Fate of pGFP-bearing Escherichia coli O157:H7 in ground beef at 2 and 10 degrees C and effects of lactate, diacetate, and citrate

Although beef has been implicated in the largest outbreaks of Escherichia coli O157:H7 infection in the United States, studies on the fate of this pathogen have been limited. Problems in such studies are associated with detection of the pathogen at levels considerably lower than the levels of the competing microorganisms. In the present study, a green fluorescent protein-expressing E. coli O157:H7 strain was used, and the stable marker allowed us to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10 degrees C. In addition, the effects of sodium salts of lactate (SL) (0.9 and 1.8%), diacetate (SDA) (0.1 and 0.2%), and buffered citrate (SC) (1 and 2%) and combinations of SL and SDA were evaluated. SC had negligible antimicrobial activity, and SL delayed microbial growth, while SDA and SL plus SDA were most inhibitory to the total-aerobe population in the meat. At 2 degrees C, the initial numbers of E. coli O157:H7 (3 and 5 log(10) CFU/g) decreased by approximately 1 log(10) CFU/g when spoilage was manifest (>7 log(10) CFU of total aerobes/g), irrespective of the treatment. There was no decline in the numbers of the pathogen during storage at 10 degrees C. Our results showed that the pathogen was resistant to the salts tested and confirmed that refrigerated meat contaminated with the pathogen remains hazardous.     

Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation

The role played by resident macrophages (Mphi) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional Mphi ablation strategy to determine the role of resident peritoneal Mphi in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional Mphi ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive Mphi populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident Mphi ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic Mphi. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal Mphi in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.     

Real-time kinetic analysis of hCG-monoclonal antibody interaction using radiolabeled hCG probe: presence of two forms of Ag-mAb complex as revealed by solid phase dissociation studies

Real-time kinetics of ligand-ligate interaction has predominantly been studied by either fluorescence or surface plasmon resonance based methods. Almost all such studies are based on association between the ligand and the ligate. This paper reports our analysis of dissociation data of monoclonal antibody-antigen (hCG) system using radio-iodinated hCG as a probe and nitrocellulose as a solid support to immobilize mAb. The data was analyzed quantitatively for a one-step and a two-step model. The data fits well into the two-step model. We also found that a fraction of what is bound is non-dissociable (tight-binding portion (TBP)). The TBP was neither an artifact of immobilization nor does it interfere with analysis. It was present when the reaction was carried out in homogeneous solution in liquid phase. The rate constants obtained from the two methods were comparable. The work reported here shows that real-time kinetics of other ligand-ligate interaction can be studied using nitrocellulose as a solid support.     

Proteomic View of Interactions of Shiga Toxin-Producing Escherichia coli with the Intestinal Environment in Gnotobiotic Piglets

Background

Shiga toxin (Stx)-producing Escherichia coli cause severe intestinal infections involving colonization of epithelial Peyer’s patches and formation of attachment/effacement (A/E) lesions. These lesions trigger leukocyte infiltration followed by inflammation and intestinal hemorrhage. Systems biology, which explores the crosstalk of Stx-producing Escherichia coli with the in vivo host environment, may elucidate novel molecular pathogenesis aspects.

Methodology/Principal Findings

Enterohemorrhagic E. coli strain 86–24 produces Shiga toxin-2 and belongs to the serotype O157:H7. Bacterial cells were scrapped from stationary phase cultures (the in vitro condition) and used to infect gnotobiotic piglets via intestinal lavage. Bacterial cells isolated from the piglets’ guts constituted the in vivo condition. Cell lysates were subjected to quantitative 2D gel and shotgun proteomic analyses, revealing metabolic shifts towards anaerobic energy generation, changes in carbon utilization, phosphate and ammonia starvation, and high activity of a glutamate decarboxylase acid resistance system in vivo. Increased abundance of pyridine nucleotide transhydrogenase (PntA and PntB) suggested in vivo shortage of intracellular NADPH. Abundance changes of proteins implicated in lipopolysaccharide biosynthesis (LpxC, ArnA, the predicted acyltransferase L7029) and outer membrane (OM) assembly (LptD, MlaA, MlaC) suggested bacterial cell surface modulation in response to activated host defenses. Indeed, there was evidence for interactions of innate immunity-associated proteins secreted into the intestines (GP340, REG3-γ, resistin, lithostathine, and trefoil factor 3) with the bacterial cell envelope.

Significance

Proteomic analysis afforded insights into system-wide adaptations of strain 86–24 to a hostile intestinal milieu, including responses to limited nutrients and cofactor supplies, intracellular acidification, and reactive nitrogen and oxygen species-mediated stress. Protein and lipopolysaccharide compositions of the OM were altered. Enhanced expression of type III secretion system effectors correlated with a metabolic shift back to a more aerobic milieu in vivo. Apparent pathogen pattern recognition molecules from piglet intestinal secretions adhered strongly to the bacterial cell surface.     

Plant seed extract assisted,eco-synthesized C-ZnO nanoparticles: Characterization,chromium(VI) ion adsorption and kinetic studies

This report attempts to elucidate the potential of plant seed extract assisted synthesis of graphite-based zinc oxide nanoparticles (C-ZnO NPs) towards removal of chromium(VI) ions from water samples. The graphite-based zinc oxide (C-ZnO) composites were characterized using thermogravimetric analysis (TGA), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The C-ZnO nanocomposites have found to remove chromium from the sample through an adsorption process. The sensitivity of chromium removal through adsorption is found to be in the range of 40 to 240 mg. The adsorption behaviour was found to be fitting with Langmuir isotherm model and the adsorption reaction follows pseudo second-order kinetics.     

Non-photochemical quenching-dependent acclimation and thylakoid organization of Chlamydomonas reinhardtii to high light stress

Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000 µmol m^?2 s^?1 with faster growth and more biomass production when compared to cells at 50 µmol m^?2 s^?1 light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which is an adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein–pigment complexes which are in agreement with circular dichoism spectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII–LHCII supercomplexes. These results specify that acclimation to high light stress through NPQ mechanism by expression of LHCSR3 and also observed changes in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.

     

Zirconia-Cu(I) stabilized copper oxide mesoporous nano-catalyst: Synthesis and DNA reactivity of 1,2,4-oxadiazole-quinolinepeptidomimetics-based metal(II) complexes

Abstract

Contemporary research reveals an undemanding protocol for the catalytic synthesis of 1,2,4-oxadiazole-quinolinepeptide in the incidence of a cost-effective and reusable mesoporous ZrO₂-supported Cu₂O (Cu₂ZrO₃) catalyst. This paper depicts a unique system for peptide bond synthesis staying away from toxic solvents and reactants. The catalyst was reused for four cycles without noteworthy loss in the activity, and the catalyst was genuinely heterogeneous. The method followed a simple workup procedure, and no column chromatography was needed. Further, the synthesized 1,2,4-oxadiazole-quinolinepeptide ligand (L), and its complexes of type, [FeLCl₂] and [CuL]Cl₂ were synthesized and characterized by spectral and analytical techniques. An octahedral geometry has been projected for Fe(II) complexes, while the Cu(II) complex exhibits a square planar geometry. The binding properties of the complexes with CT-DNA were studied by absorption spectral analysis, followed by viscosity measurement and thermal denaturation studies. The photo-induced cleavage studies revealed that the complexes possess photonuclease activity against pUC19 DNA under UV–visible irradiation.