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Student engagement with the community is a cornerstone of undergraduate nursing education in Canada. Working with community from perspectives of social justice, health equity, advocacy, and political action are essential for workforce readiness. We suggest that the erosion of public health theory and clinical courses in baccalaureate nursing programs undermines the potential capability of nurses to address the intersectionality of the social determinants of health. The impact of the COVID-19 pandemic on populations further demonstrates inequities, particularly among marginalized populations. Public health nursing education supports students' understanding about the health impacts of social injustice, how systemic racism is embedded in colonial and Eurocentric structures, and practices of superiority and privileges.We, as a national group of public health nursing educators, set out to investigate how existing guidelines and competencies support public health in undergraduate education across Canada. Results from a national questionnaire of educators, and of PHN leaders on new graduate practice readiness are presented. Questionnaire responses confirm an erosion of PHN theory and practice in baccalaureate nursing education (BNE) curricula. The results of the questionnaires combined with evidence of PHN since the global pandemic provide educators and practitioners more insight to inform future directions to respond to workforce readiness.

Affinity chromatography was used to identify potential cellular targets that are responsible for neuroprotective activity of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides. Active and inactive representatives of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides bearing an extended linker were synthesized and immobilized on an agarose-based matrix. This was followed by the identification of specifically bound proteins isolated out of the whole rat brain extract. Inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) was identified as candidates for cellular targets.

Sixteen new 1-(2-methoxyphenyl)-4-(1-phenethylpiperidin-4-yl)piperazines and 1-(2-methoxyphenyl)-4-[(1-phenethylpiperidin-4-yl)methyl]piperazines were synthesized to be used as probes for mapping the dopamine D2 receptor (D2 DAR) arylpiperazine binding site. All compounds were evaluated for their affinity toward D2 DAR in an in vitro competitive displacement assay. The most active one was 1-(2-methoxyphenyl)-4-{[1-(3-nitrophenethyl)piperidin-4-yl]methyl}piperazine (25) with an affinity of Ki = 54 nM. Docking analysis was conducted on all herein described compounds, whereas molecular dynamic simulation was performed on ligand 25 to establish its mode of interaction with D2 DAR. Two possible docking orientations are proposed; the one with a salt bridge between the piperidine moiety and Asp114 of D2 DAR is more stable.

Arylpiperazine-based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2 /5-HT1A ligands, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl}-phenyl]-picolinamide (6a) and N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate-immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5-HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0-7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL-6, IL-1, and GM-CSF, TH 1 cytokine IFN-γ, TH 17 cytokine IL-17, as well as the signature transcription factors of TH 1 (T-bet) and TH 17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti-apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN-93 and neuronal cell line PC12 from mitogen-activated normal T cells or myelin basic protein-activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation. Arylpiperazine dopaminergic/serotonergic ligands reduce neurological symptoms of acute autoimmune encephalomyelitis in rats without affecting the activation of autoreactive immune response, through mechanisms involving a decrease in CNS immune infiltration, as well as direct protection of CNS from immune-mediated damage. These data indicate potential usefulness of arylpiperazine-based compounds in the treatment of neuroinflammatory disorders such as multiple sclerosis.

The ratio of affinities toward the dopamine D₂ and the 5-hydroxytryptamine 5-HT(1A) receptors is one of the important parameters that determine the efficiency of antipsychotic drugs. Here, we present the synthesis of ortho-, meta-, and para-N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides and their structure-activity relationship studies on dopamine D₂ and 5-hydroxytryptamine 5-HT(1A) receptors. It was shown that the biological activity of the described ligands strongly depends on their topology as well as on the nature of the heteroaryl group in the head of the molecules. Docking simulations together with conformational analysis revealed a rational explanation for the ligands' behavior. The molecular model of receptor-ligand interactions described herein provided us with a tool for the rational design of new compounds with a favorable D₂/5-HT(1A) profile.

