Survival of Listeria Strains and Shelf Life Determination of Fresh Blueberries (Vaccinium corymbosum) Treated with Cold Atmospheric Plasma.

Fresh blueberries are delicate, hand-picked, packaged, and refrigerated fruits vulnerable to spoilage and contamination. Cold atmospheric plasma (CAP) is a promising antimicrobial technology; therefore, this study evaluated the CAP treatment effect on acid-tolerant Listeria innocua and Listeria monocytogenes and evaluated changes in the quality of the treated fruit. Samples were spot-inoculated with pH 5.5 and 6.0 acid-adapted Listeria species. Samples were treated with gliding arc CAP for 15, 30, 45, and 60 s and evaluated after 0, 1, 4, 7, and 11 days of storage at 4 °C and 90% humidity for the following quality parameters: total aerobic counts, yeast and molds, texture, color, soluble solids, pH, and titratable acidity. CAP treatments of 30 s and over demonstrated significant reductions in pathogens under both the resistant strain and pH conditions. Sixty-second CAP achieved a 0.54 Log CFU g-1 reduction in L. monocytogenes (pH 5.5) and 0.28 Log CFU g-1 for L. monocytogenes (pH 6.0). Yeast and mold counts on day 0 showed statistically significant reductions after 30, 45, and 60 s CAP with an average 2.34 Log CFU g-1 reduction when compared to non-CAP treated samples. Quality parameters did not show major significant differences among CAP treatments during shelf life. CAP is an effective antimicrobial treatment that does not significantly affect fruit quality.

Risk factors for mortality in patients with dengue: A systematic review and meta-analysis.

OBJECTIVE: To investigate risk factors for mortality in dengue. METHODS: We performed a systematic review and meta-analysis searching MEDLINE, Embase, SciELO, LILACS Bireme, and OpenGrey databases to identify eligible observational studies of patients with dengue, of both genders, aged 14 years or older, that analysed risk factors associated with mortality and reported adjusted risk measures with their respective confidence intervals (CIs). We estimated the pooled weighted mean difference and 95% CIs with a DerSimonian and Laird random-effects model. We assessed the methodological quality using the Newcastle-Ottawa Scale. RESULTS: Of 1,170 citations reviewed, 18 papers, with a total of 25,851 patients, were included in the systematic review and 12 in the meta-analysis. Severe hepatitis (OR 29.222, 95% CI 3.876-220.314), dengue shock syndrome (OR 23.575, 95% CI 3.664-151.702), altered mental status (OR 3.76, 95% CI 1.67-8.42), diabetes mellitus (OR 3.698, 95% CI 1.196-11.433), and higher pulse rate (OR 1.039, 95% CI 1.011-1.067) are associated with mortality in patients with dengue. All studies included were classified as having a high quality. CONCLUSIONS: Proper identification and management of these risk factors should be considered to improve patient outcomes and reduce the hidden burden of this neglected tropical disease. Future well-designed studies are needed to investigate the association of other clinical, radiological, and laboratorial findings with mortality in dengue, as well as to develop prognostic models based on the risk factors found in our study.

Quercetin-Crosslinked Chitosan Films for Controlled Release of Antimicrobial Drugs.

Natural polymer-based films, due to their favorable biological and mechanical properties, have demonstrated great potential as coatings for biomedical applications. Among them, chitosan films have been widely studied both as coating materials and as controlled drug release systems. Crosslinkers are often used to tune chitosan's crosslinking degree and thus to control the drug release kinetics. For this purpose, quercetin, a plant-derived natural polyphenol, has gained attention as a crosslinker, mainly for its intrinsic anti-inflammatory, antioxidant, and antibacterial features. In this study, chitosan films crosslinked with three different concentrations of quercetin (10, 20, and 30% w/w) have been used as controlled release systems for the delivery of the antibacterial drug trimethoprim (TMP, 10% w/w). Physicochemical and antimicrobial properties were investigated. Surface wettability and composition of the films were assessed by contact angle measurements, X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectroscopy (FTIR), respectively. The release kinetic of TMP in phosphate-buffered saline (PBS) and 2-(N-morpholino) ethanesulfonic acid (MES) was studied over time. Finally, antibacterial properties were assessed on E. coli and S. aureus through Kirby-Bauer disc diffusion and micro-dilution broth assays. Results show that quercetin, at the tested concentrations, clearly increases the crosslinking degree in a dose-dependent manner, thus influencing the release kinetic of the loaded TMP while maintaining its bactericidal effects. In conclusion, this work demonstrates that quercetin-crosslinked chitosan films represent a promising strategy for the design of antibiotic-releasing coatings for biomedical applications.

