Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

BACKGROUND: Nucleic acids, RNA among them, are widely used in biomedicine and Biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. METHODS AND RESULTS: In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. DISCUSSION: QIAcube™ and semi-automated extraction methods were perceived as the best options because of their lower variability, good functionality, and lower cost (P < 0.001). These data contribute to facilitating researchers or research service platforms (Biobanks) in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

Consultas nutricionales en menores de 9 años en la región del Maule, Chile, entre 2017-2021, efecto de estallido social y pandemia COVID-19

Introducción: La consulta nutricional es la primera línea de atención en niñas y niños en salud pública que presentan condiciones de malnutrición por déficit o exceso. Sin embargo, la atención a estos niños y niñas fue afectada por las movilizaciones sociales y la pandemia por COVID-19. Objetivo: Evaluar la tendencia de las consultas realizadas a menores de 9 años por profesional nutricionista en la región del Maule, Chile, desde el año 2017 a 2021. Métodos: Estudio descriptivo de corte longitudinal histórico basado en los datos de los Registros Estadísticos Mensuales (REM) del Servicio de Salud del Maule, las tendencias fueron analizadas con coeficiente de determinación (R2) mediante la regresión de Prais-Winsten. Resultados: Se analizaron 274.377 consultas nutricionales de niños/as menores de 9 años. 53,8% en clasificación de malnutrición por exceso y 12,1% en déficit. Se registró una disminución de 56,8% en las consultas nutricionales durante las movilizaciones sociales y un 92% al inicio de pandemia. Se observó una tendencia al aumento de las consultas por déficit nutricional, especialmente en menores de 12 meses (R2 0,633, β=4,45, p<0,001). Conclusión: La situación social y epidemiológica afectaron significativamente las atenciones nutricionales en salud pública. Es necesario dar una mayor visibilidad de los profesionales nutricionistas y promover el desarrollo de estrategias innovadoras para afrontar este escenario epidemiológico.

Background: The nutritional appointments is the first line of care for children with malnutrition or overweight in public primary health, but its normal functioning was affected by social mobilizations and the COVID-19 pandemic. Objective: To evaluate trends in consultations among children under 9 years of age in the Maule region, Chile, between 2017 and 2021. Methods: Descriptive longitudinal study based on data from the Monthly Statistical Records (REM) of the Maule Health Service, the trends were analyzed with coefficient of determination (R2) using Prais-Winsten regression. Results: 274,377 nutritional consultations were analyzed, of which 53.8% were overweight and 12.1% with malnutrition. A 56.8% decrease in nutritional consultations was recorded during social mobilizations and 92% at the beginning of the pandemic. A tendency to increase consultations due to malnutrition was observed, especially in children under 12 months of age (R2 0.633, β=4.45, p<0.001). Conclusion: The social and epidemiological situations significantly affected nutritional care in public health. It is necessary to give nutrition professionals greater visibility and promote the development of innovative strategies to deal with this epidemiological scenario.

Quality assessment on the long-term cryopreservation and nucleic acids extraction processes implemented in the andalusian public biobank.

Human samples are commonly collected and long-term stored in biobanks for current and future analyses. Even though techniques for freezing human blood are well established, the storage time can compromise the cell viability as well as the yield and quality of nucleic acids (RNA and DNA) extracted from them. In this study, a protocol to obtain peripheral blood mononuclear cells (PBMCs) from 70 subjects, which were stored at - 196 °C from EDTA tubes for a long-term, was assessed. In parallel; a protocol to obtain DNA from the same subjects, which were stored at - 80 °C from citrate tubes, was also studied. Samples stored from 2008 to 2012 were studied and the results obtained showed that there were no statistically significant differences in the RNA or DNA extracted in terms of purity, integrity and functionality The freezing protocol used by the Málaga Biobank shows that viable PBMCs and DNA could be kept for a period of, at least, 10 years, with a high quality and performance. Furthermore, RNA extracted from these PBMCs presents also a good quality and performance. Therefore, the samples frozen according to the conditions of the protocols assessed in this study could be optimal for biomedical research.

Gene therapy with mesenchymal stem cells expressing IFN-ß ameliorates neuroinflammation in experimental models of multiple sclerosis.

