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Accurate characterization of tropical moist forest changes is needed to support conservation policies and to quantify their contribution to global carbon fluxes more effectively. We document, at pantropical scale, the extent and changes (degradation, deforestation, and recovery) of these forests over the past three decades. We estimate that 17% of tropical moist forests have disappeared since 1990 with a remaining area of 1071 million hectares in 2019, from which 10% are degraded. Our study underlines the importance of the degradation process in these ecosystems, in particular, as a precursor of deforestation, and in the recent increase in tropical moist forest disturbances (natural and anthropogenic degradation or deforestation). Without a reduction of the present disturbance rates, undisturbed forests will disappear entirely in large tropical humid regions by 2050. Our study suggests that reinforcing actions are needed to prevent the initial degradation that leads to forest clearance in 45% of the cases.

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Ciliates have been reported as pathogens of many species of economically important bivalves. Mussel protozoan X (MPX), is an uncharacterised intracellular ciliate of mussels and has been widely reported in Mytilus spp. around the world. In order to characterise this ciliate, Mytilus edulis samples were collected from a site on the West coast of Scotland, and four different fixatives for histological examination were tested. Fresh preparations of mussel digestive glands were also examined by laser scanning confocal microscopy. Intracellular ciliates were prepared by laser capture microdissection and partial sequences of small subunit ribosomal RNA gene and of large subunit ribosomal RNA gene were generated, using Phyllopharyngea primers. Methacarn solution proved to be the best fixative for both histological and molecular characterisation. The morphological and molecular investigations confirmed that this ciliate belongs to the class Phyllopharyngea, order Rhynchodida. However, this organism does not belong to any known family, genus or species, therefore, a new description is necessary, following further morphological analyses. Most mussel samples containing MPX displayed mild to moderate infections, with no signs of necrosis or haemocytic response, although a single sample displayed a severe infection (∼103 ciliates per section). The localisation of this ciliate in tissues other than the digestive gland, the presence of necrosis in infected tissue of the most severely infected mussel and the binary fission of this ciliate have been observed here for the first time. We also report the first observation of the live ciliate isolated from tissue. Although MPX remains of unknown significance to the mussel industry, tools and protocols described here will be useful in further characterising these and other ciliates (subclass Rhynchodia) known as pathogens for bivalves.

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Cetuximab is a chimeric monoclonal antibody that acts as a competitive antagonist, by binding to EGFR. This cell signalling pathways regulates tumor progression. The oral squamous cell carcinoma undergoes to regional spreading and distant metastasis. This study aimed to evaluate the effect of treatment with Cetuximab on cell migration and invasion in OSCC cells, by using the SCC-4 cell line. Cell migration and cell invasion assay were performed and actin cytoskeleton of control and treated with Cetuximab cells were evaluated. Differences were considered significant when p<0.05.Cetuximab inhibited the migration of SCC-4 cells at three concentrations: 1 µg/mL, 50 µg/mL and 100 µg/mL (p<0.0001) in a dose-dependent manner. The number of SCC-4 treated cells with 1 µg/mL that migrated through the membrane was statistically different from 50 µg/mL (p<0.001) and 100 µg/mL (p<0.0001), and between 50 µg/mL and 100 µg/mL (p<0.01). Cetuximab 50 µg/mL inhibited cell invasion through the MatrigelTM compared with SCC-4 control cells (p<0.01). Cetuximab 50 µg/mL affected the organization of the actin cytoskeleton. Cetuximab has an inhibitory effect on actin cytoskeleton organization, cell migration and invasion, suggesting that Cetuximab treatment can be important to avoid oral squamous cell carcinoma metastasis.

