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.

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.

Use of Multipotent Mesenchymal Stromal Cells, Fibrin, and Scaffolds in the Production of Clinical Grade Bone Tissue Engineering Products.

Tissue engineering products (TEP) are a new type of medicines resulting from the combination of cells, scaffolds, and/or signalling factors, which can be used for the regeneration of damaged tissues thus opening new avenues for the treatment of complex conditions. However, such combination of biologically active elements, particularly living cells, poses an unprecedented challenge for their production under pharmaceutical standards.In the methods presented here, we formulated two types of TEP based on the use of multipotent mesenchymal stromal cells with osteogenic potential combined with osteoinductive and osteoconductive bony particles from tissue bank embedded in a fibrin hydrogel that, altogether, can induce the generation of new tissue while adapting to the diverse architecture of bony defects. In agreement with pharmaceutical quality and regulatory requirements, procedures presented herein can be performed in compliance with current good manufacturing practices and be readily implemented in straightforward facilities at hospitals and academic institutions.

Photoplethysmographic Waveform and Pulse Rate Variability Analysis in Hyperbaric Environments.

The main aim of this work is to identify alterations in the morphology of the pulse photoplethysmogram (PPG) signal, due to the exposure of the subjects to a hyperbaric environment. Additionally, their Pulse Rate Variability (PRV) is analysed to characterise the response of their Autonomic Nervous System (ANS). To do that, 28 volunteers are introduced into a hyperbaric chamber and five sequential stages with different atmospheric pressures from 1 atm to 5 atm are performed. In this work, nineteen morphological parameters of the PPG signal are analysed: the pulse amplitude; eight parameters related to pulse width; eight parameters related to pulse area; and the two two pulse slopes. Also, classical time and frequency parameters of PRV are computed. Notable widening of the pulses width is observed in the stages analysed. The PPG area increases with pressure, with no significant changes when the initial pressure is recovered. These changes in PPG waveform may be caused by an increase in the systemic vascular resistance as a consequence of of vasoconstriction in the extremities, suggesting a sympathetic activation. However, the PRV results show an augmented parasympathetic activity and a reduction in the parameters that characterise the sympathetic response. So, only a sympathetic activation is detected in the peripheral region, as reflected by PPG morphology. The information regarding the ANS and the cardiovascular response that can be extracted from the PPG signal, as well as its compatibility with wet conditions make this signal the most suitable for studying the physiological response in hyperbaric environments.

The Antifungal Protein AfpB Induces Regulated Cell Death in Its Parental Fungus Penicillium digitatum.

Filamentous fungi produce small cysteine-rich proteins with potent, specific antifungal activity, offering the potential to fight fungal infections that severely threaten human health and food safety and security. The genome of the citrus postharvest fungal pathogen Penicillium digitatum encodes one of these antifungal proteins, namely AfpB. Biotechnologically produced AfpB inhibited the growth of major pathogenic fungi at minimal concentrations, surprisingly including its parental fungus, and conferred protection to crop plants against fungal infections. This study reports an in-depth characterization of the AfpB mechanism of action, showing that it is a cell-penetrating protein that triggers a regulated cell death program in the target fungus. We prove the importance of AfpB interaction with the fungal cell wall to exert its killing activity, for which protein mannosylation is required. We also show that the potent activity of AfpB correlates with its rapid and efficient uptake by fungal cells through an energy-dependent process. Once internalized, AfpB induces a transcriptional reprogramming signaled by reactive oxygen species that ends in cell death. Our data show that AfpB activates a self-injury program, suggesting that this protein has a biological function in the parental fungus beyond defense against competitors, presumably more related to regulation of the fungal population. Our results demonstrate that this protein is a potent antifungal that acts through various targets to kill fungal cells through a regulated process, making AfpB a promising compound for the development of novel biofungicides with multiple fields of application in crop and postharvest protection, food preservation, and medical therapies.IMPORTANCE Disease-causing fungi pose a serious threat to human health and food safety and security. The limited number of licensed antifungals, together with the emergence of pathogenic fungi with multiple resistance to available antifungals, represents a serious challenge for medicine and agriculture. Therefore, there is an urgent need for new compounds with high fungal specificity and novel antifungal mechanisms. Antifungal proteins in general, and AfpB from Penicillium digitatum in particular, are promising molecules for the development of novel antifungals. This study on AfpB's mode of action demonstrates its potent, specific fungicidal activity through the interaction with multiple targets, presumably reducing the risk of evolving fungal resistance, and through a regulated cell death process, uncovering this protein as an excellent candidate for a novel biofungicide. The in-depth knowledge on AfpB mechanistic function presented in this work is important to guide its possible future clinical and agricultural applications.

First-in-human PeriCord cardiac bioimplant: Scalability and GMP manufacturing of an allogeneic engineered tissue graft.