BACKGROUND AND PURPOSE: Recombinant IFN-ß is one of the first-line treatments in multiple sclerosis (MS), despite its lack of efficacy in some patients. In this context, mesenchymal stem cells (MSCs) represent a promising therapeutic alternative due to their immunomodulatory properties and multipotency. Moreover, by taking advantage of their pathotropism, these cells can be genetically modified to be used as carriers for delivering or secreting therapeutic drugs into injured tissues. Here, we report the therapeutic effect of systemic delivery of adipose-derived MSCs (AdMSCs), transduced with the IFN-ß gene, into mice with experimental autoimmune encephalomyelitis (EAE). EXPERIMENTAL APPROACH: Relapsing-remitting and chronic progressive EAE were induced in mice. Cells were injected i.v. Disease severity, inflammation and tissue damage were assessed clinically, by flow cytometry of spleens and histopathological evaluation of the CNS respectively. KEY RESULTS: Genetic engineering did not modify the biological characteristics of these AdMSCs (morphology, growth rate, immunophenotype and multipotency). Furthermore, the transduction of IFN-ß to AdMSCs maintained and, in some cases, enhanced the functional properties of AdMSCs by ameliorating the symptoms of MS in EAE models and by decreasing indications of peripheral and central neuro-inflammation. CONCLUSION AND IMPLICATIONS: Gene therapy was found to be more effective than cell therapy in ameliorating several clinical parameters in both EAE models, presumably due to the continuous expression of IFN-ß. Furthermore, it has significant advantages over AdMSC therapy, and also over systemic IFN-ß treatment, by providing long-term expression of the cytokine at therapeutic concentrations and reducing the frequency of injections, while minimizing dose-limiting side effects.

Recombinant soluble IFN receptor (sIFNAR2) exhibits intrinsic therapeutic efficacy in a murine model of Multiple Sclerosis.

Endogenous interferon beta (IFNß) is an important cytokine involved in several chronic inflammatory diseases, such as Multiple Sclerosis (MS). In spite of the numerous therapeutic approaches available for MS patients, the administration of recombinant IFNß continues being one of the first line treatment to these patients. The soluble form of IFNß receptor (sIFNAR2) could act as critical regulator of the endogenous and the systemically administered IFNß, but whether it functions as an agonist or antagonist of its ligand is not completely elucidated. Morover, the possible role of sIFNAR2 in autoimmune diseases like MS is still unknown and so far overlooked. Here we evaluated the efficacy of the combined therapy of IFNß and our recombinant protein analogous to human sIFNAR2 as a treatment in a chronic mice model of MS (CP-EAE). We also tested the effect of the sIFNAR2 administered as a monotherapy over these EAE-animals. The results showed that our recombinant sIFNAR2 protein potentiates the immunomodulatory effects of exogenous IFNß in CP-EAE by increasing the reduction of the induced inflammation and the tissue damage. Furthermore, we demonstrate for the first time that sIFNAR2 shows intrinsic properties by modulating the CP-EAE progression and the neuroinflammation processes related to this disease. Another intrinsic activity showed by sIFNAR2 is the inhibition of the T cells proliferation, which increase its potential as therapeutic molecule.

Actividad anticonvulsivante del extracto metanólico de tallo y raíz deKalanchoe pinnata Lam. en ratones: Comparación con diazepam / Anticonvulsant activity of methanolic extract from Kalanchoe pinnata Lam. stems and roots in mice: A comparison to diazepam

