An overview of autosomal STRs and identity SNPs in a Norwegian population using massively parallel sequencing.

In recent years, probabilistic genotyping software has been adapted for the analysis of massively parallel sequencing (MPS) forensic data. Likelihood ratios (LR) are based on allele frequencies selected from populations of interest. This study provides an outline of sequence-based (SB) allele frequencies for autosomal short tandem repeats (aSTRs) and identity single nucleotide polymorphisms (iSNPs) in 371 individuals from Southern Norway. 27 aSTRs and 94 iSNPs were previously analysed with the ForenSeq™ DNA Signature Prep Kit (Verogen). The number of alleles with frequencies less than 0.05 for sequenced-based alleles was 4.6 times higher than for length-based alleles. Consistent with previous studies, it was observed that sequence-based data (both with and without flanks) exhibited higher allele diversity compared to length-based (LB) data; random match probabilities were lower for SB alleles confirming their advantage to discriminate between individuals. Two alleles in markers D22S1045 and Penta D were observed with SNPs in the 3´ flanking region, which have not been reported before. Also, a novel SNP with a minor allele frequency (MAF) of 0.001, was found in marker TH01. The impact of the sample size on minor allele frequency (MAF) values was studied in 88 iSNPs from Southern Norway (n = 371). The findings were then compared to a larger Norwegian population dataset (n = 15,769). The results showed that the smaller Southern Norway dataset provided similar results, and it was a representative sample. Population structure was analyzed for regions within Southern Norway; FST estimates for aSTR and iSNPs did not indicate any genetic structure. Finally, we investigated the genetic differences between Southern Norway and two other populations: Northern Norway and Denmark. Allele frequencies between these populations were compared, and we found no significant frequency differences (p-values > 0.0001). We also calculated the pairwise FST values per marker and comparisons between Southern and Northern Norway showed small differences. In contrast, the comparisons between Southern Norway and Denmark showed higher FST values for some markers, possibly driven by distinct alleles that were present in only one of the populations. In summary, we propose that allele frequencies from each population considered in this study could be used interchangeably to calculate genotype probabilities.

Antitumor and antioxidant activities of polysaccharides from the seaweed Durvillaea antarctica.

The present study was carried out to determine the antitumor and antioxidant activities of the seaweed Durvillaea antarctica. Extraction and purification of polysaccharides from D. antarctica were performed. They were characterized by FT-IR and GC-MS, identifying isomers of arabinose, fucose, mannose, and galactose. The antioxidant capacity of polysaccharides was analyzed using the ABTS method (14.3 ± 0.5 µmol TE g-1 PS) and the DPPH method (21.82 ± 0.32 µmol TE g-1 PS). The antitumor capacity of polysaccharides was studied by MTT colorimetric assays in human leukemia, colon, breast, and lung cancer cell lines, obtaining the lowest IC50 in colon cancer (19.99 µg mL-1 ). In the line of healthy human gingival fibroblasts (HGF-1), an IC50 of 444.39 µg mL-1 was obtained. Flow cytometry in the HL60 cell line showed that polysaccharides at concentrations higher than IC50 inhibited cell proliferation, demonstrating a possible antitumor capacity in vitro. In the proteomic analysis with HGF-1, nine proteins involved in different biological processes were identified. In conclusion, polysaccharides from D. antarctica could be considered powerful nutraceuticals, mainly against colon cancer.

Antioxidant, Immunomodulatory and Potential Anticancer Capacity of Polysaccharides (Glucans) from Euglena gracilis G.A. Klebs

