Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model.

BACKGROUND: Nowadays, nanoparticles (NPs) have evolved as multifunctional systems combining different custom anchorages which opens a wide range of applications in biomedical research. Thus, their pharmacological involvements require more comprehensive analysis and novel nanodrugs should be characterized by both chemically and biological point of view. Within the wide variety of biocompatible nanosystems, iron oxide nanoparticles (IONPs) present mostly of the required features which make them suitable for multifunctional NPs with many biopharmaceutical applications. RESULTS: Cisplatin-IONPs and different functionalization stages have been broadly evaluated. The potential application of these nanodrugs in onco-therapies has been assessed by studying in vitro biocompatibility (interactions with environment) by proteomics characterization the determination of protein corona in different proximal fluids (human plasma, rabbit plasma and fetal bovine serum),. Moreover, protein labeling and LC-MS/MS analysis provided more than 4000 proteins de novo synthetized as consequence of the nanodrugs presence defending cell signaling in different tumor cell types (data available via ProteomeXchanges with identified PXD026615). Further in vivo studies have provided a more integrative view of the biopharmaceutical perspectives of IONPs. CONCLUSIONS: Pharmacological proteomic profile different behavior between species and different affinity of protein coating layers (soft and hard corona). Also, intracellular signaling exposed differences between tumor cell lines studied. First approaches in animal model reveal the potential of theses NPs as drug delivery vehicles and confirm cisplatin compounds as strengthened antitumoral agents.

Are Circulating Cytokines Reliable Biomarkers for Amyotrophic Lateral Sclerosis?

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that has no effective treatment. The lack of any specific biomarker that can help in the diagnosis or prognosis of ALS has made the identification of biomarkers an urgent challenge. Multiple panels have shown alterations in levels of numerous cytokines in ALS, supporting the contribution of neuroinflammation to the progressive motor neuron loss. However, none of them is fully sensitive and specific enough to become a universal biomarker for ALS. This review gathers the numerous circulating cytokines that have been found dysregulated in both ALS animal models and patients. Particularly, it highlights the opposing results found in the literature to date, and points out another potential application of inflammatory cytokines as therapeutic targets.

Pressurized hot water extraction of ß-glucans from Cantharellus tubaeformis.

Cantharellus tubaeformis was processed by pressurized hot water extraction (80-240°C) with the aim of maximizing the extraction of oligomeric fractions, ß-glucans, and the in vitro antioxidant properties of the extracts. Increased severity of treatment enhanced the extraction yields above 62% at temperatures of 210ºC or higher, corresponding to the maximum ß-glucan yields. The highest antioxidant capacity was obtained at 170ºC, although the highest content of phenolic compounds was obtained at the highest severity studied. This hydrothermal treatment can be considered a suitable process to obtain extracts with antioxidant properties and rich in ß-glucans.

Synthesis of Chiral 1,3-Dienes through Ring-Closing Metathesis of Enantioenriched Enynes: Potential Precursors of Morphane Analogs.

A simple methodology for the synthesis of enynes by indium mediated diastereoselective allylation of aromatic N-tert-butanesulfinylimines bearing alkenyl groups at ortho-position with allyl bromide has been developed. The addition of the allyl indium intermediate to the chiral imine took place with excellent diastereoselectivity. Ruthenium-catalyzed ring-closing metathesis of the resulting enynes provided the expected cyclic 1,3-dienes in good to moderate yields. These chiral dienes are potential precursors of biologically and pharmacologically active morphane derivatives.

Stereoselective Synthesis of 2-(2-Aminoalkyl)- and 1,3-Disubstituted Tetrahydro-1H-pyrido[4,3-b]- Benzofuran and Indole Derivatives.

The addition of an allenyl indium intermediate to chiral N-tert-butanesulfinyl imines 7 proceeds with high levels of diastereocontrol. The resulting homopropargylic amine derivatives 10 were transformed into 2-(2-aminoalkyl)benzofuran and indole derivatives 13 and 19, after Sonogashira coupling with o-iodophenol or o-iodoaniline, followed by formation of the heteroaromatic ring through an intramolecular cyclization. Enantioenriched tetrahydropyrido-benzofuran and indole derivatives 16 and 21 were prepared through a Pictet-Spengler condensation of the free amines derived from compounds 15 and 20, involving the nucleophilic 3-position of the benzofuran or indole moiety.

