The Response of Paracoccidioides lutzii to the Interaction with Human Neutrophils.

The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Cheminformatics-driven discovery of hit compounds against Paracoccidioides spp.

Aims: The development of safe and effective therapies for treating paracoccidioidomycosis using computational strategies were employed to discover anti-Paracoccidioides compounds. Materials & methods: We 1) collected, curated and integrated the largest library of compounds tested against Paracoccidioides spp.; 2) employed a similarity search to virtually screen the ChemBridge database and select nine compounds for experimental evaluation; 3) performed an experimental evaluation to determine the minimum inhibitory concentration and minimum fungicidal concentration as well as cytotoxicity; and 4) employed computational tools to identify potential targets for the most active compounds. Seven compounds presented activity against Paracoccidioides spp. Conclusion: These compounds are new hits with a predicted mechanisms of action, making them potentially attractive to develop new compounds.

Proteomic Profiling of Paracoccidioides brasiliensis in Response to Phenacylideneoxindol Derivative: Unveiling Molecular Targets and Pathways.

BACKGROUND: The treatment of paracoccidioidomycosis (PCM) is a challenge, and the discovery of new antifungal compounds is crucial. The phenacylideneoxindoles exhibited promising antifungal activity against Paracoccidioides spp., but their mode of action remains unknown. METHODS: Through proteomic analysis, we investigated the effects of (E)-3-(2-oxo-2-phenylethylidene)indolin-2-one on P. brasiliensis. In addition, we investigated the metabolic alterations of P. brasiliensis in response to the compound. Furthermore, the effects of the compound on the membrane, ethanol production, and reactive oxygen species (ROS) production were verified. RESULTS: We identified differentially regulated proteins that revealed significant metabolic reorganization, including an increase in ethanol production, suggesting the activation of alcoholic fermentation and alterations in the rigidity of fungal cell membrane with an increase of the ergosterol content and formation of ROS. CONCLUSIONS: These findings enhance our understanding of the mode of action and response of P. brasiliensis to the investigated promising antifungal compound, emphasizing its potential as a candidate for the treatment of PCM.

State of the art in protein-protein interactions within the fungi kingdom.

Proteins rarely exert their function by themselves. Protein-protein interactions (PPIs) regulate virtually every biological process that takes place in a cell. Such interactions are targets for new therapeutic agents against all sorts of diseases, through the screening and design of a variety of inhibitors. Here we discuss several aspects of PPIs that contribute to prediction of protein function and drug discovery. As the high-throughput techniques continue to release biological data, targets for fungal therapeutics that rely on PPIs are being proposed worldwide. Computational approaches have reduced the time taken to develop new therapeutic approaches. The near future brings the possibility of developing new PPI and interaction network inhibitors and a revolution in the way we treat fungal diseases.

Proteomic identification of metabolic changes in Paracoccidioides brasiliensis induced by a nitroheteroarylchalcone.

Aim: To access the metabolic changes caused by a chalcone derivative (LabMol-75) through a proteomic approach. Methods: Proteomic analysis was performed after 9 h of Paracoccidioides brasiliensis yeast (Pb18) cell incubation with the LabMol-75 at MIC. The proteomic findings were validated through in vitro and in silico assays. Results: Exposure to the compound led to the downregulation of proteins associated with glycolysis and gluconeogenesis, ß-oxidation, the citrate cycle and the electron transport chain. Conclusion: LabMol-75 caused an energetic imbalance in the fungus metabolism and deep oxidative stress. Additionally, the in silico molecular docking approach pointed to this molecule as a putative competitive inhibitor of DHPS.

Molecular Interactions of the Copper Chaperone Atx1 of Paracoccidioides brasiliensis with Fungal Proteins Suggest a Crosstalk between Iron and Copper Homeostasis.

