Anesthetic Activation of Central Respiratory Chemoreceptor Neurons Involves Inhibition of a THIK-1-Like Background K(+) Current

At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO(2)/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na(+)-dependent cationic current and inhibition of a background K(+) current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K(+) channel, K(2P)13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na(+), and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO(2) levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

Novel endogenous peptide agonists of cannabinoid receptors

Hemopressin (Hp), a 9-residue alpha-hemoglobin-derived peptide, was previously reported to function as a CB(1) cannabinoid receptor antagonist (1). In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hp alpha) or two (VD-Hp alpha) additional amino acids, as well as a beta-hemoglobinderived peptide with sequence similarity to that of hemopressin (VD-Hp beta). Characterization of the alpha-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB(1) cannabinoid receptor antagonist, both RVD-Hp alpha and VD-Hp alpha function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca(2+) indicate that these peptides activate a signal transduction pathway distinct from that activated by the endo-cannabinoid, 2-arachidonoylglycerol, or the classic CB(1) agonist, Hu-210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB(1) receptor is involved in the integration of signals from both lipid-and peptide-derived signaling molecules.-Gomes, I., Grushko, J. S., Golebiewska, U., Hoogendoorn, S., Gupta, A., Heimann, A. S., Ferro, E. S., Scarlata, S., Fricker, L. D., Devi, L. A. Novel endogenous peptide agonists of cannabinoid receptors. FASEB J. 23, 3020-3029 (2009). www.fasebj.org

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients

Background and Objective: Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT. Material and Methods: Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed. Results: Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits. Conclusion: Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens

Plasmodium falciparum, the causative agent of human malaria, invades host erythrocytes using several proteins on the surface of the invasive merozoite, which have been proposed as potential vaccine candidates. Members of the multi-gene PfRh family are surface antigens that have been shown to play a central role in directing merozoites to alternative erythrocyte receptors for invasion. Recently, we identified a large structural polymorphism, a 0.58 Kb deletion, in the C-terminal region of the PfRh2b gene, present at a high frequency in parasite populations from Senegal. We hypothesize that this region is a target of humoral immunity. Here, by analyzing 371 P. falciparum isolates we show that this major allele is present at varying frequencies in different populations within Senegal, Africa, and throughout the world. For allelic dimorphisms in the asexual stage antigens, Msp-2 and EBA-175, we find minimal geographic differentiation among parasite populations from Senegal and other African localities, suggesting extensive gene flow among these populations and/or immune-mediated frequency-dependent balancing selection. In contrast, we observe a higher level of inter-population divergence (as measured by F(st)) for the PfRh2b deletion, similar to that observed for SNPs from the sexual stage Pfs45/48 loci, which is postulated to be under directional selection. We confirm that the region containing the PfRh2b polymorphism is a target of humoral immune responses by demonstrating antibody reactivity of endemic sera. Our analysis of inter-population divergence suggests that in contrast to the large allelic dimorphisms in EBA-175 and Msp-2, the presence or absence of the large PfRh2b deletion may not elicit frequency-dependent immune selection, but may be under positive immune selection, having important implications for the development of these proteins as vaccine candidates. (C) 2009 Elsevier B.V. All rights reserved.

Limited global diversity of the Plasmodium vivax merozoite surface protein 4 gene

Merozoite surface proteins (MSPs) of the malaria parasites are major candidates for vaccine development targeting asexual blood stages. However, the diverse antigenic repertoire of these antigens that induce strain-specific protective immunity in human is a major challenge for vaccine design and often determines the efficacy of a vaccine. Here we further assessed the genetic diversity of Plasmodium vivax MSP4 (PvMSP4) protein using 195 parasite samples collected mostly from Thailand, Indonesia and Brazil. Overall, PvMSP4 is highly conserved with only eight amino acid substitutions. The majority of the haplotype diversity was restricted to the two short tetrapeptide repeat arrays in exon 1 and 2, respectively. Selection and neutrality tests indicated that exon 1 and the entire coding region of PvMSP4 were under purifying selection. Despite the limited nucleotide polymorphism of PvMSP4, significant genetic differentiation among the three major parasite populations was detected. Moreover, microgeographical heterogeneity was also evident in the parasite populations from different endemic areas of Thailand. (C) 2009 Elsevier B.V. All rights reserved.