There is an urgent need for surrogate biomarkers in clinical diagnostics but also preclinical in toxicology of chemicals and efficacy testing of pharmaceuticals. On the background of the emerging fields of systems biology and theranostics, the importance of time scales and the synchronization of complex biological readouts become increasingly obvious. Systemic effects such as responses to stimuli, medical intervention, cellular stress, or toxicity elicit immediate molecular changes on the protein level. Early events include phosphorylation and oxidation of proteins. These posttranslational modifications have a direct impact on enzyme activities and protein-protein interactions. Only at downstream stages is gene transcription activated. Here we outline the analytical and statistical requirements dealing with complex patterns of protein expression and the extraction of protein surrogate biomarkers defining specific early stages of biological responses. We also present successful examples from research on aging and discuss prerequisites and necessary considerations while moving candidates to validation.

In this paper the results of testing and calibration of measuring systems for very fast transients (VFT) in gas-insulated substation (GIS) are presented. The general principles for the construction of measuring systems for recording and transmission of VFT are also discussed. The paper describes in detail the constructions offour measuring probe types adjusted for VFT monitoring in 123 kV SF 6 -insulated substation. The primary construction requirement was the ability to monitor VFT in 123kV SF6-insulated substation containing frequencies ≥ 200 MHz without distortion in both frequency and voltage domains. For the purpose of the measuring system testing and calibration an experimental facility was developed based on the computer experimental modelling technique. The verified experimental facility was then used for testing and calibration of measuring probes type. During the test, various effects on the measuring system response quality were noted with respect to the applied constructional solutions, with different kinds ofdielectric material used for the low-voltage capacitor and different diameters of the sensor probe. The obtained results are explained and the possibilities offurther improvement of SF 6 VFT measuring system characteristics suggested.

Clinical properties of atypical antipsychotics are based on their interaction with D(2) dopamine receptor and serotonin 5-HT(1A) and 5-HT(2A) receptors. As a part of our research program on new antipsychotics, we synthesized various derivatives of 1-cinnamyl-4-(2-methoxyphenyl)piperazines, and evaluated their affinities for D(2), 5-HT(1A), 5-HT(2A), and adrenergic (alpha(1)) receptors using radioligand-binding assays. In addition, we performed docking analysis using models for the D(2) and 5-HT(1A) receptors. All compounds exhibited low to moderate affinity to 5-HT(1A) and 5-HT(2A) receptors, high affinity to the D(2 )receptor and large variability in affinities for the alpha(1) receptor. Docking analysis indicated that the binding to D(2) and 5-HT(1A) receptors is based on (i) interaction between protonated N1 of the piperazine ring and various aspartate residues, (ii) hydrogen bonds between various moieties of the ligand and the residues of threonine, serine, histidine or tryptophane, and (iii) edge-to-face interactions of the aromatic ring of the arylpiperazine moiety with phenylalanine or tyrosine residues. Docking data for the D(2) receptor can account for the binding properties obtained in binding assays, suggesting that the model is reliable and robust. However, docking data for the 5-HT(1A) receptor cannot account for actual binding properties, suggesting that further refinement of the model is required.

Tumor necrosis factor alpha (TNF alpha) is involved in regulation of food intake, inflammatory response, and cancer cachexia. Its actions are mediated by at least two receptors: TNFR1 and TNFR2. We show that mice lacking the TNF alpha receptors have altered levels of proteins that take part in signal transduction, stress response, protein folding, glucose and amino acid metabolism, vesicle trafficking, and cytoskeletal arrangements. This is the first time that some of them have been associated with TNF alpha signaling pathways.

