Short- and mid-term morbidity and primary-care burden due to infant respiratory syncytial virus infection: A Spanish 6-year population-based longitudinal study.

BACKGROUND: The morbidity burden of respiratory syncytial virus (RSV) in infants extends beyond hospitalization. Defining the RSV burden before implementing prophylaxis programs is essential for evaluating any potential impact on short- to mid-term morbidity and the utilization of primary healthcare (PHC) and emergency services (ES). We established this reference data using a population-based cohort approach. METHODS: Infants hospitalized for RSV from January 2016 to March 2023 were matched with non-hospitalized ones based on birthdate and sex. We defined the exposure as severe RSV hospitalization. The main study outcomes were as follows: (1) PHC and ES visits for RSV, categorized using the International Classification of Primary Care codes, (2) prescriptions for respiratory airway obstructive disease, and (3) antibacterial prescriptions. Participants were followed up from 30 days before hospitalization for severe RSV until the outcome occurrence or end of the study. Adjusted incidence rate ratios (IRRs) of the outcomes along with their 95% confidence intervals (CI) were estimated using Poisson regression models. Stratified analyses by type of PHC visit (nurse, pediatrician, or pharmacy) and follow-up period were undertaken. We defined mid-term outcomes as those taking place up to 24 months of follow-up period. RESULTS: The study included 6626 children (3313 RSV-hospitalized; 3313 non-hospitalized) with a median follow-up of 53.7 months (IQR = 27.9, 69.4). After a 3-month follow-up, severe RSV was associated with a considerable increase in PHC visits for wheezing/asthma (IRR = 4.31, 95% CI: 3.84-4.84), lower respiratory infections (IRR = 4.91, 95% CI: 4.34-5.58), and bronchiolitis (IRR = 4.68, 95% CI: 2.93-7.65). Severe RSV was also associated with more PHC visits for the pediatrician (IRR = 2.00, 95% CI: 1.96-2.05), nurse (IRR = 1.89, 95% CI: 1.75-1.92), hospital emergency (IRR = 2.39, 95% CI: 2.17-2.63), primary healthcare emergency (IRR: 1.54, 95% CI: 1.31-1.82), as well as with important increase in prescriptions for obstructive airway diseases (IRR = 5.98, 95% CI: 5.43-6.60) and antibacterials (IRR = 4.02, 95% CI: 3.38-4.81). All findings remained substantial until 2 years of post-infection. CONCLUSIONS: Severe RSV infection in infants significantly increases short- to mid-term respiratory morbidity leading to an escalation in healthcare utilization (PHC/ES attendance) and medication prescriptions for up to 2 years afterward. Our approach could be useful in assessing the impact and cost-effectiveness of RSV prevention programs.

Artificial intelligence-assisted identification and retrieval of chronic hepatitis C patients lost to follow-up in the health area of Pontevedra and O Salnés (Spain).

OBJECTIVE: Direct-acting antivirals (DAAs) to treat hepatitis C virus (HCV) infection offer an opportunity to eliminate the disease. This study aimed to identify and relink to care HCV patients previously lost to medical follow-up in the health area of Pontevedra and O Salnés (Spain) using an artificial intelligence-assisted system. PATIENTS AND METHODS: Active retrospective search of previously diagnosed HCV cases recorded in the Galician Health Service proprietary health information exchange database using the Herramientas para la EXplotación de la INformación (HEXIN) application. RESULTS AND CONCLUSIONS: Out of 99 lost patients identified, 64 (64.6%) were retrieved. Of these, 62 (96.88%) initiated DAA treatment and 54 patients (87.1%) achieved a sustained virological response. Mean time from HCV diagnosis was over 10 years. Main reasons for loss to follow-up were fear of possible adverse effects of treatment (30%) and mobility impediments (21%). Among the retrieved patients, almost one in three presented advanced liver fibrosis (F3) or cirrhosis (F4) at evaluation. In sum, HCV patients lost to follow-up can be retrieved by screening past laboratory records. This strategy promotes the achievement of HCV elimination goals.

COVID-19 vaccine effectiveness in children by age groups. A population-based study in Galicia, Spain.

