Exploring Propolis as a Sustainable Bio-Preservative Agent to Control Foodborne Pathogens in Vacuum-Packed Cooked Ham.

The search for natural food additives makes propolis an exciting alternative due to its known antimicrobial activity. This work aims to investigate propolis' behavior as a nitrite substitute ingredient in cooked ham (a ready-to-eat product) when confronted with pathogenic microorganisms of food interest. The microbial evolution of Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Clostridium sporogenes inoculated at known doses was examined in different batches of cooked ham. The design of a challenge test according to their shelf life (45 days), pH values, and water activity allowed the determination of the mesophilic aerobic flora, psychotropic, and acid lactic bacteria viability. The test was completed with an organoleptic analysis of the samples, considering possible alterations in color and texture. The cooked ham formulation containing propolis instead of nitrites limited the potential growth (δ < 0.5 log10) of all the inoculated microorganisms until day 45, except for L. monocytogenes, which in turn exhibited a bacteriostatic effect between day 7 and 30 of the storage time. The sensory analysis revealed the consumer's acceptance of cooked ham batches including propolis as a natural additive. These findings suggest the functionality of propolis as a promising alternative to artificial preservatives for ensuring food safety and reducing the proliferation risk of foodborne pathogens in ready-to-eat products.

Exploring eye-tracking data as an indicator of situational awareness in nursing students during a cardiorespiratory arrest simulation.

AIM: To examine the components of visual attention that maintain situational awareness during simulation training in undergraduate nursing students with different instruction levels. BACKGROUND: Eye-tracking can provide deep insight into the nurses' attention during simulated practice. Knowing which gaze patterns promote situational awareness can significantly improve nurse instruction. DESIGN: A comparative observational study investigated the role of visual attention on the performance quality, psychophysiological parameters (vital signs, anxiety and stress) and socioemotional competencies (cognitive workload, motivation and self-efficacy) of nursing students with various experience levels. METHODS: Thirty nursing students divided into two groups according to their academic level: first cycle (n=14) and second-cycle (n=16) faced a clinical simulation scenario to resolve a cardiorespiratory arrest event. Eye tracking-based analysis required the selection of six areas of interest. The monitorization of vital signs included measuring blood pressure, heart rate, respiratory rate and oxygen saturation before and after the simulation practice. Participants completed the socioemotional questionnaire (NASA-TLX). They answered the state subscale of the State-Trait Anxiety Inventory (STAI), the Visual Analogue Scale (VAS) of stress, the Situational Motivation Scale (SIMS) and the Baessler and Schwarzer General Self-Efficacy Scale. RESULTS: The first-cycle group displayed higher vital sign scores than the second cycle, apart from the post-simulation respiratory rate. All physiological parameters increased in mean value after the clinical simulation, except oxygen saturation. Anxiety was the only parameter in the socioemotional domain to present a statistically significant difference between the groups. First-year nursing students showed greater anxiety, stress, mental workload, identified regulation and intrinsic motivation, while second-year students showed higher levels of amotivation, external regulation and perceived self-efficacy. Eye-tracking data (revisits, gaze and duration of fixations) exhibited statistically significant differences depending on the area of interest in both groups (p =. 05). The performance outcomes showed a negative and moderate association with gaze the total number of gazes in the second-cycle group (rho = -0.640, p = 0.010). CONCLUSION: Eye-tracking-based analysis can help to predict performance quality while maintaining situational awareness during nursing instruction.

Impact of a COVID-19 Outbreak in an Elderly Care Home after Primary Vaccination.