Identification of a gammaherpesvirus belonging to the malignant catarrhal fever group of viruses in Pyrenean chamois (Rupicapra p. pyrenaica).

High prevalence (46 %) of a gammaherpesvirus was confirmed by molecular detection in the lungs of hunted Pyrenean chamois. The partial glycoprotein B sequence up to the DNA polymerase gene showed 96.6 % nucleotide sequence identity to the Rupicapra rupicapra gammaherpesvirus 1 and 81.5 % to ovine herpesvirus 2. This novel sequence clusters within sequences derived from the malignant catarrhal fever group of viruses, and the corresponding virus is tentatively named Rupicapra pyrenaica gammaherpesvirus 1 (RpHV-1). No specific histological lesions were associated with RpHV-1, nor were any detrimental effects on host health. The epidemiological, phylogenetic and histopathological results suggest that Pyrenean chamois is the natural host of RpHV-1.

Modular framework to assess the risk of African swine fever virus entry into the European Union.

BACKGROUND: The recent occurrence and spread of African swine fever (ASF) in Eastern Europe is perceived as a serious risk for the pig industry in the European Union (EU). In order to estimate the potential risk of ASF virus (ASFV) entering the EU, several pathways of introduction were previously assessed separately. The present work aimed to integrate five of these assessments (legal imports of pigs, legal imports of products, illegal imports of products, fomites associated with transport and wild boar movements) into a modular tool that facilitates the visualization and comprehension of the relative risk of ASFV introduction into the EU by each analyzed pathway. RESULTS: The framework's results indicate that 48% of EU countries are at relatively high risk (risk score 4 or 5 out of 5) for ASFV entry for at least one analyzed pathway. Four of these countries obtained the maximum risk score for one pathway: Bulgaria for legally imported products during the high risk period (HRP); Finland for wild boar; Slovenia and Sweden for legally imported pigs during the HRP. Distribution of risk considerably differed from one pathway to another; for some pathways, the risk was concentrated in a few countries (e.g., transport fomites), whereas other pathways incurred a high risk for 4 or 5 countries (legal pigs, illegal imports and wild boar). CONCLUSIONS: The modular framework, developed to estimate the risk of ASFV entry into the EU, is available in a public domain, and is a transparent, easy-to-interpret tool that can be updated and adapted if required. The model's results determine the EU countries at higher risk for each ASFV introduction route, and provide a useful basis to develop a global coordinated program to improve ASFV prevention in the EU.

Seroprevalence of paramyxoviruses in synanthropic and semi-free-range birds.

Avian paramyxoviruses (APMVs) are classified into nine different serotypes (APMV 1-9). Virulent strains of APMV-1 are already well characterized as the etiologic agent of Newcastle disease (ND), an important disease in poultry that is potentially capable of infecting all orders of avian species. However, very little is known about the other eight serotypes, the majority of which can cause disease in domestic birds. The role of synanthropic and semi-free-range birds as reservoirs of avian paramyxoviruses is not well understood and the main objective of this work was to evaluate the seroprevalence of APMV 1-9 in these kind of birds. A total of 296 sera, oropharyngeal swabs, and cloacal enemas were collected from semi-free-range birds belonging to four different species: feral pigeons (Columba livia var. domestica), hybrid ducks (Anas sp.), domestic geese (Anser anser domesticus), and white storks (Ciconia ciconia). Antibodies against NDV were found in 56.3% of domestic geese, 42.9% of feral pigeons, and 30.4% of hybrid ducks. Antibodies for other APMVs (-3, -4, -6, -7, -8, -9) were also found. Seven positive individuals were positive to real-time RT-PCR detection, all of them feral pigeons captured in 2006 and 2007. The results obtained reinforce the idea that semi-free-range birds may be good sentinels for the detection of NDV and other avian paramyxoviruses.

Two cases of pseudohermaphroditism in loggerhead sea turtles Caretta caretta.

