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Nanoparticles (NPs) have wide potential applications in the biomedical field. To promote targeted and controlled delivery of encapsulated drugs, it is fundamentally important to understand the factors regulating NP uptake by different cells. Thus, the goal of the present study is to assess the internalization rates of different NPs under normal and proinflammatory states in primary human articular chondrocytes (hACs), human umbilical vein endothelial cells (EA), and human monocytes (THP-1). Here, we compared chitosan–hyaluronic acid (Ch-HA) polymeric NPs, methoxypolyethylene glycol amine–glutathione–palmitic acid (mPEG-GSHn-PA) micelles, and cholesterol/L-α-phosphatidylcholine/DSPE-PEG-Mal (Chol/EPC/DSPE-PEG-Mal) unilamellar liposomes (LUVs). Our results reveal the importance of surface charge and chemistry in determining the levels of NP internalization. Under normal conditions, the cellular uptake was ≈30% for Ch-HA NPs and ≈100% for mPEG-GSHn-PA micelles and Chol/EPC/DSPE-PEG-Mal LUVs. A proinflammatory cell state promoted a higher uptake of the Ch-HA NPs by EA cells (93% after 24 h). Since the therapeutic efficacy of the NP-loaded cargo is dependent on trafficking routes after cellular internalization, we tested their internalization pathways. Accordingly, caveolae-mediated endocytosis or energy-independent non-endocytic pathways, which circumvent lysosomal degradation, were accomplished in hACs and EA by LUVs and in M1 polarized macrophages by micelles. The present outcomes highlight the importance of considering cellular uptake and internalization pathways by the target cell when designing functional NPs for therapeutic applications., The authors acknowledge the financial support from the FCT/ MCTES (Portuguese Foundation for Science and Technology/ Ministry of Science, Technology and Higher Education) and the FSE/POCH (European Social Fund through the Operational Program of Human Capital), and for the PhD scholarship (PD/ BD/11384/2015) to A. C. Lima (PD/59/2013). The authors also acknowledge the FCT for the projects PTDC/CTM-BIO/ 4388/2014-SPARTAN, PTDC/BTM-SAL/28882/2017- Cells4_IDs, and PTDC/BTM-ORG/28070/2017- 2MATCH, the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000023-FROnTHERA and NORTE-01-0145-FEDER-000021), and the REMIX Project funded by the European Union’s Horizon 2020 Research and Innovation Programme, under the Maria Sklodowska Curie Grant for the REMIX Project (Agreement no. 778078).

Background In Alzheimer's Disease there is an impairment of the cholinergic system, causing loss of neurons, impairment of intellectual abilities. In this context, the mesenchymal stem cells (MSC) and its applications in cell therapies become target of the research, which may contribute to the treatment of neurodegenerative diseases. Through the differentiation potential of MSCs, prospecting functional repair of the injured tissue from cholinergic neuronal cells could be a potential treatment. The aims was to evaluate the possibility of differentiation of MSCs from the human umbilical cord in nestin-positive neural precursor cells (NPCN+) through the NFBX into cholinergic ‘like’ cells. Methods The isolation of hMSCs from Wharton's jelly (WJ) was by the explant and mononuclear cells by density gradient. hMSCs were plating in natural matrix as NFBX for neurospheres production. Neural precursor cells were subjected to standard cholinergic differentiation protocol. Dissociated neurospheres, neural precursor cells and cholinergic-like cells were characterized by immunocytochemistry. The RT-PCR was done. Results hMSCs were CD73+, CD90+, CD105+, CD34- and CD45- and demonstrated the trilineage differentiation. Neurospheres and their isolated cells were nestin positive, and also expressed NESTIN, MAP2, ßIII-TUBULIN, GFAPgenes. Neural precursor cells that were differentiated in cholinergic-like cells expressed ßIII-TUBULIN protein and choline acetyltransferase enzyme. Conclusions hMSCs on the natural matrix were capable of differentiating hMSC into neurospheres, obtaining neural precursor cells without growth factors or gene transfection before cholinergic differentiation.

To assess alcohol use and perceived change in alcohol consumption (before and during the pandemic) in Brazilians during the COVID-19 pandemic, its correlates, and association with depressive, anxiety and co-occurring depressive and anxiety symptoms (DA).This is a cross-sectional study comprising 992 individuals in self-isolation. A self-reported questionnaire was used to assess whether participants were drinking during self-isolation and whether they changed their drinking behavior (drinking less, more, or no change) from before to during the pandemic. DA symptoms were assessed using the Beck Depression and Anxiety Inventories (BDI and BAI).A total of 68.5% of participants reported alcohol consumption during the pandemic, and 22.7% of these reported increased alcohol use. Smoking was positively associated with alcohol consumption during the pandemic. Alcohol consumption was associated with anxiety (OR=1.40, 95% CI 1.06 - 1.85, p0.01) and DA (OR=1.38, 95% CI 1.02 - 1.87, p=0.033) symptoms.Drinking during self-isolation was prevalent and associated with risk factors for alcohol use disorders. The long-term effects of high drinking rates and increased consumption should be proactively monitored and assessed.

Different SA concentrations (10, 100 and 1000 μM) were applied in young olive trees (Olea europaea L.) subjected to drought and rewatering. Plants treated with 10 μM exhibited a close behavior to SA-starved plants. Although both 100 and 1000 μM improved the balance between ROS production and scavenging, 100 μM was more efficient. During drought, 100 μM improved ROS detoxification and scavenging by the maintenance or overaccumulation of soluble proteins. During recovery, soluble proteins return to well-watered values and increased the investment in non-enzymatic antioxidants. 100 μM was also the most effective in plant ionome regulation, improving macro and micronutrients uptake, namely P, Fe, Mn and Zn, and changing mineral allocation patterns. Therefore, 100 μM also countered the drought-induced decline in total plant biomass accumulation. The application of suitable SA concentrations is an efficient tool to improve cellular homeostasis and growth of plants subjected to recurrent drought episodes.