Elderly care home residents are particularly vulnerable to COVID-19 due to immune-senescence, pre-existing medical conditions, and the risk of transmission from staff and visitors. This study aimed to describe the outcomes of a COVID-19 outbreak in a long-term care facility for elderly persons following the initial vaccination. A single-center, retrospective, observational design was used to analyze the variables associated with hospitalization and death rate by logistic regression. Adjusted odds ratios (aOR) and their 95% confidence intervals (CI) were calculated. Sixty-eight residents received the first dose of the COVID-19 vaccine. Despite being negative six days after vaccination, the performance of a second test 4 days later revealed 51 positives (75.0%) among residents and 18 among workers (56.3%). A total of 65 of the 68 residents (95.58%) had positive results with symptoms, whereas 34.9% required hospitalization, and 25.8% died. The best-fitting model to explain the distribution of cases reflects three points at the time of infection.. The time from vaccination to symptom onset explains the hospitalization and mortality rates since a day elapsed halves the risk of hospitalization (aOR = 0.57; CI = 0.38-0.75) and the risk of death by a quarter (aOR = 0.74; CI = 0.63-0.88). Nursing homes present an elevated risk of transmission and severity of SARS-CoV-2 infection. Although vaccination reduces the risk of hospitalization and death, extreme prevention and control measures are essential in these institutions despite the high vaccination coverage.

Antimicrobial Activity of Spanish Propolis against Listeria monocytogenes and Other Listeria Strains.

The outbreaks of Listeria associated with food consumption are increasing worldwide concurrently with public concern about the need for natural growth inhibitors. In this context, propolis seems to be a promising bioactive product collected by honeybees, due to its antimicrobial activity against different food pathogens. This study aims to evaluate the efficacy of hydroalcoholic propolis extracts for controlling Listeria under several pH conditions. The physicochemical properties (wax, resins, ashes, impurities), the bioactive compounds (phenolic and flavonoid content), and the antimicrobial activity of 31 propolis samples collected from the half North of Spain were determined. Results showed similar trends in the physicochemical composition and bioactive properties, regardless of the harvesting area. Non-limiting pH conditions (7.04, 6.01, 5.01) in 11 Listeria strains (5 from collection and 6 wild strains from meat products) exhibited MICs (Minimum inhibition concentration) and MBCs (Minimum bactericidal concentration) ranging from 39.09 to 625 µg/mL. The antibacterial activity increased under acidic pH conditions, showing a synergistic effect at pH = 5.01 (p < 0.05). These findings suggest the potential of Spanish propolis as a natural antibacterial inhibitor to control Listeria growth in food products.

Using Task-Evoked Pupillary Response to Predict Clinical Performance during a Simulation Training.

Training in healthcare skills can be affected by trainees' workload when completing a task. Due to cognitive processing demands being negatively correlated to clinical performance, assessing mental workload through objective measures is crucial. This study aimed to investigate task-evoked changes in pupil size as reliable markers of mental workload and clinical performance. A sample of 49 nursing students participated in a cardiac arrest simulation-based practice. Measurements of cognitive demands (NASA-Task Load Index), physiological parameters (blood pressure, oxygen saturation, and heart rate), and pupil responses (minimum, maximum, and difference diameters) throughout revealed statistically significant differences according to performance scores. The analysis of a multiple regression model produced a statistically significant pattern between pupil diameter differences and heart rate, systolic blood pressure, workload, and performance (R2 = 0.280; F (6, 41) = 2.660; p < 0.028; d = 2.042). Findings suggest that pupil variations are promising markers to complement physiological metrics for predicting mental workload and clinical performance in medical practice.

Biochemical Profile and Antioxidant Properties of Propolis from Northern Spain.