1-(2-Heteroarylalkyl)-4-phenylpiperazines containing methyl group in either the alpha- or the beta-position of the side alkyl chain were synthesized as racemic mixtures. They were evaluated for in vitro binding affinity at the D1 and D2 dopamine and 5-HT1A serotonin receptors using synaptosomal membranes of the bovine caudate nucleus and hippocampus, respectively, as a source of the corresponding receptors. Tritiated SCH 23390 (D1 receptor-selective), spiperone (D2 receptor-selective), and 8-OH-DPAT (5-HT1A receptor-selective) were employed as the radioligands. None of the new compounds expressed significant affinity for the D1 receptor. Introduction of the methyl group into the beta-position of the parent molecules increased the affinity for the D2 receptor (10b-13b), and decreased the affinity for the 5-HT1A receptor with the exception of imidazole (11b) which was a rather efficient displacer of 8-OH-DPAT. Most potent of the newly synthesized compounds in [3H]spiperone assay were compounds (+/-)6-[1-methyl-2- (4-phenylpiperazin-1-yl)-ethyl]-1,4-dihydroquinoxaline-2,3-dione (10b), Kd = 6.0 nM and (+/-)5-[1-methyl-2-(4-phenylpiperazin-1-yl)-ethyl]-1,3-dihydrobenzoimidazol- 2-thione (13b), Kd = 5.3 nM. However, compounds containing methyl group in alpha-position (10a-13a) of the parent molecules expressed a decreased affinity for the D2 receptor, while the affinity for the 5-HT1A receptor remained in the same range of concentrations as that of closely related achiral parent compounds (14-17) run in the same binding assays as references.

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.

Chrysospermin C is a 19-residue peptaibol capable of forming transmembrane ion channels in phospholipid bilayers. The conformation of chrysospermin C bound to dodecylphosphocholine micelles has been solved using heteronuclear NMR spectroscopy. Selective 15N-labeling and 13C-labeling of specific α-aminoisobutyric acid residues was used to obtain complete stereospecific assignments for all eight α-aminoisobutyric acid residues. Structures were calculated using 339 distance constraints and 40 angle constraints obtained from NMR data. The NMR structures superimpose with mean global rmsd values to the mean structure of 0.27 A (backbone heavy atoms) and 0.42 A (all heavy atoms). Chrysospermin C bound to decylphosphocholine micelles displays two well-defined helices at the N-terminus (residues Phe1-Aib9) and C-terminus (Aib13-Trp-ol19). A slight bend preceding Pro14, i.e. encompassing residues 10–12, results in an angle of ≈ 38° between the mean axes of the two helical regions. The bend structure observed for chrysospermin C is compatible with the sequences of all 18 long peptaibols and may represent a common ‘active’ conformation. The structure of chrysospermin C shows clear hydrophobic and hydrophilic surfaces which would be appropriate for the formation of oligomeric ion channels.

Functional proteomic methods have been developed and applied to the investigation of signal transduction systems involving platelet-derived growth factor (PDGF), endothelin and bradykinin receptors. Mouse fibroblast cells have been stimulated with PDGF or endothelin. Phosphorylation/dephosphorylation of several hundred proteins has been followed as a function of time following stimulation using 2-D gel electrophoresis and anti-phosphotyrosine or anti-phosphoserine antibodies. Up to 100 of these proteins showed strong changes in phosphorylation with minutes of receptor stimulation. Identification of some of these proteins by mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and by partial peptide sequencing with ion trap electrospray mass spectrometry has identified proteins which were previously known to be associated with PDGF signaling, proteins which have been shown to be involved in other signaling pathways, but not PDGF and proteins not previously associated with signal transduction. Parallel to these studies, new methods for rapid, single-step isolation of peptide receptors using a peptide coupled to a (dA)(30) oligonucleotide have been developed and applied to mass spectrometric studies of post-translational modifications of the endothelin B and bradykinin B-2 receptors under in vivo conditions. Both receptors have been shown to undergo extensive phosphorylation as well as palmitoylation. The patterns of post-translational modifications are more complex than previously recognized and provide new indications of possible roles for these modifications in the regulation and response of these receptors.

With an aim of creating new, high affinity dopaminergic ligands, six different 3- and 4-substituted 1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]piperidines and nine related heterocyclic congeners were synthesized and evaluated for in vitro binding affinity at D1 and D2 dopamine receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei were used as a source of the dopamine receptors. Only 4-[bis-(4-fluorophenyl)methylene]-piperidines, compounds 9e, 10d, and 11d, expressed moderate affinity for the D1 receptors, while all other compounds were inactive competitors of [3H]SCH 23390. Compounds 9c, 9d, 10c, 11a, and 11c were inactive in the D2 receptor binding assay, as well. Derivatives of 4-phenylpiperidine (9-11b) and 3-phenylpiperidine (10a) expressed a moderate to low affinity for the D2 receptors. However, racemic (+/-)-1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]-3-phenylpiperidine 9a and its enantiomer (+)-9a behaved as selective, high affinity D2 receptor ligands, the latter being some four times more active than the racemate.