The present study was carried out to determine the bioactivity of polysaccharides extracted from Euglena gracilis (EgPs). These were characterized by FT-IR and GC-MS. Cytotoxicity analyses (MTT) were performed on healthy human gingival fibroblast cell lines (HGF-1), obtaining an IC50 of 228.66 µg mL-1, and cell lines with anticancer activity for colon cancer (HCT-116), breast cancer (MCF-7), human leukemia (U-937, HL-60) and lung cancer (NCl-H460), showing that EgPs have anticancer activity, mainly in HTC-116 cells (IC50 = 26.1 µg mL-1). The immunological assay determined the immunomodulatory capacity of polysaccharides for the production of proinflammatory cytokines IL-6 and TNF-α in murine macrophages (RAW 264.7) and TNF-α in human monocytes (THP-1). It was observed that the EgPs had a stimulating capacity in the synthesis of these interleukins. The antioxidant capacity of polysaccharides and their biomass were analyzed using the ABTS method (18.30 ± 0.14% and (5.40 ± 0.56%, respectively, and the DPPH method for biomass (17.79 ± 0.57%). We quantitatively profiled HGF-1 proteins by liquid chromatography-tandem mass spectrometry analysis, coupled with 2-plex tandem mass tag labelling, in normal cells. In total, 1346 proteins were identified and quantified with high confidence, of which five were considered to be overexpressed. The data is available through ProteomeXchange, under identifier PXD029076.

MPSproto: An extension of EuroForMix to evaluate MPS-STR mixtures.

We have developed MPSproto as an extension of EuroForMix to improve handling of stutter artefacts and other typing errors that commonly occur in MPS-STR data. MPSproto implements two models for read depth: gamma and negative binomial. It differs from EuroForMix in that calibration is required before mixtures are interpreted. In this study a mixture dataset (2-4 persons) was revisited, where EuroForMix interpretation of MPS-STR mixtures using the LUS+ format was first described; the performance of this model was compared to the MPSproto models. Results indicated that, overall, the MPSproto models performed better than the conventional EuroForMix model, and the gamma model implemented in MPSproto performed best. Differences were highlighted and further investigated to establish causality. Goodness of fit tests showed that the MPSproto models were adequate for the sequence reads when a low analytical threshold was applied.

A comprehensive characterization of MPS-STR stutter artefacts.

Interpretation of DNA evidence involving mixtures is challenging when alleles from minor contributors coincide with stutters from major contributors. To accommodate this, it is important to have a good understanding of stutter sequence formation trends. Here, multiple stutter types were characterized based on massively parallel sequencing (MPS) data from 387 single source samples, using the Verogen ForenSeq™ DNA Signature Prep kit. A beta regression model was used to investigate the relationship between the stutter proportion and candidate explanatory variables. In the final model, stutter proportions were explained by the length of the parental uninterrupted stretch (PTUS), which is comparable to block length of the missing motif (BLMM). Also, different stutter types (n+1, n-1, n+2, n-2, n0) were analyzed separately per locus. The fitted stutter models were then integrated into an extended probabilistic genotyping model based on EuroForMix (MPSproto). An illustrative minor/major mock mixture example is discussed. Evaluation of multiple types of stutters on a per locus basis improved the probabilistic genotyping result compared to the conventional EuroForMix model, using the LUS+ nomenclature.

Exploring Inflammatory Status in Febrile Seizures Associated with Urinary Tract Infections: A Two-Step Cluster Approach.

BACKGROUND: Urinary tract infections (UTIs) are considered common facilitating factors, along with other infections, in triggering febrile seizures (FS). The main purpose of our study was to identify specific inflammatory patterns of UTI cases from other infections in a specific cluster, using a combination of inflammatory biomarkers to differentiate UTIs from other bacterial diseases triggering FS. METHOD: This prospective study included a number of 136 patients with 197 distinct FS events, from patients hospitalized in the Pediatric Clinical Hospital Sibiu, among which 10.2% were diagnosed with UTIs. RESULTS: In one-third of the patients with UTIs (20 cases), the symptoms were limited to fever and FS. Using two-step cluster analysis, a distinct UTI inflammatory pattern has emerged: highest platelet values (PLT), median value 331 × 103/mm3 and intermediate C-reactive protein (CRP), median value 15 mg/dL, platelet distribution width (PDW), median value 9.65%, platelet-large cell ratio (P-LCR), median value 14.45%, mean platelet volume (MPV), median value 8.60 fL and neutrophil-to-lymphocyte values (NLR), median value 3.64. Furthermore, higher PDW (median value 12.25%), P-LCR (median value 28.55%), MPV (median value 10.40 fL), CRP (median value 74.00 mg/dL) and NLR values (median value 4.11) were associated mainly (85.7%) with bacterial lower respiratory infections. UTIs were highly unlikely in these patients with significantly increased CRP values and normal values of platelet indices. CONCLUSIONS: Considering the nonspecific clinical picture of UTIs at an early age, to optimize the management of FS, a fast diagnosis of UTI is mandatory. The analysis of the inflammatory biomarker clusters (rather than individual parameters) correlated with urine leukocyte and nitrite stick evaluation for specific age groups could help in identifying even oligosymptomatic UTIs patients. The study limitation (20 UTI cases) recommends future multicentric trials on larger datasets to validate the model.

