Heart Rate Variability to Automatically Identify Hyperbaric States Considering Respiratory Component.

This study's primary objective was to identify individuals whose physiological responses deviated from the rest of the study population by automatically monitoring atmospheric pressure levels to which they are exposed and using parameters derived from their heart rate variability (HRV). To achieve this, 28 volunteers were placed in a dry hyperbaric chamber, where they experienced varying pressures from 1 to 5 atmospheres, with five sequential stops lasting five minutes each at different atmospheric pressures. The HRV was dissected into two components: the respiratory component, which is linked to respiration; and the residual component, which is influenced by factors beyond respiration. Nine parameters were assessed, including the respiratory rate, four classic HRV temporal parameters, and four frequency parameters. A k-nearest neighbors classifier based on cosine distance successfully identified the atmospheric pressures to which the subjects were exposed to. The classifier achieved an 88.5% accuracy rate in distinguishing between the 5 atm and 3 atm stages using only four features: respiratory rate, heart rate, and two frequency parameters associated with the subjects' sympathetic responses. Furthermore, the study identified 6 out of 28 subjects as having atypical responses across all pressure levels when compared to the majority. Interestingly, two of these subjects stood out in terms of gender and having less prior diving experience, but they still exhibited normal responses to immersion. This suggests the potential for establishing distinct safety protocols for divers based on their previous experience and gender.

Comparability exercise of critical quality attributes of clinical-grade human mesenchymal stromal cells from the Wharton's jelly: single-use stirred tank bioreactors versus planar culture systems.

BACKGROUND AIMS: The increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice-compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. METHODS: MSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. RESULTS: After 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 × 108 ± 1.07 × 107 cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 × 108 ± 2.36 × 108 cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. CONCLUSIONS: In summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.

Enhancing Safety in Hyperbaric Environments through Analysis of Autonomic Nervous System Responses: A Comparison of Dry and Humid Conditions.

Diving can have significant cardiovascular effects on the human body and increase the risk of developing cardiac health issues. This study aimed to investigate the autonomic nervous system (ANS) responses of healthy individuals during simulated dives in hyperbaric chambers and explore the effects of the humid environment on these responses. Electrocardiographic- and heart-rate-variability (HRV)-derived indices were analyzed, and their statistical ranges were compared at different depths during simulated immersions under dry and humid conditions. The results showed that humidity significantly affected the ANS responses of the subjects, leading to reduced parasympathetic activity and increased sympathetic dominance. The power of the high-frequency band of the HRV after removing the influence of respiration, PHF⟂¯, and the number of pairs of successive normal-to-normal intervals that differ by more than 50 ms divided by the total number of normal-to-normal intervals, pNN50¯, indices were found to be the most informative in distinguishing the ANS responses of subjects between the two datasets. Additionally, the statistical ranges of the HRV indices were calculated, and the classification of subjects as "normal" or "abnormal" was determined based on these ranges. The results showed that the ranges were effective at identifying abnormal ANS responses, indicating the potential use of these ranges as a reference for monitoring the activity of divers and avoiding future immersions if many indices are out of the normal ranges. The bagging method was also used to include some variability in the datasets' ranges, and the classification results showed that the ranges computed without proper bagging represent reality and its associated variability. Overall, this study provides valuable insights into the ANS responses of healthy individuals during simulated dives in hyperbaric chambers and the effects of humidity on these responses.

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus.

Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy-to-use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.

Production of BP178, a derivative of the synthetic antibacterial peptide BP100, in the rice seed endosperm.

