Ubiquitous atmospheric production of organic acids mediated by cloud droplets.

Atmospheric acidity is increasingly determined by carbon dioxide and organic acids1-3. Among the latter, formic acid facilitates the nucleation of cloud droplets4 and contributes to the acidity of clouds and rainwater1,5. At present, chemistry-climate models greatly underestimate the atmospheric burden of formic acid, because key processes related to its sources and sinks remain poorly understood2,6-9. Here we present atmospheric chamber experiments that show that formaldehyde is efficiently converted to gaseous formic acid via a multiphase pathway that involves its hydrated form, methanediol. In warm cloud droplets, methanediol undergoes fast outgassing but slow dehydration. Using a chemistry-climate model, we estimate that the gas-phase oxidation of methanediol produces up to four times more formic acid than all other known chemical sources combined. Our findings reconcile model predictions and measurements of formic acid abundance. The additional formic acid burden increases atmospheric acidity by reducing the pH of clouds and rainwater by up to 0.3. The diol mechanism presented here probably applies to other aldehydes and may help to explain the high atmospheric levels of other organic acids that affect aerosol growth and cloud evolution.

Transcriptomic signatures of chronic active antibody-mediated rejection deciphered by RNA sequencing of human kidney allografts.

Natural killer (NK) cells mediate spontaneous cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. This dual functionality could enable their participation in chronic active antibody-mediated rejection (CA-ABMR). Earlier microarray profiling studies have not subcategorized antibody-mediated rejection into CA-ABMR and active-ABMR, and the gene expression pattern of CA-ABMR has not been compared with that of T cell-mediated rejection (TCMR). To fill these gaps, we RNA sequenced human kidney allograft biopsies categorized as CA-ABMR, active-ABMR, TCMR, or No Rejection (NR). Among the 15,910 genes identified in the biopsies, 60, 114, and 231 genes were uniquely overexpressed in CA-ABMR, TCMR, and active-ABMR, respectively; compared to NR, 50 genes were shared between CA-ABMR and active-ABMR, and 164 genes between CA-ABMR and TCMR. The overexpressed genes were annotated to NK cells and T cells in CA-ABMR and TCMR, and to neutrophils and monocytes in active-ABMR. The NK cell cytotoxicity and allograft rejection pathways were enriched in CA-ABMR. Genes encoding perforin, granzymes, and death receptor were overexpressed in CA-ABMR versus active-ABMR but not compared to TCMR. NK cell cytotoxicity pathway gene set variation analysis score was higher in CA-ABMR compared to active-ABMR but not in TCMR. Principal component analysis of the deconvolved immune cellular transcriptomes separated CA-ABMR and TCMR from active-ABMR and NR. Immunohistochemistry of kidney allograft biopsies validated a higher proportion of CD56+ NK cells in CA-ABMR than in active-ABMR. Thus, CA-ABMR was exemplified by the overexpression of the NK cell cytotoxicity pathway gene set and, surprisingly, molecularly more like TCMR than active-ABMR.

Effects of atipamezole on selected physiologic parameters in cynomolgus macaques (Macaca fascicularis).

BACKGROUND: Atipamezole, an α-2 adrenergic receptor antagonist, reverses the α-2 agonist anesthetic effects. There is a dearth of information on the physiological effects of these drugs in cynomolgus macaques (Macaca fascicularis). We assessed atipamezole's physiologic effects. We hypothesized atipamezole administration would alter anesthetic parameters. METHODS: Five cynomolgus macaques were sedated with ketamine/dexmedetomidine intramuscularly, followed 45 min later with atipamezole (0.5 mg/kg). Anesthetic parameters (heart rate, blood pressure [systolic (SAP), diastolic (DAP), and mean (MAP) blood pressure], body temperature, respiratory rate, and %SpO2) were monitored prior to and every 10 min (through 60 min) post atipamezole injection. RESULTS: While heart rate was significantly increased for 60 min; SAP, DAP, MAP, and temperature were significantly decreased at 10 min. CONCLUSIONS: This study indicates subcutaneous atipamezole results in increased heart rate and transient blood pressure decrease. These findings are clinically important to ensure anesthetist awareness to properly support and treat patients as needed.

