Atlantic flower-invertebrate interactions: A data set of occurrence and frequency of floral visits.

Encounters between flowers and invertebrates are key events for the functioning of tropical forests. Assessing the structure of networks composed of the interactions between those partners leads to a better understanding of ecosystem functioning and the effects of environmental factors on ecological processes. Gathering such data is, however, costly and time-consuming, especially in the highly diverse tropics. We aimed to provide a comprehensive repository of available flower-invertebrate interaction information for the Atlantic Forest, a South American tropical forest domain. Data were obtained from published works and "gray literature," such as theses and dissertations, as well as self-reports by co-authors. The data set has ~18,000 interaction records forming 482 networks, each containing between one and 1061 interaction links. Each network was sampled for about 200 h or less, with few exceptions. A total of 641 plant genera within 136 different families and 39 orders were reported, with the most abundant and rich families being Asteraceae, Fabaceae, and Rubiaceae. Invertebrates interacting with these plants were all arthropods from 10 orders, 129 families, and 581 genera, comprising 2419 morphotypes (including 988 named species). Hymenoptera was the most abundant and diverse order, with at least six times more records than the second-ranked order (Lepidoptera). The complete data set shows Hymenoptera interacting with all plant orders and also shows Diptera, Lepidoptera, Coleoptera, and Hemiptera to be important nodes. Among plants, Asterales and Fabales had the highest number of interactions. The best sampled environment was forest (~8000 records), followed by pastures and crops. Savanna, grasslands, and urban environments (among others) were also reported, indicating a wide range of approaches dedicated to collecting flower-invertebrate interaction data in the Atlantic Forest domain. Nevertheless, most reported data were from forest understory or lower strata, indicating a knowledge gap about flower-invertebrate interactions at the canopy. Also, access to remote regions remains a limitation, generating sampling bias across the geographical range of the Atlantic Forest. Future studies in these continuous and hard-to-access forested areas will yield important new information regarding the interactions between flowers and invertebrates in the Atlantic Forest. There are no copyright restrictions on the data set. Please cite this data paper if the data are used in publications and teaching events.

N-acetylcysteine protects against motor, optomotor and morphological deficits induced by 6-OHDA in zebrafish larvae.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder. In addition to its highly debilitating motor symptoms, non-motor symptoms may precede their motor counterparts by many years, which may characterize a prodromal phase of PD. A potential pharmacological strategy is to introduce neuroprotective agents at an earlier stage in order to prevent further neuronal death. N-acetylcysteine (NAC) has been used against paracetamol overdose hepatotoxicity by restoring hepatic concentrations of glutathione (GSH), and as a mucolytic in chronic obstructive pulmonary disease by reducing disulfide bonds in mucoproteins. It has been shown to be safe for humans at high doses. More recently, several studies have evidenced that NAC has a multifaceted mechanism of action, presenting indirect antioxidant effect by acting as a GSH precursor, besides its anti-inflammatory and neurotrophic effects. Moreover, NAC modulates glutamate release through activation of the cystine-glutamate antiporter in extra-synaptic astrocytes. Its therapeutic benefits have been demonstrated in clinical trials for several neuropsychiatric conditions but has not been tested in PD models yet. METHODS: In this study, we evaluated the potential of NAC to prevent the damage induced by 6-hydroxydopamine (6-OHDA) on motor, optomotor and morphological parameters in a PD model in larval zebrafish. RESULTS: NAC was able to prevent the motor deficits (total distance, mean speed, maximum acceleration, absolute turn angle and immobility time), optomotor response impairment and morphological alterations (total length and head length) caused by exposure to 6-OHDA, which reinforce and broaden the relevance of its neuroprotective effects. DISCUSSION: NAC acts in different targets relevant to PD pathophysiology. Further studies and clinical trials are needed to assess this agent as a candidate for prevention and adjunctive treatment of PD.

Pre-clinical effects of metformin and aspirin on the cell lines of different breast cancer subtypes.

