[Impact of days elapsed from the onset of symptoms to hospitalization in COVID-19 in-hospital mortality: Time matters]. / Impacto de los días transcurridos desde el inicio de los síntomas hasta la hospitalización en la mortalidad hospitalaria por COVID-19: el tiempo importa.

Background: COVID-19 shows different clinical and pathophysiological stages over time. Theeffect of days elapsed from the onset of symptoms (DEOS) to hospitalization on COVID-19prognostic factors remains uncertain. We analyzed the impact on mortality of DEOS to hospital-ization and how other independent prognostic factors perform when taking this time elapsedinto account. Methods: This retrospective, nationwide cohort study, included patients with confirmed COVID-19 from February 20th and May 6th, 2020. The data was collected in a standardized online datacapture registry. Univariate and multivariate COX-regression were performed in the generalcohort and the final multivariate model was subjected to a sensitivity analysis in an earlypresenting (EP; < 5 DEOS) and late presenting (LP; ≥5 DEOS) group. Results: 7915 COVID-19 patients were included in the analysis, 2324 in the EP and 5591 in theLP group. DEOS to hospitalization was an independent prognostic factor of in-hospital mortalityin the multivariate Cox regression model along with other 9 variables. Each DEOS incrementaccounted for a 4.3% mortality risk reduction (HR 0.957; 95% CI 0.93---0.98). Regarding variationsin other mortality predictors in the sensitivity analysis, the Charlson Comorbidity Index onlyremained significant in the EP group while D-dimer only remained significant in the LP group. Conclusion: When caring for COVID-19 patients, DEOS to hospitalization should be consideredas their need for early hospitalization confers a higher risk of mortality. Different prognosticfactors vary over time and should be studied within a fixed timeframe of the disease.

Leptin decreases apoptosis and promotes the activation of primordial follicles through the phosphatidylinositol-3-kinase/protein kinase B pathway in cultured ovine ovarian tissue.

This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.

DECO: decompose heterogeneous population cohorts for patient stratification and discovery of sample biomarkers using omic data profiling.

MOTIVATION: Patient and sample diversity is one of the main challenges when dealing with clinical cohorts in biomedical genomics studies. During last decade, several methods have been developed to identify biomarkers assigned to specific individuals or subtypes of samples. However, current methods still fail to discover markers in complex scenarios where heterogeneity or hidden phenotypical factors are present. Here, we propose a method to analyze and understand heterogeneous data avoiding classical normalization approaches of reducing or removing variation. RESULTS: DEcomposing heterogeneous Cohorts using Omic data profiling (DECO) is a method to find significant association among biological features (biomarkers) and samples (individuals) analyzing large-scale omic data. The method identifies and categorizes biomarkers of specific phenotypic conditions based on a recurrent differential analysis integrated with a non-symmetrical correspondence analysis. DECO integrates both omic data dispersion and predictor-response relationship from non-symmetrical correspondence analysis in a unique statistic (called h-statistic), allowing the identification of closely related sample categories within complex cohorts. The performance is demonstrated using simulated data and five experimental transcriptomic datasets, and comparing to seven other methods. We show DECO greatly enhances the discovery and subtle identification of biomarkers, making it especially suited for deep and accurate patient stratification. AVAILABILITY AND IMPLEMENTATION: DECO is freely available as an R package (including a practical vignette) at Bioconductor repository (http://bioconductor.org/packages/deco/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Chemical composition and bioactivity potential of the new Endosequence BC Sealer formulation HiFlow.