Biologically synthesized silver nanoparticles as potent antibacterial effective against multidrug-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most worrisome infectious bacteria due to its intrinsic and acquired resistance against several antibiotics and the recalcitrance of its infections; hence, the development of novel antimicrobials effective against multidrug-resistant P. aeruginosa is mandatory. In this work, silver nanoparticles obtained by green synthesis using a leaf extract and fungi were tested against a battery of clinical strains from cystic fibrosis, pneumonia and burnt patients, some of them with multidrug resistance. Both nanoparticles showed a potent antibacterial effect, causing severe damage to the cell wall, membrane and DNA, and inducing the production of reactive oxygen species. Moreover, the nanoparticles derived from fungi showed synergistic antibacterial effects with the antibiotics meropenem and levofloxacin for some clinical strains and both kinds of nanoparticles were nontoxic for larvae of the moth Galleria mellonella, encouraging further research for their implementation in the treatment of P. aeruginosa infections.

Antibacterial properties of phenothiazine derivatives against multidrug-resistant Acinetobacter baumannii strains.

AIM: As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial compounds is necessary. One strategy is to generate treatment alternatives by drug repurposing. METHODS AND RESULTS: In this work, phenotypic microarrays were used for the screening of miscellaneous compounds against the growth and biofilm formation of Acinetobacter baumannii, an important emergent multidrug-resistant opportunistic pathogen. The results showed that the phenothiazine derivatives, such as promethazine, trifluoperazine, thioridazine, and chlorpromazine, inhibited the growth of antibiotic-sensitive and multidrug-resistant strains (showing minimal inhibitory concentrations ranging from 0·05 to 0·6 g l-1 and minimal bactericidal concentrations ranging from 0·1 to 2·5 g l-1 ). All phenothiazine derivatives were active against biofilm cells (with minimal biofilm eradication concentrations ranging from 0·5 to >3 g l-1 ). Chlorpromazine promoted reactive oxigen species (ROS) production, and cell membrane and DNA damage. Chlorpromazine showed synergy with antibiotics such as ceftazidime, meropenem, and colistin and was an effective treatment for experimentally infected Galleria mellonella when combined with ceftazidime. CONCLUSIONS: It was demonstrated that phenothiazine derivatives, especially chlorpromazine, are drugs with attractive antibacterial properties against nosocomial MDR strains of A. baumannii, by generating ROS and cell membrane and DNA damage. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study indicates that repurposing phenothiazine derivatives for treating recalcitrant infections by A. baumannii could be promising.

AI (Artificial Intelligence) and Hypertension Research.

PURPOSE OF REVIEW: This review a highlights that to use artificial intelligence (AI) tools effectively for hypertension research, a new foundation to further understand the biology of hypertension needs to occur by leveraging genome and RNA sequencing technology and derived tools on a broad scale in hypertension. RECENT FINDINGS: For the last few years, progress in research and management of essential hypertension has been stagnating while at the same time, the sequencing of the human genome has been generating many new research tools and opportunities to investigate the biology of hypertension. Cancer research has applied modern tools derived from DNA and RNA sequencing on a large scale, enabling the improved understanding of cancer biology and leading to many clinical applications. Compared with cancer, studies in hypertension, using whole genome, exome, or RNA sequencing tools, total less than 2% of the number cancer studies. While true, sequencing the genome of cancer tissue has provided cancer research an advantage, DNA and RNA sequencing derived tools can also be used in hypertension to generate new understanding how complex protein network, in non-cancer tissue, adapts and learns to be effective when for example, somatic mutations or environmental inputs change the gene expression profiles at different network nodes. The amount of data and differences in clinical condition classification at the individual sample level might be of such magnitude to overwhelm and stretch comprehension. Here is the opportunity to use AI tools for the analysis of data streams derived from DNA and RNA sequencing tools combined with clinical data to generate new hypotheses leading to the discovery of mechanisms and potential target molecules from which drugs or treatments can be developed and tested. Basic and clinical research taking advantage of new gene sequencing-based tools, to uncover mechanisms how complex protein networks regulate blood pressure in health and disease, will be critical to lift hypertension research and management from its stagnation. The use of AI analytic tools will help leverage such insights. However, applying AI tools to vast amounts of data that certainly exist in hypertension, without taking advantage of new gene sequencing-based research tools, will generate questionable results and will miss many new potential molecular targets and possibly treatments. Without such approaches, the vision of precision medicine for hypertension will be hard to accomplish and most likely not occur in the near future.

