Saccharomyces cerevisiae NineTeen Complex (NTC)-associated Factor Bud31/Ycr063w Assembles on Precatalytic Spliceosomes and Improves First and Second Step Pre-mRNA Splicing Efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a similar to 25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.

Structured Illumination Microscopy and a Quantitative Image Analysis for the Detection of Positive Margins in a Pre-Clinical Genetically Engineered Mouse Model of Sarcoma.

Intraoperative assessment of surgical margins is critical to ensuring residual tumor does not remain in a patient. Previously, we developed a fluorescence structured illumination microscope (SIM) system with a single-shot field of view (FOV) of 2.1 × 1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 μm). The goal of this study was to test the utility of this technology for the detection of residual disease in a genetically engineered mouse model of sarcoma. Primary soft tissue sarcomas were generated in the hindlimb and after the tumor was surgically removed, the relevant margin was stained with acridine orange (AO), a vital stain that brightly stains cell nuclei and fibrous tissues. The tissues were imaged with the SIM system with the primary goal of visualizing fluorescent features from tumor nuclei. Given the heterogeneity of the background tissue (presence of adipose tissue and muscle), an algorithm known as maximally stable extremal regions (MSER) was optimized and applied to the images to specifically segment nuclear features. A logistic regression model was used to classify a tissue site as positive or negative by calculating area fraction and shape of the segmented features that were present and the resulting receiver operator curve (ROC) was generated by varying the probability threshold. Based on the ROC curves, the model was able to classify tumor and normal tissue with 77% sensitivity and 81% specificity (Youden's index). For an unbiased measure of the model performance, it was applied to a separate validation dataset that resulted in 73% sensitivity and 80% specificity. When this approach was applied to representative whole margins, for a tumor probability threshold of 50%, only 1.2% of all regions from the negative margin exceeded this threshold, while over 14.8% of all regions from the positive margin exceeded this threshold.

Comparison of Wavelet Transform and Time-Domain Analysis of Second Trimester Uterine Artery Doppler Waveforms in Screening for Pre-Eclampsia

The aim of this study was to compare time-domain waveform analysis of second-trimester uterine artery Doppler using the resistance index (RI) with waveform analysis using a mathematical tool known as wavelet transform for the prediction of pre-eclampsia (PE). This was a retrospective, nested case-cohort study of 336 women, 37 of whom subsequently developed PE. Uterine artery Doppler waveforms were analysed using both RI and waveform analysis. The utility of these indices in screening for PE was then evaluated using receiver operating characteristic curves. There were significant differences in uterine artery RI between the PE women and those with normal pregnancy outcome. After wavelet analysis, significant difference in the mean amplitude in wavelet frequency band 4 was noted between the 2 groups. The sensitivity for both Doppler RI and frequency band 4 for the detection of PE at a 10% false-positive rate was 45%. This small study demonstrates the application of wavelet transform analysis of uterine artery Doppler waveforms in screening for PE. Further prospective studies are needed in order to clearly define if this analytical approach to waveform analysis may have the potential to improve the detection of PE by uterine artery Doppler screening.

Nest defense behavior against the attack of ants in colonies of pre-emergent Mischocyttarus cerberus (hymenoptera, vespidae)

The study was carried out at the UNESP Rio Claro campus (SP), where biotests consisting of simulated ant attacks were performed in colonies of Mischocyttarus cerberus. The behaviors of the wasps were recorded with a camcorder, for further analysis. This analysis was done using the Mann-Whitney U test and the Principal Component Analysis. In the pre-emergence development stage, colonies with a single foundress defend the nest only after the first larvae appear. When there are only eggs in the nest, the wasp abandons the nest. Before leaving, the wasp rubs its gaster against the nest, releasing the ant repellent secretion. When the nest contains larvae or larvae and pupae, the foundress defends the colony, vibrating its wings, pumping her abdomen and biting the ant.

Endobronchial donor pre-treatment with ventavis: is a second administration during reperfusion beneficial to optimize post-ischemic function of non-heart beating donor lungs?