A Decision-Tree Approach to Assist in Forecasting the Outcomes of the Neonatal Brain Injury.

Neonatal brain injury or neonatal encephalopathy (NE) is a significant morbidity and mortality factor in preterm and full-term newborns. NE has an incidence in the range of 2.5 to 3.5 per 1000 live births carrying a considerable burden for neurological outcomes such as epilepsy, cerebral palsy, cognitive impairments, and hydrocephaly. Many scoring systems based on different risk factor combinations in regression models have been proposed to predict abnormal outcomes. Birthweight, gestational age, Apgar scores, pH, ultrasound and MRI biomarkers, seizures onset, EEG pattern, and seizure duration were the most referred predictors in the literature. Our study proposes a decision-tree approach based on clinical risk factors for abnormal outcomes in newborns with the neurological syndrome to assist in neonatal encephalopathy prognosis as a complementary tool to the acknowledged scoring systems. We retrospectively studied 188 newborns with associated encephalopathy and seizures in the perinatal period. Etiology and abnormal outcomes were assessed through correlations with the risk factors. We computed mean, median, odds ratios values for birth weight, gestational age, 1-min Apgar Score, 5-min Apgar score, seizures onset, and seizures duration monitoring, applying standard statistical methods first. Subsequently, CART (classification and regression trees) and cluster analysis were employed, further adjusting the medians. Out of 188 cases, 84 were associated to abnormal outcomes. The hierarchy on etiology frequencies was dominated by cerebrovascular impairments, metabolic anomalies, and infections. Both preterms and full-terms at risk were bundled in specific categories defined as high-risk 75-100%, intermediate risk 52.9%, and low risk 0-25% after CART algorithm implementation. Cluster analysis illustrated the median values, profiling at a glance the preterm model in high-risk groups and a full-term model in the inter-mediate-risk category. Our study illustrates that, in addition to standard statistics methodologies, decision-tree approaches could provide a first-step tool for the prognosis of the abnormal outcome in newborns with encephalopathy.

Immunomodulatory, Antioxidant Activity and Cytotoxic Effect of Sulfated Polysaccharides from Porphyridium cruentum. (S.F.Gray) Nägeli.

Porphyridium cruentum is a unicellular microalga that can synthesize and secrete to the culture medium-high amounts of polysaccharides. In this study, the immunomodulatory, cytotoxic effect and antioxidant activity of the sulfated polysaccharides (PcSPs) were determinate. The PcSPs were precipitated with 2% Cetylpyridinium bromide hydrate and ethanol and purified by dialysis. The extract was lyophilized for its characterization by Fourier transform-Infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). The antioxidant activity of PcSPs were examined with assay 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and compared with that of the biomass, observing significant differences between the results obtained from the PcSPs and biomass. To determine their ability to induce cytokine production Tumor Necrosis Factor alpha (TNF-α) and interleukina-6 (IL-6), the immunomodulatory activity of the PcSPs has been evaluated. In the mouse macrophage cell line (RAW 264.7), PcSPs are potent inducers of IL-6 cytokines but mainly of TNF-α. The cytotoxic capacity of PcSPs was measured by the MTT colorimetric assay in colorectal carcinoma (HTC-116), human leukemia (U-937 and HL-60), breast cancer (MCF-7), lung cancer (NCI-H460) and human gingival fibroblasts (HGF-1) cell lines. The IC50 value of 2311.20 µg mL-1, 1676.74 µg mL-1, 1089.63 µg mL-1, 5498.14 µg mL-1 and 2861.49 µg mL-1 respectively in the tumor lines and 5022.55 µg mL-1 in gingival fibroblasts were obtained. Our study suggested that PcSPs from P. cruentum have a moderate immunomodulatory and cytotoxic effect. The results obtained indicate that the polysaccharides from P. cruentum are potent inducers of IL-6 cytokines and, most importantly, of TNF-α. PcSPs showed no evidence of antigenic activity or hypersensitivity when administered intraperitoneally in mice. Furthermore, the in vivo study revealed an improvement of local inflammatory response against stress in the peritoneum. These findings suggest that the PcSPs from P. cruentum might have potential as a valuable ingredient in nutraceutical products.

