Characterization of flours from the aroeira leaf (Schinus terebinthifolius Raddi), obtained by different drying methods.

The present work aimed at the development and characterization of aroeira leaf flour (Schinus terebinthifolius Raddi), obtained by lyophilization and drying in an air circulation oven. The technological, physical, physico-chemical, morphological, functional, and microbiological aspects were analyzed. Physico-chemical analysis identified the following properties with values provided respectively for fresh leaves (FOin) and flours (FES and FLIO): low water activity (0.984, 0.370, 0.387 g/100 g), moisture (64.52, 5.37, 7.97 g /100 g), ash (2.69, 6.51, and 6.89 g/100 g), pH (0.89, 4.45, 4.48 g/100 g), lipids (0.84, 1.67, 5.23 g/100 g), protein (3.29, 8.23, 14.12 g/100 g), carbohydrates (17.02, 53.12, 33.02 g/100 g), ascorbic acid (19.70, 34.20, 36.90 mg/100 g). Sources of fiber from plant leaves and flours (11.64, 25.1, 32.89 g/100 g) showed increased levels of luminosity. For NMR, the presence of aliphatic and aromatic compounds with olefinic hydrogens and a derivative of gallic acid were detected. The most abundant minerals detected were potassium and calcium. Micrographs identified the presence of irregular, non-uniform, and sponge-like particles. The main sugars detected were: fructose, glucose, and maltose. Malic, succinic, citric, lactic, and formic acids were found. Fifteen phenolic compounds were identified in the samples, highlighting: kaempferol, catechin, and caffeic acid. The values ​​found for phenolics were (447, 716.66, 493.31 mg EAG/100 g), flavonoids (267.60, 267.60, 286.26 EC/100 g). Antioxidant activity was higher using the ABTS method rather than FRAP for analysis of FOin, FES, and FLIO. Since the flours of the aroeira leaf have an abundant matrix of nutrients with bioactive properties and antioxidant activity, they have a potential for technological and functional use when added to food.

Supplementation with Moringa oleifera Lam leaf and seed flour during the pregnancy and lactation period of Wistar rats: Maternal evaluation of initial and adult neurobehavioral development of the rat progeny.

ETHNOPHARMACOLOGICAL RELEVANCE: Moringa oleifera Lam. (M. oleifera) is a tree species of Indian origin popularly known as the "tree of life". In various cultures, it is used by pregnant women to increase milk production, yet studies on its effects during pregnancy and lactation are lacking. AIM OF THE STUDY: To evaluate the nutraceutical aspects of flours produced from the leaves and seeds of M. oleifera, and to evaluate the effect of supplementation of pregnant Wistar rats during the gestation and lactation period, with the aim of studying the weight gain and neonatal parameters of the pregnant rats, as well as effects on the neurobehavioral development and memory in their offspring. MATERIALS AND METHODS: The flour supplementation was conducted at a concentration of 100 mg per kg of animal body weight. For the memory tests, the Open Field Habituation test was performed and repeated after seven days. The Object Recognition test was conducted with the animal exposed to the open field in short and long familiarization sessions. The data obtained were analyzed using Kruskal-Wallis tests for non-parametric data and one-way and two-way ANOVA for parametric data. RESULTS: Flour produced from both the leaf and seed of M. oleifera was found to contain significant amounts of nutrients (protein, fibre, carbohydrates, etc.), making them suitable for supplementation. The exposure of pregnant rats to M. oleifera leaf and seed flours did not affect weight gain, did not have harmful effects on the birth of offspring, and did not result in abortions or mutations in the offspring. Regarding the supplemented group's offspring, early maturation of the senses in the offspring compared to the control group was observed in all tests were conducted; indicating that supplementation positively impacted cognitive development. Further, the offspring of the supplemented rats presented reduced locomotion and greater exploration of new objects compared to the control group offspring, indicating positive effects on learning. CONCLUSION: This study describes for the first time the beneficial effects on pregnant Wistar rats and their offspring of maternal supplementation with flour products from the leaves and seeds of M. oleifera.

Synthesis and antifungal evaluation against Candida spp. of the (E)-3-(furan-2-yl)acrylic acid.

Infections of fungal origin are mainly caused by Candida spp. Some species, such as C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis, stand out as promoters of diseases in humans. This study evaluated the synthesis and antifungal effects of (E)-3-(furan-2-yl)acrylic acid. The synthesis of the compound showed a yield of 88%, considered high. The minimum inhibitory concentration of the synthetic compound, amphotericin B, and fluconazole isolated against four Candida species ranged from 64 to 512 µg/mL, 1 to 2 µg/mL, and 32 to 256 µg/mL, respectively. The synergistic effect of the test compound was observed when associated with amphotericin B against C. albicans and C. tropicalis, with no antagonism between the substances against any of the strains tested. The potential drug promoted morphological changes in C. albicans, decreasing the amount of resistance and virulence, and reproduction structures, such as the formation of pseudohyphae, blastoconidia, and chlamydospores. Furthermore, it was also possible to identify the fungistatic profile of the test substance by studying the growth kinetics of C. albicans. Finally, it was observed that the test compound stimulated ergosterol biosynthesis by the yeast, probably by activating microbial resistance responses.

Maternal rat prenatal and neonatal treatment with pequi pulp reduces anxiety and lipid peroxidation in brain tissue of rat offspring at adolescence.

The Pequi fruit (Caryocar Brasiliense cambess), typical of the Brazilian cerrado or savannah, is a source of essential fatty acids, carotenoids, and phenolic compounds. The aim of this study was to analyze the effects of consuming this fruit on anxiety behavior and lipid peroxidation in the brains of rats whose mothers were treated (by gavage) during pregnancy and lactation with Pequi fruit (pulp or nuts) at 2000 mg/kg of body weight. Anxiety parameters were assessed using the open field (OF), elevated plus maze (EPM), and light/dark box (LDB) tests. The brain was removed to measure malondialdehyde (MDA) levels. Data were analyzed using One-way Anova (p < 0.05). In the OF, the animals in the pulp group presented more time spent in the central area (20.37 ± 0.73 vs Control: 12.51 ± 0.39; Nuts: 8.28 ± 0.40) and increased locomotion (159.7 ± 6.10) compared to the other groups (Control: 127.3 ± 5.54; Nuts: 139.08 ± 6.57). In the EPM, the pulp group entered into the open arms (8.57 ± 0.36) and stayed more time in the central area (19.44 ± 1.17) compared to the Nuts group (7.14 ± 0.34; 13.00 ± 1.57). In the LDB the pulp group entered more (8.00 ± 0.42 vs Control: 7.16 ± 0.16 and Nuts: 7.42 ± 0.75) and stayed longer in the clear light side (92.18 ± 6.42) than all the other groups (Control: 71.44 ± 3.53; Nuts: 80.57 ± 6.50), respectively. Pulp group presented lower MDA in the brain (55.34 ± 3.04) compared to Control (72.06 ± 4.66) and Nuts (66.57 ± 2.45). We conclude that Pequi pulp consumption during pregnancy and lactation reduces lipid peroxidation in brain tissue and induces anxiolytic-like behavior in rat offspring. These effects were not observed in the Pequi nuts group.

Mix of almond baru (Dipteryx alata Vog.) and goat whey modulated intestinal microbiota, improved memory and induced anxiolytic like behavior in aged rats.

The objective was to evaluate the effects of the consumption of a mix of baru almond and goat whey on memory performance and anxiety parameters related to the intestinal health of rats treated during aging. The animals were divided into three groups and treated by gavage for 10 weeks (n = 10/each group): Control (CT) - distilled water; Baru almond (BA) - 2000 mg of baru/kg of body weight; and Baru + Whey (BW) - 2000 mg of baru + 2000 mg of goat milk whey/kg of body weight. Anxiety behavior, memory, brain fatty acid profile and fecal microbiota were measured. BA and BW realized less grooming, spent more time in the central area of the open field and the open arms, and realized more head dipping in the elevated plus maze. A higher rate of exploration of the new object in the short and long-term memory was observed in BA and BW. There was an increase in the deposition of MUFAs and PUFAs and oleic acid in the brain of BA and BW. Regarding spatial memory, BA and BW performed better, with an emphasis on BW. There was a beneficial modulation of the fecal microbiota with a reduction of the pathogenic genus Clostridia_UFC-014 in BA and BW and an increase in the abundance of metabolic pathways of interest in the brain-gut axis. Thus, consumption of the mix is efficient in beneficially altering the intestinal microbiota, improving memory and anxiolytic-like behavior in rats during aging.

Antifungal Activity, Antibiofilm and Association Studies with O-Alkylamidoximes against Cryptococcus spp.

This is the first study that describes the antifungal and anti-biofilm potential of O-alkylamidoximes against strains of Cryptococcus neoformans and Cryptococcus gattii. In vitro tests have shown that O-alkylamidoximes are capable of inhibiting fungal growth and biofilm formation of the C. neoformans and C. gattii strains, suggesting, from molecular docking, the potential for interaction with the Hsp90. The associations between O-alkylamidoximes and amphotericin B were beneficial. Therefore, O-alkylamidoximes can be a useful alternative to contribute to the limited arsenal of drugs, since they showed a powerful action against the primary agents of Cryptococcosis.

Propyl (E)-3-(furan-2-yl) Acrylate: a synthetic antifungal potential with a regulatory effect on the biosynthesis of ergosterol in Candida Albicans

Abstract The genus Candida represents the main cause of infections of fungal origin. Some species stand out as disease promoters in humans, such as C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. This study evaluated the antifungal effects of propyl (E)-3-(furan-2-yl) acrylate. The minimum inhibitory concentration of the synthetic compound, amphotericin B and fluconazole alone against four species of Candida ranged from 64 to 512 µg/mL, 1 to 2 µg/mL, and 32 to 256 µg/mL, respectively. The synergistic effect of the test substance was observed when associated with fluconazole against C. glabrata, there was no antagonism between the substances against any of the tested strains. The potential drug promoted morphological changes in C. albicans, decreasing the amount of resistance, virulence, and reproduction structures, such as the formation of pseudohyphae, blastoconidia, and chlamydospores, ensuring the antifungal potential of this substance. It was also possible to identify the fungicidal profile of the test substance through the study of the growth kinetics of C. albicans. Finally, it was observed that the test compound inhibited the ergosterol biosynthesis by yeast

Targeting extracellular matrix remodeling sensitizes glioblastoma to ionizing radiation.

Background: The median survival of Glioblastoma multiforme (GBM) patients is 14+ months due to poor responses to surgery and chemoradiation. Means to counteract radiation resistance are therefore highly desirable. We demonstrate the membrane bound matrix metalloproteinase MT1-MMP promotes resistance of GBM to radiation, and that using a selective and brain permeable MT1-MMP inhibitor, (R)-ND336, improved tumor control can be achieved in preclinical studies. Methods: Public microarray and RNA-sequencing data were used to determine MT1-MMP relevance in GBM patient survival. Glioma stem-like neurospheres (GSCs) were used for both in vitro and in vivo assays. An affinity resin coupled with proteomics was used to quantify active MT1-MMP in brain tissue of GBM patients. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP and inhibition via the MT1-MMP inhibitor (R)-ND336, were used to assess the role of MT1-MMP in radio-resistance. Results: MT1-MMP expression inversely correlated with patient survival. Active MT1-MMP was present in brain tissue of GBM patients but not in normal brain. shRNA- or (R)-ND336-mediated inhibition of MT1-MMP sensitized GSCs to radiation leading to a significant increase in survival of tumor-bearing animals. MT1-MMP depletion reduced invasion via the effector protease MMP2; and increased the cytotoxic response to radiation via induction of replication fork stress and accumulation of double strand breaks (DSBs), making cells more susceptible to genotoxic insult. Conclusions: MT1-MMP is pivotal in maintaining replication fork stability. Disruption of MT1-MMP sensitizes cells to radiation and can counteract invasion. (R)-ND336, which efficiently penetrates the brain, is therefore a novel radio-sensitizer in GBM.

Vitamin D: a potentially important secosteroid for coping with COVID-19.

COVID-19 is a disease that has caused a high number of deaths in the world, and despite being controlled, it requires attention and the search for new quick and economical therapeutic strategies. In this sense, vitamin D stands out, an immunomodulator that has shown beneficial effects in decreasing the risk and severity of acute respiratory tract infections, including COVID-19. Therefore, this review presents a number of experimental, observational and clinical studies on the importance of vitamin D against viral infections with an emphasis on COVID-19, highlighting the relationship between vitamin D, Renin-Angiotensin System and cytokine storms with decreased inflammatory lesions in patients with COVID-19. In addition, aspects of pathophysiology, metabolism, risk factors, sources and recommendations of vitamin D are described. We conclude that vitamin D plays a protective role against inflammatory lesions and can decrease the risk of infections and the severity of COVID-19. Therefore, it is essential to maintain adequate levels of vitamin D to avoid complications related to its deficiency.

Synthesis, antimicrobial evaluation and in silico studies of the (E)-3-(aryl)-5-styryl-1,2,4-oxadiazoles

In recent years, investigations in the field of oxadiazoles have been intensified due to their numerous therapeutic uses. Oxadiazoles are a class of compounds that exhibit several biological applications, citing antimicrobial, anti-inflammatory, anti-diabetic, anthelmintic, anti-tumor, among others. Encouraged by the biological potential of oxadiazoles, were carried out synthesis, antimicrobial evaluation and in silica studies of five (E)-3-(aryl)-5-styryl-1,2,4-oxadiazoles. In this way, (Z)-aryl-N'-hydroxybenzimidamides and ethyl (E)-cinnamate were synthesized, which were subjected to an O-acylamidoxime reaction after by dehydration using microwave irradiation to form the oxadiazole nucleus. The compounds were characterized by spectroscopic techniques, while in vitro antimicrobial activity was evaluated against S. aureus, E. faecalis, E. coli, P. aeruginosa, and against the fungus C. utilis using the microplate microdilution method. Thus, (Z)-aryl-N'-hydroxybenzimidamides, ethyl (E)-cinnamate, and (E)-3-(aryl)-5-styryl-1,2,4-oxadiazoles were synthesized with yields ranging from moderate to good. The (E)-3-(aryl)-5-styryl-1,2,4-oxadiazoles exhibited a reduced spectrum of action, which were active against the bacterium P. aeruginosa and for the fungus C. utilis.

Polyacetylene Glycosides: Isolation, Biological Activities and Synthesis.

Polyacetylene glycosides (PAGs) constitute a relatively small class of secondary metabolites characterized by the presence of a sugar unit anomerically connected to a polyacetylene. These compounds are found in fungi, seaweed, and more often in plants. PAGs exhibit a wide range of biological and pharmacological activities and, as a result, the literature of these compounds has grown exponentially in recent years.

Synthesis of new 1,4-disubstituted 1,2,3-triazoles using the CuAAC reaction and determination of their antioxidant activities.

This study describes the synthesis and antioxidant activity of new 1,4-disubstituted 1,2,3-triazoles. These compounds were generated semi-synthetically using the Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) reaction between ethyl 2-azidoacetate and terminal acetylenes derived from the natural products carvacrol, eugenol, isovanillin, thymol and vanillin. The products were obtained at 50 to 80% yield and characterised through several spectrographic techniques. Antioxidant activity was assayed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The products exhibited moderate antioxidant activity, with ethyl 2-(4-((4-formyl-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl) acetate showing the highest antioxidant capacity (EC50 = 75.5 µg/mL) among the generated 1,4-disubstituted 1,2,3-triazoles. In conclusion, the generation of these compounds opens new possibilities for the development of new antioxidant agents.

The immunosuppressive role of Edn3 overexpression in the melanoma microenvironment.

Endothelins are cytokines expressed in the microenvironment of several tumors. To identify which stromal cells in the melanoma microenvironment respond to endothelin, we injected murine melanoma cell lines B16F10, YUMM1.7, and YUMMER1.7 in a transgenic mouse that overexpresses endothelin 3 (Edn3) under the control of the keratin 5 promoter in the skin (K5-Edn3). All cell lines developed larger tumors in K5-Edn3 mice than in control animals. In YUMM1.7 tumors, the Edn3 receptor, endothelin receptor B (Ednrb), was expressed in several stromal cell types including immune cells. This result was validated by the identification of Ednrb-positive stromal cells in human melanoma from previously published RNA-seq data. Regulatory T cells (Tregs) and dendritic cell numbers were significantly higher in K5-Edn3 tumors when compared to control tumors. Edn3 increased Treg proliferation in vitro and the expression of FOXP3. YUMM1.7-GFP tumors in K5-Edn3 mice were sensitive to immune checkpoint inhibitor (anti-CTLA-4) as well as to Ednrb blockage (BQ-788). Our results indicate that Ednrb signaling has an important role in the melanoma microenvironment where it mediates immunosuppression resulting in escape from tumor immunity.

MT1-MMP-dependent ECM processing regulates laminB1 stability and mediates replication fork restart.

Radiotherapy remains a mainstay of treatment for a majority of cancer patients. We have previously shown that the membrane bound matrix metalloproteinase MT1-MMP confers radio- and chemotherapy resistance to breast cancer via processing of the ECM and activation of integrinß1/FAK signaling. Here, we further discovered that the nuclear envelope protein laminB1 is a potential target of integrinß1/FAK. FAK interacts with laminB1 contributing to its stability. Stable laminB1 is found at replication forks (RFs) where it is likely to allow the proper positioning of RF protection factors, thus preventing RF degradation. Indeed, restoration of laminB1 expression rescues replication fork stalling and collapse that occurs upon MT1-MMP inhibition, and reduces DNA damage in breast cancer cells. Together, these data highlight a novel mechanism of laminB1 stability and replication fork restart via MT1-MMP dependent extracelluar matrix remodeling.

Wound Healing Assay for Melanoma Cell Migration.

Cell migration is a critical process involved in morphogenesis, inflammation, and cancer metastasis. Wound healing assay is a simple, non-expensive, and highly reproducible method to study cancer cell migration in vitro. It is based on the observation that cells growing in a monolayer migrate to re-establish cell contacts after the development of an artificial wound. The assay involves creation of a wound in a monolayer, image acquisition during wound closure, and comparison of migrated area at initial and final time points.

Effects of the aqueous extract of Phyllanthus niruri Linn during pregnancy and lactation on neurobehavioral parameters of rats' offspring.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus niruri L. (Phyllanthaceae) is a plant used in traditional medicine, mainly to treat kidney stones. However, the effects of maternal exposure to P. niruri remain poorly explored. AIM OF THE STUDY: The objective of this study was to investigate the effects of administration of aqueous extract of P. niruri (AEPN) during pregnancy and lactation, in maternal toxicity, reflex maturation, and offspring memory. MATERIALS AND METHODS: Pregnant rats were divided into three groups (n = 8/group): Control (vehicle), AEPN 75, and AEPN 150 (each respectively treated with P. niruri at a dose of 75 and 150 mg/kg/day). The animals were treated via intragastric gavage during pregnancy and lactation. Weight gain, feed intake, and reproductive performance were analyzed in the mothers. In the offspring, the following tests were performed: Neonatal Reflex Ontogeny, Open Field Habituation Test and the Object Recognition Test in adulthood. RESULTS: Maternal exposure to AEPN did not influence weight gain, feed intake, or reproductive parameters. In the offspring, anticipation of reflex ontogenesis (time of completion) was observed (p < 0.05). During adulthood, the AEPN groups presented decreases in exploratory activity upon their second exposure to the Open Field Habituation Test (in a dose-dependent manner) (p < 0.05). In the Object Recognition Test, administration of the extract at 75 and 150 mg/kg induced significant dose-dependent improvements in short and long-term memory (p < 0.05). CONCLUSION: Administration of the AEPN accelerated the reflex maturation in neonates, and improved offspring memory while inducing no maternal or neonatal toxicity.

Synthesis and antifungal activity of new O-alkylamidoximes

The continuous prospection for molecules that may be useful in the development of new therapeutic agents is a highly relevant issue, mainly because the launch of new drugs on the market does not accompany the emergence of new resistant microorganisms. In this context, this work describes the synthesis of new O-alkylamidoximes and the evaluation of its antifungal activity. The new O-alkylamidoximes were prepared using easy synthetic protocols and tested against three Candida species using the broth microdilution method. The synthesized compounds were obtained in moderate to good yields in high purity and without any observable decomposition. All tested compounds shown moderate antifungal activity against at least one strain of Candida. Despite the moderate activity of the new compounds, this was the first report involving the antifungal activity of O-alkylamidoximes. In view of the low chemotherapy arsenal and the development of fungal strains resistant to traditional antifungal agents, the present study opens new possibilities for the preparation of a new class of more active antifungal agents.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

Targeting Extracellular Matrix Remodeling Restores BRAF Inhibitor Sensitivity in BRAFi-resistant Melanoma.

PURPOSE: The extracellular matrix (ECM) is an intriguing, yet understudied component of therapy resistance. Here, we investigated the role of ECM remodeling by the collagenase, MT1-MMP, in conferring resistance of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutant melanoma to BRAF inhibitor (BRAFi) therapy. EXPERIMENTAL DESIGN: Publicly available RNA-sequencing data and reverse phase protein array were used to determine the relevance of MT1-MMP upregulation in BRAFi-resistant melanoma in patients, patient-derived xenografts, and cell line-derived tumors. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP, inhibition via the selective MT1-MMP/MMP2 inhibitor, ND322, or overexpression of MT1-MMP was used to assess the role of MT1-MMP in mediating resistance to BRAFi. RESULTS: MT1-MMP was consistently upregulated in posttreatment tumor samples derived from patients upon disease progression and in melanoma xenografts and cell lines that acquired resistance to BRAFi. shRNA- or ND322-mediated inhibition of MT1-MMP synergized with BRAFi leading to resensitization of resistant cells and tumors to BRAFi. The resistant phenotype depends on the ability of cells to cleave the ECM. Resistant cells seeded in MT1-MMP uncleavable matrixes were resensitized to BRAFi similarly to MT1-MMP inhibition. This is due to the inability of cells to activate integrinß1 (ITGB1)/FAK signaling, as restoration of ITGB1 activity is sufficient to maintain resistance to BRAFi in the context of MT1-MMP inhibition. Finally, the increase in MT1-MMP in BRAFi-resistant cells is TGFß dependent, as inhibition of TGFß receptors I/II dampens MT1-MMP overexpression and restores sensitivity to BRAF inhibition. CONCLUSIONS: BRAF inhibition results in a selective pressure toward higher expression of MT1-MMP. MT1-MMP is pivotal to an ECM-based signaling pathway that confers resistance to BRAFi therapy.

Efficient method for propargylation of aldehydes promoted by allenylboron compounds under microwave irradiation.

The propargylation of aldehydes promoted by microwave irradiation using allenylboron compounds in a chemo- and regioselective way is described. The corresponding products were obtained in short reaction time, high yield and purity without the need of any solvent when allenylboronic acid pinacol ester was used, or using a minimal amount of acetone when potassium allenyltrifluoroborate was used.