Two juvenile (curved carapace lengths: 28 and 30 cm) loggerhead sea turtles Caretta caretta with precocious male external characteristics were admitted to the ARCA del Mar rescue area at the Oceanogràfic Aquarium in Valencia, Spain, in 2009 and 2010. Routine internal laparoscopic examination and subsequent histopathology confirmed the presence of apparently healthy internal female gonads in both animals. Extensive tissue biopsy and hormone induction assays were consistent with female sex. To the best of our knowledge, this is the first report of pseudohermaphroditism in loggerhead sea turtles based on sexual external characteristics and internal laparoscopic examination. Our findings suggest that the practice of using external phenotypical characteristics as the basis for gender identification in sea turtles should be reevaluated. Future research should focus on detecting more animals with sexual defects and their possible effects on the sea turtle population.

Housekeeping gene expression in myogenic cell cultures from neurodegeneration and denervation animal models.

Reliability and accuracy of real-time quantitative PCR results depend on the use of housekeeping genes which must be constitutively expressed thorough the samples of the study. In the present work, we tested the expression stability of six candidate housekeeping genes (Actb, Rn18s, Gapdh, Hprt1, Sdha and B2m) considering sex, age, muscle-type and neurodegeneration or denervation status in mouse muscle satellite cells. Their expression varied under all variables tested; therefore the ranking of the most suitable genes for the normalization is modified depending on the factors included in the analysis, especially the age of the donor. Moreover, we describe the unsuitability of Rn18s in analysis comprising samples of different ages. On the other hand, we demonstrate that the use of the two best genes in each case is enough to obtain a reliable normalization factor. In this work, we give a broad information of the best housekeeping genes in mouse myogenic cells depending on the variables included in the experimental design.

Levels of membrane fluidity in the spinal cord and the brain in an animal model of amyotrophic lateral sclerosis.

A mutant form of the copper/zinc superoxide dismutase (SOD1) protein is found in some patients with amyotrophic lateral sclerosis (ALS). Alteration of the activity of this antioxidant enzyme leads to an oxidative stress imbalance, which damages the structure of lipids and proteins in the CNS. Using fluorescence spectroscopy, we monitored membrane fluidity in the spinal cord and the brain in a widely used animal model of ALS, the SOD(G93A) mouse, which develops symptoms similar to ALS with an accelerated course. Our results show that the membrane fluidity of the spinal cord in this animal model significantly decreased in symptomatic animals compared with age-matched littermate controls. To the best of our knowledge, this is the first report showing that membrane fluidity is affected in the spinal cord of a SOD(G93A) animal model of ALS. Changes in membrane fluidity likely contribute substantially to alterations in cell membrane functions in the nervous tissue from SOD(G93A) mice.

Compassionate Use of Rectal Ozone (O3) in Severe COVID-19 Pneumonia: a Case-Control Study.

Objectives: To evaluate effect of rectal ozone in severe COVID-19 pneumonia and to compare it to standard of care (SOC). Material and Methods: In a case-control study, 14 patients with severe bilateral COVID-19 pneumonia (positive RT-PCR), treated with SOC and rectal ozone, were evaluated before-and-after treatment and compared with SOC (14 patients) in a 10-day follow-up period. Ozone protocol consisted of 8 sessions (1 session/day) of intra-rectal ozone (150 mL volume, 35 µg/mL concentration [5.25mg total dose]). The SOC protocol included O2 supply, antivirals (Remdesivir), corticosteroids (Dexamethasone/Metilprednisolone), monoclonal antibodies (Anakinra/Tocilizumab), antibiotics (Azytromicine), and anticoagulants (Enoxaparine). Primary outcome variables were the following: (a) clinical (O2 saturation and O2 supply); (b) biochemical (lymphocyte count, fibrinogen, D-dimer, urea, ferritin, LDH, IL-6, and CRP); (c) radiological Taylor's scale. Secondary outcome variables were the following: (a) hospitalization length of stay, (b) mortality rate. Results: At baseline, ozone/SOC groups were not different on age, comorbidities, O2 saturation, and O2 supply. Patients in the ozone group improved O2 saturation and decrease O2 supply. SOC maintained O2 saturation and required more O2 supply. Lymphocyte count improved only in the ozone group and with statistical difference (p<0.05). Biomarkers of inflammation (fibrinogen, D-dimer, urea, LDH, CRP, and IL-6) decreased in both groups, but only significantly in favor of the ozone group (p<0.05). Ferritin showed a significant decrease in the ozone group but an increase on the SOC group. Radiological pneumonitis decreased on both groups but the decrease was only significant in the ozone group (p<0.0001). Mortality and length of stay, although not significant, were inferior in the ozone group. Conclusion: Compassionate use of rectal ozone improved O2 saturation, reduced O2 supply, decreased inflammation biomarkers, and improved Taylor's radiological scale significantly when compared to the SOC group. Mortality and length of stay were inferior in the ozone group, but this difference was not significant.

