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.

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.

Implementación de un programa para mitigar la resistencia a los antimicrobianos en escenarios de bajos recursos / Implementation of a program to face antimicrobial resistance on a low resources hospital

Estudio cuasi-experimental desarrollado para disminuir el impacto de la resistencia a los antimicrobianos a través de un programa de prevención de infecciones y optimización del uso de antimicrobianos construido "a medida" según las posibilidades de la institución. Se implementó: vigilan-cia de colonización e infección por enterobacterias pro-ductoras de carbapenemasas (EPC); vigilancia y medidas preventivas para infecciones urinarias asociadas a sonda vesical (ITU); vigilancia e intervenciones para mejorar la higiene de manos; guías locales de tratamiento de enfer-medades infecciosas con evaluación de adherencia a las mismas y consumo de antibióticos (ATB). Resultados: Comparando periodo pre y postintervención: tasa de EPC en muestras clínicas: 1,1 a 0/días paciente; razón de tasas de incidencia (IRR: 0.00, p: 0.033); tasa de colonización: 3,3 a 0,61/días paciente (IRR: 0.18, p: 0.5). Tasa de ITU 8,9 a 7,2/1000 días catéter urinario (IRR: 0.81, p 0.5). Adherencia a higiene de manos: 77,5% a 70,38% (p 0.0067). Consumo de ATB: 376,24 a 176,82 DDD, (disminu-ción 53%). Adherencia a guías en elección de ATB: 57,1% a 95,4% (p 0.00031); duración de ATB: 92,8% a 98,4% (p 0.16); adecuación según rescate microbiológico: 57,1% a 100% (p <0.01). Conclusión: Un programa con medidas simples, a medida, con supervisión externa, redujo en un tiempo relativamente corto las infecciones por EPC, el consumo y uso apropiado de ATB en un hospital público de medianos/bajos recursos

This quasi-experimental study was developed in a public hospital with the goal of reducing the impact of antimicrobial resistance through an infection prevention and antimicrobial stewardship program. The following measures were implemented: surveillance of colonization and infection by carbapenemase-producing Enterobacteriaceae (CPE); surveillance and preventive measures for urinary catheter-associated infections (UTIs); surveillance and interventions for hand hygiene; local guidelines for treatment of infectious diseases with compliance and antibiotic (ATB) consumption metrics.Results: comparing the pre-intervention and post-intervention period, CPE rate in clinical samples 1.1 to 0/patient days, incidence rate ratio (IRR): 0.00, p: 0.033 and colonization of 3.3 to 0.61/days patient, IRR: 0.18, p-value: 0.5. UTI rate 8.9 to 7.2/1000 days urinary catheter IRR: 0.81, p 0.5. Hand Hygiene compliance: 77.5% to 70.38%, p 0.0067. ATB consumption: 376.24 to 176.82 DDD, 53% decrease. Compliance to guidelines in ATB selection: 57.1% to 95.4% p 0.00031, duration of ATB from 92.8% to 98.4% p 0.16, and adequacy to microbiological rescue of 57.1% at 100%, p <0.01. Conclusion: it is possible to reduce CPE infections, the consumption of antimicrobials and optimize their use in a public hospital in a country with medium/low resources through a program with basic and tailored measures

Small extracellular vesicle-based human melanocyte and melanoma signature.

Intercellular communication is a cell-type and stimulus-dependent event driven not only by soluble factors but also by extracellular vesicles (EVs). EVs include vesicles of different size and origin that contain a myriad of molecules. Among them, small EVs (sEV; <200 nm) have been shown to modulate not just regional cell responses but also distant organ behavior. In cancer, distant organ modulation by sEVs has been associated to disease dissemination, which is one of the main concerns in melanoma. Description of broadly conserved alterations in sEV-contained molecules represents a strategy to identify key modifications in cellular communication as well as new disease biomarkers. Here, we characterize proteomes of cutaneous melanocyte and melanoma-derived sEVs to deepen on the landscape of normal and disease-related cell communication. Results reveal the presence of unique protein signatures for melanocytes and melanoma cells that reflect cellular transformation-related profound modifications. Melanocyte-derived sEVs are enriched in oxidative metabolism (e.g., aconitase 2, ACO2) or pigmentation (e.g., tyrosinase, TYR) related proteins while melanoma-derived sEVs reflect a generalized decrease in mature melanocytic markers (e.g., melanoma antigen recognized by T-cells 1, MART-1, also known as MLANA) and an increase in epithelial to mesenchymal transition (EMT)-related adhesion molecules such as tenascin C (TNC).

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.

Potential use of heat shock protein 90 as a biomarker for the diagnosis of human diseases.