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Oral squamous cell carcinoma (OSCC) is a disease with high mortality and morbidity. Metastasis is a significant prognostic factor of the OSCC patients. The Rho GTPases are signaling proteins that controls important cellular processes in various complex mechanisms involved in carcinogenesis. This study aimed to evaluate the expression pattern of RhoC in OSCC protein by immunohistochemistry in situ. Immunohistochemical reactions were performed for RhoC by the method of avitina-biotin-peroxidase activity in samples OSCC: well differentiated (BD, n=6), moderately differentiated (MD, n=24) and poorly differentiated (PD, n=13). The morphometry was taken by QuickScore (percentage and intensity of staining) and only intensity staining. There was no statistical difference (p>0.05) through none of the modes of morphometric analysis between BD, MD and PD. And the RhoC staining was not associated with the histopathologic grading (χ2 = 4.65, p>0.05). However, the morphological evaluation of immunostained for RhoC in cases BD, MD, PD OSCC, regardless of histopathologic grading. These results suggest that there is no correlation between the RhoC immunoexpression and histopathological grading of OSCC.

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PURPOSE OF INVESTIGATION: To evaluate the distribution of GTPases RhoA, RhoB, and Cdc42 in cervical intraepithelial neoplasias (CIN) and invasive neoplasias of the uterine cervix. MATERIALS AND METHODS: samples of neoplastic lesions of the uterine cervix of 44 patients were classified in: CIN I (n = 10), CIN II (n = 10), CIN III (n = 09), and invasive carcinoma (n = 15). Antibodies anti-RhoA, anti-RhoB, and anti-Cdc42 were used and staining was classified as: negative, mild, moderate, and intense positive. RESULTS: When compared with dysplastic cells, superficial cells showed: higher expression of RhoB in CIN I (p = 0.0018), and lower expression of Cdc42 in CIN I (p = 0.0225). The authors observed higher expression of RhoA (p = 0.0002) and RhoB (p = 0.0046) in CIN dysplastic cells when compared with invasive carcinoma cells. CONCLUSIONS: GTPases Rho may be involved with the regulation of biological processes, important to the progression of cervical neoplasias. Probably, RhoA is important for maintenance of cell differentiation and RhoB protects cells from malignant cervical neoplasia.

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Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.

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AIMS: To evaluate the effectiveness of laser CO2 vaporization in high-grade cervical intraepithelial neoplasias and to assess the diagnostic reliability of cytology, colposcopy, microbiology and HPV tests in predicting recurrence in a long-term outcome. METHODS: Forty-four patients affectd by high-grade cervical intraepithelial neoplasia (HG-CIN) were submitted to laser CO2 vaporization and followed-up a minimum of five years. Vaginal smears for microbiological examination were detected. HPV testing was performed by polymerase chain reaction (PCR). RESULTS: The average age of the patients was 19.5 years (range 15-24). The cure rate after a single treatment was 95%. Two cases (5%) revealed HG-CIN persistence after three months. The five year follow-up of all cases submitted to a second laser procedure revealed negative cytologic and colposcopic findings. CONCLUSIONS: A higher degree of expertise and experience from the colposcopist and long-term follow-up proves the effectiveness of laser vaporization in the management of CIN in young women. It has been suggested that HPV infection alone may not be sufficient to promote carcinogenesis and that other cofactors could be involved. Microbiological tests are important to identify and treat any inflammation which might represent a cofactor of HPV infection in the pathogenesis of cervical dysplasia. Cytocolposcopic long-term follow-up, microbiological and HPV tests can improve regression of disease.

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BACKGROUND AND PURPOSE: Based on their proven ability, in animal models of stroke, to reduce damage to brain grey matter, many drugs have been tested in clinical trials but without success. Failure to save axons from injury and to protect functional outcome has been proposed as the major reason for this lack of success. We have previously demonstrated in two rodent models of cerebral ischaemia, that AS601245 (1,3-benzothiazol-2-yl (2-([2-(3-pyridinyl) ethyl] amino)-4 pyrimidinyl) acetonitrile), an inhibitor of the c-Jun NH(2)-terminal kinase (JNK), has neuroprotective properties. The aim of the present study was to further investigate if AS601245 in addition to its ability to protect neurons also could protect neurites and preserve memory after cerebral ischaemia, in gerbils. EXPERIMENTAL APPROACH: Using immunohistochemical techniques and a behavioural test, we studied the effect of the compound AS601245 on neurodegeneration and cognitive deficits after global cerebral ischaemia in gerbils. KEY RESULTS: At a dose of 80 mg kg(-1), i.p., AS601245 reduced damage to neurites by 67% (P<0.001 versus controls) and activation of astrocytes by 84% (P<0.001 versus controls). In addition, AS601245 (80 mg kg(-1), i.p.) prevented ischaemia-induced impairment of memory in the inhibitory avoidance task model. CONCLUSIONS AND IMPLICATIONS: The present results suggest that AS601245 reduced damage to neurites and decreased astrogliosis following global ischaemia and also improved long-term memory, supporting JNK inhibition as a promising therapeutic strategy for ischaemic insults to the CNS.