Chronic haemodialysis in infants and children less than 15 kg.

BACKGROUND: Peritoneal dialysis (PD) is the most commonly used kidney replacement therapy in infants and young children with chronic kidney disease (CKD) stage 5. Chronic haemodialysis (cHD) is the alternative treatment when PD is not possible for technical reasons; however, the difficulties that may be encountered are challenging and require clinicians with specialist training and experience. This study aims to describe the clinical history, complications and outcomes in children < 15 kg on cHD. METHODS: A retrospective, descriptive study of the clinical records of patients weighing < 15 kg on cHD for more than 3 months. The reasons for CKD stage 5, age at start of treatment, duration of haemodialysis, anthropometric and metabolic variables, as well as vascular access, complications and clinical outcome were recorded. RESULTS: Fifteen patients were included between 2006 and 2018 with a median age at start of cHD of 30 (interquartile range (IQR) 13, 39) months and median duration of 15 (IQR 7.5, 25.3) months. Five patients were younger than 2 years. The median weight at start of treatment was 11.2 (IQR 6.4, 12.8) kg. Forty-five tunneled catheters with a median survival of 106 days were used. The main cause of loss of vascular access was obstruction or displacement dysfunction (39%). The catheter-associated infection rate was 0.76 per 1000 catheter days. Ten patients received a successful kidney transplant, 4 were transferred to PD and one died from complications during abdominal surgery. CONCLUSIONS: cHD can be successfully performed in children < 15 kg by addressing specific clinical and technical issues.

Spontaneous Breathing Trials in Preterm Infants: Systematic Review and Meta-Analysis.

BACKGROUND: Few data are available on the use of spontaneous breathing trials (SBTs) in the neonatal population, despite advocacy of the practice in many neonatal ICUs. In this meta-analysis, we systematically reviewed the literature regarding the accuracy of SBTs as a predictor for extubation failure in premature infants. METHODS: Following the PRISMA recommendations, scientific articles were collected in December 2019 and January 2020 using PubMed, LILACS, Web of Science, Scopus, Google Scholar, OATD, and BDTD databases. The risk of bias in the studies included herein was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The pooled sensitivity and specificity of the studies were estimated using a mixed logistic regression model of 2 levels and a normal bivariate model. RESULTS: Six studies were included for qualitative and quantitative evaluation in this study. All SBTs were performed with endotracheal CPAP, with a total observation time of 3-5 min. The parameters for passing/failing the test were similar in 5 of the 6 studies and included bradycardia or desaturation during the test. The SBT showed a high pooled sensitivity (0.97, 95% CI 0.85-0.99), indicating proper identification of neonates "ready" for successful extubation. However, a low pooled specificity (0.40, 95% CI 0.24-0.58), with many false-positive cases, indicated inaccurate prediction of extubation failure. Heterogeneity of included studies was considerable for sensitivity and substantial for specificity. CONCLUSIONS: The SBT in premature infants can accurately predict extubation success but not extubation failure. Therefore, even though it is an attractive, practical, and easy-to-perform bedside assessment tool, there is a lack of evidence to support its use as an independent predictor of extubation failure in premature infants. Its routine use should be evaluated and monitored carefully.

Characterization and Application of Pomegranate Epicarp Extracts as Functional Ingredients in a Typical Brazilian Pastry Product.

