Single Nucleotide Polymorphisms as Biomarker Predictors of Oral Mucositis Severity in Head and Neck Cancer Patients Submitted to Combined Radiation Therapy and Chemotherapy: A Systematic Review.

Single Nucleotide Polymorphisms (SNPs) are the most common type of genetic variation found in an individual's DNA sequences. SNPs can occur in both coding and non-coding regions of the genome and can affect gene expression, protein function, and disease susceptibility. In this systematic review, we evaluate the potential of SNPs as biomarkers in the assessment of oral mucositis (OM) severity in head and neck cancer (HNC) patients treated with concomitant chemoradiation (CRT). The study selection process involved screening 66 articles from different platforms, and after removing duplicates and excluding articles that did not meet the eligibility criteria, 23 articles were included for full-text evaluation. Among them, genes from several pathways were analyzed. The DNA damage repair pathways had the highest number of genes studied. The most frequently analyzed gene was XRCC1. The proinflammatory cytokine pathways evaluated were TNF, with three articles, and NF-κB, with one article. Most included studies showed a potential association between certain SNPs and high-grade mucositis. We conclude that SNPs can be used as possible biomarkers for the assessment of OM intensity in HNC patients, and further research is needed to explore the potential of SNPs in personalized medicine for HNC treatment.

DNA methylation in regulatory elements of the FKBP5 and NR3C1 gene in mother-child binomials with depression.

BACKGROUND: The FKBP5 and NR3C1 genes play an important role in stress response, thus impacting mental health. Stress factor exposure in early life, such as maternal depression, may contribute to epigenetic modifications in stress response genes, increasing the susceptibility to different psychopathologies. The present study aimed to evaluate the DNA methylation profile in maternal-infant depression in regulatory regions of the FKBP5 gene and the alternative promoter of the NR3C1 gene. METHODS: We evaluated 60 mother-infant pairs. The levels of DNA methylation were analyzed by the MSRED-qPCR technique. RESULTS: We observed an increased DNA methylation profile in the NR3C1 gene promoter in children with depression and children exposed to maternal depression (p < 0.05). In addition, we observed a correlation of DNA methylation between mothers and offspring exposed to maternal depression. This correlation shows a possible intergenerational effect of maternal MDD exposure on the offspring. For FKBP5, we found a decrease in DNA methylation at intron 7 in children exposed to maternal MDD during pregnancy and a correlation of DNA methylation between mothers and children exposed to maternal MDD (p < 0.05). LIMITATIONS: Although the individuals of this study are a rare group, the sample size of the study was small, and we evaluated the DNA methylation of only one CpG site for each region. CONCLUSION: These results indicate changes in DNA methylation levels in regulatory regions of FKBP5 and NR3C1 in the mother-child MDD context and represent a potential target of studies to understand the depression etiology and how it occurs between generations.

In Vitro and In Vivo Relevant Antineoplastic Activity of Platinum(II) Complexes toward Triple-Negative MDA-MB-231 Breast Cancer Cell Line.

Two platinum complexes [Pt(HL3)Cl]·H2O (3) and [Pt(HL4)Cl]·H2O (4) containing α- and ß-naphthyl groups, respectively, were investigated in more detail in vitro and in vivo for antineoplastic activity. The cytotoxicity activity induced by these platinum(II) compounds against breast cancer (MDA-MB-231 and MCF-7), lung (A549), prostate (PC3), pancreas (BXPC-3), and normal peripheral blood mononuclear (PBMC) cells were evaluated by MTT assay. The cell viability MTT assay showed that complex (4) was more cytotoxic to all cancer cell lines tested and less cytotoxic against human PBMC. Therefore, complex (4) was selected to further investigate the mechanism of cytotoxic effects involved against MDA-MB-231 cell line (human triple-negative breast cancer). Sub-G1 analysis of the cell cycle showed that this complex induces cell death by apoptosis due to the cell loss of DNA content detected in flow cytometry. The cytotoxic effect induced by complex (4) was associated with the capability of the complex to induce mitochondrial membrane depolarization, as well as increase ROS levels and caspase activation, as a result of the activation of both extrinsic and intrinsic apoptosis pathways. Ultrastructural alterations were observed using scanning and transmission electron microscopy (SEM and TEM), such as membrane blebbing, filopodia reduction, empty mitochondrial matrix, and DNA fragmentation. Furthermore, complex (4) was tested in an MDA-MB-231 tumor nodule xenograft murine model and demonstrated a remarkable reduction in tumor size in BALB/c nude mice, when compared to the control animals.