BACKGROUND: Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation. METHODS: The PeriCord is a clinical-size (12-16 cm2) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth. FINDINGS: PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12·5 × 106 viable WJ-MSCs (85·4% cell viability; <0·51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area. INTERPRETATION: This preliminary report describes the development of a scalable clinical-size allogeneic PeriCord cardiac bioimplant, and its first-in-human implantation. FUNDING: La Marató de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, "La Caixa" Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund.

Biotechnological Production of the Cell Penetrating Antifungal PAF102 Peptide in Pichia pastoris.

Antimicrobial peptides (AMPs) have potent and durable antimicrobial activity to a wide range of fungi and bacteria. The growing problem of drug-resistant pathogenic microorganisms, together with the lack of new effective compounds, has stimulated interest in developing AMPs as anti-infective molecules. PAF102 is an AMP that was rationally designed for improved antifungal properties. This cell penetrating peptide has potent and specific activity against major fungal pathogens. Cecropin A is a natural AMP with strong and fast lytic activity against bacterial and fungal pathogens, including multidrug resistant pathogens. Both peptides, PAF102 and Cecropin A, are alternative antibiotic compounds. However, their exploitation requires fast, cost-efficient production systems. Here, we developed an innovative system to produce AMPs in Pichia pastoris using the oleosin fusion technology. Oleosins are plant-specific proteins with a structural role in lipid droplet formation and stabilization, which are used as carriers for recombinant proteins to lipid droplets in plant-based production systems. This study reports the efficient production of PAF102 in P. pastoris when fused to the rice plant Oleosin 18, whereas no accumulation of Cecropin A was detected. The Ole18-PAF102 fusion protein targets the lipid droplets of the heterologous system where it accumulates to high levels. Interestingly, the production of this fusion protein induces the formation of lipid droplets in yeast cells, which can be additionally enhanced by the coexpression of a diacylglycerol transferase gene that allows a three-fold increase in the production of the fusion protein. Using this high producer strain, PAF102 reaches commercially relevant yields of up to 180 mg/l of yeast culture. Moreover, the accumulation of PAF102 in the yeast lipid droplets facilitates its downstream extraction and recovery by flotation on density gradients, with the recovered PAF102 being biologically active against pathogenic fungi. Our results demonstrate that plant oleosin fusion technology can be transferred to the well-established P. pastoris cell factory to produce the PAF102 antifungal peptide, and potentially other AMPs, for multiple applications in crop protection, food preservation and animal and human therapies.

Rice Seeds as Biofactories of Rationally Designed and Cell-Penetrating Antifungal PAF Peptides.

PAFs are short cationic and tryptophan-rich synthetic peptides with cell-penetrating antifungal activity. They show potent and selective killing activity against major fungal pathogens and low toxicity to other eukaryotic and bacterial cells. These properties make them a promising alternative to fulfill the need of novel antifungals with potential applications in crop protection, food preservation, and medical therapies. However, the difficulties of cost-effective manufacturing of PAFs by chemical synthesis or biotechnological production in microorganisms have hampered their development for practical use. This work explores the feasibility of using rice seeds as an economical and safe production system of PAFs. The rationally designed PAF102 peptide with improved antifungal properties was selected for assessing PAF biotechnological production. Two different strategies are evaluated: (1) the production as a single peptide targeted to protein bodies and (2) the production as an oleosin fusion protein targeted to oil bodies. Both strategies are designed to offer stability to the PAF peptide in the host plant and to facilitate its downstream purification. Our results demonstrate that PAF does not accumulate to detectable levels in rice seeds when produced as a single peptide, whereas it is successfully produced as fusion protein to the Oleosin18, up to 20 µg of peptide per gram of grain. We show that the expression of the chimeric Ole18-PAF102 gene driven by the Ole18 promoter results in the specific accumulation of the fusion protein in the embryo and aleurone layer of the rice seed. Ole18-PAF102 accumulation has no deleterious effects on seed yield, germination capacity, or seedling growth. We also show that the Oleosin18 protein serves as carrier to target the fusion protein to oil bodies facilitating PAF102 recovery. Importantly, the recovered PAF102 is active against the fungal phytopathogen Fusarium proliferatum. Altogether, our results prove that the oleosin fusion technology allows the production of PAF bioactive peptides to assist the exploitation of these antifungal compounds.

Photoplethysmographic Waveform in Hyperbaric Environment.

