A systematic review on guanitoxin: General characteristics and ecological risks.

Guanitoxin (GNT) is a potent cyanotoxin, with a relatively low number of publications (n = 51) compared to other cyanotoxins. Among the published studies, 35 % were on the effect of the toxin in animals, mainly in rodents and in vitro testing, followed by studies that identified species of cyanobacteria that produce GNT in aquatic systems and consequently accidental poisoning in wild and domestic animals (27 %). Studies that developed or tested methods for identifying the molecule, based on colorimetric and analytical techniques, represented 14 %, while 8 % were on GNT biosynthesis. Review articles and chemical isolation (6 %) and on the stability of the molecule (4 %) were the topics with the lowest number of publications. The results show the occurrence of GNT was identified mainly in eutrophic environments with a higher incidence in the American continent. Chemical characteristics of the molecule, such as short half-life in the environment, instability in solutions with alkaline pH values, temperature >23 °C, added to the lack of an analytical standard, are factors that make it difficult to identify and quantify it. However, GNT monitoring can be performed using LC-MS-MRM methods or genes specific to the newly discovered molecule.

Toxicological effects of cyanobacterial metabolites on zebrafish larval development.

Freshwater cyanobacteria are known worldwide for their potential to produce toxins. However, these organisms are also found in marine, terrestrial and extreme environments and produce unique compounds, other than toxins. Nevertheless, their effects on biological systems are still barely known. This work tested extracts of different cyanobacterial strains against zebrafish (Danio rerio) larvae and analyzed their metabolomic profiles using liquid chromatography combined with mass spectrometry. Strains Desertifilum tharense, Anagnostidinema amphibium, and Nostoc sp. promoted morphological abnormalities such as pericardial edema, edema in the digestive system region, curvature of the tail and spine in zebrafish larvae in vivo. In contrast, Microcystis aeruginosa and Chlorogloeopsis sp. did not promote such changes. Metabolomics revealed unique compounds belonging to the classes of terpenoids, peptides, and linear lipopeptides/microginins in the nontoxic strains. The toxic strains were shown to contain unique compounds belonging to the classes of cyclic peptides, amino acids and other peptides, anabaenopeptins, lipopeptides, terpenoids, and alkaloids and derivatives. Other unknown compounds were also detected, highlighting the rich structural diversity of secondary metabolites produced by cyanobacteria. The effects of cyanobacterial metabolites on living organisms, mainly those related to potential human and ecotoxicological risks, are still poorly known. This work highlights the diverse, complex, and unique metabolomic profiles of cyanobacteria and the biotechnological potential and associated risks of exposure to their metabolites.

Different Species of Marine Sponges Diverge in Osteogenic Potential When Therapeutically Applied as Natural Scaffolds for Bone Regeneration in Rats.

A highly porous structure, and an inorganic (biosilica) and collagen-like organic content (spongin) makes marine sponges potential candidates to be used as natural scaffolds in bone tissue engineering. The aim of this study was to characterize (through SEM, FTIR, EDS, XRD, pH, mass degradation and porosity tests) scaffolds produced from two species of marine sponges, Dragmacidon reticulatum (DR) and Amphimedon viridis (AV), and to evaluate the osteogenic potential of these scaffolds by using a bone defect model in rats. First, it was shown that the same chemical composition and porosity (84 ± 5% for DR and 90 ± 2% for AV) occurs among scaffolds from the two species. Higher material degradation was observed in the scaffolds of the DR group, with a greater loss of organic matter after incubation. Later, scaffolds from both species were surgically introduced in rat tibial defects, and histopathological analysis after 15 days showed the presence of neo-formed bone and osteoid tissue within the bone defect in DR, always around the silica spicules. In turn, AV exhibited a fibrous capsule around the lesion (19.9 ± 17.1%), no formation of bone tissue and only a small amount of osteoid tissue. The results showed that scaffolds manufactured from Dragmacidon reticulatum presented a more suitable structure for stimulation of osteoid tissue formation when compared to Amphimedon viridis marine sponge species.

Radiographers' musculoskeletal health in Western Switzerland: WRMSDs symptoms prevalence and risk factors.

