Transcription Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation.

Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids transformed into Escherichia coli cells. From in vivo measurements, we found that these promoters in tandem formation can have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters' positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which is known to hamper RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation in these constructs emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to falloff. Finally, the broad spectrum of possible, externally regulated, attenuation strengths observed in our synthetic tandem promoters suggests that they could become useful as externally controllable regulators of future synthetic circuits.

A library of reporters of the global regulators of gene expression in Escherichia coli.

The topology of the transcription factor network (TFN) of Escherichia coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for green fluorescent protein (GFP) fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli. Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption.IMPORTANCECells contain thousands of genes. Many genes are involved in the control of cellular activities. Some activities require a few hundred genes to run largely synchronous transcriptional programs. To achieve this, cells have evolved global regulator (GR) proteins that can influence hundreds of genes simultaneously. We have engineered a library of Escherichia coli strains to track the levels over time of these, phenotypically critical, GRs. Each strain has a single-copy plasmid coding for a fast-maturing green fluorescent protein whose transcription is controlled by a copy of the natural GR promoter. By allowing the tracking of GR levels, with sensitivity and specificity, this library should become of wide use in scientific research on bacterial gene expression (from molecular to synthetic biology) and, later, be used in applications in therapeutics and bioindustries.

Delirium related distress experienced by nurses / Distress nos enfermeiros relacionado com o Delirium / Distrés de los enfermeros relacionada con el Delirium

Abstract Background: Delirium is described as a distressful experience by patients and their families. Also, among healthcare staff, nurses caring for patients with delirium are the most at risk of high distress related to this syndrome. Aims: To assess nurses' distress related to delirium and its specific symptoms and severity. To identify factors nurses' sociodemographic and psychological characteristics and professional experience, that may be associated to this distress. Methods: A prospective study was carried out with nurses caring older adults with delirium in Intensive Care Medicine Service. Distress related to delirium, global psychological distress and burnout in nurses were measured with Delirium Experience Questionnaire, Kessler Psychological Distress Scale and Copenhagen Burnout Inventory, respectively. Results: Eighteen nurses were included (mean age=32 years; 89% women). About 46% of the nurses classified the experience of caring for patients in delirium as moderately distressing. Disorientation and psychomotor agitation were the most distressing symptoms. High distress related to delirium was associated with greater personal (p=0.040) and work-related burnout (p=0.020), and more global psychological distress (p=0.007). Conclusion: Caring for patients with delirium is distressing for nurses, particularly if the disorientation and psychomotor agitation are present and if nurses presented high burnout or psychological distress. This study highlights the need for development of education and support strategies for all professionals dealing with these patients.

Resumo Contexto: O delirium é descrito como uma experiência angustiante pelos pacientes e famílias. Também entre os profissionais de saúde, os enfermeiros que cuidam destes doentes são os que correm maior risco de grande distress relacionado com esta síndrome. Objetivos: Avaliar o distress dos enfermeiros relacionado com o delirium, os seus sintomas específicos e gravidade. Identificar fatores sociodemográficos e psicológicos e experiência profissional dos enfermeiros, que podem estar associados a este distress. Metodologia: Foi realizado um estudo prospetivo com enfermeiros que cuidam de idosos com delirium no Serviço de Medicina Intensiva. O distress relacionado com o delirium, distress psicológico global e burnout em enfermeiros foram medidos com Questionário Experiência de Delirium, Escala Distress Psicológico de Kessler e Inventário Burnout de Copenhaga, respetivamente. Resultados: Foram incluídos dezoito enfermeiros (média=32 anos; 89% mulheres). Cerca de 46% dos enfermeiros classificaram a experiência de cuidar de pacientes em delirium como moderadamente distressing. A desorientação e a agitação psicomotora, foram os sintomas mais distressing. O elevado distress relacionado com o delirium foi associado a um maior burnout pessoal (p=0,040) e relacionado com o trabalho (p=0,020), e mais distress psicológico global (p=0,007). Conclusões: Cuidar de doentes com delirium é distressing para os enfermeiros, particularmente se desorientação e agitação psicomotora estiverem presentes e se os enfermeiros apresentarem um elevado burnout ou distress psicológico. Este estudo salienta a necessidade do desenvolvimento de estratégias de formação e apoio para todos os profissionais que lidem com estes pacientes.

