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Hippocampus is the brain area where aluminum (Al) accumulates in abundance and is widely associated with learning and memory. In the present study, we evaluate behavioral, tissue, and proteomic changes in the hippocampus of Wistar rats caused by exposure to doses that mimic human consumption of aluminum chloride (AlCl3) in urban areas. For this, male Wistar rats were divided into two groups: Control (distilled water) and AlCl3 (8.3 mg/kg/day), both groups were exposed orally for 60 days. After the Al exposure protocol, cognitive functions were assessed by the Water maze test, followed by a collection for analysis of the global proteomic profile of the hippocampus by mass spectrometry. Aside from proteomic analysis, we performed a histological analysis of the hippocampus, to the determination of cell body density by cresyl violet staining in Cornu Ammonis fields (CA) 1 and 3, and hilus regions. Our results indicated that exposure to low doses of aluminum chloride triggered a decreased cognitive performance in learning and memory, being associated with the deregulation of proteins expression, mainly those related to the regulation of the cytoskeleton, cellular metabolism, mitochondrial activity, redox regulation, nervous system regulation, and synaptic signaling, reduced cell body density in CA1, CA3, and hilus.

Objectives To evaluate the oral condition of alcohol and tobacco dependents and identify salivary protein candidates for biomarkers of oral disorders. Subjects and methods Thirty-three male volunteers were evaluated for alcohol abuse rehabilitation; nine were selected for proteomic analysis. Intraoral examination was performed, and non-stimulated saliva was collected. Salivary proteins were extracted and processed for analysis. A list of proteins identified in saliva was generated from the database and manually revised, obtaining the total number of candidate biomarkers for oral disorders. Results The mean age (n = 33) was 42.94 ± 8.61 years. Fourteen (42.4%) subjects presented with 23 oral mucosa changes, and 31 (94%) had dental plaque. A total of 282 proteins were found in saliva (n = 9), of which 26 were identified as candidates for biomarkers of oral disorders. After manual review, 21 proteins were selected. The highest number of candidates for biomarkers was associated with carcinoma of head and neck (n = 10), nasopharyngeal carcinoma (n = 6), and periodontal disease (n = 6). Conclusion Alcohol and tobacco dependents showed gingival inflammation, and less than half of them showed oral mucosa changes. Twenty-one protein candidates for biomarkers of oral disorders were identified in saliva. The two major oral disorders in number of candidates for biomarkers were head and neck cancer and periodontal disease.

Aluminum (Al) is one of the most abundant elements on Earth, and its high extraction rate and industrial use make human exposure very common. As Al may be a human toxicant, it is important to investigate the effects of Al exposure, mainly at low doses and for prolonged periods, by simulating human exposure. This work aimed to study the effects of low-dose exposure to chloride aluminum (AlCl3) on the oxidative biochemistry, proteomic profile, and morphology of the major salivary glands. Wistar male rats were exposed to 8.3 mg/kg/day of AlCl3 via intragastric gavage for 60 days. Then, the parotid and submandibular glands were subjected to biochemical assays, proteomic evaluation, and histological analysis. Al caused oxidative imbalance in both salivary glands. Dysregulation of protein expression, mainly of those related to cytoarchitecture, energy metabolism and glandular function, was detected in both salivary glands. Al also promoted histological alterations, such as acinar atrophy and an increase in parenchymal tissue. Prolonged exposure to Al, even at low doses, was able to modulate molecular alterations associated with morphological impairments in the salivary glands of rats. From this perspective, prolonged Al exposure may be a risk to exposed populations and their oral health.

Mercury (Hg) is a toxic metal that became a public health problem due to environmental contamination caused by anthropogenic activity. In this sense, oral homeostasis can undergo changes due to the toxic effects of metal on the salivary glands. Therefore, our objective was to investigate the proteomic and genotoxic changes in salivary glands after exposure to inorganic mercury (IHg). Forty Wistar rats that were divided into a control group, which received distilled water, and an exposed group, which received 0.375 mg/kg of mercury chloride for 45 days via orogastric gavage. After that, the animals were euthanized, and the parotid and submandibular glands were collected for analysis of the genotoxic effects, using the comet assay and proteome global profile assessment. The results showed that IHg promoted damage to cellular DNA associated with proteomic changes that showed events such as oxidative stress, mitochondrial dysfunction, changes in the cytoskeleton, and apoptosis. Therefore, these findings show a profile of molecular changes due to the interactions of IHg with several proteins and mechanisms inherent to the cell, which consequently may result in dysfunction of the salivary glands and impaired homeostasis of the oral cavity.