New hydroperoxycycloartane-complete chemical shifts assignment of 13C and 1H-and cytotoxicity evaluation of cycloartanes isolated from Trichilia casaretti.

New cycloartane, 22-hydroxy-25-hydroperoxycycloart-23E-en-3-one (1), along with six known analogues (2-7) and three steroids (8-10), were isolated from the leaves of Trichilia casaretti. Structures were elucidated mainly on the basis of the analysis of 1D and 2D NMR (1H and 13C) and HRESIMS spectroscopic data, involving comparison with data of the literature. The cytotoxic activities of 1-7 and 10 isolated compounds were also evaluated against human leukemia cell line Molt-4 (acute lymphoblastic) and exhibited good cytotoxic activity with IC50 values ranging from 10.62 to 21.14 µM.

Antitumor activity via apoptotic cell death pathway of water soluble copper(II) complexes: effect of the diamino unit on selectivity against lung cancer NCI-H460 cell line.

The cytotoxicity against five human tumor cell lines (THP-1, U937, Molt-4, Colo-205 and NCI-H460) of three water soluble copper(II) coordination compounds containing the ligands 3,3'-(ethane-1,2-diylbis(azanediyl))dipropanamide (BCEN), 3,3'-(piperazine-1,4-diyl)dipropanamide (BPAP) or 3,3'-and (1,4-diazepane-1,4-diyl)dipropanamide (BPAH) are reported in this work. The ligands contain different diamine units (ethylenediamine, piperazine or homopiperazine) and two propanamide units attached to the diamine centers, resulting in N2O2 donor sets. The complex containing homopiperazine unit presented the best antiproliferative effect and selectivity against lung cancer cell line NCI-H460, showing inhibitory concentration (IC50) of 58 µmol dm-3 and Selectivity Index (SI) > 3.4. The mechanism of cell death promoted by the complex was investigated by Sub-G1 cell population analysis and annexin V and propidium iodide (PI) labeling techniques, suggesting that the complex promotes death by apoptosis. Transmission electron microscopy investigations are in agreement with the results presented by mitochondrial membrane potential analysis and also show the impairment of other organelles, including endoplasmic reticulum.

Effect of the hydroxamate group in the antitumoral activity and toxicity toward normal cells of new copper(II) complexes.

The synthesis, physico-chemical characterization and cytotoxicity of four copper(II) coordination complexes, i.e. [Cu(HBPA)Cl2] (1), [Cu(BHA)2] (2), [Cu(HBPA)(BHA)Cl] CH3OH (3) and [Cu(HBPA)2]Cl2·4H2O (4), are reported. HBPA is the tridentate ligand N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)amine and HBHA is the benzohydroxamic acid. The reaction between the HBHA and CuCl2.2H2O has resulted in the new complex (2) and the reaction between complex (1) and HBHA has resulted in the new complex (3). X-ray diffraction studies for complex (3) indicated the effective coordination of HBHA as BHA-. Their cytotoxicity was evaluated against three human tumoral cell lines (Colo-205, NCI-H460 and U937) and PBMC (peripheral blood mononuclear cells), using the MTT cytotoxic assay. The results toward PBMC reveal that the new copper(II) complex (2) presents lower toxicity toward normal cells. Furthermore, complex (2) presents IC50 values lower than cisplatin toward NCI-H460 and the best selectivity index obtained towards NCI-H460 (SI = 2.2) and U937 cell lines (SI = 2.0), as a result of the presence of two molecules of HBHA in its structure. Complex (3) presents IC50 values lower than cisplatin toward NCI-H460, Colo-205 and comparable to cisplatin toward U937. The evaluation of the cell death type promoted by complexes (2) and (4) was investigated toward NCI-H460 revealing better results than the standard drug cisplatin, according to the Annexin V and propidium iodide (PI) labeling experiment. Based on the studies here performed, HBHA seems to be related to lower toxicity toward PBMC and HBPA is improving directly the cytotoxity.

