Sparse Decomposition of Heart Rate Using a Bernoulli-Gaussian Model: Application to Sleep Apnoea Detection.

In this paper, we propose a sparse decomposition of the heart rate during sleep with an application to apnoea-RERA detection. We observed that the tachycardia following an apnoea event has a quasi-deterministic shape with a random amplitude. Accordingly, we model the apnoea-perturbed heart rate as a Bernoulli-Gaussian (BG) process convolved with a deterministic reference signal that allows the identification of tachycardia and bradycardia events. The problem of determining the BG series indicating the presence or absence of an event and estimating its amplitude is a deconvolution problem for which sparsity is imposed. This allows an almost syntactic representation of the heart rate on which simple detection algorithms are applied.

TiSA: TimeSeriesAnalysis-a pipeline for the analysis of longitudinal transcriptomics data.

Improved transcriptomic sequencing technologies now make it possible to perform longitudinal experiments, thus generating a large amount of data. Currently, there are no dedicated or comprehensive methods for the analysis of these experiments. In this article, we describe our TimeSeries Analysis pipeline (TiSA) which combines differential gene expression, clustering based on recursive thresholding, and a functional enrichment analysis. Differential gene expression is performed for both the temporal and conditional axes. Clustering is performed on the identified differentially expressed genes, with each cluster being evaluated using a functional enrichment analysis. We show that TiSA can be used to analyse longitudinal transcriptomic data from both microarrays and RNA-seq, as well as small, large, and/or datasets with missing data points. The tested datasets ranged in complexity, some originating from cell lines while another was from a longitudinal experiment of severity in COVID-19 patients. We have also included custom figures to aid with the biological interpretation of the data, these plots include Principal Component Analyses, Multi Dimensional Scaling plots, functional enrichment dotplots, trajectory plots, and complex heatmaps showing the broad overview of results. To date, TiSA is the first pipeline to provide an easy solution to the analysis of longitudinal transcriptomics experiments.

Whole-Genome Sequencing Snapshot of Clinically Relevant Carbapenem-Resistant Gram-Negative Bacteria from Wastewater in Serbia.

Wastewater (WW) is considered a source of antibiotic-resistant bacteria with clinical relevance and may, thus, be important for their dissemination into the environment, especially in countries with poor WW treatment. To obtain an overview of the occurrence and characteristics of carbapenem-resistant Gram-negative bacteria (CR-GNB) in WW of Belgrade, we investigated samples from the four main sewer outlets prior to effluent into international rivers, the Sava and the Danube. Thirty-four CR-GNB isolates were selected for antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS). AST revealed that all isolates were multidrug-resistant. WGS showed that they belonged to eight different species and 25 different sequence types (STs), seven of which were new. ST101 K. pneumoniae (blaCTX-M-15/blaOXA-48) with novel plasmid p101_srb was the most frequent isolate, detected at nearly all the sampling sites. The most frequent resistance genes to aminoglycosides, quinolones, trimethroprim-sulfamethoxazole, tetracycline and fosfomycin were aac(6')-Ib-cr (55.9%), oqxA (32.3%), dfrA14 (47.1%), sul1 (52.9%), tet(A) (23.5%) and fosA (50%), respectively. Acquired resistance to colistin via chromosomal-mediated mechanisms was detected in K. pneumoniae (mutations in mgrB and basRS) and P. aeruginosa (mutation in basRS), while a plasmid-mediated mechanism was confirmed in the E. cloacae complex (mcr-9.1 gene). The highest number of virulence genes (>300) was recorded in P. aeruginosa isolates. Further research is needed to systematically track the occurrence and distribution of these bacteria so as to mitigate their threat.

Cross-Gram matrices and their use in transfer learning: Application to automatic REM detection using heart rate.