Autohydrolysis of Lentinus edodes for Obtaining Extracts with Antiradical Properties.

The autohydrolysis of Lentinus edodes was proposed for the extraction of components with antioxidant properties. Operation under non-isothermal conditions was evaluated and compared with isothermal heating. The influence of process severity was assessed in the range of 0.18 to 4.89 (temperature between 50 and 250 °C), up to 80% (d.b.) The influence of process severity during the autohydrolysis of Lentinus edodes was assessed in the range -0.3 to 4.89 (temperature between 50 and 250 °C). Up to 80% (d.b.) of the initial raw material could be solubilized at 210 °C. The different behavior of the saccharide and phenolic fractions was observed with the treatment temperature. Whereas the highest concentration of the saccharide components (mainly glucooligosaccharides) was found at 210 °C, the maximum phenolic yield was identified at 250 °C. The phenolic content and the antiradical properties of the extract showed a continuous increase with the temperature range studied, and at 250 °C, showed antiradical properties comparable to synthetic antioxidants. Autohydrolysis liquid fractions were used as solvents in the formulation of bioactive starch-based hydrogels, identifying a positive correlation between the gel softening and the extracts' bioactivity features.

Effect of Rectal Ozone (O3) in Severe COVID-19 Pneumonia: Preliminary Results.

The aim of this study is to evaluate the effectiveness of rectal ozone (O3) in COVID-19 patients with severe pneumonia admitted at Hospital Universitario Santa Cristina, Madrid. In a before-and-after study, four patients admitted with severe bilateral pneumonia due to COVID-19 were treated with rectal ozone and confirmed with (+) RT-PCR for SARS-CoV-2 and evaluated afterwards. The analyzed outcome variables were as follows: (a) clinical improvement (O2 saturation and O2 supply); (b) biochemical improvement (fibrinogen, D-dimer, urea, ferritin, LDH, IL-6, and CRP); (c) radiological improvement. The treatment protocol consisted of 5 sessions (1 session/day) of intra-rectal ozone, applied in a volume of 100 mL and a concentration of 35 µg/mL. The Protocol was previously approved by the Hospital's Health Care Ethics Committee (CEAS) (Report 15/4/2020) for compassionate use in the face of this exceptional pandemic situation, and prior informed consent was obtained from the patient/legal representative. The patients improved oxygen saturation, as observed by the lower number of desaturations and the lower supply of O2. Biomarkers of inflammation decreased (fibrinogen, D-dimer, urea, ferritin, LDH, IL-6, and CRP). Finally, the radiological signs of bilateral viral pneumonitis improved between 1 and 2 grades based on Taylor's radiological scale. Rectal ozone decreases O2 supply and improves O2 saturation, decreases inflammation biomarkers, and improves Taylor's radiological grade in patients with severe COVID-19 pneumonia. Rectal ozone is a safe, effective, cheap, and simple alternative capable of acting on the SARS-CoV-2 virus, and it is presented as an adjunctive therapeutic option to consider in the management of severe bilateral COVID-19 pneumonia.

Recovery of bioactive and gelling extracts from edible brown seaweed Laminaria ochroleuca by non-isothermal autohydrolysis.

The non-isothermal autohydrolysis temperature impact of edible brown seaweed Laminaria ochroleuca was studied to recover high valuable compounds. Extraction yield was determined, above 80% was obtained at 220 °C. The maximal fucose content (17% d.b.) was attained at 180 °C, whereas the maximal sulphate was achieved at 160 °C, and phenolic and protein content at 220 °C. The maximum sulphated fucoidan content (41.38 g fucoidan/100 g extract) was obtained at 160 °C, whereas the maximum fucose oligosaccharides was obtained at 180 °C. The antioxidant capacity was equivalent to 32 mg Trolox/g dry extract produced at 220 °C. The milder processing condition was selected to study the potentiality of the precipitated alginate in terms of viscoelastic properties determined by rheology. Alginate extraction (14.94 g/100 g extract) was determined at 160 °C. The crude fucoidan fractions were tested at 25-500 µg/mL, showed up to 50% cell growth inhibition in four selected tumoral cell lines.