Currently, there is a clear tendency to incorporate natural ingredients into food and pharmaceutical formulations. Besides being well-accepted by consumers, these ingredients have less adverse side effects than their artificial counterparts. The pomegranate processing industry produces large quantities of by-products that are discarded as bio-residues, despite containing bioactive compounds. Accordingly, the epicarp of two pomegranate varieties (Mollar de Elche and Purple Queen) was tested as a potential source of bioactive compounds with food application. The phenolic profile was identified by HPLC-DAD-ESI/MS, revealing fourteen phenolic compounds in both varieties (Purple Queen showed also three anthocyanins), with punicalagin isomers as the major compounds. Nonetheless, Mollar de Elche presented greater antioxidant and antibacterial activities. Despite this result, Purple Queen was selected to be tested as a new natural colouring and functionalizing ingredient in a Brazilian pastry product. The incorporation of the selected extract maintained the nutritional profile and provided a higher antioxidant activity compared to the traditional product. In this way, this work confirmed the possible use of pomegranate epicarp as a natural ingredient in the food industry, conferring dyeing and functionalizing effects, and anticipating a possible valorisation of this bio-residue.

Effects of vasoconstrictor use on digital nerve block: systematic review with meta-analysis. / Efeitos do uso de vasoconstritores no bloqueio de nervos digitais: revisão sistemática com metanálise.

Conventionally, the association of local anesthetics with vasoconstrictors is avoided at extremities due to the risk of ischemia. However, recent studies suggest that there is safety in the use of vasoconstrictors at extremities. Thus, we sought to evaluate the effectiveness and safety of vasoconstrictor use combined with local anesthetics in digital nerve block compared to the use of anesthetics without vasoconstrictors, through a systematic review with meta-analysis of randomized clinical trials. Until May 2019 we searched MEDLINE, LILACS, SciELO, ScienceDirect, Scopus, ClinicalTrials.gov, and gray literature databases, without date or language restrictions. The keywords were the following: digital block, vasoconstrictor, and ischemia. We included randomized clinical trials in which there was the use of local anesthetics with associated or not with vasoconstrictors in digital blocks. In the primary variables, the occurrence of ischemic complications and the duration of anesthesia were analysed; in the secondary variables, the need for anesthetic reapplication, bleeding control, and latency were observed. Ten studies were included in this review. The occurrence of ischemia was not observed, regardless of the use of vasoconstrictors or not. The use of vasoconstrictors at a concentration of 1:100,000 or less was associated with longer anesthesia duration (P<0.00001), lower need for anesthetic reapplication (P=0.02), lower need for bleeding control (P=0.00006), and lower latency (P<0.00001). We could conclude that the use of vasoconstrictors associated with local anesthetics in digital block proved to be a safe and effective technique.

Convencionalmente, a associação de anestésicos locais com vasoconstritores é evitada em extremidades pelo risco de isquemia. Entretanto, estudos recentes sugerem haver segurança no uso de vasoconstritor em extremidades. Procuramos, assim, avaliar a efetividade e segurança do uso de vasoconstritores combinados com anestésicos locais no bloqueio de nervos digitais em comparação ao uso de anestésicos plenos, através de uma revisão sistemática com metanálise de ensaios clínicos randomizados. Pesquisamos, até maio de 2019, nas bases de dados MEDLINE, LILACS, SciELO, ScienceDirect, Scopus, ClinicalTrials.gov e literatura cinzenta, sem restrições de data ou idioma, os descritores: bloqueio digital, vasoconstritor e isquemia. Foram incluídos ensaios clínicos randomizados nos quais houve a utilização de anestésicos locais associados ou não a vasoconstritores em bloqueios digitais. Nas variáveis primárias foram analisadas a ocorrência de complicações isquêmicas e a duração da anestesia, e nas variáveis secundárias foram observadas necessidade de reaplicação anestésica, de controle de sangramento e latência. Dez estudos foram incluídos nesta revisão. Não foi observada a ocorrência de isquemia, independente do uso ou não de vasoconstritores. O uso de vasoconstritores na concentração de 1:100.000 ou menor esteve associado a maior duração da anestesia (P<0,00001), menor necessidade de reaplicação anestésica (P=0,02), menor necessidade de controle de sangramento (P=0,00006) e menor latência (P<0,00001). Pudemos concluir que uso de vasoconstritores associados a anestésicos locais no bloqueio digital mostrou-se uma técnica segura e efetiva.