The antioxidant, anti-inflammatory, and antimicrobial characteristics of propolis, a bioactive compound collected from hives, have prompted its use in the food sector in recent times. This study investigated the physicochemical characteristics, phenolic profile, and antioxidant capacity of 31 propolis extracts collected from Northern Spain. The physicochemical composition (resins, waxes, ashes mineral content, and heavy metals) was within the allowable regulatory limits. The analysis of bioactive compounds enabled the identification of 51 constituents: flavonoids (apigenin, catechin, chrysin, quercetin, and pinocembrin) and phenolic acids (caffeic, ferulic, and coumaric). The mean value of total polyphenols was 42.72 ± 13.19 Pinocembrin-Galangin Equivalents/100 g, whereas a range between 1.64 ± 0.04 and 4.95 ± 0.36 Quercetin Equivalents (QE) g/100 g was found for total flavonoids content. The determination of bioactivities revealed significant antioxidant capacity using DPPH (1114.28 ± 10.39 µM Trolox Equivalents and 3487.61 ± 318.66 µM Vitamin C Equivalents). Resin content in propolis samples was positively and significantly correlated with both polyphenols (rho = 0.365; p = 0.043) and flavonoid composition (rho = 0.615; p = 0.000) as well as the antioxidant capacity TEAC DPPH (rho = 0.415; p = 0.020). A multiple regression analysis modeled the correlation between resin composition, flavonoids, and TEAC DPPH values, yielding a significant regression equation (R2 = 0.618; F (2,28) = 22.629; p < 0.000; d = 2.299). Therefore, evaluating physicochemical parameters and biological activities provides a promising framework for predicting propolis' quality and antioxidant properties, thus suggesting its potential as a functional and bioactive compound for the food industry.

Effect of Psychophysiological Stress and Socio-Emotional Competencies on the Clinical Performance of Nursing Students during a Simulation Practice.

Psychophysiological stress can affect the cognitive response and effective learning of students during medical simulation practices. This study aimed to explore the effect of psychophysiological stress and socio-emotional competencies on clinical performance during a simulation experience. A pre-test/post-test design was used to assess physiological (blood pressure, heart rate and blood oxygen saturation) and psychological parameters (stress and anxiety) as well as socio-emotional skills (cognitive load, self-efficacy and motivation) in nursing students (n = 40) before and after the simulation of a cardiopulmonary resuscitation practice. Physiological responses showed statistically significant differences between pre-test and post-test conditions for blood pressure and heart rate (p < 0.0001). Moderate and significant correlations were also observed when comparing self-efficacy with stress (r = -0.445, p = 0.004), anxiety (r = -0.467, p = 0.002) and motivation (r = -0.406, p = 0.009) measures. Similarly, cognitive-load dimensions were significantly associated with either physiological (r = -0.335, p = 0.034) or psychological (r = -0.448, p = 0.004) indicators. The analysis of multiple regression models revealed a relationship between the effectiveness of the simulated experience, post-test blood oxygen saturation, heart rate, workload and self-efficacy (R2 = 0.490; F (3, 39) = 8.305; p < 0.0001; d = 1.663). Therefore, the evaluation of psychophysiological parameters and socio-emotional skills seems to provide a promising framework for predicting the quality of simulated clinical practices.

Plasmonic Biosensors for Single-Molecule Biomedical Analysis.

The rapid spread of epidemic diseases (i.e., coronavirus disease 2019 (COVID-19)) has contributed to focus global attention on the diagnosis of medical conditions by ultrasensitive detection methods. To overcome this challenge, increasing efforts have been driven towards the development of single-molecule analytical platforms. In this context, recent progress in plasmonic biosensing has enabled the design of novel detection strategies capable of targeting individual molecules while evaluating their binding affinity and biological interactions. This review compiles the latest advances in plasmonic technologies for monitoring clinically relevant biomarkers at the single-molecule level. Functional applications are discussed according to plasmonic sensing modes based on either nanoapertures or nanoparticle approaches. A special focus was devoted to new analytical developments involving a wide variety of analytes (e.g., proteins, living cells, nucleic acids and viruses). The utility of plasmonic-based single-molecule analysis for personalized medicine, considering technological limitations and future prospects, is also overviewed.

Clinical Applications of Visual Plasmonic Colorimetric Sensing.

