Heme protonation affects iron-NO binding in the NO transport protein nitrophorin.

The nitrophorins (NPs) comprise an unusual group of heme proteins with stable ferric heme iron nitric oxide (Fe-NO) complexes. They are found in the salivary glands of the blood-sucking kissing bug Rhodnius prolixus, which uses the NPs to transport the highly reactive signaling molecule NO. Nuclear resonance vibrational spectroscopy (NRVS) of both isoform NP2 and a mutant NP2(Leu132Val) show, after addition of NO, a strong structured vibrational band at around 600 cm-1, which is due to modes with significant Fe-NO bending and stretching contribution. Based on a hybrid calculation method, which uses density functional theory and molecular mechanics, it is demonstrated that protonation of the heme carboxyl groups does influence both the vibrational properties of the Fe-NO entity and its electronic ground state. Moreover, heme protonation causes a significant increase of the gap between the highest occupied and lowest unoccupied molecular orbital by almost one order of magnitude leading to a stabilization of the Fe-NO bond.

Interest of cytology combined with Xpert® HPV and Anyplex® II HPV28 Detection human papillomavirus (HPV) typing: differential profiles of anal and cervical HPV lesions in HIV-infected patients on antiretroviral therapy.

OBJECTIVES: The aim of the study was to assess the interest to combine cytological examination and human papillomavirus (HPV) typing of anal and cervical Papanicolaou (Pap) smears of HIV-infected patients on combination antiretroviral therapy (cART), to evaluate whether differences in prevalence exist between anal and cervical squamous intraepithelial lesions in patients with high-risk oncogenic HPV infection. METHODS: Anal and/or cervical Pap smears were obtained by anoscopy and/or colposcopy in 238 subjects recruited consecutively in 2015: anal smears were obtained from 48 male and female patients [42 men; 35 men who have sex with men (MSM)] and cervical smears from 190 female patients. Cytological Bethesda classification was coupled with HPV typing. HPV typing was performed, on the same smears, using the Xpert® HPV Assay, which detects only high-risk HPV (hrHPV), and the Anyplex® II HPV28 Detection assay, which detects hrHPV and low-risk (lr) HPV. RESULTS: Our data showed clear-cut differences between the anal and cervical samples. Compared with the cervical samples, the anal samples exhibited (1) more numerous cytological lesions, which were histologically proven; (2) a higher hrHPV infection prevalence; (3) a higher prevalence of multiple hrHPV coinfections whatever HPV typing kit was used; (4) a predominance of HPV16 and HPV18/45 types. Overall, there was an almost perfect agreement between the two HPV typing assays (absolute agreement = 90.3%). CONCLUSIONS: Co-testing consisting of cytology and HPV typing is a useful screening tool in the HIV-infected population on cART. It allows detection of prevalence differences between anal and cervical HPV-related lesions. As recently recommended, anal examination should be regularly performed especially in HIV-infected MSM but also in HIV-infected women with genital hrHPV lesions.

Vasoactive intestinal peptide dampens formyl-peptide-induced ROS production and inflammation by targeting a MAPK-p47phox phosphorylation pathway in monocytes.

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47phox, p67phox, p40phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the "MAPK-p47phox phosphorylation-NOX2 activation" axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.

Parvalbumin- and vasoactive intestinal polypeptide-expressing neocortical interneurons impose differential inhibition on Martinotti cells.

Disinhibition of cortical excitatory cell gate information flow through and between cortical columns. The major contribution of Martinotti cells (MC) is providing dendritic inhibition to excitatory neurons and therefore they are a main component of disinhibitory connections. Here we show by means of optogenetics that MC in layers II/III of the mouse primary somatosensory cortex are inhibited by both parvalbumin (PV)- and vasoactive intestinal polypeptide (VIP)-expressing cells. Paired recordings revealed stronger synaptic input onto MC from PV cells than from VIP cells. Moreover, PV cell input showed frequency-independent depression, whereas VIP cell input facilitated at high frequencies. These differences in the properties of the two unitary connections enable disinhibition with distinct temporal features.

Characterization of the mixed axial ligand complex (4-cyanopyridine)(imidazole)(tetramesitylporphinato)iron(iii) perchlorate. Stabilization by synergic bonding.