Recent Northern Hemisphere stratospheric HCl increase due to atmospheric circulation changes.

The abundance of chlorine in the Earth's atmosphere increased considerably during the 1970s to 1990s, following large emissions of anthropogenic long-lived chlorine-containing source gases, notably the chlorofluorocarbons. The chemical inertness of chlorofluorocarbons allows their transport and mixing throughout the troposphere on a global scale, before they reach the stratosphere where they release chlorine atoms that cause ozone depletion. The large ozone loss over Antarctica was the key observation that stimulated the definition and signing in 1987 of the Montreal Protocol, an international treaty establishing a schedule to reduce the production of the major chlorine- and bromine-containing halocarbons. Owing to its implementation, the near-surface total chlorine concentration showed a maximum in 1993, followed by a decrease of half a per cent to one per cent per year, in line with expectations. Remote-sensing data have revealed a peak in stratospheric chlorine after 1996, then a decrease of close to one per cent per year, in agreement with the surface observations of the chlorine source gases and model calculations. Here we present ground-based and satellite data that show a recent and significant increase, at the 2σ level, in hydrogen chloride (HCl), the main stratospheric chlorine reservoir, starting around 2007 in the lower stratosphere of the Northern Hemisphere, in contrast with the ongoing monotonic decrease of near-surface source gases. Using model simulations, we attribute this trend anomaly to a slowdown in the Northern Hemisphere atmospheric circulation, occurring over several consecutive years, transporting more aged air to the lower stratosphere, and characterized by a larger relative conversion of source gases to HCl. This short-term dynamical variability will also affect other stratospheric tracers and needs to be accounted for when studying the evolution of the stratospheric ozone layer.

Assessing the relationship between organic farming practices and microbiological characteristics of organic lettuce varieties (Lactuca sativa L.) grown in Sao Paulo, Brazil.

AIMS: This study aimed to gather information on farming practices employed in organic lettuce fields in Sao Paulo, Brazil and associate these practices with the microbiological characteristics of the products. METHODS AND RESULTS: Practices were surveyed using a questionnaire applied in ten farms, where 200 heads of lettuce were collected and submitted to enumeration of total coliforms and generic Escherichia coli and tested for Salmonella spp. using culture and molecular (qPCR) methods. Based on the responses, the farms could be clustered in two groups: group 1, comprised by six farms, where chicken manure was used as fertilizer in most of them and the composting process was not performed on site; and group 2, comprised by four farms, where other types of fertilizer were used, and the composting process was performed on site. Generic E. coli was detected in 56 (28%) samples, with an average of 1·1 ± 0·7 log MPN per g. Salmonella DNA was detected in two (1%) samples by qPCR. CONCLUSIONS: The prevalence and bacterial loads of generic E. coli, and the occurrence of Salmonella, even at low populations undetectable by conventional culture methods, highlight the need for control measures during farming practices to reduce microbial contamination and risks of foodborne illnesses. These measures include the use of properly composted manure and appropriate washing procedures for leafy vegetables before consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained data contribute to a better understanding of the farming practices of organically grown lettuces in Sao Paulo, Brazil.

Development of an oxidative stress sensor in live bacteria using the optimized HyPer2 protein.

Oxidative stress is a key regulator in many cellular processes but also an important burden for living organisms. The source of oxidative damage usually is difficult to measure and assess with analytical tools or chemical indicators. One major limitation is to discriminate the presence of secondary oxidant molecules derived from the cellular metabolism after exposure to the oxidant or the scavenging capacity of reactive oxygen species by cells. Using a whole-cell reporter system based on an optimized HyPer2 protein for Escherichia coli expression, we demonstrate that, as previously shown for eukaryotic organisms, the effect at the transcriptional level of hydrogen peroxide can be monitored in vivo using flow cytometry of bacterial cells without the need of a direct analytical measurement. In this approach, we generated two different HyPer2 expression systems, one that is induced by IPTG and a second one that is induced by oxidative stress responsive promoters to control the expression of the HyPer2 protein and the exposure of higher H2O2 concentrations that has been shown to activate oxidative response genes. Both systems showed that the pathway that leads to the generation of H2O2 in vivo can be traced from H2O2 exposure. Our results indicate that hydrogen peroxide pulses can be readily detected in E. coli cells by a defined fluorescence signature that is H2O2 concentration-dependent. Our findings indicate that although less sensitive than purified protein or expressed in eukaryotic cells, HyPer2 is a good bacterial sensor for H2O2. As proof of concept, this system was used to trace the oxidative capacity of Toluidine Blue O showing that oxidative stress and redox imbalance is generated inside the cell. This system is expanding the repertoire of whole cell probes available for tracing cellular stress in bacteria.