Analysis of neonatal mortality risk factors in Brazil: a systematic review and meta-analysis of observational studies / Análise dos fatores de risco na mortalidade neonatal no Brasil: uma revisão sistemática e metanálise de estudos observacionais

Abstract Objective: To identify, using a systematic review and meta-analysis of observational studies, which risk factors are significantly associated with neonatal mortality in Brazil, and to build a comprehensive national analysis on neonatal mortality. Sources: This review included observational studies on neonatal mortality, performed between 2000 and 2018 in Brazilian cities. The MEDLINE, Elsevier, Cochrane, LILACS, SciELO, and OpenGrey databases were used. For the qualitative analysis, the Newcastle-Ottawa Scale was used. For the quantitative analysis, the natural logarithms of the risk measures and their confidence intervals were used, as well as the DerSimonian and Laird method as a random effects model, and the Mantel-Haenszel model for heterogeneity estimation. A confidence level of 95% was considered. Summary of findings: The qualitative analysis resulted in six studies of low and four studies of intermediate-low bias risk. The following exposure factors were significant: absence of partner, maternal age ≥35 years, male gender, multiple gestation, inadequate and absent prenatal care, presence of complications during pregnancy, congenital malformation in the assessed pregnancy, Apgar < 7 at the fifth minute, low and very low birth weight, gestational age ≤ 37 weeks, and caesarean delivery. Conclusion: The most significant risk factors presented in this study are modifiable, allowing aiming at a real reduction in neonatal deaths, which remain high in the country.

Resumo: Objetivo: Identificar, através de uma revisão sistemática e da metanálise de estudos observacionais, quais fatores de risco associam-se significativamente com a mortalidade neonatal no Brasil e construir uma análise nacional abrangente sobre a mortalidade neonatal. Fontes: Foram avaliados os estudos observacionais sobre mortalidade neonatal realizados entre 2000 e 2018 em cidades brasileiras. Usaram-se as bases MEDLINE, Elsevier, Cochrane, LILACS, SciELO e OpenGrey. Para a análise qualitativa, foi usada a Escala Newcastle-Ottawa. Para a análise quantitativa, foram utilizados os logaritmos naturais das medidas de risco e de seus intervalos de confiança, o método de DerSimonian e Laird como modelo de efeitos aleatórios e o modelo de Mantel-Haenszel para estimativa da heterogeneidade. Considerou-se nível de confiança de 95%. Resumo dos achados: A análise qualitativa resultou em seis estudos de baixo e quatro estudos de intermediário-baixo risco de viés. Foram significativos os seguintes fatores de exposição: ausência de companheiro, idade materna ≥ 35 anos, sexo masculino, gestação múltipla, pré-natal inadequado e ausente, presença de intercorrências durante a gestação, de malformação congênita na gestação em estudo, Apgar < 7 no quinto minuto, baixo e muito baixo peso ao nascer, idade gestacional ≤ 37 semanas e parto cesariano. Conclusão: Os fatores de risco mais significativos apresentados neste estudo são modificáveis, o que possibilita almejar uma redução real das mortes neonatais, que ainda permanecem elevadas no país.

Analysis of neonatal mortality risk factors in Brazil: a systematic review and meta-analysis of observational studies.

OBJECTIVE: To identify, using a systematic review and meta-analysis of observational studies, which risk factors are significantly associated with neonatal mortality in Brazil, and to build a comprehensive national analysis on neonatal mortality. SOURCES: This review included observational studies on neonatal mortality, performed between 2000 and 2018 in Brazilian cities. The MEDLINE, Elsevier, Cochrane, LILACS, SciELO, and OpenGrey databases were used. For the qualitative analysis, the Newcastle-Ottawa Scale was used. For the quantitative analysis, the natural logarithms of the risk measures and their confidence intervals were used, as well as the DerSimonian and Laird method as a random effects model, and the Mantel-Haenszel model for heterogeneity estimation. A confidence level of 95% was considered. SUMMARY OF FINDINGS: The qualitative analysis resulted in six studies of low and four studies of intermediate-low bias risk. The following exposure factors were significant: absence of partner, maternal age ≥35 years, male gender, multiple gestation, inadequate and absent prenatal care, presence of complications during pregnancy, congenital malformation in the assessed pregnancy, Apgar<7 at the fifth minute, low and very low birth weight, gestational age≤37 weeks, and caesarean delivery. CONCLUSION: The most significant risk factors presented in this study are modifiable, allowing aiming at a real reduction in neonatal deaths, which remain high in the country.