Paracoccidioides spp. are endemic fungi from Latin America that cause Paracoccidioidomycosis, a systemic disease. These fungi present systems for high-affinity metal uptake, storage, and mobilization, which counteract host nutritional immunity and mitigate the toxic effects of metals. Regarding Cu mobilization, the metallochaperone Atx1 is regulated according to Cu bioavailability in Paracoccidioides spp., contributing to metal homeostasis. However, additional information in the literature on PbAtx1 is scarce. Therefore, in the present work, we aimed to study the PbAtx1 protein-protein interaction networks. Heterologous expressed PbAtx1 was used in a pull-down assay with Paracoccidioides brasiliensis cytoplasmic extract. Nineteen proteins that interacted with PbAtx1 were identified by HPLC-MSE. Among them, a relevant finding was a Cytochrome b5 (PbCyb5), regulated by Fe bioavailability in Aspergillus fumigatus and highly secreted by P. brasiliensis in Fe deprivation. We validated the interaction between PbAtx1-PbCyb5 through molecular modeling and far-Western analyses. It is known that there is a relationship between Fe homeostasis and Cu homeostasis in organisms. In this sense, would PbAtx1-PbCyb5 interaction be a new metal-sensor system? Would it be supported by the presence/absence of metals? We intend to answer those questions in future works to contribute to the understanding of the strategies employed by Paracoccidioides spp. to overcome host defenses.

Exposure of Paracoccidioides brasiliensis to Mebendazole Leads to Inhibition of Fungal Energy Production.

Paracoccidioidomycosis (PCM) is a fungal disease caused by organisms of the genus Paracoccidioides spp. The treatment of the disease is lengthy and includes several adverse effects. Various methodologies focus on the search for new treatments against fungal disease, including the repositioning of drugs. Our group showed the fungicidal effect of mebendazole in P. brasiliensis cells. Thus, understanding the effect of exposing fungal cells to mebendazole is significant for further studies in order to demonstrate it as a potential drug for the treatment of PCM. A proteomic analysis of P. brasiliensis exposed to mebendazole was carried out. Analyses showed that exposure strongly affected the pathways related to energy production, such as glycolysis, fermentation, and the electron transport chain. The quantification of adenosine triphosphate (ATP) and mitochondrial activity demonstrated that the drug alters the electron chain, resulting in an increase in oxidative stress. Enzymes such as superoxide dismutase (SOD) and cytochrome c oxidase (Cyt C) were repressed in cells exposed to mebendazole. The concentration of ethanol produced by the cells under treatment demonstrated that the attempt to produce energy through fermentation is also arrested. Thus, the drug inhibits fungal growth through changes in energy metabolism, making it a promising compound for use in the treatment of PCM.

3-phenacylideneoxindoles as a new class of antifungal compounds against Paracoccidioides spp.

Aims: Considering the need to identify new compounds with antifungal action, the activity of five 3-phenacylideneoxindoles compounds was evaluated. Materials & methods: The compounds were synthesized, and their antifungal activity was elucidated through minimum inhibitory concentration tests and interaction assay with other antifungals. Potential targets of compounds were predicted in silico. Results: 3-phenacylideneoxindoles compounds inhibited fungal growth with minimum inhibitory concentration and minimum fungicidal concentration ranging from 3.05 to 12.26 µM. The compounds demonstrated high selectivity index and presented a synergistic effect with itraconazole. In silico prediction revealed the pentafunctional AROM polypeptide, enolase, superoxide dismutase, catalase and kinases as proteins targets of the compound 4a. Conclusion: The results demonstrate that 3-phenacylideneoxindoles is a potential new class of antifungal compounds for paracoccidioidomycosis treatment.

Patients affected by paracoccidioidomycosis (PCM) require long-term treatment, which commonly influences their adherence. In addition, only three drugs are in clinical use, which indicates the relevance of research in identifying new drugs for treating PCM. Thus, five drugs were tested in the laboratory to verify whether they could prevent the growth of the fungus without being toxic to humans. In addition, whether these compounds in combination with drugs used to treat PCM could be even more potent was evaluated. All compounds tested efficiently inhibited the growth of Paracoccidioides, the fungus that causes PCM. One drug was identified that, combined with itraconazole, decreased the required dose of both the discovered compound and itraconazole needed to inhibit fungal growth. Using computational tools, this work suggests how the new drug could act against the fungus. The results demonstrate a potential new treatment option, but more studies are needed to confirm the safety of these drugs.

New Methylcitrate Synthase Inhibitor Induces Proteolysis, Lipid Degradation and Pyruvate Excretion in Paracoccidioides brasiliensis.