Morin exhibits leukemic cellular apoptosis through caspase pathway.

The present study investigated the possibility of apoptosis-inducing activity in human leukemia U-937 and THP-1 cells by the flavonoid morin. The treatments were evaluated by using the MTT and LDH assays; analysis of mitochondrial membrane potential (ΔΨm) was evaluated by flow cytometry, cell death by apoptosis was confirmed by fluorescence microscopy and by assessing the activity of caspases-3 and -6. The data indicated that the flavonoid morin has promoted a decrease in cell viability in a concentration-dependent way for both of the cancerous cell lines. An increase in the percentage of cell death caused by apoptosis was associated to a potential alteration in the mitochondrial membrane (ΔΨm) suggesting the involvement of cell death in intrinsic apoptotic pathways. Activation of caspases-3 and -6 confirmed the presence of apoptotic activity from morin. The results reinforce the antileukemic potential of flavonol morin.

Cetacean Morbillivirus Infection in a Killer Whale (Orcinus orca) from Brazil.

We provide pathological, immunohistochemical and molecular evidence of cetacean morbillivirus (CeMV) infection in a live-stranded adult female killer whale (Orcinus orca), which stranded alive in Espírito Santo State, Brazil, in 2014. Although attempts were made to release the animal, it stranded again and died. The main pathological findings were severe pulmonary oedema, pleural petechiation, multifocal, lymphoplasmacytic meningoencephalitis and leptomeningomyelitis with perivascular cuffing and gliosis, chronic lymphocytic bronchointerstitial pneumonia and multicentric lymph node and splenic lymphoid depletion. Other pathological findings were associated with the 'live-stranding stress response'. Immunohistochemical analysis revealed multifocal morbilliviral antigen in neurons and astrocytes, and in pneumocytes, histiocytes and leukocytes in the lung. CeMV was detected by a novel reverse transcriptase polymerase chain reaction method in the brain and kidney. Phylogenetic analysis of part of the morbillivirus phosphoprotein gene indicates that the virus is similar to the Guiana dolphin (Sotalia guianensis) morbillivirus strain, known to affect cetaceans along the coast of Brazil. To the authors' knowledge, this is the first report of morbillivirus disease in killer whales.

In vitro and in vivo anti-proliferative activity and ultrastructure investigations of a copper(II) complex toward human lung cancer cell NCI-H460.

The aim of our study was to evaluate the in vitro and in vivo anti-proliferative potential of complex (2) [Cu (L1)Cl]Cl.2H2O, where L1 = 1-[2-hydroxybenzyl(2-pyridylmethyl)amino]-3-(1-naphthyloxy)-2-propanol on lung carcinoma cell NCI-H460. Cell viability assay determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay demonstrated that the complex (2) exhibits higher activity against the NCI-H460 cell, with an IC50 value lower than cisplatin (26.5 µM ± 1.1 and 203 ± 1.2 µM respectively). Cell death by apoptosis was investigated by flow cytometer analysis of sub-G1 populations in the cell cycle and Annexin V/Propidium Iodide assay. Changes on the cell surface and ultrastructure were detected by scanning and transmission electron microscopy. Our work revealed that complex (2) induced changes associated with apoptosis, such as plasma membrane blebbing and a lower microvilli amount, fragmentation and condensation of chromatin, alterations in mitochondria, and enlargement of the endoplasmic reticulum. Mitochondrial function of NCI-H460 cells evaluated by 5,5',6,6'-tetrachloro 1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide (JC-1) probes showed high loss of mitochondrial membrane potential when treated with complex (2). Moreover, caspase-12 measurement showed an expressive activation level, which is related to endoplasmic reticulum stress. In vivo assay using the murine model of human lung cancer cell showed that complex (2) and cisplatin has similar antineoplastic activity.

