Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease models

We present a digital atlas system that allows mapping of molecular expression patterns at cellular resolution through large series of histological sections. Using this system, we have mapped the distribution of a distinct marker, encoded by the LacZ reporter gene driven by the tetracycline-responsive prion protein promoter in double transgenic mice. The purpose is to evaluate the suitability of this promoter mouse line for targeting genes of interest to specific brain regions, essential for construction of inducible transgenic disease models. Following processing to visualize the promoter expression, sections were counterstained to simultaneously display cytoarchitectonics. High-resolution mosaic images covering entire coronal sections were collected through the mouse brain at intervals of 200 microm. A web-based application provides access to a customized virtual microscopy tool for viewing and navigation within and across the section images. For each section image, the nearest section in a standard atlas is defined, and annotations of key structures and regions inserted. Putative categorization of labeled cells was performed with use of distribution patterns, followed by cell size and shape, as parameters that were compared to legacy data. Among the ensuing results were expression of this promoter in putative glial cells in the cerebellum (and not in Purkinje cells), in putative glial cells in the substantia nigra, in pallidal glial cells or interneurons, and in distinct cell layers and regions of the hippocampus. The study serves as a precursor for a database resource allowing evaluation of the suitability of different promoter mouse lines for generating disease models.     

Intramolecular phenotypic capacitance in a modular RNA molecule

Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA’s ancient role in life suggests that phenotypic capacitance may have influenced evolution since life’s origins.Phenotypic capacitance requires mutations that reversibly alternate between hidden and revealed states in response to environmental or genetic perturbations (1, 2). In the hidden or “cryptic” state the mutations can survive selection because they do not change the phenotype, and multiple such mutations can accumulate within individual genomes in a population. Perturbation after such accumulation can reveal the combined effects of multiple mutations. By exposing the phenotypic effects of mutations that may not have been beneficial individually, capacitance provides a mechanism of generating new phenotypes—from macromolecules to morphological traits—with novel functions (1–4). The term “phenotypic capacitance” is appropriate for situations where altered phenotypes with a genetic basis can be hidden and revealed, even when no adaptive potential of the revealed phenotypes is demonstrated (2). “Evolutionary capacitance,” on the other hand, is a term reserved for instances when an adaptive role is demonstrated. Phenotypic capacitance has been known in fruit flies since the 1950s (5), but demonstrations of adaptive potential (4, 6), and the various molecular mechanisms behind it (7–11) have been reported only relatively recently.A module in a biological system is a group of system parts that interact more with each other than with parts outside of the module (12). In RNA molecules, the parts are nucleotides, and modules are units of tertiary structure that fold independently, and often perform different functions, such as binding to different proteins, RNAs, or small molecules. Modularity has been described in the telomerase RNA component (13), ribosomal RNA (14), long noncoding RNA (e.g., Xist, Hotair) (15), riboswitches (16), and self-splicing introns (17, 18). A link between modularity and phenotypic capacitance could provide a mechanism for the evolution of novel functions involving modules of RNA structure, and especially for functional innovations that require multiple simultaneous mutations. It is not known how modularity might change the potential to hide and reveal the effects of mutations in RNA structures, which is a prerequisite for phenotypic capacitance.To investigate a possible link between modularity and phenotypic capacitance in RNA, we chose to study the Azoarcus group I RNA enzyme (ribozyme). Group I ribozymes such as this have two structural modules that are functionally distinct (Fig. 1). The first of them is a scaffold module (Fig. 1, yellow) that folds rapidly, and forms a nearly identical structure even when removed from the context of the rest of the ribozyme (19, 20). The scaffolding it provides facilitates the folding of the less thermodynamically stable catalytic module (Fig. 1, blue) (21). Biochemical studies have shown that the catalytic phenotype (protein-free splicing) resides in the catalytic module, which can maintain activity if the scaffold module is deleted, but only under conditions of very high magnesium (80 mM) and extended incubation times (16 h) (22). This instability caused by deleting the scaffold supports the idea that this module acts as an intramolecular chaperone.Open in a separate windowFig. 1.The modular structure of the Azoarcus group I ribozyme. (A) The scaffold module (yellow) and the catalytic module (blue) are shown in the context of the ribozyme secondary structure. The regions that show base pairing (P) are numbered sequentially from the 5′ to the 3′ end, according to group I intron standards. The substrate for the ribozyme is written in lowercase letters. The modules are also shown in the context of the 3D crystal structure (PDB ID: 1ZZN), from a “top view” (B) and “side view” (C), with respect to the scaffold module.The diversification of RNA functions has played an important role in the evolution of extant organisms, and may have been even more important at life’s beginnings when RNA enzymes (ribozymes) played a central role as catalysts in the RNA world scenario (23). In a previous publication using the Azoarcus ribozyme, we demonstrated an adaptive role for accumulated cryptic variation that was revealed when altered enzymatic activity was required (24). Here, we focus on how the functionally distinct modules of this structure might facilitate the occurrence of environmentally conditional mutations that enable phenotypic capacitance.We aimed to identify mutations that maintain the catalytic phenotype under normal conditions but alter this phenotype under stressful conditions. The ideal stressful condition for our RNA system is a low concentration of magnesium ions. The concentration of magnesium ions inside of cells is maintained at a higher concentration than any other divalent ion due to its role in many cellular functions. Importantly for our current experiments, it is known that magnesium ions are critical for stabilizing the native structure of RNA molecules (25). Low concentrations of magnesium only allow folding of the most stable RNA structures and can lead to misfolding of less stable structures. In addition, many ribozymes, including group I introns, use highly coordinated magnesium ions in their active site (26). Based on previous reports on the magnesium dependence of the Azoarcus ribozyme (19), we here studied a stressful environment with low (2 mM) MgCl₂, a relaxed environment with high (25 mM) MgCl₂, and an intermediate environment (10 mM MgCl₂). We note that magnesium availability is often limited in natural environments, a stressful condition that organisms have evolved adaptive responses to. For example, magnesium homeostasis in bacteria is maintained by the expression of ion transporters that are sometimes regulated by cis-acting RNA regulatory elements. These “magnesium riboswitch” elements control downstream mRNA expression by conformational changes induced by altered magnesium concentrations (27). This example and others (28) highlight the widespread importance of our chosen experimental stressor.     

Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ～1.1-1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.     

Oxygen levels during negative pressure wound therapy

Aim of the studyNegative pressure wound therapy (NPWT) has become an established treatment modality when dealing with chronic and infected wounds. The underlying mechanism of action is still under discussion and remains controversial. Evidence exists showing rather hypoxic conditions as the main reason for the positive results and bacterial clearance. In an attempt to further explain the mechanism of action, we investigated oxygen levels within the foam interface of a NPWT device.Materials and methodsWe used an optical sensor based on the principle of dynamic fluorescence quenching and tested five different commonly available NPWT systems used during our daily clinical routine. All measurements were done in an in vitro experimental design for at least 24 h and multiple vacuum intensities were investigated.ResultsOxygen levels decreased as much as 22.8% and the amount of vacuum applied inversely correlated with the oxygen reduction. A stepwise increase in vacuum of 25 mmHg showed a linear mean drop of 2.75% per setting. All devices were able to maintain a constant level of negative pressure, and no significant difference between the various dressings was found (p > 0.05).ConclusionTherefore, oxygen levels are decreased within the foam of NPWT dressings, likely leading to oxygen deprivation effects in the underlying wound tissue.     

EGFR mutation detection in NSCLC��assessment of diagnostic application and recommendations of the German Panel for Mutation Testing in NSCLC

EGFR mutation testing in non-small cell lung cancer (NSCLC) is a novel and important molecular pathological diagnostic assay that is predictive of response to anti-epidermal growth factor receptor (EGFR) therapy. A comprehensive compilation of a large number of EGFR mutation analyses of the German Panel for Mutation Analyses in NSCLC demonstrates (a) a higher than previously reported mutation frequency outside the conventionally tested exons 19 and 21 and (b) an overall superiority of sequencing based assays over mutation-specific PCR. The implications for future diagnostic EGFR mutation testing are discussed.     

Partial purification of an iron-dependent L-serine dehydratase from Clostridium sticklandii

An oxygen-sensitive and highly unstable L -serine dehydratase was partially purified from the Grampositive anaerobe Clostridium sticklandii. The final active preparation contained five proteins of 27, 30, 44.5, 46, and 58 kDa as judged by SDS-PAGE. The N-terminal sequence of the 30 kDa subunit showed some similarity to the α-subunits of the iron-containing L -serine dehydratases from Clostridium propionicum and Peptostreptococcus asaccharolyticus. Oxygen-inactivated L -serine dehydratase from C. sticklandii was reactivated by incubation with Fe²⁺ under reducing conditions. Furthermore, the enzyme was inactivated by iron-chelating substances like phenanthroline and EDTA. Pyridoxal-5-phosphate (PLP) did not stimulate the activity, and known inhibitors of PLP-containing enzymes such as NaBH₄ had no effect on the activity of L -serine dehydratase from C. sticklandii.     

Tested communication strategies for providing information to patients in medical consultations: A scoping review and quality assessment of the literature

ObjectivesTo systematize the scientific knowledge of empirically tested strategies for verbally providing medical information in patient-physician consultations.MethodsA scoping review searching for terms related to physician, information, oral communication, and controlled study. Four pairs of reviewers screened articles. For each selected study, we assessed the quality and summarized aspects on participants, study, intervention, and outcomes. Information provision strategies were inductively classified by types and main categories.ResultsAfter screening 9422 articles, 39 were included. The methodological quality was moderate. We identified four differently used categories of strategies for providing information: cognitive aid (n = 13), persuasive (n = 8), relationship- (n = 3), and objectivity-oriented strategies (n = 4); plus, one “mixed” category (n = 11). Strategies were rarely theoretically derived.ConclusionsCurrent research of tested strategies for verbally providing medical information is marked by great heterogeneity in methods and outcomes, and lack of theory-driven approaches. The list of strategies could be used to analyse real life communication.Practice implicationsFindings may aid the harmonization of future efforts to develop empirically-based information provision strategies to be used in clinical and teaching settings.     

CD4+ T cells regulate glucose homeostasis independent of adipose tissue dysfunction in mice

Obesity is frequently associated with a chronic low-grade inflammation in the adipose tissue (AT) and impaired glucose homeostasis. Adipose tissue macrophages (ATMs) have been shown to accumulate in the inflamed AT either by means of recruitment from the blood or local proliferation. ATM proliferation and activation can be stimulated by TH2 cytokines, such as IL-4 and IL-13, suggesting involvement of CD4-positive T cells in ATM proliferation and activation. Furthermore, several studies have associated T cells to alterations in glucose metabolism. Therefore, we sought to examine a direct impact of CD4-positive T cells on ATM activation, ATM proliferation and glucose homeostasis using an in vivo depletion model. Surprisingly, CD4 depletion did not affect ATM activation, ATM proliferation, or insulin sensitivity. However, CD4 depletion led to a significant improvement of glucose tolerance. In line with this, we found moderate disturbances in pancreatic endocrine function following CD4 depletion. Hence, our data suggest that the effect on glucose metabolism observed after CD4 depletion might be mediated by organs other than AT and independent of AT inflammation.