BACKGROUND AND OBJECTIVES: while traditional sleep staging is achieved through the visual - expert-based - annotation of a polysomnography, it has the disadvantages of being unpractical and expensive. Alternatives have been developed over the years to relieve sleep staging from its heavy requirements, through the collection of more easily assessable signals and its automation using machine learning. However, these alternatives have their limitations, some due to variabilities among and between subjects, other inherent to their use of sub-discriminative signals. Many new solutions rely on the evaluation of the Autonomic Nervous System (ANS) activation through the assessment of the heart-rate (HR); the latter is modulated by the aforementioned variabilities, which may result in data and concept shifts between what was learned and what we want to classify. Such adversary effects are usually tackled by Transfer Learning, dealing with problems where there are differences between what is known (source) and what we want to classify (target). In this paper, we propose two new kernel-based methods of transfer learning and assess their performances in Rapid-Eye-Movement (REM) sleep stage detection, using solely the heart rate. METHODS: our first contribution is the introduction of Kernel-Cross Alignment (KCA), a measure of similarity between a source and a target, which is a direct extension of Kernel-Target Alignment (KTA). To our knowledge, KCA has currently never been studied in the literature. Our second contribution is two alignment-based methods of transfer learning: Kernel-Target Alignment Transfer Learning (KTATL) and Kernel-Cross Alignment Transfer Learning (KCATL). Both methods differ from KTA, whose traditional use is kernel-tuning: in our methods, the kernel has been fixed beforehand, and our objective is the improvement of the estimation of unknown target labels by taking into account how observations relate to each other, which, as it will be explained, allows to transfer knowledge (transfer learning). RESULTS: we compare performances with transfer learning (KCATL, KTATL) to performances without transfer using a fixed classifier (a Support Vector Classifier - SVC). In most cases, both transfer learning methods result in an improvement of performances (higher detection rates for a fixed false-alarm rate). Our methods do not require iterative computations. CONCLUSION: we observe improved performances using our transfer methods, which are computationally efficient, as they only require the computation of a kernel matrix and are non-iterative. However, some optimisation aspects are still under investigation.

Characterization of the cytokinin sensor TCSv2 in arabidopsis and tomato.

BACKGROUND: Hormones are crucial to plant life and development. Being able to follow the plants hormonal response to various stimuli and throughout developmental processes is an important and increasingly widespread tool. The phytohormone cytokinin (CK) has crucial roles in the regulation of plant growth and development. RESULTS: Here we describe a version of the CK sensor Two Component signaling Sensor (TCS), referred to as TCSv2. TCSv2 has a different arrangement of binding motifs when compared to previous TCS versions, resulting in increased sensitivity in some examined tissues. Here, we examine the CK responsiveness and distribution pattern of TCSv2 in arabidopsis and tomato. CONCLUSIONS: The increased sensitivity and reported expression pattern of TCSv2 make it an ideal TCS version to study CK response in particular hosts, such as tomato, and particular tissues, such as leaves and flowers.

Autoregulation of RCO by Low-Affinity Binding Modulates Cytokinin Action and Shapes Leaf Diversity.

Mechanisms through which the evolution of gene regulation causes morphological diversity are largely unclear. The tremendous shape variation among plant leaves offers attractive opportunities to address this question. In cruciferous plants, the REDUCED COMPLEXITY (RCO) homeodomain protein evolved via gene duplication and acquired a novel expression domain that contributed to leaf shape diversity. However, the molecular pathways through which RCO regulates leaf growth are unknown. A key question is to identify genome-wide transcriptional targets of RCO and the DNA sequences to which RCO binds. We investigate this question using Cardamine hirsuta, which has complex leaves, and its relative Arabidopsis thaliana, which evolved simple leaves through loss of RCO. We demonstrate that RCO directly regulates genes controlling homeostasis of the hormone cytokinin to repress growth at the leaf base. Elevating cytokinin signaling in the RCO expression domain is sufficient to both transform A. thaliana simple leaves into complex ones and partially bypass the requirement for RCO in C. hirsuta complex leaf development. We also identify RCO as its own target gene. RCO directly represses its own transcription via an array of low-affinity binding sites, which evolved after RCO duplicated from its progenitor sequence. This autorepression is required to limit RCO expression. Thus, evolution of low-affinity binding sites created a negative autoregulatory loop that facilitated leaf shape evolution by defining RCO expression and fine-tuning cytokinin activity. In summary, we identify a transcriptional mechanism through which conflicts between novelty and pleiotropy are resolved during evolution and lead to morphological differences between species.

Metabolomic Investigation of Staphylococcus aureus Antibiotic Susceptibility by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Staphylococcus aureus is a major human pathogen that can readily acquire antibiotic resistance. For instance, methicillin-resistant S. aureus represents a major cause of hospital- and community-acquired bacterial infections. In this chapter, we first provide a detailed protocol for obtaining unbiased and reproducible S. aureus metabolic profiles. The resulting intracellular metabolome is then analyzed in an untargeted manner by using both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry. Such analyses are done in conjunction with our in-house spectral database to identify with high confidence as many meaningful S. aureus metabolites as possible. Under these conditions, we can routinely monitor more than 200 annotated S. aureus metabolites. We also indicate how this protocol can be used to investigate the metabolic differences between methicillin-resistant and susceptible strains.

Synergistic action of auxin and cytokinin mediates aluminum-induced root growth inhibition in Arabidopsis.