Mercury is a severe environmental pollutant with neurotoxic effects, especially when exposed for long periods. Although there are several evidences regarding mercury toxicity, little is known about inorganic mercury (IHg) species and cerebellum, one of the main targets of mercury associated with the neurological symptomatology of mercurial poisoning. Besides that, the global proteomic profile assessment is a valuable tool to screen possible biomarkers and elucidate molecular targets of mercury neurotoxicity; however, the literature is still scarce. Thus, this study aimed to investigate the effects of long-term exposure to IHg in adult rats’ cerebellum and explore the modulation of the cerebellar proteome associated with biochemical and functional outcomes, providing evidence, in a translational perspective, of new mercury toxicity targets and possible biomarkers. Fifty-four adult rats were exposed to 0.375 mg/kg of HgCl2 or distilled water for 45 days using intragastric gavage. Then, the motor functions were evaluated by rotarod and inclined plane. The cerebellum was collected to quantify mercury levels, to assess the antioxidant activity against peroxyl radicals (ACAPs), the lipid peroxidation (LPO), the proteomic profile, the cell death nature by cytotoxicity and apoptosis, and the Purkinje cells density. The IHg exposure increased mercury levels in the cerebellum, reducing ACAP and increasing LPO. The proteomic approach revealed a total 419 proteins with different statuses of regulation, associated with different biological processes, such as synaptic signaling, energy metabolism and nervous system development, e.g., all these molecular changes are associated with increased cytotoxicity and apoptosis, with a neurodegenerative pattern on Purkinje cells layer and poor motor coordination and balance. In conclusion, all these findings feature a neurodegenerative process triggered by IHg in the cerebellum that culminated into motor functions deficits, which are associated with several molecular features and may be related to the clinical outcomes of people exposed to the toxicant.

Made available in DSpace on 2022-04-29T08:36:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Objective: To study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. Materials and methods: In five crossover phases, after prophylaxis, 10 volunteers in 5 consecutive days, rinsed (10 mL, 1 min) with the following solutions: deionized water (H2O- negative control- 1), 0.1 mg/mL CaneCPI-5 (2), 1.88×10−5 M StN15 (3), 1.0 mg/mL Hb (4) or their combination (5). The AEP formed after 3 min was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. Results: Rinsing with the proteins/peptide increased the amounts of proteins in the AEP. The total numbers of proteins identified after rinsing with CaneCPI-5, StN15, Hb or their combination versus water, were 131, 167, 148 and 142, respectively. The treatment with the proteins/peptide or their combination increased proteins that bind calcium, phosphate and interact with distinct proteins, as well as proteins with antimicrobial and acid-resistant properties, such as, Cornifin-B (7.7, 12.6, and 4.3-fold for CaneCPI-5, StN15 and Hb, respectively), isoforms of Cystatin (2.2–2.4-fold for CaneCPI-5 and StN15), Proline-rich-protein 4 (4.3-fold; StN15), Histatin-1 (2.8-fold; StN15) and Hemoglobin (7.7–25-fold for Hb and Combination). Immunoglobulin, Keratin and Histone were exclusively identified upon treatment with the proteins/peptide, alone or combined. Conclusion: Rinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. Clinical Relevance: Our results suggest that rinsing with the proteins/peptide or their combination increases the proteins capable of enhancing the protective function of the basal layer of AEP. Department of Biological Sciences Bauru School of Dentistry University of São Paulo Department of Genetics and Evolution São Carlos Federal University Department of Biochemistry and Technology Institute of Chemistry São Paulo State University (UNESP) Department of Biochemistry and Technology Institute of Chemistry São Paulo State University (UNESP)

Made available in DSpace on 2022-04-29T08:45:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-27 Although fluoride (F) is well-known to prevent dental caries, changes in cell processes in different tissues have been associated with its excessive exposure. Thus, this study aimed to evaluate the effects of F exposure on biochemical, proteomic, and genotoxic parameters of submandibular glands. Twenty one old rats (n = 30) were allocated into three groups: 60 days administration of drinking water containing 10 mgF/L, 50 mgF/L, or only deionized water (control). The submandibular glands were collected for oxidative biochemistry, protein expression profile, and genotoxic potential analyses. The results showed that both F concentrations increased the levels of thiobarbituric acid–reactive substances (TBARS) and reduced glutathione (GSH) and changed the proteomic profile, mainly regarding the cytoskeleton and cellular activity. Only the exposure to 50 mgF/L induced significant changes in DNA integrity. These findings reinforce the importance of continuous monitoring of F concentration in drinking water and the need for strategies to minimize F intake from other sources to obtain maximum preventive/therapeutic effects and avoid potential adverse effects. Laboratory of Functional and Structural Biology Institute of Biological Sciences Federal University of Pará Laboratory of Clinical Immunology and Oxidative Stress Faculty of Pharmacy Institute of Health Sciences Federal University of Pará Section of Parasitology Evandro Chagas Institute Laboratory of Cell Culture and Cytogenetics Environment Section Evandro Chagas Institute Department of Biological Sciences Bauru School of Dentistry University of São Paulo Department of Chemistry University of Nebraska-Lincoln Department of Preventive and Restorative Dentistry School of Dentistry São Paulo State University (UNESP) Department of Preventive and Restorative Dentistry School of Dentistry São Paulo State University (UNESP)

Gastrointestinal signs and symptoms are the first signs of toxicity due to exposure to fluoride (F). This suggests the possibility that lower levels of subchronic F exposure may affect the gut. The aim of this study was to evaluate changes in the morphology, proteome and microbiome of the ileum of rats, after subchronic exposure to F. Male rats ingested water with 0, 10, or 50mgF/L for thirty days. Treatment with F, regardless of the dose, significantly decreased the density of HuC/D-IR neurons, whereas CGRP-IR and SP-IR varicosities were significantly increased compared to the control group. Increased VIP-IR varicosities were significantly increased only in the group treated with 50mgF/L. A significant increase in thickness of the tunica muscularis, as well as in the total thickness of the ileum wall was observed at both F doses when compared to controls. In proteomics analysis, myosin isoforms were increased, and Gastrotopin was decreased in F-exposed mice. In the microbiome metagenomics analysis, Class Clostridia was significantly reduced upon exposure to 10mgF/L. At the higher F dose of 50mg/L, genus Ureaplasma was significantly reduced in comparison with controls. Morphological and proteomics alterations induced by F were marked by changes associated with inflammation, and alterations in the gut microbiome. Further studies are needed to determine whether F exposure increases inflammation with secondary effects of the gut microbiome, and/or whether primary effects of F on the gut microbiome enhance changes associated with inflammation.

