Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

RNAs and ribonucleoproteins in recognition and catalysis.

CONTENTS. 1. Did life begin with catalytic RNA?–2. Self-splicing and self-cleaving RNAs–2.1 Self-splicing of group I introns – 2.2 Self-splicing of group II introns – 2.3 Self-cleaving RNAs–3. Splicing mediated by trans-acting factors–3.1 Group III introns – 3.2 Splicing of nuclear pre-mRNAs – 3.3 Trans-splicing – 3.4 Is nuclear pre-mRNA splicing evolutionarily related to group I and group II self-splicing?– 3.5 Non-RNA mediated splicing of tRNAs–4. Processing of ribosomal precursor RNAs–5. Processing of pre-mRNA 3′ ends–5.1 Polyadenylation – 5.2 Histone pre-mRNA 3′ processing–6. Other RNPs involved in metabolic mechanisms–6.1 5′ end processing of pre-tRNAs by RNase P – 6.2 The signal recognition particle – 6.3 Telomerase – 6.4 RNA editing in trypanosomatid mitochondria–7. Why RNA?

Hybrid Contribution of JPEG200 Video Files for Professional Production Centers Reliability and Non-Reliability Transmission File Based in Image Quality Needed

ATM, SDH or satellite have been used in the last century as the contribution network of Broadcasters. However the attractive price of IP networks is changing the infrastructure of these networks in the last decade. Nowadays, IP networks are widely used, but their characteristics do not offer the level of performance required to carry high quality video under certain circumstances. Data transmission is always subject to errors on line. In the case of streaming, correction is attempted at destination, while on transfer of files, retransmissions of information are conducted and a reliable copy of the file is obtained. In the latter case, reception time is penalized because of the low priority this type of traffic on the networks usually has. While in streaming, image quality is adapted to line speed, and line errors result in a decrease of quality at destination, in the file copy the difference between coding speed vs line speed and errors in transmission are reflected in an increase of transmission time. The way news or audiovisual programs are transferred from a remote office to the production centre depends on the time window and the type of line available; in many cases, it must be done in real time (streaming), with the resulting image degradation. The main purpose of this work is the workflow optimization and the image quality maximization, for that reason a transmission model for multimedia files adapted to JPEG2000, is described based on the combination of advantages of file transmission and those of streaming transmission, putting aside the disadvantages that these models have. The method is based on two patents and consists of the safe transfer of the headers and data considered to be vital for reproduction. Aside, the rest of the data is sent by streaming, being able to carry out recuperation operations and error concealment. Using this model, image quality is maximized according to the time window. In this paper, we will first give a briefest overview of the broadcasters requirements and the solutions with IP networks. We will then focus on a different solution for video file transfer. We will take the example of a broadcast center with mobile units (unidirectional video link) and regional headends (bidirectional link), and we will also present a video file transfer file method that satisfies the broadcaster requirements.

The U5 RNA of trypanosomes deviates from the canonical U5 RNA: The Leptomonas collosoma U5 RNA and its coding gene

Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid Leptomonas collosoma revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5′ phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of Trypanosoma brucei [Dungan, J. D., Watkins, K. P. & Agabian, N. (1996) EMBO J. 15, 4016–4029]. The L. collosoma U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4/U6⋅U5 tri-small nuclear RNP complex. In contrast to T. brucei U5 RNA (62 nt), the L. collosoma homologue is longer (80 nt) and possesses a second stem–loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNACys was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5′ splice site region (positions −1 and +1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis- and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.

A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs

Hepatitis δ virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5′OH and 2′,3′-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5′ hydroxyl and 2′,3′- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

Behavioral expression of emotions and non-invasive assessment of physiological correlates in dogs (Canis familiaris)