The objective of this work is the identification of significant variations of morphological parameters of the photoplethysmographic (PPG) signal when the subjects are exposed to an increase in atmospheric pressure. To achieve this goal, the PPG signal of 26 subjects, exposed to a hyperbaric environment whose pressure increases up to 5 atm, has been recorded. From this record, segments of 4 minutes have been processed at 1 atm, 3 atm and 5 atm, both in the descending (D) and ascending (A) periods of the immersion. In total, four states (3D, 5, 3A and 1A) normalized to the basal state (1D) have been considered. In these segments, six morphological parameters of the PPG signal were studied. The width, the amplitude, the widths of the anacrotic and catacrotic phases, and the upward and downward slopes of each PPG pulse were extracted. The results showed significant increases in the three parameters related to the pulse width. This increase is significant in the four states analysed for the anacrotic phase width. Furthermore, a significant decrease in the amplitude and in both slopes (in the states 1A) was observed. These results show that the PPG width responds rapidly to the increase in pressure, indicating an activation of the sympathetic system, while amplitude and pulse slopes are decreased when the subjects are exposed to the hyperbaric environment for a considerable period of time.

Multivariable relationships between autonomic nervous system related indices in hyperbaric environments.

The main aim of this work is to model the relationships between parameters extracted from the heart rate variability (HRV) signal, which is derived from the electrocardiogram (ECG), at different stages of a simulated immersion in a hyperbaric chamber. The response of the Autonomic Nervous System is known to be affected by changes in atmospheric pressure, reflected in changes in the HRV signal. A dataset consisting of ECG signals from 17 subjects exposed to a controlled hyperbaric environment, simulating depths from 0 m to 40 m, was used. Both linear and nonlinear dependences of HRV parameters were analysed using linear regression and Mutual Information (entropy-based) techniques. Furthermore, relationships between parameters of the HRV signals, biophysical variables of the subjects, and atmospheric pressure changes were characterized by artificial neural networks. In particular, self-organizing maps (SOM) were trained for modelling and clustering all the data. In the mid-term, these models could be the basis to create predictive models of HRV parameters at high depths in order to increase the safety for divers by warning them if some abnormal body response could be expected just by processing the ECG signal at sea level before immersion.

Autonomic Nervous System Measurement in Hyperbaric Environments Using ECG and PPG Signals.

The main aim of this paper was to characterize the Autonomic Nervous System response in hyperbaric environments using electrocardiogram (ECG) and pulse-photoplethysmogram (PPG) signals. To that end, 26 subjects were introduced into a hyperbaric chamber and five stages with different atmospheric pressures (1 atm; descent to 3 and 5 atm; ascent to 3 and 1 atm) were recorded. Respiratory information was extracted from the ECG and PPG signals and a combined respiratory rate was studied. This information was also used to analyze Heart Rate Variability (HRV) and Pulse Rate Variability (PRV). The database was cleaned by eliminating those cases where the respiratory rate dropped into the low frequency band (LF: 0.04-0.15 Hz) and those in which there was a discrepancy between the respiratory rates estimated using the ECG and PPG signals. Classical temporal and frequency indices were calculated in such cases. The ECG results showed a time-related dependency, with the heart rate and sympathetic markers (normalized power in LF and LF/HF ratio) decreasing as more time was spent inside the hyperbaric environment. A dependence between the atmospheric pressure and the parasympathetic response, as reflected in the high-frequency band power (HF: 0.15-0.40 Hz), was also found, with power increasing with atmospheric pressure. The combined respiratory rate also reached a maximum in the deepest stage; thus, highlighting a significant difference between this stage and the first one. The PPG data gave similar findings and also allowed the oxygen saturation to be computed; therefore, we propose the use of this signal for future studies in hyperbaric environments.

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.

Three Antifungal Proteins From Penicillium expansum: Different Patterns of Production and Antifungal Activity.

Antifungal proteins of fungal origin (AFPs) are small, secreted, cationic, and cysteine-rich proteins. Filamentous fungi encode a wide repertoire of AFPs belonging to different phylogenetic classes, which offer a great potential to develop new antifungals for the control of pathogenic fungi. The fungus Penicillium expansum is one of the few reported to encode three AFPs each belonging to a different phylogenetic class (A, B, and C). In this work, the production of the putative AFPs from P. expansum was evaluated, but only the representative of class A, PeAfpA, was identified in culture supernatants of the native fungus. The biotechnological production of PeAfpB and PeAfpC was achieved in Penicillium chrysogenum with the P. chrysogenum-based expression cassette, which had been proved to work efficiently for the production of other related AFPs in filamentous fungi. Western blot analyses confirmed that P. expansum only produces PeAfpA naturally, whereas PeAfpB and PeAfpC could not be detected. From the three AFPs from P. expansum, PeAfpA showed the highest antifungal activity against all fungi tested, including plant and human pathogens. P. expansum was also sensitive to its self-AFPs PeAfpA and PeAfpB. PeAfpB showed moderate antifungal activity against filamentous fungi, whereas no activity could be attributed to PeAfpC at the conditions tested. Importantly, none of the PeAFPs showed hemolytic activity. Finally, PeAfpA was demonstrated to efficiently protect against fungal infections caused by Botrytis cinerea in tomato leaves and Penicillium digitatum in oranges. The strong antifungal potency of PeAfpA, together with the lack of cytotoxicity, and significant in vivo protection against phytopathogenic fungi that cause postharvest decay and plant diseases, make PeAfpA a promising alternative compound for application in agriculture, but also in medicine or food preservation.