BACKGROUND: Paracoccidioidomycosis is a systemic mycosis caused by the inhalation of conidia of the genus Paracoccidioides. During the infectious process, fungal cells use several carbon sources, leading to the production of propionyl-CoA. The latter is metabolized by the methylcitrate synthase, a key enzyme of the methylcitrate cycle. We identified an inhibitor compound (ZINC08964784) that showed antifungal activity against P. brasiliensis. METHODS: This work aimed to understand the fungal metabolic response of P. brasiliensis cells exposed to ZINC08964784 through a proteomics approach. We used a glucose-free medium supplemented with propionate in order to simulate the environment found by the pathogen during the infection. We performed pyruvate dosage, proteolytic assay, dosage of intracellular lipids and quantification of reactive oxygen species in order to validate the proteomic results. RESULTS: The proteomic analysis indicated that the fungal cells undergo a metabolic shift due to the inhibition of the methylcitrate cycle and the generation of reactive species. Proteolytic enzymes were induced, driving amino acids into degradation for energy production. In addition, glycolysis and the citric acid cycle were down-regulated while ß-oxidation was up-regulated. The accumulation of pyruvate and propionyl-CoA led the cells to a state of oxidative stress in the presence of ZINC08964784. CONCLUSIONS: The inhibition of methylcitrate synthase caused by the compound promoted a metabolic shift in P. brasiliensis damaging energy production and generating oxidative stress. Hence, the compound is a promising alternative for developing new strategies of therapies against paracoccidioidomycosis.

Paracoccidioides brasiliensis plasma membrane characterization by EPR spectroscopy and interactions with amphotericin B, miltefosine and nerolidol.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to characterize the interactions of amphotericin B (AmB), miltefosine (MIL) and nerolidol (NER) with the plasma membrane of Paracoccidioides brasiliensis. Spin-labeled analogs of stearic acid and steroid androstane distributed into the plasma membrane of the fungus treated with AmB, showed strong interactions with putative AmB/sterol complexes. The observed increase in the EPR parameter 2A// caused by AmB can be interpreted as a remarkable reduction in the spin label mobility and/or an increase in the local polarity. The 2A// parameter reduced gradually as the concentration of MIL and NER increased. The membrane-water partition coefficient (KM/W) of the three compounds under study was estimated based on the minimum concentration of the compounds that causes a change in EPR spectrum. The KM/W values indicated that the affinity of the compounds for the P. brasiliensis membrane follows the order: AmB > MIL > NER. The minimum inhibitory concentration (MIC) values were lower than the respective minimum concentrations of the compounds to cause a change in the EPR spectrum, being ∼3.5-fold lower for AmB, 3.9-fold for MIL and ∼1.4-fold for NER. Taken together, the EPR spectroscopy results suggest that the anti-proliferative effects of the three compounds studied are associated with alterations in cell membranes. One of the most likely consequences of these changes would be electrolyte leakage.Communicated by Ramaswamy H. Sarma.

Midias sociais para atração de pacientes em clínicas odontológicas privadas durante pandemia de Covid 19 / Social media for attracting patients in private dental clinics during the Covid 19 pandemic

Verificar a rotina dos cirurgiões-dentistas (CD) e a utilização das mídias sociais, como meio de divulgação profissional e de atração de pacientes em clínicas odontológicas privadas, durante a pandemia de Covid-19. Este estudo transversal qualitativo inclui questionários respondidos por CD atuantes em clínicas privadas na região sudeste do Brasil. Um questionário virtual, elaborado através da plataforma Google Forms, abordou informações sobre o perfil dos profissionais e os aspectos dos métodos publicitários e mídias sociais utilizados para o alcance de pacientes durante o período de pandemia de Covid-19. Os dados obtidos foram tabulados e submetidos à análise estatística descritiva (%). No total, 102 CD participaram do estudo, sendo que 96 questionários seguiram os critérios de inclusão para a análise dos dados. A divulgação social como meio de exposição profissional foi uma ação realizada por 75 (78,13%) CD da amostra. Considerando esses profissionais, 74 (98,67%) utilizam redes sociais e domínios virtuais para tal finalidade, sendo que 71 (95,95%) CD usufruem do Instagram. Grande parte dos participantes (71,62%) relatou não possuir assessoria de marketing especializada para fazer publicações de conteúdo profissional nas redes sociais, embora a maioria publique este tipo de conteúdo mais de uma vez por semana (58,11%). Uma grande parcela dos participantes do estudo (67,71%) notou que a pandemia de Covid-19 procovou diminuição na quantidade de pacientes nas clínicas odontológicas. A inclusão de outros equipamentos de proteção individual foi a conduta mais seguida pelos CD (56,25%) para evitar a transmissão da doença neste período. Conclui-se que os CD participantes acreditam que a pandemia de Covid-19 promoveu um impacto negativo na atração de pacientes em clínicas odontológicas privadas na região sudeste brasileira, ainda que a maioria destes profissionais tenham incluído outros equipamentos de proteção individual como medida de segurança e utilizem frequentemente redes sociais para divulgação de conteúdo profissional, tendo o Instagram como a principal mídia social. No entanto, a assessoria de marketing especializada em publicações de conteúdo profissional nas redes sociais ainda é um recurso pouco utilizado no meio odontológico(AU)