Made available in DSpace on 2019-10-04T12:30:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazilian funding agency FAPEG (Fundacao de Amparo a Pesquisa do Estado de Goias) The inhibition of arginase from Leishmania spp. is considered a promising approach to the leishmaniasis treatment. In this study, the potential of a fucogalactan isolated from the medicinal mushroom Agrocybe aegerita was evaluated against arginase (ARG) from Leishmania amazonensis. The polysaccharide was obtained via aqueous extraction, and purified by freeze thawing and precipitation with Fehling solution. Its chemical structure was established by monosaccharide composition, methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The data indicated that it is a fucogalactan (FG-Aa; M-w = 13.8 kDa), having a (1 -> 6)-linked alpha-D-Galp main-chain partially substituted in O-2 by non-reducing end-units of alpha-L-Fucp. FG-Aa showed significant inhibitory activity on ARG with IC50 potency of 5.82 +/- 0.57 mu M. The mechanism of ARG inhibition by the heterogalactan was the competitive type, with K(i)of 1.54 +/- 0.15 mu M. This is the first report of an inhibitory activity of arginase from L. amazonensis by biopolymers, which encourages us to investigate further polysaccharides as a new class of ARG inhibitors. Univ Fed Goias, Unidade Acad Especial Quim, BR-75704020 Catalao, Go, Brazil Univ Fed Goias, Inst Quim, Lab Ressonancia Magnet Nucl, Campus Samambaia, BR-74001970 Goiania, Go, Brazil Univ Estadual Paulista, Fac Ciencias Agron, Dept Engn Rural, BR-18610307 Botucatu, SP, Brazil Univ Fed Sao Carlos, Dept Quim, Rodovia Washington Luis,Km 235, BR-13565905 Sao Carlos, SP, Brazil Univ Estadual Paulista, Fac Ciencias & Tecnol, Dept Quim & Bioquim, BR-19060900 Presidente Prudente, SP, Brazil Univ Estadual Paulista, Fac Ciencias Agron, Dept Engn Rural, BR-18610307 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Ciencias & Tecnol, Dept Quim & Bioquim, BR-19060900 Presidente Prudente, SP, Brazil

Ultraviolet B radiation represents 10% of the total UV radiation that reaches the Earth's surface, being the primary responsible for the biological effects related to skin cancer and photoaging. Ilex Paraguariensis A. St. Hil., known as Yerba mate (YM), is a native tree of South America whose polyphenols in its leaves are described to exhibit photochemoprotective effect and are employed in the treatment of cancer. Additionally, the polyphenols are used to prevent lipid peroxidation and reduce the UV-induced damage, which ultimately decreases the oxidative stress. Thus, the present study aimed to characterize a new YM extract, evaluate the extract cytotoxicity and develop a formulation containing YM extract to prevent UVB-induced damage in mice skin. The YM extract showed high levels of polyphenols, flavonoids, and tannins and exhibited excellent antioxidant activity. Its main components were suggested as chlorogenic acid (1.92%) and caffeic acid (0.41%). Besides, YM extract did not exhibit cytotoxicity in fibroblasts and decreased the activity of myeloperoxidase and metalloproteinase-2 after acute UVB exposure. As a result, the formulation containing the YM extract showed a potential photochemoprotective.

Multiple sclerosis is a neurodegenerative disease causing disability in young adults. Alterations in metabolism and lipid profile have been associated with this disease. Several studies have reported changes in the metabolism of arachidonic acid and the profile of fatty acids, ceramides, phospholipids and lipid peroxidation products. Nevertheless, the understanding of the modulation of circulating lipids at the molecular level in multiple sclerosis remains unclear. In the present study, we sought to assess the existence of a distinctive lipid signature of multiple sclerosis using an untargeted lipidomics approach. It also aimed to assess the differences in lipid profile between disease status (relapse and remission). For this, we used hydrophilic interaction liquid chromatography coupled with mass spectrometry for phospholipidomic profiling of serum samples from patients with multiple sclerosis. Our results demonstrated that multiple sclerosis has a phospholipidomic signature different from that of healthy controls, especially the PE, PC, LPE, ether-linked PE and ether-linked PC species. Plasmalogen PC and PE species, which are natural endogenous antioxidants, as well as PC and PE polyunsaturated fatty acid esterified species showed significantly lower levels in patients with multiple sclerosis and patients in both remission and relapse of multiple sclerosis. Our results show for the first time that the serum phospholipidome of multiple sclerosis is significantly different from that of healthy controls and that few phospholipids, with the lowest p-value, such as PC(34:3), PC(36:6), PE(40:10) and PC(38:1) may be suitable as biomarkers for clinical applications in multiple sclerosis. published

Rheumatoid arthritis (RA) is a highly debilitating chronic inflammatory autoimmune disease most prevalent in women. The true etiology of this disease is complex, multifactorial, and is yet to be completely elucidated. However, oxidative stress and lipid peroxidation are associated with the development and pathogenesis of RA. In this case, oxidative damage biomarkers have been found to be significantly higher in RA patients, associated with the oxidation of biomolecules and the stimulation of inflammatory responses. Lipid peroxidation is one of the major consequences of oxidative stress, with the formation of deleterious lipid hydroperoxides and electrophilic reactive lipid species. Additionally, changes in the lipoprotein profile seem to be common in RA, contributing to cardiovascular diseases and a chronic inflammatory environment. Nevertheless, changes in the lipid profile at a molecular level in RA are still poorly understood. Therefore, the goal of this review was to gather all the information regarding lipid alterations in RA analyzed by mass spectrometry. Studies on the variation of lipid profile in RA using lipidomics showed that fatty acid and phospholipid metabolisms, especially in phosphatidylcholine and phosphatidylethanolamine, are affected in this disease. These promising results could lead to the discovery of new diagnostic lipid biomarkers for early diagnosis of RA and targets for personalized medicine.

The enormous health and economic challenges precipitated by the 2019 coronavirus disease (COVID-19) pandemic are comparable or even greater than those associated with previous historical world crises. Alcohol use, especially drinking to cope with stress, is a concern, as an increase in its sales has been reported in some countries during the quarantine. This study aims to provide a better understanding of what to expect in terms of alcohol consumption, risk factors for excessive use, and its potential consequences during this pandemic based on previous experiences. We investigated how traumatic events related to alcohol consumption. Studies on mass traumatic events (i.e., terrorism as 9/11), epidemic outbreaks (i.e., severe acute respiratory syndrome [SARS] in 2003), economic crises (such as 2008's Great Recession), and COVID-19 were selected. The main keywords used to select the studies were alcohol use, drinking patterns, alcohol use disorders, and alcohol-related consequences. Previous studies reported increases in alcohol use associated with those events mediated, at least partially, by anxiety and depressive symptoms, and posttraumatic stress disorder (PTSD). Being male, young, and single also seems to be associated with a higher vulnerability to develop risky drinking behavior after those tragic events. The discussion of previous risk and protective factors can contribute to elaborate more specific public health policies to mitigate the impact of the current pandemic on people's mental health, especially alcohol-related problems.

