Instituto de Biofísica Carlos Chagas Filho

The main goal of chemotherapeutic drugs is to induce massive cell death in tumors. Cisplatin is an antitumor drug widely used to treat several types of cancer. Despite its remarkable efficiency, most tumors show intrinsic or acquired drug resistance. The primary biological target of cisplatin is genomic DNA, and it causes a plethora of DNA lesions that block transcription and replication. These cisplatin-induced DNA lesions strongly induce cell death if they are not properly repaired or processed. To counteract cisplatin-induced DNA damage, cells use an intricate network of mechanisms, including DNA damage repair and translesion synthesis. In this review, we describe how cisplatin-induced DNA lesions are repaired or tolerated by cells and focus on the pivotal role of DNA repair and tolerance mechanisms in tumor resistance to cisplatin. In fact, several recent clinical findings have correlated the tumor cell status of DNA repair/translesion synthesis with patient response to cisplatin treatment. Furthermore, these mechanisms provide interesting targets for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy.

To determine the locus, full extent, and topographic organization of cortical connections of area V4 (visual area 4), we injected anterograde and retrograde tracers under electrophysiological guidance into 21 sites in 9 macaques. Injection sites included representations ranging from central to far peripheral eccentricities in the upper and lower fields. Our results indicated that all parts of V4 are connected with occipital areas V2 (visual area 2), V3 (visual area 3), and V3A (visual complex V3, part A), superior temporal areas V4t (V4 transition zone), MT (medial temporal area), and FST (fundus of the superior temporal sulcus [STS] area), inferior temporal areas TEO (cytoarchitectonic area TEO in posterior inferior temporal cortex) and TE (cytoarchitectonic area TE in anterior temporal cortex), and the frontal eye field (FEF). By contrast, mainly peripheral field representations of V4 are connected with occipitoparietal areas DP (dorsal prelunate area), VIP (ventral intraparietal area), LIP (lateral intraparietal area), PIP (posterior intraparietal area), parieto-occipital area, and MST (medial STS area), and parahippocampal area TF (cytoarchitectonic area TF on the parahippocampal gyrus). Based on the distribution of labeled cells and terminals, projections from V4 to V2 and V3 are feedback, those to V3A, V4t, MT, DP, VIP, PIP, and FEF are the intermediate type, and those to FST, MST, LIP, TEO, TE, and TF are feedforward. Peripheral field projections from V4 to parietal areas could provide a direct route for rapid activation of circuits serving spatial vision and spatial attention. By contrast, the predominance of central field projections from V4 to inferior temporal areas is consistent with the need for detailed form analysis for object vision.

Abstract Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. We present a detailed analysis of regulatory interactions involved in gap gene regulation based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. Our models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. Our models predict activation of Kr by Cad and clarify several other regulatory interactions. Our analysis suggests a central role for repressive feedback loops between complementary gap genes. We observe that repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, our models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system.

Clearance of apoptotic exudate neutrophils (efferocytosis) induces either pro- or anti-inflammatory responses in mouse macrophages depending on host genetic background. In this study, we investigated whether neutrophil efferocytosis induces a stable macrophage phenotype that could be recalled by late restimulation with LPS. Bone marrow-derived macrophages previously stimulated by pro- but not anti-inflammatory neutrophil efferocytosis expressed a regulatory/M2b phenotype characterized by low IL-12 and high IL-10 production following restimulation, increased expression of LIGHT/TNF superfamily 14, Th2-biased T cell responses, and permissive replication of Leishmania major. Induction of regulatory/M2b macrophages required neutrophil elastase activity and was partially dependent on TLR4 signaling. These results suggested that macrophage differentiation to a regulatory phenotype plays a role in resolution of inflammation but could contribute to increased humoral Ab responses and parasite persistence in the infected host.

The layout of areas in the cerebral cortex of different primates is quite similar, despite significant variations in brain size. However, it is clear that larger brains are not simply scaled up versions of smaller brains: some regions of the cortex are disproportionately large in larger species. It is currently debated whether these expanded areas arise through natural selection pressures for increased cognitive capacity or as a result of the application of a common developmental sequence on different scales. Here, we used computational methods to map and quantify the expansion of the cortex in simian primates of different sizes to investigate whether there is any common pattern of cortical expansion. Surface models of the marmoset, capuchin, and macaque monkey cortex were registered using the software package CARET and the spherical landmark vector difference algorithm. The registration was constrained by the location of identified homologous cortical areas. When comparing marmosets with both capuchins and macaques, we found a high degree of expansion in the temporal parietal junction, the ventrolateral prefrontal cortex, and the dorsal anterior cingulate cortex, all of which are high-level association areas typically involved in complex cognitive and behavioral functions. These expanded maps correlated well with previously published macaque to human registrations, suggesting that there is a general pattern of primate cortical scaling.

