aeruginosa tagged with Alexa Fluor 647 tagged to Ps. aeruginosa and on subsequent enzymatic immunological demonstration of the conjugated enzyme. In this experiment, the negative control was the Salmonella enterica strain. The two antibodies had no interaction with the negative control, instead, they were specific for E. coli and Ps. aeruginosa strains. When both strains were in the same well, the fluorescence intensity given by the presence of E. coli was 2.3 times higher than that given by Ps. aeruginosa, and the intensity of fluorescence decreased if there are both bacterial strains in the wells.

scholarly journals Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen

2021 ◽  
Vol 18 (18) ◽  
pp. 9630
Author(s):  
Pierre Nsele Mutantu ◽  
Mya Myat Ngwe Tun ◽  
Takeshi Nabeshima ◽  
Fuxun Yu ◽  
Patrick Kakoni Mukadi ◽  
...  
Keyword(s):  
Immunoglobulin G ◽  
Expression System ◽  
Negative Control ◽  
Spike Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
N Protein ◽  
E Coli ◽  
System A ◽  
Recombinant Nucleocapsid Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the assay’s performance, with the plaque reduction neutralization test and the commercial SARS-CoV-2 spike protein IgG ELISA as gold standards. The SARS-CoV-2 N truncated protein-based ELISA showed similar sensitivity (91.1% vs. 91.9%) and specificity (93.8% vs. 93.8%) between the PRNT and spike IgG ELISA, as well as also higher specificity compared to the full-length N protein (93.8% vs. 89.9%). Our ELISA can be used for the diagnosis and surveillance of COVID-19.

scholarly journals DNA Methylation of Pig FUT3 Promoter Alters mRNA Expression to Regulate E. coli F18 Susceptibility

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1586
Author(s):  
Zhengchang Wu ◽  
Dongfeng Shi ◽  
Jian Jin ◽  
Hairui Fan ◽  
Wenbin Bao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Core Promoter ◽  
Inhibitory Effect ◽  
Weaned Piglets ◽  
E Coli ◽  
The Core ◽  
Cpg Sites ◽  
Sp1 Transcription Factor ◽  
Candidate Target

Post-weaning diarrhea (PWD) is frequently associated with E. coli F18 infections in piglets. However, the underlying molecular mechanism concerning the resistance of E. coli F18 in local weaned piglets in China is not clearly understood. In the present study, by a comparative analysis of the transcriptome, a-1,3-fucosyltransferase (FUT3) was evaluated as a key candidate correlated with resistance to E. coli F18 in Sutai and Meishan piglets. Functional analysis demonstrated that FUT3 acts as a key positive regulator of E. coli F18 susceptibility in newly food accustomed piglets. However, the core promoter of FUT3 was present at −500–(−206) bp (chr.2: g.73171117–g.73171616), comprising of 9 methylated CpG sites. Among these, the methylation levels of the two CpG sites (mC-3, mC-5) located in HIF1A and Sp1 transcription factor (TF) considerably associated with mRNA expression of FUT3 (p < 0.05). Our findings indicated that the methylation of mC-3 and mC-5 sites has certain inhibitory effect on FUT3 expression and promotes the resistance of E. coli F18 in piglets. The underlined study may explore FUT3 as a new candidate target in E. coli F18 infection in Chinese local weaned piglets.

scholarly journals Recombinant Co-Expression of Collagen A1 (I) Fragment with the Prolyl 4-Hydroxylases (P4H) Subunits in Komagataella phaffii

2020 ◽  
pp. 65-74
Author(s):  
Esra Avci ◽  
Zülal Kesmen
Keyword(s):  
Target Genes ◽  
Recombinant Expression ◽  
Expression Vectors ◽  
Enzyme Linked Immunosorbent Assay ◽  
E Coli ◽  
Recombinant Strains ◽  
Komagataella Phaffii ◽  
Standard Quality ◽  
Collagen Fragment

Recombinant collagen and collagen-like products are increasingly replacing animal-sourced collagen that is difficult to produce in safe and standard quality. In this study to produce hydroxylated collagen, a 400 base pair collagen fragment of the bovine COL1A1 gene was co-expressed with prolyl-4-hydroxylase subunit α (P4Hα) and prolyl-4-hydroxylase subunit β(P4Hβ) encoding the P4H enzyme in Komagataella phaffii. For this purpose, each target gene was inserted into the pPICZαA vector and then cloned in E. coli DH5α cells. Subsequently, co-expression vectors were constructed using recombinant vectors isolated from positive clones according to the in vitro multimer ligation method. All recombinant expression and co-expression vectors were transformed into K. phaffii X33 cells by electroporation. The results of reverse transcriptase-polymerase chain reaction (PCR) proved that all target genes were transcribed by recombinant strains. The expression of recombinant proteins was performed for 96 hours by methanol-fed cultivation, and the concentration of the purified proteins from the culture medium was measured by the His-Tag enzyme-linked immunosorbent assay (ELISA) method. The concentrations of rP4Hα and rP4Hβ, and rCol1 proteins expressed individually by recombinant strains were determined to be 10.69 µg/L, 10.74 µg/L, and 8.61 µg/L, respectively, while the concentrations of co-expressed rP4Hα/β and rP4Hα/β/rCol1 proteins were 7.82 µg/L and 5.02 µg/L, respectively. These results showed that the target genes were successfully expressed and co-expressed in the recombinant K. phaffii cell.