BACKGROUND: Studies on vaccine effectiveness (VE) against COVID-19 in the pediatric population are outgoing. We aimed to quantify VE against SARS-CoV-2 in two pediatric age groups, 5-11 and 12-17-year-old, while considering vaccine type, SARS-CoV-2 variant, and duration of protection. METHODS: A population-based test-negative control study was undertaken in Galicia, Spain. Children 5-11-year-old received the Comirnaty® (Pfizer, US) vaccine, while those aged 12-17-year-old received the Comirnaty® (Pfizer, US) or SpikeVax® (ModernaTX, Inc) vaccine. Participants were categorized into unvaccinated (0 doses or one dose with <14 days since vaccination), partially vaccinated (only one dose with ≥14 days, or two doses with <14 days after the second dose administration), and fully vaccinated (two doses with ≥14 days after the second injection). Adjusted odds ratios (OR) and their 95% confidence intervals (CI) were estimated using multiple logistic regression models. VE was calculated as (1-OR) * 100. Stratified and sensitivity analyses were performed. RESULTS: In the fully vaccinated 5-11-year-old children, VE against the Omicron variant was 44.1% (95% CI: 38.2%-49.4%). In the fully vaccinated 12-17-year-old individuals, VE was 83.4% (95% CI: 81.2%-85.3%) against Delta and 74.8% (95% CI: 58.5%-84.9%) against Omicron. Comirnaty® and SpikeVax® vaccines showed a similar magnitude of VE against Delta [Comirnaty® VE: 81.9% (95% CI: 79.3%-84.1%) and SpikeVax® VE: 85.3% (95% CI: 81.9%-88.1%)]. Comirnaty® (Pfizer, US; VE: 79.7%; 95% CI: 50.7%-92.4%) showed a slightly higher magnitude of protection against Omicron than SpikeVax® (ModernaTX, Inc), yet with an overlapping CI (VE: 74.3%; 95% CI: 56.6%-84.9%). VE was maintained in all age subgroups in both pediatric populations, but it declined over time. CONCLUSIONS: In Galicia, mRNA VE was moderate against SARS-CoV-2 infections in the 5-11-year-old populations, but high in older children. VE declined over time, suggesting a potential need for booster dose schedules.

Advances in Platelet Rich Plasma-Derived Extracellular Vesicles for Regenerative Medicine: A Systematic-Narrative Review.

The use of platelet-rich plasma (PRP) has gained increasing interest in recent decades. The platelet secretome contains a multitude of growth factors, cytokines, chemokines, and other biological biomolecules. In recent years, developments in the field of platelets have led to new insights, and attention has been focused on the platelets' released extracellular vesicles (EVs) and their role in intercellular communication. In this context, the aim of this review was to compile the current evidence on PRP-derived extracellular vesicles to identify the advantages and limitations fortheir use in the upcoming clinical applications. A total of 172 articles were identified during the systematic literature search through two databases (PubMed and Web of Science). Twenty publications met the inclusion criteria and were included in this review. According to the results, the use of PRP-EVs in the clinic is an emerging field of great interest that represents a promising therapeutic option, as their efficacy has been demonstrated in the majority of fields of applications included in this review. However, the lack of standardization along the procedures in both the field of PRP and the EVs makes it extremely challenging to compare results among studies. Establishing standardized conditions to ensure optimized and detailed protocols and define parameters such as the dose or the EV origin is therefore urgent. Further studies to elucidate the real contribution of EVs to PRP in terms of composition and functionality should also be performed. Nevertheless, research on the field provides promising results and a novel basis to deal with the regenerative medicine and drug delivery fields in the future.

microRNA-based signatures obtained from endometrial fluid identify implantative endometrium.

STUDY QUESTION: Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? SUMMARY ANSWER: The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY: miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. STUDY DESIGN, SIZE, DURATION: In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS: The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. LARGE SCALE DATA: The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION: One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. WIDER IMPLICATIONS OF THE FINDINGS: We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. STUDY FUNDING/COMPETING INTEREST(S): J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.

Effectiveness of COVID-19 vaccine booster in the general population and in subjects with comorbidities. A population-based study in Spain.

