基于XGBoost方法的大肠杆菌-NC膜复合电极阻抗模型研究

将机器学习方法用于分析大批量大肠杆菌菌液浓度以及临界低阈值抗生素抑菌效果评价。采用XGBoost机器学习算法,构建金电极上大肠杆菌-NC膜贴附模型,用于检测不同浓度大肠杆菌的电化学阻抗谱。在此基础上,分析不同浓度抗生素硫酸阿米卡星作用于标准浓度大肠杆菌所对应的阻抗谱变化。根据Randles等效电路,使用ZView软件拟合阻抗曲线得到所对应的7个电化学参数,通过主成分分析法,依据选取信息量前90%的原则,提取R_s、CPE-P、CPE-T、R₁等4个参数作为XGBoost预测模型输入,分别以菌液浓度、抗生素浓度为预测值,建立大肠杆菌菌液浓度预测模型和抗生素浓度预测模型。两组实验预测结果均与实际结果吻合,菌液预测浓度平均均方根误差(RMSE)为2.18×10^-3 lg CFU/mL,每组样本预测浓度最大上下限差值在1.49×10⁷ CFU/mL之内;抗生素预测浓度平均均方根误差(RMSE)为7.45×10^-3 μL/mL,回归精度达0.01 μL/mL,实现了大肠杆菌浓度及抗生素含量快速准确预测。因此,基于XGBoost的大肠杆菌-NC膜阻抗模型可定量分析菌液浓度以及临界低阈值抗生素抑菌效果评价,从而可对电极表面的微量蛋白质和细菌等生物膜贴附后造成的长时程阻抗变化进行定量检测评估,在食品安全领域电化快速检测中具有较大的应用价值。     

Specific regions of genome plasticity and genetic diversity of the commensal Escherichia coli A0 34/86

Escherichia coli A0 34/86 (O83:K24:H31) is a commensal strain that has been used for prophylactic and therapeutic colonization of the intestine of newborn infants. To identify traits specific for E. coli A0 34/86, we used a minimal tiling set of 148 BAC clones of A0 34/86 genomic DNA, to construct restriction-digested BAC arrays. Hybridization with genomic DNA from four E. coli strains (CFT073; O157:H7; K12 and Nissle 1917) allowed selection of two BAC clones that were sequenced to identify A0 34/86-specific regions. Genes for the yersiniabactin siderophore system, several proteins homologous to Salmonella enterica serovar Typhimurium vitamin B₁₂ synthesis proteins, as well as genes necessary for the degradation of propanediol, the pix fimbriae determinant and genes coding for a putative phosphoglycerate transport system present also on pathogenicity island V of E. coli strain 536 were all identified in E. coli A0 34/86. This comparative analysis underlines the important genome heterogeneity between E. coli strains.     

Immunoreactivity of Recombinant Carcinoembryonic Antigen Proteins Expressed in Escherichia Coli

Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E. coli) were analyzed in relation to the CEA domain structure [domains N, I (A1—B1), II (A2—B2), III (A3—B3) and M). We reconstructed in a prokaryotic expression vector, pUCPL-cI, the cDNAs for CEA-N, CEA-I. CEA-II, and CEA-III-M. The latter three were expressed as fusion products with bacterial β-galactosidase. The recombinant proteins were solubilized by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution. Their molecular weights Judged from Western blotting coincided with those calculated from their cDNA sequences, respectively. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E. coli and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells. The reactivities of 5 MAbs with the recombinant proteins expressed in E. coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the epitopes identified with the recombinant CEA proteins expressed in CHO cells. The remaining 2 MAbs did not react with any recombinant protein expressed in E. coli. These results indicate that the fusion CEA-proteins expressed in E. coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested. However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E. coli.     

Immune Response of E. cuniculi Infected Mice to Aflatoxin B1

The immunomodulatory effects of aflatoxin B₁ (AFB₁) on mice experimentally infected with Encephalitozoon cuniculi (E. cuniculi) were studied. Mice inoculated intraperitoneally with spores of E. cuniculi drank a daily solution of AFB₁ (0.2 mg kg^-1 of body weight) for 27 days. Application of AFB₁ to mice demonstrated a decrease of immunocompetent cells. On the other hand, the mice infected with E. cuniculi and given AFB₁ showed significant increased number of both monocytes and CD8+ T cells, and tendency to a decrease in CD4+ T cells. Aflatoxin B₁ revealed to merely modulate systemic immune response of E. cuniculi infected mice.     