Modulating the antitumoral activity by the design of new platinum(II) compounds: Synthesis, characterization, DFT, ultrastructure and mechanistic studies.

The synthesis, physico-chemical characterization, Density functional theory (DFT) calculation and cytotoxicity against five human tumoral cell lines (THP-1, U937, Molt-4, Colo205 and H460) of four new platinum(II) coordination compounds are reported, i.e. [Pt(HL1)Cl]·H2O (1), [Pt(HL2)Cl]·H2O (2), [Pt(HL3)Cl]·H2O (3) and [Pt(HL4)Cl]·H2O (4). The ligands contain N2O donor sets. Furthermore, H2L3 and H2L4 present α and ß-naphthyl groups respectively, which are absent in HL1 and H2L2. X-ray diffraction studies were performed for complex (3), indicating the formation of a mononuclear platinum(II) complex. Complexes (3) and (4), which contain α and ß-naphthyl groups respectively, have presented lower IC50 (inhibitory concentration) values than those exhibited by complexes (1) and (2). The mechanism of cell death promoted by complexes (3) and (4) was investigated, suggesting that, toward U937 cell line, the α isomer promotes death by apoptosis and the ß isomer by necrosis. Transmission and scanning electron microscopy investigations are in agreement with the loss of mitochondrial membrane potential (ΔΨm) observed by JC-1 mitochondrial potential sensor and indicate that the activity of complex (3) against U937 cell line is mediated by an apoptotic mechanism associated with mitochondrial dysfunction. A quantification of caspases 3, 6, 8 and 9 indicated that both the intrinsic and extrinsic pathways are involved in the apoptotic stimuli. Based on DFT calculations all the Pt(II) complexes present the same coordination environment for the metal centre, indicating that the higher cytotoxic activities exhibited by complexes (3) and (4) are related to the presence of the α and ß-naphthyl groups in the ligand structure.

Myrtenal-induced V-ATPase inhibition - A toxicity mechanism behind tumor cell death and suppressed migration and invasion in melanoma.

BACKGROUND: Metastatic tumor cells have acidic extracellular pH and differential electrochemical H+ gradients generated across their cell membranes by V-type H+-ATPases. This study shows that inhibition of the V-ATPases by the plant-derived monoterpene Myrtenal results in tumor cell death and decreased metastatic dissemination in mice. METHODS: The Myrtenal anticancer toxicity was evaluated in vitro using murine (B16F0 and B16F10) and human (SkMel-5) melanoma cell lines, and in in vivo mouse metastatic dissemination model. Proton flux and extracellular acidification were directly evaluated at the surface of living cells using a non-invasive selective ion electrode approach. RESULTS: The inhibition of V-ATPases by 100 µM Myrtenal disrupted the electrochemical H+ gradient across the cell membranes, strongly induced cell death (4-5 fold), and decreased tumor cells migration and invasion in vitro. Myrtenal (15 mg/kg) also significantly reduced metastasis induced by B16F10 in vivo, further reinforcing that V-ATPase is a molecular target to halt the progression of cancers. CONCLUSIONS: These data revealed the therapeutic potential of Myrtenal as inhibitor of melanoma progression proposing a mechanism of action by which once inhibited by this monoterpene the proton pumps fail to activate cancer-related differential electrochemical gradients and H+ fluxes across the tumor cell membranes, disrupting pH signatures inherent in tumor progression, resulting in reprogrammed cell death and metastasis inhibition. GENERAL SIGNIFICANCE: The work represents a new mechanistic strategy for contention of melanoma, the most aggressive and deadly form of cutaneous neoplasm, and highlights Myrtenal, other related monoterpenes and derivatives as promising proton pump inhibitors with high chemotherapeutic potential.

