Branch Site Research Articles

Semi-fluorinated Poly(aryl ether sulfone)s via step-growth polymerization of perfluorocyclohexene with bisphenols

Perfluorocyclohexene (PFCH) undergoes step-growth polymerization and copolymerization with variable sulfone and polyaromatic containing bisphenols in the presence of triethyl amine under mild conditions affording a new class of semi-fluorinated poly(aryl ether sulfone)s. The apparent vinyl addition/elimination mechanism provides regio-selective vinyl versus allylic substituted polymers with reactive 1,2-diaryloxyvinylene linkages in the main chain. Moderate to high molecular weight (ca. 50 kDa) polymers gave tough, creaseable films from solution with excellent transparency, and variable refractive indices ranging from 1.61 to 1.65 (at 589.2 nm). The amorphous sulfone copolymers exhibited glass transition temperatures (T g ) ranging from 122 to 225 °C and thermal stabilities with decomposition temperatures (T d at 5% wt. loss) ranging from 336 to 404 °C in N 2 . Enchained PFCH vinylene ether groups provide competing but controllable branch sites during later stages of polymerization and further serve as effective targets for post-polymerization functionalization or simple thermally induced crosslinking. Semi-fluorinated poly(aryl ether sulfone) thermoplastics and tailored networks are pursued for optical, electronic, and gas separation applications. • Poly(aryl ether sulfone)s via base mediated polymerization of decafluorocyclohexene and commercial bisphenols. • Tough flexible films provide high thermal stability, excellent optical transparency, and variable refractive index. • Step-growth addition/elimination affords reactive vinylene ether enchainment available for post reactions or crosslinking.

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

Central retinal artery delineation using magnetic resonance imaging.

Few studies have explored the central retinal artery (CRA) using neuroimaging. Our study aimed to explore this using magnetic resonance imaging (MRI). A total of 81 patients with intact orbital structures and visual function underwent thin-slice contrast MRI. The identified CRAs showed highly variable morphologies on both axial and sagittal images. On the axial images, the CRAs were detected in the right orbit in 11.1% and in the left orbit in 19.8%. The distance between the site of CRA branching from the ophthalmic artery to the posterior limit of the bulb was 18.8 ± 3.9mm (12.8-24.6mm) on the right and 18.9 ± 3.3mm (14.6-26.7mm) on the left. On the sagittal images, CRAs were detected on the right in 76.5% and on the left in 85.2%. The distance between the CRA branching site and the posterior limit of the bulb was 20.4 ± 3.8mm (14.2-28.2mm) on the right and 19.2 ± 3.7mm (11.3-27.1mm) on the left. Thin-sliced, contrast sagittal MRI can be used to explore the proximal part of the CRA. In particular, serial sagittal imaging may be useful for detecting the CRAs and their relationship with relevant structures.