Effect of Freezing Colostrum on Resistance of Neonatal Lambs to Experimental Infection with Escherichia coli

Effects of freezing and thawing colostrum on resistance of neonatal lambs to experimental infection with Escherichia coli were evaluated using 16 newborn lambs. Eight sets of twins were fed colostrum from the ewe at 3.3 and 15.4 h of age. Colostrum was obtained from the ewe and divided into two equal portions. One portion was frozen in liquid nitrogen and then thawed in a water bath prior to feeding. The second portion was held at approximately 39°C in a water bath. Four sets of twins were orally inoculated with 3 ×10⁸ to 10¹¹ cfu of enterotoxigenic E. coli at 24 h of age. Blood was sampled at 0 and 24 h for IgG and differential leukocyte counts. Freezing and thawing reduced cell viability in colostrum from 43.1 to 10.1%. Neither freezing and thawing colostrum nor E. coli inoculation affected plasma IgG or total or differential leukocyte counts, fecal scores, respiration rates, rectal temperatures, fecal coliform excretion or intake. Shedding of K99+ E. coli was increased and body weight gain from 7 to 14 d was decreased when lambs were inoculated with E. coli. Results of this study suggest that freezing and thawing colostrum does not destroy components that provide resistance to E. coli challenge in newborn lambs.     

Identification and characterization of "pathoadaptive mutations" of the cadBA operon in several intestinal Escherichia coli

The dysenteric Shigella spp. and enteroinvasive Escherichia coli (EIEC) have evolved from commensal E. coli by the acquisition of a virulence plasmid and inactivation of genes of the cad locus encoding lysine decarboxylase (LDC) by so-called pathoadaptive mutation. As horizontal gene transfer and recombination occurs frequently in E. coli we were interested to see if similar pathoadaptive mutations are commonly present in other intestinal pathotypes. Therefore, we examined 140 intestinal E. coli strains of various pathotypes and the ECOR collection for their ability to decarboxylate lysine, and identified 25 strains that were unable to do so. Complementation of a Shiga toxin-producing E. coli and two enteropathogenic E. coli strains, both LDC-negative, with the intact cad locus restored LDC activity and resulted in a reduction in adherence to tissue culture cells. We investigated the cad locus for possible alterations by using hybridization and PCR techniques and compared the results with the alterations reported for Shigella spp. and EIEC strains. Interestingly, the alterations of the cad genes were similar to those previously reported, pointing towards a parallel evolution of LDC silencing in different intestinal E. coli pathotypes.     

An ultrastructural study of enteropathogenic Escherichia coli infection in human infants

Over the past 2 years, we have studied and treated 18 infants with protracted diarrhea due to an enteropathogenic Escherichia coli serogroup 0119. All patients had persistent stool escretion and jejunal overgrowth with this pathogenic E. coli. Jejunal biopsy revealed atrophy of villi with a chronic inflammatory cell infiltrate in the lamina propria. E. coli 0119 adhered to the luminal surface of enterocytes. Electron microscopy showed disappearance of glycocalyx and microvilli at the areas of bacterial adherence. Intracellular damage was indicated by dilatation of rough endoplasmic reticulum, mitochondrial changes, and cytoplasmic pallor. Similar changes in histology and ultrastructure occurred in ileal epithelial cells. Glandular crypt epithelium showed prominent subnuclear vacuolation and separation of lateral intercellular junctions throughout the small intestine. Rectal mucosal biopsy showed mucus depletion and irregular atrophy of the epithelium, with E. coli 0119 adherent to the luminal surface. Ultrasuctural damage paralleled that in the small intestine. E. coli 0119 causes damage to epithelial cells throughout the infant intestinal tract. This damage leads to atrophy of villi and a marked reduction in absorptive surface area, resulting in protracted diarrhea.     

