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.     

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.     

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.     

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.     

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...     

Simultaneous determination of theophylline, enoxacin and ciprofloxacin in human plasma and saliva by high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of theophylline, ciprofloxacin and enoxacin in plasma and saliva. The biological fluid samples were extracted with methylene chloride-isopropyl alcohol prior to isocratic chromatography on a Waters C₁₈ μBondapak column. Ultraviolet detection was carried out at 268 nm. The assay in linear for ciprofloxacin and enoxacin (0.05–10 μg/ml), and theophylline (0.1–20 μ/ml). The assay can be used to investigate the interaction of these two fluoroquinolones with theophylline.     

Spatiotemporal characterization of short versus long duration calcium transients in embryonic muscle and their role in myofibrillogenesis

Intracellular calcium (Ca(2+)) signals are essential for several aspects of muscle development, including myofibrillogenesis-the terminal differentiation of the sarcomeric lattice. Ryanodine receptor (RyR) Ca(2+) stores must be operative during this period and contribute to the production of spontaneous global Ca(2+) transients of long duration (LDTs; mean duration approximately 80 s). In this study, high-speed confocal imaging of intracellular Ca(2+) in embryonic myocytes reveals a novel class of spontaneous Ca(2+) transient. These short duration transients (SDTs; mean duration approximately 2 s) are blocked by ryanodine, independent of extracellular Ca(2+), insensitive to changes in membrane potential, and propagate in the subsarcolemmal space. SDTs arise from RyR stores localized to the subsarcolemmal space during myofibrillogenesis. While both LDTs and SDTs occur prior to myofibrillogenesis, LDT production ceases and only SDTs persist during a period of rapid sarcomere assembly. However, eliminating SDTs during this period results in only minor myofibril disruption. On the other hand, artificial extension of LDT production completely inhibits sarcomere assembly. In conjunction with earlier work, these results suggest that LDTs have at least two roles during myofibrillogenesis-activation of sarcoplasmic regulatory cascades and regulation of gene expression. The distinct spatiotemporal patterns of LDTs versus SDTs may be utilized for differential regulation of cytosolic cascades, control of nuclear gene expression, and localized activation of assembly events at the sarcolemma.