Gene expression is controlled at multiple layers, and cells may integrate different regulatory steps for coherent production of proper protein levels. We applied various microarray-based approaches ...to determine key gene-expression intermediates in exponentially growing fission yeast, providing genome-wide data for translational profiles, mRNA steady-state levels, polyadenylation profiles, start-codon sequence context, mRNA half-lives, and RNA polymerase II occupancy. We uncovered widespread and unexpected relationships between distinct aspects of gene expression. Translation and polyadenylation are aligned on a global scale with both the lengths and levels of mRNAs: efficiently translated mRNAs have longer poly(A) tails and are shorter, more stable, and more efficiently transcribed on average. Transcription and translation may be independently but congruently optimized to streamline protein production. These rich data sets, all acquired under a standardized condition, reveal a substantial coordination between regulatory layers and provide a basis for a systems-level understanding of multilayered gene-expression programs.
The effect of both temperature and composition on the rate of transformation of a nematic phase from an isotropic solution is examined from a theoretical standpoint. The kinetics of the ...transformation are presented as a time–concentration–temperature–transformation (TCTT) diagram, analogous to the time–temperature–transformation (TTT) diagrams commonly used in metallurgical process design. The transformation is regarded as a nucleated process in which the transformation rate is proportional to the number of stable nuclei, and this in turn depends on a balance between the energy gained by nematic ordering and the energy expended in forming the nematic–isotropic interface. The nematic ordering term is estimated as a function of both concentration and temperature via two different approaches: (i) the lattice theory as described by Flory and Warner and (ii) the Maier–Saupe theory of nematic ordering. Although there are differences in detail, both approaches yield the same qualitative result. The present work provides the details necessary for developing a specific example application of our recently reported generalized system-independent model of nucleation and growth. The TCTT diagram is in essence a phase diagram augmented by kinetic information for nematic ordering, and thus is expected to be a powerful graphical tool in liquid crystal process engineering and other applications.
Ubiquitination has emerged as one of the major post-translational modifications that decide on protein fate, targeting, and regulation of protein function. Whereas the ubiquitination of proteins can ...be monitored with classic biochemical methods, the mapping of modified side chains proves to be challenging. More recently, mass spectrometry has been applied to identify ubiquitinated proteins and also their sites of modification. Typically, liquid chromatography tandem mass spectrometry (LC-MS/MS) based approaches, including collision-induced fragmentation (CID), have been successfully used in the past. However, a potential difficulty arises from the unstable nature of this modification, and also that the isopeptide bond linkage between C-terminal glycine and the N(ε) lysyl side chain is susceptible to fragmentation under these conditions. Here we investigate the utility of electron-transfer dissociation (ETD)-based fragmentation to detect ubiquitination sites in proteins. Our results indicate that ETD can provide alternative fragmentation patterns that allow detection of gly-gly-modified lysyl side chains, in particular z+1 fragment ions derived from triply charged precursor ions. We subsequently applied ETD fragmentation-based analysis and detected novel ubiquitination sites on DNA polymerase B1 that were not easily observed using CID. We conclude that ETD can provide significant alternative fragmentation information that complements CID-derived data to improve the coverage when mapping ubiquitination sites in proteins.
A systematic comparison of CID and ETD ion trap MS/MS for the analysis of protein ubiquitination.
Distance-based classification of handwritten symbols Golubitsky, Oleg; Watt, Stephen M.
International journal on document analysis and recognition,
06/2010, Volume:
13, Issue:
2
Journal Article, Conference Proceeding
Peer reviewed
Open access
We study online classification of isolated handwritten symbols using distance measures on spaces of curves. We compare three distance-based measures on a vector space representation of curves to ...elastic matching and ensembles of SVM. We consider the Euclidean and Manhattan distances and the distance to the convex hull of nearest neighbors. We show experimentally that of all these methods the distance to the convex hull of nearest neighbors yields the best classification accuracy of about 97.5%. Any of the above distance measures can be used to find the nearest neighbors and prune totally irrelevant classes, but the Manhattan distance is preferable for this because it admits a very efficient implementation. We use the first few Legendre-Sobolev coefficients of the coordinate functions to represent the symbol curves in a finite-dimensional vector space and choose the optimal dimension and number of bits per coefficient by cross-validation. We discuss an implementation of the proposed classification scheme that will allow classification of a sample among hundreds of classes in a setting with strict time and storage limitations.
Involving patients as co-leaders and co-creators in research is key to reflecting the patient’s voice in decision-making. However, co-creation of patient-centered data to inform decisions is rare, ...especially in early drug development where patient input is critical to prioritizing patient-relevant outcomes and endpoints for use in clinical trials. Despite the industry's growing commitment to patient centricity, most patients are excluded from sharing their expertise in research; more inclusive methods of engaging patients as research partners are needed. We describe a collaboration between a pharmaceutical company and a patient organization in co-leading and co-creating a program to understand priorities of patients and caregivers for treatment features and outcomes in sickle cell disease to inform endpoint selection in clinical development. The results of this program will be used as a basis for continued interaction between patients and the sponsor and to inform ongoing clinical development and evidence-generation activities. This case study demonstrates an approach to meaningful collaborations between patient organizations and pharmaceutical companies aimed at including the patient's voice early in the medical product lifecycle.
As exome sequencing gives way to genome sequencing, the need to interpret the function of regulatory DNA becomes increasingly important. To test whether evolutionary conservation of cis-regulatory ...modules (CRMs) gives insight into human gene regulation, we determined transcription factor (TF) binding locations of four liver-essential TFs in liver tissue from human, macaque, mouse, rat, and dog. Approximately, two thirds of the TF-bound regions fell into CRMs. Less than half of the human CRMs were found as a CRM in the orthologous region of a second species. Shared CRMs were associated with liver pathways and disease loci identified by genome-wide association studies. Recurrent rare human disease causing mutations at the promoters of several blood coagulation and lipid metabolism genes were also identified within CRMs shared in multiple species. This suggests that multi-species analyses of experimentally determined combinatorial TF binding will help identify genomic regions critical for tissue-specific gene control.
Genomic copy number alterations are a feature of many human diseases including cancer. We have evaluated the effectiveness of an oligonucleotide array, originally designed to detect single-nucleotide ...polymorphisms, to assess DNA copy number. We first showed that fluorescent signal from the oligonucleotide array varies in proportion to both decreases and increases in copy number. Subsequently we applied the system to a series of 20 cancer cell lines. All of the putative homozygous deletions (10) and high-level amplifications (12; putative copy number >4) tested were confirmed by PCR (either qPCR or normal PCR) analysis. Low-level copy number changes for two of the lines under analysis were compared with BAC array CGH; 77% (n = 44) of the autosomal chromosomes used in the comparison showed consistent patterns of LOH (loss of heterozygosity) and low-level amplification. Of the remaining 10 comparisons that were discordant, eight were caused by low SNP densities and failed in both lines. The studies demonstrate that combining the genotype and copy number analyses gives greater insight into the underlying genetic alterations in cancer cells with identification of complex events including loss and reduplication of loci.
CONTEXT The link between long-haul air travel and venous thromboembolism is the subject of continuing debate. It remains unclear whether the reduced cabin pressure and oxygen tension in the airplane ...cabin create an increased risk compared with seated immobility at ground level. OBJECTIVE To determine whether hypobaric hypoxia, which may be encountered during air travel, activates hemostasis. DESIGN, SETTING, AND PARTICIPANTS A single-blind, crossover study, performed in a hypobaric chamber, to assess the effect of an 8-hour seated exposure to hypobaric hypoxia on hemostasis in 73 healthy volunteers, which was conducted in the United Kingdom from September 2003 to November 2005. Participants were screened for factor V Leiden G1691A and prothrombin G20210A mutation and were excluded if they tested positive. Blood was drawn before and after exposure to assess activation of hemostasis. INTERVENTIONS Individuals were exposed alternately (≥1 week apart) to hypobaric hypoxia, similar to the conditions of reduced cabin pressure during commercial air travel (equivalent to atmospheric pressure at an altitude of 2438 m), and normobaric normoxia (control condition; equivalent to atmospheric conditions at ground level, circa 70 m above sea level). MAIN OUTCOME MEASURES Comparative changes in markers of coagulation activation, fibrinolysis, platelet activation, and endothelial cell activation. RESULTS Changes were observed in some hemostatic markers during the normobaric exposure, attributed to prolonged sitting and circadian variation. However, there were no significant differences between the changes in the hypobaric and the normobaric exposures. For example, the median difference in change between the hypobaric and normobaric exposure was 0 ng/mL for thrombin-antithrombin complex (95% CI, −0.30 to 0.30 ng/mL); –0.02 nmol/L for prothrombin fragment 1 + 2 (95% CI, –0.03 to 0.01 nmol/L); 1.38 ng/mL for D-dimer (95% CI, –3.63 to 9.72 ng/mL); and –2.00% for endogenous thrombin potential (95% CI, –4.00% to 1.00%). CONCLUSION Our findings do not support the hypothesis that hypobaric hypoxia, of the degree that might be encountered during long-haul air travel, is associated with prothrombotic alterations in the hemostatic system in healthy individuals at low risk of venous thromboembolism.
NF-κB signaling plays a crucial role in regulating proliferation and differentiation in the epidermis. Alterations in the NF-κB pathway can lead to skin pathologies with a significant burden to human ...health such as psoriasis and cutaneous squamous cell carcinoma (cSCC). Caspase recruitment domain (CARD)-containing scaffold proteins are key regulators of NF-κB signaling by providing a link between membrane receptors and NF-κB transcriptional subunits. Mutations in the CARD family member, CARD14, have been identified in patients with the inflammatory skin diseases psoriasis and pityriasis rubra pilaris. Here, we describe that the gene coding for another CARD scaffold protein, CARD11, is mutated in more than 38% of 111 cSCCs, and show that novel variants outside of the coiled-coil domain lead to constitutively activated NF-κB signaling. CARD11 protein expression was detectable in normal skin and increased in all cSCCs tested. CARD11 mRNA levels were comparable with CARD14 in normal skin and CARD11 mRNA was increased in cSCC. In addition, we identified CARD11 mutations in peritumoral and sun-exposed skin, suggesting that CARD11-mediated alterations in NF-κB signaling may be an early event in the development of cSCC.
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