Vermeersiekte or &ldquo, vomiting disease&rdquo, is an economically important disease of ruminants following ingestion of Geigeria (G.) species in South Africa. Sheep are more susceptible, and poisoning is characterized by stiffness, regurgitation, bloat, paresis, and paralysis. Various sesquiterpene lactones have been implicated as the cause of poisoning. The in vitro cytotoxicity of two sesquiterpene lactones, namely, ivalin (purified from Geigeria aspera) and parthenolide (a commercially available sesquiterpene lactone), were compared using mouse skeletal myoblast (C2C12) and rat embryonic cardiac myocyte (H9c2) cell lines, representing the oesophageal, skeletal and cardiac muscles, which are affected in sheep. For 24, 48, and 72 h, both cell lines were exposed. A colorimetric viability assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was used to assess cytotoxicity. A concentration-dependent cytotoxic response was observed in both cell lines, however, the C2C12 cells were more sensitive, with the half-maximal effective concentrations (EC50s) ranging between 2.7 and 3.3 &micro, M. In addition, the effect that ivalin and parthenolide has on desmin, an important cytoskeletal intermediate filament in myocytes, was evaluated using the C2C12 myoblasts. Disorganization and aggregation of desmin were caused by both sesquiterpene lactones, which could clarify some of the ultrastructural lesions described in vermeersiekte.

Water is the most widely limiting factor for plants distribution, survival and agricultural productivity, their responses to drought and recovery being critical for their success and productivity. Olea europaea L. is a well-adapted species to cyclic drought events, still at considerable expense of carbon reserves and CO2 supply. To study the role of abscisic acid (ABA) as a promoter of drought adaptability, young potted olive trees subjected to three drought-recovery cycles were pre-treated with ABA. The results demonstrated that ABA pre-treatment allowed the delay of the drought effects on stomatal conductance (gs) and net photosynthesis (An), and under severe drought, permitted the reduction of the non-stomatal limitations to An and the relative water content decline, the accumulation of compatible solutes and avoid the decline of photosynthetic pigments, soluble proteins and total thiols concentrations and the accumulation of ROS. Upon rewatering, ABA-sprayed plants showed an early recovery of An. The plant ionome was also changed by the addition of ABA, with special influence on root K, N and B concentrations. The improved physiological and biochemical functions of the ABA-treated plants attenuated the drought-induced decline in biomass accumulation and potentiated root growth and whole-plant water use efficiency after successive drought-rewatering cycles. These changes are likely to be of real adaptive significance, with important implications for olive tree growth and productivity.

Cracking of sweet cherry (Prunus avium L.) fruits is caused by rain events close to harvest. This problem has occurred in most cherry growing regions with significant economic losses. Several orchard management practices have been applied to reduce the severity of this disorder, like the foliar application of minerals or growth regulators. In the present study, we hypothesized that preharvest spray treatments improve the physiological performance of sweet cherry trees and could also mitigate environmental stressful conditions. Effects of repeated foliar spraying of calcium (Ca), gibberellic acid (GA3), abscisic acid (ABA), salicylic acid (SA), glycine betaine (GB), and the biostimulant Ascophyllum nodosum (AN) on the physiological and biochemical performance of &lsquo, Skeena&rsquo, sweet cherry trees during two consecutive years (without Ca in 2015 and in 2016 with addition of Ca) were studied. Results showed that in general spray treatments improved the physiological performance and water status of the trees. AN and ABA sprays were demonstrated to be the best compounds for increasing yield and reducing cherry cracking as well as improving photosynthetic performance and leaf metabolites content. In conclusion, AN and ABA might be promising tools in the fruit production system.

Environmental health is increasingly compromised by persistent toxic substances, which may have serious implications in food safety and, thus, in human health. Polybrominated diphenyl ethers (PBDEs) are anthropogenic contaminants with endocrine disruption abilities and are commonly found in seafood, the main route of human exposure. Growing evidence points out that the human gut microbiota interacts with xenobiotics, which may lead to impairment of host homeostasis if functions of microbiota become compromised. The aim of this study was to ascertain if the physiological balance of human gut microbiome is affected by the presence and degree of exposure to PBDEs. Fermentation was performed in a batch closed-system using an inoculum made from fresh human stool. The volatolomic profile was analysed by solid-phase microextraction coupled to gas chromatography-mass spectrometry. Mesophilic, Gram-negative bacteria and coliforms were quantified by classic plating methods. Changes in the gut microbiome were evaluated after DNA extraction followed by deep sequencing of the 16S rDNA region. The exposure to PBDEs resulted in an imbalance in sulfur, short-chain fatty acids and aromatic organic compounds, changing the microbial volatolome in a dose- and time-dependent manner. Slight deviations in the microbial structure of human gut occurred in the presence of PBDEs, especially for high doses of exposure. For the first time, the impact of PBDEs on the microbial homeostasis of human gut microbiota was taken into consideration, revealing noteworthy modifications with serious health implications even at oral exposure doses considered as safe by worldwide regulatory entities. The research leading to these results received financial support from FEDER (Programa Operacional Competitividade e Internacionalização - COMPETE 2020), from PIDDAC through FCT/MCTES project POCI-01-0145-FEDER-028708 and from UID/QUI/50006/2019 with funding from FCT/MCTES. Rebeca Cruz thanks to FCT for the PhD grant SFRH/BD/101945/2014. António Marques thanks to FCT IF program. Miguel Faria acknowledges FCT the researcher contract. Sara C. Cunha also acknowledges FCT for the IF/01616/2015 contract.

The predicted accentuation of drought events highlights the importance of optimize plants capacity to tolerate drought, but also the capacity to recovery from it, especially in species, as olive tree (Olea europaea L.), that grows in particularly susceptible regions. Three different concentrations (10, 100 and 1000 μM) of salicylic acid (SA), a stress signaling phytohormone, was sprayed on 3-year-old potted olive trees subjected to three successive drought and rewatering events. Trees responses to SA application are concentration dependent, being 100 μM the most effective concentration to improve drought tolerance and recovery capacity. During drought events, this effectiveness was achieved by osmolytes accumulation, leaf water status maintenance, reduced photosynthetic systems drought-associated damages, and by optimizing shoot/root ratio. The better plant fitness during drought allowed a fast recovery of the physiological functions upon rewatering and reduced the necessity to invest in extra repair damages, allowing the regrowth. The intense abscisic acid (ABA) signal close to upper epidermis in stressed controls suggests a “memory” of the worst water status displayed by those plants. SA attenuated the limitation of total biomass accumulation imposed by drought, mainly in root system, increased water use efficiency and lead to a higher intense signal of indoleacetic acid (IAA) in leaves during recovery period. In summary, in a suitable concentration, SA demonstrate to be a promising tool to increase drought adaptability of olive trees.