Resumen Antecedentes: Los pacientes y sus familias describen el delirium como una experiencia angustiosa. También entre los profesionales de la salud, los enfermeros que atienden a estos pacientes son las que corren más riesgo de sufrir un gran distrés relacionado con este síndrome. Objetivos: Evaluar el distrés de los enfermeros relacionada con el delirium, sus síntomas específicos y gravedad. Identificar los factores sociodemográficos y psicológicos de los enfermeros y experiencia profesional que pueden estar asociados a este distrés. Métodos: Se realizó un estudio prospectivo con los enfermeros que atendían a los pacientes ancianos con delirium en la Unidad de Cuidados Intensivos. Distrés relacionado con el delirium, distrés psicológico global y burnout en los enfermeros se midieron con Cuestionario Experiencia de Delirio, Escala Distrés Psicológico de Kessler y Inventario Burnout de Copenhague, respectivamente. Resultados: Se incluyeron dieciocho enfermeros (media=32 años;89% mujeres). Aproximadamente el 46% de los enfermeros calificaron la experiencia de atender a pacientes con delirium como moderadamente angustiosa. Desorientación y agitación psicomotriz fueron los síntomas con más distrés. Distrés relacionado con delirium se asoció con un mayor burnout personal (p=0,040) y laboral (p=0,020), y con más distrés psicológico global (p=0,007). Conclusión: El cuidado de los pacientes con delirium es angustioso para los enfermeros, en particular si hay desorientación y agitación psicomotriz y si los enfermeros tienen un alto grado de burnout o distrés psicológico. Este estudio pone de manifiesto la necesidad de desarrollar estrategias de formación y apoyo para todos los profesionales que tratan con estos pacientes.

O conhecimento como "superpoder" que a mulher leva para o seu trabalho de parto / Childbirth preparation: Knowledge as a "superpower" women bring to their labour

Este relatório pretende descrever as etapas e eventos mais importantes na aquisição e desenvolvimento de competências enquanto Enfermeira Especialista em Enfermagem de saúde materna e Obstétrica. Descrevem-se, portanto as aprendizagens decorrentes do Ensino Clínico realizado no Hospital Pedro Hispano, em três módulos distintos: Gravidez com complicações; Trabalho de Parto e Parto; e Autocuidado no Pós-Parto e Parentalidade. Partindo-se das vivências do Ensino Clínico, exploraram-se os conceitos, intervenções resultantes de prescrição e intervenções autónomas de enfermagem implementadas durante em cada um destes módulos. Procurou-se igualmente refletir sobre as mesmas, fundamentando-se as a sua implementação na evidência científica sempre que possível, ou, refletindo-se sobre a necessidade de actualizar as práticas tendo isto presente. O paradigma actual da enfermagem baseada na evidência, justificou a inclusão de uma componente de investigação sob a forma de uma revisão integrativa da literatura com a tema: "Preparação para o parto: O conhecimento como "superpoder" que a mulher leva para o seu trabalho de parto". Da análise dos cinco artigos seleccionados conseguiu extrairse uma categoria relevante face à questão de investigação, relacionada com o Conhecimento sobre estratégias para lidar com a dor de trabalho de parto. Referindo os vários autores que as intervenções de preparação para o parto, particularmente no âmbito da dor, se refletem numa diminuição do medo do parto e da ansiedade, promovendo experiências de parto mais positivas.

This report intends to describe the most important stages and events in the acquisition and development of competences as a Specialist Nurse in Maternal and Obstetric Health Nursing. Therefore, the learning resulting from the Clinical Teaching carried out at Hospital Pedro Hispano is described, in three distinct modules: Pregnancy with complications; Labour and Childbirth; and Self-care in Postpartum and Parenting. Starting from the experiences of Clinical Teaching, the concepts, interventions resulting from prescription and autonomous nursing interventions implemented during each of these modules were explored. It was also sought to reflect on them, basing their implementation on scientific evidence whenever possible, or, reflecting on the need to update practices with this in mind. The current paradigm of evidence-based nursing justified the inclusion of a research component in the form of an integrative literature review with the theme: "Preparation for childbirth: Knowledge as a "superpower" that women bring to their labour". From the analysis of the five selected articles, a relevant topic was extracted in relation to the research question, related to knowledge about strategies to deal with labour pain. Many authors mention that childbirth preparation interventions, particularly in terms of pain, are reflected in a decrease in childbirth fear and anxiety, promoting more positive childbirth experiences.