Single nucleotide variant counts computed from RNA sequencing and cellular traffic into human kidney allografts.

Advances in bioinformatics allow identification of single nucleotide polymorphisms (variants) from RNA sequence data. In an allograft biopsy, 2 genomes contribute to the RNA pool, 1 from the donor organ and the other from the infiltrating recipient's cells. We hypothesize that imbalances in genetic variants of RNA sequence data of kidney allograft biopsies provide an objective measure of cellular infiltration of the allograft. We performed mRNA sequencing of 40 kidney allograft biopsies, selected to represent a comprehensive range of diagnostic categories. We analyzed the sequencing reads of these biopsies and of 462 lymphoblastoid cell lines from the 1000 Genomes Project, for RNA variants. The ratio of heterozygous to nonreference genome homozygous variants (Het/Hom ratio) on all autosomes was determined for each sample, and the estimation of stromal and immune cells in malignant tumors using expression data (ESTIMATE) score was computed as a complementary estimate of the degree of cellular infiltration into biopsies. The Het/Hom ratios (P = .02) and the ESTIMATE scores (P < .001) were associated with the biopsy diagnosis. Both measures correlated significantly (r = .67, P < .0001), even though the Het/Hom ratio is based on mRNA sequence variation, while the ESTIMATE score uses mRNA expression. Het/Hom ratio and the ESTIMATE score may offer unbiased and quantitative parameters for characterizing cellular traffic into human kidney allografts.

Brazilian artisanal ripened cheeses as sources of proteolytic lactic acid bacteria capable of reducing cow milk allergy.

AIM: The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. METHODS AND RESULTS: Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed αS1 -, αS2 - and ß-caseins, α-lactalbumin and ß-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that caused partial hydrolysis of αS1 - and αS2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylLL and cylLS but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. CONCLUSION: Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products.

Autosomal recessive IFT57 hypomorphic mutation cause ciliary transport defect in unclassified oral-facial-digital syndrome with short stature and brachymesophalangia.

The 13 subtypes of oral-facial-digital syndrome (OFDS) belong to the heterogeneous group of ciliopathies. Disease-causing genes encode for centrosomal proteins, components of the transition zone or proteins implicated in ciliary signaling. A unique consanguineous family presenting with an unclassified OFDS with skeletal dysplasia and brachymesophalangia was explored. Homozygosity mapping and exome sequencing led to the identification of a homozygous mutation in IFT57, which encodes a protein implicated in ciliary transport. The mutation caused splicing anomalies with reduced expression of the wild-type transcript and protein. Both anterograde ciliary transport and sonic hedgehog signaling were significantly decreased in subjects' fibroblasts compared with controls. Sanger sequencing of IFT57 in 13 OFDS subjects and 12 subjects with Ellis-Van Creveld syndrome was negative. This report identifies the implication of IFT57 in human pathology and highlights the first description of a ciliary transport defect in OFDS, extending the genetic heterogeneity of this subgroup of ciliopathies.

Ground-based detection of nighttime clouds above Manila Observatory (14.64°N, 121.07°E) using a digital camera.

Ground-based cloud detection at nighttime is achieved by using cameras, lidars, and ceilometers. Despite these numerous instruments gathering cloud data, there is still an acknowledged scarcity of information on quantified local cloud cover, especially at nighttime. In this study, a digital camera is used to continuously collect images near the sky zenith at nighttime in an urban environment. An algorithm is developed to analyze pixel values of images of nighttime clouds. A minimum threshold pixel value of 17 is assigned to determine cloud occurrence. The algorithm uses temporal averaging to estimate the cloud fraction based on the results within the limited field of view. The analysis of the data from the months of January, February, and March 2015 shows that cloud occurrence is low during the months with relatively lower minimum temperature (January and February), while cloud occurrence during the warmer month (March) increases.