Introducción: En México, la medicina tradicional emplea extractos de hojas o de la planta completa de «siempre viva» (Kalanchoe pinnata [K. pinnata] Lam.) para tratar la alferecía amarilla cuando presenta convulsiones. La actividad anticonvulsivante del tallo o de la raíz sigue sin explorarse. Métodos: El extracto metanólico de la raíz (EMR) y el del tallo (EMT) de K. pinnata Lam., fueron evaluados con el modelo de inducción de convulsiones con pentilentetrazol en ratones de la cepa Balb/C, comparado con diazepam. Las fracciones del EMT fueron subsecuentemente evaluadas. Resultados: El EMR incrementó la latencia a las crisis clónico-tónicas de forma inversamente proporcional a la dosis, observándose el mismo patrón sobre los efectos letales del pentilentetrazol. Todas las dosis evaluadas del EMT aumentaron la latencia a las mioclonías y a las crisis clónicas de forma dosis-dependiente e incrementaron la latencia a las crisis clónico-tónicas de manera semejante al diazepam con una protección del 100% contra los efectos letales del pentilentetrazol. El fraccionamiento del EMT redujo su eficacia. Al mezclar las fracciones de cloroformo y acetato de etilo, se recuperó la actividad anticonvulsivante y la protección contra los efectos letales. El análisis fitoquímico preliminar identificó alcaloides y esteroles en el EMR; esteroles y terpenos en el EMT. Conclusión: La actividad anticonvulsivante de EMR de K. pinnata Lam. disminuye aumentando la dosis y en el EMT se presenta de forma dosis-dependiente, conservándose en la mezcla de cloroformo y acetato de etilo. Se sugiere que los metabolitos responsables de estos efectos son esteroles en el EMR; esteroles y terpenos presentes en el EMT

Introduction: In ancient and current traditional medicine in México, extracts from the leaves or whole plant of ‘life leaf’ (Kalanchoe pinnata [K. pinnata]Lam) have been used to treat an entity known locally as ‘yellow epilepsy’ (alferecía amarilla) when it is accompanied by seizures. However, the anticonvulsive activity of its stems and roots remains unexplored. Methods: The anticonvulsant activity of the methanolic root extract (MER) or stem (MES) of K. pinnata Lam. was evaluated in a pentylenetetrazol-induced seizure model in Balb/C mice, and effects were compared to those of diazepam. The stem extract fractions that produced anticonvulsant activity were subsequently evaluated using the pentylenetetrazol -induced seizure model. Results: We observed increased latency of tonic-clonic seizures that was inversely proportional to the dose of MRE, with a similar impact on the lethal effects of pentylenetetrazol. Different doses of the MSE showed a dose-dependent increase in latency to myoclonus, clonus, and tonic-clonic seizures, acting similarly to diazepam and offering 100% protection against the lethal effects of pentylenetetrazol. Fractioning MSE decreased its effectiveness, but when fractions were mixed with fractions of chloroform and ethyl acetate, anticonvulsive activity was restored. The preliminary phytochemical analysis identified alkaloids and sterols in MRE, and sterols and terpenes in MSE. Conclusions: The anticonvulsant activity of K. pinnata Lam. decreases with increased doses of MRE, whereas the effect of MSE is dose-dependent and preserved in the mixture chloroform and ethyl acetate. We suggest that the metabolites responsible for these effects are sterols in MRE, and sterols and terpenes in MSE

Anticonvulsant activity of methanolic extract from Kalanchoe pinnata Lam. stems and roots in mice: A comparison to diazepam.

INTRODUCTION: In ancient and current traditional medicine in México, extracts from the leaves or whole plant of 'life leaf' (Kalanchoe pinnata [K. pinnata]Lam) have been used to treat an entity known locally as 'yellow epilepsy' (alferecía amarilla) when it is accompanied by seizures. However, the anticonvulsive activity of its stems and roots remains unexplored METHODS: The anticonvulsant activity of the methanolic root extract (MER) or stem (MES) of K. pinnata Lam. was evaluated in a pentylenetetrazol-induced seizure model in Balb/C mice, and effects were compared to those of diazepam. The stem extract fractions that produced anticonvulsant activity were subsequently evaluated using the pentylenetetrazol -induced seizure model. RESULTS: We observed increased latency of tonic-clonic seizures that was inversely proportional to the dose of MRE, with a similar impact on the lethal effects of pentylenetetrazol. Different doses of the MSE showed a dose-dependent increase in latency to myoclonus, clonus, and tonic-clonic seizures, acting similarly to diazepam and offering 100% protection against the lethal effects of pentylenetetrazol. Fractioning MSE decreased its effectiveness, but when fractions were mixed with fractions of chloroform and ethyl acetate, anticonvulsive activity was restored. The preliminary phytochemical analysis identified alkaloids and sterols in MRE, and sterols and terpenes in MSE CONCLUSIONS: The anticonvulsant activity of K. pinnata Lam. decreases with increased doses of MRE, whereas the effect of MSE is dose-dependent and preserved in the mixture chloroform and ethyl acetate. We suggest that the metabolites responsible for these effects are sterols in MRE, and sterols and terpenes in MSE.

