Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

Effect of overexpression of citrus 9-cis-epoxycarotenoid dioxygenase 3 (CsNCED3) on the physiological response to drought stress in transgenic tobacco.

9-cis-epoxycarotenoid dioxygenase (NCED) encodes a key enzyme in abscisic acid (ABA) biosynthesis. Little is known regarding the regulation of stress response by NCEDs at physiological levels. In the present study, we generated transgenic tobacco overexpressing an NCED3 ortholog from citrus (CsNCED3) and investigated its relevance in the regulation of drought stress tolerance. Wild-type (WT) and transgenic plants were grown under greenhouse conditions and subjected to drought stress for 10 days. Leaf predawn water potential (Ψwleaf), stomatal conductance (gs), net photosynthetic rate (A), transpiration rate (E), instantaneous (A/E) and intrinsic (A/gs) water use efficiency (WUE), and in situ hydrogen peroxide (H2O2) and abscisic acid (ABA) production were determined in leaves of irrigated and drought-stressed plants. The Ψwleaf decreased throughout the drought stress period in both WT and transgenic plants, but was restored after re-watering. No significant differences were observed in gs between WT and transgenic plants under normal conditions. However, the transgenic plants showed a decreased (P ≤ 0.01) gs on the 4th day of drought stress, which remained lower (P ≤ 0.001) than the WT until the end of the drought stress. The A and E levels in the transgenic plants were similar to those in WT; therefore, they exhibited increased A/gs under drought conditions. No significant differences in A, E, and gs values were observed between the WT and transgenic plants after re-watering. The transgenic plants had lower H2O2 and higher ABA than the WT under drought conditions. Our results support the involvement of CsNCED3 in drought avoidance.

TcCYS4, a cystatin from cocoa, reduces necrosis triggered by MpNEP2 in tobacco plants.

In Brazil, most cocoa bean production occurs in Southern Bahia. Witches' broom disease arrived in this area in 1989 and has since caused heavy losses in production. The disease is caused by the basidiomycete fungus Moniliophthora perniciosa, a hemibiotrophic fungus that produces the necrosis and ethylene-inducting protein (MpNEP2) during infection; this protein can activate cysteine proteases and induce programmed cell death. Cysteine proteases can be modulated by cystatin. In this study, we overexpressed TcCYS4, a cocoa cystatin, in tobacco plants and evaluated the effect on MpNEP2 in model plants. Tccys4 cDNA was cloned into the pCAMBIA 1390 vector and inserted into the tobacco plants via Agrobacterium tumefaciens. Transgene expression was analyzed by reverse transcription-quantitative PCR and Western blot analysis. Transcript and protein levels in Tcccys4:tobacco lines were 8.9- and 1.5-fold higher than in wild-type plants (wt). Tcccys4:tobacco lines showed no change in growth compared to wt plants. CO2 net assimilation (A) increased in Tcccys4:tobacco lines compared to wt plants. Only one line showed statistically significant stomatal conductance (gs) and transpiration rate (E) changes. MpNEP2 was infiltered into the foliar mesophyll of Tcccys4:tobacco lines and wt plants, and necrotic lesions were attenuated in lines highly expressing Tccys4. Our results suggest that cocoa cystatin TcCYS4 affects MpNEP2 activity related to the progression of programmed cell death in tobacco plants. This may occur through the action of cystatin to inhibit cysteine proteases activated by MpNEP2 in plant tissues. Further studies are necessary to examine cystatin in the Theobroma cacao-M. perniciosa pathosystem.

Phosphate-induced-1 gene from Eucalyptus (EgPHI-1) enhances osmotic stress tolerance in transgenic tobacco.