Cytotoxic activity of halogenated sesquiterpenes from Laurencia dendroidea.

Red algae of the genus Laurencia J. V. Lamouroux are found in tropical and subtropical regions throughout the world and are an extremely rich source of active secondary metabolites with diverse structural features. In the present study, 6 sesquiterpenes (obtusol, (-)-elatol, dendoidiol, debrome-elatol, triquinane, and obtusane) isolated from Laurencia dendroidea were investigated for their cytotoxicity, using 4 cancer cell lines (U937, Jurkat, B16F10, and Colo-205). Among all sesquiterpenes tested, obtusol and (-)-elatol showed a promising activity in the treatment of Colo-205 strain, with IC50 of 1.2 ± 1.4 and 2.5 ± 1.3 µg/ml, respectively. In addition, fluorescence microscopy results indicated that, at 100 µg/ml, obtusol induced apoptosis at 79% and (-)-elatol at 95%. Activation of Caspases 2, 4, 6, and 8 showed to be involved in (-)-elatol activity and only Caspase 6 in obtusol activity. These data demonstrated the effective apoptosis-inducing activity of the sesquiterpene (-)-elatol and obtusol in the treatment of Colo-205 strain. Therefore, more studies should be done so that the sesquiterpenes (-)-elatol and obtusol might become promising chemotherapy.

In vitro and in vivo studies of the antineoplastic activity of copper (II) compounds against human leukemia THP-1 and murine melanoma B16-F10 cell lines.

We investigated the antineoplastic activities of a previously reported copper (II) coordination compound, [Cu(BMPA)Cl2]CH3OH (1), and a new compound, [Cu(HBPA)Cl2]H2O (2), where BMPA is bis(pyridin-2-ylmethyl)amine and HBPA is (2-hydroxybenzyl)(2-pyridylmethyl)amine, using various cellular models of human leukemia (THP-1, U937, HL60, Molt-4, JURKAT) and human colon cancer (COLO 205), as well as a murine highly metastatic melanoma (B16-F10) cell line. Compound (2) was characterized using several physical and chemical techniques, including X-ray diffraction studies. The IC50 values of the copper coordination complexes in the human leukemia cell lines ranged from 87.63 ± 1.02 to ≥400 µM at high cell concentrations and from 19.17 ± 1.06 to 97.67 ± 1.23 µM at low cell concentrations. Both compounds induced cell death, which was determined by cell cycle analyses and phosphatidylserine exposure studies. THP-1 cells released cytochrome c to the cytoplasm 12 h after treatment with 400 µM of compound (2). To evaluate the apoptosis pathway induced by compound (2), we measured the activities of initiator caspases 8 and 9 and executioner caspases 3 and 6. The results were suggestive of the activation of both intrinsic and extrinsic apoptosis pathways. To investigate the activities of the compounds in vivo, we selected two sensitive cell lines from leukemia (THP-1) and solid tumor (B16-F10) lineages. BALB/c nude bearing THP-1 tumors treated with 12 mg·kg(-1) of compound (2) showed a 92.4% inhibition of tumor growth compared with the control group.

Synthesis, characterization and antitumoral activity of new cobalt(II)complexes: Effect of the ligand isomerism on the biological activity of the complexes.