To verify the routine of Dental Surgeons (DS) and the use of social media as a means of professional dissemination and patient attraction in private dental clinics during the Covid-19 pandemic. This cross-sectional qualitative study included questionnaires answered by Dental Surgeons working in private clinics in the southeastern region of Brazil. A virtual questionnaire, which was developed through the Google Forms platform, addressed information about the profile of professionals and aspects of advertising methods and social media used to reach patients during the Covid-19 pandemic period. The data were tabulated and submitted to descriptive statistical analysis (%). A total of 102 Dental Surgeons participated in the study, with 96 questionnaires meeting the inclusion criteria for data analysis. Social disclosure as a means of professional exposure was an action performed by 75 (78.13%) Dental Surgeons of the survey. Considering these professionals, 74 (98.67%) use social networks and virtual domains for this purpose and 71 (95.95%) Dental Surgeons use Instagram. A large portion of the participants (71.62%) reported not having a specialized marketing consultancy to make professional contente publications on social media, although most publish this type of content more than once a week (58.11%). A large portion of study participants (67.71%) noted that the Covid-19 pandemic caused a decrease in the amount of patients in the dental clinics. The inclusion of other personal protective equipment was the conduct most followed by the Dental Surgeons (56.25%) to avoid the transmission of the disease in this period. It can be concluded that the participating Dental Surgeons believe that the Covid-19 pandemic promoted a negative impact on attracting patients in private dental clinics in the southeastern region of Brazil, in spite of most of these professionals have included other personal protective equipment as safety measure and frequently use social networks for dissemination of professional content, with Instagram as main social media. However, a marketing consultancy specialized in publishing professional content on social networks is still a resource which is little used in the dental environment(AU)

Paracoccidioides lutzii Formamidase Contributes to Fungal Survival in Macrophages.

Nitrogen is a crucial nutrient for microorganisms that compose essential biomolecules. However, hosts limit this nutrient as a strategy to counter infections, therefore, pathogens use adaptive mechanisms to uptake nitrogen from alternative sources. In fungi, nitrogen catabolite repression (NCR) activates transcription factors to acquire nitrogen from alternative sources when preferential sources are absent. Formamidase has been related to nitrogen depletion in Aspergillus nidulans through formamide degradation to use the released ammonia as a nitrogen source. In Paracoccidioides spp., formamidase is highly expressed in transcriptomic and proteomic analyses. Here, we aim to investigate the importance of formamidase to Paracoccidioides lutzii. Thereby, we developed a P. lutzii silenced strain of fmd gene (AsFmd) by antisense RNA technology using Agrobacterium tumefaciens-mediated transformation (ATMT). The AsFmd strain led to increased urease expression, an enzyme related to nitrogen assimilation in other fungi, suggesting that P. lutzii might explore urease as an alternative route for ammonia metabolism as a nitrogen source. Moreover, formamidase was important for fungal survival inside macrophages, as fungal recovery after macrophage infection was lower in AsFmd compared to wild-type (WT) strain. Our findings suggest potential alternatives of nitrogen acquisition regulation in P. lutzii, evidencing formamidase influence in fungal virulence.

Proteomic alterations in Paracoccidioides brasiliensis caused by exposure to curcumin.