INTRODUCTION: The heat shock protein 90 (Hsp90) is a protein involved in many different biological processes and especially in cell survival. Some of these functions require the participation of other biological molecules, so Hsp90 is a chaperone that takes part in many protein-protein interactions working as a critical signaling hub protein. As a member of the heat shock protein family, Hsp90 expression is regulated under certain environmental and/or stressful situations, therefore Hsp90 concentration can be monitored and linked to these effects. AREAS COVERED: This review discusses the Hsp90 expression in samples from individuals affected by different diseases (from infectious to cancer origin), and the biological consequences of these disorders, including the potential use of Hsp90 as a biomarker for the diagnosis of human diseases. EXPERT OPINION: The potential of Hsp90 as a biomarker disease has been demonstrated in several studies in relation to infectious diseases and especially cancer. However, further research in this field is still needed, mainly to validate in statistically significant clinical studies that the detection of Hsp90 protein allows the diagnosis of some cancers at an early stage and also that it can act as a biomarker for monitoring the efficacy of their therapies.

A Novel Approach on the Use of Samples from Faecal Occult Blood Screening Kits for Metabolomics Analysis: Application in Colorectal Cancer Population.

The incidence of colorectal cancer (CRC) is increasing, and currently it is the third most common cancer. Early CRC diagnosis is still difficult and relies on an invasive colonoscopy and tissue biopsy. The globally observed tendency demands non-invasive, specific, and accurate diagnostic tools for early diagnosis and prognosis. In this work, the main aim was to evaluate for the first time the feasibility of using extracts from the non-invasive sample collection from faecal occult blood (FOB) kits for its use in metabolomics studies taking advantage in this way of the high sensitivity of this technology. Then, a cohort of 131 samples from control individuals (CTL), adenoma (AD) and CRC patients were analysed using a semitargeted approach by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry (UHPLC-ToF-MS). Multivariate and univariate statistical analysis revealed that cholesteryl esters (ChoE) with polyunsaturated fatty acids (PUFAs) together with FOB were relevant metabolites that could clearly separate CRC patients from AD and CTL individuals, whereas the metabolic profiles of CTL and AD were very similar. These results are in agreement with previous findings and reveal the advantage of using the same FOBT samples for several analyses, which would facilitate sample collection and improve direct connection between FOB measurements and metabolomics analysis. Although the sample size and the number of metabolites should be enhanced to cover a wider range of metabolites, alterations in lipid metabolism clearly point out for future perspectives.

Fístula arteriovenosa post-TIPS, un reto diagnóstico: reporte de caso / Post-TIPS arteriovenous fistula, a diagnostic challenge: case report

La hipertensión portal es una de las principales complicaciones de la cirrosis. El papel de la derivación portosistémica transyugular intrahepática (TIPS, por sus siglas en inglés), ha ganado aceptación como tratamiento efectivo en la hipertensión portal. En los últimos años su técnica se ha ido perfeccionando, disminuyendo la morbimortalidad relacionada con este procedimiento. Describimos un caso de un paciente masculino con cirrosis Child-Pugh 8 y MELD 16, con antecedente de descompensación por sangrado variceal recurrente y trombosis parcial de la vena porta, con un gradiente de presión venosa hepática (GPVH) de 20 mmHg, por lo que es llevado a TIPS como profilaxis secundaria, con un gradiente final post-TIPS de 6 mmHg. Posterior al procedimiento, presentó evolución tórpida con deterioro de las pruebas de bioquímica hepática. Se realizó una angiografía demostrando permeabilidad del TIPS sin progresión de la trombosis portal, y hallazgos anormales inespecíficos de la arteria hepática. Se decidió realizar una arteriografía selectiva, demostrando un pseudoaneurisma de la rama derecha de la arteria hepática y una fístula arteriovenosa de la arteria hepática a las colaterales portales. Se realizó embolización selectiva de la fístula con evolución satisfactoria del paciente.

Portal hypertension is a life-threatening complication of cirrhosis. The role of transyugular intrahepatic portosystemic shunt (TIPS) has gained acceptance as an effective treatment for portal hypertension. In the past few years, its technique has been improved, decreasing the mortality related with the procedure. We describe a case of a male with Child-Pugh 8 and MELD 16 cirrhosis, with previous decompensation of recurrent variceal bleeding and partial thrombosis of the portal vein. TIPS was performed due to a hepatic venous pressure gradient (HVPG) of 20 mmHg. The final measure showed HVPG of 6 mmHg. After the procedure, he presented a torpid evolution with deterioration of liver function tests. An angiography was performed demonstrating patency of the TIPS without progression of portal thrombosis and nonspecific abnormal findings of the hepatic artery. Selective arteriography was performed and revealed a pseudoaneurysm of the right branch of the hepatic artery and an arteriovenous fistula (AVF) from the hepatic artery to portal collaterals. Embolization was performed to treat the fistula with satisfactory evolution of the patient.