AIM: To evaluate in a laboratory setting the effects of Endosequence BC Sealer HiFlow (Brasseler USA, Savannah, GA, USA), a novel calcium silicate-based sealer developed for use in warm canal filling techniques, on human periodontal ligament stem cells (hPDLSCs). METHODOLOGY: Eluates of EndoSequence BC Sealer HiFlow (BCHiF) (Brasseler USA), EndoSequence BC Sealer (BCS) (Brasseler USA) and AH Plus (AHP) (Dentsply DeTrey GmbH, Konstanz, Germany) were placed in contact with hPDLSCs. The characterization of the chemical elements of the root canal sealers was assessed using scanning electron microscopy and energy-dispersive X-ray analysis (SEM-EDX). Inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine the ion release of the sealers. MTT assay and wound healing techniques were used to determine cell viability and migration, respectively. Cell morphology and cell attachment were assessed using a direct contact technique of hPDLSCs onto the surface of the sealers and analysed by SEM. The bioactivity potential was carried out with the Alizarin Red and qPCR testing methods. The statistical differences were evaluated using one-way anova and Tukey's test (P < 0.05). RESULTS: ICP-MS and EDX revealed significantly more zirconium in BCHiF than BCS (P < 0.05), whereas BCS had slightly higher levels of Ca2+ than BCHiF (P < 0.05). The cell viability assay revealed no relevant differences between BCS and BCHiF when compared with the control group (P > 0.05). Both BCS and BCHiF had similar rates of cell migration to the control group at 24 and 48 h. Cell morphology and adhesion capacity were also similar for BCS and BCHiF groups, whilst the AHP group was associated with reduced adhesion capacity. The Alizarin Red assay revealed a significant difference between the BCS and the control group (P < 0.001), as well as for the BCHiF group (P < 0.001). Finally, BCS and BCHiF promoted overexpression of osteo/cementogenic genes. CONCLUSIONS: In general, EndoSequence BC Sealer HiFlow possesses suitable biological properties to be safely used as a root canal filling material and promote increased expression of oste/cementogenic genes by hPDLSCs.

[Clinical characteristics of patients hospitalized with COVID-19 in Spain: Results from the SEMI-COVID-19 Registry]. / Características clínicas de los pacientes hospitalizados con COVID-19 en España: resultados del Registro SEMI-COVID-19.

BACKGROUND: Spain has been one of the countries most affected by the COVID-19 pandemic. OBJECTIVE: To create a registry of patients with COVID-19 hospitalized in Spain, in order to improve our knowledge of the clinical, diagnostic, therapeutic, and prognostic aspects of this disease. METHODS: A multicentre retrospective cohort study, including consecutive patients hospitalized with confirmed COVID-19 throughout Spain. Epidemiological and clinical data, additional tests at admission and at seven days, treatments administered, and progress at 30 days of hospitalization were collected from electronic medical records. RESULTS: Up to June 30th 2020, 15,111 patients from 150 hospitals were included. Their median age was 69.4 years (range: 18-102 years) and 57.2% were male. Prevalences of hypertension, dyslipidemia, and diabetes mellitus were 50.9%, 39.7%, and 19.4%, respectively. The most frequent symptoms were fever (84.2%) and cough (73.5%). High values of ferritin (73.5%), lactate dehydrogenase (73.9%), and D-dimer (63.8%), as well as lymphopenia (52.8%), were frequent. The most used antiviral drugs were hydroxychloroquine (85.6%) and lopinavir/ritonavir (61.4%); 33.1% developed respiratory distress. Overall mortality rate was 21.0%, with a marked increase with age (50-59 years: 4.7%, 60-69 years: 10.5%, 70-79 years: 26.9%, ≥ 80 years: 46.0%). CONCLUSIONS: The SEMI-COVID-19 Network provides data on the clinical characteristics of patients with COVID-19 hospitalized in Spain. Patients with COVID-19 hospitalized in Spain are mostly severe cases, as one in three patients developed respiratory distress and one in five patients died. These findings confirm a close relationship between advanced age and mortality.

Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro.

The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analyses (fresh control) or cultured in α-MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100 µM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p > 0.05), except for 100 µM kaempferol, which had less normal follicles (p < 0.05). Moreover, kaempferol at 10 µM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p < 0.05) and a percentage of TUNEL-positive cells similar to that in the fresh control and lower than other treatments (p < 0.05). LY294002 significantly inhibited primordial follicle activation stimulated by α-MEM+ and 10 µM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10 µM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.

Prevalence of carotid artery calcifications among 2,500 digital panoramic radiographs of an adult Brazilian population.