Paracoccidioides spp. are the etiological agent of paracoccidioidomycosis, a disease that causes skin lesions and affect the lungs and other organs. The current management of the disease is long and has several side effects that often lead the patient to give up the treatment, sequelae and even death. The search for new forms of treatment that minimize these drawbacks is very important. Thus, natural compounds are targets of great interest. Curcumin is one of the main components of the tubers of Curcuma longa, presenting medicinal effects well described in the literature, including the antifungal effect on Paracocidioides brasiliensis. Nevertheless, the mechanisms related to the antifungal effect of such compound are still unknown, so the objective of the present research is to understand what changes occur in the metabolism of P. brasiliensis after exposure to curcumin and to identify the main targets of the compound. Proteomic analysis as based on nanoUPLC-MS analysis and the functional classification of the identified proteins. The main metabolic processes that were being regulated were biologically validated through assays such as fluorescence microscopy, EPR and phagocytosis. Proteomic analysis revealed that curcumin regulates several metabolic processes of the fungus, including important pathways for energy production, such as the glycolytic pathway, beta oxidation and the glyoxylate cycle. Protein synthesis was down-regulated in fungi exposed to curcumin. The electron transport chain and the tricarboxylic acid cycle were also down-regulated, indicating that both the mitochondrial membrane and the mitochondrial activity were compromised. Plasma membrane and cell wall structure were altered following exposure to the compound. The fungus' ability to survive the phagocytosis process by alveolar macrophages was reduced. Thus, curcumin interferes with several metabolic pathways in the fungus that causes paracoccidioidomycosis. BIOLOGICAL SIGNIFICANCE: The challenges presented by the current treatment of paracoccidioidomycosis often contributing to patients' withdrawal from treatment, leading to sequelae or even death. Thus, the search for new treatment options against this disease is growing. The discovery that curcumin is active against Paracoccidioides was previously reported by our study group. Here, we clarify how the compound acts on the fungus causing its growth inhibition and decreased viability. Understanding the mechanisms of action of curcumin on P. brasiliensis elucidates how we can seek new alternatives and which metabolic pathways and molecular targets we should focus on in this incessant search to bring the patient a treatment with fewer adverse effects.

Iron Deprivation Modulates the Exoproteome in Paracoccidioides brasiliensis.

Fungi of the Paracoccidioides genus are the etiological agents of the systemic mycosis paracoccidioidomycosis and, when in the host, they find a challenging environment that is scarce in nutrients and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe3+ chelators), use Fe-containing host proteins as a source of the metal, and use a non-canonical reductive pathway for Fe3+ assimilation. Despite all of these findings, there are still gaps that need to be filled in the pathogen response to metal deprivation. To contribute to the knowledge related to this subject, we obtained the exoproteome of Paracoccidioides brasiliensis (Pb18) undergoing Fe deprivation and by nanoUPLC-MSE. One hundred forty-one proteins were identified, and out of these, 64 proteins were predicted to be secreted. We also identified the regulation of several virulence factors. Among the results, we highlight Cyb5 as a secreted molecule of Paracoccidioides in the exoproteome obtained during Fe deprivation. Cyb5 is described as necessary for the Fe deprivation response of Saccharomyces cerevisiae and Aspergillus fumigatus. Experimental data and molecular modeling indicated that Cyb5 can bind to Fe ions in vitro, suggesting that it can be relevant in the arsenal of molecules related to iron homeostasis in P. brasiliensis.

Bioluminescence imaging in Paracoccidioides spp.: a tool to monitor the infectious processes.

The genus Paracoccidioides comprises the species complex causing paracoccidioidomycoses (PCM). These fungi are a serious public health problem due to the long-term drug therapy, follow-up treatment, and frequent sequelae generated by the infection, such as pulmonary fibrosis. In this sense, the objective of this work was to generate bioluminescent reporter strains of Paracoccidioides spp. harboring a thermostable, red-shifted luciferase gene under the control of different constitutive promoters. The strains were generated by the integration of a species-specific codon-optimized luciferase gene upon actin or enolase promoter's control. The insertion of the constructs in Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells were performed through Agrobacterium tumefaciens-mediated transformation. The results demonstrated the presence of several transformants harboring the luciferase gene. These transformants were further confirmed by the expression of luciferase and by the presence of the hygromycin resistance gene. Moreover, the luciferase activity could be detected in in vitro bioluminescence assays and in vivo models of infection. In general, this work presents the methodology for the construction of bioluminescent strains of Paracoccidioides spp., highlighting potential promoters and proposing an in vivo model, in which those strains could be used for the systemic study of PCM.