Antibacterial activity improvement of dental glass-ceramic by incorporation of AgVO3 nanoparticles.

OBJECTIVE: This study aimed to investigate the role of the incorporation of an antibacterial nanoceramic (AgVO3) on the properties of a restorative dental glass-ceramic. METHOD: A commercially available restorative glass-ceramic, commonly designated as porcelain (IPS d.SIGN) was functionalized with an antibacterial agent (nanostructured ß-AgVO3), synthesized by a hydrothermal route. Both functionalized and pristine samples were processed according to the manufacturer's instructions. All samples were characterized by X-ray diffraction, Rietveld refinement, particle size distribution, Scanning Electron Microscopy, chemical solubility, and Inductively Coupled Plasma Spectroscopy. Their antibacterial potential (Mueller-Hinton test) was analyzed against gram-positive (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). RESULTS: The commercial glass-ceramic showed leucite (KAlSi2O6) as the only detectable crystalline phase, and, for both strains, no antibacterial activity could be detected in the Mueller-Hinton agar plates test. A monophasic, needle-shaped, and nanometric ß-AgVO3 powder was successfully synthesized by a simple hydrothermal route. After thermal treatment, glass-ceramic samples containing different percentages of ß-AgVO3 showed a second crystalline phase of microline [K0.95(AlSi3O8)]. For modified samples, inhibition halos were easily visible on the Mueller-Hinton test, which ranged from 11.1 ± 0.5 mm to 16.6 ± 0.5 mm and 12.7 ± 0.3 mm to 15.5 ± 0.3 mm in the S. aureus and E.coli cultures, respectively, showing that the halos formed were dose-dependent. Also, increasing the percentage of ß-AgVO3 promoted a significant increase in chemical solubility, from 72 µg/cm2 (samples with 1 wt% of ß-AgVO3) to 136 µg/cm2 (samples with 2 wt% of ß-AgVO3), which was associated with the silver and vanadium ions released from the glass matrix. SIGNIFICANCE: Our in vitro results indicate that IPS d.SIGN, as most of the dental glass-ceramics, do not exhibit antibacterial activity per se. Nonetheless, in this concept test, we demonstrated that it is possible to modify dental veneering materials giving them antibacterial properties by adding at least 2 wt% of ß-AgVO3, a nanomaterial easily synthesized by a simple route.

Alteration of DNA supercoiling serves as a trigger of short-term cold shock repressed genes of E. coli.

Cold shock adaptability is a key survival skill of gut bacteria of warm-blooded animals. Escherichia coli cold shock responses are controlled by a complex multi-gene, timely-ordered transcriptional program. We investigated its underlying mechanisms. Having identified short-term, cold shock repressed genes, we show that their responsiveness is unrelated to their transcription factors or global regulators, while their single-cell protein numbers' variability increases after cold shock. We hypothesized that some cold shock repressed genes could be triggered by high propensity for transcription locking due to changes in DNA supercoiling (likely due to DNA relaxation caused by an overall reduction in negative supercoiling). Concomitantly, we found that nearly half of cold shock repressed genes are also highly responsive to gyrase inhibition (albeit most genes responsive to gyrase inhibition are not cold shock responsive). Further, their response strengths to cold shock and gyrase inhibition correlate. Meanwhile, under cold shock, nucleoid density increases, and gyrases and nucleoid become more colocalized. Moreover, the cellular energy decreases, which may hinder positive supercoils resolution. Overall, we conclude that sensitivity to diminished negative supercoiling is a core feature of E. coli's short-term, cold shock transcriptional program, and could be used to regulate the temperature sensitivity of synthetic circuits.

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration.

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the gene's numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

TKTL1 Knockdown Impairs Hypoxia-Induced Glucose-6-phosphate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase Overexpression.