BACKGROUND: Work-related musculoskeletal disorders (WRMSDs) are a significant occupational health concern in radiographers. OBJECTIVE: This study aimed to describe radiographers' WRMSDs symptoms prevalence and severity, exploring associations with occupational risk factors. METHODS: A cross-sectional study was conducted to explore WRMSDs symptoms and occupational risk factors in radiographers of Western Switzerland using an online survey, based on the Nordic Musculoskeletal Questionnaire (NMQ). Descriptive statistics were conducted to analyze the collected data, and associative statistics to identify the risk factors related to symptoms. RESULTS: Participants (n = 359) presented a high prevalence of WRMSDs symptoms in the last 12 months (94.7%), with a related absenteeism rate of 15.6%. In the last 7 days, symptoms prevalence was 67.7%. The most affected anatomical regions, over both time periods, were the neck (73.0%, 36.8%) and low back (67.4%, 35.7%). Associative statistics underlined risk factors affecting significantly radiographers' health (OR >2) were the "awkward postures" (OR = 2.86; 95% CI 1.78-4.58) and "feeling anxiety/stress at work" both for low back (OR = 2.38; 95% CI 1.39-4.08), and being a woman for the neck (OR = 2.64; 95% CI 1.51-4.61). CONCLUSIONS: There is a high WRMSDs symptoms prevalence in Western Switzerland radiographers. Radiographers' work demands namely for awkward postures increases the odds for WRMSDs symptoms presence, affecting predominantly neck, upper and lower back. Our data suggest that further research is needed to implement adapted prevention to this specific context.

Métodos de extração, estabilidade e biodisponibilidade da guanitoxina em cultivos de Sphaeropermopsis torques-reginae analisadas por espectrometria de massas / Methods of extraction, stability, and bioavailability of guanitoxin in Sphaeropermopsis torques-reginae cultures analyzed by mass spectrometry

A guanitoxina (GNT) é uma neurotoxina produzida por algumas cepas de cianobactérias dos gêneros Dolichospermum e Sphaerospermopsis>. A GNT é o único organofosforado natural, capaz de causar a morte de animais selvagens e domésticos devido à inibição irreversível da acetilcolinesterase. Apesar de sua alta toxicidade, o diagnóstico da GNT em amostras biológicas ainda é um grande desafio. A dificuldade para sua detecção está diretamente ligada à sua instabilidade em altas temperaturas e pH alcalino, tornando difícil seu monitoramento em corpos d'água. Por isso, esta pesquisa objetivou estudar a estabilidade e biodisponibilidade da GNT em amostras aquosas, com intuito de obter mais informações sobre a natureza química e biológica dessa potente neurotoxina. Para realizar este estudo, a cepa ITEP-24 (S. torques-reginae) produtora de GNT foi cultivada em laboratório sob condições controladas, para obter biomassa para os experimentos de extração, semi-isolamento, estabilidade, ensaio in vitro e identificação por LC-MS/MS. Primeiramente foram realizados testes de extração da GNT partir de células liofilizadas da cepa ITEP-24 utilizando água, metanol e etanol em pH ácido. Depois utilizou-se dois métodos de extração em fase sólida (SPE) com cartuchos preenchidos com fases estacionarias C18 em fase reversa e sílica gel em fase normal, com objetivo de avaliar qual método de SPE seria melhor para extrair e concentrar a GNT. Nós também testamos métodos para lisar as células com sondas de ultrassom, misturador e centrifugação. Além dos métodos de extração, nós avaliamos a estabilidade da toxina em diferentes temperaturas, para isso a biomassa seca contendo a GNT ficou condicionada a 4 °C, 23 °C, -20 °C, -80 °C durantes seis meses, e análises de identificação foram realizadas dentro período de 150 dias em uma sequência de 30 dias. A estabilidade da toxina foi analisada também a partir de extrações em soluções com diferentes valores de pH (1,5; 3,0; 5,0; 7,0; 8,5; 10,5) e temperatura (23 ºC e 37 ºC). Depois, analisou-se a biodisponibilidade da GNT em células frescas da linhagem ITEP-24 através de teste de dissolução in vitro. O objetivo deste teste foi avaliar a liberação da toxina intracelular em meio simulado do conteúdo gástrica e intestinal com e sem enzimas digestivas para compreender e estimar a disponibilidade da GNT in vivo. Os resultados de todos experimentos descritos neste estudo, foram obtidos a partir de análises por cromatografia líquida de interação hidrofílica (HILIC) acoplado ao espectrômetro de massas do tipo triplo quadrupolo LC-QqQ-MS/MS utilizando as transições 253>58, 253>159 e 159>58 [M+H]+ utilizando coluna com fase estacionária zwitteriônica (ZIC). A identificação da GNT foi realizada também por cromatografia líquida acoplada ao espectrômetro de massas de alta resolução (LC-HR-QTOF-MS) com coluna Luna C18, Hydro-RP C18 e ZIC-HILIC. Dos protocolos de extração testados, a combinação de metanol/água (70:30 v/v) com ácido acético (0.3%) extraiu maior quantidade relativa da GNT a partir de células frescas e liofilizadas da cepa ITEP-24 e a concentração da toxina foi maior em amostras de células frescas. Em relação aos métodos de lise celular, as extrações realizadas em sonda de ultrassom com banho-maria e centrifugação por 1h foram estatisticamente significantes para liberar a toxina intracelular. Não houve diferença significativa entre os testes de SPE, no entanto, a semipurificação da toxina foi melhor com cartucho preenchido com sílica gel em fase normal e adaptação desse método em coluna aberta permitiu obter uma fração enriquecida com GNT. A GNT mostrou ser mais estável em pH ácido, sendo o pH 3,0 o melhor para manter e extrair a toxina em amostras aquosas e a toxina intracelular presente em células secas podem degradar em temperatura de 23 °C por um período de 150 dias mesmo em solução com pH 3,0. Durante os testes de extração e purificação foi observado também a degradação da toxina em processos de secagem e ressuspensão. As análises realizadas no LC-HR-QTOF-MS com diferentes métodos cromatográficos possibilitou a identificação da GNT, porém o método realizado com coluna ZIC-HILIC mostrou melhor resolução cromatográfica dos picos relativos m/z e tempo de retenção de toxina. Os resultados obtidos nos testes de dissolução in vitro mostraram que a GNT fica mais disponível no simulado gástrico com e sem a enzima pepsina, mas também pode ser absorvida no intestino. Portanto, o teste de dissolução in vitro pode ser uma ferramenta útil para a avaliação de risco de cianotoxinas in vivo, devido ao seu potencial de monitorar qualitativa e quantitativamente substâncias dissolvidas em fluidos gastrointestinais. Os resultados apresentados neste estudo fornecem informações valiosas para uma melhor compreensão da estabilidade e biodisponibilidade do GNT. Além disso, os métodos apresentados neste estudo podem ser úteis para diversas aplicações projetadas para identificar a toxina em amostras ambientais, bem como orientações para procedimentos de purificação da GNT