Stimulation of saliva production is an alternative to improve the quality of life of patients treated by radiotherapy. However, there is no information about changes in the salivary proteome of stimulated and unstimulated saliva in these patients.Thus, we evaluated the difference in the proteomic profile of stimulated and unstimulated saliva in patients with head and neck cancer (HNC) treated by radiotherapy.Stimulated and unstimulated saliva were collected from 9 patients with HNC before (BRT), during (DRT; 2-5 weeks) and after (ART; 3-4 months) treatment. Healthy patients paired by age and gender also had their saliva collected (C; control group). The stimulated and unstimulated salivary flow were evaluated (p 0.05). Salivary proteins were extracted and processed for shotgun proteomic analysis.Significant differences were observed between stimulated and unstimulated salivary flows for C and BRT (p greater than 0.001), but not for DRT and ART. Proteins involved with apoptosis, antibacterial and acid-resistance were decreased in stimulated saliva in comparison to unstimulated saliva DRT and ART. Isoforms of keratins were not identified in control and BRT.there is a marked difference in the protein profile of stimulated and unstimulated salivary flows in HNC patients treated by radiotherapy. In addition, saliva stimulation in patients with HNC decreases important proteins involved with dental protection. The unstimulated salivary flow seems to be the best alternative to search for biomarkers. Our results contribute in an unprecedented way to understand the changes in the salivary proteome of different flows in HNC patients undergoing radiotherapy treatment.

The antimicrobial and anticaries effects of CaneCPI-5 were evaluated. Ninety bovine enamel samples were treated for 60 s with either phosphate-buffered-saline (PBS), 0.12% chlorhexidine (CHX), 0.05 mg ml

Background Pulmonary sequelae (PS) in patients with chronic paracoccidioidomycosis (PCM) typically include pulmonary fibrosis and emphysema. Knowledge of the molecular pathways involved in PS of PCM is required for treatment and biomarker identification. Methodology/Principal findings This non-concurrent cohort study included 29 patients with pulmonary PCM that were followed before and after treatment. From this group, 17 patients evolved to mild/ moderate PS and 12 evolved severe PS. Sera from patients were evaluated before treatment and at clinical cure, serological cure, and apparent cure. A nanoACQUITY UPLC-Xevo QT MS system and PLGS software were used to identify serum differentially expressed proteins, data are available via ProteomeXchange with identifier PXD026906. Serum differentially expressed proteins were then categorized using Cytoscape software and the Reactome pathway database. Seventy-two differentially expressed serum proteins were identified in patients with severe PS compared with patients with mild/moderate PS. Most proteins altered in severe PS were involved in wound healing, inflammatory response, and oxygen transport pathways. Before treatment and at clinical cure, signaling proteins participating in wound healing, complement cascade, cholesterol transport and retinoid metabolism pathways were downregulated in patients with severe PS, whereas signaling proteins in gluconeogenesis and gas exchange pathways were upregulated. At serological cure, the pattern of protein expression reversed. At apparent cure pathways related with tissue repair (fibrosis) became downregulated, and pathway related oxygen transport became upregulated. Additionally, we identified 15 proteins as candidate biomarkers for severe PS. Conclusions/Significance Development of severe PS is related to increased expression of proteins involved in glycolytic pathway and oxygen exchange), indicative of the greater cellular activity and replication associated with early dysregulation of wound healing and aberrant tissue repair. Our findings provide new targets to study mechanisms of PS in PCM, as well as potential biomarkers., Author summary Pulmonary fibrosis is the main sequel of paracoccidioidomycosis (PCM), a fungal disease that affects mainly men, rural workers. The development of pulmonary fibrosis is complex and involves several mechanisms that culminate in aberrant collagen production and deposition in the lungs making it became stiff and blocking the air passages. These changes lead to difficulty in breathing and in PCM patients dyspnea in response to high or low levels of exertion is common. Therefore, these patients show incapacity to work and the decreased quality of life. With the possibility of identifying some marker, for example, it could help the indication of respiratory physiotherapy, professional rehabilitation, or therapeutic intervention. This is the first study to examine the pulmonary sequelae (PS) in patients with paracoccidioidomycosis using an approach combining proteomics with bioinformatics. Here, we identify the specific proteome pattern found in PCM patients with severe sequelae that distinguishes these patients from that with mild/moderate sequelae. Our results showed that time points immediately before treatment and at clinical cure are key moments at which PS can progress to severe PS due a dysregulation in wound healing with consequent delayed in the healing processes resulting in an aberrant scar. As such, we suggest that the prognoses for severe PS should be considered as soon as possible and as early as diagnosis of PCM. Furthermore, we used proteomics to identify possible serum biomarkers with which to predict the likely development of severe PS, to be validated in future studies.