Auxin acts synergistically with cytokinin to control the shoot stem-cell niche, while both hormones act antagonistically to maintain the root meristem. In aluminum (Al) stress-induced root growth inhibition, auxin plays an important role. However, the role of cytokinin in this process is not well understood. In this study, we show that cytokinin enhances root growth inhibition under stress by mediating Al-induced auxin signaling. Al stress triggers a local cytokinin response in the root-apex transition zone (TZ) that depends on IPTs, which encode adenosine phosphate isopentenyltransferases and regulate cytokinin biosynthesis. IPTs are up-regulated specifically in the root-apex TZ in response to Al stress and promote local cytokinin biosynthesis and inhibition of root growth. The process of root growth inhibition is also controlled by ethylene signaling which acts upstream of auxin. In summary, different from the situation in the root meristem, auxin acts with cytokinin in a synergistic way to mediate aluminum-induced root growth inhibition in Arabidopsis.

Imaging TCSn::GFP, a Synthetic Cytokinin Reporter, in Arabidopsis thaliana.

Cytokinins are classical plant hormones that control numerous developmental processes throughout the plant life cycle. Cytokinin-responsive cells activate transcription via a phospho-relay signaling network. Type-B nuclear RESPONSE REGULATOR (RR) proteins mediate transcriptional activation as the final step in the signaling cascade. They bind to promoters of immediate-early target genes via a conserved Myb-related DNA-binding domain. To monitor transcriptional activation in response to a cytokinin stimulus, we have constructed a synthetic promoter, TCS (two-component signaling sensor) that harbors the concatemerized binding motifs for activated type-B RR in an optimized configuration. Here, we describe our protocols for imaging TCSn::GFP expression in transgenic Arabidopsis plants. The use of the fluorescent reporter GFP allows the visualization of cytokinin-responding cells by fluorescent microscopy without the need for tissue processing steps, or staining reactions. This method is fast and with a low risk of artifacts. However, since cytokinin signaling integrates various environmental information including light, nutrient status, and biotic and abiotic stress, special care needs to be devoted to the control of growth conditions.

Plant development regulated by cytokinin sinks.

Morphogenetic signals control the patterning of multicellular organisms. Cytokinins are mobile signals that are perceived by subsets of plant cells. We found that the responses to cytokinin signaling during Arabidopsis development are constrained by the transporter PURINE PERMEASE 14 (PUP14). In our experiments, the expression of PUP14 was inversely correlated to the cytokinin signaling readout. Loss of PUP14 function allowed ectopic cytokinin signaling accompanied by aberrant morphogenesis in embryos, roots, and the shoot apical meristem. PUP14 protein localized to the plasma membrane and imported bioactive cytokinins, thus depleting apoplastic cytokinin pools and inhibiting perception by plasma membrane-localized cytokinin sensors to create a sink for active ligands. We propose that the spatiotemporal cytokinin sink patterns established by PUP14 determine the cytokinin signaling landscape that shapes the morphogenesis of land plants.

Bronchial fistula closure with negative pressure wound therapy: a feasible and cost-effective treatment.

Treatment of bronchial fistula (BF) after pulmonary lobectomy is a challenge. Often, patients require long hospital stay, have recurrent empyema and pneumonia, are susceptible to sepsis, often need broad-spectrum antibiotics, as well as various surgical approaches. With the advent and growing evidence of the benefits of negative pressure therapy (NPT), its use in some patients with BF has been reported with encouraging results concerning its feasibility and cost-effectiveness. The aim of this study was to demonstrate the application of NPT as a resource for BF treatment and comparatively analyze the overall cost of treatment. RESUMO O tratamento de fístula brônquica (FB) após lobectomia pulmonar é um desafio. Muitas vezes, o paciente demanda longo tempo de internação, apresenta recidivas de empiema e pneumonia, pode evoluir para sepse, frequentemente necessita de antibioticoterapia de amplo espectro, bem como de várias abordagens cirúrgicas. Com o advento e acúmulo de evidências dos benefícios da terapia por pressão negativa (TPN), seu uso em alguns pacientes com FB tem sido relatado com resultados animadores relativos à sua viabilidade e ao seu custo-efetividade. O objetivo deste estudo foi demonstrar a aplicação de TPN como recurso para tratamento da FB e analisar comparativamente o custo global do seu tratamento.

Annotation of the Staphylococcus aureus Metabolome Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry and Application to the Study of Methicillin Resistance.