Increased expression of transketolase (TKT) and its isoform transketolase-like-1 (TKTL1) has been related to the malignant leukemia phenotype through promoting an increase in the non-oxidative branch of the pentose phosphate pathway (PPP). Recently, it has also been described that TKTL1 can have a role in survival under hypoxic conditions and in the acquisition of radio resistance. However, TKTL1's role in triggering metabolic reprogramming under hypoxia in leukemia cells has never been characterized. Using THP-1 AML cells, and by combining metabolomics and transcriptomics techniques, we characterized the impact of TKTL1 knockdown on the metabolic reprogramming triggered by hypoxia. Results demonstrated that TKTL1 knockdown results in a decrease in TKT, glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities and impairs the hypoxia-induced overexpression of G6PD and GAPDH, all having significant impacts on the redox capacity of NADPH- and NADH-related cells. Moreover, TKTL1 knockdown impedes hypoxia-induced transcription of genes encoding key enzymes and transporters involved in glucose, PPP and amino acid metabolism, rendering cells unable to switch to enhanced glycolysis under hypoxia. Altogether, our results show that TKTL1 plays a key role in the metabolic adaptation to hypoxia in THP-1 AML cells through modulation of G6PD and GAPDH activities, both regulating glucose/glutamine consumption and the transcriptomic overexpression of key players of PPP, glucose and amino acids metabolism.

Sequence-dependent model of genes with dual σ factor preference.

Escherichia coli uses σ factors to quickly control large gene cohorts during stress conditions. While most of its genes respond to a single σ factor, approximately 5% of them have dual σ factor preference. The most common are those responsive to both σ70, which controls housekeeping genes, and σ38, which activates genes during stationary growth and stresses. Using RNA-seq and flow-cytometry measurements, we show that 'σ70+38 genes' are nearly as upregulated in stationary growth as 'σ38 genes'. Moreover, we find a clear quantitative relationship between their promoter sequence and their response strength to changes in σ38 levels. We then propose and validate a sequence dependent model of σ70+38 genes, with dual sensitivity to σ38 and σ70, that is applicable in the exponential and stationary growth phases, as well in the transient period in between. We further propose a general model, applicable to other stresses and σ factor combinations. Given this, promoters controlling σ70+38 genes (and variants) could become important building blocks of synthetic circuits with predictable, sequence-dependent sensitivity to transitions between the exponential and stationary growth phases.

Analytical kinetic model of native tandem promoters in E. coli.

Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites 'dTSS', pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS ≤ 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.

Exploring the cancer patients' experiences during external radiotherapy: A systematic review and thematic synthesis of qualitative and quantitative evidence.

PURPOSE: To investigate the subjective experience of adult cancer patients undergoing external radiotherapy and provide evidence for better practices in radiotherapy services. METHODS: A systematic review was performed according to the PRISMA Statement Guidelines. Qualitative and mixed studies were identified through five electronic databases (CINAHL, PsychINFO, Medline, Scopus and Web of Science), between March and April 2020, using defined criteria. Methodological quality assessment was conducted, and the data integrated into a thematic synthesis. RESULTS: Of the 886 studies identified, 13 met our inclusion criteria. Patients experiences were described into four main themes: (1) Time, (2) Physical Environment, (3) Treatment Concerns and (4) Radiotherapy Team. Time refers to waiting time and treatment time; Physical Environment states temperature in the treatment room and equipment; Treatment Concerns included side effects, daily activities, positioning and immobilization and treatment preparation (e.g., bladder filling); radiotherapy team comprised patients support; response to patients needs and recognized team. The generating analytical phase of thematic analysis allowed us to recognize that the effectiveness of the radiotherapy team operates as a facilitator of the patients' experience. In contrast, time in the waiting room, the treatment preparation when is required, and the positioning and immobilization, specifically for the neck and head patients, act as barriers. CONCLUSION: Four distinct themes were identified to be positively and negatively associated with patients' experience during external radiotherapy. The evidence provides valuable recommendations to improved radiotherapy services organization, as well as to the delivery of more patient-centred care adjusted to the concerns and needs of patients.

Efficiency and Robustness of Processes Driven by Nucleoid Exclusion in Escherichia coli.

The internal spatial organization of prokaryotic organisms, including Escherichia coli, is essential for the proper functioning of processes such as cell division. One source of this organization in E. coli is the nucleoid, which causes the exclusion of macromolecules - e.g. protein aggregates and the chemotaxis network - from midcell. Similarly, following DNA replication, the nucleoid(s) assist in placing the Z-ring at midcell. These processes need to be efficient in optimal conditions and robust to suboptimal conditions. After reviewing recent findings on these topics, we make use of past data to study the efficiency of the spatial constraining of Z-rings, chemotaxis networks, and protein aggregates, as a function of the nucleoid(s) morphology. Also, we compare the robustness of these processes to nonoptimal temperatures. We show that Z-rings, Tsr clusters, and protein aggregates have temperature-dependent spatial distributions along the major cell axis that are consistent with the nucleoid(s) morphology and the volume-exclusion phenomenon. Surprisingly, the consequences of the changes in nucleoid size with temperature are most visible in the kurtosis of these spatial distributions, in that it has a statistically significant linear correlation with the mean nucleoid length and, in the case of Z-rings, with the distance between nucleoids prior to cell division. Interestingly, we also find a negative, statistically significant linear correlation between the efficiency of these processes at the optimal condition and their robustness to suboptimal conditions, suggesting a trade-off between these traits.