BACKGROUND: The aim of the present study was to analyze the epidemiological data of digital panoramic radiographs revealing suggestive images of carotid artery calcifications (CAC) from a Northeast Brazilian population. MATERIAL AND METHODS: A cross-sectional retrospective study was conducted with 2,500 digital panoramic radiographs obtained from a single imaging reference center in Northeast Brazil. Images from individuals of both sexes and older than 18 years were included and those that did not cover the region of cervical vertebrae or presented low radiographic quality were excluded. Data were analyzed regarding prevalence, location (bilateral, right or left), sex, and age using the Chi-square test at the significance level of 5%. RESULTS: An amount of 96 (4%) patients presented suggestive images of CAC. The female sex (p=0.003) and individuals aged up to 70 years (p=0.002) were statically significant. 40.4% were found bilaterally, 37.6% on the right side (p<0.001) and 22% on the left side. CONCLUSIONS: In conclusion, this study showed a low prevalence of suggestive images of CAC in digital panoramic radiographs from a Northeast Brazilian population. It was observed a higher prevalence of CAC associated with female sex, older patients, and right side location.

The role of epigenetic modifiers in extended cultures of functional hepatocyte-like cells derived from human neonatal mesenchymal stem cells.

The development of predictive in vitro stem cell-derived hepatic models for toxicological drug screening is an increasingly important topic. Herein, umbilical cord tissue-derived mesenchymal stem cells (hnMSCs) underwent hepatic differentiation using an optimized three-step core protocol of 24 days that mimicked liver embryogenesis with further exposure to epigenetic markers, namely the histone deacetylase inhibitor trichostatin A (TSA), the cytidine analogue 5-azacytidine (5-AZA) and dimethyl sulfoxide (DMSO). FGF-2 and FGF-4 were also tested to improve endoderm commitment and foregut induction during Step 1 of the differentiation protocol, being HHEX expression increased with FGF-2 (4 ng/mL). DMSO (1%, v/v) when added at day 10 enhanced cell morphology, glycogen storage ability, enzymatic activity and induction capacity. Moreover, the stability of the hepatic phenotype under the optimized differentiation conditions was examined up to day 34. Our findings showed that hepatocyte-like cells (HLCs) acquired the ability to metabolize glucose, produce albumin and detoxify ammonia. Global transcriptional analysis of the HLCs showed a partial hepatic differentiation degree. Global analysis of gene expression in the different cells revealed shared expression of gene groups between HLCs and human primary hepatocytes (hpHeps) that were not observed between HepG2 and hpHeps. In addition, bioinformatics analysis of gene expression data placed HLCs between the HepG2 cell line and hpHeps and distant from hnMSCs. The enhanced hepatic differentiation observed was supported by the presence of the hepatic drug transporters OATP-C and MRP-2 and gene expression of the hepatic markers CK18, TAT, AFP, ALB, HNF4A and CEBPA; and by their ability to display stable UGT-, EROD-, ECOD-, CYP1A1-, CYP2C9- and CYP3A4-dependent activities at levels either comparable with or even higher than those observed in primary hepatocytes and HepG2 cells. Overall, an improvement of the hepatocyte-like phenotype was achieved for an extended culture time suggesting a role of the epigenetic modifiers in hepatic differentiation and maturation and presenting hnMSC-HLCs as an advantageous alternative for drug discovery and in vitro toxicology testing.

Is manual ultrasonographic measurement of carotid intima-media thickness a reproducible cardiovascular biomarker? / Es la medida ecográfica manual del grosor íntima-media carotídeo un biomarcador cardiovascular reproducible?

To establish the ability of a potential biomarker to discriminate between diseased and healthy subjects, it is essential to know the intrinsic variability of the methods used to measure the biomarker. This aspect is especially important in the ultrasonographic measurement of carotid intima-media thickness, where submillimetric variations due largely to the unrecognized lack of precision in the ultrasonographic technique can have a significant clinical impact on the classification of cardiovascular risk. This article aims to present the evidence about the reproducibility of carotid intima-media thickness measured by ultrasonography to discuss whether the variability inherent in the technique can influence the potential of this parameter as a biomarker.

Visible light communication using InGaN optical sources with AlInGaP nanomembrane down-converters.