Agenesias dentárias atípicas: relato de caso clínico / Atypical dental agenesis: clinical case report

A agenesia dentaria é uma anomalia de desenvolvimento caracterizada pela determinação congênita de menor numero de dentes, podendo estar associada a síndromes genéticas ou ocorrer isoladamente. Embora seja considerada uma das anomalias mais frequentes quando envolve terceiros molares, segundo pré-molares e incisivos laterais, sua ocorrência em dentes estáveis, tais como o canino e o primeiro molar permanente é rara. Desta forma, este relato apresentará o caso de uma paciente do sexo feminino, 8 anos idade, que iniciou tratamento de rotina na Clínica de Odontopediatria da UNIFENAS. Na anamnese não foi relatada pela responsável a ocorrência de alterações sistêmicas, nem queixa ou histórico odontológico relevante. No exame clínico odontológico, notou-se o não irrompimento do primeiro molar permanente superior direito (16). Diante dos exames de imagem, foi confirmada a agenesia do dente 16 e também do canino permanente superior direito (13). A agenesia, sobretudo de dentes estáveis e relevantes no arco dentário, pode comprometer o desenvolvimento adequado da oclusão, mastigação, fonação e estética. Desta maneira, é importante o diagnóstico precoce desta ocorrência com o objetivo de favorecer a elaboração de um adequado plano de tratamento e, minimizar as sequelas destas agenesias atípicas(AU)

Dental agenesis is a developmental anomaly characterized by the congenital determination of fewer teeth, may be associated with genetic syndromes or occur in an isolated form. Although it is considered one of the most frequent anomalies when it involves third molars, second lateral premolars and incisors, the occurrence in stable teeth, such as the permanent canine and the permanent first molar is rare. This report will present the case of an 8-year-old female patient who started routine treatment at the Pediatric Dentistry Clinic of UNIFENAS. In the anamnesis, the responsible person did not report the occurrence of systemic alterations, or a relevant dental complaint or history. On dental clinical examination, it was noticed the non-rupture of the permament maxillar right first molar (16). In view of the imaging tests, the agenesis of tooth 16 and also of the permanent maxillary right canine (13) was confirmed. Agenesis, especially of stable and relevant teeth in the dental arch, may compromise the proper development of occlusion, chewing, phonation and aesthetics. Thus, it is of paramount importance to early diagnosis of this occurrence in order to favor the development of an appropriate treatment plan in order to minimize the sequelae of these atypical agenesis(AU)

A structure-based approach for the discovery of inhibitors against methylcitrate synthase of Paracoccidioides lutzii.

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in Latin America, caused by fungi of the genus Paracoccidioides. The treatment of PCM is complex, requiring a long treatment period, which often results in serious side effects. The aim of this study was to screen for inhibitors of a specific target of the fungus that is absent in humans. Methylcitrate synthase (MCS) is a unique enzyme of microorganisms and is responsible for the synthesis of methylcitrate at the beginning of the propionate degradation pathway. This pathway is essential for several microorganisms, since the accumulation of propionyl-CoA can impair virulence and prevent the development of the pathogen. We performed the modeling and molecular dynamics of the structure of Paracoccidioides lutzii MCS (PlMCS) and performed a virtual screening on 89,415 compounds against the active site of the enzyme. The compounds were selected according to the affinity and efficiency criteria of in vitro tests. Six compounds were able to inhibit the enzymatic activity of recombinant PlMCS but only the compound ZINC08964784 showed fungistatic and fungicidal activity against Paracoccidioides spp. cells. The analysis of the interaction profile of this compound with PlMCS showed its effectiveness in terms of specificity and stability when compared to the substrate (propionyl-CoA) of the enzyme. In addition, this compound did not show cytotoxicity in mammalian cells, with an excellent selectivity index. Our results suggest that the compound ZINC08964784 may become a promising alternative antifungal against Paracoccidioides spp. Communicated by Ramaswamy H. Sarma.