The development of new approaches that allow early assessment of which cases of COVID-19 will likely become critical and the discovery of new therapeutic targets are urgent demands. In this cohort study, we performed proteomic and laboratorial profiling of plasma from 163 patients admitted to Bauru State Hospital (Bauru, SP, Brazil) between May 4th and July 4th, 2020, who were diagnosed with COVID-19 by RT-PCR nasopharyngeal swab samples. Plasma samples were collected upon admission for routine laboratory analyses and shotgun quantitative label-free proteomics. Based on the course of the disease, the patients were further divided into 3 groups: a) mild symptoms, discharged without admission to an intensive care unit (ICU) (n=76); b) severe symptoms, discharged after admission to an ICU (n=56); c) critical, died after admission to an ICU (n=31). White cells and neutrophils were significantly higher in severe and critical patients compared to mild ones. Lymphocytes were significantly lower in critical patients compared to mild ones and platelets were significantly lower in critical patients compared to mild and severe ones. Ferritin, TGO, urea and creatinine were significantly higher in critical patients compared to mild and severe ones. Albumin, CPK, LDH and D-dimer were significantly higher in severe and critical patients compared to mild ones. PCR was significantly higher in severe patients compared to mild ones. Proteomic analysis revealed marked changes between the groups in plasma proteins related to complement activation, blood coagulation, antimicrobial humoral response, acute inflammatory response, and endopeptidase inhibitor activity. Higher levels of IREB2, GELS, POLR3D, PON1 and ULBP6 upon admission to hospital were found in patients with mild symptoms, while higher levels of Gal-10 were found in critical and severe patients. This needs to be validated in further studies. If confirmed, pathways involving these proteins might be potential new therapeutic targets for COVID-19.

This work was supported by Pro-Reitoria ? de Pesquisa da UFPA (PROPESP, UFPA, Brazil), Brazilian National Council for Scientific and Technological Development (CNPq, Grant n. 435093/2018-5) and Coordena?ao ? de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)- Finance Code 001. Federal University of Par?. Institute of Biological Sciences. Laboratory of Functional and Structural Biology. Bel?m, PA, Brazil. Federal University of Par?. Institute of Biological Sciences. Laboratory of Functional and Structural Biology. Bel?m, PA, Brazil. Federal University of Par?. Institute of Biological Sciences. Laboratory of Functional and Structural Biology. Bel?m, PA, Brazil. Federal University of Par?. Institute of Biological Sciences. Laboratory of Functional and Structural Biology. Bel?m, PA, Brazil. University of S?o Paulo. Bauru Dental School. Department of Biological Sciences. Bauru, SP, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura Celular e Citogen?tica. Ananindeua, PA, Brasil. University of S?o Paulo. Bauru Dental School. Department of Biological Sciences. Bauru, SP, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura Celular e Citogen?tica. Ananindeua, PA, Brasil. Federal University of Par?. Institute of Biological Sciences. Laboratory of Molecular Pharmacology. Bel?m, PA, Brazil. Federal University of Par?. Institute of Biological Sciences. Laboratory of Functional and Structural Biology. Bel?m, PA, Brazil. Long-term exposure to high concentrations of fluoride (F) can damage mineralized and soft tissues such as bones, liver, kidney, intestine, and nervous system of adult rats. The high permeability of the blood?brain barrier and placenta to F during pregnancy and lactation may be critical to neurological development. Therefore, this study aimed to investigate the effects of F exposure during pregnancy and lactation on molecular processes and oxidative biochemistry of offspring rats? hippocampus. Pregnant Wistar rats were randomly assigned into 3 groups in accordance with the drinking water received: G1 ? deionized water (control); G2 ? 10 mg/L of F and G3 ? 50 mg/L of F. The exposure to fluoridated water began on the first day of pregnancy and lasted until the 21st day of breastfeeding (when the offspring rats were weaned). Blood plasma samples of the offspring rats were collected to determine F levels. Hippocampi samples were collected for oxidative biochemistry analyses through antioxidant capacity against peroxyl (ACAP), lipid peroxidation (LPO), and nitrite (NO2 ? ) levels. Also, brainderived neurotrophic factor (BDNF) gene expression (RT-qPCR) and proteomic profile analyses were performed. The results showed that exposure to both F concentrations during pregnancy and lactation increased the F bioavailability, triggered redox imbalance featured by a decrease of ACAP, increase of LPO and NO2 ? levels, BDNF overexpression and changes in the hippocampus proteome. These findings raise novel questions regarding potential repercussions on the hippocampus structure and functioning in the different cognitive domains.

