Transcription factor induction of vascular blood stem cell niches in vivo.

The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

Quantitative sensory testing profiles in children, adolescents and young adults (6-20 years) with cerebral palsy: Hints for a neuropathic genesis of pain syndromes.

INTRODUCTION: Many patients with cerebral palsy (CP) suffer chronic pain as one of the most limiting factors in their quality of life. In CP patients, pain mechanisms are not well understood, and pain therapy remains a challenge. Quantitative sensory testing (QST) might provide unique information about the functional status of the somatosensory system and therefore better guide pain treatment. OBJECTIVES: To understand better the underlying pain mechanisms in pediatric CP patients, we aimed to assess clinical and pain parameters, as well as QST profiles, which were matched to the patients' cerebral imaging pathology. PATIENTS AND METHODS: Thirty CP patients aged 6-20 years old (mean age 12 years) without intellectual impairment underwent standardized assessments of QST. Cerebral imaging was reassessed. QST results were compared to age- and sex-matched controls (multiple linear regression; Fisher's exact test; linear correlation analysis). RESULTS: CP patients were less sensitive to all mechanical and thermal stimuli than healthy controls but more sensitive to all mechanical pain stimuli (each p < 0.001). Fifty percent of CP patients showed a combination of mechanical hypoesthesia, thermal hypoesthesia and mechanical hyperalgesia; 67% of CP patients had periventricular leukomalacia (PVL), which was correlated with mechanic (r = 0.661; p < 0.001) and thermal (r = 0.624; p = 0.001) hypoesthesia. CONCLUSION: The combination of mechanical hypoesthesia, thermal hypoesthesia and mechanical hyperalgesia in our CP patients implicates lemniscal and extralemniscal neuron dysfunction in the thalamus region, likely due to PVL. We suspect that extralemniscal tracts are involved in the original of pain in our CP patients, as in adults.

Tomo-seq: A method to obtain genome-wide expression data with spatial resolution.

To improve our understanding of pattern formation during development and disease we heavily rely on the identification of novel regulators and pathways. While RNA sequencing yields genome-wide expression data that suit this purpose, it lacks spatial resolution. Such spatial resolution can be obtained by microscopy-based methods like in situ hybridization, but these fail to provide information on more than a few genes at a time. Here, we describe tomo-seq, a technique that combines the advantages of the above-mentioned approaches and provides genome-wide expression data with spatial information. The tomo-seq technique is based on cryosectioning of an embryo or tissue of interest and performing RNA-seq on individual sections. Using this method, we have generated genome-wide transcriptomics with high spatial resolution of the whole zebrafish embryo at various stages of development (Junker et al., 2014) and of adult zebrafish hearts after injury (Wu et al., 2016).

Sensitivity of commercial prothrombin time reagents to detect coagulation factor deficiencies in equine plasma.

The sensitivity of commercial prothrombin time (PT) tests was assessed based on a dilution series of equine pooled plasma (EPP) (experiment 1) and on 40 equine plasma samples with reduced activity of coagulation factors II, V, VII and X (experiment 2). Two different PT reagents (reagent 1, human placental thromboplastin; reagent 2, recombinant human tissue factor) were used according to the manufacturers' instructions (standard test, PT([ST])) and compared to a modified test procedure (modified test, PT([MT])) using sample dilution and fibrinogen addition. In all samples, sensitivity was lower (P<0.01) when using PT([ST]) with reagent 2 (0.20) than when using either PT([ST]) with reagent 1 (0.65) or PT([MT]) with both reagents (reagent 1, 0.60-0.75, reagent 2, 0.58-0.70, depending on sample dilution). The highest sensitivity was found for PT([MT]) when using a 1:20 sample dilution. In those samples in which at least one coagulation factor activity was decreased (by 20%; n=18), the sensitivity of PT([ST]) with reagent 2 (0.33) was found to be inadequate, in contrast to all other test procedures (0.83-0.94). This low sensitivity corresponded to shorter time intervals between different coagulation activity levels prepared by EPP dilution. The results indicate that adequate sensitivity of PT measurements in equine plasma can be achieved using a standard test procedure as long as a suitable reagent is used.

Influence of substrate binding on the mechanical stability of mouse dihydrofolate reductase.

We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase using single-molecule force spectroscopy by atomic force microscopy. We find that under mechanical forces dihydrofolate reductase unfolds via a metastable intermediate with lifetimes on the millisecond timescale. Based on the measured length increase of approximately 22 nm we suggest a structure for this intermediate with intact substrate binding sites. In the presence of the substrate analog methotrexate and the cofactor NADPH lifetimes of this intermediate are increased by up to a factor of two. Comparing mechanical and thermodynamic stabilization effects of substrate binding suggests mechanical stability is dominated by local interactions within the protein structure. These experiments demonstrate that protein mechanics can be used to probe the substrate binding status of an enzyme.