BACKGROUND: Lung retrieval from non-heart-beating donors (NHBD) has been introduced into clinical practice successfully. However, because of potentially deleterious effects of warm ischemia on microvascular integrity, use of NHBD lungs is limited by short tolerable time periods before preservation. Recently, improvement of NHBD graft function was demonstrated by donor pre-treatment using aerosolized Ventavis (Schering Inc., Berlin, Germany). Currently, there is no information whether additional application of this approach in reperfusion can further optimize immediate graft function. MATERIAL AND METHODS: Asystolic pigs (n = 5/group) were ventilated for 180-min of warm ischemia (groups 1-3). In groups 2 and 3, 100 microg Ventavis were aerosolized over 30-min using an ultrasonic nebulizer (Optineb). Lungs were then retrogradely preserved with Perfadex and stored for 3-h. After left lung transplantation and contralateral lung exclusion, grafts were reperfused for 6-h. Only in group 3, another dose of 100 microg Ventavis was aerosolized during the first 30-min of reperfusion. Hemodynamics, pO2/FiO2 and dynamic compliance were monitored continuously and compared to controls. Intraalveolar edema was quantified stereologically, and extravascular-lung-water-index (EVLWI) was measured. Statistics comprised ANOVA analysis with repeated measurements. RESULTS: Dynamic compliance was significantly lower in both Ventavis groups, but additional administration did not result in further improvement. Oxygenation, pulmonary hemodynamics, EVLWI and intraalveolar edema formation were comparable between groups. CONCLUSIONS: Alveolar deposition of Ventavis in NHBD lungs before preservation significantly improves dynamic lung compliance and represents an important strategy for improvement of preservation quality and expansion of warm ischemic intervals. However, additional application of this method in early reperfusion is of no benefit.

The "Image" of the Pharaoh in Judahite and Israelite Society According to the Glyptic Evidence

Among the many thousand scarabs, scaraboids and other stamp-seal amulets unearthed in Iron Age contexts in Cis- and Transjordan, there are many such seals showing royal Egyptian imagery on their bases. Focusing mainly on Pharaonic motifs, the paper aims to catalogue the principal iconemes, to trace their development throughout the Iron Ages and to extrapolate their significance vis-à-vis the contemporary glyptic assemblages. As will be shown, the royal imagery of the Egyptian king underwent considerable changes during pre-monarchic and monarchic times in Israel/Judah. This allows – to some extent – deducing the perception of the ‘image’ of the Egyptian king in this part of the Southern Levant at the close of the second and during the first centuries of the first millennium BCE. While the local seal production not only vividly copied earlier and contemporary Egyptian prototypes, it also developed idiosyncratic ‘Pharaonic’ motifs that were produced for the local market. On the other hand, imported Egyptian glyptic goods – such as scarabs and other amulet types – reveal further facets of the consumer behavior. They, too, shed light upon the ideological and religious preferences of the local population and illuminate the development of the vernacular attitude towards the Pharaonic symbols of power – including their obvious political and sacred connotations.

Radiomics of NSCLC: Quantitative CT Image Feature Characterization and Tumor Shrinkage Prediction

Radiomics is the high-throughput extraction and analysis of quantitative image features. For non-small cell lung cancer (NSCLC) patients, radiomics can be applied to standard of care computed tomography (CT) images to improve tumor diagnosis, staging, and response assessment. The first objective of this work was to show that CT image features extracted from pre-treatment NSCLC tumors could be used to predict tumor shrinkage in response to therapy. This is important since tumor shrinkage is an important cancer treatment endpoint that is correlated with probability of disease progression and overall survival. Accurate prediction of tumor shrinkage could also lead to individually customized treatment plans. To accomplish this objective, 64 stage NSCLC patients with similar treatments were all imaged using the same CT scanner and protocol. Quantitative image features were extracted and principal component regression with simulated annealing subset selection was used to predict shrinkage. Cross validation and permutation tests were used to validate the results. The optimal model gave a strong correlation between the observed and predicted shrinkages with . The second objective of this work was to identify sets of NSCLC CT image features that are reproducible, non-redundant, and informative across multiple machines. Feature sets with these qualities are needed for NSCLC radiomics models to be robust to machine variation and spurious correlation. To accomplish this objective, test-retest CT image pairs were obtained from 56 NSCLC patients imaged on three CT machines from two institutions. For each machine, quantitative image features with concordance correlation coefficient values greater than 0.90 were considered reproducible. Multi-machine reproducible feature sets were created by taking the intersection of individual machine reproducible feature sets. Redundant features were removed through hierarchical clustering. The findings showed that image feature reproducibility and redundancy depended on both the CT machine and the CT image type (average cine 4D-CT imaging vs. end-exhale cine 4D-CT imaging vs. helical inspiratory breath-hold 3D CT). For each image type, a set of cross-machine reproducible, non-redundant, and informative image features was identified. Compared to end-exhale 4D-CT and breath-hold 3D-CT, average 4D-CT derived image features showed superior multi-machine reproducibility and are the best candidates for clinical correlation.