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specific probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

2',6'-Dihydroxy-4'-methoxychalcone (DMC) was purified from the dichloromethane extract of Piper aduncum inflorescences. DMC showed significant activity in vitro against promastigotes and intracellular amastigotes of Leishmania amazonensis, with 50% effective doses of 0.5 and 24 micrograms/ml, respectively. Its inhibitory effect on amastigotes is apparently a direct effect on the parasites and is not due to activation of the nitrogen oxidative metabolism of macrophages, since the production of nitric oxide by both unstimulated and recombinant gamma interferon-stimulated macrophages was decreased rather than increased with DMC. The phagocytic activity of macrophages was functioning normally even with DMC concentrations as high as 80 micrograms/ml, as seen by electron microscopy and by the uptake of fluorescein isothiocyanate-labeled beads. Ultrastructural studies also showed that in the presence of DMC the mitochondria of promastigotes were enlarged and disorganized. Despite destruction of intracellular amastigotes, no disarrangement of macrophage organelles were observed, even at 80 micrograms of DMC/ml. These observations suggest that DMC is selectively toxic to the parasites. Its simple structure may well enable it to serve as a new lead compound for the synthesis of novel antileishmanial drugs.

In this study, we show the leishmanicidal effects of a chloroform fraction (CLF) and a purified indole alkaloid obtained from crude stem extract of Peschiera australis against Leishmania amazonensis, a causative agent of cutaneous leishmaniasis in the New World. In a bioassay-guided chemical fractionation, the leishmanicidal activity in CLF completely and irreversibly inhibited promastigote growth. This fraction was also active against amastigotes in infected murine macrophages. Chemical analysis of CLF identified an iboga-type indole alkaloid coronaridine as one of its major compounds. Coronaridine showed potent antileishmanial activity, inhibiting promastigote and amastigote growth. Promastigotes and amastigotes treated with CLF or coronaridine showed pronounced alterations in their mitochondria as assessed by transmission electron microscopy.

Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against beta-tubulin III, glial fibrillary acidic protein, and 2',3'-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.

The effects of capsular polysaccharides, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM), from acapsular (GXM negative) and encapsulate strains of Cryptococcus neoformans were investigated in RAW 264.7 and peritoneal macrophages. Here, we demonstrate that GalXM and GXM induced different cytokines profiles in RAW 264.7 macrophages. GalXM induced production of TNF-alpha, NO and iNOS expression, while GXM predominantly induced TGF-beta secretion. Both GalXM and GXM induced early morphological changes identified as autophagy and late macrophages apoptosis mediated by Fas/FasL interaction, a previously unidentified mechanism of virulence. GalXM was more potent than GXM at induction of Fas/FasL expression and apoptosis on macrophages in vitro and in vivo. These findings uncover a mechanism by which capsular polysaccharides from C. neoformans might compromise host immune responses.

Epimastigote forms of the pathogenic parasite Trypanosoma cruzi were used to study the endocytic process in a protozoon. These elongated unicellular organisms are highly polarized cells: endocytosis occurs only at the anterior region through the cytostome and the flagellar pocket membrane, areas of the plasma membrane where the cell cytoskeleton, formed by sub-pellicular microtubules, is absent. When the cells were incubated at 4 degrees C or 28 degrees C with gold-labeled transferrin, fixed and processed for routine transmission electron microscopy our observations show that this ligand initially binds to the cytosome and the membrane lining the flagellar pocket and is subsequently ingested through a clathrin-independent receptor-mediated endocytotic process, with formation of uncoated pits and vesicles. Ingested complexes are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the posterior end of the cell. Immunocytochemical data from Lowicryl-embedded cells demonstrated that the reservosomes are acidic compartments (pH 6.0, as shown using DAMP as a pH probe) with no acid phosphatase or typical lysosome-associated membrane proteins (LAMP 1, LAMP 2 and lgp 120), but rich in cysteine proteinase. These data suggest that the reservosome is a pre-lysosomal compartment. Since cysteine proteinase of T. cruzi contains no phosphorylated mannose residues and the cation-independent mannose 6-phosphate receptor could not be immunocytochemically detected in the reservosomes, it is possible that lysosomal enzymes in the epimastigote forms of T. cruzi are targeted to compartments related to the endocytic pathway through a mechanism different from that which occurs in other eukaryotic cells.