Colorimetric analysis has become of great importance in recent years to improve the operationalization of plasmonic-based biosensors. The unique properties of nanomaterials have enabled the development of a variety of plasmonics applications on the basis of the colorimetric sensing provided by metal nanoparticles. In particular, the extinction of localized surface plasmon resonance (LSPR) in the visible range has permitted the exploitation of LSPR colorimetric-based biosensors as powerful tools for clinical diagnostics and drug monitoring. This review summarizes recent progress in the biochemical monitoring of clinical biomarkers by ultrasensitive plasmonic colorimetric strategies according to the distance- or the morphology/size-dependent sensing modes. The potential of colorimetric nanosensors as point of care devices from the perspective of naked-eye detection is comprehensively discussed for a broad range of analytes including pharmaceuticals, proteins, carbohydrates, nucleic acids, bacteria, and viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The practical suitability of plasmonic-based colorimetric assays for the rapid visual readout in biological samples, considering current challenges and future perspectives, is also reviewed.

Recent Progress in Plasmonic Biosensing Schemes for Virus Detection.

The global burden of coronavirus disease 2019 (COVID-19) to public health and global economy has stressed the need for rapid and simple diagnostic methods. From this perspective, plasmonic-based biosensing can manage the threat of infectious diseases by providing timely virus monitoring. In recent years, many plasmonics' platforms have embraced the challenge of offering on-site strategies to complement traditional diagnostic methods relying on the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). This review compiled recent progress on the development of novel plasmonic sensing schemes for the effective control of virus-related diseases. A special focus was set on the utilization of plasmonic nanostructures in combination with other detection formats involving colorimetric, fluorescence, luminescence, or Raman scattering enhancement. The quantification of different viruses (e.g., hepatitis virus, influenza virus, norovirus, dengue virus, Ebola virus, Zika virus) with particular attention to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was reviewed from the perspective of the biomarker and the biological receptor immobilized on the sensor chip. Technological limitations including selectivity, stability, and monitoring in biological matrices were also reviewed for different plasmonic-sensing approaches.

Effect of Facial Skin Temperature on the Perception of Anxiety: A Pilot Study.

The extent of anxiety and psychological stress can impact upon the optimal performance of simulation-based practices. The current study investigates the association between differences in skin temperature and perceived anxiety by under- (n = 21) and post-graduate (n = 19) nursing students undertaking a cardiopulmonary resuscitation (CPR) training. Thermal facial gradients from selected facial regions were correlated with the scores assessed by the State-Trait Anxiety Inventory (STAI) and the chest compression quality parameters measured using mannequin-integrated accelerometer sensors. A specific temperature profile was obtained depending on thermal facial variations before and after the simulation event. Statistically significant correlations were found between STAI scale scores and the temperature facial recordings in the forehead (r = 0.579; p < 0.000), periorbital (r = 0.394; p < 0.006), maxillary (r = 0.328; p < 0.019) and neck areas (r = 0.284; p < 0.038). Significant associations were also observed by correlating CPR performance parameters with the facial temperature values in the forehead (r = 0.447; p < 0.002), periorbital (r = 0.446; p < 0.002) and maxillary areas (r = 0.422; p < 0.003). These preliminary findings suggest that higher anxiety levels result in poorer clinical performance and can be correlated to temperature variations in certain facial regions.

Low-Fouling Substrates for Plasmonic Sensing of Circulating Biomarkers in Biological Fluids.

The monitoring of biomarkers in body fluids provides valuable prognostic information regarding disease onset and progression. Most biosensing approaches use noninvasive screening tools and are conducted in order to improve early clinical diagnosis. However, biofouling of the sensing surface may disturb the quantification of circulating biomarkers in complex biological fluids. Thus, there is a great need for antifouling interfaces to be designed in order to reduce nonspecific adsorption and prevent inactivation of biological receptors and loss of sensitivity. To address these limitations and enable their application in clinical practice, a variety of plasmonic platforms have been recently developed for biomarker analysis in easily accessible biological fluids. This review presents an overview of the latest advances in the design of antifouling strategies for the detection of clinically relevant biomarkers on the basis of the characteristics of biological samples. The impact of nanoplasmonic biosensors as point-of-care devices has been examined for a wide range of biomarkers associated with cancer, inflammatory, infectious and neurodegenerative diseases. Clinical applications in readily obtainable biofluids such as blood, saliva, urine, tears and cerebrospinal and synovial fluids, covering almost the whole range of plasmonic applications, from surface plasmon resonance (SPR) to surface-enhanced Raman scattering (SERS), are also discussed.