Guanitoxin (GNT) is a neurotoxin produced by some strains of cyanobacteria of the genus Dolichospermum and Sphaerospermopsis. GNT is the only natural organophosphate, capable of causing the death of animals from wild and domestic animals due to irreversible inhibition of acetylcholinesterase. Despite its high toxicity, the diagnosis of GNT in biological samples is still a significant challenge. The difficulty in its detection is directly linked to its instability at high temperatures and alkaline pH, making it difficult to monitor in bodies of water. Therefore, this research aimed to study the stability and bioavailability of GNT in aqueous samples to provide more information about the chemical and biological nature of this molecule. The strain ITEP-24 (S. torques-reginae) producing GNT was grown in the laboratory under controlled conditions to obtain biomass for the extraction, semi-isolation, stability, in vitro tests, and toxin identification by LC-MS/MS. Firstly, tests were carried out to extract GNT from lyophilized cells strain ITEP-24 using water, methanol, and ethanol at acidic pH and, two SPE methods in cartridges with stationary phases of C18 reverse phase and normal phase gel silica, to evaluate which would be better to extract and concentrate the GNT. We also tested different methods of cell lysis, such as ultrasound probes, mixers, and centrifugation. In addition to the extraction methods, the stability of the toxin was evaluated at different temperatures, for this, the dry biomass containing the toxin was conditioned at 4 °C, 23 °C, -20 °C, -80 °C for 150 days and analysis of the identification of the GNT was carried out within that period in a sequence of 30 days. The toxin stability was also analyzed from extractions in solutions with different pH values (1.5; 3.0; 5.0; 7.0; 8.5; 10.5) and temperature (23 ºC and 37 ºC). In addition, we performed dissolution tests with fresh cells of the ITEP-24 strain to evaluate the bioavailability of GNT in simulated gastric and intestinal fluids with and without digestive enzymes to understand and estimate the availability of GNT in vivo. The results of all experiments described in this study were obtained from analyzes by hydrophilic interaction liquid chromatography (HILIC) coupled to the LC-QqQ-MS/MS triple quadrupole mass spectrometer using the transitions m/z 253> 58, m/z 253> 159 and m/z 159> 58 [M + H]+ using a column with the zwitterionic stationary phase (ZIC). Liquid chromatography coupled to the high-resolution mass spectrometer (LC-HR-QTOF-MS) with Luna column C18, Hydro-RP C18, and ZIC-HILIC carried out the identification of the GNT. From the extraction protocols tested, the combination of methanol/water (70:30 v/v) with acetic acid (0.3%) extracted a greater relative amount of GNT from fresh and lyophilized ITEP-24 cells, and the concentration of the toxin is higher previously fresh. Concerning cellular methods, the ultrasound probe with a water bath and centrifugation for 1h ware statistically significant to release the intracellular toxin. There was no significant difference between the SPE tests. However, the semi-purification of the toxin was better with a cartridge filled with gel silica in the normal phase and adaptation of this method in an open column allowed to obtain a fraction enriched with GNT. GNT was more stable at acid pH, with pH 3.0 being the best to maintain and the intracellular toxin present in dry cells can degrade at a temperature at 23 °C for 150 days even in pH 3.0 solution. The toxin can also hydrolyze in the drying and resuspension processes. The analyzes carried out in LC-HR-QTOF-MS with different chromatographic methods made it possible to identify the GNT itself, however, the ZIC-HILIC column method showed excellent chromatographic resolution of the relative m/z peaks and toxin retention time. The results obtained in the in vitro dissolution tests showed that GNT is more available in the gastric simulation with and without the enzyme pepsin, but it can also be absorbed in the intestine. Thus, in vitro dissolution tests can be used as a useful tool for the risk assessment of cyanotoxins in vivo due to their potential to qualitatively and quantitatively monitor substances dissolved in gastrointestinal fluids. The results presented in this study provide valuable information for a better understanding of the stability and bioavailability of GNT. Besides, the methods presented in this study can be useful for various applications designed to identify the toxin in environmental samples, as well as guidance on procedures for purifying GNT