The synthesis, physico-chemical characterization and cytotoxicity against five human tumoral cell lines (THP-1, U937, Molt-4, Colo205 and H460) of three new cobalt(II) coordination compounds are reported (i.e. Co(HL1)Cl (1), Co(HL2)Cl (2) and [Co(HL3)Cl]0.0.5 (CH3)2CHOH (3)). H2L2 (2-{[[2-hydroxy-3-(1-naphthyloxy)propyl](pyridin-2-ylmethyl)amino]methyl}phenol) and H2L3 (2-{[[2-hydroxy-3-(2-naphthyloxy)propyl](pyridin-2-ylmethyl)amino]methyl}phenol) present α and ß-naphthyl groups respectively, which is absent in H2L1 (N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)[(3-chloro)(2-hydroxy)]propylamine. These compounds were characterized by a range of physico-chemical methods. X-ray diffraction studies were performed for complex (3), indicating the formation of a mononuclear complex. Complexes (2) and (3), which contain α and ß-naphthyl groups respectively, have presented lower IC50 values than those exhibited by complex (1). Complex (3) presents IC50 values lower than cisplatin against Colo205 (90 and 196µmolL(-1), respectively) and H460 (147 and 197µmolL(-1), respectively). These human neoplastic cells under investigation were also more susceptible toward complex (3) than peripheral blood mononuclear cells. Transmission electron microscopy investigations are in agreement with the loss of mitochondrial membrane potential (ΔΨm) observed by JC-1 mitochondrial potential sensor and indicate that the activity of complex (3) against leukemic cell line (U937) is mediated by an apoptotic mechanism associated with mitochondrial dysfunction (intrinsic pathway).

Rapid tree carbon stock recovery in managed Amazonian forests.

While around 20% of the Amazonian forest has been cleared for pastures and agriculture, one fourth of the remaining forest is dedicated to wood production. Most of these production forests have been or will be selectively harvested for commercial timber, but recent studies show that even soon after logging, harvested stands retain much of their tree-biomass carbon and biodiversity. Comparing species richness of various animal taxa among logged and unlogged forests across the tropics, Burivalova et al. found that despite some variability among taxa, biodiversity loss was generally explained by logging intensity (the number of trees extracted). Here, we use a network of 79 permanent sample plots (376 ha total) located at 10 sites across the Amazon Basin to assess the main drivers of time-to-recovery of post-logging tree carbon (Table S1). Recovery time is of direct relevance to policies governing management practices (i.e., allowable volumes cut and cutting cycle lengths), and indirectly to forest-based climate change mitigation interventions.

Morpho-pathological and ultrastructural analysis of the murinemelanoma B16F10 and B16F0 cell lines and derived tumors / Anáalise morfo-patolóogica e ultraestrutural de linhagens celulares B16F10 E B16F0 e melanoma decamundongo e tumores derivados

Murine B16 cell lines of tumorigenic melanocytes have been widely used as cellular model for biochemicaland molecular studies in melanoma research. Despite such importance there is still a lack of information onthe microanatomy and cytopathology of these B16 models. The aim of this study was to shed light on themorpho-pathological specificities of the murine melanoma cell lines B16F10 and B16F0, and derived tumors,and correlate the findings with distinct metastatic potential. Cyto- and histopathological analysis carried outby optical and transmission electron microscopy (TEM) revealed that both tumor cell lines exhibit typicalfeatures of carcinogenic cells such as atypical mitoses, multiple nucleoli and enlarged nucleus. In agreementwith their highest metastatic potential, B16F10 cells and tumors exhibited more aberrant morphology thanthat observed in B16F0 samples. Also the histopathological analysis revealed that only the B16F10 tumor cellscould invade adjacent muscle tissues. Under TEM, B16F0 cell line presented mitochondrial hyperplasia andhypertrophia and a large number of melanosomes, while B16F10 showed a larger number of mitochondria ina wider range of shapes and sizes, numerous polyribosomes, cellular peripheral fibrillar material, and abnormalclusters of mitochondria. These morphological features related to the differential metastatic potentials ofB16F10 and B16F0, provide new insights on the cellular changes involved in melanoma metastatic progressionand also would be instrumental to expand the application of these cell lines as models in melanoma research.