scholarly journals High-throughput functional analysis of IncRNA core promoters elucidatesrules governing tissue-specificity

2018 ◽  
Author(s):  
Kaia Mattioli ◽  
Pieter-Jan Volders ◽  
Chiara Gerhardinger ◽  
James C. Lee ◽  
Philipp G. Maass ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Tissue Specificity ◽  
Core Promoter ◽  
Expression Profiles ◽  
Nucleotide Polymorphisms ◽  
Promoter Regions ◽  
Single Nucleotide ◽  
Tissue Specific ◽  
Core Promoters ◽  
Motif Density

AbstractTranscription initiates at both coding and non-coding genomic elements, including mRNA and long non-coding RNA (lncRNA) core promoters and enhancer RNAs (eRNAs). However, each class has different expression profiles with lncRNAs and eRNAs being the most tissue-specific. How these complex differences in expression profiles and tissue-specificities are encoded in a single DNA sequence, however, remains unresolved. Here, we address this question using computational approaches and massively parallel reporter assays (MPRA) surveying hundreds of promoters and enhancers. We find that both divergent lncRNA and mRNA core promoters have higher capacities to drive transcription than non-divergent lncRNA and mRNA core promoters, respectively. Conversely, lincRNAs and eRNAs have lower capacities to drive transcription and are more tissue-specific than divergent genes. This higher tissue-specificity is strongly associated with having less complex TF motif profiles at the core promoter. We experimentally validated these findings by testing both engineered single-nucleotide deletions and human single-nucleotide polymorphisms (SNPs) in MPRA. In both cases, we observe that single nucleotides associated with many motifs are important drivers of promoter activity. Thus, we suggest that high TF motif density serves as a robust mechanism to increase promoter activity at the expense of tissue-specificity. Moreover, we find that 22% of common SNPs in core promoter regions have significant regulatory effects. Collectively, our findings show that high TF motif density provides redundancy and increases promoter activity at the expense of tissue specificity, suggesting that specificity of expression may be regulated by simplicity of motif usage.

scholarly journals Regulation and Molecular Mechanism of TLR5 on Resistance to Escherichia coli F18 in Weaned Piglets

Animals ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 735 ◽  
Author(s):  
Chaohui Dai ◽  
Li Yang ◽  
Jian Jin ◽  
Haifei Wang ◽  
Shenglong Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanism ◽  
Promoter Region ◽  
Cellular Localization ◽  
Cpg Island ◽  
Cell Damage ◽  
Core Promoter ◽  
Weaned Piglets ◽  
E Coli ◽  
Tlr5 Expression

Toll-like receptor 5 (TLR5) plays an important role in immune system. In this study, we performed transcriptome analysis of the duodenum in E. coli F18-resistant and -sensitive Sutai weaned piglets and analyzed the differential expression of TLR5. The cellular localization of TLR5 was investigated, and the effect of TLR5 expression on E. coli invasion was evaluated after pig small intestinal epithelial cell lines (IPEC-J2) were stimulated by E. coli. The results showed that TLR5 expression level in duodenum and jejunum were significantly higher in E. coli F18-sensitive than in E. coli F18-resistant piglets. TLR5 protein was mainly expressed in the cytoplasm and cell membrane. The expression of genes associated with the TLR5 signaling pathway were significantly higher in TLR5-overexpressed cells than in control cells. Bacterial adhesion was higher in TLR5-overexpressed cells than in blank cells and lower in TLR5 interference than in blank cells. The core promoter region of TLR5 included two CpG islands and 16 acting elements. The methylation of the mC-6 site in the second CpG island of the promoter region had a regulatory effect on TLR5 expression. Therefore, TLR5 plays an important regulatory role on E. coli invasion. Low expression of TLR5 inhibited the immune response and decreased cell damage, which was conducive to the resistance to E. coli stimulation. In conclusion, this study preliminarily revealed the molecular mechanism of TLR5 gene regulating the resistance of piglets to Escherichia coli, and provided a new candidate gene for screening Escherichia coli resistance markers in pigs.

19 Dietary Supplementation of Botanicals Enhanced Growth Performance and Disease Resistance of Weaned Pigs Experimentally Infected with a Pathogenic E. coli

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 29-29
Author(s):  
Braden T Wong ◽  
Yijie He ◽  
Kwangwook Kim ◽  
Sharon Xu ◽  
Christopher Lingga ◽  
...  
Keyword(s):  
Body Weight ◽  
Growth Performance ◽  
Block Design ◽  
Average Daily Gain ◽  
Dietary Supplementation ◽  
Negative Control ◽  
High Dose ◽  
Weaned Piglets ◽  
Weaned Pigs ◽  
E Coli