Background Breast cancer is highly prevalent among women worldwide. It is classified into three main subtypes: estrogen receptor positive (ER+), human epidermal growth factor receptor 2 positive (HER2+), and triple negative breast cancer (TNBC). This study has evaluated the effects of aspirin and metformin, isolated or in a combination, in breast cancer cells of the different subtypes. Methods The breast cancer cell lines MCF-7, MDA-MB-231, and SK-BR-3 were treated with aspirin and/or metformin (0.01 mM - 10 mM); functional in vitro assays were performed. The interactions with the estrogen receptors (ER) were evaluated in silico. Results Metformin (2.5, 5 and 10 mM) altered the morphology and reduced the viability and migration of the ER+ cell line MCF-7, whereas aspirin triggered this effect only at 10 mM. A synergistic effect for the combination of metformin and aspirin (2.5, 5 or 10 mM each) was observed in the TNBC cell subtype MDA-MB-231, according to the evaluation of its viability and colony formation. Partial inhibitory effects were observed for either of the drugs in the HER2+ cell subtype SK-BR-3. The effects of metformin and aspirin partly relied on cyclooxygenase-2 (COX-2) upregulation, without the production of lipoxins. In silico, metformin and aspirin bound to the ERα receptor with the same energy. Conclusion We have provided novel evidence on the mechanisms of action of aspirin and metformin in breast cancer cells, showing favorable outcomes for these drugs in the ER+ and TNBC subtypes.

Transient Disruption of Adenosine Signaling During Embryogenesis Triggers a Pro-epileptic Phenotype in Adult Zebrafish.

Adenosinergic signaling has important effects on brain function, anatomy, and physiology in both late and early stages of development. Exposure to caffeine, a non-specific blocker of adenosine receptor, has been indicated as a developmental risk factor. Disruption of adenosinergic signaling during early stages of development can change the normal neural network formation and possibly lead to an increase in susceptibility to seizures. In this work, morpholinos (MO) temporarily blocked the translation of adenosine receptor transcripts, adora1, adora2aa, and adora2ab, during the embryonic phase of zebrafish. It was observed that the block of adora2aa and adora2aa + adora2ab transcripts increased the mortality rate and caused high rate of malformations. To test the susceptibility of MO adora1, MO adora2aa, MO adora2ab, and MO adora2aa + adora2ab animals to seizure, pentylenetetrazole (10 mM) was used as a convulsant agent in larval and adult stages of zebrafish development. Although no MO promoted significant differences in latency time to reach the seizures stages in 7-day-old larvae, during the adult stage, all MO animals showed a decrease in the latency time to reach stages III, IV, and V of seizure. These results indicated that transient interventions in the adenosinergic signaling through high affinity adenosine receptors during embryonic development promote strong outcomes on survival and morphology. Additionally, long-term effects on neural development can lead to permanent impairment on neural signaling resulting in increased susceptibility to seizure.

Nickel exposure alters behavioral parameters in larval and adult zebrafish.

Nickel is a heavy metal that, at high concentrations, leads to environmental contamination and causes health problems. We evaluated the effects of NiCl2 exposure on cognition and behavior in larval and adult zebrafish. Larval and adult zebrafish were exposed to NiCl2 concentrations (0.025, 2.0, 5.0, and 15.0mg/L) or water (control) in two treatment regimens: acute and subchronic. Larvae were exposed to NiCl2 for 2h (acute treatment: 5-day-old larvae treated for 2h, tested after treatment) or 11days (subchronic treatment: 11-day-old larvae treated since fertilization, tested at 5, 8 and 11days post-fertilization, dpf). Adults were exposed for 12h (acute treatment) or 96h (subchronic treatment) and were tested after the treatment period. In both regimens, exposed zebrafish showed concentration-dependent increases in body nickel levels compared with controls. For larvae, delayed hatching, decreased heart rate and morphological alterations were observed in subchronically treated zebrafish. Larvae from subchronic treatment tested at 5dpf decrease distance and mean speed at a low concentration (0.025mg/L) and increased at higher concentrations (5.0 and 15.0mg/L). Subchronic treated larvae decrease locomotion at 15.0mg/L at 8 and 11dpf, whereas decreased escape responses to an aversive stimulus was observed at 2.0, 5.0 and 15.0mg/L in all developmental stages. For adults, the exploratory behavior test showed that subchronic nickel exposure induced anxiogenic-like behavior and decrease aggression, whereas impaired memory was observed in both treatments. These results indicate that exposure to nickel in early life stages of zebrafish leads to morphological alterations, avoidance response impairment and locomotor deficits whereas acute and subchronic exposure in adults resulst in anxiogenic effects, impaired memory and decreased aggressive behavior. These effects may be associated to neurotoxic actions of nickel and suggest this metal may influence animals' physiology in doses that do not necessarily impact their survival.