Stochastic models coupling gene expression and partitioning in cell division in Escherichia coli.

Regulation of future RNA and protein numbers is a key process by which cells continuously best fit the environment. In bacteria, RNA and proteins exist in small numbers and their regulatory processes are stochastic. Consequently, there is cell-to-cell variability in these numbers, even between sister cells. Traditionally, the two most studied sources of this variability are gene expression and RNA and protein degradation, with evidence suggesting that the latter is subject to little regulation, when compared to the former. However, time-lapse microscopy and single molecule fluorescent tagging have produced evidence that cell division can also be a significant source of variability due to asymmetries in the partitioning of RNA and proteins. Relevantly, the impact of this noise differs from noise in production and degradation since, unlike these, it is not continuous. Rather, it occurs at specific time points, at which moment it can introduce major fluctuations. Several models have now been proposed that integrate noise from cell division, in addition to noise in gene expression, to mimic the dynamics of RNA and protein numbers of cell lineages. This is expected to be particularly relevant in genetic circuits, where significant fluctuations in one component protein, at specific time moments, are expected to perturb near-equilibrium states of the circuits, which can have long-lasting consequences. Here we review stochastic models coupling these processes in Escherichia coli, from single genes to small circuits.

A comprehensive look into the volatile exometabolome of enteroxic and non-enterotoxic Staphylococcus aureus strains.

Staphylococcal food poisoning is a disease that originates significant health and economic losses and is caused by Staphylococcus aureus strains able to produce enterotoxins. The aim of this work is to go further on the study of the volatile exometabolome of S. aureus using an advanced gas chromatographic technique. Enterotoxic and non-enterotoxic strains were assessed. The volatile exometabolome profile comprised 240 volatiles belonging to ten chemical families. This volatiles were mainly by-products of branched-chain amino acids and methionine degradation, pyruvate metabolism, diacetyl pathway, oxidative stress and carotenoid cleavage. Metabolites released by the first two pathways were produced in higher contents by the enterotoxic strains. This study add further insights to S. aureus volatile exometabolome, and also shows that by applying it, it is possible to distinguish strains of S. aureus by the number of produced enterotoxins, which is especially important from the food safety point of view.

TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT.

Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.

Evaluation of resistance development and viability recovery by toxigenic and non-toxigenic Staphylococcus aureus strains after repeated cycles of high hydrostatic pressure.

In this work, the development of resistance and the recovery of growth after several consecutive cycles of high hydrostatic pressure (HPP) were for the first time evaluated in different strains of Staphylococcus aureus. Three strains of this important and highly resilient to HPP foodborne pathogen were used: a non-enterotoxigenic ATCC 6538 strain, treated with 600 MPa for 30 min at 20 °C, and the toxigenic strains 2153 MA (with enterotoxin A) and 2065 MA (with the enterotoxins A, G and I), treated with 600 MPa for 15 min at 20 °C. After the first treatment, surviving colonies were used to produce new bacterial cultures. This procedure was repeated nine times more for each bacterium or until total inactivation occurred. The inactivation profile of non-enterotoxic strain and the two enterotoxic strains did not change after consecutive cycles, but the toxic strain with three enterotoxins was completely inactivated after the fourth cycle. The three strains did not recover their viability after 14 days. The results indicate that HPP effectively inactivates non-toxigenic and toxigenic strains of S. aureus after a single treatment. The surviving bacteria did not develop resistance after 10 cycles of pressurization and did not recover their viability after 14 days of incubation.

A microcosm approach to evaluate the degradation of tributyltin (TBT) by Aeromonas molluscorum Av27 in estuarine sediments.

