Uncovering cis Regulatory Codes Using Synthetic Promoter Shuffling

Revealing the spectrum of combinatorial regulation of transcription at individual promoters is essential for understanding the complex structure of biological networks. However, the computations represented by the integration of various molecular signals at complex promoters are difficult to decipher in the absence of simple cis regulatory codes. Here we synthetically shuffle the regulatory architecture — operator sequences binding activators and repressors — of a canonical bacterial promoter. The resulting library of complex promoters allows for rapid exploration of promoter encoded logic regulation. Among all possible logic functions, NOR and ANDN promoter encoded logics predominate. A simple transcriptional cis regulatory code determines both logics, establishing a straightforward map between promoter structure and logic phenotype. The regulatory code is determined solely by the type of transcriptional regulation combinations: two repressors generate a NOR: NOT (a OR b) whereas a repressor and an activator generate an ANDN: a AND NOT b. Three-input versions of both logics, having an additional repressor as an input, are also present in the library. The resulting complex promoters cover a wide dynamic range of transcriptional strengths. Synthetic promoter shuffling represents a fast and efficient method for exploring the spectrum of complex regulatory functions that can be encoded by complex promoters. From an engineering point of view, synthetic promoter shuffling enables the experimental testing of the functional properties of complex promoters that cannot necessarily be inferred ab initio from the known properties of the individual genetic components. Synthetic promoter shuffling may provide a useful experimental tool for studying naturally occurring promoter shuffling

Cardiovascular magnetic resonance physics for clinicians: part I

There are many excellent specialised texts and articles that describe the physical principles of cardiovascular magnetic resonance (CMR) techniques. There are also many texts written with the clinician in mind that provide an understandable, more general introduction to the basic physical principles of magnetic resonance (MR) techniques and applications. There are however very few texts or articles that attempt to provide a basic MR physics introduction that is tailored for clinicians using CMR in their daily practice. This is the first of two reviews that are intended to cover the essential aspects of CMR physics in a way that is understandable and relevant to this group. It begins by explaining the basic physical principles of MR, including a description of the main components of an MR imaging system and the three types of magnetic field that they generate. The origin and method of production of the MR signal in biological systems are explained, focusing in particular on the two tissue magnetisation relaxation properties (T1 and T2) that give rise to signal differences from tissues, showing how they can be exploited to generate image contrast for tissue characterisation. The method most commonly used to localise and encode MR signal echoes to form a cross sectional image is described, introducing the concept of k-space and showing how the MR signal data stored within it relates to properties within the reconstructed image. Before describing the CMR acquisition methods in detail, the basic spin echo and gradient pulse sequences are introduced, identifying the key parameters that influence image contrast, including appearances in the presence of flowing blood, resolution and image acquisition time. The main derivatives of these two pulse sequences used for cardiac imaging are then described in more detail. Two of the key requirements for CMR are the need for data acquisition first to be to be synchronised with the subject's ECG and to be fast enough for the subject to be able to hold their breath. Methods of ECG synchronisation using both triggering and retrospective gating approaches, and accelerated data acquisition using turbo or fast spin echo and gradient echo pulse sequences are therefore outlined in some detail. It is shown how double inversion black blood preparation combined with turbo or fast spin echo pulse sequences acquisition is used to achieve high quality anatomical imaging. For functional cardiac imaging using cine gradient echo pulse sequences two derivatives of the gradient echo pulse sequence; spoiled gradient echo and balanced steady state free precession (bSSFP) are compared. In each case key relevant imaging parameters and vendor-specific terms are defined and explained

Computational Analysis of Constraints on Noncoding Regions, Coding Regions and Gene Expression in Relation to Plasmodium Phenotypic Diversity

Malaria-causing Plasmodium species exhibit marked differences including host choice and preference for invading particular cell types. The genetic bases of phenotypic differences between parasites can be understood, in part, by investigating constraints on gene expression and genic sequences, both coding and regulatory.We investigated the evolutionary constraints on sequence and expression of parasitic genes by applying comparative genomics approaches to 6 Plasmodium genomes and 2 genome-wide expression studies. We found that the coding regions of Plasmodium transcription factor and sexual development genes are relatively less constrained, as are those of genes encoding CCCH zinc fingers and invasion proteins, which all play important roles in these parasites. Transcription factors and genes with stage-restricted expression have conserved upstream regions and so do several gene classes critical to the parasite's lifestyle, namely, ion transport, invasion, chromatin assembly and CCCH zinc fingers. Additionally, a cross-species comparison of expression patterns revealed that Plasmodium-specific genes exhibit significant expression divergence.Overall, constraints on Plasmodium's protein coding regions confirm observations from other eukaryotes in that transcription factors are under relatively lower constraint. Proteins relevant to the parasite's unique lifestyle also have lower constraint on their coding regions. Greater conservation between Plasmodium species in terms of promoter motifs suggests tight regulatory control of lifestyle genes. However, an interspecies divergence in expression patterns of these genes suggests that either expression is controlled via genomic or epigenomic features not encoded in the proximal promoter sequence, or alternatively, the combinatorial interactions between motifs confer species-specific expression patterns

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

Avaliação da função cardíaca por ressonância magnética com seqüências em equilíbrio estável: segmentadas × tempo real Assessment of cardiac function by magnetic resonance imaging: segmented x real-time steady-state free precession sequences

OBJETIVO: Comparar os índices de função sistólica ventricular obtidos entre as seqüências de cine-ressonância magnética em equilíbrio estável, em tempo real e acoplada ao eletrocardiograma, em pacientes com ritmo regular ou não. MATERIAIS E MÉTODOS: Foram comparados a fração de ejeção e os volumes diastólico e sistólico finais, em 31 pacientes, 11 com ritmo cardíaco irregular e 20 com ritmo cardíaco sinusal regular, utilizando-se seqüências segmentadas acopladas ao eletrocardiograma e em tempo real. O tratamento estatístico foi feito através da correlação de Pearson e a concordância de Bland-Altman, com p < 0,01. RESULTADOS: As aquisições em tempo real demonstraram borramento dos contornos endocárdicos, mas ambas as seqüências tiveram forte correlação positiva entre os valores obtidos: fração de ejeção, r = 0,94; volume diastólico final, r = 0,93; volume sistólico final, r = 0,98. A análise dos 11 pacientes com ritmo irregular não demonstrou diferença estatisticamente significativa, apesar da menor relação de contraste sangue-miocárdio. CONCLUSÃO: Seqüências em tempo real podem ser utilizadas para a análise da função cardíaca, independente do ritmo cardíaco dos pacientes.<br>OBJECTIVE: To compare ventricular systolic parameters on segmented and real-time steady-state free precession cine-MRI sequences and ECG-gated MRI in patients presenting or not with cardiac arrhythmias. MATERIALS AND METHODS: Ejection fraction and end-diastolic/end-systolic volumes have been compared in 31 patients, 11 presenting with cardiac arrhythmias, and 20 with regular sinus rhythm, using ECG-gated segmented and real-time sequences. The statistical analysis was performed using Pearson's correlation and Bland-Altman agreement plot, with p < 0.01. RESULTS: Real-time acquisitions demonstrated endocardial borders blurring effects, but both sequences presented a clear, positive correlation: ejection fraction r = 0.94; end-diastolic volume r = 0.93 and end-systolic volume r = 0.98. The assessment of 11 patients with arrhythmias has not demonstrated a statistically significant difference, despite the lower blood pool-myocardial contrast ratio. CONCLUSION: Real-time sequences may be utilized for cardiac function assessment, regardless the patient's cardiac rhythm