Characterization and Biological Performance of Marine Sponge Collagen

Abstract This study characterized the morphological aspects of marine collagen - spongin (SPG) extract from marine sponges, as well as, evaluating its in vitro and in vivo biological performance. Aplysina fulva marine sponge was used for the SPG extraction. It was investigated the physicochemical and morphological properties of SPG by using scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and compared to PMMA and bovine collagen. Additionally, the SPG cytotoxicity and its influence on cell proliferation, through in vitro tests. Moreover, the in vivo biological response was investigated using an experimental model of tibial bone defect. The results demonstrated that SPG presented an irregular granular aspect, with a composition of OH, C=O, NH, CN and an amorphous profile. Also, in vitro viability results for the L929 and MC3T3 cell lines cultured with SPG extracts demonstrated normal growth in comparison to controls, except for MC3T3 viability at day 3. For in vivo analysis, using tibial bone defects in rats, SPG treated animals presented an increased rate of material resorption and higher granulation and bone formation deposition in the region of the defect, mainly after 45 days. As a conclusion, SPG was successfully extracted. The in vitro and in vivo studies pointed out that SPG samples produced an increase in L929 and MC3T3 viability and improved the performance in tibial bone defects. It can be concluded that SPG can be used as a bone graft for bone regeneration.

Availability of Guanitoxin in Water Samples Containing Sphaerospermopsis torques-reginae Cells Submitted to Dissolution Tests.

Guanitoxin (GNT) is a potent neurotoxin produced by freshwater cyanobacteria that can cause the deaths of wild and domestic animals. Through reports of animal intoxication by cyanobacteria cells that produce GNT, this study aimed to investigate the bio-accessibility of GNT in simulated solutions of the gastrointestinal content in order to understand the process of toxicosis promoted by GNT in vivo. Dissolution tests were conducted with a mixture of Sphaerospermopsis torques-reginae (Cyanobacteria; Nostocales) cultures (30%) and gastrointestinal solutions with and without proteolytic enzymes (70%) at a temperature of 37 °C and rotation at 100 rpm for 2 h. The identification of GNT was performed by LC-QqQ-MS/MS through the transitions [M + H]+m/z 253 > 58 and [M + H]+m/z 253 > 159, which showed high concentrations of GNT in simulated gastric fluid solutions (p-value < 0.001) in comparison to simulated solutions of intestinal content. The gastric solution with pepsin promoted the stability of GNT (p-value < 0.05) compared to the simulated solution of gastric fluid at the same pH without the enzyme. However, the results showed that GNT is also available in intestinal fluids for a period of 2 h, and solutions containing the pancreatin enzyme influenced the bio-accessibility of the toxin more compared to the intestinal medium without enzyme (p-value < 0.05). Therefore, the bio-accessibility of the toxin must be considered both in the stomach and in the intestine, and may help in the diagnosis and prediction of exposure and risk in vivo through the oral ingestion of GNT-producing cyanobacteria cells.

In vitro toxicity of isolated strains and cyanobacterial bloom biomasses over Paramecium caudatum (ciliophora): Lessons from a non-metazoan model organism.

