Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples.

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples.

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

Saúde mental e restrição de liberdade: relato de experiência como médica psiquiatra em centro de detenção de refugiados / Mental health and deprivation of liberty: experience report as a psychiatrist in a refugee detention center

RESUMO O relato de experiência refere-se ao trabalho da autora, por seis meses, como psiquiatra por Médicos Sem Fronteiras (MSF) em centro de detenção de refugiados localizado em Nauru, um país insular da Oceania. O estudo traz descrições do trabalho de campo e reflexões teóricas. Discute-se a temática do suicídio e a especificidade dessa questão para o grupo de refugiados e solicitantes de asilo atendidos, assim como relata-se a descoberta de um novo diagnóstico clínico chamado síndrome de resignação. Busca-se ainda refletir sobre o papel dos profissionais de saúde mental diante de situações de privação de liberdade e discutem-se impasses éticos, vividos em campo, com relação à política de imigração australiana, sua importância econômica para Nauru e o impacto na população de refugiados; impasses esses vividos até a expulsão da equipe de MSF pelo governo local em 05 de outubro de 2018.

ABSTRACT The experience report refers to the author's work, for six months, as a psychiatrist for Médecins Sans Frontières (Doctors Without Borders - MSF) in a refugee detention center in Nauru, an island country in Oceania. The report provides descriptions of the field work and theoretical reflections. The author discusses the issue of suicide and the specificity of such issue for the group of refugees and asylum seekers attended, as well as the discovery of a new clinical diagnosis called resignation syndrome. It's also sought to reflect on the role of mental health professionals in dealing with deprivation of liberty and discusses ethical challenges experienced in the field, regarding Australia's refugee policy, its economic importance to Nauru and the impact on the refugee population; obstacles encountered until MSF team was expelled by the local government on October 5th, 2018.

Violência urbana e saúde mental: por narrativas em primeira pessoa / Urban violence and mental health: first person narratives

Resumo O artigo apresenta um estudo qualitativo acerca da temática violência urbana e saúde mental através das narrativas de pacientes de uma clínica da família localizada em área muito violenta. Empregou-se a metodologia da Medicina Narrativa e foram entrevistadas três pacientes da clínica da família, selecionadas pelo seu pedido de cuidado em saúde mental e intensa experiência com violência. São analisadas as narrativas, que foram gravadas, e depreendidos elementos que podem indicar os agravos proporcionados pela experiência violenta à saúde mental das entrevistadas, a saber, trauma, "desterritorialização" afetiva, prolongamento do luto, intervenção do poder dos códigos da violência na autonomia subjetiva e identitária.

Abstract The article presents a qualitative study about the theme of urban violence and mental health through the narratives of patients of a family clinic located in a very violent area. The Narrative Medicine methodology was used, and three patients from the family clinic were interviewed, selected for their request for mental health care and intense experience with violence. The narratives are analyzed, recorded, and pointed the elements that may indicate the harms provided by the violent experience to the mental health of the interviewees, namely trauma, affective "deterritorialization", prolongation of mourning, and intervention of the power of violence codes in subjective and identity autonomy.

Viajando não, só sonhando! / I'm just dreaming

O artigo traz um relato de caso, com discussão teórica, abordando o percurso de um jovem psicótico através de um Centro de Reabilitação Psicossocial (Centro de Atenção Psicossocial Infanto-Juvenil - CAPSIJ/CARIM/IPUB/UFRJ). O jovem tem história de longa institucionalização psiquiátrica, após a qual começou um outro tratamento oferecido pelo Centro e caracterizado por uma proposta de atenção diária. São apresentados momentos desse trabalho e as falas do jovem, sempre com uma articulação teórica, feita a partir de referenciais psicanalíticos, filosóficos e de psicoterapia institucional. Estes momentos ilustram a riqueza de seu quadro, demonstrando que a longa institucionalização do jovem foi, absolutamente, desnecessária.(AU)