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Bacterial vaginosis (BV) is a condition that seldom occurs in prepuberal girls or postmenopausal women, suggesting a hormonal component in its aetiology. The precise mechanisms by which BV arises are not fully understood. One proposed mechanism suggests that carcinogenic nitrosamines act either independently or via human papilloma virus (HPV). Human papillomavirus is known to be associated with the development of squamous intraepithelial lesion (SIL). Still today the relationship between BV and SIL is debated. Many confounding factors regarding the relationship between BV and SIL include the presence of HPV and/or other sexually transmitted diseases. In a case-controlled study the correlation between BV, SIL and the presence of HPV was evaluated. BV was diagnosed according to standard criteria: vaginal pH > 4.5; positive amine test or 'whiff' test; presence of clue cells and abnormal discharge. High risk-HPV testing by PCR was performed. X2 Pearson analysis was applied for statistical evaluation of data. The results of the study have shown that BV is not associated with SIL.

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Extracellular glutamate levels were measured by microdialysis in the prefrontal cortex (PFC) of anaesthetised rats in response to a short, experimenter-provoked mechanical movement of the animal head. Movement caused significant, nerve impulse-independent elevations of glutamate levels (maximum increase, 300+/-30% of baseline). This study reveals a possible artifact in the measurement of extracellular glutamate concentrations by microdialysis and suggests that, in awake animals, treatments associated with stimulation of motor activity can cause non-specific efflux of glutamate in the PFC.

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Ethanol withdrawal is a physiopathological state associated with increased number and function of N-methyl-D-aspartate glutamate receptors. We assessed the effect of N-methyl-D-aspartate receptor stimulation on the extracellular levels of glutamate in vivo by the focal application of N-methyl-D-aspartate in the striatum of dependent rats following withdrawal from chronic treatment with ethanol. In control, chronic sucrose-treated rats, 800 microM N-methyl-D-aspartate increased glutamate levels to 268% of baseline values. In ethanol-withdrawn animals, 12 h after interruption of the chronic treatment, the application of N-methyl-D-aspartate increased glutamate levels to 598% of baseline values. In ethanol-intoxicated rats N-methyl-D-aspartate was ineffective. Concentration-response curves showed that in ethanol withdrawn animals N-methyl-D-aspartate was five-fold more potent than in controls. In withdrawn animals, the non-competitive N-methyl-D-aspartate receptor antagonist dizocilpine (1.0 mg/kg i.p.) or ethanol (5 g/kg i.g.) markedly reduced the N-methyl-D-aspartate-induced increase in glutamate levels. These results are consistent with the up-regulation of N-methyl-D-aspartate receptors by chronic ethanol and add biochemical evidence for the presence of N-methyl-D-aspartate receptors facilitating glutamate release through a positive feedback mechanism. The glutamate-induced, N-methyl-D-aspartate receptor-mediated elevations of extracellular glutamate may constitute a neurochemical substrate for the neuropathological alterations associated with alcoholism.