Cyanobacteria have been considered a major global threat because of their widespread ability to proliferate and contaminate inland and marine waters with toxic metabolites. For this reason, to avoid risks to humans and environmental health, regulatory legislation and guidelines have been established based on extensive toxicological data. However, most of what is known in this field come from works on microcystin (MC) variants, which effects were almost exclusively tested in metazoan models. In this work, we used acute end-point toxicological assays and high-resolution hybrid quadrupole time-of-flight mass spectrometer coupled with electrospray ionization source (ESI-Q-TOF-MS) analyses to evaluate the deleterious impact of aqueous extracts prepared from cultures of cyanobacteria and environmental bloom biomasses over a non-metazoan model organism, the cosmopolitan fresh/brackish water unicellular microeukaryote, Paramecium caudatum (Ciliophora). Our data suggest that all extracts produced time-dependent effects on P. caudatum survival, irrespective of their metabolite profile; and that this ciliate is more sensitive to extracts containing microginins than to extracts with only MCs, stressing that more toxicological investigations should be performed on the environmental impact of neglected cyanotoxins. Further, our data provide evidence that P. caudatum may be more sensitive to cyanotoxins than vertebrates, indicating that guidelines values, set on metazoans are likely to be inaccurate to protect organisms from basal food web positions. Thus, we highly recommend the widespread use of microeukaryotes, such as ciliates in environmental risk assessment frameworks for the establishment of more reliable cyanotoxin monitoring guideline values.

Marine spongin incorporation into Biosilicate® for tissue engineering applications: An in vivo study.

Biomaterials and bone grafts, with the ability of stimulating tissue growth and bone consolidation, have been emerging as very promising strategies to treat bone fractures. Despite its well-known positive effects of biosilicate (BS) on osteogenesis, its use as bone grafts in critical situations such as bone defects of high dimensions or in non-consolidated fractures may not be sufficient to stimulate tissue repair. Consequently, several approaches have been explored to improve the bioactivity of BS. A promising strategy to reach this aim is the inclusion of an organic part, such as collagen, in order to mimic bone structure. Thus, the present study investigated the biological effects of marine spongin (SPG)-enriched BS composites on the process of healing, using a critical experimental model of cranial bone defect in rats. Histopathological and immunohistochemistry analyzes were performed after two and six weeks of implantation to investigate the effects of the material on bone repair (supplemental material-graphical abstract). Histological analysis demonstrated that for both BS and BS/SPG, similar findings were observed, with signs of material degradation, the presence of granulation tissue along the defect area and newly formed bone into the area of the defect. Additionally, histomorphometry showed that the control group presented higher values for Ob.S/BS (%) and for N.Ob/T.Ar (mm2) (six weeks post-surgery) compared to BS/SPG and higher values of N.Ob/T.Ar (mm2) compared to BS (two weeks post-surgery). Moreover, BS showed higher values for OV/TV (%) compared to BS/SPG (six weeks post-surgery). Also, VEGF immunohistochemistry was increased for BS (two weeks post-surgery) and for BS/SPG (six weeks) compared to CG. TGFb immunostaining was higher for BS compared to CG. The results of this study demonstrated that the BS and BS/SPG scaffolds were biocompatible and able to support bone formation in a critical bone defect in rats. Moreover, an increased VEGF immunostaining was observed in BS/SPG.

Evaluation of the In Vivo Biological Effects of Marine Collagen and Hydroxyapatite Composite in a Tibial Bone Defect Model in Rats.

One of the most promising strategies to improve the biological performance of bone grafts is the combination of different biomaterials. In this context, the aim of this study was to evaluate the effects of the incorporation of marine spongin (SPG) into Hydroxyapatite (HA) for bone tissue engineering proposals. The hypothesis of the current study is that SPG into HA would improve the biocompatibility of material and would have a positive stimulus into bone formation. Thus, HA and HA/SPG materials were produced and scanning electron microscopy (SEM) analysis was performed to characterize the samples. Also, in order to evaluate the in vivo tissue response, samples were implanted into a tibial bone defect in rats. Histopathological, immunohistochemistry, and biomechanical analyses were performed after 2 and 6 weeks of implantation to investigate the effects of the material on bone repair. The histological analysis demonstrated that composite presented an accelerated material degradation and enhanced newly bone formation. Additionally, histomorphometry analysis showed higher values of %BV/TV and N.Ob/T.Ar for HA/SPG. Runx-2 immunolabeling was higher for the composite group and no difference was found for VEGF. Moreover, the biomechanical analysis demonstrated similar values for all groups. These results indicated the potential of SPG to be used as an additive to HA to improve the biological performance for bone regeneration applications. However, further long-term studies should be carried out to provide additional information regarding the material degradation and bone regeneration.

Bioglass/PLGA associated to photobiomodulation: effects on the healing process in an experimental model of calvarial bone defect.