Fine mapping and functional analysis of the multiple sclerosis risk gene CD6.

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2(nd) SRCR domain with susceptibility to MS (P max(T) permutation = 1×10(-4)). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. - CD4(+) naïve cells, P = 0.0001; CD8(+) naïve cells, P<0.0001; CD4(+) and CD8(+) central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4(+) and CD8(+) T cells.

Replication study of 10 genes showing evidence for association with multiple sclerosis: validation of TMEM39A, IL12B and CBLB [correction of CLBL] genes.

BACKGROUND AND OBJECTIVES: Ten genes previously showing different evidence of association with multiple sclerosis have been selected to validate. METHODS: Eleven polymorphisms were genotyped with the iPLEX™ Sequenom in a well-powered collection of Spanish origin including 2863 multiple sclerosis cases and 2930 controls. RESULTS: Replication extended to the following polymorphisms: PKN2 (rs305217), GTF2B (rs7538427), EPHA4 (rs1517440), YTHDF3 (rs12115114), ANKFN1 (rs17758761) and PTPRM (rs4798571), which did not reach the threshold of significance in a follow-up of the first genome-wide association study (GWAS) conducted in multiple sclerosis; TMEM39A (rs1132200), which appeared as a newly identified susceptibility gene in the same study; a gene previously reaching GWAS significance in Italy, CBLB (rs9657904); IL12B (rs6887695, rs10045431), a susceptibility gene shared by diverse autoimmune diseases and, finally, another gene showing inconclusive association with multiple sclerosis, CNR1 (rs1049353). CONCLUSIONS: Pooled analysis corroborated the effect on MS predisposition of three genes: TMEM39A [rs1132200: p(M-H)=0.001; OR(M-H) (95% CI)= 0.84 (0.75-0.93)], IL12B [rs6887695: p(M-H)=0.03; OR(M-H) (95% CI)= 1.09 (1.01-1.17)] and CBLB [rs9657904: p(M-H)=0.01; OR(M-H) (95% CI)= 0.89 (0.81-0.97)].

Gene expression in IFNß signalling pathway differs between monocytes, CD4 and CD8 T cells from MS patients.

IFNß exerts its activity through the interaction with IFNAR, through activation of the JAK/STAT pathway. We analyzed the changes in IFNAR1, IFNAR2, STAT1, STAT2, Tyk2, JAK1, IRF9 and MxA gene expressions after prolonged IFNß treatment, in isolated mononuclear-cell subpopulations from MS patients, by real time PCR. The effect of IFNß on gene expression differed depending on the subpopulation assessed. The data suggest that CD8+ T cells are the most influenced by prolonged IFNß therapy as IFNAR2, Tyk2, IRF9 and Jak1 expressions were decreased, whereas MxA expression was increased in these cells.

The autoimmune disease-associated KIF5A, CD226 and SH2B3 gene variants confer susceptibility for multiple sclerosis.

Genome-wide association studies (GWAS) have revealed that different diseases share susceptibility variants. Twelve single-nucleotide polymorphisms (SNPs) previously associated with different immune-mediated diseases in GWAS were genotyped in a Caucasian Spanish population of 2864 multiple sclerosis (MS) patients and 2930 controls. Three SNPs were found to be associated with MS: rs1678542 in KIF5A (P=0.001, odds ratio (OR)=1.13, 95% confidence interval (CI)=1.05-1.23); rs3184504 in SH2B3 (P=0.00001, OR=1.19, 95% CI=1.10-1.27) and rs763361 in CD226 (P=0.00007, OR=1.16, 95%CI=1.08-1.25). These variants have previously been associated with rheumatoid arthritis and type 1 diabetes. The SH2B3 polymorphism has additionally been associated with systemic lupus erythematosus. Our results, in addition to validating some of these loci as risk factors for MS, are consistent with shared genetic mechanisms underlying different immune-mediated diseases. These data may help to shape the contribution of each pathway to different disorders.