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Using double in situ hybridization, we found extensive coexpression of dopamine D1 and D3 receptor (D1R and D3R) mRNAs in neurons of the island of Calleja major (ICjM) and ventromedial shell of nucleus accumbens (ShV), respectively. Thus, at least 79 and 63% of D3R mRNA-expressing neurons in ICjM and ShV also expressed the D1R mRNA. Coexpression of D1R and D3R mRNAs was found to occur in substance P (SP) mRNA-expressing neurons in both areas, suggesting SP mRNA as a marker of the activity of coexpressing neurons. Administration of SKF 38393, a D1R receptor agonist, increased c-fos mRNA in ICjM, whereas administration of quinpirole, a D2R/D3R agonist, decreased it; SCH 23390, a D1 R antagonist and nafadotride, a preferential D3R antagonist, given alone, had effects opposite to those of the corresponding agonists. These data indicate that basal c-fos expression in ICjM is maintained by endogenous dopamine acting tonically upon two receptor subtypes subserving opposite effects on the same cell. However, in ShV, whereas SKF 38393 also increased c-fos mRNA, quinpirole had no effect, a difference presumably reflecting the lower fraction of neurons coexpressing D1R and D3R in this area. In contrast, in ShV from reserpine-treated rats, SKF 38393 increased SP mRNA and quinpirole potentiated this effect. These contrasting interactions of D1R- and D3R-mediated signalling events, i.e. in either opposite or synergistic directions, most likely occurring at the single cell level, may serve to increase the dopamine response threshold of the target cells in ICjM and to maintain a strong tonic activity of ShV neurons.

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In rats with unilateral lesions of the nigrostriatal dopamine pathway with 6-hydroxydopamine, the motor stimulating effects of levodopa, an indirect dopamine receptor agonist, evidenced by contraversive rotations, become enhanced upon repeated intermittent administration. However, the mechanisms of this behavioral sensitization are essentially unknown. We show that development of sensitization is accompanied by a progressive appearance of D3 receptor mRNA and binding sites, visualized by in situ hybridization and 7-[3H] hydroxy-N,N-di-n-propyl-2-aminotetralin autoradiography, respectively, occurring in the denervated caudate putamen, a brain area from which this receptor subtype is normally absent. Development and decay of these two processes occur with closely parallel time courses, whereas there were no marked changes in D1 or D2 receptor mRNAs. D3 receptor induction by levodopa is mediated by repeated D1 receptor stimulation, since it is prevented by the antagonist SCH 33390 and mimicked by the agonist SKF 38393, but not by two D2 receptor agonists. The enhanced behavioral response to levodopa is mediated by the newly synthesized D3 receptor, since it is antagonized by nafadotride, a preferential D3 receptor antagonist, in low dosage, which has no such effect before D3 receptor induction. D3 receptor induction and behavioral sensitization are also accompanied by a sustained enhancement of prodynorphin mRNA level and a progressively decreasing expression of the preprotachykinin gene. We propose that imbalance between dynorphin and substance P release from the same striatonigral motor efferent pathway, related to D3 receptor induction, is responsible for behavioral sensitization.

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Extracellular glutamate was measured by microdialysis in the striatum of ethanol-dependent, freely behaving rats following withdrawal from chronic ethanol treatment. Within 12 h from withdrawal, extracellular glutamate rose to 255% of that in control, chronic sucrose-treated rats. Glutamate output remained elevated for the subsequent 12 h and returned to control levels within 36 h from the interruption of the treatment. The changes in glutamate were time-locked to the overt physical signs of withdrawal. In 12-h ethanol-withdrawn rats an ethanol challenge suppressed the withdrawal signs and reduced the extracellular glutamate. The NMDA receptor antagonist, dizocilpine, reduced both the physical signs of withdrawal and glutamate output. In contrast, diazepam reduced the withdrawal signs but failed to change the glutamate levels. These findings suggest that the increased extraneuronal glutamate reflects overactivity of excitatory neurotransmission during withdrawal. Furthermore, they provide a biochemical rationale for the use of NMDA receptor antagonists and ethanol itself in the treatment of ethanol withdrawal syndrome.

