Comparing morphological and secretory aspects of cephalic glands among the New World coral snakes brings novel insights on their biological roles.

Oral and other cephalic glands have been surveyed by several studies with distinct purposes. Despite the wide diversity and medical relevance of the New World coral snakes, studies focusing on understanding the biological roles of the glands within this group are still scarce. Specifically, the venom glands of some coral snakes were previously investigated but all other cephalic glands remain uncharacterized. In this sense, performing morphological and molecular analysis of these glands may help better understand their biological role. Here, we studied the morphology of the venom, infralabial, rictal, and harderian glands of thirteen species of Micrurus and Micruroides euryxanthus. We also performed a molecular characterization of these glands from selected species of Micrurus using transcriptomic and proteomic approaches. We described substantial morphological variation in the cephalic glands of New World coral snakes and structural evidence for protein-secreting cells in the inferior rictal glands. Our molecular analysis revealed that the venom glands, as expected, are majorly devoted to toxin production, however, the infralabial and inferior rictal glands also expressed some toxin genes at low to medium levels, despite the marked morphological differences. On the other hand, the harderian glands were dominated by the expression of lipocalins, but do not produce toxins. Our integrative analysis, including the prediction of biological processes and pathways, helped decipher some important traits of cephalic glands and better understand their biology.

Nanog, Stat-3, and Sox-5 involvement in human fetal temporomandibular joint late development.

Background and aim: The temporomandibular joint (TMJ) is a synovial joint that allows the complex movements essential for life. It connects the jawbone to the skull, working as a sliding hinge. Moreover, pluripotent stem cells are a source of precursors and tissue-specific cells in developing organisms, however, their biodistribution in developing fetal tissues is weakly studied. The aim of our study was analyse immunohistochemical expression of Nanog, Oct-4, Sox-2 and Stat-3 and Sox-5, in TMJ tissue samples from human fetuses aged between the 12th and 20th weeks of intrauterine life. Materials and methods: We fixed and processed TMJ tissue samples from human fetuses, histological sections and immunohistochemical procedures were carried out. Results: TMJ histological studies examination did not reveal any difference in the tissue organization between the samples in the studied periods. Immunohistochemical analysis demonstrated that Oct-4 and Sox-2 lack their expression in TMJ. In contrast, Nanog was expressed in nucleous of proliferative layer of mandibular condyle, Stat-3 was expressed in nuclear cells of articular disc, Stat-3 and Sox-5 showed positive nuclear and cytoplasmic immunostaining in codrocyte layers and in ossification areas. Conclusions: Nanog acts in maintanence of pluripotency, Stat-3 in articular disc acts as a transcriptional factor. Stat-3 and Sox-2 act in chondrocyte and osteoblast diferentiation. Distribution of the cells, which express Nanog, Stat-3, and Sox-5 in TMJ tissue during fetal development, can help further understand its physiology, pathology, and repairing capacities.

Comparing morphological and secretory aspects of cephalic glands among the New World coral snakes brings novel insights on their biological roles

Oral and other cephalic glands have been surveyed by several studies with distinct purposes. Despite the wide diversity and medical relevance of the New World coral snakes, studies focusing on understanding the biological roles of the glands within this group are still scarce. Specifically, the venom glands of some coral snakes were previously investigated but all other cephalic glands remain uncharacterized. In this sense, performing morphological and molecular analysis of these glands may help better understand their biological role. Here, we studied the morphology of the venom, infralabial, rictal, and harderian glands of thirteen species of Micrurus and Micruroides euryxanthus. We also performed a molecular characterization of these glands from selected species of Micrurus using transcriptomic and proteomic approaches. We described substantial morphological variation in the cephalic glands of New World coral snakes and structural evidence for protein-secreting cells in the inferior rictal glands. Our molecular analysis revealed that the venom glands, as expected, are majorly devoted to toxin production, however, the infralabial and inferior rictal glands also expressed some toxin genes at low to medium levels, despite the marked morphological differences. On the other hand, the harderian glands were dominated by the expression of lipocalins, but do not produce toxins. Our integrative analysis, including the prediction of biological processes and pathways, helped decipher some important traits of cephalic glands and better understand their biology.