Naphthoquinones are the most commonly occurring type of quinones in nature. They are a diverse family of secondary metabolites that occur naturally in plants, lichens and various microorganisms. This subgroup is constantly being expanded through the discovery of new natural products and by the synthesis of new compounds via innovative techniques. Interest in quinones and the search for new biological activities within the members of this class have intensified in recent years, as evidenced by the evaluation of the potential antimicrobial activities of quinones. Among fungi of medical interest, yeasts of the genus Candida are of extreme importance due to their high frequency of colonization and infection in humans. The objective of this review is to describe the development of naphthoquinones as antifungals for the treatment of Candida species and to note the most promising compounds. By using certain criteria for selection of publications, 68 reports involving both synthetic and natural naphthoquinones are discussed. The activities of a large number of substances were evaluated against Candida albicans as well as against 7 other species of the genus Candida. The results discussed in this review allowed the identification of 30 naphthoquinones with higher antifungal activities than those of the currently used drugs.

Burkholderia kururiensis M130 is one of the few rice endophytic diazotrophic bacteria identified thus far which is able to enhance growth of rice. To date, very little is known of how strain M130 and other endophytes enter and colonize plants. Here, we identified genes of strain M130 that are differentially regulated in the presence of rice plant extract. A genetic screening of a promoter probe transposon mutant genome bank and RNAseq analysis were performed. The screening of 10,100 insertions of the genomic transposon reporter library resulted in the isolation of 61 insertions displaying differential expression in response to rice macerate. The RNAseq results validated this screen and indicated that this endophytic bacterium undergoes major changes in the presence of plant extract regulating 27.7% of its open reading frames. A large number of differentially expressed genes encode membrane transporters and secretion systems, indicating that the exchange of molecules is an important aspect of bacterial endophytic growth. Genes related to motility, chemotaxis, and adhesion were also overrepresented, further suggesting plant–bacteria interaction. This work highlights the potential close signaling taking place between plants and bacteria and helps us to begin to understand the adaptation of an endophyte in planta.

Germ line inactivating mutations in BRCA1 confer susceptibility for breast and ovarian cancer. However, the relevance of the many missense changes in the gene for which the effect on protein function is unknown remains unclear. Determination of which variants are causally associated with cancer is important for assessment of individual risk. We used a functional assay that measures the transactivation activity of BRCA1 in combination with analysis of protein modeling based on the structure of BRCA1 BRCT domains. In addition, the information generated was interpreted in light of genetic data. We determined the predicted cancer association of 22 BRCA1 variants and verified that the common polymorphism S1613G has no effect on BRCA1 function, even when combined with other rare variants. We estimated the specificity and sensitivity of the assay, and by meta-analysis of 47 variants, we show that variants with <45% of wild-type activity can be classified as deleterious whereas variants with >50% can be classified as neutral. In conclusion, we did functional and structure-based analyses on a large series of BRCA1 missense variants and defined a tentative threshold activity for the classification missense variants. By interpreting the validated functional data in light of additional clinical and structural evidence, we conclude that it is possible to classify all missense variants in the BRCA1 COOH-terminal region. These results bring functional assays for BRCA1 closer to clinical applicability.

BACKGROUND: Trypanosoma cruzi uses several strategies to survive in different hosts. A key step in the life-cycle of this parasite is metacyclogenesis, which involves various morphological, biochemical, and genetic changes that induce the differentiation of non-pathogenic epimastigotes into pathogenic metacyclic trypomastigotes. During metacyclogenesis, T. cruzi displays distinct morphologies and ultrastructural features, which have not been fully characterized. RESULTS: We performed a temporal description of metacyclogenesis using different microscopy techniques that resulted in the identification of three intermediate forms of T. cruzi: intermediates I, II and III. Such classification was based on morphological and ultrastructural aspects as the location of the kinetoplast in relation to the nucleus, kinetoplast shape and kDNA topology. Furthermore, we suggested that metacyclic trypomastigotes derived from intermediate forms that had already detached from the substrate. We also found that changes in the kinetoplast morphology and kDNA arrangement occurred only after the repositioning of this structure toward the posterior region of the cell body. These changes occurred during the later stages of differentiation. In contrast, changes in the nucleus shape began as soon as metacyclogenesis was initiated, while changes in nuclear ultrastructure, such as the loss of the nucleolus, were only observed during later stages of differentiation. Finally, we found that kDNA networks of distinct T. cruzi forms present different patterns of DNA topology. CONCLUSIONS: Our study of T. cruzi metacyclogenesis revealed important aspects of the morphology and ultrastructure of this intriguing cell differentiation process. This research expands our understanding of this parasite's fascinating life-cycle. It also highlights the study of T. cruzi as an important and exciting model system for investigating diverse aspects of cellular, molecular, and evolutionary biology.

Hyperglycemia is a common feature of diabetes mellitus, considered as a risk factor for cancer. However, its direct effects in cancer cell behavior are relatively unexplored. Herein we show that high glucose concentration induces aberrant glycosylation, increased cell proliferation, invasion and tumor progression of colon cancer. By modulating the activity of the rate-limiting enzyme, glutamine-fructose-6-phosphate amidotransferase (GFAT), we demonstrate that hexosamine biosynthetic pathway (HBP) is involved in those processes. Biopsies from patients with colon carcinoma show increased levels of GFAT and consequently aberrant glycans' expression suggesting an increase of HBP flow in human colon cancer. All together, our results open the possibility that HBP links hyperglycemia, aberrant glycosylation and tumor malignancy, and suggest this pathway as a potential therapeutic target for colorectal cancer.

Eugenol, a methoxyphenol component of clove oil, suppresses cyclooxygenase-2 expression, while eugenol dimers prevent nuclear factor-kappaB (NF-kappaB) activation and inflammatory cytokine expression in lipopolysaccharide-stimulated macrophages. Our aim was to examine the in vivo anti-inflammatory effects of eugenol. BALB/c mice were divided into four groups. Mice received saline [0.05 ml intratracheally (it), control (Ctrl) and eugenol (Eug) groups] or Escherichia coli LPS (10 microg it, LPS and LPSEug groups). After 6 h, mice received saline (0.2 ml ip, Ctrl and LPS groups) or eugenol (160 mg/kg ip, Eug and LPSEug groups). Twenty-four hours after LPS injection, pulmonary resistive (DeltaP1) and viscoelastic (DeltaP2) pressures, static elastance (E(st)), and viscoelastic component of elastance (DeltaE) were measured. Lungs were prepared for histology. In parallel mice, bronchoalveolar lavage fluid was collected 24 h after LPS injection. TNF-alpha was determined by ELISA. Lung tissue expression of NF-kappaB was determined by EMSA. DeltaP1, DeltaP2, E(st), and DeltaE were significantly higher in the LPS group than in the other groups. LPS mice also showed significantly more alveolar collapse, collagen fibers, and neutrophil influx and higher TNF-alpha levels and NF-kappaB expression than the other groups. Eugenol treatment reduced LPS-induced lung inflammation, improving lung function. Our results suggest that eugenol exhibits in vivo anti-inflammatory action in LPS-induced lung injury.

Cortical projections to the middle temporal (MT) visual area were studied by injecting the retrogradely transported fluorescent tracer Fast Blue into MT in adult New World monkeys (Cebus apella). Injection sites were selected based on electrophysiological recordings, and covered eccentricities from 2-70 deg, in both the upper and lower visual fields. The position and laminar distribution of labeled cell bodies were correlated with myeloarchitectonic boundaries and displayed in flat reconstructions of the neocortex. Topographically organized projections were found to arise mainly from the primary, second, third, and fourth visual areas (V1, V2, V3, and V4). Coarsely topographic patterns were observed in transitional V4 (V4t), in the parieto-occipital and parieto-occipital medial areas (PO and POm), and in the temporal ventral posterior area (TVP). In addition, widespread or nontopographic label was found in visual areas of the superior temporal sulcus (medial superior temporal, MST, and fundus of superior temporal, FST), annectent gyrus (dorsointermediate area, DI; and dorsomedial area, DM), intraparietal sulcus (lateral intraparietal, LIP; posterior intraparietal, PIP; and ventral intraparietal, VIP), and in the frontal eye field (FEF). Label in PO, POm, and PIP was found only after injections in the representation of the peripheral visual field (> 10 deg), and label in V4 and FST was more extensive after injections in the central representation. The projections from V1 and V2 originated predominantly from neurons in supragranular layers, whereas those from V3, V4t, DM, DI, POm, and FEF consisted of intermixed patches with either supragranular or infragranular predominance. All of the other projections were predominantly infragranular. Invasion of area MST by the injection site led to the labeling of further pathways, including substantial projections from the dorsal prelunate area (DP) and from an ensemble of areas located along the medial wall of the hemisphere. In addition, weaker projections were observed from the parieto-occipital dorsal area (POd), area 7a, area prostriata, the posterior bank of the arcuate sulcus, and areas in the anterior part of the lateral sulcus. Despite the different nomenclatures and areal boundaries recognized by different models of simian cortical organization, the pattern of projections to area MT is remarkably similar among primates. Our results provide evidence for the existence of many homologous areas in the extrastriate visual cortex of New and Old World monkeys.

Abstract SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 h after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2.

Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients' sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients' lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.