Enhancing the Effects of Neurofeedback Training: The Motivational Value of the Reinforcers.

The brain activity that is measured by electroencephalography (EEG) can be modified through operant conditioning, specifically using neurofeedback (NF). NF has been applied to several disorders claiming that a change in the erratic brain activity would be accompanied by a reduction of the symptoms. However, the expected results are not always achieved. Some authors have suggested that the lack of an adequate response may be due to an incorrect application of the operant conditioning principles. A key factor in operant conditioning is the use of reinforcers and their value in modifying behavior, something that is not always sufficiently taken into account. This work aims to clarify the relevance of the motivational value versus the purely informational value of the reinforcer. In this study, 113 subjects were randomly assigned two different reinforcer conditions: a selected reinforcer-the subjects subjectively selected the reinforcers-or an imposed reinforcer-the reinforcers were assigned by the experimenter-and both groups undertook NF sessions to enhance the sensorimotor rhythm (SMR). In addition, the selected reinforcer group was divided into two subgroups: one receiving real NF and the other one sham NF. There were no significant differences between the groups at baseline in terms of SMR amplitude. After the intervention, only those subjects belonging to the selected reinforcer group and receiving real NF increased their SMR. Our results provide evidence for the importance of the motivational value of the reinforcer in Neurofeedback success.

Adherencia a la dieta mediterránea en tutores y residentes de una Unidad Docente Multiprofesional de Atención Familiar y Comunitaria de Tenerife / Adherence to the Mediterranean diet in tutors and residents of a Multiprofessional Teaching Unit for Family and Community Care in Tenerife

INTRODUCCIÓN: Son múltiples los beneficios en salud de la dieta mediterránea. Existen pocos estudios que hayan valorado su adherencia en profesionales sanitarios. OBJETIVOS: Valorar la adherencia a la dieta mediterránea (DM) en tutores y residentes de una Unidad Docente Multiprofesional de Atención Familiar y Comunitaria (UDMAFyC) y su asociación con la edad, género, condición de tutor o residente, profesión y país de origen. MÉTODOS: Se diseñó un estudio descriptivo transversal. La población de estudio la formaron tutores y residentes de Medicina y Enfermería Familiar y Comunitaria de la UDMAFyC Tenerife zona I. Aprovechando un encuentro formativo, cumplimentaron un cuestionario que contenía las variables siguientes: adherencia a la dieta mediterránea (cuestionario MEDAS-14, de 14 ítems; alta adherencia: ≥ 9 puntos, baja adherencia: < 9 puntos), edad, género, ser tutor o residente, profesión (médico o enfermero) y país de origen. RESULTADOS: Participaron 136 profesionales sanitarios, 76 tutores, 56 de Medicina Familiar y Comunitaria (MFyC) y 20 de Enfermería Familiar y Comunitaria (EFyC) y 60 residentes (55 de MFyC y 5 de EFyC). La adherencia a la DM fue alta en 96 profesionales (70,6%), y baja en 40 (29,4%). La adherencia media fue 9,46 (DT: 1,92). No se observaron diferencias estadísticamente significativas entre ésta y el resto de variables. Sí se detectaron diferencias en el cumplimiento de los siguientes ítems del cuestionario en tutores y residentes: consumo de dos o más cucharadas de aceite de oliva al día (73,7% frente a 53,3%; p= 0,014), consumo de tres o más raciones a la semana de pescado y marisco (40,8% frente a 21,7%; p= 0,018) y consumo preferente de carne blanca (84,2% frente a 98,3%; p= 0,005). CONCLUSIONES: Los tutores y residentes de MFyC y EFyC de esta Unidad Docente tienen un grado de adherencia alto a la dieta mediterránea