Cervical intramedullar schistosomiasis as a rare cause of acute tetraparesis.

INTRODUCTION: The trematode infection schistosomiasis affects at least 200 million people in endemic areas. Granulomas cause the typical manifestations of urogenital, intestinal and hepatolienal schistosomiasis. Involvement of other organs especially the central nervous system (CNS) is uncommon. CASE REPORT: We describe a 40-year old male with a history of repeated contact with schistosome contaminated water. After having suffered from flu-like symptoms with fever and arthralgias, he first presented with a polyradiculopathy of unknown origin. Then 4 weeks later an acute tetraparesis occurred. Spinal magnetic resonance imaging (MRI) revealed a spinal stenosis and query medullary hyperintensities at C6-C8 without contrast-enhancement. Serologic testing was positive for schistosomiasis. The intraoperative appearance at decompressive laminectomy revealed a myelitic form of schistosomiasis. Under therapy with praziquantel, initially high dose cortisone and intensive physiotherapy, symptoms slowly improved over months. On follow-up 1 year later, the patient presented with a spastic distally marked tetraparesis and sensory impairment from C6 downwards. CONCLUSION: Cervical intramedullar schistosomiasis is a rare cause of acute tetra- or para-paresis in patients, who have had contact with schistosomes. Early diagnosis is essential because of the excellent prognosis with specific therapy.

Global perspectives on proteins: comparing genomes in terms of folds, pathways and beyond.

The sequencing of complete genomes provides us with a global view of all the proteins in an organism. Proteomic analysis can be done on a purely sequence-based level, with a focus on finding homologues and grouping them into families and clusters of orthologs. However, incorporating protein structure into this analysis provides valuable simplification; it allows one to collect together very distantly related sequences, thus condensing the proteome into a minimal number of 'parts.' We describe issues related to surveying proteomes in terms of structural parts, including methods for fold assignment and formats for comparisons (eg top-10 lists and whole-genome trees), and show how biases in the databases and in sampling can affect these surveys. We illustrate our main points through a case study on the unique protein properties evident in many thermophile genomes (eg more salt bridges). Finally, we discuss metabolic pathways as an even greater simplification of genomes. In comparison to folds these allow the organization of many more genes into coherent systems, yet can nevertheless be understood in many of the same terms.

Structure determination of a key intermediate of the enantioselective Pd complex catalyzed allylic substitution reaction

The structure of a catalytic intermediate with important implications for the interpretation of the stereochemical outcome of the palladium complex catalyzed allylic substitution with phosphino-oxazoline (PHOX) ligands is determined by liquid state NMR. The complex displays a novel structure that is highly distorted compared with other palladium eta2-olefin complexes known so far. The structure has been determined from nuclear overhauser data (NOE), scalar coupling constants, and long range projection angle restraints derived from dipole dipole cross-correlated relaxation of multiple quantum coherence. The latter restraints have been implemented into a distance geometry protocol. The projection angle restraints yield a higher precision in the determination of the relative orientation of the two molecular moieties and are essential to provide an exact structural definition of the olefinic part of the catalytic intermediate with respect to the ligand.

Computer-assisted constitutional assignment of large molecules: COCON analysis of ascomycin.

[structure: see text] The computer-assisted constitutional assignment of ascomycin (1) is discussed. This example demonstrates that the NMR-based structure generator COCON is able to analyze data sets of organic molecules covering the full known range in size and complexity. The structural proposals of ascomycin were validated by calculating the 13C NMR chemical shifts using the computer program SpecEdit. The enhanced calculation time of COCON achieved by preorganizing the input data is also demonstrated for the data sets of aflatoxin B1 (2), 11-hydroxyrotenone (3), and haemoventosin (4).

Extended junctional sarcoplasmic reticulum of avian cardiac muscle contains functional ryanodine receptors.

The ryanodine receptor (RYR)/Ca2+ release channel of avian cardiac muscle was localized by immunocytochemical techniques and biochemically characterized using isolated membrane and receptor protein fractions. Monoclonal antibody C3-33 raised against the canine cardiac RYR bound to the junctional sarcoplasmic reticulum of pigeon and finch hearts, both at peripheral couplings and at extended junctional sarcoplasmic reticulum (EJSR). Immunoblots of sarcoplasmic reticulum vesicles from pigeon and finch hearts showed this antibody recognized a single high molecular weight protein, which co-migrated with the canine M(r) 565,000 RYR/Ca2+ release channel polypeptide. The pigeon heart RYR bound [3H]ryanodine with high affinity in a Ca(2+)-dependent manner, comparable to the canine cardiac RYR. Purification of the pigeon RYR yielded a 30 S protein complex, which bound the maximum calculated amount of [3H]ryanodine ((440 +/- 60) pmol/mg protein), assuming one high affinity site/tetrameric 30 S RYR comprised of M(r) 565,000 polypeptides. Autoradiography of isolated finch cardiac myocytes indicated [3H]ryanodine binding throughout the cells. These results suggest that avian heart contains a single population of RYRs, and thereby support the hypothesis that avian EJSR contains functional calcium release channels which, because of the absence of transverse tubules, can be located micrometers away from the surface membrane in avian heart.