This article presents the trajectory of a psychotic adolescent in a Psychosocial Rehabilitation Service (CAPSIJ / CARIM / IPUB / UFRJ). He lived for a long time in a Mental Health Institution and, after this period he initiated a new treatment in the CAPSIJ. This treatment proposed a daily care and in the article some important moments of his trajectory are presented and discussed through psychoanalytic and philosophical aspects and through institutional psychotherapy ideas. These moments presented show the importance and the beauty of his trajectory and also emphasize how the long time he passed in a Mental Health Institution was completely unnecessary .(AU)

Viajando não, só sonhando! / I'm just dreaming

O artigo traz um relato de caso, com discussão teórica, abordando o percurso de um jovem psicótico através de um Centro de Reabilitação Psicossocial (Centro de Atenção Psicossocial Infanto-Juvenil - CAPSIJ/CARIM/IPUB/UFRJ). O jovem tem história de longa institucionalização psiquiátrica, após a qual começou um outro tratamento oferecido pelo Centro e caracterizado por uma proposta de atenção diária. São apresentados momentos desse trabalho e as falas do jovem, sempre com uma articulação teórica, feita a partir de referenciais psicanalíticos, filosóficos e de psicoterapia institucional. Estes momentos ilustram a riqueza de seu quadro, demonstrando que a longa institucionalização do jovem foi, absolutamente, desnecessária.

This article presents the trajectory of a psychotic adolescent in a Psychosocial Rehabilitation Service (CAPSIJ / CARIM / IPUB / UFRJ). He lived for a long time in a Mental Health Institution and, after this period he initiated a new treatment in the CAPSIJ. This treatment proposed a daily care and in the article some important moments of his trajectory are presented and discussed through psychoanalytic and philosophical aspects and through institutional psychotherapy ideas. These moments presented show the importance and the beauty of his trajectory and also emphasize how the long time he passed in a Mental Health Institution was completely unnecessary .

Intestinal levels of anandamide and oleoylethanolamide in food-deprived rats are regulated through their precursors.

The anorectic lipid oleoylethanolamide and the orexigenic lipid anandamide both belong to the group of N-acylethanolamines that are generated by the enzyme N-acylphosphatidylethanolamine-hydrolyzing phospholipase D. The levels of the two bioactive lipids were investigated in rat intestines after 24 h of starvation as well as after 1 and 4 h of re-feeding. Total levels of precursor phospholipids and N-acylethanolamines were decreased upon food-deprivation whereas the level of the anandamide precursor molecule was significantly increased. The level of 2-arachidonoyl-glycerol was unchanged as was the activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolyzing phospholipase D, and fatty acid amide hydrolase upon starvation and re-feeding. It is concluded that remodeling of the amide-linked fatty acids of N-acylphosphatidylethanolamine is responsible for the opposite effects on levels of anandamide and oleoylethanolamide in intestines of food-deprived rats and not an alternative biochemical route for anandamide synthesis. Furthermore, linoleoylethanolamide, which accounted for more than 50 mol% of the endogenous pool of N-acylethanolamines, was found not to have the same inhibitory effect on food intake, as did oleoylethanolamide following oral administration.

Presence and potential signaling function of N-acylethanolamines and their phospholipid precursors in the yeast Saccharomyces cerevisiae.

N-acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs) are trace constituents of vertebrate cells and tissues and much is known about their metabolism and possible function in animals. Here we report for the first time the identification and quantification of NAEs and NAPEs in several strains of the yeast Saccharomyces cerevisiae. Gas chromatography-mass spectrometry of appropriate derivatives revealed 16:0, 16:1, 18:0 and 18:1 N-acyl groups in both NAE and NAPE whose levels, in wild-type cells, were 50 to 90 and 85 to 750 pmol/micromol lipid P, respectively (depending on the phase of growth). NAPE levels were reduced by 45 to 60% in a strain lacking three type B phospholipases, suggesting their involvement in NAPE synthesis by their known transacylation activity. A yeast strain lacking the YPL103c gene, which codes for a protein with 50.3% homology to human NAPE-specific phospholipase D, exhibited a 60% reduction in NAE, compared to wild-type controls. The exposure of various yeast strains to peroxidative stress, by incubation in media containing 0.6 mM H(2)O(2), resulted in substantial increases in NAE. Because yeast cells lack polyunsaturated fatty acids, they offer a useful system for the study of NAE generation and its potential signaling and cytoprotective effects in the absence of polyunsaturated ("endocannabinoid") congeners.