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We have studied the effects of middle cerebral artery (MCA) occlusion in rats on polyamine efflux in the parietal cortex using the microdialysis technique. Dialysis probe implantation itself provoked a delayed, prolonged and vigorous release of spermidine and putrescine. Spermidine release returned to stable baseline levels within 48 hours. Putrescine release also returned to lower levels within this time period but putrescine levels in the dialysate fluctuated dramatically in individual animals. Because of the underlying effects of the dialysis probe (likely a reflection of traumatic cerebral damage and stimulation of polyamine metabolism and release within the immediate vicinity of the dialysis probe), MCA occlusion was performed 48 hours after probe implantation. MCA occlusion persistently (5/5 animals) resulted in a significant increase in cortical spermidine efflux, although the onset, magnitude and duration of this increased release was variable. Putrescine efflux was significantly increased in 2/5 animals with MCA occlusion but the increase in release was similar to the spontaneous fluctuations observed in control animals. Spermine was not detectable in cortical dialysates of control or MCA occluded groups. Spermidine, but not spermine or putrescine is consistently released from the parietal cortex following permanent focal ischaemia and may contribute to ischaemic neuropathology either through its effects at the N-methyl-D-aspartate (NMDA) receptor or via direct, and as yet uncharacterised, neurotoxic effects.

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Activity of the mesolimbic dopaminergic system was investigated in rats withdrawn from chronic ethanol administration by single-cell extracellular recordings from dopaminergic neurons of the ventrotegmental area, coupled with antidromic identification from the nucleus accumbens, and by microdialysis-technique experiments in the nucleus accumbens. Spontaneous firing rates, spikes per burst, and absolute burst firing but not the number of spontaneously active neurons were found drastically reduced; whereas absolute and relative refractory periods increased in rats withdrawn from chronic ethanol treatment as compared with chronic saline-treated controls. Consistently, dopamine outflow in the nucleus accumbens and its acid metabolites were reduced after abruptly stopping chronic ethanol administration. All these changes, as well as ethanol-withdrawal behavioral signs, were reversed by ethanol administration. This reversal suggests that the abrupt cessation of chronic ethanol administration plays a causal role in the reduction of mesolimbic dopaminergic activity seen in the ethanol-withdrawal syndrome. Results indicate that during the ethanol-withdrawal syndrome the mesolimbic dopaminergic system is tonically reduced in activity, as indexed by electrophysiological and biochemical criteria. Considering the role of the mesolimbic dopaminergic system in the reinforcing properties of ethanol, the depressed activity of this system during the ethanol-withdrawal syndrome may be relevant to the dysphoric state associated with ethanol withdrawal in humans.

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The administration of ethanol (2 g/kg, i.p.) or of the non-competitive antagonist(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloepten-5,1 0-imine maleate (MK-801; 1 mg/kg, i.p.) induced a decrease in the extracellular concentrations of glutamate, as studied by microdialysis in the striatum of awake rats. Moreover, ethanol and MK-801 completely prevented the increase in extraneuronal glutamate concentration induced by the focal application of N-methyl-D-aspartate (NMDA). The present results suggest that ethanol suppresses glutamate release through an inhibition of NMDA glutamate receptors in the rat striatum.

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Dialysis probe implantation in the rat parietal cortex results in delayed, prolonged and biphasic increases in the efflux of putrescine and spermidine with primary and secondary efflux peaks 6-8 h and 20-24 h after implantation. Putrescine and spermidine efflux remain elevated for at least 30 h after implantation. The primary efflux peak is attenuated by the continual infusion via the dialysis probe of either the ornithine decarboxylase inhibitor difluoromethylornithine or by the NMDA antagonist 2-APV. The secondary peak is resistant to either of these treatments. These changes in polyamine outflow are likely related to the traumatic brain damage associated with dialysis probe implantation which may be a useful model to study the effects of local brain trauma.

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Withdrawal of rats from chronic ethanol (2-5 g/kg, every 6 hr for 6 days) resulted in withdrawal symptomatology and dramatic fall in extracellular dopamine (DA) in the ventral striatum as measured by microdialysis. The changes in DA output paralleled the withdrawal symptomatology and both phenomena were reversed by a challenge ethanol dose (5 g/kg orally). The results suggest that the decrease in DA output may be responsible for the aversive symptoms of withdrawal.

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