The aim of this thesis was to validate the use of infrared thermography (IRT) to non-invasively measure emotional reactions to different situations in pet dogs (Canis familiaris). A preliminary test, aimed to evaluate the correlation between eye-temperature and rectal temperature in dog, was performed. Then, in three different situations, negative (veterinary visit), positive (palatable food rewards), and mildly stressing followed by mildly positive (separation from and reunion with the owner), variations in heat emitted from lacrimal caruncle (referred to as eye temperature) were measured with an infrared thermographic camera. In addition, heart rate and heart rate variability parameters were collected using a non-invasive heart rate monitor designed for human use and validated on dogs. All experiments were video recorded to allow behavioral coding. During the negative situation dogs’ level of activity and stress related behaviors varied across compared to the baseline and dogs showed an increase in eye temperature despite having a significant decrease in the level of activity. The positive situation was characterized by a peak in eye temperature and mean HR and dogs engaged in behaviors indicating a positive arousal, focusing on food treats and tail wagging but there were not variations in HRV during stimulation but only an increment in SDNN immediately after the stimulus. In the separation from and reunion with the owner dogs’ eye temperature and mean HR did not vary neither in the stressful nor in the positive situations, RMSSD increased after the positive episode, SDNN dropped during the two stimulations and it increased after the stimulations. During the separation from the owner dogs were mainly directed to the door or to the experimenter while during the reunion with the owner dogs were focused mainly on the owner and on the environment, exhibiting safe base effect. A different approach was used to assess the welfare of shelter dogs. Dogs were implanted with a telemeter and after implantation dogs were housed in sequence in four different situations lasting 1 week: alone, alone with toys and a stretch cot for sleeping, with an unknown, spayed, female, and alone with a daily 2-hours interaction with an experimenter. Two different approaches were tried: partially random extracted fragments from every week, behaviors from 8 a.m. to 4 p.m. were continuous during baseline and the female situation. Results showed different reactions by dogs to the different situations and interestingly not all enrichments were enjoyed by the dogs improving their welfare. Overall results suggest that IRT may represent a useful tool to investigate emotional reactions in dogs. Nevertheless, further research is needed to establish the specificity and sensivity of IRT in this context and to assess how different dogs’ characteristics, breed, previous experience and the valence and arousal elicited by the stimulus could influence the magnitude and type of the response. The role of HRV in understanding emotional valence and the one of telemeters in understanding long-term effects on sheltered dogs’ welfare is also discussed.

Whole genome analysis reveals a high incidence of non-optimal codons in secretory signal sequences of Escherichia coli

Translational pausing may occur due to a number of mechanisms, including the presence of non-optimal codons, and it is thought to play a role in the folding of specific polypeptide domains during translation and in the facilitation of signal peptide recognition during see-dependent protein targeting. In this whole genome analysis of Escherichia coli we have found that non-optimal codons in the signal peptide-encoding sequences of secretory genes are overrepresented relative to the mature portions of these genes; this is in addition to their overrepresentation in the 5'-regions of genes encoding non-secretory proteins. We also find increased non-optimal codon usage at the 3' ends of most E. coli genes, in both non-secretory and secretory sequences. Whereas presumptive translational pausing at the 5' and 3' ends of E. coli messenger RNAs may clearly have a general role in translation, we suggest that it also has a specific role in sec-dependent protein export, possibly in facilitating signal peptide recognition. This finding may have important implications for our understanding of how the majority of non-cytoplasmic proteins are targeted, a process that is essential to all biological cells. (C) 2004 Elsevier Inc. All rights reserved.

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E-coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms. (c) 2005 Elsevier Ltd. All rights reserved.

Pseudo-messenger RNA: Phantoms of the transcriptome

The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo-messenger RNA to be RNA molecules that resemble protein- coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo - messenger RNAs ( approximately half of which are transposonassociated) among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein- coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense- mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non- standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level.

Structure and dynamics of a gene network model incorporating small RNAs

As advances in molecular biology continue to reveal additional layers of complexity in gene regulation, computational models need to incorporate additional features to explore the implications of new theories and hypotheses. It has recently been suggested that eukaryotic organisms owe their phenotypic complexity and diversity to the exploitation of small RNAs as signalling molecules. Previous models of genetic systems are, for several reasons, inadequate to investigate this theory. In this study, we present an artificial genome model of genetic regulatory networks based upon previous work by Torsten Reil, and demonstrate how this model generates networks with biologically plausible structural and dynamic properties. We also extend the model to explore the implications of incorporating regulation by small RNA molecules in a gene network. We demonstrate how, using these signals, highly connected networks can display dynamics that are more stable than expected given their level of connectivity.