BACKGROUND: BP178 peptide is a synthetic BP100-magainin derivative possessing strong inhibitory activity against plant pathogenic bacteria, offering a great potential for future applications in plant protection and other fields. Here we report the production and recovery of a bioactive BP178 peptide using rice seeds as biofactories. RESULTS: A synthetic gene encoding the BP178 peptide was prepared and introduced in rice plants. The gene was efficiently expressed in transgenic rice under the control of an endosperm-specific promoter. Among the three endosperm-specific rice promoters (Glutelin B1, Glutelin B4 or Globulin 1), best results were obtained when using the Globulin 1 promoter. The BP178 peptide accumulated in the seed endosperm and was easily recovered from rice seeds using a simple procedure with a yield of 21 µg/g. The transgene was stably inherited for at least three generations, and peptide accumulation remained stable during long term storage of transgenic seeds. The purified peptide showed in vitro activity against the bacterial plant pathogen Dickeya sp., the causal agent of the dark brown sheath rot of rice. Seedlings of transgenic events showed enhanced resistance to the fungal pathogen Fusarium verticillioides, supporting that the in planta produced peptide was biologically active. CONCLUSIONS: The strategy developed in this work for the sustainable production of BP178 peptide using rice seeds as biofactories represents a promising system for future production of peptides for plant protection and possibly in other fields.

Calcium-dependent protein kinase OsCPK10 mediates both drought tolerance and blast disease resistance in rice plants.

Plant growth and productivity is negatively affected by different stresses. Most stresses trigger calcium signals that initiate acclimation responses in plants. The multigene family of plant calcium-dependent protein kinases (CPKs) functions in multiple stress responses by transducing calcium signals into phosphorylation events. This work reports that the OsCPK10 isoform positively mediates tolerance to different stresses in rice plants by enhancing their antioxidant capacity and protecting them from reactive oxygen species (ROS) damage, with the uncontrolled generation of ROS being a common feature of these stresses. Here, we show that the constitutive accumulation of an HA-tagged OsCPK10 full-length protein enhances the hydrogen peroxide detoxifying capacity of rice plants during desiccation. This is achived by modulating the accumulation of catalase proteins, which reduces the extent of lipid peroxidation and protects the integrity of cell membranes, resulting in drought tolerance. OsCPK10HA accumulation also confers blast disease resistance by interfering with fungal necrotrophic growth via a reduction in the accumulation of hydrogen peroxide. Furthermore, we show by bimolecular complementation assays that OsCPK10 is a plasma membrane protein that physically interacts in vivo with catalase A. OsCPK10 therefore appears to be a good molecular target to improve tolerance to abiotic stresses as well as to blast disease, which limit rice crop productivity.

Assessment of biodistribution using mesenchymal stromal cells: Algorithm for study design and challenges in detection methodologies.

BACKGROUND AIMS: Biodistribution of candidate cell-based therapeutics is a critical safety concern that must be addressed in the preclinical development program. We aimed to design a decision tree based on a series of studies included in actual dossiers approved by competent regulatory authorities, noting that the design, execution and interpretation of pharmacokinetics studies using this type of therapy is not straightforward and presents a challenge for both developers and regulators. METHODS: Eight studies were evaluated for the definition of a decision tree, in which mesenchymal stromal cells (MSCs) were administered to mouse, rat and sheep models using diverse routes (local or systemic), cell labeling (chemical or genetic) and detection methodologies (polymerase chain reaction [PCR], immunohistochemistry [IHC], fluorescence bioimaging, and magnetic resonance imaging [MRI]). Moreover, labeling and detection methodologies were compared in terms of cost, throughput, speed, sensitivity and specificity. RESULTS: A decision tree was defined based on the model chosen: (i) small immunodeficient animals receiving heterologous MSC products for assessing biodistribution and other safety aspects and (ii) large animals receiving homologous labeled products; this contributed to gathering data not only on biodistribution but also on pharmacodynamics. PCR emerged as the most convenient technique despite the loss of spatial information on cell distribution that can be further assessed by IHC. DISCUSSION: This work contributes to the standardization in the design of biodistribution studies by improving methods for accurate assessment of safety. The evaluation of different animal models and screening of target organs through a combination of techniques is a cost-effective and timely strategy.

Enhancing blast disease resistance by overexpression of the calcium-dependent protein kinase OsCPK4 in rice.

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium-dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops.

Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM)-derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use. METHODS: BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 ± 10 × 10(6) viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining. RESULTS: A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 ± 17.8 mL of BM containing 2.53 ± 0.92 × 10(9) viable NC were used as starting material, yielding 38.8 ± 5.3 × 10(6) viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases. DISCUSSION: Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use.

Evaluation of a cell-banking strategy for the production of clinical grade mesenchymal stromal cells from Wharton's jelly.