Linhas de células B16 de melanócitos tumorais de camundongos têm sido amplamente utilizadascomo modelo celular para estudos bioquímicos e moleculares na pesquisa de melanoma. Apesardessa importância, ainda há uma falta de informação sobre a micro anatomia e citopatologia destesmodelos B16. O objetivo deste estudo foi pesquisar sobre as especificidades morfo-patológicas das linhasde células de melanoma de camundongos B16F10 e B16F0 e tumores derivados, e correlacionaros achados com potencial metastático distinto. Cito e análise histopatológica realizada por microscopiaóptica e eletrônica de transmissão (TEM) revelou que ambas as linhagens celulares tumoraisexibem características típicas de células cancerígenas, como mitoses atípicas, múltiplos nucléolos enúcleo aumentado. De acordo com seu potencial mais elevado, as células B16F10 e tumores metastáticosexibiram morfologia mais aberrante do que a observada em amostras de B16F0. A análisehistopatológica também revelou que apenas as células tumorais B16F10 poderiam invadir os tecidosmusculares adjacentes. De acordo com TEM, a linhagem de células B16F0 apresentou hiperplasiae hipertrofia mitocondrial e um grande número de melanossomas, enquanto B16F10 demonstrouum maior número de mitocôndrias em uma ampla gama de formas e tamanhos, numerosos polirribossomas,material fibrilar celular periférico e aglomerados de mitocôndrias anormais. Estascaracterísticas morfológicas relacionadas aos diferenciais pote nciais metastáticos de B16F10 e B16F0forneceram novas compreensões sobre as mudanças celulares envolvidas na progressão metastáticade melanoma e também seria fundamental para ampliar a aplicação dessas linhagens celulares comomodelo na pesquisa melanoma.

Morpho-pathological and ultrastructural analysis of the murinemelanoma B16F10 and B16F0 cell lines and derived tumors / Anáalise morfo-patolóogica e ultraestrutural de linhagens celulares B16F10 E B16F0 e melanoma decamundongo e tumores derivados

Murine B16 cell lines of tumorigenic melanocytes have been widely used as cellular model for biochemicaland molecular studies in melanoma research. Despite such importance there is still a lack of information onthe microanatomy and cytopathology of these B16 models. The aim of this study was to shed light on themorpho-pathological specificities of the murine melanoma cell lines B16F10 and B16F0, and derived tumors,and correlate the findings with distinct metastatic potential. Cyto- and histopathological analysis carried outby optical and transmission electron microscopy (TEM) revealed that both tumor cell lines exhibit typicalfeatures of carcinogenic cells such as atypical mitoses, multiple nucleoli and enlarged nucleus. In agreementwith their highest metastatic potential, B16F10 cells and tumors exhibited more aberrant morphology thanthat observed in B16F0 samples. Also the histopathological analysis revealed that only the B16F10 tumor cellscould invade adjacent muscle tissues. Under TEM, B16F0 cell line presented mitochondrial hyperplasia andhypertrophia and a large number of melanosomes, while B16F10 showed a larger number of mitochondria ina wider range of shapes and sizes, numerous polyribosomes, cellular peripheral fibrillar material, and abnormalclusters of mitochondria. These morphological features related to the differential metastatic potentials ofB16F10 and B16F0, provide new insights on the cellular changes involved in melanoma metastatic progressionand also would be instrumental to expand the application of these cell lines as models in melanoma research.AU