Clopidogrel, independent of the vascular P2Y(12) receptor, improves arterial function in small mesenteric arteries from Angll-hypertensive rats

The P2Y(12) receptor antagonist clopidogrel blocks platelet aggregation, improves systemic endothelial nitric oxide bioavailability and has anti-inflammatory effects. Since P2Y(12) receptors have been identified in the vasculature, we hypothesized that clopidogrel ameliorates Angll (angiotensin II)-induced vascular functional changes by blockade of P2Y(12) receptors in the vasculature. Male Sprague Dawley rats were infused with Angll (60 ng/min) or vehicle for 14 days. The animals were treated with clopidogrel (10 mg . kg(-1) of body weight . day(-1)) or vehicle. Vascular reactivity was evaluated in second-order mesenteric arteries. Clopidogrel treatment did not change systolic blood pressure [(mmHg) control-vehicle, 117 +/- 7.1 versus control-clopidogrel, 125 +/- 4.2; Angll vehicle, 197 +/- 10.7 versus Angll clopidogrel, 198 +/- 5.2], but it normalized increased phenylephrine-induced vascular contractions [(%KCI) vehicle-treated, 182.2 +/- 18% versus clopidogrel, 133 +/- 14%), as well as impaired vasodilation to acetylcholine [(%) vehicle-treated, 71.7 +/- 2.2 versus clopidogrel, 85.3 +/- 2.8) in Angll-treated animals. Vascular expression of P2Y(12) receptor was determined by Western blot. Pharmacological characterization of vascular P2Y(12) was performed with the P2Y(12) agonist 2-MeS-ADP [2-(methylthio) adenosine 5`-trihydrogen diphosphate trisodium]. Although 2-MeS-ADP induced endothelium-dependent relaxation [(Emax %) = 71 +/- 12%) as well as contractile vascular responses (Emax % = 83 +/- 12%), these actions are not mediated by P2Y(12) receptor activation. 2-MeS-ADP produced similar vascular responses in control and Angll rats. These results indicate potential effects of clopidogrel, such as improvement of hypertension-related vascular functional changes that are not associated with direct actions of clopidogrel in the vasculature, supporting the concept that activated platelets contribute to endothelial dysfunction, possibly via impaired nitric oxide bioavailability.

Notch signaling controls the balance of ciliated and secretory cell fates in developing airways

Although there is accumulated evidence of a role for Notch in the developing lung, it is still unclear how disruption of Notch signaling affects lung progenitor cell fate and differentiation events in the airway epithelium. To address this issue, we inactivated Notch signaling conditionally in the endoderm using a Shh-Cre deleter mouse line and mice carrying floxed alleles of the Pofut1 gene, which encodes an O-fucosyltransferase essential for Notch-ligand binding. We also took the same conditional approach to inactivate expression of Rbpjk, which encodes the transcriptional effector of canonical Notch signaling. Strikingly, these mutants showed an almost identical lung phenotype characterized by an absence of secretory Clara cells without evidence of cell death, and showed airways populated essentially by ciliated cells, with an increase in neuroendocrine cells. This phenotype could be further replicated in cultured wild-type lungs by disrupting Notch signaling with a gamma-secretase inhibitor. Our data suggest that Notch acts when commitment to a ciliated or non-ciliated cell fate occurs in proximal progenitors, silencing the ciliated program in the cells that will continue to expand and differentiate into secretory cells. This mechanism may be crucial to define the balance of differentiated cell profiles in different generations of the developing airways. It might also be relevant to mediate the metaplastic changes in the respiratory epithelium that occur in pathological conditions, such as asthma and chronic obstructive pulmonary disease.

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R(free)=21.2%) at 1.70 angstrom resolution and 15.81% (R(free) = 19.2%) at 1.85 angstrom resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly beta-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity. (C) 2008 Elsevier Ltd. All rights reserved.