Assessing the effect of sodium dichloroisocyanurate concentration on transfer of Salmonella enterica serotype Typhimurium in wash water for production of minimally processed iceberg lettuce (Lactuca sativa L.).

UNLABELLED: This study evaluated the impact of sodium dichloroisocyanurate (5, 10, 20, 30, 40, 50 and 250 mg l(-1) ) in wash water on transfer of Salmonella Typhimurium from contaminated lettuce to wash water and then to other noncontaminated lettuces washed sequentially in the same water. Experiments were designed mimicking the conditions commonly seen in minimally processed vegetable (MPV) processing plants in Brazil. The scenarios were as follows: (1) Washing one inoculated lettuce portion in nonchlorinated water, followed by washing 10 noninoculated portions sequentially. (2) Washing one inoculated lettuce portion in chlorinated water followed by washing five noninoculated portions sequentially. (3) Washing five inoculated lettuce portions in chlorinated water sequentially, followed by washing five noninoculated portions sequentially. (4) Washing five noninoculated lettuce portions in chlorinated water sequentially, followed by washing five inoculated portions sequentially and then by washing five noninoculated portions sequentially in the same water. Salm. Typhimurium transfer from inoculated lettuce to wash water and further dissemination to noninoculated lettuces occurred when nonchlorinated water was used (scenario 1). When chlorinated water was used (scenarios 2, 3 and 4), no measurable Salm. Typhimurium transfer occurred if the sanitizer was ≥10 mg l(-1) . Use of sanitizers in correct concentrations is important to minimize the risk of microbial transfer during MPV washing. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the impact of sodium dichloroisocyanurate in the wash water on transfer of Salmonella Typhimurium from inoculated lettuce to wash water and then to other noninoculated lettuces washed sequentially in the same water was evaluated. The use of chlorinated water, at concentration above 10 mg l(-1) , effectively prevented Salm. Typhimurium transfer under several different washing scenarios. Conversely, when nonchlorinated water was used, Salm. Typhimurium transfer occurred in up to at least 10 noninoculated batches of lettuce washed sequentially in the same water.

Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins.

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and ß-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

The experiences of cancer survivors while transitioning from tertiary to primary care.

PURPOSE: In current fiscally constrained health care systems, the transition of cancer survivors to primary care from tertiary care settings is becoming more common and necessary. The purpose of our study was to explore the experiences of survivors who are transitioning from tertiary to primary care. METHODS: One focus group and ten individual telephone interviews were conducted. Data saturation was reached with 13 participants. All sessions were audio-recorded, transcribed verbatim, and analyzed using a qualitative descriptive approach. RESULTS: Eight categories relating to the main content category of transition readiness were identified in the analysis. Several factors affected participant transition readiness: how the transition was introduced, perceived continuity of care, support from health care providers, clarity of the timeline throughout the transition, and desire for a "roadmap." Although all participants spoke about the effect of their relationships with health care providers (tertiary, transition, and primary care), their relationship with the primary care provider had the most influence on their transition readiness. CONCLUSIONS: Our study provided insights into survivor experiences during the transition to primary care. Transition readiness of survivors is affected by many factors, with their relationship with the primary care provider being particularly influential. Understanding transition readiness from the survivor perspective could prove useful in ensuring patient-centred care as transitions from tertiary to primary care become commonplace.

Urinary cell mRNA profiles and differential diagnosis of acute kidney graft dysfunction.