Nanog, Stat-3, and Sox-5 involvement in human fetal temporomandibular joint late development

The temporomandibular joint (TMJ) is a synovial joint that allows the complex movements essential for life. It connects the jawbone to the skull, working as a sliding hinge. Moreover, pluripotent stem cells are a source of precursors and tissue-specific cells in developing organisms, however, their biodistribution in developing fetal tissues is weakly studied. The aim of our study was analyse immunohistochemical expression of Nanog, Oct-4, Sox-2 and Stat-3 and Sox-5, in TMJ tissue samples from human fetuses aged between the 12th and 20th weeks of intrauterine life.

The immunomodulatory potential role of mesenchymal stem cells in diseases of the central nervous system

Several studies indicate the role of mesenchymal stem cells (MSCs) as an important tool in regenerative medicine associated with injuries that affect the central nervous system (CNS). The MSCs have the capacity to differentiate into cells of the embryonal tissue, such as the mesoderm. So, these cells can be found in a variety of tissues. Also, the MSCs can release immunomodulatory and neurotrophic factors performance as inflammation mediators operating in injured tissue the regeneration. Furthermore, they can differentiate into neural, like cells in vitro. Thereby, because of the high immunomodulatory role of MSCs, this review sought to describe the main immunomodulatory mechanisms performed by MSCs in CNS recovery after tissue injury or neurodegenerative diseases. Methods: PubMed and ScienceDirect were searched between January 2011 to March 2021 and 43 articles met the criteria of the review. Results: This systematic review indicate that MSCs were used in vivo experimental Multiple Sclerosis (MS), Parkinson's disease (PA), Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS), Ischemic Stroke (IS) and Traumatic Brain Injury (TBI). The treatment MSCs were usually from human origin, derived from bone marrow and administered intravenously. Discussion/Conclusion: It was shown that MSCs, independent from origin or administration pathway, can reduce inflammation and help in the recovery and preservation of injured neural tissue. Thus, the use of MSCs represents a potential therapeutic option in the treatment of neurological disorders mediated by inflammatory processes.

Murine osteoclastogenesis suppression using conditioned media produced by melanoma or activated and non-activated Jurkat-E6 cells.

Conditioned medium (CM) (cell secretome) is a cocktail of growth factors, cytokines, and other soluble mediators secreted by cells into a culture medium. These growth factors are fundamental in many cellular processes such as cell growth, differentiation, and others and the composition of these factors is individual for each cell type. Osteoclasts are large multinucleated cells that are responsible for bone resorption. Immune and cancer cells are known to produce different growth factors, which are able to induce or inhibit osteoclast differentiation. Herein, we evaluated the effect of CM obtained from the supernatant of activated and non-activated Jukart-E6 cells, as well as from one murine (B16-F10) and one human melanoma cell line (SK-MEL-28). To induce osteoclast differentiation, murine bone marrow mononuclear cells were cultured in the presence and absence of differentiation factors (DF), such as macrophage colony-stimulating factor, prostaglandin E2, receptor activator of nuclear factor-κB ligand, and CM. We measured the concentration of interleukin 6, tumor necrosis factor-α and interferon γ (IFN-γ) in CM that can inhibit or induce osteoclastogenesis. Our study demonstrated that CM obtained from each cell line suppresses or inhibits osteoclasts formation at early and intermediate stages of differentiation in the absence or presence of DF. CM obtained from activated Jurkat-E6 cells demonstrates a stronger effect when compared with CM from naïve Jurkat-E6 cells or human and murine melanoma cells. Moreover, CM obtained from activated Jurkat-E6 cells shows higher secretion of IFN-γ, which is an inhibitor of osteoclastogenesis, in comparison with CM obtained from the three other cell lines. On the other hand, CM derived from B16-F10 cells showed a smaller inhibitory effect when compared with CM derived from the other cells.