The overall folding of an RNA molecule is reflected in its base pairing pattern. The identification of that pattern provides a first step towards the determination of the structure of an RNA molecule. We show that the application of heteronuclear NMR methods at 750 MHz to E. coli 5S rRNA (120 nucleotides) selectively labelled with 15N in guanine and uridine allows observation of base pairing patterns for a larger RNA molecule. We also present evidence that the fold of the E-domain of the 5S rRNA (nt 79–97) as a contiguous part of the 5S rRNA and as an isolated molecule is virtually the same.

Bovine lung endothelin-B receptor has been isolated in good yield with a new procedure involving the use of endothelin-1 coupled to iminobiotin with a long spacer and avidin-agarose affinity chromatography. Contrary to previous reports, evidence has been obtained that the native form of this receptor corresponds to the full-length transcript expected on the basis of cDNA clones. The binding of endothelin to a variety of shortened fragments of the full receptor suggests that the long N-terminal sequence of this receptor has very little influence on the binding of endothelin and that the main determinants of the endothelin binding site might be constituted by residues in the sixth, and possibly the seventh, transmembrane helices.

With an aim of creating new, high affinity dopaminergic ligands, six different 3- and 4-substituted 1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]piperidines and nine related heterocyclic congeners were synthesized and evaluated for in vitro binding affinity at D1 and D2 dopamine receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei were used as a source of the dopamine receptors. Only 4-[bis-(4-fluorophenyl)methylene]-piperidines, compounds 9e, 10d, and 11d, expressed moderate affinity for the D1 receptors, while all other compounds were inactive competitors of [3H]SCH 23390. Compounds 9c, 9d, 10c, 11a, and 11c were inactive in the D2 receptor binding assay, as well. Derivatives of 4-phenylpiperidine (9-11b) and 3-phenylpiperidine (10a) expressed a moderate to low affinity for the D2 receptors. However, racemic (+/-)-1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]-3-phenylpiperidine 9a and its enantiomer (+)-9a behaved as selective, high affinity D2 receptor ligands, the latter being some four times more active than the racemate.

A series of substituted 4-[2-(5-benzimidazolelethyl)-arylpiperazines was synthesized by introducing different substituents into position 2 of benzimidazole ring of 4-(2-(N,N-di-n-propyl-amino)ethyl)-1,2-diaminobenzenes. They were evaluated for in vitro binding affinity at the D 1 and D 2 dopamine and- 3-HT 1A serotonin receptors using synaptosomal membranes of the bovine caudate nuclei and hippocampi, respectively. Tritiated SCH 23390 (D 1 receptor-selective), spiperone (D 2 receptor selective) and- 8 OH-DPAT (5-HT 1A receptor selective) were employed as the radioligands. Only compound 6 expressed a moderate binding affinity at the dopamine D 1 receptor, while the remaining ligands were inefficient or weak competitors of [ 3 H]SCH 23390. Compound 12 was an absolutely inactive competitor of all three radioligands.Also, compound 7 was an inefficient displacer of [ 3 H]-8-OH-DPAT. Compound 19 with a K i value of 3.5 nM was the most potent competitor of [ 3 H]spiperone and compound 13(K i = 3.3 nM) was the most efficient in displacing [ 3 H]-8-OH-DPAT from the 5-HT 1A serotonin receptor. Ligands 5,6,8-11, and 13-2O expressed mixed dopaminergic/serotonergic activity in nanomolar range of concentrations with varying affinity ratios which strongly depended on the properties of the substituents introduced into position 2 of benzimidazole ring of the parent compounds.