An Exploration of the Sympathetic Component of the Recurrent Laryngeal Nerve

INTRODUCTIONHigh interest in the recurrent laryngeal nerve (RLN) morphology has been dictated by the frequent iatrogenic injures that can result in multifaceted functional losses such as dysphagia, voice alteration, and breathing. Thus, RLN has been extensively studied on the macroscopic and histological levels. According to the literature, RLN is a mixed nerve carrying motor, sensory, and preganglionic parasympathetic fibers to the larynx and displays asymmetry in branching from the vagus nerve. Even though gross anastomoses between RLN and the cervical sympathetic trunk have been observed, the presence of sympathetic postganglionic fibers within RLN have not been addressed. The present study was conducted to determine if catecholaminergic fibers are present within the RLN using immunohistochemical analysis for tyrosine hydroxylase (TH).METHODSRLN samples were obtained from 19 formalin‐preserved adult human cadavers (male n=11, female n=8). Anastomoses with sympathetic structures were noted and documented before obtaining tissue samples for histological examination. Samples were collected one centimeter distal to the RLN branching site. All sections were prepared using Luxol fast blue staining to reveal the presence of myelinated and unmyelinated fibers and TH antibody staining was used to determine the presence of catecholaminergic fibers. Histological slides were then photographed and quantitative analysis of the images was conducted with ImageJ software by measuring of the effective surface area (ESA), TH‐positive, myelinated, and unmyelinated. We reported results as mean ± SEM.RESULTSNeural communications between right RLNs and sympathetic ganglia were observed in two whole‐body donors. No statistically significant differences in the number of the fascicles, ESA, myelinated, and unmyelinated fibers between right and left RLNs were found. TH‐positive fibers were detected bilaterally or unilaterally in 89.5% of examined donors. Bilateral absence of TH activity throughout the effective surface area was observed in two cases. TH‐positive nerve fiber area within the RLNs varied from 0% to 22.14% (Figure 1). The average of TH‐positive fibers on the right was 0.045 ± 0.011 mm2 (4.47% ± 1.1% of ESA) and 0.05 ± 0.015 mm2 (5.03±1.5% of ESA) on the left. A positive correlation between the number of unmyelinated fibers and TH‐positive component was found (P=0.022 and P=0.0026 on the right and left, respectfully). All parameters, such as fascicles, ESA, myelinated, unmyelinated, and TH‐positive fibers, occurred independently of age, gender, or conditions (p>0.05).CONCLUSIONSympathetic postganglionic fibers within RLN nerve exist; however, there is a considerable difference in their number between individuals. However, the origin and functions of this component are ambiguous.SIGNIFICANCEKnowledge of the sympathetic component of RLN could assist in better understanding the physiological effects and adverse responses of vagus nerve stimulation therapy and the sequala of the RLN injury.

Implementation of a transitions of care elective course at a pharmacy school with branch sites

Background: The Center for Advancement of Pharmacy Education educational outcomes include an objective regarding managing patient's healthcare needs during transitions of care (TOC). Pharmacy school curriculums should be designed to teach student pharmacists to provide TOC services. Methods: A TOC focused elective was implemented across three sites of a pharmacy school. The course involved lectures, interactive activities, flip-the-classroom activities, and group projects. Results: A total of 51 students completed course evaluation surveys over two course offerings. Most students, 92.2% (n = 47), agreed or strongly agreed that the course objectives were met. Similarly, 96.1% (n = 49) agreed or strongly agreed that the course was well organised. Conclusion: An assessment of course evaluations demonstrated positive student perceptions of the course and successful course implementation. Schools of pharmacy should consider implementing a TOC focused elective to provide students with foundational knowledge for caring for patients throughout the continuum of care.

Anatomy of the proximal septal vein in patients with focal intramural ventricular arrhythmias.

Focal ventricular arrhythmias (VAs) originating from the intramural myocardium of the basal septum are difficult to localize and ablate. Proximal septal veins emptying into the great cardiac vein can reach close to the origin of intramural arrhythmias. To assess characteristics of proximal septal coronary veins in patients with intramural VAs. Among 84 consecutive patients with intramural VAs, 29 patients (age 60 ± 11years, 16 males, ejection fraction 47 ± 13%) underwent preprocedural cardiac computed tomographic angiography (CTA). In 14 of these patients, the intramural site of origin (SOO) was identified with multipolar catheters. The intramural SOO could not be accessed with mapping catheters in the other 15 patients while mapping the coronary venous system. The CTA identified sizable proximal septal veins in all patients in whom the SOO could be accessed with mapping catheters. In the patients in whom the intramural SOO was not identified, the proximal septal veins were often either small (<2 mm atthe branching site) or non-existent (n = 9, p = .001). The proximal septal veins in patients in whom the SOO was identified were larger than in the patients in whom the SOO could not be identified (3.0 ± 0.6 mm vs. 2.1 ± 0.9 mm, p = .01). Preprocedural imaging with CTAs can be beneficial in identifying the anatomy of proximal septal coronary veins that allow adequate mapping of patients with suspected intramural VAs.

Diversity and complexity of the cavotricuspid isthmus in rabbits: A novel scheme for classification and geometrical transformation of anatomical structures.