INTRODUCTION: There are multiple health benefits of the Mediterranean diet. There are few studies that have evaluated its adherence in health professionals. OBJECTIVES: To assess the adherence to the Mediterranean diet in tutors and residents of a Multiprofessional Teaching Unit for Family and Community Care and its association with age, gender, the condition of tutor or resident, their profession and the country of origin. METHODS: A descriptive cross-sectional study was designed. The study population were tutors and residents of Family and Community Medicine and Nursing of the Multiprofessional Teaching Unit of Family and Community Attention of Tenerife zone I. Taking advantage of a training meeting, they completed a questionnaire that contained the following variables: adherence to the Mediterranean diet (MEDAS-14 questionnaire, of 14 items; high adherence: ≥9 points, low adherence: <9 points), age, gender, being a tutor or resident, profession (doctor or nurse) and country of origin. RESULTS: 136 health professionals participated, 76 of them tutors (56 from Family Medicine and 20 from Family Nursing) and 60 residents (55 from Family Medicine and 5 from Family Nursing). Adherence to the Mediterranean diet was high in 96 healthcare professionals (70.6%), and lowin 40 (29.4%). The mean adherence was 9.46 (SD: 1.92). No statistically significant differences were observed between this and the rest of the study variables. Differences were detected in the adherence of the following items of the questionnaire in tutors and residents: consumption of two or more tablespoons of olive oil per day, whose compliance frequency was 73.7% in tutors and 53.3% in residents (p = 0.014), consumption of three or more servings per week of fish and shellfish, which compliance was affirmed by 40.8% of the tutors and by 21.7% of the residents (p = 0.018 ) and preferential consumption of white meat, whose adherence was higher in residents (tutors:84.2; residents: 98.3%; p = 0.005). CONCLUSIONS: Despite the fact that the tutors and residents of Family Medicine and Family Nursing of this Teaching Unit have a high degree of adherence to DM, the consumption of fish or shellfish, legumes and fruit in these professionals can be improved

Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance.

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.

The invention of CRISPR-Cas9 genome editing has unlocked a greater understanding of the human genome. Researchers can use this system to make targeted cuts in any gene in the genome, forcing the cell to perform a rapid repair at the cut site. These repairs often introduce mutations into the damaged area, adding or removing DNA letters and disrupting the gene. This allows researchers to study what happens to cells when specific genes are missing, which can help to uncover what each gene is for. One of the most comprehensive ways to use this technique is to perform a CRISPR-Cas9 screen, which disrupts each gene in the genome one by one. For a CRISPR-Cas9 screen to work well, a cell needs to survive the cuts to its genome. But there is a crucial gene that can stop this happening. Often described as the 'guardian of the genome', this gene codes for a protein called p53, a tumour suppressor that helps to stop a cell turning cancerous when its DNA becomes damaged. This protein activates when the cell senses a cut in its genetic material and can kill the cell if it fails to make a successful repair. Recent work has shown that the presence of a working copy of the gene for the p53 protein might limit the ability of CRISPR-Cas9 to edit genes. But the evidence was inconclusive. So, Bowden, Morales-Juarez et al. performed two parallel CRISPR-Cas9 screens in human cells with and without p53 to find out more. This revealed that CRISPR-Cas9 can inactivate genes in both normal cells and cells lacking the p53 protein, but that it works better in cells without p53. This was because, when p53 was active, the cells initiated a protective response against the CRISPR-Cas9 cuts. This changed the patterns of genes successfully inactivated by the screen, but it did not make the results unusable. Careful experimental design and thorough data analysis made it possible to get useful results even in cells with functional p53 protein. The gene for p53 has mutations in around half of human cancers. So, understanding how it affects CRISPR-Cas9 screens could influence the design of future experiments. It is possible that the effects of the p53 protein could vary from cell type to cell type, and with different p53 mutations. Comparisons like the one performed here could help to further unpick how the cell's DNA repair systems might interfere with future CRISPR experiments.