Murine osteoclastogenesis suppression using conditioned media produced by melanoma or activated and non-activated jurkat-E6 cells

Conditioned medium (CM) (cell secretome) is a cocktail of growth factors, cytokines, and other soluble mediators secreted by cells into a culture medium. These growth factors are fundamental in many cellular processes such as cell growth, differentiation, and others and the composition of these factors is individual for each cell type. Osteoclasts are large multinucleated cells that are responsible for bone resorption. Immune and cancer cells are known to produce different growth factors, which are able to induce or inhibit osteoclast differentiation. Herein, we evaluated the effect of CM obtained from the supernatant of activated and non-activated Jukart-E6 cells, as well as from one murine (B16-F10) and one human melanoma cell line (SK­MEL­28). To induce osteoclast differentiation, murine bone marrow mononuclear cells were cultured in the presence and absence of differentiation factors (DF), such as macrophage colony-stimulating factor, prostaglandin E2, receptor activator of nuclear factor-capaB ligand, and CM. We measured the concentration of interleukin 6, tumor necrosis factor-a and interferon gama (IFN-gama) in CM that can inhibit or induce osteoclastogenesis. Our study demonstrated that CM obtained from each cell line suppresses or inhibits osteoclasts formation at early and intermediate stages of differentiation in the absence or presence of DF. CM obtained from activated Jurkat-E6 cells demonstrates a stronger effect when compared with CM from naïve Jurkat-E6 cells or human and murine melanoma cells. Moreover, CM obtained from activated Jurkat-E6 cells shows higher secretion of IFN-?, which is an inhibitor of osteoclastogenesis, in comparison with CM obtained from the three other cell lines. On the other hand, CM derived from B16-F10 cells showed a smaller inhibitory effect when compared with CM derived from the other cells.

Differential Macrophage Subsets in Muscle Damage Induced by a K49-PLA2 from Bothrops jararacussu Venom Modulate the Time Course of the Regeneration Process.

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation's inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-α and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies.

Differential macrophage subsets in muscle damage induced by a K49-PLA2 from Bothrops jararacussu venom modulate the time course of the regeneration process

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation’s inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-a and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies

The amphibian diacylglycerol O-acyltransferase 2 (DGAT2): a 'paleo-protein' with conserved function but unique folding

Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians’ skin, and no antibody recognition on fish and mammals’ skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a ‘paleo-protein’. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding—as indicated by the molecular models and immunohistochemistry analyses—a consequence of the different cysteine pattern but with conserved biological function.

Analysis of the toxicity and histopathology induced by the oral administration of Pseudanabaena galeata and Geitlerinema splendidum (cyanobacteria) extracts to mice.

Cyanobacteria are common members of the freshwater microbiota in lakes and drinking water reservoirs, and are responsible for several cases of human intoxications in Brazil. Pseudanabaena galeata and Geitlerinema splendidum are examples of the toxic species that are very frequently found in reservoirs in Sao Paulo, which is the most densely populated area in Brazil. In the search for toxic strains collected from water reservoirs and maintained in the Cyanobacterial Culture Collection (CCIBt) of the Institute of Botany of Brazil, the acetic acid extracts (AE) of P. galeata CCIBt 3082 and G. splendidum CCIBt 3223 were analyzed by planar chromatography, which indicated the absence of cyanotoxins. Animal tests were then carried out, and both extracts were found to induce toxic effects in mice when administered intraperitoneally. The present study aimed to investigate whether the oral ingestion of the above mentioned cyanobacteria extracts would also induce toxic effects in mice. Necropsy and histopathological studies were conducted using tissue samples from the animals, which were euthanized one week after the administration of the extracts. The AE of P. galeata did not cause death but did induce transient symptoms, including eyebrow ptosis, straub tail, and pain. The euthanized animals presented hemorrhage in the liver, whereas the histological analysis showed disorganization of the hepatic parenchyma, necrosis, hyperemia, and proximity of the centrilobular vein in the liver. In addition, alterations in the convoluted tubules of the kidneys were observed, and the lungs were unaffected. The AE of G. splendidum caused only one death, and induced transient symptoms, such as dyspnea, paralysis, and pain, in the other mice. The necropsy of the euthanized mice showed hemorrhage in the lungs and liver. The lungs presented hemorrhagic focuses, alveolar collapse, and granulomatous foci. The liver presented hemorrhagic and enlarged sinusoids, hyperemia, proximity of the centrilobular vein, and disorganization of the hepatic parenchyma. Some areas also exhibited an inflammatory infiltrate and calcified tissue inside blood vessels. Necrosis and rupture of the convoluted tubule cells were observed in the kidneys. Further analysis of the both extracts indicated the lack of hemolytic activity, and the presence of two unknown anti-AChE substances in the AE of G. splendidum. Thus, P. galeata and G. splendidum are producers of novel toxins that affect mammals when administered orally.