BACKGROUND AIMS: Umbilical cord (UC) has been proposed as a source of mesenchymal stromal cells (MSCs) for use in experimental cell-based therapies provided that its collection does not raise any risk to the donor, and, similar to bone marrow and lipoaspirates, UC-MSCs are multipotent cells with immuno-modulative properties. However, some of the challenges that make a broader use of UC-MSCs difficult include the limited availability of fresh starting tissue, time-consuming processing for successful derivation of cell lines, and the lack of information on identity, potency and genetic stability in extensively expanded UC-MSCs, which are necessary for banking relevant cell numbers for preclinical and clinical studies. METHODS: Factors affecting the success of the derivation process (namely, time elapsed from birth to processing and weight of fragments), and methods for establishing a two-tiered system of Master Cell Bank and Working Cell Bank of UC-MSCs were analyzed. RESULTS: Efficient derivation of UC-MSCs was achieved by using UC fragments larger than 7 g that were processed within 80 h from birth. Cells maintained their immunophenotype (being highly positive for CD105, CD90 and CD73 markers), multi-potentiality and immuno-modulative properties beyond 40 cumulative population doublings. No genetic abnormalities were found, as determined by G-banding karyotype, human telomerase reverse transcriptase activity was undetectable and no toxicity was observed in vivo after intravenous administration of UC-MSCs in athymic rats. DISCUSSION: This works demonstrates the feasibility of the derivation and large-scale expansion of UC-MSCs from small and relatively old fragments of UC typically discarded from public cord blood banking programs.

[Interventions for improving adherence to treatment in patients with multiple pathologies: overview of systematic reviews]. / Intervenciones para la mejora de la adherencia al tratamiento en pacientes pluripatológicos: resumen de revisiones sistemáticas.

OBJECTIVE: To assess the available scientific evidence regarding the efficacy of interventions aimed to enhance medication adherence in patients with multiple chronic conditions (PMCC). DESIGN: Overview of systematic reviews. DATA SOURCES: The following databases were consulted (September 2013): Pubmed, EMBASE, the Cochrane Library, CRD and WoS to identify interventions aimed to enhance medication adherence in PMCC, or otherwise, patients with chronic diseases common in the PMCC, or polypharmacy. STUDY SELECTION: Systematic reviews of clinical trials focused on PMCC or similar were included. They should compare the efficacy of any intervention aimed to improve compliance to prescribed and self-administered medications with clinical practice or other interventions. DATA EXTRACTION: Information about the study population, nature of intervention and efficacy in terms of improved adherence was extracted. RESULTS: 566 articles were retrieved of which 9 systematic reviews were included. None was specifically focused on PMCC but considered patients with chronic diseases common in the PMCC, patients with more than one chronic disease and polypharmacy. The overall effectiveness of interventions was modest without relevant differences between behavioural, educational and combined interventions. Some components of these interventions including patient counselling and regimen simplification appear to be effective tools in improving adherence in this population group. CONCLUSION: There is a large heterogeneity of interventions aimed to improve adherence with modest efficacy, none in PMCC.

Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation.

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage.

Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides.

There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 µM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories.

Production of cecropin A antimicrobial peptide in rice seed endosperm.

BACKGROUND: Cecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds. RESULTS: Transgenic rice plants expressing a codon-optimized synthetic cecropin A gene drived by an endosperm-specific promoter, either the glutelin B1 or glutelin B4 promoter, were generated. The signal peptide sequence from either the glutelin B1 or the glutelin B4 were N-terminally fused to the coding sequence of the cecropin A. We also studied whether the presence of the KDEL endoplasmic reticulum retention signal at the C-terminal has an effect on cecropin A subcellular localization and accumulation. The transgenic rice plants showed stable transgene integration and inheritance. We show that cecropin A accumulates in protein storage bodies in the rice endosperm, particularly in type II protein bodies, supporting that the glutelin N-terminal signal peptides play a crucial role in directing the cecropin A to this organelle, independently of being tagged with the KDEL endoplasmic reticulum retention signal. The production of cecropin A in transgenic rice seeds did not affect seed viability or seedling growth. Furthermore, transgenic cecropin A seeds exhibited resistance to infection by fungal and bacterial pathogens (Fusarium verticillioides and Dickeya dadantii, respectively) indicating that the in planta-produced cecropin A is biologically active. CONCLUSIONS: Rice seeds can sustain bioactive cecropin A production and accumulation in protein bodies. The system might benefit the production of this antimicrobial agent for subsequent applications in crop protection and food preservation.