The well established M1 selective muscarinergic antagonist Pirenzepine 11-[2-(4-methyl-piperazin-1-yl)-acetyl]-5,11-dihydro-benzo[e]pyrido[3,2-b][1,4]diazepin-6-one (1) exhibits an unusual behaviour in vivo, which cannot be explained with M1 antagonism exclusively. One of the aspects discussed is a specific interaction with poly ADP-ribose polymerase (PARP-1). 1 undergoes metabolism to form LS 75 5,11-dihydro-benzo[e]pyrido[3,2-b][1,4]diazepin-6-one (2). In order to study deviations in Pirenzepine efficacy from pure M1 binding in vivo using PET, appropriate positron emitter labelled analogues of 1 and 2 were synthesised. Non-radioactive reference compounds 3 and 4 were tested for PARP-1 inhibition. The n-octanol–water partition coefficients of compounds 1, 2, 3 and 4 at pH 7.4 (logD7.4) were determined. Both, 3 and 4 were labelled with 18F via 2-[18F]fluoroalkylation in position 5 of the benzodiazepinone moiety to obtain N5-[18F]fluoroethyl Pirenzepine [18F]-3 and N5-[18F]fluoroethyl LS 75 [18F]-4. Radiotracers [18F]-3 and [18F]-4 were obtained in radiochemical yields of 15±4 % and 30±5% after 120 and 110 min, respectively. Metabolism of both compounds was investigated in vitro in human and rat plasma, respectively. Compound 3 did not show activity as an inhibitor of PARP-1. Contrary, 4 displays moderate PARP-1 inhibition potency. The new radiotracer [18F]-4 can be applied for molecular imaging using autoradiography and PET. Copyright © 2009 John Wiley & Sons, Ltd.

Neuroinflammation is an important mechanism of Alzheimer’s disease (AD)-associated neurodegeneration. Accumulation and deposition of Aβ around neurovasculature is found in the majority of AD patients. However, little is known whether Aβ deposition would result in chronic vascular inflammation and insufficiency, which may contribute to neurodegeneration. We showed previously that Aβ1-40 induces strong expression of inflammatory genes in primary cultured human brain endothelial cells (HBEC) by semi-quantitative RT-PCR analyses, including IL-1β, MCP-1, TNF-α, IL-8 and ICAM-1. In the present study, we have profiled cytokine expression at the protein level and shown that Aβ-treated HBEC secrete immunoreactive cytokines into media, including MCP-1, MCP-2, GRO, IL6, IL-8, etc. In order to determine whether these cytokines are expressed in AD brain, we have carried out real-time Q-PCR analyses using AD and age-matched nondemented control (ND) brain samples. Increased expression of inflammatory genes, IL-1β, MCP-1, IL-6, and GRO were observed in AD compared to ND brain samples. These results validate our findings in cultured HBEC. We next investigated transcriptional regulation of inflammatory genes in Aβ-treated HBEC. HBEC were treated with Aβ1-40 or scrambled Aβ40-1 for 8hr. Nuclear extracts were prepared and TranSignal Protein/DNA Array blots carrying 56 different transcription factors (TF) were used for the analyses (Panomics). Each TF was spotted four times on the blots and the experiments were repeated. In comparison with controls, five TFs were increased over two fold (AP-1, CREB, GATA, NFATc, GRE) and three TFs were down-regulated over two fold (p53, TR, MEF-1) in Aβ1-40-treated HBEC. Increased levels of AP-1, CREB and GATA were confirmed in AD brain samples by the same array analyses. Sequence analyses show that the promoter regions of many inflammatory genes carry AP-1 or/and CREB binding sites. GATA and NFATc are believed to complex or associate with AP-1 or/and CREB to regulate cytokine expression in cells. Our studies demonstrate that Aβ can induce inflammatory gene expression in HBEC and some of these genes are also up-regulated in AD brain and suggest that several TFs (AP-1, CREB, GATA or NFATc) may be involved in transcriptional regulation of the inflammatory genes in Aβ-treated HBEC and/or in AD brain., 10th International Conference on Alzheimer's Disease and Related Disorders, July 15 - 20, 2006; Madrid, Spain