Profile of Chicken Macrophage Functions After Exposure to Catecholamines In Vitro

The effects of catecholamines (CA) on various chicken macrophage functions were examined. Macrophage monolayers were exposed to .01, .1, .25, 1, 2, and 5 (μg/mL of dopamine (DA), norepinephrine (NE) and epinephrine (E) for 1 hr. All CA were toxic for macrophages at 1 -5 μg dose range resulting in 25-50% cell death. All CA at the .1 and .25 μg/mL level increased E. coli and sheep red blood cells (SRBC) phagocytosis by macrophages. the percentage of Fc-receptor positive macrophages increased after CA exposure. Prolonged exposure of macrophages (3 hr) reduced SRBC phagocytosis by DA-treated but not in NE-and E-treated macrophages. However, after 1 hr exposure and 3 hr recovery period, CA-induced changes were reversed in all but DA-treated cultures. Apomorphine and metoclopromide blocked DA whereas propranolol blocked NE and E effects suggesting specificity of the observed effects via catecholaminergic receptors on chicken macrophages. Dopamine and NE (.25 μg/mL) did not affect but E exposure enhanced LPS-induced tumoricidal factor production. These findings suggest that CA modulate chicken macrophage effector functions.     

Efficient recovery of a functional extracellular domain of bovine IgG2 Fc receptor (boFcgamma2R) from inclusion bodies by a rapid dilution refolding system

The extracellular domain of the boFcγ2R gene was constructed and cloned into the Escherichia coli expression vector pET-28a. The recombinant protein was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine hydrochloride and refolded by rapid dilution. After renaturation, the purity of the recovered recombinant protein was up to 95%. ELISA assay showed that the renatured recombinant protein could inhibit bovine IgG2 binding to expressed boFcγ2R on the COS-7 cell surface with an IC50 value of 0.68 μM. The overall yield of the active rsbo2R was up to 20 mg/l of culture. Crystals of the rsbo2R were grown at 293 K by the hanging-drop vapour diffusion method showed weak diffraction.     

Differential Effect of Mixed D1/D2 and Selective D2 Dopaminergic Antagonists on Mouse T and B Lymphocyte Proliferation and Interleukin Production In Vitro

The effects of some dopaminergic antagonists were investigated on mouse lymphocyte proliferative responses in vitro. The mixed D₁/D₂ dopaminergic antagonists chlorpromazine, haloperidol and fIupentixol inhibited ³H-Thymidine incorporation into adult BALB/c mouse spleen cells stimulated by concanavalin A, lipopolysaccharide from Escherichia coli, and allogenic cells in a mixed lymphocyte reaction. The inhibition was achieved at concentrations greater than 10^-6M. It was not accounted for by decreased cell viability and it was no longer demonstrable when the compound was added 24h or 48h after the mitogenic stimulus. Conversely selective D₂ dopaminergic antagonists sulpiride, metoclopramide and domperidone had no inhibitory effect at concentrations ranging from 10^-9 to 10^-5 or 10^-4M. The three mixed D_l/D₂ antagonists inhibited the mitogenic effect of interleukin-1 on concanavalin A-stimulated thymocytes, but not the activity of interleukin-2 on the proliferaiton of the CTLL-2 cell line. The mixed D_l/D₂ antagonists interfered with the production of interleukin-2 but not with that of interleukin-1. These results indicate that dopaminergic antagonists may differerentially affect lymphocyte proliferative responses to T or B cell mitogens or alloantigens. The mechanisms involved in terms of receptor specific or non specific phenomenons are discussed.     

Differentiation of fecal Escherichia coli from poultry and free-living birds by (GTG)5-PCR genomic fingerprinting

Determination of the non-point sources of fecal pollution is essential for the assessment of potential public health risk and development of appropriate management practices for prevention of further contamination. Repetitive extragenic palindromic-PCR coupled with (GTG)₅ primer [(GTG)₅-PCR] was performed on 573 Escherichia coli isolates obtained from the feces of poultry (chicken, duck and turkey) and free-living (Canada goose, hawk, magpie, seagull and songbird) birds to evaluate the efficacy of (GTG)₅-PCR genomic fingerprinting in the prediction of the correct source of fecal pollution. A discriminant analysis with the jack-knife algorithm of (GTG)₅-PCR DNA fingerprints revealed that 95%, 94.1%, 93.2%, 84.6%, 79.7%, 76.7%, 75.3% and 70.7% of magpie, hawk, turkey, seagull, Canada goose, chicken, duck and songbird fecal E. coli isolates classified into the correct host source, respectively. The results of this study indicate that (GTG)₅-PCR can be considered to be a complementary molecular tool for the rapid determination of E. coli isolates identity and tracking the non-point sources of fecal pollution.     