The reaction of [Fe(TMP)(OClO3)], where TMP is the dianion of tetramesitylporphyrin, with a combination of a strong π-acceptor ligand and a π-donating imidazole can lead to the preparation of mixed-ligand complexes [Fe(Porph)(4-CNPy)(L)]+ where L is imidazole itself or 1-acetylimidazole and 4-cyanopyridine is the strong π acceptor ligand. The stability of the new mixed-ligand pair is the presumed result of synergic bonding between the two axial ligands. The molecular structure and other characterization of the new mixed axial ligand complex, [Fe(TMP)(4-CNPy)(HIm)]ClO4 is described. The axial ligands have a relative perpendicular arrangement with Fe-N(imidazole) = 1.945 Å and Fe-N(pyridine) = 2.021 Å The average equatorial Fe-Np distance is 1.963 Å, which is consistent with the S4-ruffled TMP core. Despite the relative perpendicular arrangement of axial ligands, the EPR spectrum of the complex is a rhombic signal and not a large gmax signal. The EPR g-values are g1 = 3.05, g2 = 2.07, and g3 = 1.22. A quadrupole doublet was seen in the Mössbauer spectrum with an isomer shift of 0.197 mm/s and quadrupole splitting of 1.935 mm/s. Two crystalline forms of [Fe(TMP)(4-CNPy)(HIm)]ClO4 have been characterized; the two forms differ only in the solvent content of the lattice. Crystal data for form A: a = 15.432 (12) Å, b = 20.696 (2) Å, c = 19.970 (5) Å, and ß = 99.256 (14)°, monoclinic, space group P21/n, V = 6295 (2) Å3, Z = 4, formula FeCl3O4N8C69H69, 8397 observed data, R1 = 0.086, wR2 = 0.210, refinement on F2. Crystal data for form B: a = 15.267 (3) Å, b = 20.377 (6) Å, c = 19.670 (4) Å, and ß = 98.14 (1)°, monoclinic, space group P21/n, V = 6058 (4) Å3, Z = 4, formula C65.25H60.5Cl1.5FeN8O4, 5464 observed data, R1 = 0.096, wR2 = 0.112, refinement on F.

Determinants of macroscopic anal cancer and precancerous lesions in 1206 HIV-infected screened patients.

AIM: Anal screening is recommended in HIV-positive patients, especially men who have sex with men (MSM), due to an increased incidence of anal cancer. The optimal screening methods are not generally agreed. METHOD: Screening for anal lesions by anorectal examination, including anoscopy, was offered to HIV-positive outpatients in a tertiary care university hospital regardless of gender or sexual orientation. RESULTS: Among the 1206 screened patients (701 MSM, 247 heterosexual men, 258 women), 311 (26%) had histologically proven lesions related to human papilloma virus (HPV) (34% MSM, 14% heterosexual men, 14% women); 123 (10%) had low-grade dysplasia and 70 (6%) high-grade dysplasia. Seven anal cancers were also diagnosed. Determinants of any lesion were age < 45 years [OR = 1.56 (95% CI, 1.16-2.11)], a CD4 count of < 200/mm3 [OR = 2.54 (1.71-3.78)], receptive anal intercourse [OR =3.03 (2.06-4.47)], sub-Saharan African origin [OR = 0.53 (0.33-0.85)], and history of HPV-related lesion [OR = 1.84 (1.35-2.51)]. These determinants were similar for all different grades of dysplasia. In patient subgroup analysis, receptive anal intercourse, the CD4 cell count and a history of HPV lesions were determinants of HPV-positivity in all patients, whereas age was only a determinant in men. CONCLUSION: Anoscopy is an alternative method for anal screening in an HIV-positive population. This screening has to be compared with other tools in populations at high risk of anal cancer.

Combined ampakine and BDNF treatments enhance poststroke functional recovery in aged mice via AKT-CREB signaling.

Cerebral ischemia results in damage to neuronal circuits and lasting impairment in function. We have previously reported that stimulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors with the ampakine, CX1837, increases brain-derived neurotrophic factor (BDNF) levels and affords significant motor recovery after stroke in young mice. Here, we investigated whether administration of CX1837 in aged (24 months old) mice was equally effective. In a model of focal ischemia, administration of CX1837 from 5 days after stroke resulted in a small gain of motor function by week 6 after stroke. Mice that received a local delivery of BDNF via hydrogel implanted into the stroke cavity also showed a small gain of function from 4 to 6 weeks after stroke. Combining both treatments, however, resulted in a marked improvement in motor function from 2 weeks after insult. Assessment of peri-infarct tissue 2 weeks after stroke revealed a significant increase in p-AKT and p-CREB after the combined drug treatment. Using the pan-AKT inhibitor, GSK-690693, or deletion of CREB from forebrain neurons using the CREB-flox/CAMKii-cre mice, we were able to block the recovery of motor function. These data suggest that combined CX1837 and local delivery of BDNF are required to achieve maximal functional recovery after stroke in aged mice, and is occurring via the AKT-GSK3-CREB signaling pathway.