Tributyltin (TBT) is a biocide extremely toxic to a wide range of organisms, which has been used for decades in antifouling paints. Despite its global ban in 2008, TBT is still a problem of great concern due to the high levels trapped in sediments. Aeromonas molluscorum Av27 is a TBT degrading bacterium that was isolated from an estuarine system. We investigated the ability and the role of this bacterium on TBT degradation in this estuarine system, using a microcosm approach in order to mimic environmental conditions. The experiment was established and followed for 150 days. Simultaneously, changes in the indigenous bacterial community structure were also investigated. The results revealed a maximum TBT degradation rate of 28% accompanied by the detection of the degradation products over time. Additionally, it was observed that TBT degradation was significantly enhanced by the presence of Av27. In addition a significantly higher TBT degradation occurred when the concentration of Av27 was higher. TBT degradation affected the bacterial community composition as revealed by the changes in the prevalence of Proteobacteria subdivisions, namely the increase of Deltaproteobacteria and the onset of Epsilonproteobacteria. However, the addition of Av27 strain did not affect the dominant phylotypes. Total bacterial number, bacterial biomass productivity, 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analyses also indicated alterations on the bacterial community structure over time, with bacteria non-tolerant to pollutants increasing their representativeness, as, for instance, the increase of the number of Alphaproteobacteria clones from 6% in the beginning to 12% at the end of the experiment. The work herein presented confirms the potential of Av27 strain to be used in the decontamination of TBT-polluted environments.

Contribution of chemical water properties to the differential responses of bacterioneuston and bacterioplankton to ultraviolet-B radiation.

The surface microlayer (SML) is characterized by different physicochemical properties from underlying waters (UW). However, whether these differences in abiotic factors underlie the distinct sensitivity of bacterioneuston (i.e. SML bacteria) and bacterioplankton to environmental stressors remains to be addressed. We investigated the contribution of abiotic factors to the UV-B sensitivity of bacterioneuston and bacterioplankton. Nutrients (especially nitrogen and phosphate) emerged as important determinants of bacterial UV-B sensitivity. The role of particles, nutrients, and dissolved organic components on bacterial UV-B sensitivity was further evaluated using dilution cultures. Filtered samples were twofold more UV sensitive than unfiltered samples, suggesting a UV-protective effect of particles. High nutrient concentrations attenuated bacterial UV-B sensitivity (up to 40%), compared with unamended conditions, by influencing bacterial physiology and/or community composition. Suspending cells in natural water, particularly from the SML, also attenuated UV-B sensitivity (up to 23%), compared with suspension in an artificial mineral solution. Bioassays using Pseudomonas sp. strain NT5I1.2B revealed that chemical water properties influence UV-induced oxidative damage. UV-B sensitivity was associated with high cell-specific activities. The chemical environment of the SML and UW influences UV-B effects on the corresponding bacterial communities. Maintaining low cell activities might be advantageous in stressful environments, like the SML.

Can volatile organic metabolites be used to simultaneously assess microbial and mite contamination level in cereal grains and coffee beans?

A novel approach based on headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-ToFMS) was developed for the simultaneous screening of microbial and mite contamination level in cereals and coffee beans. The proposed approach emerges as a powerful tool for the rapid assessment of the microbial contamination level (ca. 70 min versus ca. 72 to 120 h for bacteria and fungi, respectively, using conventional plate counts), and mite contamination (ca. 70 min versus ca. 24 h). A full-factorial design was performed for optimization of the SPME experimental parameters. The methodology was applied to three types of rice (rough, brown, and white rice), oat, wheat, and green and roasted coffee beans. Simultaneously, microbiological analysis of the samples (total aerobic microorganisms, moulds, and yeasts) was performed by conventional plate counts. A set of 54 volatile markers was selected among all the compounds detected by GC×GC-ToFMS. Principal Component Analysis (PCA) was applied in order to establish a relationship between potential volatile markers and the level of microbial contamination. Methylbenzene, 3-octanone, 2-nonanone, 2-methyl-3-pentanol, 1-octen-3-ol, and 2-hexanone were associated to samples with higher microbial contamination level, especially in rough rice. Moreover, oat exhibited a high GC peak area of 2-hydroxy-6-methylbenzaldehyde, a sexual and alarm pheromone for adult mites, which in the other matrices appeared as a trace component. The number of mites detected in oat grains was correlated to the GC peak area of the pheromone. The HS-SPME/GC×GC-ToFMS methodology can be regarded as the basis for the development of a rapid and versatile method that can be applied in industry to the simultaneous assessment the level of microbiological contamination and for detection of mites in cereals grains and coffee beans.