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (l-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis of l-arginine to l-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K_M and V_max values of 23.9(±0.96) mM and 192.3 μmol/min mg protein (±14.3), respectively, for the native enzyme. For the recombinant counterpart, K_M was 21.5(±0.90) mM and V_max was 144.9(±8.9) μmol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy.     

Modulation of cellular immune response by orbifloxacin in noninfected and E. coli-infected mice

The studies were conducted on noninfected and Escherichia (E) coli-infected mice treated with orbifloxacin administered orally 10 times at 24-hr intervals at a dose of 2.5 mg/kg. Orbifloxacin did not change the activity of peritoneal macrophages in noninfected mice. Administration of orbifloxacin in E. coli-infected mice modulated the effects of infection on the percentage of phagocyting macrophages, the percentage of NBT-positive cells, and nitric oxide production. Orbifloxacin did not affect the synthesis and release of interleukin-1 by macrophages. Orbifloxacin exerted a modulating effect on the subsets of lymphocytes in thymus, spleen, and mesenteric lymph node cells in noninfected and E. coli-infected mice.     

Effect of Saccharomyces boulardii and Bacillus cereus var. toyoi on the humoral and cellular response of mice to vaccines

The effect of Saccharomyces boulardii and Bacillus cereus var. toyoi on the humoral and cellular response of mice to the simultaneous inoculation of a tetravalent E. coli bacterin and a viable vaccine against Canine Parvovirus is reported. Thirty-six isogenic BALB-c female mice, seven weeks old, were vaccinated with 1/20th of the dose used for pigs and dogs, respectively, and randomly divided into three groups. Group 1 received non-supplemented feed, group 2 was supplemented with S. boulardii, and group 3 with Bacillus cereus var. toyoi. The humoral response was quantified through ELISAs using specific antigens, and the cellular response quantifying IL-4 and IFNg through ELISA. Means of seroconversions to the bacterin were always higher in supplemented mice than in controls, varying from 1.6-1.8 for group 2 and from 1.2-1.4 for group 3. Seroconversions against Parvovirus for group 2 were 5.2-12 times higher, and those of group 3 were 6.8-9.1 times higher than those of controls. IL-4 was produced by spleen cells of S. boulardii supplemented mice stimulated with E. coli fimbriae.     

Programmed aging or error catastrophe? An exmination by two-dimensional polyacrylamide gel electrophoresis

We have examined newly synthesized proteins in the young adult and in older populations of the nematode Caenorhabditis elegans using two-dimensional polyacrylamide gel electrophoresis (2D PAGE). A temperature-sensitive mutant strain, DH26, with a mean life span of about 15 days, under our conditions, was used to block progeny development. Nematodes of several different ages were pulse-labeled for 5 h, in vivo, with ³⁵S-labeled E. coli, A subsequent 30-min chase with unlabeled E. coli served to rid the worms of endogenous labeled E. coli proteins. We resolve 700 or more proteins by 2D PAGE polyacrylamide gel electrophoresis of extracts of young nematodes. The patterns of these proteins are highly reproducible in comparisons of independent repeats of identical experiments. No new major proteins are synthesized at any time during the adult phase (4–22 days) nor are any of the most abundant proteins not made during this period. At our level of detectability (estimated as a satellite spot containing 4% of the amount of label in a major spot) we see no misincorporation of radioactive amino acids into newly synthesized proteins. These data are inconsistent with predictions by any one of several, so called, “error catastrophe” models of senescence and also show that modulation of the highest abundancy classes of proteins are also not involved in senescence.     

A Two Step Purification of Recombinant Human Interleukin-1β Expressed in E. Coli

Recombinant human interleukin-1β has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1β is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1β (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 × 10⁷ units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1β sera.     