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) / Enfermedad del hígado graso asociado al metabolismo: de la simple esteatosis a la cirrosis hepática y sus posibles complicaciones. Actas de la Tercera Reunión de Hepatología Traslacional, organizada por la Asociación Española para el Estudio del Hígado (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).(AU)

Este es el resumen de la 3ª Reunión de Hepatología Traslacional celebrada en Alicante, España, en octubre de 2021. La reunión, organizada por la Asociación Española para el Estudio del Hígado (AEEH), ofreció una actualización de los recientes avances en el campo de la Hepatología básica y traslacional, con un enfoque en los mecanismos moleculares y celulares y las dianas terapéuticas implicadas en la enfermedad del hígado graso asociada a disfunción metabólica (MAFLD), la esteatohepatitis asociada a disfunción metabólica (MASH), y las etapas terminales como la cirrosis y el carcinoma hepatocelular (HCC).(AU)

Top-down modulation impairs priming susceptibility in complex decision-making with social implications.

Could social context variables prime complex decisions? Could top-down processes impair this priming susceptibility? Complex decisions have been mainly studied from economic and moral perspectives, and Dual Process Theories provide evidence of how these processes could be affected. To address these issues from a political perspective, online experiments were conducted. Participants (n = 252) were asked to choose a face from 4 options, each associated with different frequencies (repetition priming) or with phrases with different emotional valence (emotional priming), for an unspecified task (UST group) or an important task (IMT group). The most repeated face was chosen most in the UST group, and was associated with lower response times. Positive faces were equally chosen by both groups. To compare results in a more ecological situation, a social study was conducted during the 2019 Argentine Presidential Election, including online surveys (n = 3673) and analysis of news media mentioning candidates. The familiarity and trust to each candidate explained the voting-probability for most of them, as well as correlated with their frequency of mentions in the news, their positive associations, and election results. Our results suggest complex decision-making is susceptible to priming, depending on top-down modulation.

miR-200c-3p, miR-222-5p, and miR-512-3p Constitute a Biomarker Signature of Sorafenib Effectiveness in Advanced Hepatocellular Carcinoma.

BACKGROUND: Sorafenib constitutes a suitable treatment alternative for patients with advanced hepatocellular carcinoma (HCC) in whom atezolizumab + bevacizumab therapy is contraindicated. The aim of the study was the identification of a miRNA signature in liquid biopsy related to sorafenib response. METHODS: miRNAs were profiled in hepatoblastoma HepG2 cells and tested in animal models, extracellular vesicles (EVs), and plasma from HCC patients. RESULTS: Sorafenib altered the expression of 11 miRNAs in HepG2 cells. miR-200c-3p and miR-27a-3p exerted an anti-tumoral activity by decreasing cell migration and invasion, whereas miR-122-5p, miR-148b-3p, miR-194-5p, miR-222-5p, and miR-512-3p exerted pro-tumoral properties by increasing cell proliferation, migration, or invasion, or decreasing apoptosis. Sorafenib induced a change in EVs population with an increased number of larger EVs, and promoted an accumulation of miR-27a-3p, miR-122-5p, miR-148b-3p, miR-193b-3p, miR-194-5p, miR-200c-3p, and miR-375 into exosomes. In HCC patients, circulating miR-200c-3p baseline levels were associated with increased survival, whereas high levels of miR-222-5p and miR-512-3p after 1 month of sorafenib treatment were related to poor prognosis. The RNA sequencing revealed that miR-200c-3p was related to the regulation of cell growth and death, whereas miR-222-5p and miR-512-3p were related to metabolic control. CONCLUSIONS: The study showed that Sorafenib regulates a specific miRNA signature in which miR-200c-3p, miR-222-5p, and miR-512-3p bear prognostic value and contribute to treatment response.

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.

Does the plasticity of neural stem cells and neurogenesis make them biosensors of disease and damage?

Postnatal and adult neurogenesis takes place in the dentate gyrus of the hippocampus in the vast majority of mammals due to the persistence of a population of neural stem cells (NSCs) that also generate astrocytes and more NSCs. These are highly plastic and dynamic phenomena that undergo continuous modifications in response to the changes brain homeostasis. The properties of NSCs as well as the process of neurogenesis and gliogenesis, are reshaped divergently by changes in neuronal activity and by different types of disease and damage. This richness of plastic responses identifies NSCs and newborn neurons as biosensors of the health state of the hippocampus, detecting and providing useful information about processes such as neuronal and network hyperexcitation, excitotoxicity, neurodegeneration, and neuroinflammation. Learning to gather and use this information is a challenge worth of our attention.