Tratamento não farmacológico da hipertensão arterial / Nonpharmacologic treatment of arterial hypertension

A hipertensão arterial sistêmica (HAS) é uma doença multifatorial caracterizada por níveis elevados e sustentados de pressão arterial (PA). Vários são os fatores de risco implicados em sua expressão clínica, dentre eles: herança genética, idade, gênero, etnia, excesso de peso, ingestão excessiva de sal e de álcool, sedentarismo, entre outras. Nesta revisão são abordadas as modificações no estilo de vida (MEV) como fatores associados não apenas à prevenção, mas ao próprio tratamento de uma moléstia muito prevalente na população geral, de grande impacto socioeconômico e responsável por significativa morbimortalidade cardiovascular

Systemic arterial hypertension (SAH) is a multifactorial disease characterized by elevated and sustained blood pressure (BP) levels. There are several risk factors involved in its clinical expression, among them: genetic inheritance, age, gender, ethnicity, excess weight, excessive intake of salt and alcohol, sedentary lifestyle, among others. In this review, lifestyle modifications are addressed as factors associated not only with prevention, but also with the treatment of a very prevalent disease in the general population, with a high socioeconomic impact and responsible for significant cardiovascular morbidity and mortality.

Efeitos do uso de vasoconstritores no bloqueio de nervos digitais: revisão sistemática com metanálise / Effects of vasoconstrictor use on digital nerve block: systematic review with meta-analysis

RESUMO Convencionalmente, a associação de anestésicos locais com vasoconstritores é evitada em extremidades pelo risco de isquemia. Entretanto, estudos recentes sugerem haver segurança no uso de vasoconstritor em extremidades. Procuramos, assim, avaliar a efetividade e segurança do uso de vasoconstritores combinados com anestésicos locais no bloqueio de nervos digitais em comparação ao uso de anestésicos plenos, através de uma revisão sistemática com metanálise de ensaios clínicos randomizados. Pesquisamos, até maio de 2019, nas bases de dados MEDLINE, LILACS, SciELO, ScienceDirect, Scopus, ClinicalTrials.gov e literatura cinzenta, sem restrições de data ou idioma, os descritores: bloqueio digital, vasoconstritor e isquemia. Foram incluídos ensaios clínicos randomizados nos quais houve a utilização de anestésicos locais associados ou não a vasoconstritores em bloqueios digitais. Nas variáveis primárias foram analisadas a ocorrência de complicações isquêmicas e a duração da anestesia, e nas variáveis secundárias foram observadas necessidade de reaplicação anestésica, de controle de sangramento e latência. Dez estudos foram incluídos nesta revisão. Não foi observada a ocorrência de isquemia, independente do uso ou não de vasoconstritores. O uso de vasoconstritores na concentração de 1:100.000 ou menor esteve associado a maior duração da anestesia (P<0,00001), menor necessidade de reaplicação anestésica (P=0,02), menor necessidade de controle de sangramento (P=0,00006) e menor latência (P<0,00001). Pudemos concluir que uso de vasoconstritores associados a anestésicos locais no bloqueio digital mostrou-se uma técnica segura e efetiva.