The aim of this study was to describe the morphology of the cavotricuspid isthmus (CTI) in detail and introduce a comprehensive scheme to describe the topology of this region based on functional considerations. This may lead to a better understanding of isthmus-dependent flutter and fibrillation and to improved intervention strategies. We used images of the cavotricuspid isthmus from 52 rabbits of both sexes with a median weight of 3.40 ± 0.93 kg. The area of the CTI was 124.25 ± 42.14 mm2 with 53.28 ± 21.13 mm2 covered by pectinate muscles connecting the terminal crest and the vestibule. Isthmus length decreased from inferolateral (13.09 ±2.14 mm) to central (9.85 ± 2.14 mm) to paraseptal (4.88 ± 1.96 mm) resembling the overall human geometry. Ramification sites of pectinate muscles were identified and six levels dividing the CTI from posterior to anterior were introduced. This allowed the classification of pectinate muscle segments based on the connected ramification level. To account for the high inter-individual variations in size and shape, the CTI was projected onto a normalized reference frame using bilinear transformation. Furthermore, two measures of complexity were introduced: (i) the ramification index, which reflects the total number of muscle segments connected to a ramification site and (ii) the complexity index, which reflects the type of ramification (branching or merging site). Topological analysis showed that the complexity of the pectinate muscle network decreases from inferolateral to paraseptal and that the number of electrically uncoupled parallel pathways increases in the central section between the terminal crest and the vestibule which introduces potential reentry pathways.

Functional Hyperbranched Polythioamides Synthesized from Catalyst‐free Multicomponent Polymerization of Elemental Sulfur†

Comprehensive SummaryHyperbranched polymers with unique topological structures, large number of branching sites and terminal groups have attracted much attention, and are expected to possess advanced functionalities compared with their linear polymer counterparts. The development of hyperbranched polymers is hence highly desired but challenging, especially for sulfur‐containing polymers which are attractive metal absorbents, optical materials, dielectric materials, and self‐healing materials. In this work, six hyperbranched polythioamides with various topological structures, well‐defined repeating units, satisfying yields (up to 99%), and high molecular weights (up to 101400 g/mol) were successfully designed and synthesized from the catalyst‐free multicomponent polymerization of elemental sulfur, aromatic alkynes and aliphatic amines, through different monomer combination strategies based on the designed three‐ or four‐functional alkyne and amine monomers. The hyperbranched polythioamides possess unique luminescence property, and strong affinity toward Hg2+, which can be utilized in the fluorescence detection of Hg2+, as well as mercury removal from aqueous solutions with high efficiency of 99.99% and low mercury residue of 0.1 ppb. Hyperbranched polythioamides with unique structures may be developed to a group of fascinating materials and find their potential applications as metal absorbents and optoelectronic materials.

Structural basis of branch site recognition by the human spliceosome.

Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17S U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)–dependent remodeling and binding to the pre–messenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.

Phylogeny and conservation of plant U2A/U2A', a core splicing component in U2 spliceosomal complex.

This study systematically identifies 112 U2A genes from 80 plant species by combinatory bioinformatics analysis, which is important for understanding their phylogenetic history, expression profiles and for predicting specific functions. In eukaryotes, a pre-mRNA can generate multiple transcripts by removing certain introns and joining corresponding exons, thus greatly expanding the transcriptome and proteome diversity. The spliceosome is a mega-Dalton ribonucleoprotein (RNP) complex that is essential for the process of splicing. In spliceosome components, the U2 small nuclear ribonucleoprotein (U2 snRNP) forms the pre-spliceosome by association with the branch site. An essential component that promotes U2 snRNP assembly, named U2A, has been extensively identified in humans, yeast and nematodes. However, studies examining U2A genes in plants are scarce. In this study, we performed a comprehensive analysis and identified a total of 112 U2A genes from 80 plant species representing dicots, monocots, mosses and algae. Comparisons of the gene structures, protein domains, and expression patterns of 112 U2A genes indicated that the conserved functions were likely retained by plant U2A genes and important for responses to internal and external stimuli. In addition, analysis of alternative transcripts and splice sites of U2A genes indicated that the fifth intron contained a conserved alternative splicing event that might be important for its molecular function. Our work provides a general understanding of this splicing factor family in terms of genes and proteins, and it will serve as a fundamental resource that will contribute to further mechanistic characterization in plants.