BACKGROUND: Research on the effectiveness of COVID-19 booster-based vaccine schedule is ongoing and real-world data on vaccine effectiveness (VE) in comorbid patients are limited. We aimed to estimate booster dose VE against SARS-CoV-2 infection and COVID-19 severity in the general population and in comorbid patients. METHOD: A retrospective test-negative control study was undertaken in Galicia-Spain (December 2020-November 2021). VE and 95% confidence interval (CI) were estimated using multivariate logistic regression models. RESULTS: 1,512,415 (94.13%) negative and 94,334 (5.87%) positive SARS-CoV-2 test results were included. A booster dose of COVID-19 vaccine is associated with substantially higher protection against SARS-CoV-2 infection than vaccination without a booster [VEboosted = 87% (95%CI: 83%; 89%); VEnon-boosted = 66% (95%CI: 65%; 67%)]. The high VE was observed in all ages, but was more pronounced in subjects older than 65 years. VE against COVID-19 severity was analyzed in a mixed population of boosted and non-boosted individuals and considerable protection was obtained [VE: hospitalization = 72% (95%CI: 68%; 75%); intensive care unit administration = 83% (95%CI: 78%; 88%), in-hospital mortality = 66% (95%CI: 53%; 75%)]. Boosted comorbid patients are more protected against SARS-CoV-2 infection than those who were non-boosted. This was observed in a wide range of major diseases including cancer (81% versus 54%), chronic obstructive pulmonary disease (84% versus 61%), diabetes (84% versus 65%), hypertension (82% versus 65%) and obesity (91% versus 67%), among others. CONCLUSIONS: A booster dose of COVID-19 vaccine increases the protection against SARS-CoV-2 infection and COVID-19 severity in the general population and in comorbid patients.

Histological and biochemical evaluation of plasma rich in growth factors treatment for grade II muscle injuries in sheep.

The purpose of this study was to perform a histological and biochemical evaluation of the influence of plasma rich in growth factors (PRGF) on muscle regeneration process after a surgically induced grade II muscle laceration. A randomized, single blind, controlled experimental research was conducted including twenty-one adult healthy sheep, randomly divided in three groups (n = 7). A grade II surgical section was performed in the biceps femoris muscle of both hindlimbs. After two days (basal time), intralesional infiltration of autologous PRGF or Saline solution was randomly administered in both hindlimbs. Treatment was repeated once a week. Animal groups were euthanized at 1 (T1), 2 (T2) or 4 (T4) weeks. Histological assessment showed that PRGF intralesional injection induced a significant decrease of inflammatory cells density, significant higher centrally nucleated fibers percentage and significantly smaller fibrotic areas compared to Saline-treated muscles at T1, T2 and T4. Also, lower vascular density, with lower capillaries cross-sectional area, in PRGF group compared to Saline was observed. Biochemical analysis revealed a significant higher expression level of MYOD1, MYF5 and MYOG genes in PRGF groups at T1 compared to Saline treated muscles. At ultrastructural level, PRGF groups presented scarce edema and loss of connective tissue structure, as well as higher mitochondrial density adequately associated to the sarcomere unit in contrast to the Saline group. In conclusion, histological, biochemical, and ultrastructural results showed that PRGF treatment improved muscle regeneration process leading to more mature histological aspect in newly formed muscle tissue after a surgically induced grade II muscle injury.

Metabolic-associated fatty liver disease: From simple steatosis toward liver cirrhosis and potential complications. Proceedings of the Third Translational Hepatology Meeting, organized by the Spanish Association for the Study of the Liver (AEEH).

This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, Spain, in October 2021. The meeting, which was organized by the Spanish Association for the Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and translational hepatology, with a particular focus on the molecular and cellular mechanisms and therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC).

Extracellular Vesicles in the Fungi Kingdom.

Extracellular vesicles (EVs) are membranous, rounded vesicles released by prokaryotic and eukaryotic cells in their normal and pathophysiological states. These vesicles form a network of intercellular communication as they can transfer cell- and function-specific information (lipids, proteins and nucleic acids) to different cells and thus alter their function. Fungi are not an exception; they also release EVs to the extracellular space. The vesicles can also be retained in the periplasm as periplasmic vesicles (PVs) and the cell wall. Such fungal vesicles play various specific roles in the lives of these organisms. They are involved in creating wall architecture and maintaining its integrity, supporting cell isolation and defence against the environment. In the case of pathogenic strains, they might take part in the interactions with the host and affect the infection outcomes. The economic importance of fungi in manufacturing high-quality nutritional and pharmaceutical products and in remediation is considerable. The analysis of fungal EVs opens new horizons for diagnosing fungal infections and developing vaccines against mycoses and novel applications of nanotherapy and sensors in industrial processes.