Cyanobacteria are known for their extensive and highly visible blooms in rivers or dams in Africa. One of the most important cyanobacteria is Microcystis aeruginosa which can synthesise various microcystins that may affect the health of humans and animals. Accurate and efficient detection of microcystins in water is thus important for public and veterinary health. Two enzyme-linked immunosorbent assays (ELISA), a commercially-available ELISA kit (Abraxis) and a newly-developed Norwegian ELISA (putatively cheaper and more robust) were used to detect microcystins in fresh water in South Africa. Water samples were collected monthly at two sites, the Hartbeespoort Dam and a crocodile breeding dam. Extremely high microcystin concentrations (exceeding 360 μg L−1) were detected in the Hartbeespoort Dam during January 2015, whereas the microcystin concentrations in the crocodile breeding dam peaked during March–April 2015. Both ELISAs were positively correlated when analysing water samples ‘as is’ and following resin adsorption and methanol extraction. However, following resin adsorption and methanol extraction of the water samples, the correlation between the two assays was much stronger. These results suggests that the two ELISAs provide comparable results. If the Norwegian-developed ELISA can be packaged and made available as a user-friendly kit, it could be used successfully in surveillance programmes to monitor microcystin concentrations in fresh water bodies in Africa.

Knowledge about short-term climate change adaptation strategies for Mediterranean vineyards is needed in order to improve grapevine physiology and yield-quality attributes. We investigated effects of kaolin-particle film suspension on water relations, photosynthesis and oxidative stress of field-grown grapevines in the Douro region (northern Portugal) in 2012 and 2013. Kaolin suspension decreased leaf temperature by 18% and increased leaf water potential (up to 40.7% in 2013). Maximum photochemical quantum efficiency of PSII was higher and the minimal chlorophyll fluorescence was lower in the plants sprayed by kaolin. Two months after application, net photosynthesis and stomatal conductance at midday increased by 58.7 and 28.4%, respectively, in treated plants. In the same period, kaolin treatment increased photochemical reflectance, photosynthetic pigments, soluble proteins, soluble sugars, and starch concentrations, while decreased total phenols and thiobarbituric acid-reactive substances. Kaolin application can be an operational tool to alleviate summer stresses, which ameliorates grapevine physiology and consequently leads to a higher yield.

Olive pomace, an olive oil processing byproduct, can be upcycled and meet the current demand for natural and sustainable food ingredients. In this work, a patented process was used to obtain a functional ingredient from different olive pomaces. The nutritional, chemical and antioxidant profiles, as well as the antimicrobial activity against S. aureus, E. coli and C. albicans, were investigated for the first time. The amount of phenolics ranged between 3.1 and 3.8 g gallic acid eq./100 g in all samples and flavonoids between 2.0 and 3.2 g catechin eq/100 g. No significant differences were found regarding the antioxidant activity. The total fat varied between 5 and 11%, α-tocopherol being the major vitamer and oleic acid the main fatty acid. The protein and ash contents were 1–4% and 10–17%, respectively. The functional ingredient with a higher hydroxytyrosol content (220 mg/100 g) also presented the best minimal inhibitory concentration against the tested bacteria. No activity against C. albicans was verified. This new functional ingredient presents the potential to be used as a natural preservative or as a nutritional profile enhancer. Moreover, it can be an advantageous ingredient in food products, since it comprises specific lipid and hydrophilic bioactive compounds usually not present in other plant extracts.

BackgroundObesity is often associated with iron deficiency in children and adolescents. We aimed to study the effect of an 8-month physical exercise (PE) intervention on hepcidin and other markers of inflammation and on iron status in overweight/obese children and adolescents.MethodsSeventy-three overweight/obese children and adolescents participated in the 8-month-long longitudinal study. They were divided into two groups according to their participation in an after-school PE program: the PE group (n=44) and the control group (n=29). Hepcidin, interleukin (IL)-6, C-reactive protein (CRP), iron, ferritin, transferrin, and soluble transferrin receptor (sTfR) were evaluated.ResultsAt baseline, IL-6 correlated positively with hepcidin and negatively with iron and transferrin saturation, suggesting that increasing adiposity associates with increasing IL-6 and hepcidin synthesis, reducing iron availability. After 8 months, the PE group showed a decrease in BMI z-score (P=0.003), body fat mass (P=0.012), CRP (P=0.002), IL-6 (P=0.048), ferritin (P=0.013), hepcidin (P=0.040), and sTfR (P=0.010), and an increase in iron concentration (P=0.002). Moreover, the PE group, when compared with the control group, showed lower weight (P=0.026), BMI (P=0.040), waist circumference (P=0.010), and waist-to-height ratio (P=0.046).ConclusionWe showed that an 8-month-long intervention at school allowed a reduction in BMI z-score and an improvement in inflammation, reducing hepcidin levels and the disturbances in iron status.

Objective: The aim of this study is to evaluate the adhesion of an endodontic sealer (AHPlus in root canals after the use of different protocols of 17% EDTA and the use of intracanal medication (ICM) based on calcium hydroxide in aqueous vehicle. Material and Methods: For this, 72 single-rooted human teeth were instrumented up to #50 and divided into six groups (n = 12). Group 1: EDTA for 3 min; Group 2: 3 mL of EDTA + 3 min of EDTA; Group 3: 3 mL of EDTA + 3 min of EDTA + 30 seconds of ultrasonic agitation; Group 4: EDTA for 3 min + ICM; Group 5: 3 mL of EDTA + 3 min of EDTA + ICM; Group 6: 3 mL of EDTA + 3 min of EDTA + 30 seconds of ultrasonic agitation + ICM. The root canals were filled with endodontic sealer after each protocol and after 7 days they were prepared to the push-out test. The data were analyzed using ANOVA two factors (p < 0.05). Results: It was observed no statistically difference in bond strength in the 3 different 17% EDTA protocols. However, the use of ICM increased significantly the resistance adhesion. Conclusion: Intracanal medication based on calcium hydroxide improved the bond strength of AHPlus to dentin walls, regardless of the EDTA protocol.KeywordsCalcium hydroxide; EDTA; Dentin.