Calorie restriction increases lipopolysaccharide-induced neuropeptide Y immunolabeling and reduces microglial cell area in the arcuate hypothalamic nucleus.

Calorie restriction (CR) increases longevity and elicits many health promoting benefits including delaying immunosenescence and reducing the incidence of age-related diseases. Although the mechanisms underlying the health-enhancing effects of CR are not known, a likely contributing factor is alterations in immune system functioning. CR suppresses lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines, blocks LPS-induced fever, and shifts hypothalamic signaling pathways to an anti-inflammatory bias. Furthermore, we have recently shown that CR attenuates LPS-stimulated microglial activation in the hypothalamic arcuate nucleus (ARC), a brain region containing neurons that synthesize neuropeptide Y (NPY), an orexigenic neuropeptide that is upregulated by a CR diet and has anti-inflammatory properties. To determine if increased NPY expression in the ARC following CR was associated with changes in microglial activation, a set of brain sections from mice that were exposed to 50% CR or ad libitum feeding for 28 days before being injected with LPS were immunostained for NPY. The density of NPY-immunolabeling was assessed across the rostrocaudal extent of the ARC and hypothalamic paraventricular nucleus (PVN). An adjacent set of sections were immunostained for ionized calcium-binding adapter molecule-1 (Iba1) and immunostained microglia in the ARC were digitally reconstructed to investigate the effects of CR on microglial morphology. We demonstrated that exposure to CR increased NPY expression in the ARC, but not the PVN. Digital reconstruction of microglia revealed that LPS increased Iba1 intensity in ad libitum fed mice but had no effect on Iba1 intensity in CR mice. CR also decreased the size of ARC microglial cells following LPS. Correlational analyses revealed strong associations between NPY and body temperature, and body temperature and microglia area. Together these results suggest that CR-induced changes in NPY are not directly involved in the suppression of LPS-induced microglial activation, however, NPY may indirectly affect microglial morphology through changes in body temperature.

Can rectal endoscopic sonography be used to predict infiltration depth in patients with deep infiltrating endometriosis of the rectum?

OBJECTIVES: To evaluate the diagnostic accuracy of rectal endoscopic sonography (RES) in the prediction of the infiltration depth of rectal endometriosis and to ascertain whether RES could be used to choose between segmental bowel resection and a more conservative approach, such as shaving or discoid resection. METHODS: In this retrospective study, 38 consecutive patients with symptomatic deep infiltrating endometriosis of the rectum who underwent laparoscopic colorectal resection were included. RES results for infiltration depth of rectal endometriosis were compared with results of pathological examination. The sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively), positive and negative likelihood ratios (LRs) and test accuracy were calculated for the presence of infiltration of the muscularis layers and submucosal/mucosal layers, as demonstrated by RES and confirmed by histopathological analysis. RESULTS: For the detection of muscularis layer infiltration by endometriosis, the PPV of RES was 100%, whereas for the detection of submucosal/mucosal layer involvement, the sensitivity was 89%, specificity was 26%, PPV was 55%, NPV was 71%, test accuracy was 58% and positive and negative LRs were 1.21 and 0.40, respectively. CONCLUSIONS: RES is a valuable tool for detecting rectal endometriosis as endometriotic infiltration of the muscularis layer can be predicted accurately. However, RES is less accurate in detecting submucosal/mucosal layer involvement and cannot, therefore, be used to choose between bowel resection and a more conservative approach.

Production and significance of CCAAT enhancer binding proteins alpha and beta in sinonasal inverted papilloma.

Sinonasal inverted papilloma (SIP) is a rare benign tumor featuring increased cell proliferation, a tendency toward squamous differentiation, recurrence and malignant transformation. The CCAAT enhancer binding proteins, C/EBPs, are transcription factors regulating the proliferation and differentiation of various types of cells, including epithelial cells. We prospectively investigated the production of these transcription factors and the related proliferation and differentiation targets, keratin-10, keratin-15 and cyclin-D1, in 26 SIP patients and 8 sinonasal polyposis cases suspected for SIP. Ten of these patients had one or more recurrences over follow-up periods of one to eight years. C/EBP-alpha and C/EBP-beta proteins were not found in normal-looking sinonasal epithelial cells. The proteins and RNAs were detected in SIP and, occasionally, in polyposis tissues. The production of these factors was not significantly correlated with age, sex, site, tumor size or medical history. By contrast, correlations were found between the levels of C/EBP-alpha and keratin-10 levels and between those of C/EBP-beta and keratin-15. C/EBP-alpha levels were also significantly correlated with cyclin-D1 levels. These data suggested that the C/EBPs are implicated in the regulation of cell proliferation and differentiation in SIP. Finally, recurrent SIP produced significantly larger amounts of C/EBP-alpha than non- recurrent tumors. These results implicate CCAAT enhancer binding proteins in the pathogenesis of SIP and highlight the role of C/EBP-alpha as a candidate marker for tumor recurrence.

Tyrosine triad at the interface between the Rieske iron-sulfur protein, cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus.

A triad of tyrosine residues (Y152-154) in the cytochrome c(1) subunit (C1) of the Rhodobacter capsulatus cytochrome bc(1) complex (BC1) is ideally positioned to interact with cytochrome c(2) (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations-A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.

Validation of a new method for building a three-dimensional physical model of the skull and dentition.

We present a new method for replicating the skull and occlusal surface with an accurate physical model that could be used for planning orthognathic surgery. The investigation was made on 6 human skulls, and a polyvinyl splint was fabricated on the dental cast of the maxillary dentition in each case. A cone beam computed tomogram (CBCT) was taken of each skull and a three-dimensional replica produced. The distorted dentition (as a result of magnification errors and streak artefacts) was removed from the three-dimensional model and replaced by new plaster dentition that was fabricated using the polyvinyl splint and a transfer jig replication technique. To verify the accuracy of the method the human skulls and the three dimensional replica model, with the new plaster dentition in situ, were scanned using a laser scanner. The three-dimensional images produced were superimposed to identify the errors associated with the replacement of the distorted occlusal surface with the new plaster dentition. The overall mean error was 0.72 and SD was (0.26)mm. The accuracy of the method encouraged us to use it clinically in a case of pronounced facial asymmetry.

Evaluation of an improved orthognathic articulator system. 2. Accuracy of occlusal wafers.

The errors produced by occlusal wafers constructed on casts of the teeth mounted on a standard articulator and an improved orthognathic articulator were investigated by carrying out simulated orthognathic surgery on plastic skulls. The wafers were used to relocate the position of the maxillae of the skulls. The vertical and horizontal displacements of the maxillae were determined from measurements of the positions of markers on the skull and teeth. Comparison of the magnitudes of the actual and intended movements showed that wafers constructed on the standard articulator had systematic prediction errors of up to 5mm, but the improved orthognathic articulator showed much smaller random errors. There was a statistically significant improvement in overall accuracy in predicting maxillary Le Fort I position with the use of the improved orthognathic articulator which the authors recommend for clinical use.

Evaluation of an improved orthognathic articulator system: 1. Accuracy of cast orientation.

A systematic study was carried out using plastic model skulls to quantify the accuracy of the transfer of face bow registration to the articulator. A standard Dentatus semi-adjustable articulator system was compared to a purpose built orthognathic articulator system by measuring the maxillary occlusal plane angles of plastic model skulls and of dental casts mounted on the two different types of articulators. There was a statistically significant difference between the two systems; the orthognathic system showed small random errors, but the standard system showed systematic errors of up to 28°.

Oxidation and loss of heme in soluble guanylyl cyclase from Manduca sexta.

Oxidation and loss of heme in soluble guanylyl/guanylate cyclase (sGC), the nitric oxide receptor, is thought to be a major contributor to cardiovascular disease and is the target of compounds BAY 58-2667 and HMR1766. Using spectroelectrochemical titration, we found a truncated sGC to be highly stable in the ferrous state (234 mV) and to bind ferrous heme tightly even in the presence of NO, despite the NO-induced release of the proximal histidine. In contrast, oxidized sGC readily loses ferric heme to myoglobin (0.47 ± 0.02 h(-1)). Peroxynitrite, the presumed cellular oxidant, readily oxidizes sGC in 5 mM glutathione.