Recent developments on the extraction and application of ursolic acid. A review.

Ursolic acid (UA) is a pentacyclic triterpenoid widely found in herbs, leaves, flowers and fruits; update information on the major natural sources or agro-industrial wastes is presented. Traditional (maceration, Soxhlet and heat reflux) and modern (microwave-, ultrasound-, accelerated solvent- and supercritical fluid) extraction and purification technologies of UA, as well as some patented process, are summarized. The great interest in this bioactive compound is related to the beneficial effects in human health due to antioxidant, antimicrobial, anti-inflammatory, hepatoprotective, immunomodulatory, anti-tumor, chemopreventive, cardioprotective, antihyperlipidemic and hypoglycemic activities, and others. UA may augment the resistance of the skin barrier to irritants, prevent dry skin and could be suitable to develop antiaging products. The development of nanocrystals and nanoparticle-based drugs could reduce the side effects of high doses of UA in organisms, and increase its limited solubility and poor bioavailability of UA which limit the potential of this bioactive and the further applications. Commercial patented applications in relation to cosmetical and pharmaceutical uses of UA and its derivatives are surveyed.

Algae Polysaccharides' Chemical Characterization and their Role in the Inflammatory Process.

Polysaccharides are abundant components in marine macroalgae with potential applications in different areas such as pharmaceutical, biomedical, cosmetics and nutrition. The current interest in these compounds is due to their known bioactivities, conferred by their antiallergic, neuroprotective, gastroprotective, cardioprotective, cytotoxic, anticoagulant/antithrombotic, antiviral, antilipidemic, antinociceptive, and immunomodulatory properties, making them promising bioactive products and biomaterials. The inflammatory process is a complex event mediated by the immune system that culminates in the neutralization and elimination of the offending insult protecting the host and restoring tissue homeostasis. This review focuses on the extraction procedures and chemical characterization of polysaccharides from different classes of algae (Phaeophyceae, Rhodophyceae and Chlorophyceae) and on the studies on their inflammatory process modulatory effect.

Importance of Ecological Factors and Colony Handling for Optimizing Health Status of Apiaries in Mediterranean Ecosystems.

We analyzed six apiaries in several natural environments with a Mediterranean ecosystem in Madrid, central Spain, in order to understand how landscape and management characteristics may influence apiary health and bee production in the long term. We focused on five criteria (habitat quality, landscape heterogeneity, climate, management and health), as well as 30 subcriteria, and we used the analytic hierarchy process (AHP) to rank them according to relevance. Habitat quality proved to have the highest relevance, followed by beehive management. Within habitat quality, the following subcriteria proved to be most relevant: orographic diversity, elevation range and important plant species located 1.5 km from the apiary. The most important subcriteria under beehive management were honey production, movement of the apiary to a location with a higher altitude and wax renewal. Temperature was the most important subcriterion under climate, while pathogen and Varroa loads were the most significant under health. Two of the six apiaries showed the best values in the AHP analysis and showed annual honey production of 70 and 28 kg/colony. This high productivity was due primarily to high elevation range and high orographic diversity, which favored high habitat quality. In addition, one of these apiaries showed the best value for beehive management, while the other showed the best value for health, reflected in the low pathogen load and low average number of viruses. These results highlight the importance of environmental factors and good sanitary practices to maximize apiary health and honey productivity.

An in vitro study of hematopoietic progenitor cells from peripheral blood of rabbits.

The purpose of this study was to isolate and cultivate a subpopulation of pluripotent stem cells (PSCs) from the peripheral blood of rabbits, which are frequently used in veterinary research as an animal model. Pluripotent stem cells, as described in human beings, are fibroblast-like cells that exhibit a CD34 marker, specific from other hematopoietic stem cells. Commonly used human commercial media has been researched for culturing rabbit PSCs. These findings allow us to contemplate the direct application of this simple and standardized methodology in several areas of study, such as of the pharmacological effect of many drugs on hematopoietic cells, veterinary practice, and even the study of new strategies in cellular therapy for some human diseases.

Time-Point Dependent Activation of Autophagy and the UPS in SOD1G93A Mice Skeletal Muscle.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.