Environmental stresses such as drought, freezing, and high salinity induce osmotic stress in plant cells. The plant response to osmotic stress involves a number of physiological and developmental changes, which are made possible, in part, by the modulation of the expression of specific genes. Phosphate-induced-1 gene (PHI-1) was first isolated from phosphate-treated phosphate-starved tobacco cell cultures as a stress-inducible gene, which is presumably related to intracellular pH maintenance; however, the role of the PHI-1 gene product has not yet been clarified. A gene encoding a predicted protein with high similarity to tobacco PHI-1, named EgPHI-1, was previously identified in Eucalyptus by comparative transcriptome analysis of xylem cells from species of contrasting phenotypes for wood quality and growth traits. Here, we show that the overexpression of EgPHI-1 in transgenic tobacco enhances tolerance to osmotic stress. In comparison with wild-type plants, EgPHI-1 transgenic plants showed a significant increase in root length and biomass dry weight under NaCl-, polyethylene glycol, and mannitol-induced osmotic stresses. The enhanced stress tolerance of transgenic plants was correlated with increased endogenous protein levels of the molecular chaperone binding protein BiP, which in turn was correlated with the EgPHI-1 expression level in the different transgenic lines. These results provide evidence about the involvement of EgPHI-1 in osmotic stress tolerance via modulation of BiP expression, and pave the way for its future use as a candidate gene for engineering tolerance to environmental stresses in crop plants.

Comparative study of putative 9-cis-epoxycarotenoid dioxygenase and abscisic acid accumulation in the responses of Sunki mandarin and Rangpur lime to water deficit.

Abscisic acid is a plant hormone that participates in essential plant physiological processes, especially during adaptation to many environmental stresses, such as water deficit. The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase (CCD) genes was investigated in the pot-cultivated leaves and roots of the 'Rangpur' lime and 'Sunki Maravilha' mandarin plants. Transpiration, stomatal resistance and leaf growth were evaluated when these genotypes were subjected to continuous water deficit. Under water deficit conditions, the 'Rangpur' lime extracts used greater amounts of water when compared to the 'Sunki Maravilha' plants, which reached the greatest stomatal resistance 5 days before 'Rangpur' lime. When subjected to water deficit, the roots and leaves of 'Sunki Maravilha' showed a progressive increase in ABA accumulation; however, in 'Rangpur' lime, alternations between high and low ABA concentrations were observed. These results suggest a retroactive feeding regulation by ABA. In 'Rangpur' lime the NCED2, NCED3 and CCD4a genes were expressed at the highest levels in the roots, and NCED5 was highly expressed in the leaves; in 'Sunki Maravilha', the NCED2 and NCED5 genes were most highly expressed in the roots, and NCED2 was most highly expressed in the leaves. However, for both genotypes, the transcription of these genes only correlated with ABA accumulation during the most severe water deficit conditions. The 'Rangpur' lime behaved as a vigorous rootstock; the leaf growth remained unaltered even when water was scarce. However, 'Sunki Maravilha' adaptation was based on the equilibrium of the response between the root and the aerial tissues due to water restriction. The use of the Sunki mandarin in combination with a scion with similar characteristics as its own, which responds to water deficit stress by accumulating ABA in the leaves, may display good drought tolerance under field conditions.

Proteomic response of Moniliophthora perniciosa exposed to pathogenesis-related protein-10 from Theobroma cacao.

TcPR-10, a member of the pathogenesis-related protein 10 family, was identified in EST library of interactions between Theobroma cacao and Moniliophthora perniciosa. TcPR-10 has been shown to have antifungal and ribonuclease activities in vitro. This study aimed to identify proteins that are differentially expressed in M. perniciosa in response to TcPR-10 through a proteomic analysis. The fungal hyphae were subjected to one of four treatments: control treatment or 30-, 60- or 120-min treatment with the TcPR-10 protein. Two-dimensional maps revealed 191 differentially expressed proteins, 55 of which were identified by mass spectrometry. The proteins identified in all treatments were divided into the following classes: cell metabolism, stress response, zinc binding, phosphorylation mechanism, transport, autophagy, DNA repair, and oxidoreductases. The predominant class was stress-response proteins (29%), such as heat shock proteins; these proteins exhibited the highest expression levels relative to the control treatment and are known to trigger defense mechanisms against cytotoxic drugs as well as TcPR-10. Oxidoreductases (25%) were overexpressed in the control and in 30-min treatments but exhibited reduced expression at 120 min. These proteins are involved in the repair of damage caused by oxidative stress due to the contact with TcPR- 10. Consistent with the antifungal activity of TcPR-10, several proteins identified were related to detoxification, autophagy or were involved in mechanisms for maintaining fungal homeostasis, such as ergosterol biosynthesis. These results show that the sensitivity of the fungus to TcPR-10 involves several biochemical routes, clarifying the possible modes of action of this antifungal protein.