Objective Salivary proteomic analysis may help to understand physiopathological changes in crack cocaine dependents. This study aimed to compare the salivary protein profile between crack cocaine dependents and non-drug users. Design Nine heavy smokers and alcohol consumers men admitted to rehab due to crack cocaine abuse and nine non-drug users age-matched men were evaluated. Unstimulated whole saliva was collected. Proteomic analysis was performed by mass spectrometer. Data were processed using ProteinLynx GlobalServer software. Results were obtained by searching the Homo sapiens database from the UniProt catalog. The search tool IBI-IMIM was used to identify proteins candidates for biomarkers. Results The mean age of crack cocaine and control groups was 36.89 ± 7.78 and 35.78 ± 6.68 years, respectively. 458 salivary proteins were identified in both groups; 305 proteins in the crack cocaine group. Among the 68 proteins presented in both groups, 29 were down-regulated (i.e. “Statherin” and “Transforming growth factor-beta-induced protein ig-h3” were down-regulated at least 10-fold) and 27 up-regulated (i.e. “Negative elongation factor” was up-regulated 19-fold) in the crack cocaine group compared to controls. 90 out of the 458 proteins found in the proteomic analysis were identified as candidates for biomarkers of diseases. Among these, 65 (72.22 %) were detected in the crack cocaine group. Conclusion Crack cocaine dependents with chronic alcohol and tobacco use have a higher number of proteins in saliva compared to non-drug users. 22.3 % of salivary proteins present in crack cocaine dependents were present in controls; 3.9 % of them were expressed in similar quantity.

Objectives To compare in vitro the effect of a toothpaste containing fluoride (F), calcium silicate (CaSi) and sodium phosphate salts to conventional toothpaste (NaF) on human enamel specimens submitted to erosive and abrasive challenges. Methods 48 sound and 48 enamel samples pre-treated with 1% citric acid were divided into 4 groups (n = 12): Group 1- Non-fluoride toothpaste; Group 2- NaF toothpaste (1450 ppmF); Group 3- CaSi toothpaste (1450 ppmF; MFP); Group 4- Erosion only. The samples were subjected to pH cycling (3 cycles/day; 90s; 1% citric acid, pH 3.6) and to abrasion for 7 days. After the 1st and the last cycle, they were submitted to abrasion (15s, 1.5N load), using a brushing machine, soft toothbrush and toothpaste slurry (1:3; 15ml/sample) and then immersed in the slurry for 45s. Samples were immersed in artificial saliva between the challenges. Enamel loss was evaluated using profilometry on days 3 and 7. Data were analysed by ANOVA and Tukey's test (p < 0.05). Results For sound enamel at baseline, mean (±SD) enamel loss (μm) for groups 1–4 on day 3 was 2.15 ± 0.35a, 1.20 ± 0.22b, 0.95 ± 0.19b and 1.98 ± 0.32a; on day 7 was 3.05 ± 0.40a, 2.07 ± 0.32b, 1.36 ± 0.33c and 3.69 ± 0.27d respectively. For acid-softened enamel at baseline, enamel loss on day 3 was 3.16 ± 0.19a, 2.17 ± 0.14b, 1.70 ± 0.11c and 3.04 ± 0.19a; on day 7 was 3.92 ± 0.25a, 3.07 ± 0.13b, 2.09 ± 0.15c and 3.87 ± 0.25a respectively. Conclusions Both F toothpastes led to significantly higher enamel protection from short-term erosion and abrasion in comparison to the non-F toothpaste and erosion only. In the longer term, CaSi toothpaste conferred significantly higher protection than NaF toothpaste. Clinical significance The results showed that for the longer term the CaSi toothpaste provided significantly higher protection than the NaF toothpaste, which indicates a good potential of the former to help prevent erosive tooth wear., Enamel; Erosion; Abrasion; Toothbrushing; Toothpaste.

Lead (Pb) is a toxic metal with great neurotoxic potential. The aim of this study was to investigate the effects of a long-term Pb intoxication on the global proteomic profile, oxidative biochemistry and neuronal density in motor cortex of adult rats, and the possible outcomes related to motor functions. For this, Wistar rats received for 55 days a dose of 50 mg/Kg of Pb acetate by intragastric gavage. Then, the motor abilities were evaluated by open field and inclined plane tests. To investigate the possible oxidative biochemistry modulation, the levels of pro-oxidant parameters as lipid peroxidation and nitrites were evaluated. The global proteomic profile was evaluated by ultraefficiency liquid chromatography system coupled with mass spectrometry (UPLC/MS) followed by bioinformatic analysis. Moreover, it was evaluated the mature neuron density by anti-NeuN immunostaining. The statistical analysis was performed through Student’s t -test, considering p < 0.05 . We observed oxidative stress triggering by the increase in malonaldehyde and nitrite levels in motor cortex. In the proteomic analysis, the motor cortex presented alterations in proteins associated with neural functioning, morphological organization, and neurodegenerative features. In addition, it was observed a decrease in the number of mature neurons. These findings, associated with previous evidences observed in spinal cord, cerebellum, and hippocampus under the same Pb administration protocol, corroborate with the motor deficits in the rats towards Pb. Thus, we conclude that the long-term administration to Pb in young Wistar rats triggers impairments at several organizational levels, such as biochemical and morphological, which resulted in poor motor performance.