Staphylococcus aureus can cause a variety of severe disease patterns and can readily acquire antibiotic resistance; however, the mechanisms by which this commensal becomes a pathogen or develops antibiotic resistance are still poorly understood. Here we asked whether metabolomics can be used to distinguish bacterial strains with different antibiotic susceptibilities. Thus, an efficient and robust method was first thoroughly implemented to measure the intracellular metabolites of S. aureus in an unbiased and reproducible manner. We also placed special emphasis on metabolome coverage and annotation and used both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry in conjunction with our spectral database developed in-house to identify with high confidence as many meaningful S. aureus metabolites as possible. Overall, we were able to characterize up to 210 metabolites in S. aureus, which represents a substantial ∼50% improvement over previously published data. We then preliminarily compared the metabolic profiles of 10 clinically relevant methicillin-resistant and susceptible strains harvested at different time points during the exponential growth phase (without any antibiotic exposure). Interestingly, the resulting data revealed a distinct behavior of "slow-growing" resistant strains, which show modified levels of several precursors of peptidoglycan and capsular polysaccharide biosynthesis.

Phage amplification and immunomagnetic separation combined with targeted mass spectrometry for sensitive detection of viable bacteria in complex food matrices.

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.

Chronic welfare restrictions and adrenal gland morphology in broiler chickens.

Gait problems constitute an important and chronic welfare restriction for broiler chickens. The objective of the present study was to determine if adrenal gland morphology indicates chronic welfare restrictions in broiler chickens, using gait problems as the stressor. Sixty-six birds raised on a commercial unit were selected at 40 d of age and separated into groups according to gait score. One group was apparently healthy birds (AH) with gait scores of 0 to 2, and the other group had birds with gait problems (GP) that showed gait scores of 4 to 5. Birds were slaughtered and weighed, and their adrenal glands were measured and weighed; proportions of medullary and adrenocortical tissues, and lymphatic tissue and blood vessels were studied. GP birds had lower BW when compared to AH birds, and when adrenal gland weight values were adjusted to BW, a greater relative adrenal weight was observed for the GP group. Adrenals from GP birds also presented a higher proportion of blood vessels when compared to AH birds. These results might indicate increased adrenal activity and evidence of the inflammatory process as a consequence of chronic stress. Results showed that gait problems caused significant adrenal gland changes, suggesting a possible role for the study of adrenal gland morphology as an indicator of chronic welfare problems in broiler chickens.

Burns and epilepsy--review and case report.

Decompensation of epilepsy in burned patients may be caused by several factors. Burn is a classic etiology of systemic inflammatory response syndrome, and evolves into two physiological phases. The first 48h after injury corresponds to the first phase involving severe hypovolemic shock. The second phase corresponds to the hypermetabolic response to burns. Altered pharmacokinetics of anticonvulsant drugs is observed. Albumin and other plasma proteins are reduced, leading to increased free fraction of phenytoin, resulting in greater clearance and a lower total drug concentration. Associated with metabolic changes of burned patient, this fact predisposes to seizures in epileptic burned patients. The authors present the case of an epileptic 36-year-old-woman who developed recurrent seizures after a thermal injury, despite using the same medications and doses of anticonvulsant drugs of last 12 years, with controlled epilepsy.

Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

Perfil microbiológico da unidade de queimaduras da EPM/UNIFESP, São Paulo, Brasil / Microbiological surveillance of burn unit of EPM/UNIFESP in São Paulo, Brazil

Introdução: Apesar dos grandes avanços em seu tratamento, infecção de pele com queimadura continua a ser um grande desafio. O objetivo deste estudo é avaliar os aspectos microbiológicos do primeiro ano de funcionamento de uma unidade de queimadura em um Hospital Universitário. Métodos: Estudo retrospectivo. Dados microbiológicos foram coletados e analisados a partir de pacientes internados na Unidade de Queimadura (UTQ) do Hospital São Paulo, Hospital Universitário da Escola Paulista de Medicina (EPM) da Universidade Federal de São Paulo (UNIFESP), entre junho de 2009 e julho de 2010. Resultados: O tempo médio de permanência hospitalar foi de 13,8 dias, com uma taxa de mortalidade de 5,9%. A média da superfície corpórea queimada foi de 10,3%. Avaliou-se 159 culturas de 101 pacientes. Culturas de sangue foram as mais solicitadas (41%). Também foram acessadas 245 culturas de vigilância, coletadas de 75 pacientes. A análise microbiológica revelou um índice de positividade total de 34,5%. Os agentes mais prevalentes foram Staphylococcus coagulase-negativo - CoNS - (33%), Pseudomonas aeruginosa (24%), Acinetobacter spp. (22%) e Klebsiella pneumoniae (5%). Conclusão: A avaliação microbiológica do primeiro ano de funcionamento da UTQ da EPM/ UNIFESP revelou que, embora o agente mais prevalente tenha sido a CoNS, bacilos Gram negativos ainda são muito prevalentes, como a Pseudomonas aeruginosa e a Acinetobacter baumannii. Apesar de pouco tempo de operação, observou-se um grande número de microrganismos multirresistentes, que pode ser explicado por longa exposição a agentes antimicrobianos e alta taxa de transferência de outros hospitais.