Targeted polyelectrolyte complex micelles treat vascular complications in vivo

Vascular disease is a leading cause of morbidity and mortality in the United States and globally. Pathological vascular remodeling, such as atherosclerosis and stenosis, largely develop at arterial sites of curvature, branching, and bifurcation, where disturbed blood flow activates vascular endothelium. Current pharmacological treatments of vascular complications principally target systemic risk factors. Improvements are needed. We previously devised a targeted polyelectrolyte complex micelle to deliver therapeutic nucleotides to inflamed endothelium invitro by displaying the peptide VHPKQHR targeting vascular cell adhesion molecule 1 (VCAM-1) on the periphery of the micelle. This paper explores whether this targeted nanomedicine strategy effectively treats vascular complications invivo. Disturbed flow-induced microRNA-92a (miR-92a) has been linked to endothelial dysfunction. We have engineered a transgenic line (miR-92aEC-TG /Apoe-/- ) establishing that selective miR-92a overexpression in adult vascular endothelium causally promotes atherosclerosis in Apoe-/- mice. We tested the therapeutic effectiveness of the VCAM-1-targeting polyelectrolyte complex micelles to deliver miR-92a inhibitors and treat pathological vascular remodeling invivo. VCAM-1-targeting micelles preferentially delivered miRNA inhibitors to inflamed endothelial cells invitro and invivo. The therapeutic effectiveness of anti-miR-92a therapy in treating atherosclerosis and stenosis in Apoe-/- mice is markedly enhanced by the VCAM-1-targeting polyelectrolyte complex micelles. These results demonstrate a proof of concept to devise polyelectrolyte complex micelle-based targeted nanomedicine approaches treating vascular complications invivo.

Rare NOTCH3 Variants in a Chinese Population-Based Cohort and Its Relationship With Cerebral Small Vessel Disease.

Researches on rare variants of NOTCH3 in the general Chinese population are lacking. This study aims to describe the spectrum of rare NOTCH3 variants by whole-exome sequencing in a Chinese community-based cohort and to investigate the association between rare NOTCH3 variants and age-related cerebral small vessel disease. The cross-sectional study comprised 1065 participants who underwent whole-exome sequencing and brain magnetic resonance imaging. NOTCH3 variants with minor allele frequency<1% in all 4 public population databases (1000 Genomes, ESP6500siv2_ALL, GnomAD_ALL, and GnomAD_EAS) were defined as rare variants. Multivariable linear and logistic regressions were used to investigate the associations between rare NOTCH3 variants and volume of white matter hyperintensities and cerebral small vessel disease burden. Clinical and imaging characteristics of rare NOTCH3 variant carriers were summarized. Sixty-five rare NOTCH3 variants were identified in 147 of 1065 (13.8%) participants, including 57 missense single nucleotide polymorphisms (SNPs), 5 SNPs in splice branching sites, and 3 frameshift deletions. A significantly higher volume of white matter hyperintensities and heavier burden of cerebral small vessel disease was found in carriers of rare NOTCH3 EGFr (epidermal growth factor-like repeats)-involving variants, but not in carriers of EGFr-sparing variants. The carrying rate of rare EGFr-involving NOTCH3 variants in participants with dementia or stroke was significantly higher than those without dementia or stroke (12.4% versus 6.6%, P=0.041). Magnetic resonance imaging signs suggestive of CADASIL were found in 3.4% (5/145) rare EGFr cysteine-sparing NOTCH3 variant carriers but not in 2 cysteine-altering NOTCH3 variant carriers. Carriers of rare NOTCH3 variants involving the EGFr domain may be genetically predisposed to age-related cerebral small vessel disease in the general Chinese population.

Phylogenetic Analysis of the Plant U2 snRNP Auxiliary Factor Large Subunit A Gene Family in Response to Developmental Cues and Environmental Stimuli.

In all organisms, splicing occurs through the formation of spliceosome complexes, and splicing auxiliary factors are essential during splicing. U2AF65 is a crucial splicing cofactor, and the two typical RNA-recognition motifs at its center recognize and bind the polypyrimidine sequence located between the intron branch site and the 3′-splice site. U2AF65A is a member of the U2AF65 gene family, with pivotal roles in diseases in mammals, specifically humans; however, few studies have investigated plant U2AF65A, and its specific functions are poorly understood. Therefore, in the present study, we systematically identified U2AF65A in plant species from algae to angiosperms. Based on 113 putative U2AF65A sequences from 33 plant species, phylogenetic analyses were performed, followed by basic bioinformatics, including the comparisons of gene structure, protein domains, promoter motifs, and gene expression levels. In addition, using rice as the model crop, we demonstrated that the OsU2AF65A protein is localized to the nucleus and cytoplasm, and it is involved in responses to various stresses, such as drought, high salinity, low temperature, and heavy metal exposure (e.g., cadmium). Using Arabidopsis thaliana and rice mutants, we demonstrated that U2AF65A is involved in the accumulation of plant biomass, growth of hypocotyl upon thermal stimulation, and reduction of tolerance of high temperature stress. These findings offer an overview of the U2AF65 gene family and its stress response functions, serving as the reference for further comprehensive functional studies of the essential specific splicing cofactor U2AF65A in the plant kingdom.

Directed homoenzymatic fragmentation of the plant protopectin complex: Assessment criteria

Introduction. The functional basis of protopectin complex can be represented as a network of regions that consist of homogalacturonan sequences and a base of rhamnogalacturonans-I, i.e. rhamnosyl-containing branching sites. Enzymatic isolation of these regions is possible only at a certain minimal native degree of polymerization. The research objective was to develop a system of criteria for assessing the potential applicability of the enzymatic transformation of plant protopectin complex.&#x0D; Study objects and methods. The research featured the polymerization degree of the homogalacturonan regions of the protopectin complex and produced a system of assessment criteria for the enzymatic fragmentation potential of the protopectin complex. The theoretical calculations were based on the values of the mass fractions of rhamnosyl and galacturonide residues in plant cell walls. The result was a new polymerization degree analytical function.&#x0D; Results and discussion. The ratio of the mass fractions of rhamnosyl and galacturonide residues in the water-insoluble plant tissue served as a dimensionless criterion of applicability. The rational condition for the dimensionless criterion of applicability was based on the fundamental constraint for homogalacturonan regions in the protopectin complex. It was expressed by a fundamental inequation. The rational area for determining the numerical values of the applicability criterion was presented as . The functional dependence was reduced to a two-dimensional criteria space as “width of rhamnosyl branches vs. the criterion of applicability”, where each pectin-containing raw material was given a single uniquely defined position. The boundary conditions for the criteria space were determined analytically. &#x0D; Conclusion. The new approach offers an assessment of the enzymatic fragmentation potential of the plant protopectin complex by homoenzyme preparations. The approach is in fact the second stage of the decision tree in the science-based technology for pectin and its products.

Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila.

SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and–H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system.

Computational Modeling of Catheter-based Radiofrequency Renal Denervation with Patient-specific Model.

Renal sympathetic denervation (RDN) is an effective approach for uncontrolled hypertension. Although several studies have compared the ablation characteristics at various locations, there is no direct comparative study on the effect of ablation in main and branch renal artery (RAs) and different electrode materials. The study aims to investigate the effect of different electrode materials (copper, gold, and platinum) and positions (proximal, middle, or distal site) on ablation. A 3D patient-specific renal artery model and a unipolar model (470 kHz) were constructed to mimic RDN. Two therapeutic strategies, including main (site 1 and 2) and branch (site 3) ablations were simulated with three electrode materials. The finite element method was used to calculate the coupled electric-thermal-flow field. Maximum lesion depth, width, area, and lesion angle were analyzed. The results showed that the difference in lesion width and depth was no mere than 0.5 mm, and the maximum difference value in lesion area is 0.683 mm2 among three electrode materials. The lesion angle of proximal site 1 versus middle site 2 was 58.39 ° and 52.23 °, but the difference between distal site 3 and site 1, or site 2 was 29.19 ° and 35.35 ° respectively. There is no significant difference in the use of the three electrode materials, and ablation at the distal site of the artery is more effective.Clinical Relevance-This provides a reference for the selection of RF electrode materials and ablation locations.