Ingestion of large quantities of Geigeria species by sheep causes “vermeersiekte”, an economically important poisoning in southern Africa. The toxic principles are several sesquiterpene lactones, such as vermeerin, geigerin and ivalin. These sesquitepene lactones are myotoxic and the disease is characterized by microscopic and ultrastructural lesions in skeletal and cardiac muscle. Murine myoblast cells (C2C12) were exposed to 2.0, 2.5 and 5.0 mM geigerin for 24, 48 and 72 h to evaluate its effect on cytoskeletal proteins and filaments using immunocytochemistry and immunofluorescence staining. A concentration-dependent cytotoxic response was observed in desmin-expressing murine myoblasts under the light microscope, evidenced by disorganization and dot-like perinuclear aggregation of desmin filaments in the cells. β-Tubulin, other desmin-associated proteins (αB-crystallin and synemin) as well as the microfilament F-actin were unaffected. The disorganization and aggregation of desmin following exposure to increasing geigerin concentrations is significant and can explain some of the striated muscle lesions observed in “vermeersiekte”.

Autoimmune diseases (AID) are a heterogeneous group of disorders that have in common a chronic inflammation and dysregulation of the immune system. Systemic lupus erythematosus (SLE) is one of the most frequent systemic autoimmune diseases characterized by autoimmune phenomena in multiple organs. The tests used for evolution and prognosis assessment are either non-specific or non-sensitive, impairing an adequate therapeutics. To face this drawback, lipidomics is being used to provide more knowledge and insights regarding autoimmune disorders. Through lipidomic approaches using MS, it is possible to identify and quantify the level of lipid molecular species in the biological system and this could be useful to identify biomarkers and to better understand the pathophysiology of autoimmune diseases. There are some evidence that lipids and oxidized lipids can play a key role in AID pathogenesis. Although this field has been scarcely explored, there are some studies that reported variations on the lipid profile at a molecular level using lipidomic approaches based on MS in SLE. The results gathered herein showed changes mainly in the level of phospholipids, with decrease of some plasmenyl lipids, fatty acids, with reduction of PUFA, and sphingolipids, with changes in fatty acyl chain composition. These changes may be the result of lipids` modifications due to oxidation and increase of ROS. Some alterations can be associated with changes in membrane of lymphocytes and with the deregulation of the immune system. Thus, exploring the knowledge from modern lipidomic approaches in the study of the role of lipids and oxidized lipids, in oxidative stress and in inflammatory diseases, could contribute for the identification of new lipid biomarkers. Lipid biomarkers are promising tools to prognosis and treatment monitoring, tailored for the best therapeutic response and highest safety to ensure better patient care and to be used for personalized medicine. published

Made available in DSpace on 2022-04-29T08:28:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Acetylcholinesterase (AChE) is responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh) leading to acetate and choline (Ch) release. The inhibition of AChE produces a generalized synaptic collapse that can lead to insect death. Herein we report for the first time the isolation of two AChEs from Atta sexdens which were purified by sulphate ammonium precipitation followed by ion exchange chromatography. AsAChE-A and AsAChE-B enzymes have optimum pH of 9.5 and 9.0 and higher activities in 30/50°C and 20°C, respectively, using acetylthiocholine (ATCh) as substrate. Immobilized capillary enzyme reactors (ICERs) were obtained for both enzymes (AsAChE-A-ICER and AsAChE-B-ICER) and their activities were measured by LC-MS/MS through hydrolysis product quantification of the natural substrate ACh. The comparison of activities by LC-MS/MS of both AChEs using ACh as substrate showed that AsAChE-B (free or immobilized) had the highest affinity. The inverse result was observed when the colorimetric assay (Elman method) was used for ATCh as substrate. Moreover, by mass spectrometry and phylogenetic studies, AsAChE-A and AsAChE-B were classified as belonging to AChE-2 and AChE-1 classes, respectively. Federal University of São Carlos Department of Chemistry São Paulo University Instituto de Ciências Biomédicas (ICB) São Paulo State University Center for the Study of Social Insects São Paulo State University Center for the Study of Social Insects

Phosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of divalent metal ion, and dependent of adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP). PEPCK is an important enzyme in the metabolism of some organisms, such as Trypanosoma cruzi, being suggested as a potential drug target to treat Chagas’ disease. Its catalytic activity is, classically, measured by coupled assays. Herein, a direct assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying PEP, in co-elution with OAA by the differentiation obtained by the mass spectra, is reported. The developed assay was used throughout the purification protocol in order to measure the activity of PEPCK of T. cruzi, which was expressed in Escherichia coli. The purified enzyme was kinetically characterized by the developed method with Michaelis-Menten constant (KMapp) values of 96 ± 4 and 275 ± 18 µmol L-1 to OAA and ATP as substrates, respectively. The developed assay was also used for ligand screening and proved to be able to identify very low inhibitions for small molecules (50 µmol L-1).

The aim of this study was to describe energy partitioning in dairy crossbreed bulls fed tropical forage-based diets supplemented with different additives. Twenty F1 crossbred bulls (Holstein x Gyr) with initial and final live weight (LW) averages of 190 ± 17 and 275 ± 20 kg were fed sorghum (Sorghum bicolour) and Tanzania grass (Panicum maximum cv. Tanzania) silage (70:30 DM basis) with supplemented concentrate at a forage to concentrate ratio of 50:50. The bulls were allocated to four treatment: control groups (without additives), monensin [22 mg/kg monensin dry matter (DM)] (M), virginiamycin (30 mg/kg virginiamycin DM) (V), and combination (22 mg/kg DM of monensin and 30 mg/kg DM of virginiamycin) (MV), in a completely randomised design. The intake of gross energy (GE, MJ/d), digestible energy (DE, MJ/d), metabolizable energy (ME, MJ/d), as well as energy losses in the form of faeces, urine, methane, heat production (HE), and retained energy (RE) were measured. Faecal output was measured in apparent digestibility trial. Right after the apparent digestibility trial, urine samples were collected in order to estimate the daily urinary production of the animals. Heat and methane production were measured in an open circuit respirometry chamber. The intake of GE, DE, and ME of the animals receiving monensin and virginiamycin alone or in combination (MV) showed no differences (P>0.05) from the control treatment. However, the MV treatment reduced (P0.05) the utilization efficiency of ME for weight gain, RE and net gain energy. This study showed that for cattle fed tropical forages, the combination of virginiamycin and monensin as feed additives affected their energy metabolism by a reduction in the energy lost as methane.

Intoxication by Moraea pallida Bak. (yellow tulp) in livestock is of great importance in South Africa, ranking top among all plant-induced cardiac glycoside toxicosis. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. Treatment of poisoning is challenging and affected livestock often succumbs due to the stress of handling. Manipulating animals to resist poisoning is a potential management strategy. The goal of this study was to explore the potential to develop a vaccine against epoxyscillirosidine by raising antibodies against epoxyscillirosidine in sheep and to assess the neutralization ability of the antibodies in vitro. Epoxyscillirosidine was successfully conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA) rendering them immunogenic. The sheep, vaccinated with epoxyscillirosidine-KLH conjugate (n = 4) and KLH (n = 2) with Montanide, developed antibodies as determined with an indirect enzyme linked immunosorbent assay (ELISA). Total immunoglobulins from sera of vaccinated and control sheep that were purified and concentrated using ammonium sulphate precipitation were 11,940 and 7850 μg, respectively. The in vitro neutralization assay using the methyl blue tetrazolium bromide (MTT) cell viability assay indicated no significant difference (p > 0.05) between anti-epoxyscillirosidine-KLH and KLH antibodies. Rather, the antibodies seemed to enhance the cytotoxicity of epoxyscillirosidine in H9c2 cells. Thus, it is necessary to develop improved vaccination methods to generate antibodies capable of neutralizing the functional group responsible for epoxyscillirosidine toxicity.

The purpose of this study was to determine the energy partition of pregnant F1 Holstein x Gyr with average initial body weight (BW) of 515.6 kg and Gyr cows with average initial BW of 435.1 kg at 180, 210 and 240 days of gestation, obtained using respirometry. Twelve animals in two groups (six per genetic group) received a restricted diet equivalent to 1.3 times the net energy for maintenance (NEm). The proportion of gross energy intake (GEI) lost as feces did not differ between the evaluated breeds and corresponded to 28.65% on average. The daily methane production (L/d) was greater for (P0.05). The daily loss of energy as urine (mean of 1.42 Mcal/d) did not differ (P>0.05) between groups and ranged from 3.87 to 5.35% of the GEI. The metabolizable energy intake (MEI) of F1 HxG animals was greater (P < 0.05) at all gestational stages compared to Gyr cows when expressed in Mcal/d. However, when expressed in kcal/kg of metabolic BW (BW0,75), the F1 HxG cows had MEI 11% greater (P0,75. Gyr cows showed no change in MEI over time (P>0.05), with a mean of 146.66 kcal/kg BW0. 75. The ME used by the conceptus was calculated by deducting the metabolizable energy for maintenance (MEm) from the MEI, which was obtained in a previous study using the same cows prior to becoming pregnant. The values of NEm obtained in the previous study with similar non-pregnant cows were 92.02 kcal/kg BW0.75 for F1 HxG, and 76.83 kcal/kg BW0.75 for Gyr (P = 0.06). The average ME for pregnancy (MEp) was 5.33 Mcal/d for F1 HxG and 4.46 Mcal/d for Gyr. The metabolizability ratio, averaging 0.60, was similar among the evaluated groups (P>0.05). The ME / Digestible Energy (DE) ratio differed between groups and periods evaluated (Pp) was 2.76 Mcal/d and did not differ between groups and gestational stages (P>0.05).

In T. cruzi, a causative agent of Chagas disease, phosphoenolpyruvate carboxykinase (TcPEPCK) is associated with carbohydrate catabolism. Due to its importance in the metabolism of the parasite, it has become a promising target for the development of new drugs against Chagas disease. Aiming to investigate different approaches for ligands screening, TcPEPCK was immobilized on amine-terminated magnetic beads (TcPEPCK-MB) and kinetically characterized by liquid chromatography tandem mass spectrometry activity assay with a KMapp value of 10 ± 1 μM to oxaloacetate as substrate. Natural products library affords highly diverse molecular frameworks through their secondary metabolites, herein a ligand fishing TcPEPCK-MB assay is described for prospecting ligands in four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora (Vochysiaceae), Diospyros burchellii (Ebenaceae), Anadenanthera falcata (Fabaceae) and Byrsonima coccolobifolia (Malpighiaceae). The chemical characterization of eleven identified ligands was carried out by liquid chromatography tandem high-resolution mass spectrometry experiments. Senecic acid, syneilesinolide A, phytosphingosine and vanillic acid 4-glucopyranoside are herein reported for the first time for Q. grandiflora, D. burchellii, A. falcata, respectively. In addition, the specificity of the assay was observed since only catechin was fished out from the ethanolic extract of B. coccolobifolia leaves, despite the presence of epicatechin epimer.

In this paper, we present the optimization of porous anodic alumina membranes for ultrafiltration prepared by anodically oxidized aluminum foils. The membranes were characterized by field-emission scanning electron microscopy to measure the pore diameter and the membrane thicknesses. The liquid fluxes were estimated through gas permeability measurements using Darcy's and Forchheimerś equations. A 2(3) factorial design we used to optimize the membrane properties: pore diameter, membrane thickness, and liquid flux using as control variables the applied current density, solution composition and concentration. It was observed that the most import variables to control the pore diameter were current density and electrolyte composition. After the anodization both, metallic aluminum substrate and the barrier layer of alumina were removed using adequate solutions to obtain the free standing membrane. Then, Escherichia coli a common bacterial contamination of drinking water was removed using these PAA membranes with 100% of efficiency to obtain bacteria-free water.

Extreme conditions, such as drought, high temperature, and solar irradiance intensity, are major factors limiting growth and productivity of grapevines. In a field experiment, kaolin particle film application on grapevine leaves was examined during two different summer conditions (in 2012 and 2013) with the aim to evaluate benefits of this practice against stressful conditions hindering photochemical processes. We used chlorophyll a fluorescence to investigate attached leaves. Two months after the application, during the hottest midday, the kaolin-treated plants showed by the JIP test significantly higher quantum yield of PSII photochemistry, flux ratios, maximum trapped excitation flux of PSI, absorption flux, electron transport flux, maximum trapped energy flux per cross section, and performance index than plants under control conditions in the warmer year. On the contrary, the treated plants showed a lower initial slope of relative variable fluorescence and a decrease in the absorption and electron transport per cross section. The JIP test showed higher efficiency of PSII in the plants treated with kaolin mainly in 2013 (higher temperature and drought). Our results supported the hypothesis that the accumulation of active PSII reaction centres was associated with decreased susceptibility to photoinhibition in the kaolin-treated plants and with more efficient photochemical quenching. Grapevines in the Douro Region seems to profit from the kaolin application.

The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60°C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of KM, Vmax and kcat were 0.65 mg·mL-1, 1800 μmol·min-1·mg-1 and 35.97 s-1, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0; otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.

Objective: To evaluate the effects of 1% and 2.5% sodium hypochlorite (NaOCl) solutions at different periods of time on the composition of gutta-percha cones using Energy Dispersive Spectroscopy (EDS).Material and Methods: 110 master gutta-percha points were divided into 3 control groups and 8 experimental groups. Cones were immersed in 1% and 2.5% NaOCl for 30 minutes, 6, 12, 24 hours. Then, composition of the samples was analyzed by EDS. Statistical analysis was performed using the ANOVA and Tukey tests (p

Multiple sclerosis is a chronic inflammatory and neurodegenerative disease of the central nervous system, and it is one of the most common neurological cause of disability in young adults. It is known that several factors contribute to increase the risk of development and pathogenesis of multiple sclerosis, nonetheless, but the true etiology of this pathology remains unknown. Similar to other inflammatory diseases, oxidative stress and lipid peroxidation are also associated to multiple sclerosis. Alterations in the lipid profile seem to be a hallmark of this pathology which can contribute to the dysregulation of lipid homeostasis and lipid metabolism in multiple sclerosis. Lipidomic studies analysed in this review clearly demonstrate the role of lipids in inflammatory processes, in immunity, and in the onset and development of multiple sclerosis. Several investigations reported alterations of some molecular lipid species, in particular, with decrease of fatty acids (FA) 18:2 and 20:4 and total polyunsaturated FA, with compensatory increases of saturated FA with shorter carbon chains. Oxidized phospholipids were reported in few studies as well. Also, it was shown that clinical lipidomics has potential as a tool to aid both in multiple sclerosis diagnosis and therapeutics by allowing a detailed lipidome profiling of the patients suffering with this disease.

Phenylketonuria (PKU) is the most prevalent inborn error of amino acid metabolism. The disease is due to the deficiency of phenylalanine (Phe) hydroxylase activity, which causes the accumulation of Phe. Early diagnosis through neonatal screening is essential for early treatment implementation, avoiding cognitive impairment and other irreversible sequelae. Treatment is based on Phe restriction in the diet that should be maintained throughout life. High dietary restrictions can lead to imbalances in specific nutrients, notably lipids. Previous studies in PKU patients revealed changes in levels of plasma/serum lipoprotein lipids, as well as in fatty acid profile of plasma and red blood cells. Most studies showed a decrease in important polyunsaturated fatty acids, namely DHA (22:6n-3), AA (20:4n-6) and EPA (20:5n-6). Increased oxidative stress and subsequent lipid peroxidation have also been observed in PKU. Despite the evidences that the lipid profile is changed in PKU patients, more studies are needed to understand in detail how lipidome is affected. As highlighted in this review, mass spectrometry-based lipidomics is a promising approach to evaluate the effect of the diet restrictions on lipid metabolism in PKU patients, monitor their outcome, namely concerning the risk for other chronic diseases, and find possible prognosis biomarkers. published

Laccases produced by Leucoagaricus gongylophorus act in lignocellulose degradation and detoxification processes. Therefore, the use of L. gongylophorus laccase (Lac1Lg) was proposed in this work for degradation of anthracene and others polycyclic aromatic hydrocarbons without the use of mediators. Degradation reactions were performed in buffer aqueous solution with 10 ppm of anthracene and other PAHs, Tween-20 in 0.25% v/v and a laccase preparation of 50 U. The optimum condition (pH 6.0 and 30 °C) was determined by response surface methodology with an excellent coefficient of determination (R2) of 0.97 and an adjusted coefficient of determination (R2adj) of 0.93. In addition, the employment of the mediator ABTS decreased the anthracene biodegradation from 44 ± 1% to 30 ± 1%. This optimum pH of 6.0 suggests that the reaction occurs by a hydrogen atom transfer mechanism. Additionally, in 24 h Lac1Lg biodegraded 72 ± 1% anthracene, 40 ± 3% fluorene and 25 ± 3% phenanthrene. The yellow laccase from L. gongylophorus biodegraded anthracene and produced anthrone and anthraquinone, which are interesting compounds for industrial applications. Moreover, this enzyme also biodegraded the PAHs phenanthrene and fluorene justifying the study of Lac1Lg for bioremediation of these compounds in the environment.

The aim of this study was to determine the energy metabolism and partition of lactating Gyr and F1 Holstein x Gyr (F1 HxG) cows in different planes of nutrition. Six F1 HxG and six Gyr cows with 130 days in milking (DIM) fed corn silage and concentrate were evaluated. The experiment consisted of four periods with different levels of feeding: 1st ad libitum dry matter intake (DMI) and the others with 5, 10 and 20% restricted DMI, related to the first one. An apparent digestibility assay was performed before measurements in the respiration chamber. Total feces were collected for three days. The cows were confined for 24h in the chamber in each period to determine methane and heat production (HP). F1 HxG had higher gross energy intake (GEI), metabolisable energy intake (MEI) and digestible energy intake (DEI). GE lost in feces was higher in F1 HxG (23.7% GEI) than in Gyr (20.5%) cows. Energy lost as methane and urine was similar between the groups. The metabolisability (q) was 0.67, and the efficiency of converting ME to NE (k) was 0.56. There was no difference in the energy requirements for maintenance between breeds (426.6 MJ/kg BW0,75 average value). The energy requirements for lactation were higher in F1 HxG animals due to the higher volume of milk produced, since there was no difference in energy requirements for production of one kg of milk.

Apesar do uso de criopreservacao de tecido ovariano para restaurar a fertilidade, nao ha estudos publicados abordando os efeitos desta tecnica no metabolismo osseo. Este estudo tem como objetivos avaliar os resultados da pega do enxerto de tecido ovariano apos vitrificacao e reimplante no omento de ratas, comparar os niveis de estradiol nessas ratas em diferentes etapas de acordo com a presenca ou nao de tecido ovariano e avaliar os efeitos sobre o metabolismo osseo de tal reimplante O presente estudo foi realizado em 27 ratas com idade entre 11 e 12 semanas e pesando 200-300g, periodo no qual ha estabelecimento de maturidade hormonal. Todos os animais foram submetidos a ooforectomia bilateral e os ovarios foram vitrificados. O autotransplante ocorreu em dois periodos distintos de acordo com cada grupo: apos 1 semana (grupo A, n = 8) ou apos 1 mes (grupo B, n = 19) contados a partir da primeira intervencao (antes da ooforectomia). Foram realizados ensaios de estradiol no soro e analise histologica do tecido ovariano e da tibia: antes da ooforectomia (basal), no final do periodo de castracao cirurgica (apos ooforectomia) e um mes apos o transplante (reimplante). Nos dois grupos houve piora dos parâmetros osseos apos a castracao e melhoria dos mesmos apos o reimplante ovariano, a saber: as espessuras corticais medias da tibia foram 0,463 ± 0,14mm (media ± DP) na linha de base, 0,360 ± 0,14mm apos ooforectomia e 0,445 ± 0,17mm em 1 mes apos o reimplante (p < 0,005). Alem disso, as espessuras medias trabecular foram 0.050 ± 0,08mm (media ± DP) na linha de base, 0,022 ± 0,08mm apos ooforectomia e 0.049 ± 0,032mm em 1 mes apos o reimplante (p < 0,005). Quanto a mensuracao de estradiol serico, nos dois grupos houve queda apos a castracao e aumento apos o reimplante. Entretanto nao houve diferenca estatisticamente significante entre a variacao dos niveis de estradiol nos dois grupos de estudo (p=0.819). Nos animais com castracao cirurgica precoce apesar da variacao dos niveis basais (25.6±0.8ng/dl) para o status castrado (19.0±1.1ng/dl) e pos-reimplante de um mes (25.6±3.9ng/dl) nao houve diferenca significante entre os tres momentos de analise de estradiol (p=0.140). Porem, o grupo com castracao cirurgica tardia apresentou uma reducao significante dos niveis de estradiol do pre-operatorio (28.0±1.1ng/dl) para o status castrado (19.7±1.6ng/dl) e recuperacao dos niveis normais um mes apos o reimplante (25.8±0.5ng/dl) (p=0.001). Houve sucesso de pega de enxerto, ou seja, observou-se presenca de foliculos atresicos, foliculos primordiais e de corpo luteo em cerca de 81,5 % dos animais (grupo A: n = 7/8, 87%, grupo B: n = 15/19, 78,9%, p = 0,080) demonstrando que o local de reimplante e cirurgicamente adequado. Valores semelhantes sao observados em outros trabalhos experimentais de referencia. De acordo com estes resultados, o transplante de tecido ovariano criopreservado nao so restaurou a funcao hormonal ovariana, mas restabeleceu os parâmetros iniciais do osso. Estes resultados podem sugerir que reimplantacao ovariana em outros mamiferos, como em mulheres, tenha efeitos beneficos no metabolismo osseo. Palavras-chave: Tecido ovariano. Autotransplante. Menopausa. Metabolismo osseo.

Cyanobacteria or blue green algae are known for their extensive and highly visible blooms in eutrophic, stagnant freshwater bodies. Climate change and global warming have also contributed to a rise in toxic cyanobacterial blooms. One of the most important cyanobacteria is Microcystis aeruginosa, which can synthesize various microcystins that can affect the health of terrestrial and aquatic animals. Commercial Nile crocodile (Crocodylus niloticus) farming in South Africa is based on keeping breeders (adult males and females) in big dams on farms (captive-bred approach). Unfortunately, cyanobacterial blooms in the breeder dams are a concern to farm owners, managers and veterinarians. The main objectives of this research project were to determine if microcystins were present in the contents of crocodile eggs and the liver and yolk of dead hatchlings, and to determine if the reduced hatchability on commercial farms might be caused by these toxins. Furthermore, the concentration of microcystins in the breeder dam was monitored on a monthly basis spanning the ovulation and egg laying period. During the hatching season microcystin concentrations in unfertilised eggs, egg shell membranes and in the yolk and liver of dead hatchlings were determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Microcystins were detected in Nile crocodile egg and hatchling samples. Microcystin (MC-LR, MC-RR, MC-YR) concentrations in the crocodile egg and hatchling samples collected from clutches with a good hatching rate (≥90%) ranged between 0 and 1.76 ng g−1, with the highest concentration in the egg shell membranes. Microcystin concentrations in samples collected from clutches with a bad hatching rate (≤10%) ranged from 0 – 1.63 ng g−1 with the highest concentration detected in the hatchling yolk. However, the concentrations were probably underestimated as the percentage recovery from spiked samples was very low with the extraction method employed. Bayesian analysis suggests that the liver, yolk and unfertilised egg all have similar microcystin concentrations, while the membranes have (with moderate to high certainty) higher microcystin concentrations. There appears to be no difference in microcystin concentrations among good and bad clutches across all tissue types or within a specific tissue type, but due to the small sample size, it was not possible to determine whether microcystin affected the hatchability of Nile crocodile eggs. However, vertical transmission of microcystin variants to the Nile crocodile egg does occur and the possible implications for the survival of wild Nile crocodile populations should be ascertained.

Background The mesenchymal stem cell therapy has proven to be an effective option in the treatment of skin injuries. The combination of these cells with nanostructured membranes seems to be the future for tissues recovery. The aim of this project was to use biomolecules of polysaccharides to be incorporated on regenerated cellulose membranes and to prospect the improvement as bioactive wound dressings with mesenchymal stem cells. Methods The biocomposites were obtained after defibrillation with the use of never-dried bacterial cellulose to form a pulp, and, after the films were regenerated, in the presence of gellan gum with or without fluconazole. Membrane atomic force microscopy was performed for comparison of their structures. Results Adipose-derived mesenchymal stem cells were obtained from human adipose tissue liposuction in accordance with Zuk et al. The flow cytometric analysis and induction tests for adipocytes and osteocytes were performed. In vitro assays were performed on different membranes to evaluate the ability of these cells to adhere at 2 hours and proliferate at 7 days; the results were obtained by use of the MTT cell counting technique. In vivo testing allowed us to observe cell migration and participation in wound-healing by fluorescence labeling of the cells with BrdU. The bioactive curative, seeded with cells, was tested in skin burned in a murine model. Conclusions The bacterial cellulose with gelan gum membrane incorporated with fluconazole presented the best performance in adhesion and proliferation tests. The cells can be identified in burned host tissue after occurrence of the wound.

Medicinal plants have been the subject of research in several countries such as Brazil. The Duguetia furfuracea A. St.-Hil., popularly known as araticum-bravo, ata-brava and ata de lobo, has been used in folk medicine as anti-rheumatic drugs, for the treatment of renal dysfunction, spinal pain and stomach, and against pediculosis. This work aimed to analyze the antibacterial effect of the crude extract and fractions obtained from the leaves of D. furfuracea. The characterization of secondary metabolites was carried out through phytochemical prospection, being checked for the presence of tannins, flavonoids and alkaloids. The minimum inhibitory concentration (MIC) was determined by using broth microdilution method and its modulating activity of antibiotic activity in sub-inhibitory (MIC/8) concentration. When standard bacterial strain is used for the MIC and multidrug-resistant strains of modulation, all samples had a MIC ≥ 1024 µg/ml. The samples when combined with aminoglycosides demonstrated synergistic activity against the Escherichia coli 27 and Staphylococcus aureus 358. The results of this study indicate the species D. furfuracea as a promising source in combating bacterial multidrug resistance, increasing the potential of antibiotics. Key words: Duguetia furfuracea A. St.-Hil., modulation, antibacterial activity, aminoglycosides.