Bioactive glasses (BG) are known for their ability to bond to bone tissue. However, in critical situations, even the osteogenic properties of BG may be not enough to induce bone consolidation. Thus, the enrichment of BG with polymers such as Poly (D, L-lactic-co-glycolic) acid (PLGA) and associated to photobiomodulation (PBM) may be a promising strategy to promote bone tissue healing. The aim of the present study was to investigate the in vivo performance of PLGA supplemented BG, associated to PBM therapy, using an experimental model of cranial bone defect in rats. Rats were distributed in 4 different groups (Bioglass, Bioglass/PBM, Bioglas/PLGA and BG/PLGA/PBM). After the surgical procedure to induce cranial bone defects, the pre-set samples were implanted and PBM treatment (low-level laser therapy) started (808 nm, 100 mW, 30 J/cm2). After 2 and 6 weeks, animals were euthanized, and the samples were retrieved for the histopathological, histomorphometric, picrosirius red staining and immunohistochemistry analysis. At 2 weeks post-surgery, it was observed granulation tissue and areas of newly formed bone in all experimental groups. At 6 weeks post-surgery, BG/PLGA (with or without PBM) more mature tissue around the biomaterial particles. Furthermore, there was a higher deposition of collagen for BG/PLGA in comparison with BG/PLGA/PBM, at second time-point. Histomorphometric analysis demonstrated higher values of BM.V/TV for BG compared to BG/PLGA (2 weeks post-surgery) and N.Ob/T.Ar for BG/PLGA compared to BG and BG/PBM (6 weeks post-surgery). This current study concluded that the use of BG/PLGA composites, associated or not to PBM, is a promising strategy for bone tissue engineering.

Incorporation of collagen and PLGA in bioactive glass: in vivo biological evaluation.

Bioactive glasses (BG) are known for their unique ability to bond to bone tissue. However, in critical situations, even the osteogenic properties of BG may be not sufficient to produce bone consolidation. The use of composite materials may constitute an optimized therapeutical intervention for bone stimulation. The aim of this study was to characterize BG/collagen/poly (d,l-lactic-co-glycolic) acid (BG/COL/PLGA) composites, in vitro biocompatibility and in vivo biological properties. MC3T3-E1 cells were evaluated by cell proliferation, ALP activity, cell adhesion and morphology. Qualitative histology and immunohistochemistry were performed in a calvarial bone defect model in rats. The in vitro study demonstrated, after 3 and 6 days of culture, a significant increase of proliferation was observed for BG/PLGA compared to BG/COL and BG/COL/PLGA. BG/COL/PLGA presented a higher value for ALP activity after 3 days of culture compared to BG/PLGA. For in vivo analysis, 6 weeks post-surgery, BG/PLGA showed a more mature neoformed bone tissue. As a conclusion, the in vitro and in vivo studies pointed out that BG/PLGA samples improved biological properties in calvarial bone defects, highlighting the potential of BG/PLGA composites to be used as a bone graft for bone regeneration applications.

Toxicity of Cyanopeptides from Two Microcystis Strains on Larval Development of Astyanax altiparanae.

Absorption and accumulation of bioavailable cyanobacterial metabolites (including cyanotoxins) are likely in fish after senescence and the rupturing of cells during bloom episodes. We determined the toxicity of cyanopeptides identified from two strains of Microcystis (M. panniformis MIRS-04 and M. aeruginosa NPDC-01) in a freshwater tropical fish, Astyanax altiparanae (yellowtail tetra, lambari). Aqueous extracts of both Microcystis strains were prepared in order to simulate realistic fish exposure to these substances in a freshwater environment. Both strains were selected because previous assays evidenced the presence of microcystins (MCs) in MIRS-04 and lack of cyanotoxins in NPDC-01. Identification of cyanobacterial secondary metabolites was performed by LC-HR-QTOF-MS and quantification of the MC-LR was carried out by LC-QqQ-MS/MS. MIRS-04 produces the MCs MC-LR, MC-LY and MC-HilR as well as micropeptins B, 973, 959 and k139. NPCD-01 biosynthetizes microginins FR1, FR2/FR4 and SD-755, but does not produce MCs. Larval fish survival and changes in morphology were assessed for 96 h exposure to aqueous extracts of both strains at environmentally relevant concentrations from 0.1 to 0.5 mg (dry weight)/mL, corresponding to 0.15 to 0.74 µg/mL of MC-LR (considering dried amounts of MIRS-04 for comparison). Fish mortality increased with concentration and time of exposure for both strains of Microcystis. The frequencies of morphological abnormalities increased with concentration in both strains, and included abdominal and pericardial oedema, and spinal curvature. Results demonstrate that toxicity was not solely caused by MCs, other classes of cyanobacterial secondary metabolites contributed to the observed toxicity.