Simultaneous Quantification of Steroid Hormones Using hrLC-MS in Endocrine Tissues of Male Rats and Human Samples.

Steroid hormones play a vital role in the regulation of cellular processes, and dysregulation of these metabolites can provoke or aggravate pathological issues, such as autoimmune diseases and cancer. Regulation of steroid hormones involves different organs and biological compartments. Therefore, it is important to accurately determine their levels in tissues and biofluids to monitor changes after challenge or during disease. In this work, we have developed and optimized the extraction and quantification of 11 key members of the different steroid classes, including androgens, estrogens, progestogens and corticoids. The assay consists of a liquid/liquid extraction step and subsequent quantification by high-resolution liquid chromatography coupled time-of-flight mass spectrometry. The recoveries range between 74.2 to 126.9% and 54.9 to 110.7%, using a cell culture or urine as matrix, respectively. In general, the signal intensity loss due to matrix effect is no more than 30%. The method has been tested in relevant steroidogenic tissues in rat models and it has also been tested in human urine samples. Overall, this assay measures 11 analytes simultaneously in 6 min runtime and it has been applied in adrenal gland, testis, prostate, brain and serum from rats, and urine and extracellular vesicles from humans.

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.

Extracellular vesicles from thyroid cancer harbor a functional machinery involved in extracellular matrix remodeling.

Extracellular vesicles (EVs) participate in cell-stroma crosstalk within the tumor microenvironment and fibroblasts (Fb) contribute to tumor promotion in thyroid cancer. However, the role of tumor-stroma derived EVs still needs to be deciphered. We hypothesized that the interaction of thyroid tumor cells with Fb would liberate EVs with a specific proteomic profile, which would have an impact on EV-functionality in thyroid tumor progression-related events. Tumor (TPC-1, 8505c) and non-tumor (NThyOri) thyroid cells were co-cultured with human Fb. EVs, obtained by ultracentrifugation of conditioned media, were characterized by nanoparticle tracking analysis and western blotting. EV-proteomic analysis was performed by mass-spectrometry, and metalloproteinases (MMPs) were studied by zymography. EV-exchange was evaluated using immunofluorescence, confocal microscopy and FACS. EVs expressed classical exosome markers, with EVs from thyroid tumor cell-Fb co-cultures showing a proteomic profile related to extracellular matrix (ECM) remodeling. Bidirectional crosstalk between Fb and TPC-1 cells produced significantly more EVs than their isolated cells, and potentiated EV-functionality. In line with this, Fb-TPC-1 derived EVs induced MMP2 activation in NThyOri supernatants, and MMP2 activity could be evidenced in Fb and TPC-1 contact-independent co-cultures. Besides, MMP2 interactors allowed us to discriminate between EVs from thyroid tumoral and non-tumoral milieus. Interestingly, Fb internalized more EVs from TPC-1 than from NThyOri producing cells. Fb and thyroid tumor cell crosstalk produces specialized EVs with an ECM remodeling proteomic profile, enabling activation of MMP2 and possibly facilitating ECM-degradation, which is potentially linked with thyroid tumor progression.

A Comprehensive Metabolomics Analysis of Fecal Samples from Advanced Adenoma and Colorectal Cancer Patients.

Accurate diagnosis of colorectal cancer (CRC) still relies on invasive colonoscopy. Noninvasive methods are less sensitive in detecting the disease, particularly in the early stage. In the current work, a metabolomics analysis of fecal samples was carried out by ultra-high-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). A total of 1380 metabolites were analyzed in a cohort of 120 fecal samples from patients with normal colonoscopy, advanced adenoma (AA) and CRC. Multivariate analysis revealed that metabolic profiles of CRC and AA patients were similar and could be clearly separated from control individuals. Among the 25 significant metabolites, sphingomyelins (SM), lactosylceramides (LacCer), secondary bile acids, polypeptides, formiminoglutamate, heme and cytidine-containing pyrimidines were found to be dysregulated in CRC patients. Supervised random forest (RF) and logistic regression algorithms were employed to build a CRC accurate predicted model consisting of the combination of hemoglobin (Hgb) and bilirubin E,E, lactosyl-N-palmitoyl-sphingosine, glycocholenate sulfate and STLVT with an accuracy, sensitivity and specificity of 91.67% (95% Confidence Interval (CI) 0.7753-0.9825), 0.7 and 1, respectively.