Advances in nanoplasmonic biosensors for clinical applications.

Biomarkers are unquestionable biological indicators for diagnosis and therapeutic interventions providing appropriate classification of a wide range of health disorders and risk factors. Nonetheless, the detection and quantification of biomarkers need to be tested with sufficient reliability by robust analytical methods in order to assure clinical performance in health care settings. Since the analytical performance is determined by the sensitivity and specificity of the method employed, techniques have been intensively refined in order to avoid the misinterpretation of results and undesirable bias. Although biomarkers can be detected with the existing analytical techniques, to reproducibly quantify them in decentralized settings or remote locations with the required accuracy is still a challenge. Currently, only a few point-of-care devices for biomarker evaluation are commercially available. Thus, more focused research efforts are needed to overcome these limitations in order to provide universal patient-centered care platforms. To this end, plasmonic biosensors can be conveniently used as portable diagnostic devices for attaining timely and cost-effective clinical outcomes. The development of enhanced performance based on nanoplasmonics technology opens the way for sensor miniaturization, multiplexing and point of care testing. This review covers recent advances and applications of plasmonic and nanoplasmonic biosensors intended for biomarker diagnosis in clinical practice, including cancer, cardiovascular and neurodegenerative diseases. The review specially focuses on: (i) recent progress in plasmonics development including the design of singular nanostructured surfaces, (ii) novel chemical functionalization strategies for the appropriate incorporation of bioreceptors and (iii) plasmonic applications as real operative devices in the clinical field. Future prospects in the use of nanoplasmonic sensor platforms for personalised quantification and management of biomarkers directly in body fluids will also be discussed.

Effects of dietary supplementation with lemon verbena extracts on serum inflammatory markers of multiple sclerosis patients.

INTRODUCTION: Inflammation is one of the main contributory factors to the etiopathogenesis of multiple sclerosis (MS). Dietary interventions with Lipia citriadora (lemon verbena) extracts have been proved to be effective in the prevention of inflammatory diseases. OBJECTIVES: The aim of this study is to evaluate the effect of lemon verbena supplementation in pro- and anti- inflammatory serum biomarkers of patients with different clinical subtypes of multiple sclerosis. METHODS: The effect of lemon verbena supplementation (10% w/w verbascoside) was evaluated in a randomized, double-blinded placebo-controlled study with 30 participants classified in relapsing-remitting (n=10), primary progressive (n=5) and secondary progressive (n=15) MS presentations. Serum cytokine and C reactive protein levels were assessed in intervention and control groups for each MS clinical subtype after 28 days of dietary supplementation. RESULTS: Serum levels of C reactive protein and 8 cytokines/ inflammatory (IFN-γ, IL-12, IL-23, IL-6, TNF-α, TGF-ß, IL-4 and IL-10) markers were studied. Secondary progressive MS- supplemented patients showed C reactive protein concentrations significantly lower compared to the placebo group (p.

Introducción: La inflamación es uno de los principales factores que contribuyen en la etiopatogénesis de la esclerosis múltiple (EM). Se ha demostrado que las intervenciones en la dieta con extractos de Lipia citriadora (hierbaluisa) son efectivas en la prevención de las enfermedades inflamatorias. Objectivos: El objetivo de este estudio es evaluar el efecto de la suplementación con extractos de hierbaluisa en los biomarcadores de inflamación en suero de pacientes con diferentes subtipos clínicos de esclerosis múltiple. Métodos: El efecto de la suplementación con hierbaluisa (10 % p/p verbascósido) se evaluó mediante un estudio aleatorizado de doble ciego controlado con grupo placebo, constituido por 30 participantes clasificados según la forma de presentación de EM en: remitentes-recaídas (n=10), primaria progresiva (n=5) y secundaria progresiva (n=15). Los niveles de citoquinas y proteína C reactiva en suero se valoraron en los grupos intervención y control de cada uno de los subtipos clínicos de EM después de 28 días de suplementación en la dieta. Resultados: Se estudiaron los niveles en suero de proteína C reactiva y de 8 citoquinas como biomarcadores de inflamación (IFN-, IL-12, IL-23, IL-6, TNF-, TGF-, IL-4 e IL-10). Los pacientes del grupo de intervención con EM secundaria progresiva presentaron concentraciones de proteína C reactiva significativamente más bajos comparados con el grupo placebo (p.