[Pharmacological treatment conciliation methodology in patients with multiple conditions]. / Metodología de conciliación del tratamiento farmacológico en pacientes pluripatológicos.

OBJECTIVE: To carry out a bibliographic review in order to identify the different methodologies used along the reconciliation process of drug therapy applicable to polypathological patients. DESIGN: We performed a literature review. Data sources The bibliographic review (February 2012) included the following databases: Pubmed, EMBASE, CINAHL, PsycINFO and Spanish Medical Index (IME). The different methodologies, identified on those databases, to measure the conciliation process in polypathological patients, or otherwise elderly patients or polypharmacy, were studied. Study selection Two hundred and seventy three articles were retrieved, of which 25 were selected. Data extraction Specifically: the level of care, the sources of information, the use of registration forms, the established time, the medical professional in charge and the registered variables such as errors of reconciliation. RESULTS: Most of studies selected when the patient was admitted into the hospital and after the hospital discharge of the patient. The main sources of information to be highlighted are: the interview and the medical history of the patient. An established time is not explicitly stated on most of them, nor the registration form is used. The main professional in charge is the clinical pharmacologist. Apart from the home medication, the habits of self-medication and phytotherapy are also identified. The common errors of reconciliation vary from the omission of drugs to different forms of interaction with other medicinal products (drugs interactions). CONCLUSIONS: There is a large heterogeneity of methodologies used for reconciliation. There is not any work done on the specific figure of the polypathological patient, which precisely requires a standardized methodology due to its complexity and its susceptibility to errors of reconciliation.

[Suitability of pharmacological treatment in patients with multiple chronic conditions]. / Adecuación del tratamiento farmacológico en pacientes pluripatológicos.

OBJECTIVE: To identify tools for measuring the appropriateness of drug therapy useful in patients with multiple chronic conditions. DESIGN: We performed a literature review. DATA SOURCES: The following database were consulted (December 2009): Pubmed, EMBASE, CINAHL, PsycINFO and Spanish Medical Index (IME) to detect tools for measuring the appropriateness of treatment in patients with multiple chronic conditions, or otherwise elderly or polypharmacy. STUDY SELECTION: Studies were identified both qualitative and quantitative methodology, both theoretical and field work, both original and revised work and included work from all areas of the health system. 108 articles were retrieved, of which we selected 59. The consultation of their references include 20 jobs allowed, resulting in a total of 59 articles. DATA EXTRACTION: Of all the tools identified, the researchers performed a selection of those with possible utility for classified PP. The articles were classified into implicit and explicit methods and the characteristics of the field works were tabulated. RESULTS: We identified two implicit methods (MAI and Hamdy) and 6 explicit methods (Beers criteria, IPET, STOPP/START, ACOVE, CRIME and NORGEP). None was specific to patients with multiple chronic conditions. The questionnaire MAI, the Beers criteria and its modifications are most often used in literature. The advantages of explicit criteria means that many of them have been developed recently. CONCLUSION: There are several tools to measure the appropriateness and none of them has been designed for a population of patients with multiple chronic conditions yet, which by its nature requires a specific approach spreads.

Impact of the PPG Sampling Rate in the Pulse Rate Variability Indices Evaluating Several Fiducial Points in Different Pulse Waveforms.