A new peptide-affinity tag for the detection and affinity purification of recombinant proteins with a monoclonal antibody

A monoclonal anti-peptide antibody (2E11) was raised against the synthetic peptide 38 (C-L-D-K-S-G-L-P-S-D-R-F-F-A) representing a part of the variable region of the Vβ 6.2 T-cell receptor. This mAb (IgG₁, κ light chain) bound very specifically to peptide 38 as shown by ELISA but did not recognize the corresponding native Vβ 6.2 T-cell receptor on T-cells. For epitope analysis, overlapping peptides of 4–10 amino acids in length corresponding to the sequence of peptide 38 were synthesized and assayed by SPOT synthesis on cellulose sheets. The shortest peptide recognized was L-P-S-D-R. The specificity of mAb 2E11 was examined with 100 different peptides comprising other parts of the different variable Vβ domains of the human T-cell receptor that do not include the epitope region L-P-S-D-R. None of these peptides were recognized. The chemical synthesis of a peptide with the sequence L-P-S-D-R on Sepharose™ beads allowed to efficiently purify the mAb 2E11 in a single step by affinity chromatography. An equilibrium binding constant of 4.9×10⁶ l/mol was determined for mAb 2E11 by using rhodamine-green-labelled peptide 38 in fluorescence correlation spectroscopy. In order to demonstrate that peptide 38 can be used as an affinity-tag, it was fused to the carboxyl-terminus of interferon regulatory factor-1 (IRF-1). It could be shown that in vitro translated peptide 38 tagged IRF-1 was immunoprecipitated by the mAb 2E11 and that the fusion protein could be purified by immunoaffinity chromatography. Additionally peptide 38 was fused to the amino-terminus of the Taq polymerase. This recombinant protein was expressed in E. coli and specifically detected in a Dot blot and Western blot using mAb 2E11.     

Genetic selection for aflatoxin B1 resistance influences chicken T-cell and thymocyte proliferation.

Studies were conducted with two lines of chickens that were selected for high and low plasma protein concentrations in response to aflatoxin B₁ (AFB₁) exposure. The experiments were designed to determine genetic differences in the responses of T cells and thymocytes to the toxin. Chicks were orally administered AFB₁ at a rate of 0, 100, or 500 μg/kg body weight up to 21 days of age. At 4 weeks of age, concanavalin A (Con A, 2.5 μg/mL) stimulated T-cell proliferation was similar for untreated chicks from the low line (LL) and the high line (HL). However, AFB₁ reduced the responses of T cells with HL cells being more sensitive. In a second experiment, immature chickens were bled and peripheral blood lymphocytes were cultured with Con A and either 0, 3.125, 6.25, 12.5, or 25 μg/mL AFB₁. T cells from LL had greater responses to Con A than those from HL, and LL T-cells were also more resistant to in vitro AFB₁ exposure. Furthermore, thymocyte proliferation was greater for LL chicks; but when thymocytes were cultured with 25 μg/mL AFB₁, ³H-thymidine incorporation was similarly reduced in both lines. Cell cycle analysis indicated that there were more LL thymocytes in S phase, and the percentages for both lines decreased with AFB₁ treatment. Although there were no differences between the lines for percent G₂/M cells, AFB₁ treatment increased the percentages of thymocytes in G₂/M. These studies showed that selection for plasma protein response also changed T-cell and thymocyte proliferative activity.     

Strain specificity in antimicrobial activity of silver and copper nanoparticles

The antimicrobial properties of silver and copper nanoparticles were investigated using Escherichia coli (four strains), Bacillus subtilis and Staphylococcus aureus (three strains). The average sizes of the silver and copper nanoparticles were 3 nm and 9 nm, respectively, as determined through transmission electron microscopy. Energy-dispersive X-ray spectra of silver and copper nanoparticles revealed that while silver was in its pure form, an oxide layer existed on the copper nanoparticles. The bactericidal effect of silver and copper nanoparticles were compared based on diameter of inhibition zone in disk diffusion tests and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of nanoparticles dispersed in batch cultures. Bacterial sensitivity to nanoparticles was found to vary depending on the microbial species. Disk diffusion studies with E. coli and S. aureus revealed greater effectiveness of the silver nanoparticles compared to the copper nanoparticles. B. subtilis depicted the highest sensitivity to nanoparticles compared to the other strains and was more adversely affected by the copper nanoparticles. Good correlation was observed between MIC and MBC (r² = 0.98) measured in liquid cultures. For copper nanoparticles a good negative correlation was observed between the inhibition zone observed in disk diffusion test and MIC/MBC determined based on liquid cultures with the various strains (r² = −0.75). Although strain-specific variation in MIC/MBC was negligible for S. aureus, some strain-specific variation was observed for E. coli.