Lithium-ion batteries towards circular economy: A literature review of opportunities and issues of recycling treatments.

Nowadays, Lithium-ion batteries are widely used in advanced technological devices and Electric and Hybrid Vehicles, due to their high energy density for weight, reduced memory effect and significant number of supported charging/discharging cycles. As a consequence, the production and the use of Lithium-ion batteries will continuously increase in the near future, focusing the global attention on their End-of-Life management. Unfortunately, wasted Lithium-ion batteries treatments are still under development, far from the optimization of recycling processes and technologies, and currently recycling represents the only alternative for the social, economic and environmental sustainability of this market, able to minimize toxicity of End-of-Life products, to create a monetary gain and to lead to the independence from foreign resources or critical materials. This paper analyses the current alternatives for the recycling of Lithium-ion batteries, specifically focusing on available procedures for batteries securing and discharging, mechanical pre-treatments and materials recovery processes (i.e. pyro- and hydrometallurgical), and it highlights the pros and cons of treatments in terms of energy consumption, recovery efficiency and safety issues. Target metals (e.g. Cobalt, Nickel and Lithium) are listed and prioritized, and the economic advantage deriving by the material recovery is outlined. An in-depth literature review was conducted, analysing the existing industrial processes, to show the on-going technological solutions proposed by research projects and industrial developments, comparing best results and open issues and criticalities.

Cartilage regeneration using a novel autologous growth factors-based matrix for full-thickness defects in sheep.

PURPOSE: To investigate the chondrogenic-regenerative properties of a novel autologous-made matrix composed of hyaline cartilage chips combined with a growth factors-based clot for full-thickness defects in sheep. METHODS: A full-thickness, 8-mm diameter cartilage defect was created in the weight-bearing area of the medial femoral condyle in 6 sheep. Treatment consisted of surgical implantation of an autologous-based matrix of hyaline cartilage chips combined with a clot of plasma poor in platelets and intraarticular injection of plasma rich in growth factors. Outcome measures at 1, 3 and 6 months included macroscopic International Cartilage Repair Society (ICRS) score, histological and immunohistochemical analysis for collagen expression, and transmission electron microscopy study. RESULTS: The 6-month macroscopic evaluation showed nearly normal (11.1 ± 0.7) cartilage repair assessment. The ICRS score was significantly higher at 6 months compared to 3 months (5.5 ± 1.3; p < 0.0001) and 1 (1.1 ± 0.4; p < 0.0001) month. At 6 months, hyaline cartilage tissue filling the defect was observed with adequate integration of the regenerated cartilage at the surrounding healthy cartilage margin. At 6 months, mature chondrons and cartilage matrix contained collagen fibers with masked fibrillary structure, and the expression of collagen in the newly formed cartilage was similar in intensity and distribution pattern compared to the healthy adjacent cartilage. CONCLUSIONS: This novel treatment enhanced chondrogenesis and regenerated hyaline cartilage at 6 months with nearly normal macroscopic ICRS assessment. Histological analysis showed equivalent structure to mature cartilage tissue in the defect and a collagen expression pattern in the newly formed cartilage similar to that found in adjacent healthy articular cartilage. The present technique may have clinical application for chondral injuries in humans because this procedure is cheap (no need for allograft, or expensive instrumentation/biomaterials/techniques), easy and fast-performing through a small arthrotomy, and safe (no rejection possibility because the patients' own tissue, cells, and plasma are used).

The First Dose of Fingolimod Affects Circulating Extracellular Vesicles in Multiple Sclerosis Patients.

Extracellular vesicles (EVs) are membrane-bound particles involved in intercellular communication. They carry proteins, lipids, and nucleotides such as microRNAs (miRNAs) from the secreting cell that can modulate target cells. We and others have previously described the presence of EVs in peripheral blood of multiple sclerosis (MS) patients and postulated them as novel biomarkers. However, their immune function in MS pathogenesis and the effect during the onset of new immunomodulatory therapies on EVs remain elusive. Here, we isolated plasma EVs from fingolimod-treated MS patients in order to assess whether EVs are affected by the first dose of the treatment. We quantified EVs, analyzed their miRNA cargo, and checked their immune regulatory function. Results showed an elevated EV concentration with a dramatic change in their miRNA cargo 5 h after the first dose of fingolimod. Besides, EVs obtained prior to fingolimod treatment showed an increased immune regulatory activity compared to EVs obtained 5 h post-treatment. This work suggests that EVs are implicated in the mechanism of action of immunomodulatory treatments from the initial hours and opens a new avenue to explore a potential use of EVs for early treatment monitoring.

Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.

Methionine and S-adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis.

BACKGROUND & AIMS: Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe non-alcoholic fatty liver disease. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism. METHODS: We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice. RESULTS: We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameliorated liver steatosis. CONCLUSIONS: These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis.

Microarray-based identification of lectins for the purification of human urinary extracellular vesicles directly from urine samples.

As cellular-derived vesicles largely maintain the biomolecule composition of their original tissue, exosomes, which are found in nearly all body fluids, have enormous potential as clinical disease markers. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. We employed lectin arrays to identify potential lectins as probes for affinity-based isolation of exosomes from the urinary matrix. We found three lectins that showed specific interactions to vesicles and no (or only residual) interaction with matrix proteins. Based on these findings a bead-based method for lectin-based isolation of exosomes from urine was developed as a sample preparation step for exosome-based biomarker research.

Characterization of Extracellular Vesicles from Human Saliva: Effects of Age and Isolation Techniques.

Salivary extracellular vesicles (EVs) represent an attractive source of biomarkers due to the accessibility of saliva and its non-invasive sampling methods. However, the lack of comparative studies assessing the efficacy of different EV isolation techniques hampers the use of salivary EVs in clinical settings. Moreover, the effects of age on salivary EVs are largely unknown, hindering the identification of salivary EV-associated biomarkers across the lifespan. To address these questions, we compared salivary EV concentration, size mode, protein concentration, and purity using eight EV isolation techniques before and after magnetic bead immunocapture with antibodies against CD9, CD63, and CD81. The effects of age on salivary EVs obtained with each isolation technique were further investigated. Results showed higher expression of CD63 on isolated salivary EVs compared to the expression of CD81 and flotillin-1. Overall, magnetic bead immunocapture was more efficient in recovering salivary EVs with Norgen's Saliva Exosome Purification Kit and ExoQuick-TC ULTRA at the cost of EV yield. Regardless of age, Invitrogen Total Exosome Isolation Solution showed the highest level of protein concentration, whereas Izon qEVOriginal-70nm columns revealed the highest purity. This study provides the first comprehensive comparison of salivary EVs in younger and older adults using different EV isolation techniques, which represents a step forward for assessing salivary EVs as a source of potential biomarkers of tissue-specific diseases throughout the life cycle.

Integration of proteomic and metabolomic analysis reveal distinct metabolic alterations of prostate cancer-associated fibroblasts compared to normal fibroblasts from patient's stroma samples.

The prostate gland is a complex and heterogeneous organ composed of epithelium and stroma. Whilst many studies into prostate cancer focus on epithelium, the stroma is known to play a key role in disease with the emergence of a cancer-associated fibroblasts (CAF) phenotype associated upon disease progression. In this work, we studied the metabolic rewiring of stromal fibroblasts following differentiation to a cancer-associated, myofibroblast-like, phenotype. We determined that CAFs were metabolically more active compared to normal fibroblasts. This corresponded with a heightened lipogenic metabolism, as both reservoir species and building block compounds. Interestingly, lipid metabolism affects mitochondria functioning yet the mechanisms of lipid-mediated functions are unclear. Data showing oxidised fatty acids and glutathione system are elevated in CAFs, compared to normal fibroblasts, strengthens the hypothesis that increased metabolic activity is related to mitochondrial activity. This manuscript describes mechanisms responsible for the altered metabolic flux and shows that prostate cancer-derived extracellular vesicles can increase basal respiration in normal fibroblasts, mirroring that of the disease-like phenotype. This indicates that extracellular vesicles derived from prostate cancer cells may drive an altered oxygen-dependent metabolism associated to mitochondria in CAFs.

Implementing precision oncology in Latin America to improve patient outcomes: the status quo and a call to action for key stakeholders and decision-makers.