In vitro antileishmanial and cytotoxic activities of nerolidol are associated with changes in plasma membrane dynamics.

The sesquiterpene nerolidol is a membrane-active compound that has demonstrated antitumor, antibacterial, antifungal and antiparasitic activities. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and biophysical parameters determined via cell culture assays to study the mechanisms underlying the in vitro antileishmanial activity of nerolidol. The EPR spectra of a spin-labeled stearic acid indicated notable interactions of nerolidol with the cell membrane of Leishmania amazonensis amastigotes. The nerolidol IC50 values in L. amazonensis amastigotes and promastigotes were found to depend on the cell concentration used in the assay. This dependence was described by an equation that considers various cell suspension parameters, such as the 50% inhibitory concentrations of nerolidol in the cell membrane (cm50) and the aqueous phase (cw50) and the membrane-water partition coefficient of nerolidol (KM/W). Via cytotoxicity (CC50) and hemolytic potential (HC50) data, these parameters were also determined for nerolidol in macrophages and erythrocytes. With a cw50 of 125 µM, macrophages were less sensitive to nerolidol than amastigotes and promastigotes, which had mean cw50 values of 56 and 74 µM, respectively. The estimated cm50 values of nerolidol for amastigotes and promastigotes and macrophages were between 2.6 and 3.0 M, indicating substantial accumulation of nerolidol in the cell membrane. In addition, the spin-label EPR data indicated that membrane dynamic changes occurred in L. amazonensis amastigotes at concentrations similar to the nerolidol IC50 value.

Neurotoxic effects of sublethal concentrations of cyanobacterial extract containing anatoxin-a(s) on Nauphoeta cinerea cockroaches.

The detection of cyanotoxins, such as the anatoxin-a(s), is essential to ensure the biological safety of water environments. Here, we propose the use of Nauphoeta cinerea cockroaches as an alternative biological model for the biomonitoring of the activity of anatoxin-a(s) in aquatic systems. In order to validate our proposed model, we compared the effects of a cyanobacterial extract containing anatoxin-a(s) (CECA) with those of the organophosphate trichlorfon (Tn) on biochemical and physiological parameters of the nervous system of Nauphoeta cinerea cockroaches. In brain homogenates from cockroaches, CECA (5 and 50 µg/g) inhibited acetylcholinesterase (AChE) activity by 53 ±â€¯2% and 51 ±â€¯7%, respectively, while Tn (5 and 50 µg/g) inhibited AChE activity by 35 ±â€¯4% and 80 ±â€¯9%, respectively (p < 0.05; n = 6). Moreover, CECA at concentrations of 5, 25, and 50 µg/g decreased the locomotor activity of the cockroaches, diminishing the distance travelled and increasing the frequency and duration of immobile episodes similarly to Tn (0.3 µg/g) (p < 0.05, n = 40, respectively). CECA (5, 25 and 50 µg/g) induced an increase in the leg grooming behavior, but not in the movement of antennae, similarly to the effect of Tn (0.3 µg/g). In addition, both CECA (50 µg/200 µl) and Tn (0.3 µg/200 µl) induced a negative chronotropism in the insect heart (37 ±â€¯1 and 47 ±â€¯8 beats/min in 30 min, respectively) (n = 9, p > 0.05). Finally, CECA (50 µg/g), Tn (0.3 µg/g) and neostigmine (50 µg/g) caused significant neuromuscular failure, as indicated by the monitoring of the in vivo neuromuscular function of the cockroaches, during 100 min (n = 6, p < 0.05, respectively). In conclusion, sublethal doses of CECA provoked entomotoxicity. The Tn-like effects of CECA on Nauphoeta cinerea cockroaches encompass both the central and peripheral nervous systems in our insect model. The inhibitory activity of CECA on AChE boosts a cascade of signaling events involving octopaminergic/dopaminergic neurotransmission. Therefore, this study indicates that this insect model could potentially be used as a powerful, practical, and inexpensive tool to understand the impacts of eutrophication and for orientating decontamination processes.

Vacuumed collagen-impregnated bioglass scaffolds: Characterization and influence on proliferation and differentiation of bone marrow stromal cells.