The main aim of this work is to study the effect of the sampling rate of the photoplethysmographic (PPG) signal for pulse rate variability (PRV) analysis. Forehead and finger PPG signals were recorded at 1000 Hz during a rest state, with red and infrared wavelengths, simultaneously with the electrocardiogram (ECG). The PPG sampling rate has been reduced by decimation, obtaining signals at 500 Hz, 250 Hz, 125 Hz, 100 Hz, 50 Hz and 25 Hz. Five fiducial points were computed: apex, up-slope, medium, line-medium and medium interpolate point. The medium point is located in the middle of the up-slope of the pulse. The medium interpolate point is a new proposal as fiducial point that consider the abrupt up-slope of the PPG pulse, so it can be recovered by linear interpolation when the sampling rate is reduced. The error performed in the temporal location of the fiducial points was computed. Pulse period time interval series were obtained from all PPG signals and fiducial points, and compared with the RR intervals obtained from the ECG. Heart rate variability and PRV signals were estimated and classical time and frequency domain indices were computed. The results showed that the medium interpolate point of the PPG pulse was the most accurate fiducial point under different PPG morphologies and sensor locations, when sampling rate was reduced. Being able to reduce the sampling rate to 50 Hz without causing significant changes in time and frequency indices, when medium interpolate point was used as fiducial point.

Autonomic Nervous System characterization in hyperbaric environments considering respiratory component and non-linear analysis of Heart Rate Variability.

OBJECTIVES: an evaluation of Principal Dynamic Mode (PDM) and Orthogonal Subspace Projection (OSP) methods to characterize the Autonomic Nervous System (ANS) response in three different hyperbaric environments was performed. METHODS: ECG signals were recorded in two different stages (baseline and immersion) in three different hyperbaric environments: (a) inside a hyperbaric chamber, (b) in a controlled sea immersion, (c) in a real reservoir immersion. Time-domain parameters were extracted from the RR series of the ECG. From the Heart Rate Variability signal (HRV), classic Power Spectral Density (PSD), PDM (a non-linear analysis of HRV which is able to separate sympathetic and parasympathetic activities) and OSP (an analysis of HRV which is able to extract the respiratory component) methods were used to assess the ANS response. RESULTS: PDM and OSP parameters follows the same trend when compared to the PSD ones for the hyperbaric chamber dataset. Comparing the three hyperbaric scenarios, significant differences were found: i) heart rate decreased and RMSSD increased in the hyperbaric chamber and the controlled dive, but they had the opposite behavior during the uncontrolled dive; ii) power in the OSP respiratory component was lower than power in the OSP residual component in cases a and c; iii) PDM and OSP methods showed a significant increase in sympathetic activity during both dives, but parasympathetic activity increased only during the uncontrolled dive. CONCLUSIONS: PDM and OSP methods could be used as an alternative measurement of ANS response instead of the PSD method. OSP results indicate that most of the variation in the heart rate variability cannot be described by changes in the respiration, so changes in ANS response can be assigned to other factors. Time-domain parameters reflect vagal activation in the hyperbaric chamber and in the controlled dive because of the effect of pressure. In the uncontrolled dive, sympathetic activity seems to be dominant, due to the effects of other factors such as physical activity, the challenging environment, and the influence of breathing through the scuba mask during immersion. In sum, a careful description of the changes in all the possible factors that could affect the ANS response between baseline and immersion stages in hyperbaric environments is needed for better interpretation of the results.

AtCPK1 calcium-dependent protein kinase mediates pathogen resistance in Arabidopsis.

In mammals, lipid bodies play a key role during pathological and infectious diseases. However, our knowledge on the function of plant lipid bodies, apart from their role as the major site of lipid storage in seed tissues, remains limited. Here, we provide evidence that a calcium-dependent protein kinase (CPK) mediates pathogen resistance in Arabidopsis. AtCPK1 expression is rapidly induced by fungal elicitors. Loss-of-function mutants of AtCPK1 exhibit higher susceptibility to pathogen infection compared to wild-type plants. Conversely, over-expression of AtCPK1 leads to accumulation of salicylic acid (SA) and constitutive expression of SA-regulated defence and disease resistance genes, which, in turn, results in broad-spectrum protection against pathogen infection. Expression studies in mutants affected in SA-mediated defence responses revealed an interlocked feedback loop governing AtCPK1 expression and components of the SA-dependent signalling pathway. Moreover, we demonstrate the dual localization of AtCPK1 in lipid bodies and peroxisomes. Overall, our findings identify AtCPK1 as a component of the innate immune system of Arabidopsis plants.

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6.

Mitogen-activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen-induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR-like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.