Noninvasive tests to differentiate the basis for acute dysfunction of the kidney allograft are preferable to invasive allograft biopsies. We measured absolute levels of 26 prespecified mRNAs in urine samples collected from kidney graft recipients at the time of for-cause biopsy for acute allograft dysfunction and investigated whether differential diagnosis of acute graft dysfunction is feasible using urinary cell mRNA profiles. We profiled 52 urine samples from 52 patients with biopsy specimens indicating acute rejection (26 acute T cell-mediated rejection and 26 acute antibody-mediated rejection) and 32 urine samples from 32 patients with acute tubular injury without acute rejection. A stepwise quadratic discriminant analysis of mRNA measures identified a linear combination of mRNAs for CD3ε, CD105, TLR4, CD14, complement factor B, and vimentin that distinguishes acute rejection from acute tubular injury; 10-fold cross-validation of the six-gene signature yielded an estimate of the area under the curve of 0.92 (95% confidence interval, 0.86 to 0.98). In a decision analysis, the six-gene signature yielded the highest net benefit across a range of reasonable threshold probabilities for biopsy. Next, among patients diagnosed with acute rejection, a similar statistical approach identified a linear combination of mRNAs for CD3ε, CD105, CD14, CD46, and 18S rRNA that distinguishes T cell-mediated rejection from antibody-mediated rejection, with a cross-validated estimate of the area under the curve of 0.81 (95% confidence interval, 0.68 to 0.93). Incorporation of these urinary cell mRNA signatures in clinical decisions may reduce the number of biopsies in patients with acute dysfunction of the kidney allograft.

Glutathione metabolism modeling: a mechanism for liver drug-robustness and a new biomarker strategy.

BACKGROUND: Glutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to simulate perturbations observed when human liver derived THLE cells, transfected with human cytochrome P452E1 (THLE-2E1 cells), were exposed to paracetamol (acetaminophen). METHODS: Human liver derived cells containing extra human cytochrome P4502E1 were treated with paracetamol at various levels of methionine and in the presence and absence of an inhibitor of glutamyl-cysteine synthetase (GCS). GCS activity was also measured in extracts. Intracellular and extracellular concentrations of substances involved in glutathione metabolism were measured as was damage to mitochondria and proteins. A bottom up mathematical model was made of the metabolic pathways around and including glutathione. RESULTS: Our initial model described some, but not all the metabolite-concentration and flux data obtained when THLE-2E1 cells were exposed to paracetamol at concentrations high enough to affect glutathione metabolism. We hypothesized that the lack of correspondence could be due to upregulation of expression of glutamyl cysteine synthetase, one of the enzymes controlling glutathione synthesis, and confirmed this experimentally. A modified model which incorporated this adaptive response adequately described the observed changes in the glutathione pathway. Use of the adaptive model to analyze the functioning of the glutathione network revealed that a threshold input concentration of methionine may be required for effective detoxification of reactive metabolites by glutathione conjugation. The analysis also provided evidence that 5-oxoproline and ophthalmic acid are more useful biomarkers of glutathione status when analyzed together than when analyzed in isolation, especially in a new, model-assisted integrated biomarker strategy. CONCLUSION: A robust mathematical model of the dynamics of cellular changes in glutathione homeostasis in cells has been developed and tested in vitro. GENERAL SIGNIFICANCE: Mathematical models of the glutathione pathway that help examine mechanisms of cellular protection against xenobiotic toxicity and the monitoring thereof, can now be made.

Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese.

AIM: Evaluate technologically relevant properties from bacteriocin-producing strains to use as starter/adjunct cultures in cheese making. METHODS AND RESULTS: Eight isolates obtained from Pico cheese produced in Azores (Portugal) were found to produce bacteriocins against Listeria monocytogenes and three isolates against Clostridium perfringens. They were identified as Lactococcus lactis and Enterococcus faecalis and submitted to technological tests: growth at different conditions of temperature and salt, acid production, proteolysis, lipolysis, coexistence, enzymatic profile and autolytic capacity. Safety evaluation was performed by evaluating haemolytic, gelatinase and DNase activity, resistance to antibiotics and the presence of virulence genes. Some isolates presented good technological features such as high autolytic activity, acid and diacetyl production. Lactococcus lactis was negative for all virulence genes tested and inhibit the growth of all Lactic acid bacteria (LAB) isolates. Enterococci were positive for the presence of some virulence genes, but none of the isolates were classified as resistant to important antibiotics. CONCLUSIONS: The bacteriocin-producing Lc. lactis present good potential for application in food as adjunct culture in cheese production. The study also reveals good technological features for some Enterococcus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocin-producing strains presented important technological properties to be exploited as new adjunct culture for the dairy industry, influencing flavour development and improve safety.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.