We report free space visible light communication using InGaN sources, namely micro-LEDs and a laser diode, down-converted by a red-emitting AlInGaP multi-quantum-well nanomembrane. In the case of micro-LEDs, the AlInGaP nanomembrane is capillary-bonded between the sapphire window of a micro-LED array and a hemispherical sapphire lens to provide an integrated optical source. The sapphire lens improves the extraction efficiency of the color-converted light. For the case of the down-converted laser diode, one side of the nanomembrane is bonded to a sapphire lens and the other side optionally onto a dielectric mirror; this nanomembrane-lens structure is remotely excited by the laser diode. Data transmission up to 870 Mb/s using pulse amplitude modulation (PAM) with fractionally spaced decision feedback equalizer is demonstrated for the micro-LED-integrated nanomembrane. A data rate of 1.2 Gb/s is achieved using orthogonal frequency division multiplexing (ODFM) with the laser diode pumped sample.

Post-transcriptional silencing of the SGE1 gene induced by a dsRNA hairpin in Fusarium oxysporum f. sp cubense, the causal agent of Panama disease.

Fusarium oxysporum f. sp cubense (Foc), the causal agent of Panama disease, is responsible for economic losses in banana crops worldwide. The identification of genes that effectively act on pathogenicity and/or virulence may contribute to the development of different strategies for disease control and the production of resistant plants. The objective of the current study was to analyze the importance of SGE1 gene expression in Foc virulence through post-transcriptional silencing using a double-stranded RNA hairpin. Thirteen transformants were selected based on different morphological characteristics, and sporulation in these transformants was significantly reduced by approximately 95% (P < 0.05) compared to that of the wild-type strain. The relative SGE1 expression levels in the transformant strains were reduced by 27 to 47% compared to those in the wild-type strain. A pathogenicity analysis revealed that the transformants were able to reach the rhizomes and pseudostems of the inoculated banana plants. However, the transformants induced initial disease symptoms in the banana plants approximately 10 days later than that by the wild-type Foc, and initial disease symptoms persisted even at 45 days after inoculation. These results indicate that the SGE1 gene is directly involved in the virulence of Foc. Therefore, SGE1 may be a potential candidate for host-induced gene silencing in banana plants.

Comparative study of the primary cilia in thyrocytes of adult mammals.

Since their discovery in different human tissues by Zimmermann in 1898, primary cilia have been found in the vast majority of cell types in vertebrates. Primary cilia are considered to be cellular antennae that occupy an ideal cellular location for the interpretation of information both from the environment and from other cells. To date, in mammalian thyroid gland, primary cilia have been found in the thyrocytes of humans and dogs (fetuses and adults) and in rat embryos. The present study investigated whether the existence of this organelle in follicular cells is a general event in the postnatal thyroid gland of different mammals, using both immunolabeling by immunofluorescence and electron microscopy. Furthermore, we aimed to analyse the presence of primary cilia in various thyroid cell lines. According to our results, primary cilia are present in the adult thyroid gland of most mammal species we studied (human, pig, guinea pig and rabbit), usually as a single copy per follicular cell. Strikingly, they were not found in rat or mouse thyroid tissues. Similarly, cilia were also observed in all human thyroid cell lines tested, both normal and neoplastic follicular cells, but not in cultured thyrocytes of rat origin. We hypothesize that primary cilia could be involved in the regulation of normal thyroid function through specific signaling pathways. Nevertheless, further studies are needed to shed light on the permanence of these organelles in the thyroid gland of most species during postnatal life.

Dorsal periaqueductal gray post-stimulation freezing is counteracted by neurokinin-1 receptor antagonism in the central nucleus of the amygdala in rats.

