Detection of pathogenic bacteria in food samples using highly-dispersed carbon particles

There is an unmet need for detection methods that can rapidly and sensitively detect food borne pathogens. A flow through immunoassay system utilizing highly dispersed carbon particles and an amperometric technique has been developed and optimized. A sandwich immunoassay format was utilized in which pathogenic cells were captured by antibodies immobilized onto activated carbon particles, and labeled with horseradish peroxidase (HRP) conjugated antibodies. Flow of the peroxidase substrates resulted in an amperometric signal that is proportional to the number of captured cells. Factors influencing the analytical performance of the system, such as the quantity of carbon particles and concentrations of capture antibody, enzyme labeled antibody, and enzyme substrates, were investigated and optimized. Detection and quantification of Escherichia coli, Listeria monocytogenes and Campylobacter jejuni were demonstrated with low detection limits of 50, 10, and 50 cells/ml, respectively, and an overall assay time of 30 min. Milk and chicken extract samples were spiked with various concentrations of these pathogens and were used to challenge the system. The system design is flexible enough to allow its application to the detection of viruses and proteins.     

Stability of domain 4 of the anthrax toxin protective antigen and the effect of the VWA domain of CMG2 on stability

The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von‐Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic stability may improve storage and long‐term stability of PA for use as a vaccine. In order to understand the origin of this increase in stability, we have isolated the receptor binding domain of PA, domain 4 (D4), and have studied the effect of the addition of CMG2 on thermodynamic stability. We are able to determine a binding affinity between D4 and CMG2 (～300 nM), which is significantly weaker than that between full‐length PA and CMG2 (170–300 pM). Unlike full‐length PA, we observe very little change in stability of D4 on binding to CMG2, using either fluorescence or ¹⁹F‐NMR experiments. Because in previous experiments we could observe a stabilization of both domain 4 and domain 2, the mechanism of stabilization of PA by CMG2 is likely to involve a mutual stabilization of these two domains.     

Adaptation to high temperature mitigates the impact of water deficit during combined heat and drought stress in C3 sunflower and C4 maize varieties with contrasting drought tolerance

Heat and drought stress frequently occur together, however, their impact on plant growth and photosynthesis (P_N) is unclear. The frequency, duration and severity of heat and drought stress events are predicted to increase in the future, having severe implications for agricultural productivity and food security. To assess the impact on plant gas exchange, physiology and morphology we grew drought tolerant and sensitive varieties of C3 sunflower (Helianthus annuus) and C4 maize (Zea mays) under conditions of elevated temperature for 4 weeks prior to the imposition of water deficit. The negative impact of temperature on P_N was most apparent in sunflower. The drought tolerant sunflower retained ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RubisCO) activity under heat stress to a greater extent than its drought sensitive counterpart. Maize exhibited no varietal difference in response to increased temperature. In contrast to previous studies, where a sudden rise in temperature induced an increase in stomatal conductance (G_s), we observed no change or a reduction in G_s with elevated temperature, which alongside lower leaf area mitigated the impact of drought at the higher temperature. The drought tolerant sunflower and maize varieties exhibited greater investment in root‐systems, allowing greater uptake of the available soil water. Elevated temperatures associated with heat‐waves will have profound negative impacts on crop growth in both sunflower and maize, but the deleterious effect on P_N was less apparent in the drought tolerant sunflower and both maize varieties. As C4 plants generally exhibit water use efficiency (WUE) and resistance to heat stress, selection on the basis of tolerance to heat and drought stress would be more beneficial to the yields of C3 crops cultivated in drought prone semi‐arid regions.     

Spontaneous Genomic Alterations in a Chimeric Model of Colorectal Cancer Enable Metastasis and Guide Effective Combinatorial Therapy

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and β-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.     

Dual effects of glucagon and cyclic AMP on DNA synthesis in cultured rat hepatocytes: stimulatory regulation in early G1 and inhibition shortly before the S phase entry

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.     

The slow S to M rise of chlorophyll a fluorescence reflects transition from state 2 to state 1 in the green alga Chlamydomonas reinhardtii

Photosynthesis Research - The green alga Chlamydomonas (C.) reinhardtii is a model organism for photosynthesis research. State transitions regulate redistribution of excitation energy between...     

Anaerobiosis Induced State Transition: A Non Photochemical Reduction of PQ Pool Mediated by NDH in Arabidopsis thaliana

Background

Non photochemical reduction of PQ pool and mobilization of LHCII between PSII and PSI are found to be linked under abiotic stress conditions. The interaction of non photochemical reduction of PQ pool and state transitions associated physiological changes are critically important under anaerobic condition in higher plants.

Methodology/Findings

The present study focused on the effect of anaerobiosis on non-photochemical reduction of PQ pool which trigger state II transition in Arabidopsis thaliana. Upon exposure to dark-anaerobic condition the shape of the OJIP transient rise is completely altered where as in aerobic treated leaves the rise is unaltered. Rise in F _o and F _J was due to the loss of oxidized PQ pool as the PQ pool becomes more reduced. The increase in F_o′ was due to the non photochemical reduction of PQ pool which activated STN7 kinase and induced LHCII phosphorylation under anaerobic condition. Further, it was observed that the phosphorylated LHCII is migrated and associated with PSI supercomplex increasing its absorption cross-section. Furthermore, evidences from crr2-2 (NDH mutant) and pgr5 mutants (deficient in non NDH pathway of cyclic electron transport) have indicated that NDH is responsible for non photochemical reduction of the PQ pool. We propose that dark anaerobic condition accelerates production of reducing equivalents (such as NADPH by various metabolic pathways) which reduce PQ pool and is mediated by NDH leading to state II transition.

Conclusions/Significance

Anaerobic condition triggers non photochemical reduction of PQ pool mediated by NDH complex. The reduced PQ pool activates STN7 kinase leading to state II transition in A. thaliana.     

Ante-mortem detection of chronic wasting disease in recto-anal mucosa-associated lymphoid tissues from elk (Cervus elaphus nelsoni) using real-time quaking-induced conversion (RT-QuIC) assay: A blinded collaborative study

Prion diseases are transmissible spongiform encephalopathies (TSEs) characterized by fatal, progressive neurologic diseases with prolonged incubation periods and an accumulation of infectious misfolded prion proteins. Antemortem diagnosis is often difficult due to a long asymptomatic incubation period, differences in the pathogenesis of different prions, and the presence of very low levels of infectious prion in easily accessible samples. Chronic wasting disease (CWD) is a TSE affecting both wild and captive populations of cervids, including mule deer, white-tailed deer, elk, moose, muntjac, and most recently, wild reindeer. This study represents a well-controlled evaluation of a newly developed real-time quaking-induced conversion (RT-QuIC) assay as a potential CWD diagnostic screening test using rectal biopsy sections from a depopulated elk herd. We evaluated 69 blinded samples of recto-anal mucosa-associated lymphoid tissue (RAMALT) obtained from USDA Veterinary Services. The results were later un-blinded and statistically compared to immunohistochemical (IHC) results from the USDA National Veterinary Services Laboratories (NVSL) for RAMALT, obex, and medial retropharyngeal lymph node (MRPLN). Comparison of RAMALT RT-QuIC assay results with the IHC results of RAMALT revealed 92% relative sensitivity (95% confidence limits: 61.52–99.8%) and 95% relative specificity (95% confidence limits: 85.13–99%). Collectively, our results show a potential utility of the RT-QuIC assay to advance the development of a rapid, sensitive, and specific prion diagnostic assay for CWD prions.