Endocannabinoid metabolism in human glioblastomas and meningiomas compared to human non-tumour brain tissue.

The endogenous levels of the two cannabinoid receptor ligands 2-arachidonoyl glycerol and anandamide, and their respective congeners, monoacyl glycerols and N-acylethanolamines, as well as the phospholipid precursors of N-acylethanolamines, were measured by gas chromatography-mass spectrometry in glioblastoma (WHO grade IV) tissue and meningioma (WHO grade I) tissue and compared with human non-tumour brain tissue. Furthermore, the metabolic turnover of N-acylethanolamines was compared by measurements of the enzymatic activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolysing phospholipase D and fatty acid amide hydrolase in the same three types of tissue. Glioblastomas were characterized by enhanced levels of N-acylethanolamines (eightfold, 128 +/- 59 pmol/micromol lipid phosphorus) including anandamide (17-fold, 4.6 +/- 3.1 pmol/micromol lipid phosphorus) and several species of N-acylphosphatidylethanolamines (three to eightfold). This was accompanied by a more than 60% reduction in the enzyme activities of N-acylphosphatidylethanolamine-hydrolysing phospholipase D and fatty acid amide hydrolase. By contrast, meningiomas were characterized by a massively enhanced level of 2-monoacyl glycerols (20-fold, 2293 +/- 361 pmol/micromol lipid phosphorus) including 2-arachidonoyl glycerol (20-fold, 1524 +/- 361 pmol/micromol lipid phosphorus). This was accompanied by an enhanced in vitro conversion of phosphatidylcholine to monoacyl glycerol (fivefold). The enhanced level of the 2-arachidonoyl glycerol, anandamide and other N-acylethanolamines detected in the two types of tumour tissue may possibly act as endogenous anti-tumour mediators by stimulation of both cannabinoid and non-cannabinoid receptor-mediated mechanisms.

Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines.

In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mouse, rat and human [Okamoto, Morishita, Tsuboi, Tonai and Ueda (2004) J. Biol. Chem. 279, 5298-5305]. However, it remained unclear whether NAPE-PLD acts on endogenous NAPEs contained in the membrane of living cells. To address this question, we stably transfected two mammalian cell lines (HEK-293 and CHO-K1) with mouse NAPE-PLD cDNA, and investigated the endogenous levels and compositions of NAPEs and NAEs in these cells, compared with mock-transfected cells, with the aid of GC-MS. The overexpression of NAPE-PLD caused a decrease in the total amount of NAPEs by 50-90% with a 1.5-fold increase in the total amount of NAEs, suggesting that the recombinant NAPE-PLD utilizes endogenous NAPE as a substrate in the cell. Since the compositions of NAEs and NAPEs of NAPE-PLD-overexpressing cells and mock-transfected cells were very similar, the enzyme did not appear to discriminate among the N-acyl groups of endogenous NAPEs. These results confirm that overexpressed NAPE-PLD is capable of forming NAEs, including anandamide, in living cells.

Institutionalization and psychological suffering: notes on the mental health of institutionalized adolescents in Brazil.

This article examines the relationship between the institutionalization of needy children and adolescents and the resulting psychological suffering. Individuals who have been institutionalized for long periods suffer from increased fragility in psychological structure, becoming more vulnerable and at risk as a direct consequence of the treatment they receive in such institutions. The work described here is part of the Research Program on Adolescents, Mental Health and Culture, which is being conducted at the Child and Youth Care and Rehabilitation Center at the Institute of Psychiatry of the Federal University of Rio de Janeiro in Brazil (CARIM/IPUB/UFRJ).

Caffeine inhibits cell proliferation by G0/G1 phase arrest in JB6 cells.

Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.

Massive accumulation of N-acylethanolamines after stroke. Cell signalling in acute cerebral ischemia?

We investigated levels and compositions of N-acylethanolamines (NAEs) and their precursors, N-acyl phosphatidylethanolamines (N-acyl PEs), in a rat stroke model applying striatal microdialysis for glutamate assay. Rats (n = 18) were treated with either intravenous saline (control), NMDA receptor antagonist MK801 (1 mg/kg), or CB1 receptor antagonist SR141716A (1 mg/kg) 30 min after permanent middle cerebral artery occlusion (MCAO). MK801 significantly attenuated the release of glutamate in the infarcted striatum (79 +/- 22 micromol/L) as compared with controls (322 +/- 104 micromol/L). The administration of CB1 antagonist SR141716A had no statistically significant effect on glutamate release (340 +/- 89 micromol/L), but reduced infarct volume at 5 h after MCAO significantly by approximately 40%, whereas MK801 treatment resulted in a non-significant (18%) reduction of infarct volume. In controls, striatal and cortical NAE concentrations were about 30-fold higher in the infarcted than in the non-infarcted hemisphere, whereas ipsilateral N-acyl phosphatidylethanolamine (N-acyl PE) levels exceeded contralateral levels by only a factor of two to three. Treatment with MK801 or SR141716A, or glutamate release in the infarcted tissue, had no significant effect on these levels. NAE accumulation during acute stroke may be due to increased synthesis as well as decreased degradation, possibly by inhibition of fatty acid amide hydrolase (FAAH).

Marine alkaloid polycarpine and its synthetic derivative dimethylpolycarpine induce apoptosis in JB6 cells through p53- and caspase 3-dependent pathways.

PURPOSE: Polycarpine from ascidian Polycarpa aurata was previously found to be active against different human tumor cells. In this study, we investigated the antitumor mechanisms of polycarpine and its synthetic derivative, desmethoxyethoxy-polycarpine (dimethylpolycarpine), through the induction of apoptosis. This new knowledge regarding the proapoptotic action of polycarpine and dimethylpolycarpine should lead to a better understanding of their effects and development of a new class of anticancer drugs. METHODS: Apoptosis was clearly observed by flow cytometry and Western blotting using an antibody against cleaved caspase-3 as an apoptotic marker. RESULTS: Polycarpines differentially activated p38 kinase, JNKs, and ERKs in JB6 Cl 41 cells. The polycarpines-induced apoptosis was decreased in cells expressing a dominant-negative mutant of JNK. Both compounds stimulated p53-dependent transcriptional activity and phosphorylation. Induction of p53-phosphorylation at serine 15 was suppressed in JNKI and JNK2 knockout cells. Furthermore, polycarpines were unable to induce apoptosis in p53-deficient MEFs in contrast to a strong induction of apoptosis in wild type MEFs, suggesting that p53 is involved in apoptosis induced by polycarpines. The p53 phosphorylation in turn was mediated by activated JNKs. CONCLUSIONS: These results indicate that all three MAPK signaling pathways are involved in the response of JB6 cells to treatment with polycarpines. Evidence also supports a proapoptotic role of the JNKs signaling pathway in vivo and clearly indicates that JNKs are required for phosphorylation of c-Jun, activation of p53, and subsequent apoptosis induced by polycarpines.

Region-dependent changes in endocannabinoid transmission in the brain of morphine-dependent rats.

