Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.     

Role of native topology investigated by multiple unfolding simulations of four SH3 domains

The relative importance of amino acid sequence and native topology in the unfolding process of two SH3 domains and two circular permutants was investigated by 120 molecular dynamics runs at 375 K for a total simulation time of 0.72 micros. The alpha-spectrin (aSH3) and src SH3 (sSH3) domains, which have the same topology and a sequence identity of only 34%, show similar unfolding pathways. The disappearance of the three-stranded antiparallel beta-sheet is the last unfolding event, in agreement with a large repertoire of kinetic data derived from point mutations as well as glycine insertions and disulfide crosslinks. Two alternative routes of beta-sheet unfolding have emerged from the analysis of the trajectories. One is statistically preferred in aSH3 (n-src loop breaks before distal hair-pin) and the inverse in sSH3. An elongation of the beta2-beta3 hairpin was observed during the unfolding of sSH3 at 375 K and in 300 K simulations started from the putative transition state of sSH3 in accord with unusual kinetic data for point mutations at the n-src loop. The change of connectivity in the permutants influenced the sequence of unfolding events mainly at the permutation site. Regions where the connectivity remained unaffected showed the same chronology of contact disappearance. Taken together with previous folding simulations of two designed three-stranded antiparallel beta-sheet peptides, these results indicate that, at least for small beta-sheet proteins, the folding mechanism is primarily defined by the native state topology, whilst specific interactions determine the statistically predominant folding route.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

High-Dose Benzodiazepine Dependence: A Qualitative Study of Patients’ Perceptions on Initiation,Reasons for Use,and Obtainment

Background

High-dose benzodiazepine (BZD) dependence is associated with a wide variety of negative health consequences. Affected individuals are reported to suffer from severe mental disorders and are often unable to achieve long-term abstinence via recommended discontinuation strategies. Although it is increasingly understood that treatment interventions should take subjective experiences and beliefs into account, the perceptions of this group of individuals remain under-investigated.

Methods

We conducted an exploratory qualitative study with 41 adult subjects meeting criteria for (high-dose) BZD-dependence, as defined by ICD-10. One-on-one in-depth interviews allowed for an exploration of this group’s views on the reasons behind their initial and then continued use of BZDs, as well as their procurement strategies. Mayring’s qualitative content analysis was used to evaluate our data.

Results

In this sample, all participants had developed explanatory models for why they began using BZDs. We identified a multitude of reasons that we grouped into four broad categories, as explaining continued BZD use: (1) to cope with symptoms of psychological distress or mental disorder other than substance use, (2) to manage symptoms of physical or psychological discomfort associated with somatic disorder, (3) to alleviate symptoms of substance-related disorders, and (4) for recreational purposes, that is, sensation-seeking and other social reasons. Subjects often considered BZDs less dangerous than other substances and associated their use more often with harm reduction than as recreational. Specific obtainment strategies varied widely: the majority of participants oscillated between legal and illegal methods, often relying on the black market when faced with treatment termination.

Conclusions

Irrespective of comorbidity, participants expressed a clear preference for medically related explanatory models for their BZD use. We therefore suggest that clinicians consider patients’ motives for long-term, high-dose BZD use when formulating treatment plans for this patient group, especially since it is known that individuals are more compliant with approaches they perceive to be manageable, tolerable, and effective.     

Structure-based tailoring of compound libraries for high-throughput screening: discovery of novel EphB4 kinase inhibitors

High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.     

The C. elegans Male Exercises Directional Control during Mating through Cholinergic Regulation of Sex-Shared Command Interneurons

Background

Mating behaviors in simple invertebrate model organisms represent tractable paradigms for understanding the neural bases of sex-specific behaviors, decision-making and sensorimotor integration. However, there are few examples where such neural circuits have been defined at high resolution or interrogated.