Neutralizing antibodies against IFN beta in patients with multiple sclerosis: a comparative study of two cytopathic effect tests (CPE) for their detection.

Neutralizing antibodies (NABs) against IFN beta should be measured in specialized laboratories, using a test of inhibition of the cytopathic effect (bioassay or CPE test), based on the capacity of IFNss to block the infection of live monolayer-cultured cells by a virus, depending on the presence or absence of NABs. The European Federation of Neurological Societies (EFNS) considers this assay to be the gold standard. However, the various different ways to perform this assay complicate comparison of the results between laboratories. The World Health Organization (WHO) has published several recommendations to perform this assay using the A549 cell line and the murine encephalomyocarditis virus (EMCV). In order to validate the results previously obtained in our laboratory with HEP2/VSV, we undertook a comparative analysis of the two bioassays, HEP2/VSV and A549/EMCV, to assess whether the use of different cell lines and viruses influences sensitivity. We also calibrated the A549/EMCV assay with a reference IFNss. Our results confirm that the bioassay with HEP2/VSV is as sensitive as the assay with A549/EMCV and that a significant association and correlation exist in the results between both assays. Thus, past results with HEP2/VSV in our laboratory could be comparable with those obtained with A549/EMCV in both our laboratory and others.

Astigmatism induced by intrastromal corneal suture after small incision phacoemulsification.

PURPOSE: To evaluate the course of astigmatic evolution and complications after clear corneal incisions using an intrastromal corneal suture. SETTING: Instituto Oftalmologico de Alicante, University of Alicante, Spain. METHODS: Eighty eyes of 62 patients had endocapsular phacoemulsification. A foldable intraocular lens was implanted through a 4.0 mm clear corneal incision. A 10-0 nylon intrastromal corneal suture was used in all eyes. Change sin corneal astigmatism were calculated by vector analysis; follow-up was 6 months. Early and late suture-related complications were also evaluated. RESULTS: Mean induced cylinder was 1.25 diopters (D) +/- 1.24 (SD) with the wound 1 day postoperatively and 0.19 +/- 0.81 D against the wound at 6 months. There were no incision- or suture-related complications postoperatively. CONCLUSION: Use of the intrastromal corneal suture led to astigmatically neutral closure of multiplanar corneal incisions.

Naproxen sódico en el tratamiento de laringitis obstructiva en niños: estudio doble ciego / Naproxen sodium in the treatment of obstructive laryngitis in children: double blind study

Se efectuó estudio doble ciego con 80 casos de laringitis obstructiva, distribuidos al azar en 2 grupos de tratamiento de 40 pacientes cada uno: grupo A con naxopren sódico en supositorios y grupo B con placebo en supositorios. Recibieron además atmósfera húmeda. Fueron evaluados en forma periódica de acuerdo a un protocolo que consideraba: tiraje, disfonía, estridor, tiempo uso de Croupette, días de estada y complicaciones. Los pacientes tratados con naxopren sódico, mostraron una mayor rapidez en la mejoría de los síntomas evaluados, pero estos resultados no fueron estadísticamente significativos (p > 0.05). Sin embargo, el análisis de estos síntomas, según gravedad el ingreso, demostró que el naxopren sódico en pacientes con laringitis obstructiva grado II, fue de gran utilidad en la mejoría de ellos, con resultados estadísticamente significativos para el síntoma disfonía (p < 0.05). No ocurrió lo mismo, en pacientes con laringitis grado I

Divertículo uretral en la mujer / Urethral diverticulum in woman

Se hace un breve recuento histórico. Se analizan los distintos aspectos patogénicos de la afección; se comentan aspectos clínicos y de diagnóstico de la misma, y se exponen las posibilidades que ofrecen los estudios radiológicos, principalmente la uretrocistografía miccional y la diverticulografía; se plantea la conducta terapéutica seguida en nuestra serie. Se efectúa una revisión de 5 casos de divertículo uretral en la mujer, tratados en el servicio de urología del hospital docente "Dr. Salvador Allende" en el período comprendido entre junio de 1974 y julio de 1977 (AU)