Multimodal non-rigid image registration methods for therapy and surgery planning

La planificación pre-operatoria se ha convertido en una tarea esencial en cirugías y terapias de marcada complejidad, especialmente aquellas relacionadas con órgano blando. Un ejemplo donde la planificación preoperatoria tiene gran interés es la cirugía hepática. Dicha planificación comprende la detección e identificación precisa de las lesiones individuales y vasos así como la correcta segmentación y estimación volumétrica del hígado funcional. Este proceso es muy importante porque determina tanto si el paciente es un candidato adecuado para terapia quirúrgica como la definición del abordaje a seguir en el procedimiento. La radioterapia de órgano blando es un segundo ejemplo donde la planificación se requiere tanto para la radioterapia externa convencional como para la radioterapia intraoperatoria. La planificación comprende la segmentación de tumor y órganos vulnerables y la estimación de la dosimetría. La segmentación de hígado funcional y la estimación volumétrica para planificación de la cirugía se estiman habitualmente a partir de imágenes de tomografía computarizada (TC). De igual modo, en la planificación de radioterapia, los objetivos de la radiación se delinean normalmente sobre TC. Sin embargo, los avances en las tecnologías de imagen de resonancia magnética (RM) están ofreciendo progresivamente ventajas adicionales. Por ejemplo, se ha visto que el ratio de detección de metástasis hepáticas es significativamente superior en RM con contraste Gd–EOB–DTPA que en TC. Por tanto, recientes estudios han destacado la importancia de combinar la información de TC y RM para conseguir el mayor nivel posible de precisión en radioterapia y para facilitar una descripción precisa de las lesiones del hígado. Con el objetivo de mejorar la planificación preoperatoria en ambos escenarios se precisa claramente de un algoritmo de registro no rígido de imagen. Sin embargo, la gran mayoría de sistemas comerciales solo proporcionan métodos de registro rígido. Las medidas de intensidad de voxel han demostrado ser criterios de similitud de imágenes robustos, y, entre ellas, la Información Mutua (IM) es siempre la primera elegida en registros multimodales. Sin embargo, uno de los principales problemas de la IM es la ausencia de información espacial y la asunción de que las relaciones estadísticas entre las imágenes son homogéneas a lo largo de su domino completo. La hipótesis de esta tesis es que la incorporación de información espacial de órganos al proceso de registro puede mejorar la robustez y calidad del mismo, beneficiándose de la disponibilidad de las segmentaciones clínicas. En este trabajo, se propone y valida un esquema de registro multimodal no rígido 3D usando una nueva métrica llamada Información Mutua Centrada en el Órgano (Organ-Focused Mutual Information metric (OF-MI)) y se compara con la formulación clásica de la Información Mutua. Esto permite mejorar los resultados del registro en áreas problemáticas incorporando información regional al criterio de similitud, beneficiándose de la disponibilidad real de segmentaciones en protocolos estándares clínicos, y permitiendo que la dependencia estadística entre las dos modalidades de imagen difiera entre órganos o regiones. El método propuesto se ha aplicado al registro de TC y RM con contraste Gd–EOB–DTPA así como al registro de imágenes de TC y MR para planificación de radioterapia intraoperatoria rectal. Adicionalmente, se ha desarrollado un algoritmo de apoyo de segmentación 3D basado en Level-Sets para la incorporación de la información de órgano en el registro. El algoritmo de segmentación se ha diseñado específicamente para la estimación volumétrica de hígado sano funcional y ha demostrado un buen funcionamiento en un conjunto de imágenes de TC abdominales. Los resultados muestran una mejora estadísticamente significativa de OF-MI comparada con la Información Mutua clásica en las medidas de calidad de los registros; tanto con datos simulados (p<0.001) como con datos reales en registro hepático de TC y RM con contraste Gd– EOB–DTPA y en registro para planificación de radioterapia rectal usando OF-MI multi-órgano (p<0.05). Adicionalmente, OF-MI presenta resultados más estables con menor dispersión que la Información Mutua y un comportamiento más robusto con respecto a cambios en la relación señal-ruido y a la variación de parámetros. La métrica OF-MI propuesta en esta tesis presenta siempre igual o mayor precisión que la clásica Información Mutua y consecuentemente puede ser una muy buena alternativa en aplicaciones donde la robustez del método y la facilidad en la elección de parámetros sean particularmente importantes. Abstract Pre-operative planning has become an essential task in complex surgeries and therapies, especially for those affecting soft tissue. One example where soft tissue preoperative planning is of high interest is liver surgery. It involves the accurate detection and identification of individual liver lesions and vessels as well as the proper functional liver segmentation and volume estimation. This process is very important