Proteomic Response of Paracoccidioides brasiliensis Exposed to the Antifungal 4-Methoxynaphthalene-N-acylhydrazone Reveals Alteration in Metabolism.

Background: Paracoccidioidomycosis is a neglected mycosis with a high socioeconomic impact that requires long-term treatment with antifungals that have limitations in their use. The development of antifungals targeting essential proteins that are present exclusively in the fungus points to a potentially promising treatment. Methods: The inhibitor of the enzyme homoserine dehydrogenase drove the synthesis of N'-(2-hydroxybenzylidene)-4-methoxy-1-naphthohydrazide (AOS). This compound was evaluated for its antifungal activity in different species of Paracoccidioides and the consequent alteration in the proteomic profile of Paracoccidioides brasiliensis. Results: The compound showed a minimal inhibitory concentration ranging from 0.75 to 6.9 µM with a fungicidal effect on Paracoccidioides spp. and high selectivity index. AOS differentially regulated proteins related to glycolysis, TCA, the glyoxylate cycle, the urea cycle and amino acid metabolism, including homoserine dehydrogenase. In addition, P. brasiliensis inhibited protein synthesis and stimulated reactive oxygen species in the presence of AOS. Conclusions: AOS is a promising antifungal agent for the treatment of PCM, targeting important metabolic processes of the fungus.

Atraso de escolaridade e dificuldade de socialização de pacientes com fissura labiopalatina não sindrômica / Scholar delay and socialization dificulty of patients with non syndromic cleft lip and palate

Dentre as principais consequências da fissura labial e/ou palatina não sindrômica (FL/ PNS) estão dificuldades com fonação e autoestima, a primeira uma questão funcional e a segunda um problema social derivado não raro de contextos de bullying que, dentre outros, podem levar o indivíduo à evasão escolar. O objetivo deste estudo foi avaliar o atraso de escolaridade e a dificuldade de socialização de pacientes com FL/PNS quando comparados a uma população não afetada da mesma faixa etária de 7 a 20 anos, atendidos na Universidade José do Rosário Vellano ­UNIFENAS, campus de Alfenas. Os sujeitos foram agrupados em duas categorias de indivíduos, o grupo caso composto por indivíduos com FL/PNS em tratamento no Centro Pró-Sorriso da UNIFENAS; e o grupo controle composto por indivíduos sem FL/PNS em tratamento nas clínicas de Odontopediatria e Integrada da UNIFENAS. Os resultados demonstraram que a proporção de pacientes com FL/PNS atrasados na escola foi de quase 5 vezes maior que o número de pacientes sem fissuras (p<0,01). Constatou-se que a presença da FL/PNS pode ser o ponto de partida para outros contribuintes, com interferências psicológicas e/ou sociais, interferindo negativamente no processo de socialização (bullying) do paciente (p=0,0018). Portanto devem ser tratadas com abordagem multidisciplinar, incluindo diversos profissionais, dentre eles pedagogos, psicólogos e odontólogos(AU)

Among the main consequences of Non Syndromic Cleft Lip and Palate (NSCLP) are the difficulties with phonation and self estime, the first being a functional issue na the later being social that is derived from, not rarely, bullying contexts, that among other things, may lead na individual to school evasion. The objective of this study was to avaluate the levels of scholarity of patients with NSCLP when compared to a non affected population of the same age in individuals from 7 to 20 years old, attended the Pediatric and Integrated Pediatric Clinic of UNIFENAS, Alfenas campus. The subjects were grouped into two categories of individuals, the case group was composed of individuals with FL/PNS with treatment at the ProSmile center at UNIFENAS. The control group was composed of individuals without FL/PNS in treatment at the clinics of pediatric and integrated denistry at UNIFENAS. The results demonstrated the number of patients with FL/PNS that presented scholar delay were almost 5 times the number of patients that didn't present FL/PNS (p<0,01). The presence of NFL/PNS may be the starting point for other contributors with psychological and/or social interferences, interfering negatively with the socialization process (bullying) of the patient (p=0,0018). They should be treated with a multidisciplinary manner, including multiple professionals, among them pedagogues, psychologist and dentist(AU)