The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions.     

Hydrogen peroxide and catalase are inversely related in adult patients undergoing cardiopulmonary bypass: implications for antioxidant protection

Adult patients undergoing cardiopulmonary bypass (CPB) surgery are subjected to increased oxidative stress and show a spectrum of lung injury. Increased levels of hydrogen peroxide (H2O2) are often seen during episodes of oxidative stress, such as the use of high FiO2s, and this molecule plays a key role in the formation of highly damaging oxidants such as the hydroxyl radical. Oxidative damage to plasma proteins was assessed by measuring free thiol groups, and antioxidant protection against H2O2 by measuring catalase activity. CPB patients (n = 39) receiving either 100% or 50% oxygen at the end of bypass were studied by measuring levels of H2O2 in breath condensate and levels of catalase in their plasma, and comparing these to pre-bypass levels. Post-bypass, all CPB patients exhaled significantly lower levels of H2O2 (P < 0.0001) at a time when they had significantly increased activity (0.809 +/- 0.11 versus 1.688 +/- 0.18 U/mg protein) of catalase in their plasma. There were no significant differences in these parameters between the 100% and 50% oxygen groups. At a time when oxidative stress is greatest, there appears to be a corresponding plasma increase in the antioxidant catalase. Whether this change is fortuitous or a response to oxidative stress is at present under consideration.     

Optic chiasm presentation of Semaphorin6D in the context of Plexin-A1 and Nr-CAM promotes retinal axon midline crossing

At the optic chiasm, retinal ganglion cells (RGCs) project ipsi- or contralaterally to establish the circuitry for binocular vision. Ipsilateral guidance programs have been characterized, but contralateral guidance programs are not well understood. Here, we identify a tripartite molecular system for contralateral RGC projections: Semaphorin6D (Sema6D) and Nr-CAM are expressed on midline radial glia and Plexin-A1 on chiasm neurons, and Plexin-A1 and Nr-CAM are also expressed on contralateral RGCs. Sema6D is repulsive to contralateral RGCs, but Sema6D in combination with Nr-CAM and Plexin-A1 converts repulsion to growth promotion. Nr-CAM functions as a receptor for Sema6D. Sema6D, Plexin-A1, and Nr-CAM are all required for efficient RGC decussation at the optic chiasm. These findings suggest a mechanism by which a complex of Sema6D, Nr-CAM, and Plexin-A1 at the chiasm midline alters the sign of Sema6D and signals Nr-CAM/Plexin-A1 receptors on RGCs to implement the contralateral RGC projection.     

Stage‐specific probabilistic phenology model of Cydia pomonella (Lepidoptera: Tortricidae) using laboratory maximum likelihood parameter estimates

In this study, a probabilistic degree‐day phenology model has been developed for the codling moth, Cydia pomonella, and calibrated using data from laboratory growth studies. The model is further used to predict the succession and overlapping of certain biological events of C. pomonella in probabilistic‐physiological time scale in northern Greece fruit orchards. The model satisfactorily predicts the stage‐specific pest population dynamics, including egg laying and hatching, the occurrence of larvae and pupae stages and the emergence of adults. According to the model projections for the adult flights, there is a very high probability, p = 0.999, of observing adults of the first flight generation until 333 degree‐days (DD), but a very low probability of finding adults of the second flight generation. Moreover, at 575 DD, the probability of finding an individual to lay eggs is p = 0.15. However, there is nearly the same probability of egg hatch, p = 0.36, and larval completion p = 0.313, while at the same time, the probability of pupal completion is very low, p = 0.001. The above model predictions were validated using field data for the adult stage emergence as well as for the percentage of larval damage providing satisfactory results considering that larval emergence prediction was close to actual fruit damage observed in field. This information is very important considering that IPM programs rely on the use of biorational compounds, such as IGRs and bio‐toxins which are stage selective and often have a shorter residual activity than the preceding broad‐spectrum insecticides.     

Potential of the learning vector quantizer in the cell classification of endometrial lesions in postmenopausal women

OBJECTIVE: To investigate the potential of artificial neural networks for cell identification in endometrial lesions from postmenopausal women. STUDY DESIGN: The study was performed on cytologic material obtained by the Gynoscann endometrial cell samplerfrom 12 cases of atrophic endometrium, 48 cases of hyperplasia without cytologic atypia (18 cases of simple hyperplasia and 30 cases of complex hyperplasia), 12 cases of hyperplasia with cytologic atypia (complex atypical hyperplasia) and 48 cases of adenocarcinoma (30 cases of well-differentiated, 12 cases of moderately differentiated and 6 cases of poorly differentiated carcinoma). From each case approximately 100 cells were examined using a custom image analysis system. A learning vector quantizer (LVQ) identified the collected data. RESULTS: Investigation of cells from Endometrial Alterations with LVQ proved that according to the nuclear characteristics, as expressed by morphometric and textural measures, the endometrial cells from postmenopausal women may be identified as belonging to one of thefollowing three groups: atrophy, hyperplasia without cytologic atypia (simple and complex hyperplasia) and malignant neoplastic lesions (atypical complex and adenocarcinoma). CONCLUSION: The role of nuclear morphologic features in the cytologic diagnosis of endometrial alterations was confirmed. The overlap in thefeature space observed indicates that cell characteristics do not form strictly separate clusters. Thatfact explains the difficulty that morphologists have with the reproducible identification of cells from endometrial lesions in postmenopausal women. Application of LVQ offers a good classification at the cell level and promises to be a powerful toolfor classification on the individual patient level andfor the clarification of the natural history of endometrial pathology.     

Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms

Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.     

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.     

Functional analysis of an arthritogenic synovial fibroblast

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellullar matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.     

Molecular DNA identity of the mouflon of Cyprus (Ovis orientalis ophion,Bovidae): Near Eastern origin and divergence from Western Mediterranean conspecific populations

The mouflon population of Cyprus (Ovis orientalis ophion) comprises historically preserved feral descendants of sheep domesticated during the Neolithic. We determined genetic identity of this taxon in order to elucidate its systematic placement and enforce its protection. We used 12 loci of microsatellite DNA to infer genetic relationships between the Cypriot mouflon and either long-time isolated (Corsica, Sardinia) or recently introduced (central Italy) European mouflons (O. o. musimon). We also sequenced the mitochondrial DNA (mtDNA) Cytochrome-b gene to infer the origin of the Cypriot mouflon including many National Centre for Biotechnology Information (NCBI) entries of European and Near Eastern conspecifics. Microsatellites disclosed net divergence between Western Mediterranean and Cypriot mouflon. The latter was included in the highly heterogeneous Near Eastern O. orientalis mtDNA group, Iran representing the most credited region as the source for its ancient introduction to Cyprus. Both international and national legislation protect the mouflon of Cyprus as a wild taxon (O. o. ophion). However, the IUCN Red List of Threatened Species and NCBI include the Cypriot mouflon as subspecies of its respective domestic species, the sheep (O. aries). Unfortunately, people charged with crime against protected mouflon may benefit from such taxonomic inconsistency between legislation and databases, as the latter can frustrate molecular DNA forensic outcomes. Until a definitive light can be shed on Near Eastern O. orientalis systematics, we suggest that the Cypriot mouflon should be unvaryingly referred to as O. o. ophion in order not to impair conservation in the country where it resides.