Efficient production and characterization of the novel and highly active antifungal protein AfpB from Penicillium digitatum.

Filamentous fungi encode distinct antifungal proteins (AFPs) that offer great potential to develop new antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum, the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene (afpB), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P. digitatum strains. Biotechnological production and secretion of AfpB were achieved in P. digitatum with the use of a P. chrysogenum-based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed antifungal activity higher than the P. chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P. digitatum, which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi.

Preservación de la fertilidad en cáncer de mama: caso clínico / Preservation of fertility in breast cancer: a clinical case

El cáncer de mama es la neoplasia más frecuente en la mujer. Actualmente estas pacientes tienen altas tasas de supervivencia, aunque derivado del tratamiento quimioterápico sufren riesgo de fallo ovárico prematuro y/o pérdida de reserva folicular. La preservación de la fertilidad debe ser ofertada a las pacientes oncológicas jóvenes con deseos genésicos. La técnica de elección es la vitrificación de ovocitos, ya que se trata de una técnica consolidada y se evita la generación de embriones con un futuro incierto (AU)

Breast cancer is the most frequent tumor in women; nowadays these patients have high survival rates, although due to chemotherapy they are at risk of premature ovary failure. Preservation of fertility should be offered to young oncological patients who desire to have children. The elective technique is the oocyte vitrification, since it is considered an established technique and it avoids the creation of embryos with unknown future (AU)

Guía metodológica para la evaluación de la eficacia y la seguridad de nuevos fármacos: implementación de las recomendaciones de EUnetHTA / Methodological guideline for the efficacy and safety assessment of new pharmaceuticals: implementation of EUnetHTA's recommendations

La colaboración European Network for Health Technology Assessment (EUnetHTA) es la red colaborativa de agencias y organismos públicos de evaluación de tecnologías sanitarias de la Unión Europea. En este marco se han elaborado guías metodológicas y procedimientos comunes que han dado lugar al denominado HTA Core Model®. La Agencia de Evaluación de Tecnologías Sanitarias de Andalucía (AETSA), miembro de la Red Española de Agencias de Evaluación de Tecnologías Sanitarias y Prestaciones del Sistema Nacional de Salud, y de la colaboración EUnetHTA, participa en la recién iniciada Tercera Acción Conjunta (Joint Action 3) de EUnetHTA (2016-2019). Adicionalmente, la AETSA cuenta con una línea de evaluación de medicamentos. Parte del trabajo se integra en la elaboración de informes de posicionamiento terapéutico (IPT) sobre fármacos que han recibido recientemente la autorización de comercialización, que coordina la Agencia Española de Medicamentos y Productos Sanitarios. Como apoyo a este trabajo, la AETSA elabora «Informes de síntesis de evidencia: medicamentos», en los que se realiza una evaluación comparada de la eficacia y la seguridad de los fármacos de los que va a elaborarse un IPT. La AETSA ha diseñado un proceso para la elaboración de dichos informes, basado en el HTA Core Model® y en las guías metodológicas de EUnetHTA. En este trabajo se describe la metodología empleada en la elaboración de la guía realizada por la AETSA para la elaboración de estos informes y se presentan los distintos apartados en los que esta se estructura (AU)

The European network for Health Technology Assessment (EUnetHTA) is the network of public health technology assessment (HTA) agencies and entities from across the EU. In this context, the HTA Core Model®, has been developed. The Andalusian Agency for Health Technology Assessment (AETSA) is a member of the Spanish HTA Network and EUnetHTA collaboration In addition, AETSA participates in the new EUnetHTA Joint Action 3 (JA, 2016–2019). Furthermore, AETSA works on pharmaceutical assessments. Part of this work involves drafting therapeutic positioning reports (TPRs) on drugs that have recently been granted marketing authorisation, which is overseen by the Spanish Agency of Medicines and Medical Devices (AEMPS). AETSA contributes by drafting ‘Evidence synthesis reports: pharmaceuticals’ in which a rapid comparative efficacy and safety assessment is performed for drugs for which a TPR will be created. To create this type of report, AETSA follows its own methodological guideline based on EUnetHTA guidelines and the HTA Core Model®. In this paper, the methodology that AETSA has developed to create the guideline for ‘Evidence synthesis reports: pharmaceuticals’ is described. The structure of the report itself is also presented (AU)

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.