Statistical mechanics analysis of LDPC coding in MIMO Gaussian channels

Using analytical methods of statistical mechanics, we analyse the typical behaviour of a multiple-input multiple-output (MIMO) Gaussian channel with binary inputs under low-density parity-check (LDPC) network coding and joint decoding. The saddle point equations for the replica symmetric solution are found in particular realizations of this channel, including a small and large number of transmitters and receivers. In particular, we examine the cases of a single transmitter, a single receiver and symmetric and asymmetric interference. Both dynamical and thermodynamical transitions from the ferromagnetic solution of perfect decoding to a non-ferromagnetic solution are identified for the cases considered, marking the practical and theoretical limits of the system under the current coding scheme. Numerical results are provided, showing the typical level of improvement/deterioration achieved with respect to the single transmitter/receiver result, for the various cases. © 2007 IOP Publishing Ltd.

Template model for blur coding: the role of early nonlinearity in edge segmentation

We describe a template model for perception of edge blur and identify a crucial early nonlinearity in this process. The main principle is to spatially filter the edge image to produce a 'signature', and then find which of a set of templates best fits that signature. Psychophysical blur-matching data strongly support the use of a second-derivative signature, coupled to Gaussian first-derivative templates. The spatial scale of the best-fitting template signals the edge blur. This model predicts blur-matching data accurately for a wide variety of Gaussian and non-Gaussian edges, but it suffers a bias when edges of opposite sign come close together in sine-wave gratings and other periodic images. This anomaly suggests a second general principle: the region of an image that 'belongs' to a given edge should have a consistent sign or direction of luminance gradient. Segmentation of the gradient profile into regions of common sign is achieved by implementing the second-derivative 'signature' operator as two first-derivative operators separated by a half-wave rectifier. This multiscale system of nonlinear filters predicts perceived blur accurately for periodic and aperiodic waveforms. We also outline its extension to 2-D images and infer the 2-D shape of the receptive fields.

A deficit in encoding the order of visual sequences in developmental dyslexia: a non -phonological deficit

The present thesis tested the hypothesis of Stanovich, Siegel, & Gottardo (1997) that surface dyslexia is the result of a milder phonological deficit than that seen in phonological dyslexia coupled with reduced reading experience. We found that a group of adults with surface dyslexia showed a phonological deficit that was commensurate with that shown by a group of adults with phonological dyslexia (matched for chronological age and verbal and non-verbal IQ) and normal reading experience. We also showed that surface dyslexia cannot be accounted for by a semantic impairment or a deficit in the verbal learning and recall of lexical-semantic information (such as meaningful words), as both dyslexic subgroups performed the same. This study has replicated the results of our published study that surface dyslexia is not the consequence of a mild retardation or reduced learning opportunities but a separate impairment linked to a deficit in written lexical learning, an ability needed to create novel lexical representations from a series of unrelated visual units, which is independent from the phonological deficit (Romani, Di Betta, Tsouknida & Olson, 2008). This thesis also provided evidence that a selective nonword reading deficit in developmental dyslexia persists beyond poor phonology. This was shown by finding a nonword reading deficit even in the presence of normal regularity effects in the dyslexics (when compared to both reading and spelling-age matched controls). A nonword reading deficit was also found in the surface dyslexics. Crucially, this deficit was as strong as in the phonological dyslexics despite better functioning of the sublexical route for the former. These results suggest that a nonword reading deficit cannot be solely explained by a phonological impairment. We, thus, suggested that nonword reading should also involve another ability relating to the processing of novel visual orthographic strings, which we called 'orthographic coding'. We then investigated the ability to process series of independent units within multi-element visual arrays and its relationship with reading and spelling problems. We identified a deficit in encoding the order of visual sequences (involving both linguistic and nonlinguistic information) which was significantly associated with word and nonword processing. More importantly, we revealed significant contributions to orthographic skills in both dyslexic and control individuals, even after age, performance IQ and phonological skills were controlled. These results suggest that spelling and reading do not only tap phonological skills but also order encoding skills.