Linhas de células B16 de melanócitos tumorais de camundongos têm sido amplamente utilizadascomo modelo celular para estudos bioquímicos e moleculares na pesquisa de melanoma. Apesardessa importância, ainda há uma falta de informação sobre a micro anatomia e citopatologia destesmodelos B16. O objetivo deste estudo foi pesquisar sobre as especificidades morfo-patológicas das linhasde células de melanoma de camundongos B16F10 e B16F0 e tumores derivados, e correlacionaros achados com potencial metastático distinto. Cito e análise histopatológica realizada por microscopiaóptica e eletrônica de transmissão (TEM) revelou que ambas as linhagens celulares tumoraisexibem características típicas de células cancerígenas, como mitoses atípicas, múltiplos nucléolos enúcleo aumentado. De acordo com seu potencial mais elevado, as células B16F10 e tumores metastáticosexibiram morfologia mais aberrante do que a observada em amostras de B16F0. A análisehistopatológica também revelou que apenas as células tumorais B16F10 poderiam invadir os tecidosmusculares adjacentes. De acordo com TEM, a linhagem de células B16F0 apresentou hiperplasiae hipertrofia mitocondrial e um grande número de melanossomas, enquanto B16F10 demonstrouum maior número de mitocôndrias em uma ampla gama de formas e tamanhos, numerosos polirribossomas,material fibrilar celular periférico e aglomerados de mitocôndrias anormais. Estascaracterísticas morfológicas relacionadas aos diferenciais pote nciais metastáticos de B16F10 e B16F0forneceram novas compreensões sobre as mudanças celulares envolvidas na progressão metastáticade melanoma e também seria fundamental para ampliar a aplicação dessas linhagens celulares comomodelo na pesquisa melanoma.(AU)

Lepidotrichilins A and B, new protolimonoids with cytotoxic activity from Trichilia lepidota (Meliaceae).

Two novel protolimonoids, named lepidotrichilins A (1) and B (2), four known protolimonoids, 21,23-epoxy-7α-21α-dihydroxyapotirucalla-14,24-dien-3-one (3), 21,23-epoxy-7α-21ß-dihydroxyapotiru-calla-14,24-dien-3-one (4), dysorone D (5), deoxy-flindissone (6), and the two steroids ß-sitosterol (7) and stigmasterol (8) were identified in leaves of Trichilia lepidota subsp. schumanniana (Harms) T.D. Pennington. From wood the coumarin scopoletin (9) was isolated. The structures were established by NMR (1D (1)H and (13)C-NMR and 2D (1)H-(1)H COSY, HMQC and HMBC), mass spectroscopy and infrared (IR) spectral data. The hexane and methanol extracts of the leaves, the protolimonoids lepidotrichilins A (1) and B (2) (IC50 42.7 µg mL(-1)) and the protolimonoid deoxy-flindissone (6; IC50 9.3 µgmL(-1)) exhibited significant cytotoxic activity against the MOLT-4 and U937 leukemic cell lines.

Bothropic antivenom based on monoclonal antibodies, is it possible?

Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5 mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48 h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future.

Growth inhibition of Staphylococcus aureus and escherichia coli strains by neutralizing IgY antibodies from ostrich egg yolk

Ostrich raising around the world have some key factors and farming profit depend largely on information and ability of farmers to rear these animals. Non fertilized eggs from ostriches are discharged in the reproduction season. Staphylococcus aureus and Escherichia coli are microorganisms involved in animal and human diseases. In order to optimize the use of sub products of ostrich raising, non fertilized eggs of four selected birds were utilized for development of polyclonal IgY antibodies. The birds were immunized (200ug/animal) with purified recombinant staphylococcal enterotoxin C (recSEC) and synthetic recRAP, both derived from S. aureus, and recBFPA and recEspB involved in E. coli pathogenicity, diluted in FCA injected in the braquial muscle. Two subsequent immunization steps with 21 days intervals were repeated in 0,85% saline in FIA. Blood and eggs samples were collected before and after immunization steps. Egg yolk immunoglobulins were purified by precipitation with 19% sodium sulfate and 20% ammonium sulphate methodologies. Purified IgY 50µL aliquots were incubated in 850µL BHI broth containing 50µL inoculums of five strains of S. aureus and five strains of E.coli during four hours at 37ºC. Growth inhibition was evaluated followed by photometry reading (DO550nm). Egg yolk IgY preparation from hiperimmunized birds contained antibodies that inhibited significantly (p<0,05) growth of strains tested. Potential use of ostrich IgY polyclonal antibodies as a diagnostic and therapeutic tool is proposed for diseased animals.