Use of growth indices from radiometric culture for quantification of sheep strains of Mycobacterium avium subsp. paratuberculosis

A simple method for using growth indices from radiometric BACTEC cultures was evaluated for the enumeration of Australian sheep strains of Mycobacterium avium subsp. paratuberculosis. The numbers of viable organisms in inocula were determined by end-point titration in BACTEC cultures. Growth indices were measured by using a BACTEC 460 machine. There was a linear relationship between the number of days taken for the cumulative growth index to reach 1,000 (dCGI1000) and log(10) inoculum size. The use of dCGI1000 was shown to be as effective as the use of growth index data from the entire growth cycle for the estimation of inoculum size. For particular isolates characterized by end-point titration, the dCGI1000 of a single BACTEC vial provided estimates of viable numbers within narrow prediction limits. Predictive relationships were also established for the enumeration of M. avium subsp. paratuberculosis from field samples by using the dCGI1000 of a single BACTEC vial, with prediction limits of +/-1 to 2 log units. Organisms from feces or contaminated soil grew more slowly than those from cultures or tissues, and separate equations were developed for enumeration from these sources.     

Proteomic signatures of serum albumin-bound proteins from stroke patients with and without endovascular closure of PFO are significantly different and suggest a novel mechanism for cholesterol efflux

Background

The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation – altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure.

Methods

The method used in this study combined albumin immuno-enrichment with high resolution LC-MS in order to specifically capture and quantify the albumin-bound proteins. Subsequently, we measured cholesterol and HDL in a larger, separate cohort of PFO stroke patients, pre and post closure.

Results

The results demonstrated that a number of proteins were specifically associated with albumin in samples with and without endovascular closure of the PFO, and that the protein profiles were very different. Eight proteins, typically associated with HDL were common to both sample sets and quantitatively differently abundant. Pathway analysis of the MS results suggested that enhanced cholesterol efflux and reduced lipid oxidation were associated with PFO closure. Measurement of total cholesterol and HDL in a larger cohort of PFO closure samples using a colorimetric assay was consistent with the proteomic predictions.

Conclusions

The collective data presented in this study demonstrate that analysis of albumin-bound proteins could provide a valuable tool for biomarker discovery on the effects of PFO endovascular closure. In addition, the results suggest that PFO endovascular closure can potentially have effects on HDL, cholesterol and albumin-bound ApoA-I abundance, therefore possibly providing benefits in cardioprotective functions.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-12-2) contains supplementary material, which is available to authorized users.     

Focal adhesion kinase: an essential kinase in the regulation of cardiovascular functions

Recent studies using animal models have demonstrated an important role for FAK in the cardiovascular system. In particular, FAK is essential for angiogenesis in the embryo, functions in heart development and modulates the response of cardiomyocytes to pressure overload in adult mice. FAK function at the cellular level is discussed to provide insight into the mechanisms regulating these biological events and the role of FAK in controlling endothelial junctions and responses to mechanical stimulation are discussed.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Focal adhesion kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.     

Benzyl prolinate derivatives as novel selective KCC2 blockers

The discovery and optimization of a novel class of selective submicromolar KCC2 blockers is described. Details of synthesis and SAR are given together with ADME properties of selected compounds. A methylsulfone residue on the R¹ phenyl group improved the overall general profile of these prolinate derivatives.     

Seco diterpenoids from Excoecaria agallocha L

Chemical examination of the ethyl acetate solubles of the CH(3)OH:CH(2)Cl(2) (1:1) extract of the roots of Excoecaria agallocha L. collected from Godavary estuary resulted in the isolation of three more new diterpenoids, agallochins M-O (1-3). The structures of the new diterpenoids were elucidated by a study of their physical and spectral data as methyl ent-13-epi-8,13-epoxy-4,6alpha-dihydroxy-3,4-secolabd-14-en-3-oate (1), methyl ent-13-epi-8,13-epoxy-2,3-secolabd-14-en-2,11-olid-3-oate (2), and methyl ent-17-hydroxy-3,4-secokaura-4(19),15-dien-3-oate (3).     

Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli

Coenzyme A (CoA) and its thioester derivatives are important precursor molecules for many industrially useful compounds such as esters, PHBs, lycopene and polyketides. Previously, in our lab we could increase the intracellular levels of CoA and acetyl-Coenzyme A (acetyl-CoA) by overexpressing one of the upstream rate-controlling enzymes pantothenate kinase with a concomitant supplementation of the precursor pantothenic acid to the cell culture medium. In this study, we showed that the CoA/acetyl-CoA manipulation system could be used to increase the productivity of industrially useful compounds derived from acetyl-CoA. We chose the production of isoamyl acetate as a model system. Isoamyl acetate is an important flavor component of sake yeast and holds a great commercial value. Alcohol acetyl transferase (AAT) condenses isoamyl alcohol and acetyl-CoA to produce isoamyl acetate. The gene ATF2, coding for this AAT was cloned and expressed in Escherichia coli. This genetic engineered E. coli produces isoamyl acetate, an ester, from intracellular acetyl-CoA when isoamyl alcohol is added externally to the cell culture medium. In the current study, we showed that in a strain bearing ATF2 gene, an increase in intracellular CoA/acetyl-CoA by overexpressing panK leads to an increase in isoamyl acetate production. Additionally, the cofactor manipulation technique was combined with more traditional approach of competing pathway deletions to further increase isoamyl acetate production. The acetate production pathway competes with isoamyl acetate production for the common intracellular metabolite acetyl-CoA. Earlier we have shown that acetate pathway deletion (ackA-pta) increases isoamyl acetate production. The acetate production pathway was inactivated under elevated CoA/acetyl-CoA conditions, which lead to a further increase in isoamyl acetate production.