Introduction: Despite great advances in treatment, burned skin infection remains a major challenge. The aim of this study is to evaluate the microbiological aspects of the first year's operation of a Burn Unit in a University Hospital. Methods: Retrospective study. Microbiological data were collected and analyzed from patients admitted to the Burn Unit of São Paulo Hospital, a University Hospital of the Paulista Medical School (EPM) of the Federal University of São Paulo (UNIFESP) from June 2009 to July 2010. Results: The average length of stay was 13.8 days with a mortality rate of 5.9%, and median of TBSA was 10.3%. Evaluated 159 cultures from 101 patients. Blood cultures were the most requested (41%). It was also accessed 245 surveillance cultures collected from 75 patients. The microbiological analysis revealed a total positivity rate of 34,5%. The most prevalent agents were Coagulase-negative Staphylococcus - CoNS - (33%), Pseudomonas aeruginosa (24%), Acinetobacter spp. (22%) and Klebsiella pneumoniae (5%). Conclusion: The microbiological evaluation of the first year's activity of EPM/UNIFESP Burn Care Unit revealed that, although the most prevalent agent was CoNS, Gram negative bacilli are still very prevalent, such as Pseudomonas aeruginosa and Acinetobacter baumannii. Despite the short time of operation, was observed large number of multiresistant microorganisms which can be explained by long exposure to antimicrobials and high transfer rate from other hospitals.

Bacterial detection using unlabeled phage amplification and mass spectrometry through structural and nonstructural phage markers.

According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. E. coli at 1 × 10(5), 5 × 10(5), and 1 × 10(6) CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria-Bertani broth, orange juice, and French bean stew ("cassoulet") matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection.

Theoretical and experimental evidence indicates that there is no detectable auxin gradient in the angiosperm female gametophyte.

The plant life cycle alternates between a diploid sporophytic and a haploid gametophytic generation. The female gametophyte (FG) of flowering plants is typically formed through three syncytial mitoses, followed by cellularisation that forms seven cells belonging to four cell types. The specification of cell fates in the FG has been suggested to depend on positional information provided by an intrinsic auxin concentration gradient. The goal of this study was to develop mathematical models that explain the formation of this gradient in a syncytium. Two factors were proposed to contribute to the maintenance of the auxin gradient in Arabidopsis FGs: polar influx at early stages and localised auxin synthesis at later stages. However, no gradient could be generated using classical, one-dimensional theoretical models under these assumptions. Thus, we tested other hypotheses, including spatial confinement by the large central vacuole, background efflux and localised degradation, and investigated the robustness of cell specification under different parameters and assumptions. None of the models led to the generation of an auxin gradient that was steep enough to allow sufficiently robust patterning. This led us to re-examine the response to an auxin gradient in developing FGs using various auxin reporters, including a novel degron-based reporter system. In agreement with the predictions of our models, auxin responses were not detectable within the FG of Arabidopsis or maize, suggesting that the effects of manipulating auxin production and response on cell fate determination might be indirect.

Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase.

A single strategy to select RNA polymerase from bacteriophage T7 (T7 RNAP) mutants in Escherichia coli with enhanced thermostability or enzymatic activity is described. T7 RNAP has the ability to specifically transcribe genes under control of T7 phage promoter. By using random mutagenesis of the T7 RNAP gene in combination with an appropriate screening at 25 and 42°C, we have generated and selected E.coli clones with temperature-sensitive phenotype in the presence of chloramphenicol. The resistance to chloramphenicol used to select these clones results from expression control of the chloramphenicol acetyl transferase gene by the T7 promoter. In a second phase, and using the thermosensitive T7 RNAP variants as template, a new round of random mutagenesis was performed. Combined to an appropriate screening strategy, 11 mutations (second-site T7 RNAP revertants) that restore the initial resistance to chloramphenicol at 42°C were identified. Nine of these mutations increase the thermal resistance of the wild-type T7 RNA. They include the five mutations previously described using different approaches and four novel mutations. One improves T7 RNA catalytic activity and one has no positive effect on the natural enzyme but increases the activity of some combined mutants. Additive effects of mutations amount to an increase of as much as 10°C in T1/2 compared with the wild-type enzyme and up to a 2-fold activity enhancement.