This study evaluated physical-chemical characteristics of a vacuumed collagen-impregnated bioglass (BG) scaffolds and bone marrow stromal cells (BMSCs) behavior on those composites. scanning electron microscope and energy dispersive x-ray spectroscope demonstrated collagen (Col) was successfully introduced into BG. Vacuum impregnation system has showed efficiency for Col impregnation in BG scaffolds (approximately 20 wt %). Furthermore, mass weight decreasing and more stabilized pH were observed over time for BG/Col upon incubation in phosphate buffered saline compared to plain BG under same conditions. Calcium evaluation (Ca assay) demonstrated higher calcium uptake for BG/Col samples compared to BG. In addition, BG samples presented hydroxyapatite crystals formation on its surface after 14 days in simulated body fluid solution, and signs of initial degradation were observed for BG and BG/Col after 21 days. Fourier transform infrared spectroscopy spectra for both groups indicated peaks for hydroxyapatite formation. Finally, a significant increase of BMSCs viability for both composites was observed compared to control group, but no increase of osteogenic differentiation-related gene expressions were found. In summary, BG/Col scaffolds have improved degradation, pH equilibrium and Ca mineralization over time, accompanied by hydroxyapatite formation. Moreover, both BG and BG/Col scaffolds were biocompatible and noncytotoxic, promoting a higher cell viability compared to control. Future investigations should focus on additional molecular and in vivo studies in order to evaluate biomaterial performance for bone tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 211-222, 2019.

Photobiomodulation guided healing in a sub-critical bone defect in calvarias of rats.

BACKGROUND: Photobiomodulation presents stimulatory effects on tissue metabolism, constituting a promising strategy to produce bone tissue healing. OBJECTIVE: the aim of the present study was to investigate the in vivo performance of PBM using an experimental model of cranial bone defect in rats. MATERIAL AND METHODS: rats were distributed in 2 different groups (control group and PBM group). After the surgical procedure to induce cranial bone defects, PBM treatment initiated using a 808 nm laser (100 mW, 30 J/cm2, 3 times/week). After 2 and 6 weeks, animals were euthanized and the samples were retrieved for the histopathological, histomorphometric, picrosirius red staining and immunohistochemistry analysis. RESULTS: Histology analysis demonstrated that for PBM most of the bone defect was filled with newly formed bone (with a more mature aspect when compared to CG). Histomorphomeric analysis also demonstrated a higher amount of newly formed bone deposition in the irradiated animals, 2 weeks post-surgery. Furthermore, there was a more intense deposition of collagen for PBM, with ticker fibers. Results from Runx-2 immunohistochemistry demonstrated that a higher immunostaining for CG 2 week's post-surgery and no other difference was observed for Rank-L immunostaining. CONCLUSION: This current study concluded that the use of PBM was effective in stimulating newly formed bone and collagen fiber deposition in the sub-critical bone defect, being a promising strategy for bone tissue engineering.

[Prevalence and factors associated with alcohol use during pregnancy in a maternity hospital in Goiás, Central Brazil]. / Prevalência e fatores associados ao uso de álcool durante a gestação em uma maternidade de Goiás, Brasil Central.

The scope of this article is to estimate the prevalence and factors associated with alcohol use during pregnancy. It involved a cross-sectional study in a sample of 361 pregnant women in a reference service for gynecological and prenatal care. The data related to socio-demographic characteristics, alcohol use and potential associated factors were collected through face-to-face interviews. Poisson regression with robust variance was used to identify factors associated with the outcome analyzed. The consumption of alcohol in the sample was 17.7% (95% CI: 95% CI: 14.1% to 22.0%). A history of pre-gestational or gestational diabetes, suicidal ideation and tobacco use in the last 30 days was associated with alcohol use during pregnancy (p < 0.05). The study showed a high prevalence of alcohol use during the current pregnancy and its association with important factors. Actions such as screening for alcohol and advice on problems associated with the use of this substance, especially during the prenatal period, can contribute to effective reduction of alcohol use in pregnant women and related maternal and fetal injuries.

O objetivo deste artigo é estimar a prevalência e os fatores associados ao uso de álcool durante a gestação. Estudo de corte transversal em uma amostra de 361 gestantes de um serviço de referência à assistência ginecológica e pré-natal. Os dados relacionados às características sociodemográficas, uso de álcool e potenciais fatores associados foram coletados por meio de entrevista face a face. Análise de regressão de Poisson com variância robusta foi utilizada para verificar os fatores associados ao desfecho analisado. O consumo de álcool na amostra estudada foi de 17,7% (IC 95%: IC 95%: 14,1-22,0%). Antecedentes de diabetes pré-gestacional ou gestacional, ideação suicida e uso de tabaco nos últimos 30 dias foi associado ao uso de álcool durante a gestação (p < 0,05). O estudo apontou alta prevalência de uso de álcool na gestação atual e a sua associação com importantes fatores. Ações como rastreio para o álcool e aconselhamentos sobre os problemas associados ao uso dessa substância principalmente no pré-natal podem contribuir para redução efetiva ou anulação do seu uso em gestantes e agravos materno-fetais relacionados.