EP.WE.78Variation in the Branching Pattern of the Anterior Branches of the External Carotid Artery

Abstract Aims The external carotid artery (ECA) shows a variable branching pattern of its stemming vessels. The aim of this study is to investigate the origin of the three primary anterior branches of the external carotid artery (ECA): the superior thyroid (STA), lingual (LA), and facial arteries (FA). Methods The branching pattern of the external carotid artery was studied on 15 Thiel embalmed cadavers of Scottish population (7 males and 8 females). The carotid triangle was dissected bilaterally in order to uncover the common carotid (CCA), internal carotid (ICA) and external carotid arteries (ECA). Moreover, the origin sites of the main anterior branches of the ECA (STA, LA and FA) were assessed in relation to the carotid bifurcation (CB) and with regard to sex and side. Results The STA was found to arise more frequently from the ECA (60%) than from the CCA (40%). Regarding the assessment of the branching patterns of the external carotid artery, the specimens where the STA, LA and FA emerged as individual branches were 90% of cases. The distances from the vessel origin site to the CB were found to be (8.11 ± 2.77), (19.38 ± 8.85) and (27.95 ± 10.15), for the STA, LA, and FA respectively. Conclusions In-depth knowledge of the branching pattern of the external carotid artery is of great importance. The current findings have confirmed that the ECA branching pattern is highly variable. Therefore, considering some radiological imaging before conducting any invasive procedure in the neck region could be vital to prevent iatrogenic injuries.

Identification and in silico characterization of structural and functional impacts of genetic variants in milk protein genes in the Zebu breeds Guzerat and Gyr.

Whole genome sequencing of bovine breeds has allowed identification of genetic variants in milk protein genes. However, functional repercussion of such variants at a molecular level has seldom been investigated. Here, the results of a multistep Bioinformatic analysis for functional characterization of recently identified genetic variants in Brazilian Gyr and Guzerat breeds is described, including predicted effects on the following: (i) evolutionary conserved nucleotide positions/regions; (ii) protein function, stability, and interactions; (iii) splicing, branching, and miRNA binding sites; (iv) promoters and transcription factor binding sites; and (v) collocation with QTL. Seventy-one genetic variants were identified in the caseins (CSN1S1, CSN2, CSN1S2, and CSN3), LALBA, LGB, and LTF genes. Eleven potentially regulatory variants and two missense mutations were identified. LALBA Ile60Val was predicted to affect protein stability and flexibility, by reducing the number the disulfide bonds established. LTF Thr546Asn is predicted to generate steric clashes, which could mildly affect iron coordination. In addition, LALBA Ile60Val and LTF Thr546Asn affect exonic splicing enhancers and silencers. Consequently, both mutations have the potential of affecting immune response at individual level, not only in the mammary gland. Although laborious, this multistep procedure for classifying variants allowed the identification of potentially functional variants for milk protein genes.

A broad analysis of splicing regulation in yeast using a large library of synthetic introns.

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5' donor site, the branch site, and the 3' acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3' splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns' 3' ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism's splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

Deep Recursive Bayesian Tracking for Fully Automatic Centerline Extraction of Coronary Arteries in CT Images.

Extraction of coronary arteries in coronary computed tomography (CT) angiography is a prerequisite for the quantification of coronary lesions. In this study, we propose a tracking method combining a deep convolutional neural network (DNN) and particle filtering method to identify the trajectories from the coronary ostium to each distal end from 3D CT images. The particle filter, as a non-linear approximator, is an appropriate tracking framework for such thin and elongated structures; however, the robust ‘vesselness’ measurement is essential for extracting coronary centerlines. Importantly, we employed the DNN to robustly measure the vesselness using patch images, and we integrated softmax values to the likelihood function in our particle filtering framework. Tangent patches represent cross-sections of coronary arteries of circular shapes. Thus, 2D tangent patches are assumed to include enough features of coronary arteries, and the use of 2D patches significantly reduces computational complexity. Because coronary vasculature has multiple bifurcations, we also modeled a method to detect branching sites by clustering the particle locations. The proposed method is compared with three commercial workstations and two conventional methods from the academic literature.