Abstract The objective of this study was to investigate the effects of dietary botanical supplementation on growth performance and frequency of diarrhea of weaned piglets experimentally infected with a pathogenic F18 Escherichia coli (E. coli). Sixty weaned piglets (around 21 days old; 7.15 ± 0.97 kg) were individually housed and randomly allotted to one of five dietary treatments (n = 12): negative control (NC), positive control (PC), high dose of botanicals blend 1 (BB1, 100 ppm), low dose of botanicals blend 2 (BB2, 50 ppm), and high dose of botanicals blend 2 (BB2, 100 ppm). The experiment lasted 28 days: from day -7 to +21 relative to E. coli inoculation. All piglets except the pigs in the NC group were orally inoculated with F18 E. coli (1010 cfu per dose, 3 doses) for 3 consecutive days. Growth performance was recorded throughout the experiment and diarrhea scores were recorded daily. Data were analyzed by ANOVA using PROC MIXED of SAS with a randomized complete block design. E. coli challenge reduced (P &lt; 0.05) pig body weight and growth rate throughout the experiment. Pigs supplemented with high dose BB1 or BB2 tended (P &lt; 0.10) to have greater body weight (19.52 and 19.10 vs. 18.00 kg) on d 21 PI and greater average daily gain from d 0 to 21 PI (554 and 557 vs. 515 g/d) than PC. No differences were observed in pig performance between high dose BB1 or BB2 in comparison with NC. Supplementation of high dose BB1 or BB2 also reduced (P &lt; 0.05) frequency and severity of diarrhea of challenged pigs during the entire experimental period. In conclusion, dietary supplementation of botanicals reduced diarrhea and tended to improve growth performance of weaned pigs infected with E. coli.

Nuclear receptors in disease: the oestrogen receptors

2004 ◽  
Vol 40 ◽  
pp. 157-167 ◽  
Author(s):  
Maria Nilsson ◽  
Karin Dahlman-Wright ◽  
Jan-Åke Gustafsson
Keyword(s):  
Nuclear Receptors ◽  
Target Genes ◽  
Receptor Subtype ◽  
Target Tissue ◽  
Oestrogen Receptors ◽  
Nucleotide Polymorphisms ◽  
Cell Type ◽  
Single Nucleotide ◽  
Beneficial Effects ◽  
The Family

For several decades, it has been known that oestrogens are essential for human health. The discovery that there are two oestrogen receptors (ERs), ERalpha and ERbeta, has facilitated our understanding of how the hormone exerts its physiological effects. The ERs belong to the family of ligand-activated nuclear receptors, which act by modulating the expression of target genes. Studies of ER-knockout (ERKO) mice have been instrumental in defining the relevance of a given receptor subtype in a certain tissue. Phenotypes displayed by ERKO mice suggest diseases in which dysfunctional ERs might be involved in aetiology and pathology. Association between single-nucleotide polymorphisms (SNPs) in ER genes and disease have been demonstrated in several cases. Selective ER modulators (SERMs), which are selective with regard to their effects in a certain cell type, already exist. Since oestrogen has effects in many tissues, the goal with a SERM is to provide beneficial effects in one target tissue while avoiding side effects in others. Refined SERMs will, in the future, provide improved therapeutic strategies for existing and novel indications.

The Order in Which Antibiotics are Administered Affects the Fitness Costs of Adherent-Invasive E. coli (AIEC) Strains

2019 ◽  
Author(s):  
Jordan B Gregg
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Combination Therapy ◽  
Agar Plate ◽  
Sequential Therapy ◽  
Negative Control ◽  
Clinical Settings ◽  
Fitness Costs ◽  
Inflammatory Gene Expression ◽  
E Coli

AIEC-LF82 is a strain of bacteria that is surmised to have a role in causing IBD and Crohn’s disease by activating pro-inflammatory gene expression in organisms. Using antibiotics via combination therapy has been a technique used in clinical settings in an attempt to treat the strains, however, the attempts have not been that effective nor efficient in terms of completely halting the growth and colonization of AIEC to treat IBD and Crohn's disease patients. Research has shown that regarding hindering or preventing the colonization bacterial colonies, sequential therapy tends to be more effective and time-efficient than combination therapy, with fewer adverse effects. To test if this is also the case with the AIEC-LF82 strain of bacteria, I first tested AIEC’s response to combination therapy using the Penicillin-Streptomycin, Kanamycin-Chloramphenicol, antimicrobial peptide (AMP), Kanamycin, SPE phase and LB agar plates, all of which were experimental plates other than the LB agar plate that acted as the negative control. I then tested AIEC-LF82’s response to sequential therapy using the LB+ Kan + Spe, LB + AMP + Spe, LB+ Kan/Cam + Spe, LB + P/S + Spe, LB + P/S + Kan and LB + P/S + AMP and one LB agar plate acting as the negative control. The only differences between sets a and b were the order in which antibiotics were administered in the six aforementioned treatment sets. Ultimately, I found that set b of sequential therapy, strong-weak antibiotic treatments, was the most effective treatment but that set a regarding sequential therapy was actually the least effective of all of the treatments. In conclusion, using strong-weak sequential antibiotic therapy treatments appears to be a potentially promising option to treat patients suffering from Crohn's disease and IBD.

Sign in / Sign up

Export Citation Format

Share Document