Presenilin-1 Targeted Morpholino Induces Cognitive Deficits, Increased Brain Aß1-42 and Decreased Synaptic Marker PSD-95 in Zebrafish Larvae.

Presenilins are transmembrane proteases required for the proteolytic cleavage of Notch and also act as the catalytic core of the γ-secretase complex, which is responsible for the final cleavage of the amyloid precursor protein into Amyloid-ß (Aß) peptides of varying lengths. Presenilin-1 gene (psen1) mutations are the main cause of early-onset autosomal-dominant Familial Alzheimer Disease. Elucidating the roles of Presenilin-1 and other hallmark proteins involved in Alzheimer's disease is crucial for understanding the disease etiology and underlying molecular mechanisms. In our study, we used a morpholino antisense nucleotide that targets exon 8 splicing site of psen1 resulting in a dominant negative protein previously validated to investigate behavioral and molecular effects in 5 days post fertilization (dpf) zebrafish larvae. Morphants showed specific cognitive deficits in two optomotor tasks and morphological phenotypes similar to those induced by suppression of Notch signaling pathway. They also had increased mRNA levels of neurog1 at 5 dpf, confirming the potential interaction of Presenilin-1 and Notch in our model. We also evaluated levels of apoptotic markers including p53, PAR-4, Caspase-8 and bax-alpha and found only bax-a decreased at 5dpf. Western Blot analysis showed an increase in Aß1-42 and a decrease in the selective post-synaptic marker PSD-95 at 5 dpf. Our data demonstrates that psen1 splicing interference induces phenotypes that resemble early-stage AD, including cognitive deficit, Aß1-42 accumulation and synaptic reduction, reinforcing the potential contribution of zebrafish larvae to studies of human brain diseases.

Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme.

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van't Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

Manganese(II) chloride alters behavioral and neurochemical parameters in larvae and adult zebrafish.

Manganese (Mn) is an essential metal for organisms, but high levels can cause serious neurological damage. The aim of this study was to evaluate the effects of MnCl2 exposure on cognition and exploratory behavior in adult and larval zebrafish and correlate these findings with brain accumulation of Mn, overall brain tyrosine hydroxylase (TH) levels, dopamine (DA) levels, 3,4-dihydroxyphenylacetic acid (DOPAC) levels and cell death markers in the nervous system. Adults exposed to MnCl2 for 4days (0.5, 1.0 and 1.5mM) and larvae exposed for 5days (0.1, 0.25 and 0.5mM) displayed decreased exploratory behaviors, such as distance traveled and absolute body turn angle, in addition to reduced movement time and an increased number of immobile episodes in larvae. Adults exposed to MnCl2 for 4days showed impaired aversive long-term memory in the inhibitory avoidance task. The overall brain TH levels were elevated in adults and larvae evaluated at 5 and 7 days post-fertilization (dpf). Interestingly, the protein level of this enzyme was decreased in larval animals at 10dpf. Furthermore, DOPAC levels were increased in adult animals exposed to MnCl2. Protein analysis showed increased apoptotic markers in both the larvae and adult nervous system. The results demonstrated that prolonged exposure to MnCl2 leads to locomotor deficits that may be associated with damage caused by this metal in the CNS, particularly in the dopaminergic system.

Retalho deslocado coronalmente com a utilização de enxerto conjuntivo subepitelial e Emdogain para recobrimento radicular ­ controle clínico de cinco anos / Coronally repositioned flap with subepithelial connective tissue graft and Emdogain for root coverage ­ a 5 year follow-up case report

Uma situação desafiadora na Odontologia é a recessão gengival. Seu tratamento deve ser capaz de promover um completo recobrimento do defeito da recessão com mínima profundidade de sondagem pós-tratamento, além de promover resultados de cor e textura na área recoberta compatível com a dos tecidos moles adjacentes. Algumas situações clínicas prévias devem ser observadas quando do exame do paciente, dentre elas: a etiologia da lesão, o grau de classificação e a existência de lesões cariosas na raiz exposta. Uma abordagem previsível para o recobrimento radicular é o retalho deslocado coronalmente associado ao enxerto de tecido conjuntivo subepitelial. As proteínas derivadas da matriz do esmalte vêm sendo utilizadas associadas a técnicas de recobrimento com o intuito de aumentar o potencial regenerativo da região. Neste relato, com controle clínico de cinco anos, foi realizada a recuperação do tecido gengival perdido na região do dente 31, com o uso da técnica do retalho deslocado coronalmente associado ao enxerto de tecido conjuntivo subepitelial e ao Emdogain. Esta associação resultou em um importante ganho de inserção, além da melhora estética na região para o paciente.

A challenging situation in dentistry is gingival recession. Its treatment should be able to promote a complete coverage of the recession defect with minimum post-treatment probing depth, in addition to promoting color and texture results in the covered area compatible with the adjacent soft tissue. Some previous medical conditions must be observed when examining the patient, among them the etiology, the degree of classifi cation and the existence of caries on the exposed root. A predictable approach to root coverage is the coronally repositioned flap associated with subepithelial connective tissue grafting. The enamel derived matrix has been associated with coverage techniques in order to increase the regenerative potential. In this 5 year case report, the lost gingival tissue was recovered at the region of tooth 31 using a coronally advanced fl ap associated to STG and Emdogain. There were esthetic improvements and better attachment levels observed in the clinical results.

2-(Quinolin-4-yloxy)acetamides Are Active against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains.

2-(Quinolin-4-yloxy)acetamides have been described as potent in vitro inhibitors of Mycobacterium tuberculosis growth. Herein, additional chemical modifications of lead compounds were carried out, yielding highly potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.05 µM. Further, the synthesized compounds were active against drug-resistant strains and were devoid of apparent toxicity to Vero and HaCat cells (IC50s ≥ 20 µM). In addition, the 2-(quinolin-4-yloxy)acetamides showed intracellular activity against the bacilli in infected macrophages with action similar to rifampin, low risk of drug-drug interactions, and no sign of cardiac toxicity in zebrafish (Danio rerio) at 1 and 5 µM. Therefore, these data indicate that this class of compounds may furnish candidates for future development to, hopefully, provide drug alternatives for tuberculosis treatment.

Brain intraventricular injection of amyloid-ß in zebrafish embryo impairs cognition and increases tau phosphorylation, effects reversed by lithium.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder with no effective treatment and commonly diagnosed only on late stages. Amyloid-ß (Aß) accumulation and exacerbated tau phosphorylation are molecular hallmarks of AD implicated in cognitive deficits and synaptic and neuronal loss. The Aß and tau connection is beginning to be elucidated and attributed to interaction with different components of common signaling pathways. Recent evidences suggest that non-fibrillary Aß forms bind to membrane receptors and modulate GSK-3ß activity, which in turn phosphorylates the microtubule-associated tau protein leading to axonal disruption and toxic accumulation. Available AD animal models, ranging from rodent to invertebrates, significantly contributed to our current knowledge, but complementary platforms for mechanistic and candidate drug screenings remain critical for the identification of early stage biomarkers and potential disease-modifying therapies. Here we show that Aß1-42 injection in the hindbrain ventricle of 24 hpf zebrafish embryos results in specific cognitive deficits and increased tau phosphorylation in GSK-3ß target residues at 5dpf larvae. These effects are reversed by lithium incubation and not accompanied by apoptotic markers. We believe this may represent a straightforward platform useful to identification of cellular and molecular mechanisms of early stage AD-like symptoms and the effects of neuroactive molecules in pharmacological screenings.

Sustained behavioral effects of lithium exposure during early development in zebrafish: involvement of the Wnt-ß-catenin signaling pathway.

Lithium has been the paradigmatic treatment for bipolar disorder since 1950s, offering prophylactic and acute efficacy against maniac and depressive episodes. Its use during early pregnancy and the perinatal period remains controversial due to reports of negative consequences on the newborn including teratogenic and neurobehavioral effects generally referred as Floppy baby syndrome. The mechanisms underlying lithium therapeutic action are still elusive but exacerbation of Wnt signaling pathway due to GSK-3 inhibition is believed to represent its main effect. In this study we evaluated the impact of lithium exposure during zebrafish embryonic and early development including behavioral and molecular characterization of Wnt-ß-catenin pathway components. Wild-type zebrafish embryos were individually treated for 72 hpf with LiCl at 0.05, 0.5 and 5mM. No significant teratogenic and embryotoxic effects were observed. At the end of treatment period western blot analysis of selected Wnt-ß-catenin system components showed increased ß-catenin and decreased N-cadherin protein levels, without significant changes in Wnt3a, supporting GSK-3 inhibition as lithium's main target. At 10 dpf 0.5 and 5mM lithium-treated larvae showed a dose-dependent decrease in locomotion among other exploratory parameters, resembling lithium-induced Floppy baby syndrome neurobehavioral symptoms in humans. At this later period previously altered proteins returned to control levels in treated groups, suggesting that the neurobehavioral effects are a lasting consequence of lithium exposure during early development. RT-qPCR analysis of ß-catenin and N-cadherin gene expression showed no effects of lithium at 3 or 10 dpf, suggesting that protein fluctuations were likely due to post-transcriptional events. Other Wnt target genes were evaluated and only discrete alterations were observed. These results suggest that zebrafish may be a valuable model for investigation of early effects of lithium that may be mediated by effects on the Wnt signaling pathway.

Cannabidiol normalizes caspase 3, synaptophysin, and mitochondrial fission protein DNM1L expression levels in rats with brain iron overload: implications for neuroprotection.

We have recently shown that chronic treatment with cannabidiol (CBD) was able to recover memory deficits induced by brain iron loading in a dose-dependent manner in rats. Brain iron accumulation is implicated in the pathogenesis of neurodegenerative diseases, including Parkinson's and Alzheimer's, and has been related to cognitive deficits in animals and human subjects. Deficits in synaptic energy supply have been linked to neurodegenerative diseases, evidencing the key role played by mitochondria in maintaining viable neural cells and functional circuits. It has also been shown that brains of patients suffering from neurodegenerative diseases have increased expression of apoptosisrelated proteins and specific DNA fragmentation. Here, we have analyzed the expression level of brain proteins involved with mitochondrial fusion and fission mechanisms (DNM1L and OPA1), the main integral transmembrane protein of synaptic vesicles (synaptophysin), and caspase 3, an apoptosis-related protein, to gain a better understanding of the potential of CBD in restoring the damage caused by iron loading in rats. We found that CBD rescued iron-induced effects, bringing hippocampal DNM1L, caspase 3, and synaptophysin levels back to values comparable to the control group. Our results suggest that iron affects mitochondrial dynamics, possibly trigging synaptic loss and apoptotic cell death and indicate that CBD should be considered as a potential molecule with memory-rescuing and neuroprotective properties to be used in the treatment of cognitive deficits observed in neurodegenerative disorders.

Antiepileptic drugs prevent changes in adenosine deamination during acute seizure episodes in adult zebrafish.

Adenosine is an endogenous modulator of brain functions, which presents anticonvulsant properties. In addition, its levels can be increased during neural injury. The modulation of extracellular adenosine levels by ectonucleotidase and adenosine deaminase (ADA) activities may represent a key mechanism in the control of epileptogenesis. In the present study, we investigated the effects of acute seizure episodes and antiepileptic drug (AED) treatments on ectonucleotidases and ADA activities in adult zebrafish brain. Our data have demonstrated that pentylenetetrazole (PTZ)-induced seizures did not alter ATP, ADP, and AMP hydrolysis in brain membrane fractions. However, there was a significant increase on ecto-ADA and soluble ADA activities in PTZ-treated animals immediately after a clonus-like convulsion and loss of posture, which are typical behavioral changes observed in Stage 3. Furthermore, our results have demonstrated that AED pretreatments prevented the stimulatory effect promoted by PTZ exposure on ADA activities. The PTZ and AED treatments did not promote alterations on ADA gene expression. Interestingly, when exposed to PTZ, animals pretreated with AEDs showed longer latency to reach the clonus-like seizure status, which is an effect that matches the suppression of the increase of ADA activity promoted by the AEDs. These data suggest that the adenosine deamination could be involved in the control of seizure development in zebrafish and may be modulated by AED treatments.