Effects of a low-fat diet with antioxidant supplementation on biochemical markers of multiple sclerosis long-term care residents.

INTRODUCTION: Multiple sclerosis (MS) treatment options are primarily limited to immunomodulatory therapies in MS non-progressive forms. Nutrition intervention studies suggest that diet may be considered as a complementary treatment to control disease progression. Therefore, dietary intervention may help to improve wellness and ameliorate symptoms of MS patients. OBJECTIVES: To assess the effect of a low-fat diet with antioxidant supplementation on biochemical markers of institutionalized patients with progressive forms of multiple sclerosis. METHODS: A randomized prospective placebo-controlled study involving 9 participants, 5 of them assigned to the intervention group (low-fat diet and antioxidant supplementation) and the other 4 to the placebo group (low-fat diet). The effect of the dietary intervention, involving diet modification and antioxidant supplementation, was examined for 42 days by measuring anthropometric, biochemical parameters and oxidative stress markers in blood at baseline (day 0), intermediate (day 15) and end (day 42) stages of the treatment. RESULTS: The intervention group obtained C reactive protein levels significantly lower than those observed in the corresponding placebo group at the end of the study. Oxidative stress and inflammatory markers isoprostane 8-iso-PGF2α and interleukine IL-6 values also diminished after dietary intervention in the intervention group. Catalase activity increased significantly in the intervention group prior antioxidant supplementation. No significant differences were observed in other oxidative stress markers. CONCLUSIONS: The results suggest that diet and dietary supplements are involved in cell metabolism modulation and MS-related inflammatory processes. Consequently, low fat diets and antioxidant supplements may be used as complementary therapies for treatment of multiple sclerosis.

Introducción: Las posibilidades de tratamiento de la esclerosis múltiple (EM) se encuentran limitadas principalmente a terapias con inmumoduladores en las formas no progresivas de EM. Los estudios de intervención nutricional sugieren que la dieta puede considerarse como un tratamiento alternativo para controlar la progresión de la enfermedad. Por esta razón, las intervenciones en la dieta pueden ayudar a mejorar el bienestar y mejorar los síntomas de los pacientes con EM. Objetivos: Valorar el efecto de una dieta pobre en grasas con suplementación de antioxidantes en los marcadores bioquímicos de pacientes institucionalizados que presentan formas progresivas de EM. Métodos: Se realizó un estudio prospectivo aleatorizado controlado por placebo con 9 participantes, 5 de los cuales se asignan al grupo de intervención (dieta baja en grasas y suplementación antioxidante) y los 4 restantes al grupo placebo (dieta baja en grasas). Se evaluó el efecto de la intervención dietética que supone modificación de la dieta e introducción de antioxidantes durante 42 días mediante valoraciones de parámetros antropométricos y bioquímicos y marcadores del estrés oxidativo en sangre y orina en las etapas inicial (día 0), intermedia (día 15) y final (día 42) del tratamiento. Resultados: Se obtuvieron niveles de proteína C reactiva significativamente inferiores en el grupo de intervención con respecto al grupo placebo al final del estudio. Los marcadores de estrés oxidativo e inflamación: isoprostanos 8-iso-PGF2e interleucina IL-6 también disminuyeron en el grupo de intervención después de la intervención dietética. La actividad de la enzima catalasa aumentó de forma significativa en el grupo de intervención antes de la suplementación con antioxidantes. No se observaron diferencias significativas en otros marcadores de estrés oxidativo. Conclusiones: Los resultados obtenidos sugieren que la dieta y los suplementos dietéticos están involucrados en la modulación del metabolismo celular y los procesos de inflamación de la EM. En consecuencia, las dietas bajas en grasas y los suplementos antioxidantes podrían ser utilizados como terapias alternativas en el tratamiento de la EM.