Made available in DSpace on 2021-06-25T11:03:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-10 Arterial stiffness, frequently associated with hypertension, is associated with disorganization of the vascular wall and has been recognized as an independent predictor of all-cause mortality. The identification of the molecular mechanisms involved in aortic stiffness would be an emerging target for hypertension therapeutic intervention. This study evaluated the effects of perindopril on pulse wave velocity (PWV) and on the differentially expressed proteins in aorta of spontaneously hypertensive rats (SHR), using a proteomic approach. SHR and Wistar rats were treated with perindopril (SHRP) or water (SHRc and Wistar rats) for 8 weeks. At the end, SHRC presented higher systolic blood pressure (SBP, +70%) and PWV (+31%) compared with Wistar rats. SHRP had higher values of nitrite concentration and lower PWV compared with SHRC. From 21 upregulated proteins in the aortic wall from SHRC, most of them were involved with the actin cytoskeleton organization, like Tropomyosin and Cofilin-1. After perindopril treatment, there was an upregulation of the GDP dissociation inhibitors (GDIs), which normally inhibits the RhoA/Rho-kinase/cofilin-1 pathway and may contribute to decreased arterial stiffening. In conclusion, the results of the present study revealed that treatment with perindopril reduced SBP and PWV in SHR. In addition, the proteomic analysis in aorta suggested, for the first time, that the RhoA/Rho-kinase/Cofilin-1 pathway may be inhibited by perindopril-induced upregulation of GDIs or increases in NO bioavailability in SHR. Therefore, we may propose that activation of GDIs or inhibition of RhoA/Rho-kinase pathway could be a possible strategy to treat arterial stiffness. Joint Graduate Program in Physiological Sciences Federal University of Sao Carlos and São Paulo State University UFSCar/UNESP Department of Biological Sciences Bauru School of Dentistry University of São Paulo Post-Graduate Program in Movement Sciences São Paulo State University Department of Physical Education School of Sciences São Paulo State University Joint Graduate Program in Physiological Sciences Federal University of Sao Carlos and São Paulo State University UFSCar/UNESP Post-Graduate Program in Movement Sciences São Paulo State University Department of Physical Education School of Sciences São Paulo State University

Made available in DSpace on 2022-04-29T08:45:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-01 Changes in the proteomic profile of the acquired enamel pellicle (AEP) formed for 3 min or 2 h after rinsing with a peptide containing the 15 N-terminal residues of statherin, with serines 2 and 3 phosphorylated (StatpSpS), were evaluated. Nine volunteers participated in 2 consecutive days. Each day, after professional tooth cleaning, they rinsed for 1 min with 10 mL of phosphate buffer containing 1.88 × 10-5 M StatpSpS or phosphate buffer only (control). The acquired pellicle formed on enamel after 3 min or 2 h was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. In the 3-min AEP, 19 and 131 proteins were uniquely identified in the StatpSpS and control groups, respectively. Proteins typically found in the AEP were only found in the latter. Only 2 proteins (neutrophil defensins) were increased upon treatment with StatpSpS, while 65 proteins (among which are several typical AEP proteins) were decreased. In the 2-h AEP, 50 and 108 proteins were uniquely found in StatpSpS and control groups, respectively. Hemoglobin subunits and isoforms of keratin were only found in the StatpSpS group, while cystatin-C, cathepsin D, and cathepsin G, isoforms of heat shock 70 and protocadherin were exclusively found in the control group. In addition, 23 proteins were increased upon treatment with StatpSpS, among which are histatin-1, serum albumin, and isoforms of neutrophil defensin and keratin, while 77 were decreased, most of them were typical AEP proteins. In both evaluated periods, rinsing with StatpSpS profoundly changed the proteomic profile of the AEP, which might impact the protective role of this integument against carious or erosive demineralization. This study provides important insights on the dynamics of the protein composition of the AEP along time, after rinsing with a solution containing StatpSpS. Department of Biological Sciences Bauru School of Dentistry University of São Paulo Chemistry Institute Paulista State University Chemistry Institute Paulista State University

Made available in DSpace on 2021-06-25T10:35:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Objectives: To evaluate, in vivo: 1) proteomic alterations in the acquired enamel pellicle (AEP) after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), statherin-derived peptide (StN15) or their combination before the formation of the AEP and subsequent erosive challenge; 2) the protection of these treatments against erosive demnineralization. Materials and methods: In 5 crossover phases, after prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with: deionized water-1, 0.1 mg/mL CaneCPI-5-2, 1.0 mg/mL HB-3, 1.88 × 10-5 M StN15-4 or their combination-5. AEP was formed (2 h) and enamel biopsy (10 μL, 1%citric acid, pH 2.5, 10 s) was performed on one incisor for calcium analysis. The same acid was applied on the vestibular surfaces of the remaining teeth. The acid-resistant proteins within the remaining AEP were collected. Samples were quantitatively analyzed by label-free proteomics. Results: Treatment with the proteins/peptide, isolated or combined, increased several acid-resistant proteins in the AEP, compared with control. The highest increases were seen for PRPs (32-fold, StN15), profilin (15-fold, combination), alpha-amylase (9-fold; StN15), keratins (8-fold, CaneCPI-5 and HB), Histatin-1 (7-fold, StN15), immunoglobulins (6.5-fold, StN15), lactotransferrin (4-fold, CaneCPI-5), cystatins, lysozyme, protein S-100-A9 and actins (3.5-fold, StN15), serum albumin (3.5-fold, CaneCPI-5 and HB) and hemoglobin (3-fold, StN15). Annexin, calmodulin, keratin, tubulin and cystatins were identified exclusively upon treatment with the proteins/peptide, alone or combined. Groups 2, 3 and 4 had significantly lower Ca released from enamel compared to group 1 (Kruskal-Wallis/Dunn's, p < 0.05). Conclusions: Treatment with CaneCPI-5, HB or StN15 remarkably increases acid-resistant proteins in the AEP, protecting against erosion. Clinical significance: Our results show, for the first time, that treatment with proteins/peptide remarkably increases acid-resistant proteins in the AEP, protecting against erosive demineralization. These findings open an avenue for a new preventive approach for erosive demineralization, employing acquired pellicle engineering procedures that may in the future be incorporated into dental products. Department of Biological Sciences Bauru School of Dentistry University of São Paulo Department of Biochemistry and Technology Institute of Chemistry São Paulo State University (UNESP) Department of Genetics and Evolution São Carlos Federal University Department of Preventive and Restorative Dentistry School of Dentistry Araçatuba São Paulo State University (UNESP) Department of Biochemistry and Technology Institute of Chemistry São Paulo State University (UNESP) Department of Preventive and Restorative Dentistry School of Dentistry Araçatuba São Paulo State University (UNESP) FAPESP: 2018/12041-7 FAPESP: 2019/16254-8 CNPq: 407853/2018-9

