Validation of a novel expressed sequence tag (EST) clustering method and development of a phylogenetic annotation pipeline for livestock gene families

Prediction of functions of genes in a genome is a key step in all genome sequencing projects. Sequences that carry out important functions are likely to be conserved between evolutionarily distant species and can be identified using cross-species comparisons. In the absence of completed genomes and the accompanying high-quality annotations, expressed sequence tags (ESTs) from random cDNA clones are the primary tools for functional genomics. EST datasets are fragmented and redundant, necessitating clustering of ESTs into groups that are likely to have been derived from the same genes. EST clustering helps reduce the search space for sequence homology searching and improves the accuracy of function predictions using EST datasets. This dissertation is a case study that describes clustering of Bos taurus and Sus scrofa EST datasets, and utilizes the EST clusters to make computational function predictions using a comparative genomics approach. We used a novel EST clustering method, TAMUClust, to cluster bovine ESTs and compare its performance to the bovine EST clusters from TIGR Gene Indices (TGI) by using bovine ESTs aligned to the bovine genome assembly as a gold standard. This comparison study reveals that TAMUClust and TGI are similar in performance. Comparisons of TAMUClust and TGI with predicted bovine gene models reveal that both datasets are similar in transcript coverage. We describe here the design and implementation of an annotation pipeline for predicting functions of the Bos taurus (cattle) and Sus scrofa (pig) transcriptomes. EST datasets were clustered into gene families using Ensembl protein family clusters as a framework. Following clustering, the EST consensus sequences were assigned predicted function by transferring annotations of the Ensembl vertebrate protein(s) they are grouped to after sequence homology searches and phylogenetic analysis. The annotations benefit the livestock community by helping narrow down the gamut of direct experiments needed to verify function

TENASCIN CYTOTACTIN EGF-LIKE REPEATS - NOVEL MATRIKINE LIGANDS FOR THE EPIDERMAL GROWTH FACTOR RECEPTOR

Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits significantly higher mobility in Ten14-EGFR complex compared to the EGF-EGFR complex; we attributed this to loss of key high-affinity interactions within the Ten14-EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant KD of 74µM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability to induce degradation of receptor, or undergo EGFR-mediated internalization. This transient interaction of Ten14 with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling. We investigated whether Ten14-mediated surface restriction of EGFR resulted in altered cellular responses compared to EGF. Activation of PLCã and m-calpain, molecules associated with migration, were noted even at sub-saturating doses of Ten14. However, activation of ERK/MAPK, p90RSK and Elk1, factors affecting proliferation, remained low even at high Ten14 concentrations. Similar activation profiles were observed for EGF-treated cells at 4°C, a maneuver that limits receptor internalization. We demonstrated a direct concurrent effect of such altered signaling on overall biophysical responses - sustained migration was observed at lower levels of Ten14 that activated PLCã, but proliferation remained basal. We present a novel class of EGFR ligands that can potentially signal as a part of the matrix, triggering select signaling cascades leading to a directed cellular response from an otherwise pleiotropic receptor

Heptaphyrins: expanded porphyrins with seven heterocyclic rings

Expanded porphyrins containing seven pyrrole/heterocyclic rings linked in a cyclic fashion are termed heptaphyrins. The number of π-electrons in heptaphyrins depends on the number ofmeso carbon bridges used to link the heterocyclic rings, accordingly heptaphyrins with 28π-electrons and 30π-electrons are reported to date. Both condensation reactions of the appropriate precursors and acid-catalysed oxidative coupling reactions have been utilized to synthesise the heptaphyrins. The 30π heptaphyrins exhibit rich structural diversity where some of the heterocyclic rings in the macrocycle undergo a 180° ring flipping. An overview of the synthetic methods employed for the synthesis of heptaphyrins, their spectroscopic properties, structural behaviour and aromatic properties are highlighted in this paper

Core modified oxybenziporphyrins: new aromatic ligands for metalcarbon bond activation

Successful syntheses of two new aromatic core modified oxybenziporphyrins by a simple 3 + 1 methodology and the first aromatic core modified oxybenziporphyrin palladium complex are reported

Inverted porphyrins and expanded porphyrins: an overview

Porphyrins and metallopophyrins have attracted the attention of chemists for the past 100 years or more owing to their widespread involvement in biology. More recently, synthetic porphyrins and porphyrin-like macrocycles have attracted the attention of researchers due to their diverse applications as sensitizers for photodynamic therapy, MRI contrasting agents, and complexing agents for larger metal ions and also for their anion binding abilities. The number of π-electrons in the porphyrin ring can be increased either by increasing the numberof conjugated double bonds between the pyrrole rings or by increasing the number of heterocyclic rings. Thus, 22π sapphyrins, 26π rubyrins, 30π heptaphyrins, 34π octaphyrins and higher cyclic polypyrrole analogues containing 40π, 48π, 64π, 80π and 96π systems have recently been reported in the literature. These macrocycles show rich structural diversity where normal and different kinds of inverted structures have been identified. In this review, an attempt has been made to collect the literature of the inverted porphyrins and expanded porphyrins reported until December 2001. Since themeso aryl expanded porphyrins have tendency to form both inverted and non-inverted structures more emphasis has been given to meso aryl expanded porphyrins

Efficacy of beveled tip aspiration catheter in mechanical thrombectomy for acute ischemic stroke

BACKGROUND: Direct aspiration thrombectomy techniques use large bore aspiration catheters for mechanical thrombectomy. Several aspiration catheters are now available. We report a bench top exploration of a novel beveled tip catheter and our experience in treating large vessel occlusions (LVOs) using next-generation aspiration catheters. METHODS: A retrospective analysis from a prospectively maintained database comparing the bevel shaped tip aspiration catheter versus non-beveled tip catheters was performed. Patient demographics, periprocedural metrics, and discharge and 90-day modified Rankin Scale (mRS) scores were collected. Patients were divided into two groups based on which aspiration catheter was used. RESULTS: Our data showed no significant difference in age, gender, IV tissue plasminogen activator administration, admission NIH Stroke Scale score, baseline mRS, or LVO location between the beveled tip and flat tip groups. With the beveled tip, Thrombolysis in Cerebral Infarction (TICI) 2C or better recanalization was more frequent overall (93.2% vs 74.2%, p=0.017), stent retriever usage was lower (9.1% vs 29%, p=0.024), and patients had lower mRS on discharge (median 3 vs 4, p \u3c 0.001) and at 90 days (median 2 vs 4, p=0.008). CONCLUSION: Patients who underwent mechanical thrombectomy with the beveled tip catheter had a higher proportion of TICI 2C or better and had a significantly lower mRS score on discharge and at 90 days

Development and Application of Bovine and Porcine Oligonucleotide Arrays with Protein-Based Annotation

The design of oligonucleotide sequences for the detection of gene expression in species with disparate volumes of genome and EST sequence information has been broadly studied. However, a congruous strategy has yet to emerge to allow the design of sensitive and specific gene expression detection probes. This study explores the use of a phylogenomic approach to align transcribed sequences to vertebrate protein sequences for the detection of gene families to design genomewide 70-mer oligonucleotide probe sequences for bovine and porcine. The bovine array contains 23,580 probes that target the transcripts of 16,341 genes, about 72% of the total number of bovine genes. The porcine array contains 19,980 probes targeting 15,204 genes, about 76% of the genes in the Ensembl annotation of the pig genome. An initial experiment using the bovine array demonstrates the specificity and sensitivity of the array

The Brainstem in Emotion: A Review

Emotions depend upon the integrated activity of neural networks that modulate arousal, autonomic function, motor control, and somatosensation. Brainstem nodes play critical roles in each of these networks, but prior studies of the neuroanatomic basis of emotion, particularly in the human neuropsychological literature, have mostly focused on the contributions of cortical rather than subcortical structures. Given the size and complexity of brainstem circuits, elucidating their structural and functional properties involves technical challenges. However, recent advances in neuroimaging have begun to accelerate research into the brainstem’s role in emotion. In this review, we provide a conceptual framework for neuroscience, psychology and behavioral science researchers to study brainstem involvement in human emotions. The “emotional brainstem” is comprised of three major networks – Ascending, Descending and Modulatory. The Ascending network is composed chiefly of the spinothalamic tracts and their projections to brainstem nuclei, which transmit sensory information from the body to rostral structures. The Descending motor network is subdivided into medial projections from the reticular formation that modulate the gain of inputs impacting emotional salience, and lateral projections from the periaqueductal gray, hypothalamus and amygdala that activate characteristic emotional behaviors. Finally, the brainstem is home to a group of modulatory neurotransmitter pathways, such as those arising from the raphe nuclei (serotonergic), ventral tegmental area (dopaminergic) and locus coeruleus (noradrenergic), which form a Modulatory network that coordinates interactions between the Ascending and Descending networks. Integration of signaling within these three networks occurs at all levels of the brainstem, with progressively more complex forms of integration occurring in the hypothalamus and thalamus. These intermediary structures, in turn, provide input for the most complex integrations, which occur in the frontal, insular, cingulate and other regions of the cerebral cortex. Phylogenetically older brainstem networks inform the functioning of evolutionarily newer rostral regions, which in turn regulate and modulate the older structures. Via these bidirectional interactions, the human brainstem contributes to the evaluation of sensory information and triggers fixed-action pattern responses that together constitute the finely differentiated spectrum of possible emotions