Electrical stimulation of the dorsal periaqueductal gray (dPAG) in rats generates defensive responses that are characterized by freezing and escape behaviors, followed by post-stimulation freezing that resembles symptoms of panic attacks. dPAG post-stimulation freezing involves the processing of ascending aversive information to prosencephalic centers, including the amygdala, which allows the animal to evaluate the consequences of stressful situations. The basolateral nucleus of the amygdala (BLA) is thought to act as a filter for innate and learned aversive information that is transmitted to higher structures. The central (CeA) and medial (MeA) nuclei of the amygdala constitute an output for the expression of fear reactions through projections to limbic and brainstem regions. Neurokinin (NK) receptors are abundant in the CeA, MeA, and BLA, but their role in the expression of defensive responses and processing of aversive information that is evoked by electrical stimulation of the dPAG is still unclear. In the present study, we examined the role of NK1 receptors in these amygdala nuclei in the expression of defensive responses induced by electrical stimulation of the dPAG in rats and fear memory of this aversive stimulation. Rats were implanted with an electrode into the dPAG for electrical stimulation and one cannula in the CeA, MeA, or BLA for injections of vehicle (phosphate-buffered saline) or the NK1 receptor antagonist spantide (SPA; 100 pmol/0.2 µl). Injections of SPA into the CeA but not BLA or MeA reduced the duration of post-stimulation freezing evoked by electrical stimulation of the dPAG, without changing the aversive thresholds of freezing or escape. Twenty-four hours later, exploratory behavior was evaluated in the elevated plus maze test (EPM) in the CeA group of rats. Electrical stimulation of the dPAG rats that received vehicle exhibited higher aversion to the open arms of the EPM than sham rats that did not receive any dPAG stimulation. SPA injections into the CeA prevented the proaversive effects of electrical stimulation of the dPAG assessed in the EPM 24 h later. The present results suggest that neurokininergic modulation via NK1 receptors in the CeA but not BLA or MeA is involved in the processing of aversive information derived from dPAG stimulation. The long-lasting consequences of electrical stimulation of the dPAG may be prevented by NK1 receptor antagonism in the CeA.

Sodium calcium exchanger operates in the reverse mode in metastatic human melanoma cells.

Cytosolic Ca2+ ([Ca2+]cyt) is important in the regulation of several cellular functions involved in metastasis. We hypothesize that distinct [Ca2+]cyt regulation explains the acquisition of a more metastatic phenotype. To test this hypothesis, we used highly and lowly metastatic human melanoma cells and [Ca2+]cyt was monitored using Fura—2AM and fluorescence spectroscopy. Stimulation with ATP elicited a sustained increase in [Ca2+]cyt in highly metastatic cells, but a transient increase in lowly metastatic cells. Na+ substitution revealed Na+/Ca2+ exchanger (NCX) activity in reverse mode in highly, but not in lowly metastatic cells. In highly metastatic cells, addition of Na+ in the plateau phase of [Ca2+]cyt increase elicited with ATP, in the absence of Na+, resulted in a rapid return to basal, indicating that NCX can operate in both reverse and forward modes. Inhibition and knockdown of NCX, using KB—R7943 and siRNA NCX—1 respectively, supported the significance of NCX in [Ca2+]cyt regulation in highly metastatic cells. Stimulation with UTP triggered a rapid increase in highly metastatic cells [Ca2+]cyt, but not in lowly metastatic cells suggesting that highly and lowly metastatic cells exhibit distinct purinergic receptors. These data indicate that following agonist—stimulation, NCX operates preferentially in the reverse mode to enable a sustained [Ca2+]cyt increase in highly metastatic cells. The forward mode of NCX operation to extrude Ca2+ is preferred in lowly metastatic cells. The acquisition of a more metastatic phenotype involves a switch in NCX activity from forward to reverse mode that is favorable to maintain elevated [Ca2+]cyt in response to agonist stimulation.

Screening for fragile X syndrome in males from specialized institutions in the northeast region of Brazil.

The objective of this study was to perform a study of fragile X syndrome (FXS) in São Luís, Maranhão, in males residing in five specialized institutions. Two hundred thirty-eight males with intel-lectual disability of unknown etiology participated in this study. Blood samples were processed and stored until DNA extraction. Screening for FMR1 gene mutations was performed using non-isotopic polymerase chain reaction amplification and DNA sequencing using an ABI Prism 3130 automated sequencer. Two individuals (0.84%) were positive for FMR1 mutations. One had a mutation due to expansion of the CGG repeat beyond normal levels and the other had a deletion in exon 1 of the FMR1 gene, which was confirmed by sequencing. Both probands were over 18 years old, which demonstrates the late diagnosis of the condition in these individuals and reinforces the need to implement ef-fective programs for early diagnosis of FXS in the state of Maranhão. We found that FXS might be transmitted in the families of the two indi-viduals bearing the mutation, and that it is important to understand the mutation dynamics to provide better counseling to the family members of these two individuals.

The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation.

Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3-kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin-activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho-aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho-aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle.