Blocking Pannexin 1 Channels Alleviates Peripheral Inflammatory Pain but not Paclitaxel-Induced Neuropathy.

BACKGROUND: Pannexin1 (Panx1) is a membrane channel expressed in different cells of the nervous system and is involved in several pathological conditions, including pain and inflammation. At the central nervous system, the role of Panx1 is already well-established. However, in the periphery, there is a lack of information regarding the participation of Panx1 in neuronal sensitization. The dorsal root ganglion (DRG) is a critical structure for pain processing and modulation. For this reason, understanding the molecular mechanism in the DRG associated with neuronal hypersensitivity has become highly relevant to discovering new possibilities for pain treatment. Here, we aimed to investigate the role of Panx1 in acute nociception and peripheral inflammatory and neuropathic pain by using two different approaches. METHODS: Rats were treated with a selective Panx1 blocker peptide (10Panx) into L5-DRG, followed by ipsilateral intraplantar injection of carrageenan, formalin, or capsaicin. DRG neuronal cells were pre-treated with 10Panx and stimulated by capsaicin to evaluate calcium influx. Panx1 knockout mice (Panx1-KO) received carrageenan or capsaicin into the paw and paclitaxel intraperitoneally. The von Frey test was performed to measure the mechanical threshold of rats' and mice's paws before and after each treatment. RESULTS: Pharmacological blockade of Panx1 in the DRG of rats resulted in a dose-dependent decrease of mechanical allodynia triggered by carrageenan, and nociception decreased in the second phase of formalin. Nociceptive behavior response induced by capsaicin was significantly lower in rats treated with Panx1 blockade into DRG. Neuronal cells with Panx1 blockage showed lower intracellular calcium response than untreated cells after capsaicin administration. Accordingly, Panx1-KO mice showed a robust reduction in mechanical allodynia after carrageenan and a lower nociceptive response to capsaicin. A single dose of paclitaxel promoted acute mechanical pain in wildtype (WT) but not in Panx1-KO mice. Four doses of chemotherapy promoted chronic mechanical allodynia in both genotypes, although Panx1-KO mice had significant ablation in the first eight days. CONCLUSION: Our findings suggest that Panx1 is critical for developing peripheral inflammatory pain and acute nociception involving transient receptor potential vanilloid subtype 1 (TRPV1) but is not essential for neuropathic pain chronicity.

Automated syndrome diagnosis by three-dimensional facial imaging.

PURPOSE: Deep phenotyping is an emerging trend in precision medicine for genetic disease. The shape of the face is affected in 30-40% of known genetic syndromes. Here, we determine whether syndromes can be diagnosed from 3D images of human faces. METHODS: We analyzed variation in three-dimensional (3D) facial images of 7057 subjects: 3327 with 396 different syndromes, 727 of their relatives, and 3003 unrelated, unaffected subjects. We developed and tested machine learning and parametric approaches to automated syndrome diagnosis using 3D facial images. RESULTS: Unrelated, unaffected subjects were correctly classified with 96% accuracy. Considering both syndromic and unrelated, unaffected subjects together, balanced accuracy was 73% and mean sensitivity 49%. Excluding unrelated, unaffected subjects substantially improved both balanced accuracy (78.1%) and sensitivity (56.9%) of syndrome diagnosis. The best predictors of classification accuracy were phenotypic severity and facial distinctiveness of syndromes. Surprisingly, unaffected relatives of syndromic subjects were frequently classified as syndromic, often to the syndrome of their affected relative. CONCLUSION: Deep phenotyping by quantitative 3D facial imaging has considerable potential to facilitate syndrome diagnosis. Furthermore, 3D facial imaging of "unaffected" relatives may identify unrecognized cases or may reveal novel examples of semidominant inheritance.

Quinolone-N-acylhydrazone hybrids as potent Zika and Chikungunya virus inhibitors.

This work reports the synthesis of quinolone-N-acylhydrazone hybrids, namely 6-R-N'-(2-hydxoxybenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (R = H: 5a, F: 5b, Cl: 5c and Br: 5d), which exhibited excellent activity against arbovirus Zika (ZIKV) and Chikungunya (CHIKV). In vitro screening towards ZIKV and CHIKV inhibition revealed that all substances have significant antiviral activity, most of them being more potent than standard Ribavirin (5a-d: EC50 = 0.75-0.81 µM, Ribavirin: EC50 = 3.95 µM for ZIKV and 5a-d: 1.16-2.85 µM, Ribavirin: EC50 = 2.42 µM for CHIKV). The quinolone-N-acylhydrazone hybrids were non-toxic against Vero cells, in which compounds 5c and 5d showed the best selectivities (SI = 1410 and 630 against ZIKV and CHIKV, respectively). Antiviral activity was identified by inhibition of viral RNA production in a dose-dependent manner. In the evaluation of the time of addition of the compounds, we observed that 5b and 5c remain with strong effect even in the addition for 12 h after infection. The above results indicate that quinolone-N-acylhydrazones represent a new and promising class to be further investigated as anti-ZIKV and anti-CHIKV agents.

Oral thermosensing by murine trigeminal neurons: modulation by capsaicin, menthol and mustard oil.

KEY POINTS: Orosensory thermal trigeminal afferent neurons respond to cool, warm, and nociceptive hot temperatures with the majority activated in the cool range. Many of these thermosensitive trigeminal orosensory afferent neurons also respond to capsaicin, menthol, and/or mustard oil (allyl isothiocyanate) at concentrations found in foods and spices. There is significant but incomplete overlap between afferent trigeminal neurons that respond to oral thermal stimulation and to the above chemesthetic compounds. Capsaicin sensitizes warm trigeminal thermoreceptors and orosensory nociceptors; menthol attenuates cool thermoresponses. ABSTRACT: When consumed with foods, mint, mustard, and chili peppers generate pronounced oral thermosensations. Here we recorded responses in mouse trigeminal ganglion neurons to investigate interactions between thermal sensing and the active ingredients of these plants - menthol, allyl isothiocyanate (AITC), and capsaicin, respectively - at concentrations found in foods and commercial hygiene products. We carried out in vivo confocal calcium imaging of trigeminal ganglia in which neurons express GCaMP3 or GCAMP6s and recorded their responses to oral stimulation with thermal and the above chemesthetic stimuli. In the V3 (oral sensory) region of the ganglion, thermoreceptive neurons accounted for ∼10% of imaged neurons. We categorized them into three distinct classes: cool-responsive and warm-responsive thermosensors, and nociceptors (responsive only to temperatures ≥43-45 °C). Menthol, AITC, and capsaicin also elicited robust calcium responses that differed markedly in their latencies and durations. Most of the neurons that responded to these chemesthetic stimuli were also thermosensitive. Capsaicin and AITC increased the numbers of warm-responding neurons and shifted the nociceptor threshold to lower temperatures. Menthol attenuated the responses in all classes of thermoreceptors. Our data show that while individual neurons may respond to a narrow temperature range (or even bimodally), taken collectively, the population is able to report on graded changes of temperature. Our findings also substantiate an explanation for the thermal sensations experienced when one consumes pungent spices or mint.

Mechanism of metformin action in MCF-7 and MDA-MB-231 human breast cancer cells involves oxidative stress generation, DNA damage, and transforming growth factor ß1 induction.

The participation of oxidative stress in the mechanism of metformin action in breast cancer remains unclear. We investigated the effects of clinical (6 and 30 µM) and experimental concentrations of metformin (1000 and 5000 µM) in MCF-7 and in MDA-MB-231 cells, verifying cytotoxicity, oxidative stress, DNA damage, and intracellular pathways related to cell growth and survival after 24 h of drug exposure. Clinical concentrations of metformin decreased metabolic activity of MCF-7 cells in the MTT assay, which showed increased oxidative stress and DNA damage, although cell death and impairment in the proliferative capacity were observed only at higher concentrations. The reduction in metabolic activity and proliferation in MDA-MB-231 cells was present only at experimental concentrations after 24 h of drug exposition. Oxidative stress and DNA damage were induced in this cell line at experimental concentrations. The drug decreased cytoplasmic extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT and increased nuclear p53 and cytoplasmic transforming growth factor ß1 (TGF-ß1) in both cell lines. These findings suggest that metformin reduces cell survival by increasing reactive oxygen species, which induce DNA damage and apoptosis. A relationship between the increase in TGF-ß1 and p53 levels and the decrease in ERK1/2 and AKT was also observed. These findings suggest the mechanism of action of metformin in both breast cancer cell lineages, whereas cell line specific undergoes redox changes in the cells in which proliferation and survival signaling are modified. Taken together, these results highlight the potential clinical utility of metformin as an adjuvant during the treatment of luminal and triple-negative breast cancer.

New insights into 3-(aminomethyl)naphthoquinones: Evaluation of cytotoxicity, electrochemical behavior and search for structure-activity correlation.

Herein we describe the structure-activity relationship of a large library of Mannich bases (MBs) synthesized from 2-hydroxy-1,4-naphthoquinone. In general, the compounds have shown high to moderate activity against the HL-60 (promyelocytic leukaemia) cell line with IC50=1.1-19.2µM. Our results suggest that the nature of the aryl moiety introduced in the structure of MBs by the aldehyde component is crucial to the cytotoxicity, and although the group originated from the primary amine has a lesser influence, aromatic ones were found to suppress the activity. Thus, MBs derived from salicylaldehydes or 2-pyridinecarboxaldehyde and aliphatic amines are the most active compounds. A satisfactory correlation of the EpIIc versus IC50 (µM) in dimethylsulfoxide was observed. The most cytotoxic MBs (Series a-c, derived from salicylaldehydes) showed the least negative EpIIc values. Noteworthy, however, Series d (derived from 2-pyridinecarboxaldehyde) did not follow this correlation. They exhibited both the lowest IC50 and the most negative EpIIc values, thus suggesting that other factors also influence the cytotoxicity of the MBs, such as lipophilicity, electronic distribution and hydrogen bonding.

Merozoite surface protein-1 genetic diversity in Plasmodium malariae and Plasmodium brasilianum from Brazil.

BACKGROUND: The merozoite surface protein 1 (MSP1) gene encodes the major surface antigen of invasive forms of the Plasmodium erythrocytic stages and is considered a candidate vaccine antigen against malaria. Due to its polymorphisms, MSP1 is also useful for strain discrimination and consists of a good genetic marker. Sequence diversity in MSP1 has been analyzed in field isolates of three human parasites: P. falciparum, P. vivax, and P. ovale. However, the extent of variation in another human parasite, P. malariae, remains unknown. This parasite shows widespread, uneven distribution in tropical and subtropical regions throughout South America, Asia, and Africa. Interestingly, it is genetically indistinguishable from P. brasilianum, a parasite known to infect New World monkeys in Central and South America. METHODS: Specific fragments (1 to 5) covering 60 % of the MSP1 gene (mainly the putatively polymorphic regions), were amplified by PCR in isolates of P. malariae and P. brasilianum from different geographic origin and hosts. Sequencing of the PCR-amplified products or cloned PCR fragments was performed and the sequences were used to construct a phylogenetic tree by the maximum likelihood method. Data were computed to give insights into the evolutionary and phylogenetic relationships of these parasites. RESULTS: Except for fragment 4, sequences from all other fragments consisted of unpublished sequences. The most polymorphic gene region was fragment 2, and in samples where this region lacks polymorphism, all other regions are also identical. The low variability of the P. malariae msp1 sequences of these isolates and the identification of the same haplotype in those collected many years apart at different locations is compatible with a low transmission rate. We also found greater diversity among P. brasilianum isolates compared with P. malariae ones. Lastly, the sequences were segregated according to their geographic origins and hosts, showing a strong genetic and geographic structure. CONCLUSIONS: Our data show that there is a low level of sequence diversity and a possible absence of allelic dimorphism of MSP1 in these parasites as opposed to other Plasmodium species. P. brasilianum strains apparently show greater divergence in comparison to P. malariae, thus P. malariae could derive from P. brasilianum, as it has been proposed.

Synthetic aminomethylnaphthoquinones inhibit the in vitro replication of bovine herpesvirus 5.

Bovine herpesvirus type 5 (BoHV-5) is an etiologic agent of meningoencephalitis in cattle. The aim of this study was to evaluate the antiviral potential of a series of synthetic Mannich bases derived from lawsone and to investigate at which stage of the BoHV-5 replicative cycle the compounds might be acting. The most potent and selective inhibitor exhibited CC50 and EC50 values of 1867 µM ± 8.3 and 3.8 µM ± 1.2, respectively (ACV: 989 µM ± 2 and 166 µM ± 2, respectively).

Dosage-dependent effects of FGFR2W290R mutation on craniofacial shape and cellular dynamics of the basicranial synchondroses.

Craniosynostosis is a common yet complex birth defect, characterized by premature fusion of the cranial sutures that can be syndromic or nonsyndromic. With over 180 syndromic associations, reaching genetic diagnoses and understanding variations in underlying cellular mechanisms remains a challenge. Variants of FGFR2 are highly associated with craniosynostosis and warrant further investigation. Using the missense mutation FGFR2W290R , an effective mouse model of Crouzon syndrome, craniofacial features were analyzed using geometric morphometrics across developmental time (E10.5-adulthood, n = 665 total). Given the interrelationship between the cranial vault and basicranium in craniosynostosis patients, the basicranium and synchondroses were analyzed in perinates. Embryonic time points showed minimal significant shape differences. However, hetero- and homozygous mutant perinates and adults showed significant differences in shape and size of the cranial vault, face, and basicranium, which were associated with cranial doming and shortening of the basicranium and skull. Although there were also significant shape and size differences associated with the basicranial bones and clear reductions in basicranial ossification in cleared whole-mount samples, there were no significant alterations in chondrocyte cell shape, size, or orientation along the spheno-occipital synchondrosis. Finally, shape differences in the cranial vault and basicranium were interrelated at perinatal stages. These results point toward the possibility that facial shape phenotypes in craniosynostosis may result in part from pleiotropic effects of the causative mutations rather than only from the secondary consequences of the sutural defects, indicating a novel direction of research that may shed light on the etiology of the broad changes in craniofacial morphology observed in craniosynostosis syndromes.

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).

Intravenous administration of mesenchymal stem cells reduces Tau phosphorylation and inflammation in the 3xTg-AD mouse model of Alzheimer's disease.

Mesenchymal stem cell (MSC) administration is a novel and promising therapeutic approach for Alzheimer's disease (AD). Focusing on an intervention easily translatable into clinical practice, we administered allogeneic bone marrow-derived MSCs intravenously in a mouse model of AD (3xTg-AD). We systematically evaluated the effects of a single-dose and multiple-doses of MSCs in young and old mice (5 or 10 months old), comparing the short-term and long-term effects after 1, 2, or 7 months of treatment. A single dose of MSCs in young mice attenuated neuroinflammation 1 and 7 months after injection, whereas multiple-doses did not show any effect. Multiple-doses of MSCs (administered at 5 to 12 mo, or 10 to 12 mo) reduced the ß-secretase cleavage of the amyloid precursor protein, although levels of Aß-42 did not change. Most interestingly, multiple doses of MSCs affected tau hyperphosphorylation. MSCs administered in young mice for 7 months decreased the pathological tau phosphorylation at T205, S214, and T231. MSCs administered in old mice for 2 months decreased tau phosphorylation at S396. Our findings show how different timing and frequency of MSC injections can affect and modulate several aspects of the AD-like neuropathology in the 3xTg-AD mouse model, strengthening the concept of fine-tuning MSC therapy for Alzheimer's disease.

Cytotoxicity evaluation and DNA interaction of RuII-bipy complexes containing coumarin-based ligands.

Although there are various treatment options for cancer, this disease still has caused an increasing number of deaths, demanding more efficient, selective and less harmful drugs. Several classes of ruthenium compounds have been investigated as metallodrugs for cancer, mainly after the entry of imidazolH [trans-RuCl4-(DMSO-S)(imidazole)] (NAMI-A) and indazolH [trans-RuCl4-(Indazol)2] (KP1019) in clinical trials. In this sense, RuII complexes with general formula [Ru(L1-3)(bipy)2]PF6 (1-3) (L1 = ethyl 3-(6-methyl-2-oxo-2H-chromen-3-yl)-3-oxopropanoate, L2 = ethyl 3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-3-oxopropanoate, L3 = ethyl 3-(8-methoxy-2-oxo-2H-chromen-3-yl)-3-oxopropanoate and bipy = bipyridine) have been synthesized. The crystal structure of 2 revealed that the RuII atom lies on a distorted octahedral geometry with the deprotonated ligand (L2-) coordinated through ß-ketoester group oxygen atoms. In vitro cytotoxic activity of the compounds was evaluated against 4T1 (murine mammary carcinoma) and B16-F10 (murine metastatic melanoma) tumor cells, and the non-tumor cell line BHK-21 (baby hamster kidney). Coordination with RuII resulted in expressive enhancement of cytotoxic activity. The precursors were inactive below 100 µM and the final RuII complexes (1-3) showed IC50 ranging from 2.0 to 12.8 µM; 2 being the most potent compound. DNA interaction studies revealed a greater capacity of the complexes to interact with DNA than the ligands, where, 2 exhibited the highest Kb constant of 2.2 × 104 M-1. Fluorescence investigation demonstrated that 1-3 are capable of quenching the fluorescence emission of the EtdBr-DNA complex up to 40%. Molecular docking showed that the interaction of 1-3 between the DNA base pairs from the coumarin portion was with scores of 67.28, 68.62 and 64.88, respectively, and 75.45 for ellipticine, suggesting an intercalative mode of binding. Our findings show that the RuII complexes are eligible for continuing to be investigated as potential antitumor compounds.

Peripheral Inflammatory Hyperalgesia Depends on P2X7 Receptors in Satellite Glial Cells.

Peripheral inflammatory hyperalgesia depends on the sensitization of primary nociceptive neurons. Inflammation drives molecular alterations not only locally but also in the dorsal root ganglion (DRG) where interleukin-1 beta (IL-1ß) and purinoceptors are upregulated. Activation of the P2X7 purinoceptors by ATP is essential for IL-1ß maturation and release. At the DRG, P2X7R are expressed by satellite glial cells (SGCs) surrounding sensory neurons soma. Although SGCs have no projections outside the sensory ganglia these cells affect pain signaling through intercellular communication. Therefore, here we investigated whether activation of P2X7R by ATP and the subsequent release of IL-1ß in DRG participate in peripheral inflammatory hyperalgesia. Immunofluorescent images confirmed the expression of P2X7R and IL-1ß in SGCs of the DRG. The function of P2X7R was then verified using a selective antagonist, A-740003, or antisense for P2X7R administered in the L5-DRG. Inflammation was induced by CFA, carrageenan, IL-1ß, or PGE2 administered in rat's hind paw. Blockage of P2X7R at the DRG reduced the mechanical hyperalgesia induced by CFA, and prevented the mechanical hyperalgesia induced by carrageenan or IL-1ß, but not PGE2. It was also found an increase in P2X7 mRNA expression at the DRG after peripheral inflammation. IL-1ß production was also increased by inflammatory stimuli in vivo and in vitro, using SGC-enriched cultures stimulated with LPS. In LPS-stimulated cultures, activation of P2X7R by BzATP induced the release of IL-1ß, which was blocked by A-740003. In summary, our data suggest that peripheral inflammation leads to the activation of P2X7R expressed by SGCs at the DRG. Then, ATP-induced activation of P2X7R mediates the release of IL-1ß from SGC. This evidence places the SGC as an active player in the establishment of peripheral inflammatory hyperalgesia and highlights the importance of the events in DRG for the treatment of inflammatory diseases.

Diabetes-induced Neuropathic Mechanical Hyperalgesia Depends on P2X4 Receptor Activation in Dorsal Root Ganglia.

Peripheral diabetic neuropathy (PDN) manifests in 50-60% of type I and II diabetic patients and is the major cause of limb amputation. Adequate therapy for PDN is a current challenge. There are evidences that the activation of the P2X4 receptor (P2X4R) expressed on microglial cells of the central nervous system takes part in the development of neuropathic pain. However, there is an open question: Is P2X4R activation on dorsal root ganglia (DRG) involved in the development of neuropathic pain? To answer this question, this study verified the involvement of P2X4R expressed in DRG cells on diabetes-induced neuropathic mechanical hyperalgesia in rats. We found that intrathecal or ganglionar (L5-DRG) administration of a novel P2X4R antagonist (PSB-15417) or intrathecal administration of oligodeoxynucleotides (ODN)-antisense against the P2X4R reversed diabetes-induced neuropathic mechanical hyperalgesia. The DRG of the diabetic neuropathic rats showed an increase in P2X4R expression, and the DRG immunofluorescence suggested that P2X4R is expressed mainly in satellite glial cells (SGC). Finally, our study showed a functional expression of P2X4R in SGCs of the rat's DRG, because the P2X4R agonist BzATP elicits an increase in intracellular calcium concentration in SGCs, which was reduced by PSB-15417. These findings indicate that P2X4R activation in DRG is essential to diabetes-induced neuropathic mechanical hyperalgesia. Therefore, this purinergic receptor in DRG could be an interesting therapeutic target for quaternary P2X4R antagonists that do not cross the hematoencephalic barrier, for the control of neuropathic pain, preserving central nervous system functions.

Participation of satellite glial cells of the dorsal root ganglia in acute nociception.

At dorsal root ganglia, neurons and satellite glial cells (SGC) can communicate through ATP release and P2X7 receptor activation. SGCs are also interconnected by gap junctions and have been previously implicated in modulating inflammatory and chronic pain.We now present evidence that SGCs are also involved in processing acute nociception in rat dorsal root ganglia. Using primary dorsal root ganglia cultures we observed that calcium transients induced in neurons by capsaicin administration were followed by satellite glial cells activation. Only satellite glial cells response was reduced by administration of the P2X7 receptor antagonist A740003. In vivo, acute nociception induced by intraplantar injection of capsaicin in rats was inhibited by A740003 or by the gap junction blocker carbenoxolone administered at the dorsal root ganglia (L5 level). Both drugs also reduced the second phase of the formalin test. These results suggest that communication between neurons and satellite glial cells is not only involved in inflammatory or pathological pain, but also in the transmission of the nociceptive signal, possibly in situations involving C-fiber activation.

Early menarche and teenager pregnancy as risk factors for morbid obesity among reproductive-age women: A case-control study.

OBJECTIVES: The aim of this study was to evaluate potential risk factors, including non-communicable diseases, for morbid obesity in women between 20 and 49 years of age. METHODS: We performed a case-control study with 110 morbidly obese women and 110 women with adequate weight who were matched by age and with a 1:1 case to control ratio. All women were between 20 to 49 years old and non-menopausal. Possible risk factors were evaluated through a self-report questionnaire assessing socio-demographic, obstetric and gynecological characteristics, presence of non-communicable diseases and habits. Multiple logistic regression was used to estimate the odds ratio with respective confidence intervals. RESULTS: Menarche under 12 years old, teenage pregnancy and lower educational level were shown to be risk factors for morbid obesity among women of reproductive age. Incidences of non-communicable diseases (diabetes, hypertension, dyslipidemia, liver disease, lung disease, thyroid dysfunction, and joint pain) were increased in women with morbid obesity. CONCLUSIONS: Early menarche, teenage pregnancy and low education level are risk factors for the occurrence of morbid obesity in women of reproductive age. Some non-communicable diseases were already more prevalent in women with morbid obesity even before 50 years of age.

Novel CoIII complexes containing fluorescent coumarin-N-acylhydrazone hybrid ligands: Synthesis, crystal structures, solution studies and DFT calculations.

A series of new CoIII complexes of the type [Co(dien)(L1-L3)]ClO4 (1-3), containing fluorescent coumarin-N-acylhydrazonate hybrid ligands, (E)-N'-(1-(7-oxido-2-oxo-2H-chromen-3-yl)ethylidene)-4-R-benzohydrazonate [where R=H (L12-), OCH3 (L22-) or Cl (L32-)], were obtained and isolated in the low spin CoIII configuration. Single-crystal X-ray diffraction showed that the coumarin-N-acylhydrazones act as tridentate ligands in their deprotonated form (L2-). The cation (+1) complexes contain a diethylenetriamine (dien) as auxiliary ligand and their structures were calculated by DFT studies which were also performed for the CoII (S=1/2 and S=3/2) configurations. The LS CoII (S=1/2) concentrated the spin density on the O-Co-O axis while the HS CoII (S=3/2) exhibited a broad spin density distribution around the metallic center. Cyclic voltammetry studies showed that structural modifications made in the L2- ligands caused a slight influence on the electronic density of the metal center, and the E1/2 values for the CoIII/CoII redox couple increased following the electronic effect of the R-substituent, in the order: 2 (R=OCH3)<1 (R=H)<3 (R=Cl). The theoretical redox potentials (E°) of the process CoIII→CoII were calculated for both CoII spin states (S=1/2 and S=3/2) and a better correlation was found for CoIII→CoII (S=1/2), compared with experimental values vs SHE (E°calc=-0.37, -0.36 and -0.32V vs E°exp.=-0.371, -0.406 and -0.358V, for 1-3 respectively). Complexes 1-3 exhibited a very intense absorption band around 470nm, assigned by DFT calculations as π-π* transitions from the delocalized coumarin-N-acylhydrazone system. 1-3 were very stable in MeOH for several days. Likewise, 1-3 were stable in phosphate buffer containing sodium ascorbate after 15h, which was attributed to the high chelate effect and σ-donor ability of the L2- and dien ligands.

Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

Malaria outside the Amazon region: natural Plasmodium infection in anophelines collected near an indigenous village in the Vale do Rio Branco, Itanhaém, SP, Brazil.

A few cases of Plasmodium vivax malaria in which anophelines of subgenus Kerteszia were incriminated as vectors have been reported outside the Amazon region, in the Atlantic Forest. This study was carried out near an indigenous Guarani village in the Curucutu reserve, an environmental protection area in the municipality of Itanhaém in the state of São Paulo, Brazil, on November 30, 2009, February 18, 2010, April 29, 2010 and May 26, 2010. Mosquitoes were collected along the route to the Guarani village where the edge of the Branco river floodplain meets the forests on the mountain slopes. Adult forms were collected with CO(2)-baited CDC traps and Shannon traps from twilight to 10:00 P.M. Anopheles cruzii predominated in both traps. The other species collected in the CDC traps were An. pseudomaculipes/maculipes, An. fluminensis and An. mediopunctatus/forattinii/costai. In addition to the latter three species, An. apicimacula/intermedius and An. strodei were also found in the Shannon traps. All but An. cruzii and An. strodei belong to subgenus Anopheles. A total of 506 mosquitoes were assayed by PCR to detect natural infection by Plasmodium species. In the CDC traps, An. fluminensis and An. pseudomaculipes/maculipes were positive for Plasmodium malariae, while in the Shannon traps An. pseudomaculipes/maculipes was positive for Plasmodium vivax and Plasmodium malariae and An. cruzii was positive for P. malariae, resulting in a minimum infection rate of 0.24%. Our findings suggest that An. cruzii may be incriminated in the transmission of malaria between monkeys and humans, as this species was found to be infected by P. malariae. They also highlight the need for an understanding of the role of anophelines from outside subgenus Kerteszia in the transmission of malaria in the Atlantic Forest, as these were also found to be naturally infected by P. vivax and P. malariae.