Methodology/Principal Findings

Here we exploit the simplicity of the nematode Caenorhabditis elegans to define the neural circuits underlying the male’s decision to initiate mating in response to contact with a mate. Mate contact is sensed by male-specific sensilla of the tail, the rays, which subsequently induce and guide a contact-based search of the hermaphrodite’s surface for the vulva (the vulva search). Atypically, search locomotion has a backward directional bias so its implementation requires overcoming an intrinsic bias for forward movement, set by activity of the sex-shared locomotory system. Using optogenetics, cell-specific ablation- and mutant behavioral analyses, we show that the male makes this shift by manipulating the activity of command cells within this sex-shared locomotory system. The rays control the command interneurons through the male-specific, decision-making interneuron PVY and its auxiliary cell PVX. Unlike many sex-shared pathways, PVY/PVX regulate the command cells via cholinergic, rather than glutamatergic transmission, a feature that likely contributes to response specificity and coordinates directional movement with other cholinergic-dependent motor behaviors of the mating sequence. PVY/PVX preferentially activate the backward, and not forward, command cells because of a bias in synaptic inputs and the distribution of key cholinergic receptors (encoded by the genes acr-18, acr-16 and unc-29) in favor of the backward command cells.

Conclusion/Significance

Our interrogation of male neural circuits reveals that a sex-specific response to the opposite sex is conferred by a male-specific pathway that renders subordinate, sex-shared motor programs responsive to mate cues. Circuit modifications of these types may make prominent contributions to natural variations in behavior that ultimately bring about speciation.     

Spontaneous formation of detergent micelles around the outer membrane protein OmpX

The structure and flexibility of the outer membrane protein X (OmpX) in a water-detergent solution and in pure water are investigated by molecular dynamics simulations on the 100-ns timescale and compared with NMR data. The simulations allow for an unbiased determination of the structure of detergent micelles and the protein-detergent mixed micelle. The short-chain lipid dihexanoylphosphatidylcholine, as a detergent, aggregates into pure micelles of approximately 18 molecules, or alternatively, it binds to the protein surface. The detergent binds in the form of a monolayer ring around the hydrophobic beta-barrel of OmpX rather than in a micellar-like oblate; approximately 40 dihexanoylphosphatidylcholine lipids are sufficient for an effective suppression of water from the surface of the beta-barrel region. The phospholipids bind also on the extracellular, protruding beta-sheet. Here, polar interactions between charged amino acids and phosphatidylcholine headgroups act as condensation seed for detergent micelle formation. The polar protein surface remains accessible to water molecules. In total, approximately 90-100 detergent molecules associate within the protein-detergent mixed micelle, in agreement with experimental estimates. The simulation results indicate that OmpX is not a water pore and support the proposed role of the protruding beta-sheet as a "fishing rod".     

A double-headed cathepsin B inhibitor devoid of warhead

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.     

Morphological and molecular characterization of populations of plains bristlegrass (<i>Setaria macrostachya</i> Kunth) in Chihuahua,México

Plains bristlegrass (Setaria macrostachya Kunth) is a native grass with forage value. However, due to the lack of grazing management practices, populations and thus genetic diversity, have been reduced. Morphological and genetic variability were analyzed on 44 populations of plains bristlegrass in the State of Chihuahua. Plants were transplanted in a common area under natural conditions. Two years later, morphological characterization was evaluated measuring nine variables, and genetic variability using AFLP molecular markers. The principal components analysis (PC) showed that the three first principal components explained 73.74% of the variation. The variables with the greatest contribution to the variance in PC1 were plant height and inflorescence length; in CP2, tiller number and leaf width; and in PC3, tiller thickness. Application of four pairs of primers, presented 186 total bands, from which 87.10% showed polymorphism and 12.90% monomorphism. The combination of EcoRI-AGG MseI-CAG primers detected the highest percentage (93%) of polymorphism with 40 polymorphic bands. The cluster analysis and Dice coefficient indicated that populations clump into two groups. The wide genetic variability and morphological characteristics detected among populations represent the basis for the selection of populations that could be used with different purposes in the rehabilitation of ecosystems. In addition, this study will allow establishment of in situ conservation strategies.