ABSTRACT Conventionally, the association of local anesthetics with vasoconstrictors is avoided at extremities due to the risk of ischemia. However, recent studies suggest that there is safety in the use of vasoconstrictors at extremities. Thus, we sought to evaluate the effectiveness and safety of vasoconstrictor use combined with local anesthetics in digital nerve block compared to the use of anesthetics without vasoconstrictors, through a systematic review with meta-analysis of randomized clinical trials. Until May 2019 we searched MEDLINE, LILACS, SciELO, ScienceDirect, Scopus, ClinicalTrials.gov, and gray literature databases, without date or language restrictions. The keywords were the following: digital block, vasoconstrictor, and ischemia. We included randomized clinical trials in which there was the use of local anesthetics with associated or not with vasoconstrictors in digital blocks. In the primary variables, the occurrence of ischemic complications and the duration of anesthesia were analysed; in the secondary variables, the need for anesthetic reapplication, bleeding control, and latency were observed. Ten studies were included in this review. The occurrence of ischemia was not observed, regardless of the use of vasoconstrictors or not. The use of vasoconstrictors at a concentration of 1:100,000 or less was associated with longer anesthesia duration (P<0.00001), lower need for anesthetic reapplication (P=0.02), lower need for bleeding control (P=0.00006), and lower latency (P<0.00001). We could conclude that the use of vasoconstrictors associated with local anesthetics in digital block proved to be a safe and effective technique.

On the developmental self-regulatory dynamics and evolution of individuated multicellular organisms.

Changes in gene expression are thought to regulate the cell differentiation process intrinsically through complex epigenetic mechanisms. In fundamental terms, however, this assumed regulation refers only to the intricate propagation of changes in gene expression or else leads to non-explanatory regresses. The developmental self-regulatory dynamics and evolution of individuated multicellular organisms also lack a unified and falsifiable description. To fill this gap, I computationally analyzed publicly available high-throughput data of histone H3 post-translational modifications and mRNA abundance for different Homo sapiens, Mus musculus, and Drosophila melanogaster cell-type/developmental-period samples. My analysis of genomic regions adjacent to transcription start sites generated a profile from pairwise partial correlations between histone modifications controlling for the respective mRNA levels for each cell-type/developmental-period dataset. I found that these profiles, while explicitly uncorrelated with the respective transcriptional "identities" by construction, associate strongly with cell differentiation states. This association is not expected if cell differentiation is, in effect, regulated by epigenetic mechanisms. Based on these results, I propose a general, falsifiable theory of individuated multicellularity, which relies on the synergistic coupling across the extracellular space of two explicitly uncorrelated "self-organizing" systems constraining histone modification states at the same sites. This theory describes how the simplest multicellular individual-understood as an intrinsic, higher-order constraint-emerges from proliferating undifferentiated cells, and could explain the intrinsic regulation of gene transcriptional changes for cell differentiation and the evolution of individuated multicellular organisms.

The Specification of Cortical Subcerebral Projection Neurons Depends on the Direct Repression of TBR1 by CTIP1/BCL11a.

The acquisition of distinct neuronal fates is fundamental for the function of the cerebral cortex. We find that the development of subcerebral projections from layer 5 neurons in the mouse neocortex depends on the high levels of expression of the transcription factor CTIP1; CTIP1 is coexpressed with CTIP2 in neurons that project to subcerebral targets and with SATB2 in those that project to the contralateral cortex. CTIP1 directly represses Tbr1 in layer 5, which appears as a critical step for the acquisition of the subcerebral fate. In contrast, lower levels of CTIP1 in layer 6 are required for TBR1 expression, which directs the corticothalamic fate. CTIP1 does not appear to play a critical role in the acquisition of the callosal projection fate in layer 5. These findings unravel a key step in the acquisition of cell fate for closely related corticofugal neurons and indicate that differential dosages of transcriptions factors are critical to specify different neuronal identities.

Current measurement by Faraday effect on GEPOPU.

The design of an optical current sensor to be used in a pulsed power generator is presented. The current sensor is based on the polarization rotation by the Faraday effect. GEPOPU is a pulsed power generator, 110 kA, 120 ns double transit time, 1.5 Ω coaxial geometry, and current rise time of 50 ns. Two different optical geometries surrounding the conductor were tried, using Amici roof prism and pentaprism to go around the current once, as a way to preserve the state of polarization along the optical path by means of complementary reflections within the sensing element. We believe this to be the first time that such large and rapidly varying currents have been measured with this configuration. The values obtained for both geometries agree with the values obtained with a Rogowski coil. The traces obtained are completely noise-free and no significant time lag has been observed between the current determined from the Faraday rotation and the current measured using a Rogowski coil.