because it determines whether the patient is a suitable candidate for surgical therapy and the type of procedure. Soft tissue radiation therapy is a second example where planning is required for both conventional external and intraoperative radiotherapy. It involves the segmentation of the tumor target and vulnerable organs and the estimation of the planned dose. Functional liver segmentations and volume estimations for surgery planning are commonly estimated from computed tomography (CT) images. Similarly, in radiation therapy planning, targets to be irradiated and healthy and vulnerable tissues to be protected from irradiation are commonly delineated on CT scans. However, developments in magnetic resonance imaging (MRI) technology are progressively offering advantages. For instance, the hepatic metastasis detection rate has been found to be significantly higher in Gd–EOB–DTPAenhanced MRI than in CT. Therefore, recent studies highlight the importance of combining the information from CT and MRI to achieve the highest level of accuracy in radiotherapy and to facilitate accurate liver lesion description. In order to improve those two soft tissue pre operative planning scenarios, an accurate nonrigid image registration algorithm is clearly required. However, the vast majority of commercial systems only provide rigid registration. Voxel intensity measures have been shown to be robust measures of image similarity, and among them, Mutual Information (MI) is always the first candidate in multimodal registrations. However, one of the main drawbacks of Mutual Information is the absence of spatial information and the assumption that statistical relationships between images are the same over the whole domain of the image. The hypothesis of the present thesis is that incorporating spatial organ information into the registration process may improve the registration robustness and quality, taking advantage of the clinical segmentations availability. In this work, a multimodal nonrigid 3D registration framework using a new Organ- Focused Mutual Information metric (OF-MI) is proposed, validated and compared to the classical formulation of the Mutual Information (MI). It allows improving registration results in problematic areas by adding regional information into the similitude criterion taking advantage of actual segmentations availability in standard clinical protocols and allowing the statistical dependence between the two modalities differ among organs or regions. The proposed method is applied to CT and T1 weighted delayed Gd–EOB–DTPA-enhanced MRI registration as well as to register CT and MRI images in rectal intraoperative radiotherapy planning. Additionally, a 3D support segmentation algorithm based on Level-Sets has been developed for the incorporation of the organ information into the registration. The segmentation algorithm has been specifically designed for the healthy and functional liver volume estimation demonstrating good performance in a set of abdominal CT studies. Results show a statistical significant improvement of registration quality measures with OF-MI compared to MI with both simulated data (p<0.001) and real data in liver applications registering CT and Gd–EOB–DTPA-enhanced MRI and in registration for rectal radiotherapy planning using multi-organ OF-MI (p<0.05). Additionally, OF-MI presents more stable results with smaller dispersion than MI and a more robust behavior with respect to SNR changes and parameters variation. The proposed OF-MI always presents equal or better accuracy than the classical MI and consequently can be a very convenient alternative within applications where the robustness of the method and the facility to choose the parameters are particularly important.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast.

Temperature-sensitive alleles in four genes (slu7-1, prp16-2, prp17-1, and prp18-1) are known to confer a specific block to the second chemical step of pre-mRNA splicing in vivo in the yeast Saccharomyces cerevisiae. Previous studies showed that Prp16p and Prp18p are required solely for the second step in vitro. The RNA-dependent ATPase, Prp16p, functions at a stage in splicing when ATP is required, whereas Prp18p functions at an ATP-independent stage. Here we use immunodepletion to show that the roles of Slu7p and Prp17p are also confined to the second step of splicing. We find that extracts depleted of Prp17p require both Prp17p and ATP for slicing complementation, whereas extracts depleted of Slu7p require only the addition of Slu7p. These different ATP requirements suggest that Prp16p and Prp17p function before Prp18p and Slu7p. Although SLU7 encodes an essential gene product, we find that a null allele of prp17 is temperature-sensitive for growth and has a partial splicing defect in vitro. Finally, high-copy suppression experiments indicate functional interactions between PRP16 and PRP17, PRP16 and SLU7, and SLU7 and PRP18. Taken together, the results suggest that these four factors may function within a multi-component complex that has both an ATP-dependent and an ATP-independent role in the second step of pre-mRNA splicing.