Background: The advent of precision oncology (PO) has revolutionised diagnostic and follow up strategies and improved clinical outcomes for cancer patients. However, socio-economic inequalities in the level of implementation of PO in different countries is a prevailing issue. To improve this situation, the Latin America Patients Academy has gathered the recommendations of healthcare professionals and social civil members experienced in cancer management from Mexico, Guatemala, Costa Rica, Dominican Republic, Panama, Colombia, Chile, Ecuador, Peru and Argentina regarding the areas that need to be prioritised to improve the access to PO in Latin American (LATAM) countries. Methods: This manuscript is the culmination of a series of educational campaigns and panel discussion aimed at improving the implementations of PO in LATAM that took place from June 2021 to January 2022. The status of PO in Latin America: the level of PO implementation is generally low with some exceptions. The number of clinical trials and articles published with keywords related to PO from LATAM countries is drastically lower than in Europe and the United States. Despite sharing many complex challenges, progress is taking place in some countries in the region. Focus areas defined by the expert panel: The expert panel determined the areas of PO that should be improved by LATAM countries to improve its implementation through cancer care plans, educational programs and collaborative strategies. These initiatives should increase awareness about PO in the region and eventually improve cancer control in the region.

Three-Dimensional Hepatocyte Spheroids: Model for Assessing Chemotherapy in Hepatocellular Carcinoma.

BACKGROUND: Three-dimensional cellular models provide a more comprehensive representation of in vivo cell properties, encompassing physiological characteristics and drug susceptibility. METHODS: Primary hepatocytes were seeded in ultra-low attachment plates to form spheroids, with or without tumoral cells. Spheroid structure, cell proliferation, and apoptosis were analyzed using histological staining techniques. In addition, extracellular vesicles were isolated from conditioned media by differential ultracentrifugation. Spheroids were exposed to cytotoxic drugs, and both spheroid growth and cell death were measured by microscopic imaging and flow cytometry with vital staining, respectively. RESULTS: Concerning spheroid structure, an active outer layer forms a boundary with the media, while the inner core comprises a mass of cell debris. Hepatocyte-formed spheroids release vesicles into the extracellular media, and a decrease in the concentration of vesicles in the culture media can be observed over time. When co-cultured with tumoral cells, a distinct distribution pattern emerges over the primary hepatocytes, resulting in different spheroid conformations. Tumoral cell growth was compromised upon antitumoral drug challenges. CONCLUSIONS: Treatment of mixed spheroids with different cytotoxic drugs enables the characterization of drug effects on both hepatocytes and tumoral cells, determining drug specificity effects on these cell types.

Assessment of effectiveness and impact of universal prophylaxis with nirsevimab for prevention of hospitalizations due to respiratory syncytial virus in infants. The NIRSE-GAL study protocol.

Nirsevimab has been recently licensed for universal RSV prophylaxis in infants. NIRSE-GAL is a three-year population-based study initiated in Galicia in September 2023. It aims to evaluate nirsevimab effectiveness against RSV-related hospitalizations lower respiratory tract infections (LRTI), severe RSV, all-cause LRTI, and all-cause hospitalization. NIRSE-GAL also aims to estimate nirsevimab impact on primary healthcare use in the short and mid-term, children's wheezing and asthma, and medical prescriptions for RSV. The immunization campaigns will be scheduled based on the expected start week for the RSV season and will last the whole season. Immunization will be offered to: i) infants born during the campaign (seasonal), ii) infants < 6 months at the start of the campaign (catch-up), and iii) infants with high-risk factors, aged 6-24 months at the start of the campaign (high-risk). The follow-up period will start: i) the immunization date for all immunized infants, ii) the start of the campaign, for the non-immunized catch-up or high-risk groups, or iii) the birthdate for the non-immunized seasonal group. Infants will be followed up until outcome occurrence, death, or end of study. Nirsevimab effectiveness will be estimated using Poisson and Cox regression models. Sensitivity and stratified analyses will be undertaken. The number of averted cases and the number needed to immunize will be estimated. Immunization failure and nirsevimab safety will be monitored. NIRSE-GAL was approved by the ethics committee of Galicia (CEIC 2023-377) and registered in ClinicalTrials.gov (ID: NCT06180993). Findings will be mainly shared via peer-reviewed publications and scientific conferences.