AlterORF: a database of alternate open reading frames.

AlterORF is a searchable database that contains information regarding alternate open reading frames (ORFs) for over 1.5 million genes in 481 prokaryotic genomes. The objective of the database is to provide a platform for improving genome annotation and to serve as an aid for the identification of prokaryotic genes that potentially encode proteins in more than one reading frame. The AlterORF Database can be accessed through a web interface at www.alterorf.cl.

Bioinformatic prediction and experimental verification of Fur-regulated genes in the extreme acidophile Acidithiobacillus ferrooxidans.

The gamma-proteobacterium Acidithiobacillus ferrooxidans lives in extremely acidic conditions (pH 2) and, unlike most organisms, is confronted with an abundant supply of soluble iron. It is also unusual in that it oxidizes iron as an energy source. Consequently, it faces the challenging dual problems of (i) maintaining intracellular iron homeostasis when confronted with extremely high environmental loads of iron and (ii) of regulating the use of iron both as an energy source and as a metabolic micronutrient. A combined bioinformatic and experimental approach was undertaken to identify Fur regulatory sites in the genome of A. ferrooxidans and to gain insight into the constitution of its Fur regulon. Fur regulatory targets associated with a variety of cellular functions including metal trafficking (e.g. feoPABC, tdr, tonBexbBD, copB, cdf), utilization (e.g. fdx, nif), transcriptional regulation (e.g. phoB, irr, iscR) and redox balance (grx, trx, gst) were identified. Selected predicted Fur regulatory sites were confirmed by FURTA, EMSA and in vitro transcription analyses. This study provides the first model for a Fur-binding site consensus sequence in an acidophilic iron-oxidizing microorganism and lays the foundation for future studies aimed at deepening our understanding of the regulatory networks that control iron uptake, homeostasis and oxidation in extreme acidophiles.

Thermophiles like hot T.

A plethora of mechanisms confer protein stability in thermophilic microorganisms and, recently, it was suggested that these mechanisms might be divided along evolutionary lines. Here, a multi-genome comparison shows that there is a statistically significant increase in the proportion of NTN codons correlated with increasing optimal growth temperature for both Bacteria and Archaea. NTN encodes exclusively non-polar, hydrophobic amino acids and indicates a common underlying use of hydrophobicity for stabilizing proteins in Bacteria and Archaea that transcends evolutionary origins. However, some microorganisms do not follow this trend, suggesting that alternate mechanisms (e.g. intracellular electrolytes) might be used for protein stabilization. These studies highlight the usefulness of large-scale comparative genomics to uncover novel relationships that are not immediately obvious from protein structure studies alone.

Large-scale, multi-genome analysis of alternate open reading frames in bacteria and archaea.

Analysis of over 300,000 annotated genes in 105 bacterial and archaeal genomes reveals an unexpectedly high frequency of large (>300 nucleotides) alternate open reading frames (ORFs). Especially notable is the very high frequency of alternate ORFs in frames +3 and -1 (where the annotated gene is defined as frame +1). The occurrence of alternate ORFs is correlated with genomic G+C content and is strongly influenced by synonymous codon usage bias. The frequency of alternate ORFs in frame -1 is also influenced by the occurrence of codons encoding leucine and serine in frame +1. Although some alternate ORFs have been shown to encode proteins, many others are probably not expressed because they lack appropriate signals for transcription and translation. These latter can be mis-annotated by automatic gene finding programs leading to errors in public databases. Especially prone to mis-annotation is frame -1, because it exhibits a potential codon usage and theoretical capacity to encode proteins with an amino acid composition most similar to real genes. Some alternate ORFs are conserved across bacterial or archaeal species, and can give rise to misannotated "conserved hypothetical" genes, while others are unique to a genome and are misidentified as "hypothetical orphan" genes, contributing significantly to the orphan gene paradox.

Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis.

BACKGROUND: Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism. RESULTS: Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates. CONCLUSIONS: A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis.