Liliana Silva

Changes in serotoninergic and noradrenergic descending pain pathways during painful diabetic neuropathy: the preventive action of IGF1.

Painful diabetic neuropathy (PDN) induces neuronal hyperactivity at the spinal cord and periaqueductal gray (PAG), a key area in descending nociceptive modulation. Since the PAG uses relay stations at serotoninergic and noradrenergic brainstem areas, we determined the serotonin and noradrenaline levels at the spinal cord of streptozotocin-diabetic rats and at those brainstem areas (serotoninergic rostroventromedial medulla and noradrenergic A(5) and A(7) cell groups). Since, during diabetes, the levels of insulin growth factor 1 (IGF1) decrease, reducing its neurotrophic effect in the brain, we also studied the effects of IGF1 treatment. One week after diabetes induction, subcutaneous injections of IGF1 (2.5mg/kg) were performed during 3 weeks. Body weights, glycemia, and mechanical nociception were weekly evaluated until the end of the study, the time when the animals were subjected to a modified formalin test to study chemical allodynia. Serotonin and noradrenaline levels were quantified by ELISA at the spinal cord, whereas at the brainstem, the quantification was performed by immunohistochemistry against, respectively, tryptophan hydroxylase (TpH) or tyrosine hydroxylase (TH). STZ-diabetic rats exhibited mechanical hyperalgesia and chemical allodynia, along with higher spinal levels of serotonin and noradrenaline and higher numbers of neurons expressing TpH at the RVM and TH at the A(5) noradrenergic cell group. Treatment with IGF1 prevented the behavioral signs of PDN and reversed the neuronal hyperactivity at the spinal cord and ventrolateral PAG and the neurochemical changes at the spinal cord and at the brainstem. Based on the facilitatory role of serotoninergic and noradrenergic descending modulation during chronic pain, the increased serotonin and noradrenaline innervation of the dorsal horn in STZ-diabetic rats may probably account for enhanced pain during PDN. The benefits of IGF1 in PDN are probably due to blockade of the increased peripheral input to the somatosensory system, but direct central actions cannot be discarded. The value of IGF1 in PDN treatment deserves further evaluation.

Unsuitability of MALDI-TOF MS to discriminate Acinetobacter baumannii clones under routine experimental conditions.

MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) is now in the forefront for routine bacterial species identification methodologies, being its value for clonality assessment controversial. In this work we evaluated the potential of MALDI-TOF MS for assisting infection control by depicting Acinetobacter baumannii clones. Mass spectra of 58 A. baumannii clinical isolates belonging to the worldwide spread lineages (ST98, ST103, ST208, and ST218) isolated in our country, were obtained and analyzed with several chemometric tools (pseudo gel views, peakfind function, and partial least squares discriminant analysis). The clonal lineages were obtained using the “Oxford” scheme, belonging ST98, ST208, and ST218 to the international clone II and ST103 to an epidemic clonal lineage (SG5). Additionally, mass spectra of a highly diverse international collection of 38 isolates belonging to 22 sequence types (STs) were obtained for further comparisons. Pseudo gel views and direct peak pattern analysis did not allow the discrimination of A. baumannii isolates belonging to ST98, ST103, ST208, or ST218. Moreover, a partial least square discriminant analysis of the mass spectra considering two spectral ranges (2–20 kDa and 4–10 kDa) revealed a poor degree of discrimination with only 64.6 and 65.8% of correct ST assignments, respectively. Also, mass spectra of the international isolates (n = 38, 22STs) revealed a very congruent peak pattern among them as well as among the four lineages included in this work. Despite the increasing interest of MALDI-TOF MS for bacterial typing at different taxonomical levels, we demonstrated, using routine experimental conditions, the unsuitability of this methodology for A. baumannii clonal discrimination.

Development of a FTIR-ATR based model for typing clinically relevant Acinetobacter baumannii clones belonging to ST98, ST103, ST208 and ST218

In this work we developed and validated a FTIR-ATR (Fourier transform infrared spectroscopy with attenuated total reflectance) based model for typing Acinetobacter baumannii clinical isolates belonging to ST98, ST208 and ST218 included into the worldwide spread clonal complex (CC) 92 and ST103. FTIR-ATR spectra of seventy-seven previously characterized isolates (Multi Locus Sequence Type-MLST, Pulsed-Field Gel Electrophoresis-PFGE and carbapenem-hydrolyzing class D β-lactamase-CHDL) were acquired and modeled by partial least squares discriminant analysis (PLSDA). The model was tested and successfully validated with a diverse collection of isolates (n=148) recovered from different countries and periods of time belonging to modeled and non-modeled STs.

Fibroblast growth factor 23 is associated with left ventricular hypertrophy, not with uremic vasculopathy in peritoneal dialysis patients.

AIMS Cardiovascular (CV) events are the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD), including those patients on peritoneal dialysis (PD). Fibroblast growth factor 23 (FGF23) has been associated with left ventricular hypertrophy (LVH) and mortality in patients with CKD. However, the role of FGF23 in uremic vasculopathy remains unclear. In this study, we aimed to assess the relationship between FGF23 and LVH, endothelial dysfunction, vascular calcification, and arterial stiffness in 48 stable PD patients. METHODS Left ventricular mass index (LVMI) was assessed using 2-D echocardiography. Intact FGF23 blood levels were evaluated using an ELISA kit (Immutopics, Inc., San Clemente, CA, USA). Reactive hyperemia index (RHI) is a surrogate marker of endothelial dysfunction and the augmentation index (AI) is a surrogate marker of arterial stiffness. Both were assessed using peripheral arterial tonometry (EndoPAT 2000). Vascular calcification (VC) was assessed using the Adragão score. RESULTS In unadjusted analysis; FGF23 was positively correlated with serum Pi (r = 0.487, p < 0.001), serum urea (r = 0.351, p = 0.015), serum creatinine (r = 0.535, p < 0.001), dialysis vintage (r = 0.309, p = 0.033), and LVMI (r = 0.369, p = 0.027) and was negatively correlated with age (r = -0.343, p = 0.017), residual renal function (r = -0.359, p < 0.012), and AI (r = -0.304, p = 0.038). In multivariate adjusted analysis, FGF23 was associated with LVMI (β = 0.298, p = 0.041), serum Pi (β = 0.345, p = 0.018), and age (β = -0.372, p = 0.007) independent of dialysis vintage, gender, residual renal function (RRF), albumin, C-reactive protein and systolic blood pressure. There were no associations found between FGF23 and RHI, AI, or VC in multivariable- adjusted models. CONCLUSIONS Our results show that FGF23 is associated with LVH but not with endothelial dysfunction, arterial stiffness, or vascular calcification in PD patients.

Whole-Genome Identification, Phylogeny, and Evolution of the Cytochrome P450 Family 2 (CYP2) Subfamilies in Birds

The cytochrome P450 (CYP) superfamily defends organisms from endogenous and noxious environmental compounds, and thus is crucial for survival. However, beyond mammals the molecular evolution of CYP2 subfamilies is poorly understood. Here, we characterized the CYP2 family across 48 avian whole genomes representing all major extant bird clades. Overall, 12 CYP2 subfamilies were identified, including the first description of the CYP2F, CYP2G, and several CYP2AF genes in avian genomes. Some of the CYP2 genes previously described as being lineage-specific, such as CYP2K and CYP2W, are ubiquitous to all avian groups. Furthermore, we identified a large number of CYP2J copies, which have been associated previously with water reabsorption. We detected positive selection in the avian CYP2C, CYP2D, CYP2H, CYP2J, CYP2K, and CYP2AC subfamilies. Moreover, we identified new substrate recognition sites (SRS0, SRS2_SRS3, and SRS3.1) and heme binding areas that influence CYP2 structure and function of functional importance as under significant positive selection. Some of the positively selected sites in avian CYP2D are located within the same SRS1 region that was previously linked with the metabolism of plant toxins. Additionally, we find that selective constraint variations in some avian CYP2 subfamilies are consistently associated with different feeding habits (CYP2H and CYP2J), habitats (CYP2D, CYP2H, CYP2J, and CYP2K), and migratory behaviors (CYP2D, CYP2H, and CYP2J). Overall, our findings indicate that there has been active enzyme site selection on CYP2 subfamilies and differential selection associated with different life history traits among birds.

PHASE ANGLE PREDICTS ARTERIAL STIFFNESS AND VASCULAR CALCIFICATION IN PERITONEAL DIALYSIS PATIENTS

Objectives: Fluid overload (FO) is frequently present in peritoneal dialysis (PD) patients and is associated with markers of malnutrition, inflammation, and atherosclerosis/calcification (MIAC) syndrome. We examined the relationships in stable PD patients between phase angle (PhA) and the spectrum of uremic vasculopathy including vascular calcification and arterial stiffness and between PhA and changes in serum fetuin-A levels. Methods: Sixty-one stable adult PD patients were evaluated in a cross-sectional study (ST1). Phase angle was measured by multifrequency bioimpedance analysis (InbodyS10, Biospace, Korea) at 50 kHz. Augmentation index (AI), a surrogate marker of arterial stiffness, was assessed by digital pulse amplitude tonometry (Endo PAT, Itamar Medical, Caesarea, Israel). Vascular calcification was assessed by simplified calcification score (SCS). Serum fetuin-A levels were measured by ELISA (Thermo scientific; Waltham, MA, USA). Serum albumin was used as a nutritional marker, and serum C-reactive protein (CRP) was used as an inflammatory marker. The same assessments were carried out longitudinally (ST2) in the first 33 patients who completed 1 year of evaluation in ST1. Results: In ST1, patients with PhA < 6° had higher CRP levels, AI, and SCS and lower serum albumin and fetuin-A levels, in comparison with patients with PhA ≥ 6°. In addition, PhA was a predictor of both AI (β = -0.351, p = 0.023) and SCS ≥ 3 (EXP (B) = 0.243, p = 0.005). In ST2, the increase of PhA over time was associated with decreases in both AI (r = -0.378, p = 0.042) and CRP levels (r = -0.426, p = 0.021), as well as with the increase in serum fetuin-A levels (r = 0.411, p = 0.030). Conclusions: Phase angle predicts both arterial stiffness and vascular calcification in stable PD patients.

Vomeronasal Receptors in Vertebrates and the Evolution of Pheromone Detection

Pheromones were identified as chemical signals used for intraspecific communication in insects (e.g., sexual attraction) in the 1950s. However, only almost 40 years later the vomeronasal receptors type-1 (V1R) and type-2 (V2R) were identified, usually associated with the presence of a vomeronasal organ (VNO). VRs are widespread in amphibians, reptiles, and mammals, but birds lost the VNO. Similarly, fishes lack VRs and a VNO but can still detect pheromones, instead using the olfactory receptors related to class A and class C G protein-coupled receptors. Here, we review recent evidence on VR repertoire contraction/expansion in vertebrates. We assess the association between VNO development and VR repertoire size. Phylogenetic relationships and selective pressures illuminate the dynamic evolutionary history of the VRs in vertebrates.

I-3. COLONIZAÇÃO ORAL POR STAPHYLOCOCCUS EM DOENTES RENAIS CRÓNICOS EM DIÁLISE PERITONEAL

apos escovagem foi registada como ppm ou mg, recorrendo a metodos potenciometricos com auxilio de um eletrodo de luor. Todos os resultados foram indicados como media e intervalo de conianca (IC) de 95% e analisados estatisticamente com o Teste t de Student, ANOVA e post hoc (Bonferroni) consoante apropriado. Resultados: A escovagem dentaria com os tres dentifricos nao induz alteracoes estatisticamente signiicativas no luxo salivar e, embora o padrao de cinetica de libertacao do luor para a saliva seja semelhante, existe um pico de libertacao aos 5 minutos iniciais (0,1057 mg±0,03007), estatisticamente superior para a Colgate Duraphat (P<0,05). Apesar de a pasta Colgate Duraphat ser aplicada na escova em menor quantidade, 0,5885 g±0,08447 (P<0,05), apresenta maior quantidade de luor presente, 2,9424 mg±0,4223 (P<0,05) e libertado na saliva, 0,1948 mg±0,04772 (P<0,05) comparativamente aos outros dentifricos estudados. Considerando o potencial de toxicidade cronica atraves da escovagem, e necessario escovar 11 vezes por dia, com a Colgate Duraphat, ou a degluticao diaria de 1,20 g da mesma, para ocorrencia de toxicidade cronica. Fenomenos de toxicidade aguda podem surgir apos ingestao de 36 g de Colgate Duraphat. Conclusoes: O padrao de cinetica de libertacao de luor para a saliva foi semelhante em todos os dentifricos embora superior na Colgate Duraphat. Nao existe risco de toxicidade sistemica em adultos se as indicacoes do Medico Dentista forem respeitadas, independentemente das concentracoes dos dentifricos em estudo.

579 ADMINISTRATION OF LOW DOSES OF INSULIN AND IGF-1 IMPROVES PAINFUL DIABETIC NEUROPATHY IN STREPTOZOTOCIN-DIABETIC RATS

Results: At the last follow-up control (t45) both groups reported a good pain relief (VAS≤3). More specific: in group A at t30 VAS 4±0.98 at t45 VAS 2±1.17; while in group B at t30 VAS 4 ±0.75 at t45 VAS 2±1.21. Our study failed to show a statistic significative difference (p < 0.05). 3 subjects of group A with DN related sleep difficulties reported the greatest pain relief with improved sleep quality. 2 subjects of group B reported urinary retention and decrease of libido after the treatment. Discussion: According to our data Duloxetin may be considered an effective treatment for neuropatic pain with better tolerability than usual antidepressants.