Pathophysiological mechanisms linking depression and atherosclerosis: an overview.

It is well recognized that depression is independently associated with cardiovascular events. However, uncertainties remain on the pathophysiological pathways underlying the association between depression and coronary heart disease. In addition to the traditional cardiovascular risk factors, autonomic nervous system (ANS), low grade of inflammation, platelet and hypothalamic-pituitary-adrenal axis function and genetic factors may adversely impact the endothelium of the arterial wall. We provide an overview of the pathophysiological mechanisms and indices which seem to have a role in promoting and accelerating atherosclerosis and its complications due to plaque rupture and thrombosis. Given that the relationship between depression and atherosclerosis cannot be fully explained by single mechanisms, which seem at least partially interrelated, the depression-related dysfunctions in the ANS and hypothalamic-pituitary-adrenal axis seem to play a major role, promoting chronic inflammation, endothelial dysfunction and platelet activation and aggregation, which in turn are key steps in the development of atherosclerosis and its complications.

Comparing Prediction of Early TBI Mortality with Multilayer Perceptron Neural Network and Convolutional Neural Network.

In this work, we compare the performance of a multilayer perceptron neural network and convolutional networks for the prediction of 14-day mortality in patients with TBI, using a database obtained in a low-and middle-income country, with 529 records and 16 predictor variables. The missing values of several variables were filled in with techniques such as decision tree, random forest, k-nearest-neighbor and linear regression. In the simulation of neural networks, several optimization methods were used, such as RMSProp, Adam, Adamax and SGDM. The best results obtained for the prediction rate were an accuracy of 0.845 and an area under the ROC curve of 0.911. Clinical Relevance- This proposes the prediction of early mortality in patients with TBI with an area under ROC curve of 0.911.

Physical location of the carotenoid biosynthesis genes Psy and ß-Lcy in Capsicum annuum (Solanaceae) using heterologous probes from Citrus sinensis (Rutaceae).

Carotenoids are responsible for a range of fruit colors in different hot pepper (Capsicum) varieties, from white to deep red. Color traits are genetically determined by three loci, Y, C1, and C2, which are associated with carotenogenic genes. Although such genes have been localized on genetic maps of Capsicum and anchored in Lycopersicon and Solanum, physical mapping in Capsicum has been restricted to only a few clusters of some multiple copy genes. Heterologous probes from single copy genes have been rarely used. Fluorescent in situ hybridization was performed in Capsicum annuum varieties with different fruit colors, using heterologous probes of Psy and ß-Lcy genes obtained from a BAC library of the sweet orange (Citrus sinensis). The probes hybridized in the terminal portion of a chromosome pair, confirming the location of these genes in genetic maps. The hybridized segments showed variation in size in both chromosomes.

A New Approach to Classify Cardiac Arrythmias Using 2D Convolutional Neural Networks.

Cardiovascular diseases are the number one cause of death worldwide. Detecting cardiovascular diseases in its early stages could effectively reduce the mortality rate by providing timely treatment. In this study, we propose a new methodology to detect arrythmias, using 2D Convolutional Neural Networks. The main characteristic of the proposed methodology is the use of 15 x15 pixels gray-level images, containing the values of a heartbeat of the ECG signal. This work aims to detect 17 arrythmias. To validate and test the proposed methodology, MIT-BIH database, the main benchmark database available in literature, was used. When compared to other results previously published, the obtained precision, 92.31%, is in the state-of-the-art.Clinical Relevance- The presented work provides an automatic method to detect arrythmias in ECG signals by a new methodology.

Prospective evaluation of intraoperative hemodynamics in liver transplantation with whole, partial and DCD grafts.

The interaction of systemic hemodynamics with hepatic flows at the time of liver transplantation (LT) has not been studied in a prospective uniform way for different types of grafts. We prospectively evaluated intraoperative hemodynamics of 103 whole and partial LT. Liver graft hemodynamics were measured using the ultrasound transit time method to obtain portal (PVF) and arterial (HAF) hepatic flow. Measurements were recorded on the native liver, the portocaval shunt, following reperfusion and after biliary anastomosis. After LT HAF and PVF do not immediately return to normal values. Increased PVF was observed after graft implantation. Living donor LT showed the highest compliance to portal hyperperfusion. The amount of liver perfusion seemed to be related to the quality of the graft. A positive correlation for HAF, PVF and total hepatic blood flow with cardiac output was found (p = 0.001). Portal hypertension, macrosteatosis >30%, warm ischemia time and cardiac output, independently influence the hepatic flows. These results highlight the role of systemic hemodynamic management in LT to optimize hepatic perfusion, particularly in LDLT and split LT, where the highest flows were registered.

Lung Region Segmentation in Chest X-Ray Images using Deep Convolutional Neural Networks.

Lung cancer is, by far, the leading cause of cancer death in the world. Tools for automated medical imaging analysis development of a Computer-Aided Diagnosis method comprises several tasks. In general, the first one is the segmentation of region of interest, for example, lung region segmentation from Chest X-ray imaging in the task of detecting lung cancer. Deep Convolutional Neural Networks (DCNN) have shown promising results in the task of segmentation in medical images. In this paper, to implement the lung region segmentation task on chest X-ray images, was evaluated three different DCNN architectures in association with different regularization (Dropout, L2, and Dropout + L2) and optimization methods (SGDM, RMSPROP and ADAM). All networks were applied in the Japanese Society of Radiological Technology (JSRT) database. The best results were obtained using Dropout + L2 as regularization method and ADAM as optimization method. Considering the Jaccard Coefficient obtained (0.97967 ± 0.00232) the proposal outperforms the state of the art.Clinical Relevance- The presented method reduces the time that a professional takes to perform lung segmentation, improving the effectiveness.

Automatic Bacillus Detection in Light Field Microscopy Images Using Convolutional Neural Networks and Mosaic Imaging Approach.

Tuberculosis (TB) is one of the top 10 causes of death worldwide. The diagnosis and treatment of TB in its early stages is fundamental to reducing the rate of people affected by this disease. In order to assist specialists in the diagnosis in bright field smear images, many studies have been developed for the automatic Mycobacterium tuberculosis detection, the causative agent of Tb. To contribute to this theme, a method to bacilli detection associating convolutional neural network (CNN) and a mosaic-image approach was implemented. The propose was evaluated using a robust image dataset validated by three specialists. Three CNN architectures and 3 optimization methods in each architecture were evaluated. The deeper architecture presented better results, reaching accuracies values above 99%. Other metrics like precision, sensitivity, specificity and F1-score were also used to assess the CNN models performance.

Using Convolutional Neural Networks for Classification of Bifurcation Regions in IVOCT Images.

Optical Coherence Tomography (OCT) technology enabled the experts to analyze coronary lesions from high-resolution intravascular images. Studies have shown the relationship between bifurcation regions and a higher occurrence of wall thickening and lesions in these areas. Some level of automation could benefit experts, since examining pullback frames is a laborious and time-consuming task. Although Convolutional Neural Networks (CNN) have shown promising results in classification tasks of medical images, we did not identify the use of CNN's in IVOCT images to classify bifurcation regions in the literature. In this work, we evaluated a CNN architecture in the bifurcation classification task trained with IVOCT images from 9 pullbacks from 9 different patients. We used data augmentation to balance the dataset, due to the low amount of bifurcation-labeled frames. Our classification results are comparable to other works in the literature, presenting better result in AUC (99.70%).

The liver transplant recipient with cardiac disease.

Liver transplantation is a stressful condition for the cardiovascular system of patients with advanced hepatic disease. The underlying hemodynamic and cardiac status of patients with cirrhosis is crucial to determine which patients should became recipients. Generally preoperative cardiovascular testing is performed on potential candidates who are more than 45 years old, or have diabetes mellitus, or peripheral vascular disease, or more than two standard cardiac risk factors. Recent data suggest that the prevalence of coronary artery disease among patients with cirrhosis is much greater than previously believed; it likely mirrors or exceeds the prevalence rate in the healthy population. The morbidity and mortality of patients with coronary artery disease who undergo orthotopic liver transplantation (OLT) without treatment are unacceptably high. In conclusion, accurate preoperative cardiac evaluation according to the new American Heart Association & American College of Cardiology should lead to detect and treat coronary artery disease before liver transplantation. In case of alcohol-related cardiomyopathy, portopulmonary hypertension, and hypertrophic cardiomyopathy, there should be a case-by-case discussion by the hepatologist and cardiologist to consider the patient for liver transplantation. No robust data are available on the impact of decompensated dilated heart failure in this setting. If a recipient with cardiac disease is scheduled for OLT, we strongly suggest advanced intra- and postoperative hemodynamic monitoring plus transesophageal echocardiography.

Lumen Segmentation in Optical Coherence Tomography Images using Convolutional Neural Network.

Lumen segmentation in Optical Coherence Tomography (OCT) images is a very important step to analyze points of interest that may help on atherosclerosis diagnostic and treatment. Past studies use many different methods to segment the lumen in IVOCT images, like level set, morphological reconstruction, Markov random fields, and Otsu binarization. Despite Convolutional Neural Networks (CNN) have shown promising results in the image processing area, we did not identify, in the literature, works applying CNN in IVOCT images. In this paper, we present the lumen segmentation using CNN. We evaluated three different CNN architectures. The CNNs were evaluated using three versions from the image dataset, differing from each other by image size (768x768 pixels and 192x192 pixels), and by coordinate system representation (Cartesian and polar). The best results, Accuracy, Dice index and Jaccard index of over 99%, 98% and 97%, respectively, were obtained with the smallest size images represented by polar coordinate system.

Hemodynamics during liver transplantation.

Assessing the optimal volemia in the perioperative course of liver transplantation is a challenge for the anesthesiologist. Traditional estimates of intravascular volume status, such as pulmonary artery occlusion pressure (PAOP), have been widely shown to poorly correlate with changes in cardiac output among critically ill patients. Hence, there has been recent interest in alternative, catheter-related, bedside device volume estimates using thermodilution. Continuous end diastolic volume (CEDVI) showed better correlations with cardiac performance than cardiac filling pressures in studies performed in critically ill patients. When compared with conventional pressure-derived data, preload monitoring estimated as intrathoracic blood volume index (ITBVI) with the PiCCO system based on an integrated transpulmonary thermodilution technique better reflected left ventricular filling both in critically ill patients and those who underwent liver transplantation. Moreover, in liver transplantation, the use of transoesophageal echocardiography (TEE) has been increasing for it provides rapid visualization of the dimension and function of heart chambers as well as the left ventricular end diastolic area index (EDAI) that seem to correlate with graded acute hypovolemia, although its validity as on preload index is still under discussion.

Urgency priority in kidney transplantation in Rio Grande do Sul.

In 2002, it was established a system of urgency priority for kidney transplantations in cases with no vascular or peritoneal access for dialysis. The aims of this article are to describe the system in the organ donation and procurement agency (CNCDO) as well as to show the results to date. We reviewed cases of urgency priority request for kidney transplantation addressed to the CNCDO from May 2002 to August 2005. Within this period the CNCDO received 35 urgency priority requests for kidney transplantation (mean, 1 every 1.2 months). Thirty-one (88%) were accepted as urgent, and only 4 (11%) were refused. Among the 31 accepted, 26 (83%) had the transplantation performed in an average time of 19.6 days (range, 1-90), representing only 3.2% of all cadaveric kidney transplantations during that period.

Organ donation in suicides.

INTRODUCTION: There are few reports in the literature analyzing brain death epidemiology in suicides, or the rate of donation and family authorization in such situations. OBJECTIVE: The objectives of this study were to analyze the frequency of suicide as a cause of brain death and to compare the donation rates among this population with other causes of brain death. PATIENTS AND METHODS: We reviewed records from 2627 potential donors between 1988 and 2004. RESULTS: Within that period, 101 (3.8%) cases of brain death were recorded as suicides. The mean age was significantly lower (P < .05) in cases of suicide than for other causes (26.2 + 11.1 vs 34.4 + 16.5 years); there was a male prevalence (76.2% vs 60.8%). As to suicides, the donation rate was significantly higher than in other situations (62.3% vs 43.8%). This was due to a lower rate of negative family responses (17.8% vs 32.1%). CONCLUSIONS: Suicide is a frequent cause of brain death (3.8%), mainly among young men. The donation rate in this group is higher than that due to other causes of death because of a lower negative response rate by the family. The explanation remains to be clarified for such a low refusal rate for organ donation by the relatives of potential donors due to suicide.