Distribution of gap junctions in dog and rat ventricle studied with a double-label technique.

To assess the distribution of gap junctions in relation to the cardiac myocyte surface in paraffin sections of dog and rat ventricle, the sarcolemma was labeled with wheat germ agglutinin (WGA1) and gap junctions were labeled with antibodies to cardiac muscle gap junction protein connexin43. WGA labeled all of the myocyte sarcolemma, including that in intercalated discs and transverse tubules. Sarcolemmal WGA labeling was often interrupted at the sites of gap junctions, which were found both at the extreme ends of myocytes and along the length of adjacent myocytes. Small gap junctions predominated at plicate transverse portions of the intercalated disc; larger and sometimes ribbon-like gap junctions predominated at longitudinal portions. The longitudinal portions of the intercalated disc often extended over multiple sarcomere lengths, with ribbon-like gap junctions and linear arrays of smaller gap junctions arranged in parallel overlying successive sarcomeres. Morphometric study showed that ribbon-like gap junctions were relatively infrequent in both dog and rat left ventricular epimyocardium, and that animals with larger myocytes tended to have smaller gap junctions. In dog left ventricular epimyocardium, neither myocytes nor their larger gap junctions were randomly oriented with respect to perimysial separations; myocytes were usually somewhat flattened with their maximal diameters parallel to the separations, whereas large gap junctions were least often oriented parallel or perpendicular to the separations. Overall, the data indicate that myocyte geometry influences gap junction size and distribution; the double-label technique is ideally suited for the further exploration of that influence.

To excite a heart: a bird's view.

Ultrastructural investigations of avian cardiac muscle, including ratite hearts, have provided great insights into the mechanisms as to how excitation leads to contraction in the heart. The geometry of the conduction fibers of ratite hearts confirms earlier observations on birds showing that the geometry of the conduction system and its component cells is adapted to hearts of different sizes and rates of contraction so as to maintain a differential in conduction velocities between the conduction system and the working fibers. The study of the ratite conduction fibers bears out the idea of an inverse relationship between the size of the gap junctions and the input resistance of cardiac cells. The anomalous extended junctional SR typical of all avian hearts, proscribes the notion of direct contact transduction into calcium release for contraction of an excitatory signal propagating at the cell surface. Couplings appear well suited to maintain direct, if transitory, connections to the extracellular space in addition to harboring channels for intracellular calcium release.

Influences upon reported health promotion by family physicians.

Disease prevention and the promotion of healthy life-styles have received increasing attention over the past two decades. The purpose of this study was to determine the current level of health promotional activity as reported by family physicians. In addition, the study addressed various factors which may influence the level of physician promotion of healthy life-styles. This was accomplished by means of a survey of 815 active members of the American Academy of Family Physicians. A total of 521 questionnaires was returned, providing a 64 percent response rate. The results of this survey indicate that the level of physician personal health activity tends to influence their reported professional promotion of healthy habits. In addition, residency faculty and physicians working for health maintenance organizations were significantly more likely to report offering a higher frequency of health promotional activity than physicians in private practice. Finally, age and family practice residency training appear to have no influence on self-reported physician health promotional activity.

Childhood farm injuries: a neglected aspect of patient and resident education.

Since family physicians are often the primary health providers for children residing in rural areas, one might expect family practice residencies to include in their curricula some teaching in regard to the prevention of childhood farm injuries. To assess how family practice residencies are currently responding to childhood farm injuries, the authors undertook a survey of the program directors of the nation's 380 residency programs. Of the 332 returned questionnaires, only three (0.9%) reported any formal instruction in the prevention of childhood farm injuries, despite the fact that 102 programs (30.7%) stated that at least 50% of their graduates practiced in rural communities. In addition to the survey, specific proposals are made regarding what residencies can do to initiate teaching in the prevention of farm injuries in children.

Immunohistochemical determination of inducibility phenotype with a monoclonal antibody to a methylcholanthrene-inducible isozyme of cytochrome P-450.

A monoclonal antibody (MAb) to a methylcholanthrene (MC)-induced cytochrome P-450, designated MAb 1-7-1, was used for immunohistochemical staining of formalin-fixed tissues from oil- and MC-treated C57BL/6, DBA/2, and [(C57BL/6 X DBA/2) F1 X DBA/2] F2 mice. An avidin-biotin-peroxidase complex immunohistochemical technique was used. For controls, the tissues were also exposed to MAbs 1-48-5 and HyHel-9 (to egg white lysozyme). In liver, MAb 1-7-1 specifically stained the cytoplasm of centrilobular hepatocytes of C57BL/6 mice treated with MC (80 mg/kg) 48 h before kill; staining was not observed with vehicle-treated C57BL/6 mice, with oil- or MC-treated DBA/2 mice, or with comparable antibody concentrations of control MAbs 1-48-5 or HyHel-9. In the F2 mice, about 50% were expected to be MC inducible (AhbAhd). Inducibility phenotype was determined by measuring the conversion of [14C]MC to oxidized and conjugated products by liver homogenates. In freshly fixed material from MC-treated mice, those livers shown by the determination of phenotype to be inducible also stained with MAb 1-7-1, whereas those not induced were immunohistochemically negative. Furthermore, there was a significant positive correlation between degree of staining and the level of MC-metabolizing activity measured biochemically. The immunohistochemical procedure was also accurate in determination of inducibility phenotype of livers that had been in paraffin blocks for up to 2 yr if more concentrated antibody was used. In lung, MAb 1-7-1 stained specifically the alveolar walls and endothelium of blood vessels in MC-induced C57BL/6 mice only; the control MAbs and other mice gave negative results. Similarly, in kidney MAb 1-7-1 stained only glomeruli and interstitial tissue of MC-induced C57BL/6 mice and only endothelium of blood vessels in the colons of these mice. These observations are consistent with induction of the cytochrome P-450 recognized by MAb 1-7-1 in the endothelial cells of extrahepatic tissue. Immunohistochemical staining with MAb thus shows great promise for highly specific localization of particular species of cytochromes P-450 in tissues, for in situ quantification of these enzymes, and for determination of inducibility phenotype with fixed material.

Effect of adhesion factors fibronectin, laminin, and type IV collagen on spreading and growth of transformed and control rat liver epithelial cells.

To examine the specific effects of individual basement membrane components on the behavior of transformed cells of epithelial origin, ethionine-transformed cells and control cells at low and high passage levels were seeded on glass that had been coated with fibronectin, laminin, or type IV collagen. The cells used were sublines of the liver-derived TRL 1215 epithelial cell line, a line in which transformation has been shown to be accompanied by increased cell-substrate adhesion and cell spreading. Cell spreading on the different basement membrane components was determined by morphometry, and growth (proliferation) was measured by protein and DNA analyses. Laminin increased spreading and growth in transformed and control sublines. Laminin also induced changes in cell shape that were indicative of increased cell motility. For the control cells, fibronectin and also type IV collagen were less effective than laminin in stimulating cell spreading and growth. However, for the ethionine-transformed cells, fibronectin was as effective as laminin in stimulating cell spreading. With the exception of the spreading response to fibronectin, the ethionine-transformed cells were less sensitive to the defined substrata than were the control sublines. Moreover, only the ethionine-transformed cells were able to proliferate in serum-free medium. Thus, greater autonomy is characteristic of transformation for these epithelial cells and is exemplified by the reduced influence of and dependence on exogenous factors, both substrate-bound and soluble, for spreading and growth.

Role of cytoskeleton changes and expression of the H-ras oncogene during promotion of neoplastic transformation in mouse epidermal JB6 cells.

The relationship between cytoskeletal changes and oncogene expression in initiated cells during exposure to a tumor promoter was investigated in the phorbol ester-sensitive murine epidermis-derived cell line JB6 (P+ cells) and its promotion-insensitive variant (P- cells) using immunocytochemical methods, soft agar assays, and tumorigenicity tests in nude mice. Cytoskeletal changes in P+ and P- cells induced by short-term incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) were similar. Prolonged incubation with TPA allowed P- cells to regain their original appearance and resulted in growth inhibition; however, the extended presence of TPA produced in P+ cells persistent alterations in the distribution of actin, vinculin, and fibronectin. P+ cells proceeded to develop multilayered foci. Using monoclonal antibodies, we detected the H-ras oncogene-encoded Mr 21,000 protein (p21) exclusively in focus-forming cells. Both the observed morphological changes and the expression of p21 were reversible in P+ cells when TPA exposure was terminated soon after foci had developed. In order for TPA-treated P+ cells to grow as tumors in nude mice, multiple cycles of exposure to TPA in conjunction with clonal expansion in agar were necessary. The results indicate that there exists during promotion of the P+ JB6 cells a relationship between expression of the H-ras gene product p21 and enhanced proliferation with focus formation and that both expression of p21 and focus formation depend on the continuous presence of the promoting agent.