Dimensions of the cranium and of the cranial cavity andintracranial volume in goats (Capra hircus LINNAEUS, 1758)

The aim of this project is to determine the dimensions of the cranium and the cranial cavity and the intracranialvolume in goats, using 64 adults. The dimensions of the cranium and cranial cavity were measured throughmetric tape and paquimeter, considering the intervals of the largest distances. To determine the intracranialvolume, balloons of latex were introduced in the cranial cavity, through the magnum foramen, later on, filledwith water that was transferred for graduate test tube. The average and the standard deviation of length, widthand height of the cranium, in millimeters, were respectively: 218.01 ± 6.96, 120.17 ± 10.01 and 108.14 ± 4.46.The average and the standard deviation of length, width, height, in millimeters, and of the volume of thecranial cavity, in cubic centimeters, were respectively: 109.31 ± 7.25, 61.36 ± 4.51, 63.85 ± 2.88 and119.31 ± 12.21. It was observed that, the width of the cranium possesses positive significant correlationswith the length (r = 0.6865), with the height (r = 0.5644) and with the intracranial volume (r = 0.5436).They were still established, positive significant correlations among the height of the cranial cavity, with thelength (r = 0.5682) and with the intracranial volume (r = 0.5473). Differences were evidenced between malesand females, in relation to the dimensions of the cranium and cranial cavity. There wasn’t difference of theintracranial volume in function of the sex of the goats.

FoxP3 overexpression and CD1a+ and CD3+ depletion in anal tissue as possible mechanisms for increased risk of human papillomavirus-related anal carcinoma in HIV infection.

AIM: We analysed local cellular and humoral immunity factors in the anal mucosa in an attempt to explain how HIV infection increases the risk of anal cancer in HPV-infected patients. METHOD: HIV-positive cases and matched HIV-negative controls with more than one recurrence of condylomas were included in a prospective study following treatment of the initial lesions. Patients were followed every 3 to 6 months for the development of anal intraepithelial neoplasia (AIN3) and cancer for up to 60 months. Tissue CD1a(+), CD3(+), CD4(+), CD8(+) cells and mRNAs of selected cytokines and chemokines were quantified and compared in patients with or without AIN3 or cancer using morphometric or immunohistochemistry analysis and qRT-PCR. RESULTS: Sixty-six individuals (22 patients and 44 controls) were included. In the case group, CD1a(+) and CD3(+) cell counts were significantly lower in biopsies from AIN3 and cancer specimens compared with those from AIN 1-2 or normal biopsies (P < 0.0001). A CD1a(+) count of < 10/mm was predictive of AIN3 and cancer (Odds ratio = 9.4, 95% CI: 5.4-18.3, P < 0.0001). IL-8 and IL23 levels were significantly higher in cancer than in non-cancer tissues regardless of HIV status (P = 0.02). FoxP3 expression was significantly higher in HIV-infected cases than in controls with AIN3/cancer (P < 0.04). CONCLUSION: Depletion of CD1a(+) and CD3(+) cells and overexpression of FoxP3 in the anal mucosa appear likely to contribute to the risk of HPV-related anal cancer in HIV-infected patients. Furthermore, overexpression of IL-8 and IL-23 in the anal mucosa might be responsible for the development of this cancer regardless of HIV status.

Repeated social defeat selectively increases δFosB expression and histone H3 acetylation in the infralimbic medial prefrontal cortex.

Exposure to social stress has been linked to the development and maintenance of mood-related psychopathology; however, the underlying neurobiological changes remain uncertain. In this study, we examined numbers of δFosB-immunoreactive cells in the forebrains of rats subjected to 12 episodes of social defeat. This was achieved using the social conflict model whereby animals are introduced into the home cage of older males ("residents") trained to attack and defeat all such "intruders"; importantly, controls were treated identically except that the resident was absent. Our results indicated that the only region in which δFosB-positive cells were found in significantly higher numbers in intruders than in controls was the infralimbic medial prefrontal cortex (mPFC). This same effect was not apparent using another psychological stressor, noise stress. Cells of the infralimbic mPFC also displayed evidence of chromatin remodeling. We found that exposure to repeated episodes of social defeat increased numbers of cells immunoreactive for histone H3 acetylation, but not for histone H3 phosphoacetylation, in the infralimbic mPFC. Collectively, these findings highlight the importance of the infralimbic mPFC in responding to social stress-a finding that provides insight into the possible neurobiological alterations associated with stress-induced psychiatric illness.