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum.

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.

The heterotrimeric Galpha protein pga1 regulates biosynthesis of penicillin, chrysogenin and roquefortine in Penicillium chrysogenum.

We have studied the role of the pga1 gene of Penicillium chrysogenum, encoding the alpha subunit of a heterotrimeric G protein, in secondary metabolite production. The dominant activating pga1(G42R) mutation caused an increase in the production of the three secondary metabolites penicillin, the yellow pigment chrysogenin and the mycotoxin roquefortine, whereas the dominant inactivating pga1(G203R) allele and the deletion of the pga1 gene resulted in a decrease of the amount of produced penicillin and roquefortine. Chrysogenin is produced in solid medium as a yellow pigment, and its biosynthesis is clearly enhanced by the presence of the dominant activating pga1(G42R) allele. Roquefortine is produced associated with mycelium during the first 3 days in submerged cultures, and is released to the medium afterwards; dominant activating and inactivating pga1 mutations result in upregulation and downregulation of roquefortine biosynthesis recpectively. Pga1 regulates penicillin biosynthesis by controlling expression of the penicillin biosynthetic genes; the three genes pcbAB, pcbC and penDE showed elevated transcript levels in transformants expressing the pga1(G42R) allele, whereas in transformants with the inactivating pga1(G203R) allele and in the pga1-deleted mutant their transcript levels were lower than those in the parental strains. Increase of intracellular cAMP levels had no effect on penicillin production. In summary, the dominant activating pga1(G42R) allele upregulates the biosynthesis of three secondary metabolites in Penicillium chrysogenum to a different extent.

Post-translational enzyme modification by the phosphopantetheinyl transferase is required for lysine and penicillin biosynthesis but not for roquefortine or fatty acid formation in Penicillium chrysogenum.

NRPSs (non-ribosomal peptide synthetases) and PKSs (polyketide synthases) require post-translational phosphopantetheinylation to become active. This reaction is catalysed by a PPTase (4'-phosphopantetheinyl transferase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% similarity with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild-type and high-penicillin-producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase-defective mutant (Wis54-PPT(-)) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of roquefortine. The biosynthesis of roquefortine does not appear to involve PPTase-mediated modification of the synthesizing enzymes. The PPT(-) mutant did not require fatty acids, which indicates that activation of the fatty acid synthase is performed by a different PPTase. Complementation of Wis54-PPT(-) with the ppt gene restored lysine biosynthesis, pigmentation and penicillin production, which demonstrates the wide range of processes controlled by this gene.

Part per trillion determination of atrazine in natural water samples by a surface plasmon resonance immunosensor.

A new immunoassay for continuously monitoring atrazine in water has been developed. It uses a portable biosensor platform based on surface plasmon resonance (SPR) technology. This immunoassay is based on the binding inhibition format with purified polyclonal antibodies, with the analyte derivative covalently immobilized on a gold sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed on the gold-coated sensor surface in order to obtain a reusable sensing surface. The low detection limit for the optimized assay, calculated as the concentration that produces a 10% decrease in the blank signal, is 20 ng/L. A complete assay cycle, including regeneration, is accomplished in 25 min. Additionally, a study of the matrix effects of different types of wastewater was performed. All measurements were carried out with the SPR sensor system (beta-SPR) commercialised by the company Sensia, S.L. (Spain). The small size and low response time of the beta-SPR platform would allow it to be used in real contaminated locations. The immunosensor was evaluated and validated by measuring the atrazine content of 26 natural samples collected from Ebro River. Solid-phase extraction followed by gas chromatography coupled to mass spectrometric detection (SPE-GC-MS) was used to validate the new immunoassay.