It has been suggested recently that the endocannabinoid system might be a component of the brain reward circuitry and thus play a role not only in cannabinoid tolerance/dependence, but also in dependence/withdrawal to other drugs of abuse. Here we have examined the changes in endocannabinoid ligands and their receptors in different brain regions, with particular attention to those areas related to reinforcement processes, during dependence on the powerful addictive drug, morphine. Thus, we analysed the brain contents of N-arachidonoylethanolamine (anandamide, AEA), the first discovered endocannabinoid, in rats subjected to daily injections of increasing doses of morphine, according to a schedule designed to render the animals opiate-dependent. Although evidence of physical dependence was assured by the appearance of somatic and neurovegetative responses in these animals after an acute challenge with naloxone, there were no changes in the contents of this endocannabinoid in any of the brain regions analysed. By contrast, we observed a significant decrease in the specific binding for CB(1) receptors in the midbrain and the cerebral cortex of morphine-dependent rats, with no changes in the other regions. The decrease in the cerebral cortex was, however, accompanied by a rise in the activation of signalling mechanisms by CB(1) receptor agonists, as revealed by WIN-55,212-2-stimulated [(35)S]GTPgammaS binding, whereas a reduction in this parameter was measured in the brainstem of morphine-dependent rats. In summary, the present data are indicative of the existence of an alteration of the endocannabinoid transmission during morphine dependence in rats, although the changes observed were region-dependent and affected exclusively CB(1) receptors with no changes in endocannabinoid levels. Because the changes occurred in regions of the midbrain, the cerebral cortex and the brainstem, which have been implicated in drug dependence, our data suggest that pharmacological manipulation of the endocannabinoid system might be a novel tool to reduce morphine addiction.

Endocannabinoids induce ileitis in rats via the capsaicin receptor (VR1).

Intraluminal administration of the endocannabinoids N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) causes inflammation similar to that caused by Clostridium difficile toxin A in the rat ileum. The effects of anandamide and 2-AG were significantly inhibited by pretreatment with the specific capsaicin receptor (vanilloid receptor subtype 1; VR1) antagonist capsazepine. Pretreatment with the CB1 and CB2 cannabinoid receptor antagonists N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716) and N-[1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) did not affect the responses to anandamide. It has previously been shown that intraluminal toxin A stimulates substance P (SP) release from primary sensory neurons and that pretreatment with SP receptor [neurokinin (NK)-1 receptor] antagonists inhibits the inflammatory effects of toxin A. Anandamide stimulated SP release and this was blocked by capsazepine pretreatment. Also, pretreatment with the specific NK-1 receptor antagonist (2S,3S)-3-([3,5-bis[trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine (L-733,060) significantly inhibited the inflammatory effects of both toxin A and anandamide. Toxin A increased tissue concentrations of anandamide and 2-AG in the ileum, and these effects were enhanced after pretreatment with inhibitors of fatty acid amide hydrolase, a major endocannabinoid-degrading enzyme. The toxin A-stimulated release of anandamide but not 2-AG was selective over their congeners. These results demonstrate that the endocannabinoids anandamide and 2-AG stimulate intestinal primary sensory neurons via the capsaicin VR1 receptor to release SP, resulting in enteritis, and that endocannabinoids may mediate the inflammatory effects of toxin A.

Anandamide and other N-acylethanolamines in human tumors.

Long-chain N-acylethanolamines (NAE), including the endocannabinoid, anandamide, accumulate in mammalian tissues under a variety of pathological conditions. They have also been shown to inhibit the growth of various cancer cell lines in vitro. Here, we report the presence, in widely differing amounts (3.88-254.46 pmol/micromol lipid P), of NAE and their precursor phospholipids in various human tumors and some adjacent unaffected tissues. Anandamide ranged from 1.5 to 48% of total NAE, and incubation of tissue homogenates suggested possible NAE biosynthesis by both the established transacylation-phosphodiesterase pathway via N-acyl PE and by direct N-acylation of ethanolamine.

Cell signaling by endocannabinoids and their congeners: questions of selectivity and other challenges.

The major endocannabinoids, anandamide (N-arachidonoylethanolamide, 20:4n-6 N-acylethanolamine) and 2-arachidonoylglycerol (2-AG) are structurally and functionally similar, but they are produced by different metabolic pathways and their levels must therefore be regulated by different mechanisms. Both endocannabinoids are accompanied by cannabinoid receptor-inactive, saturated and mono- or di-unsaturated congeners which can influence their metabolism and function. Here we review published data on the presence and production of anandamide and 2-AG and their congeners in mammalian cells and discuss this information in terms of their proposed signaling functions.