Measuring beyond the microscope field of view using digital images in squamous cell carcinoma of the lip

OBJECTIVE: To study the impact of using digital images for measuring the size of the tumors, assisting with the prognostic evaluation of carcinomas of the oral mucosa. STUDY DESIGN: The depth of invasion of 12 cases of squamous cell carcinoma of the lower lip was assessed through a microscope and through digital images. All measurements of depth of invasion were assessed in a direction orthogonal to the lip surface. First, assessment of depth of invasion was done at the microscope, using an eyepiece reticule with an engraved scale. Second, depth of invasion was assessed by digital images, using a program module developed to assist pathologists with linear measuring. When the depth of the tumor was larger than the field of view at the proper magnification, several images were taken to include the whole area of invasion. The images were finally mounted in a single image and the depth of invasion measured. RESULTS: The results show positive and negative differences between assessments when assessing depths of < 2 mm. At greater depths (> or = 2 mm), the difference was always negative, showing that for deep invasion, measurements of longer distances were always performed on digital images. CONCLUSION: Measurements with digital images beyond the field of view at proper magnification could sig nificantly alter the diagnostic and prognostic assessment made using the microscope.     

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

Source of Chagas disease in Arcádia, state of Rio de Janeiro, Brazil

Following the report of triatomine nymphs in a house in Arcadia, Miguel Pereira, state of Rio de Janeiro, Brazil, the infested dwelling was checked. Several eggs and 46 specimens of Triatoma vitticeps (Stal, 1859) were collected. Among them, adults and nymphal instars accounted for 43.5% and 56.5%, respectively. Analysis of blood meals showed the ecletism of this species; 24 (52.2%) were single feeds, 18 insects (39.1%) fed on two hosts and 4 (8.7%) on three hosts. Trypanosoma cruzi infection rate of examined specimens was 13%. Finally one of the residents of the house was positive for anti-T. cruzi antibodies using indirect immunofluorescence.     

Plasmodium chabaudi chabaudi (AS): differential cellular responses to infection in resistant and susceptible mice

The infection with blood stages of Plasmodium chabaudi chabaudi (AS) was followed in BALB/c and DBA/2 mice. Both strains show a peak parasitemia by 7-9 days after infection, display splenic hypercellularity of T and B cells, thymic atrophy, nearly complete depletion of B cells in the bone marrow, and mount comparable polyclonal IgM and IgG responses in the serum. In contrast, these strains diverge in some aspects of the immune response and susceptibility to infection: while BALB/c survive, 70-80% of DBA/2 die within 2 weeks; BALB/c but not DBA/2 show marked increases in the levels of splenic gamma/delta and regulatory T cells, dendritic cells and macrophages and parasite-specific IgM and IgG levels; however, lower levels of TNF-alpha and IL-12 were observed. These results suggest the relevance of different cell populations that are known to participate/regulate specific antibody responses and cytokine production in the susceptibility to infection.     

Evaluation of different ways of presenting LipL32 to the immune system with the aim of developing a recombinant vaccine against leptospirosis

Leptospirosis, caused by bacteria of the genus Leptospira, is a direct zoonosis with wide geographical distribution. The implications in terms of public health and the economical losses caused by leptospirosis justify the use of a vaccine against Leptospira in human or animal populations at risk. In this study, we used the external membrane protein LipL32 as a model antigen, as it is highly immunogenic. The LipL32 coding sequence was cloned into several expression vectors: (i) pTarget, to create a DNA vaccine; (ii) pUS973, pUS974, and pUS977 for expression in BCG (rBCG); and (iii) pAE, to express the recombinant protein in Escherichia coli, for a subunit vaccine. Mice were immunized with the various constructs, and the immune response was evaluated. The highest humoral immune response was elicited by the subunit vaccine (rLipL32). However, with rBCG, the titer was still rising at the end of the experiment. The serum of vaccinated animals was able to recognize LipL32 on the membrane of the Leptospira, detected by indirect immunofluorescence. A monoclonal antibody anti-LipL32 was shown to inhibit the growth of Leptospira in vitro, indicating potential protection induced by the LipL32 antigen.     

Resource Conflicts: Challenges to Fisheries Management at the São Francisco River,Brazil

The paper describes factors influencing artisanal fisheries at the São Francisco River in Brazil as an example of the challenges of managing socially and economically valued common-pool resource systems. A rapid assessment of problems affecting São Francisco River fisheries in 10 communities was carried out in 2003, representing the upper, middle, and lower river portions. Field visits, interviews, focus group discussions and a literature survey allowed us to map socioeconomic and environmental factors important to the fisheries, including conflicts and tensions between stakeholders. Federal, state, and municipal governments, industries, farmers, hydroelectric companies, and urban and rural populations all have a stake in river use. Traditional fishers are the most disadvantaged of these stakeholders. With declining fish populations, most of the fishing communities surveyed are now poor, socially excluded, and with few alternative livelihood options. The stakeholders involved in access and use conflicts are artisanal fishers, professional fishers, sport fishers, farmers, enforcement and regulatory agencies, and hydroelectric companies. Traditional fishers have close ties to the river and its environment and they are usually not invited to contribute to resource management decisions. We recommend changes to management structures involving the fishing communities that are essential to resolve the major conflicts and to improve equity and sustainability of artisanal fisheries.     

A cysteine endopeptidase from tick (Rhipicephalus (Boophilus) microplus) larvae with vitellin digestion activity

The hard tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite. R. microplus free-living stage comprises egg development, hatching, and subsequent larval development until encountering a host. In order to complete the embryological development, this tick relies on yolk reserve substances, mainly vitellin (Vt), which is still present in the larval stage. The present study demonstrates presence and digestion of Vt in unfed R. microplus larvae. An increasing proteolytic activity is observed in larval development, as well as a decrease in total protein and in Vt content. Partial purification and characterization of a R. microplus larval cysteine endopeptidase (RmLCE) with Vt-degrading activity is also described. RmLCE has optimal activity at 37 °C at pH 5.0, being unstable at pH ≥ 7.5. This enzyme is active upon fluorogenic peptide substrates and is able to degrade Vt, its putative natural substrate. These results indicate that RmLCE has a role in supporting the nutritional needs of unfed R. microplus larva through Vt proteolysis, allowing survival until the first blood meal.     

Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein–protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.     

Adaptive Evolution and Divergence of SERPINB3: A Young Duplicate in Great Apes

A series of duplication events led to an expansion of clade B Serine Protease Inhibitors (SERPIN), currently displaying a large repertoire of functions in vertebrates. Accordingly, the recent duplicates SERPINB3 and B4 located in human 18q21.3 SERPIN cluster control the activity of different cysteine and serine proteases, respectively. Here, we aim to assess SERPINB3 and B4 coevolution with their target proteases in order to understand the evolutionary forces shaping the accelerated divergence of these duplicates. Phylogenetic analysis of primate sequences placed the duplication event in a Hominoidae ancestor (∼30 Mya) and the emergence of SERPINB3 in Homininae (∼9 Mya). We detected evidence of strong positive selection throughout SERPINB4/B3 primate tree and target proteases, cathepsin L2 (CTSL2) and G (CTSG) and chymase (CMA1). Specifically, in the Homininae clade a perfect match was observed between the adaptive evolution of SERPINB3 and cathepsin S (CTSS) and most of sites under positive selection were located at the inhibitor/protease interface. Altogether our results seem to favour a coevolution hypothesis for SERPINB3, CTSS and CTSL2 and for SERPINB4 and CTSG and CMA1. A scenario of an accelerated evolution driven by host-pathogen interactions is also possible since SERPINB3/B4 are potent inhibitors of exogenous proteases, released by infectious agents. Finally, similar patterns of expression and the sharing of many regulatory motifs suggest neofunctionalization as the best fitted model of the functional divergence of SERPINB3 and B4 duplicates.     

Inhibition of Maize Caffeate 3-O-Methyltransferase by Nitecapone as a Possible Approach to Reduce Lignocellulosic Biomass Recalcitrance

Plant Molecular Biology Reporter - Maize (Zea mays) caffeate 3-O-methyltransferase (ZmaysCOMT, EC 2.1.1.68), a key enzyme of the phenylpropanoid pathway, catalyzes the O-methylation of caffeic acid...