An Evaluation of”Middle Ages Dead or Live?”The first interactive exhibition at the National Museum of History

This is a study conducted at, and for, the National Museum of History in Stockholm. The aim of the study was to confirm or disconfirm the hypothesis that visitors in a traditional museum environment might not take part in interactivity in an interactive exhibition. And if they do the visitors might skip the texts and objects on display. To answer this and other questions a multiple method was used. Both non participant observations and exit interviews were conducted. After a description of the interactive exhibits, theory of knowledge and learning is presented before the gathered data is presented. All together 443 visitors were observed. In the observations the visitors were timed on how much time they spent in the room, the time spent on the interactivity, texts and objects. In the 40 interviews information about visitors’ participation in the interactivity was gathered. What interactivity the visitor found easiest, hardest, funniest and most boring.The result did not confirm the hypothesis. All kinds of visitors, children and adults, participated in the interactivities. The visitors took part in the texts and objects and the interactive exhibits.

Digital spiral analysis for objective assessment of fine motor timing variability in Parkinson's disease

OBJECTIVES: To develop a method for objective assessment of fine motor timing variability in Parkinson’s disease (PD) patients, using digital spiral data gathered by a touch screen device. BACKGROUND: A retrospective analysis was conducted on data from 105 subjects including65 patients with advanced PD (group A), 15 intermediate patients experiencing motor fluctuations (group I), 15 early stage patients (group S), and 10 healthy elderly subjects (HE) were examined. The subjects were asked to perform repeated upper limb motor tasks by tracing a pre-drawn Archimedes spiral as shown on the screen of the device. The spiral tracing test was performed using an ergonomic pen stylus, using dominant hand. The test was repeated three times per test occasion and the subjects were instructed to complete it within 10 seconds. Digital spiral data including stylus position (x-ycoordinates) and timestamps (milliseconds) were collected and used in subsequent analysis. The total number of observations with the test battery were as follows: Swedish group (n=10079), Italian I group (n=822), Italian S group (n = 811), and HE (n=299). METHODS: The raw spiral data were processed with three data processing methods. To quantify motor timing variability during spiral drawing tasks Approximate Entropy (APEN) method was applied on digitized spiral data. APEN is designed to capture the amount of irregularity or complexity in time series. APEN requires determination of two parameters, namely, the window size and similarity measure. In our work and after experimentation, window size was set to 4 and similarity measure to 0.2 (20% of the standard deviation of the time series). The final score obtained by APEN was normalized by total drawing completion time and used in subsequent analysis. The score generated by this method is hence on denoted APEN. In addition, two more methods were applied on digital spiral data and their scores were used in subsequent analysis. The first method was based on Digital Wavelet Transform and Principal Component Analysis and generated a score representing spiral drawing impairment. The score generated by this method is hence on denoted WAV. The second method was based on standard deviation of frequency filtered drawing velocity. The score generated by this method is hence on denoted SDDV. Linear mixed-effects (LME) models were used to evaluate mean differences of the spiral scores of the three methods across the four subject groups. Test-retest reliability of the three scores was assessed after taking mean of the three possible correlations (Spearman’s rank coefficients) between the three test trials. Internal consistency of the methods was assessed by calculating correlations between their scores. RESULTS: When comparing mean spiral scores between the four subject groups, the APEN scores were different between HE subjects and three patient groups (P=0.626 for S group with 9.9% mean value difference, P=0.089 for I group with 30.2%, and P=0.0019 for A group with 44.1%). However, there were no significant differences in mean scores of the other two methods, except for the WAV between the HE and A groups (P<0.001). WAV and SDDV were highly and significantly correlated to each other with a coefficient of 0.69. However, APEN was not correlated to neither WAV nor SDDV with coefficients of 0.11 and 0.12, respectively. Test-retest reliability coefficients of the three scores were as follows: APEN (0.9), WAV(0.83) and SD-DV (0.55). CONCLUSIONS: The results show that the digital spiral analysis-based objective APEN measure is able to significantly differentiate the healthy subjects from patients at advanced level. In contrast to the other two methods (WAV and SDDV) that are designed to quantify dyskinesias (over-medications), this method can be useful for characterizing Off symptoms in PD. The APEN was not correlated to none of the other two methods indicating that it measures a different construct of upper limb motor function in PD patients than WAV and SDDV. The APEN also had a better test-retest reliability indicating that it is more stable and consistent over time than WAV and SDDV.

Bleeding: A Collection of Poetry and Short Fiction

Poetry Short Stories

Imperial shoe palace: a collection of poems

Poems