Pluripotent stem cell transcription factors during human odontogenesis.

Stem cells are capable of generating various cell lines and can be obtained from adult or embryonic tissues for clinical therapies. Stem cells from deciduous dental pulp are among those that are easily obtainable from adult tissues and have been widely studied because of their ability to differentiate into a variety of cell lines in the presence of various chemical mediators. We have analyze the expression of several proteins related to the differentiation and proliferative potential of cell populations that compose the tooth germ of human fetuses. We evaluate 20 human fetuses of both genders. After being paraffin-embedded, cap and bell stages of tooth germ development were subjected to immunohistochemistry for the following markers: Oct-4, Nanog, Stat-3 and Sox-2. The studied antibodies showed nuclear or cytoplasmic immunnostaining within various anatomical structures and with various degrees of expression, indicating the action of these proteins during tooth development. We conclude that the interrelationship between these transcription factors is complex and associated with self-renewal and cell differentiation. Our results suggest that the expression of Oct-4, Nanog, Sox-2 and Stat-3 are related to differentiation in ameloblasts and odontoblasts.

Biologically active compounds from cyanobacteria extracts: in vivo and in vitro aspects

An investigation was directed towards the antiacetylcholinesterase activity of the acid aqueous and methanolic extracts of five cyanobacterial taxa, which encompasses an enzymatic inhibition essay and the evaluation of the physiological responses of mice to cyanobacterial extracts along with toxicological observations. The strains Calothrix sp. CCIBt 3320, Tolypothrix sp. CCIBt 3321, Phormidium cf. amoenum CCIBt 3412, Phormidium sp. CCIBt 3265, and Geitlerinema splendidum CCIBt 3223 were from the São Paulo Botanical Institute Cyanobacterial Culture Collection and all of them showed inhibitory effect on acetylcholinesterase activity (in vitro) and caused systemic effects similar to those described for anticholinesterase drugs (in vivo). With the exception of G. splendidum and Tolypothrix sp. strains, all extracts produced reversible antiacetylcolinesterase effects in mice. Complementary histopathological studies were carried out on tissues from animals administered with Phormidium sp. and P. cf. amoenum.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip.

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, ß1 subunits of α3ß1 heterodimer and ß4 subunit of integrin α6ß4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of ß1, ß4 and α3 integrins, and SISCCs lacked ß1, ß4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin ß1, ß4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype.

Allogenous bone grafts improved by bone marrow stem cells and platelet growth factors: clinical case reports.

In order to increase the amount of available bone where dental implants must be placed, the present study has associated platelet-rich plasma (PRP) and mononuclear cells (MNCs) from bone marrow aspirate and bone scaffold (BS) in 32 patients aged between 45 and 75 years old. The MNC attainment and the adherence to the BS were confirmed through histology, cell culture, and scanning electron microscopy. The clinical results, analyzed by computed tomography, have showed that the scaffolds were well integrated and adapted to the cortical bone. We can conclude that the process of healing observed in the patients was due to the presence of mesenchymal stem cell in MNC fraction in the bone grafts.

Assessment of c-Jun, c-Fos and cyclin D1 in premalignant and malignant oral lesions.

UNLABELLED: Some oral cancers are known to develop from dysplastic oral epithelium. In the present study, the expression of c-Jun, c-Fos, and cyclin D1 proteins in oral epithelial lesions with different degrees of dysplasia, and in oral squamous cell carcinomas (OSCCs) was evaluated. Eighteen cases of mild dysplasia, 23 cases of moderate to severe dysplasia and 24 OSCCs were studied immunohistochemically. Additionally, 15 sections of oral mucosa without any evidence of dysplasia were included in the study. RESULTS: c-Jun expression increased according to the degree of oral dysplasia, with the greatest expression found in OSCC. c-Fos expression was intense in normal mucosa, reduced in mild dysplasia and high in moderate to severe dysplasia and in OSCCs. Cyclin D1 was expressed in only a few cases of moderate to severe dysplasia and in most of the OSCCs. Statistical analysis showed a correlation between the three proteins and the degree of epithelial alteration. The present results indicate a possible role of c-Jun and c-Fos in malignant transformation of oral mucosa.