Although the literature does not provide evidence of health risks from exposure to fluoride (F) in therapeutic doses, questions remain about the effects of long-term and high-dose use on the function of the central nervous system. The objective of this study was to investigate the effect of long-term exposure to F at levels similar to those found in areas of artificial water fluoridation and in areas of endemic fluorosis on biochemical, proteomic, cell density, and functional parameters associated with the cerebellum. For this, mice were exposed to water containing 10 mg F/L or 50 mg F/L (as sodium fluoride) for 60 days. After the exposure period, the animals were submitted to motor tests and the cerebellum was evaluated for fluoride levels, antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (MDA), and nitrite levels (NO). The proteomic profile and morphological integrity were also evaluated. The results showed that the 10 mg F/L dose was able to decrease the ACAP levels, and the animals exposed to 50 mg F/L presented lower levels of ACAP and higher levels of MDA and NO. The cerebellar proteomic profile in both groups was modulated, highlighting proteins related to the antioxidant system, energy production, and cell death, however no neuronal density change in cerebellum was observed. Functionally, the horizontal exploratory activity of both exposed groups was impaired, while only the 50 mg F/L group showed significant changes in postural stability. No motor coordination and balance impairments were observed in both groups. Our results suggest that fluoride may impair the cerebellar oxidative biochemistry, which is associated with the proteomic modulation and, although no morphological impairment was observed, only the highest concentration of fluoride was able to impair some cerebellar motor functions.

Salivary glands are affected during radiotherapy in the head and neck region, leading to a reduction in salivary flow and changes its composition. Besides negatively affecting the oral soft tissues, this can also lead to dental impairment. Thus, we evaluated the effect of radiotherapy in the proteomic profile of the saliva in patients with head and neck cancer (HNC).HNC patients had their saliva collected before (BRT), during (2-5 weeks; DRT), and after (3-4 months; ART) radiotherapy. Saliva was also collected from healthy volunteers (control; C). Samples were processed for proteomic analysis.In total, 1055 proteins were identified, among which 46 were common to all groups, while 86, 86, 286, and 395 were exclusively found in C, BRT, DRT, and ART, respectively. Remarkably, alpha-enolase was increased 35-fold DRT compared with BRT, while proline-rich proteins were decreased. ART there was a 16-fold increase in scaffold attachment factor-B1 and a 3-fold decrease in alpha-enolase and several cystatins. When compared with C, salivary proteins of BRT patients showed increases cystatin-C, lysozyme C, histatin-1, and proline-rich proteins CONCLUSION/CLINICAL REVELANCE: Both HNC and radiotherapy remarkably change the salivary protein composition. Altogether, our results, for the first time, suggest investigating alpha-enolase levels in saliva DRT in future studies as a possible biomarker and strategy to predict the efficiency of the treatment. Moreover, our data provide important insights for designing dental products that are more effective for these patients and contribute to a better understanding of the progressive changes in salivary proteins induced by radiotherapy. Graphical abstract.

Lead (Pb) is an environmental and occupational neurotoxicant after long-term exposure. This study aimed to investigate the effects of systemic Pb exposure in rats from adolescence to adulthood, evaluating molecular, morphologic and functional aspects of hippocampus. For this, male Wistar rats were exposed to 50 mg/kg of Pb acetate or distilled water for 55 days by intragastric gavage. For the evaluation of short-term and long-term memories, object recognition and step-down inhibitory avoidance tests were performed. At the end of the behavioral tests, the animals were euthanized and the hippocampus dissected and processed to the evaluation of: Pb content levels in hippocampal parenchyma, Trolox equivalent antioxidant capacity (TEAC), glutathione (GSH) and malondialdehyde (MDA) levels as parameters of oxidative stress and antioxidant status, global proteomic profile and neuronal degeneration by anti-NeuN immunohistochemistry analysis. Our results show the increase of Pb levels in the hippocampus of adult rats exposed from adolescence, increased MDA and GSH levels, modulation of proteins related to neural structure and physiology and reduced density of neurons, hence a poor cognitive performance on short and long-term memories. Then, the long-term exposure to Pb in this period of life may impair several biologic organizational levels of the hippocampal structure associated with functional damages.

Lead (Pb) is an environmental contaminant that presents a high risk for human health. We aimed to investigate the possible alterations triggered by the exposure to Pb acetate for a long period in motor performance and the possible relationship with biochemical, proteomic and morphological alterations in the cerebellum of rats. Male Wistar rats were exposed for 55 days, at 50 mg/Kg of Pb acetate, and the control animals received distilled water. Open field (OF) and rotarod tests, biochemistry parameters (MDA and nitrite), staining/immunostaining of Purkinje cells (PC), mature neurons (MN), myelin sheath (MS) and synaptic vesicles (SYN) and proteomic profile were analyzed. Pb deposition on the cerebellum area and this study drove to exploratory and locomotion deficits and a decrease in the number of PC, MN, SYN and MS staining/immunostaining. The levels of MDA and nitrite remained unchanged. The proteomic profile showed alterations in proteins responsible for neurotransmitters release, as well as receptor function and second messengers signaling, and also proteins involved in the process of apoptosis. Thus, we conclude that the long-term exposure to low Pb dose promoted locomotion and histological tracings, associated with alterations in the process of cell signaling, as well as death by apoptosis.

Methylmercury (MeHg) is an important toxicant that causes cognitive dysfunctions in humans. This study aimed to investigate the proteomic and biochemical alterations of the hippocampus associated with behavioural consequences of low doses of MeHg in a long-term exposure model, and to realistically mimic in vivo the result of human exposure to this toxicant. Adult Wistar male rats were exposed to a dose of MeHg at 0.04 mg kg-1 day-1 by gavage for 60 days. Total mercury (Hg) content was significantly increased in the hippocampal parenchyma. The increase in the Hg levels was capable of reducing neuron and astrocyte cell density in the CA1, CA3, hilus and dentate gyrus regions, increasing both malondialdehyde and nitrite levels and decreasing antioxidant capacity against peroxyl radicals. The proteomic analysis detected 1041 proteins with altered expression due to MeHg exposure, including 364 proteins with no expression, 295 proteins with de novo expression and 382 proteins with up- or down-regulated expression. This proteomic approach revealed alterations in pathways related to chemical synapses, metabolism, amino acid transport, cell energy, neurodegenerative processes and myelin maintenance. Therefore, even at low doses of MeHg exposure, it is possible to cause hippocampal damage in adult rats at many organisational levels, triggering oxidative stress and proteome misbalance, featuring a neurodegenerative process and culminating in long- and short-term memory and learning deficits.

Fluoride (F) can induce changes in the expression of several liver proteins. It is suggested that these changes are dose- and time-dependent. The objective of this study was to analyze the effect of different F concentrations and exposure times to this ion on the pattern of protein expression in the liver of rats. Thirt-six 21-day-old male Wistar rats were divided into 2 groups (n = 18) according to the treatment duration (20 or 60 days). Each of these groups was then divided into 3 subgroups (n = 6) according to the concentration of F administered in drinking water, as follows: 0 mg/L (control), 15 mg/L or 50 mg/L. After the experiment periods, the animals were anesthetized and the liver and blood were collected. F was analyzed in plasma and liver. Part of the liver was fixed for histological analysis. Liver proteins were extracted and prepared for quantitative label-free mass spectrometry analysis. F concentrations in plasma and liver were significantly higher in the group treated with 50 mg /L in comparison with control, regardless the time of exposure. Histological alterations in the liver were more evident in the subgroups treated for 20 days. The proteomic analysis revealed changes in proteins related to endoplasmic reticulum and mitochondrial oxidative stress, mitochondrial alteration, apoptosis and cellular respiration upon exposure to F. The results reinforce previous findings showing that the effects of F in the liver are dose- and time-dependent and provide the molecular basis for understanding the evolution of these effects.

The effect of duration of chronic treatment with fluoride (F, 50 mg/L as NaF) on the lipid profile, lipid droplets and triglycerides (TG) in liver was evaluated in mice with nonalcoholic fatty liver disease (NAFLD) previously induced by hyperlipidic diet and in animals fed normocaloric diet. In addition, the effect of F administered for a short period (20 days) was evaluated on de novo lipogenesis, by nuclear magnetic resonance. GRP78, Apo-E, and sterol regulatory element-binding protein (SREBP) were quantified by Western blotting. Our data indicate that F interferes in lipid metabolism and lipid droplets, having a different action depending on the exposure time and type of diet administered. F improved lipid parameters and reduced steatosis only when administered for a short period of time (up to 20 days) to animals fed normocaloric diet. However, when NAFLD was already installed, lipid parameters were only slightly improved at 20 days of treatment, but no effect was observed on the degree of steatosis. In addition, lipid profile was in general impaired when the animals were treated with F for 30 days, regardless of the diet. Moreover, F did not alter de novo lipogenesis in animals with installed NAFLD. Furthermore, hyperlipidic diet increased F accumulation in the body. GRP78 increased, while Apo-E and SREBP decreased in the F-treated groups. Our results provide new insights on how F affects lipid metabolism depending on the available energy source.