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Background: Primary ciliary dyskinesia (PCD) is a congenital disorder characterized by chronic respiratory morbidity. To date, there is no information on PCD-specific preference-based quality of life measures such as health utilities (HU). We cross-sectionally assessed HU in adult PCD patients and explored relationships with genotype, phenotype and quality of life (QOL)-PCD scales.Methods: Diagnostic testing was performed according to international guidelines, while participants completed the visual analog scale (VAS), time trade off (TTO), standard gamble (SG), and EuroQol 5 dimensions (EQ5D) HU instruments, as well as the QOL-PCD questionnaire. Hierarchical regression was used to identify the QOL-PCD scales that are most predictive of HU.Results: Among 31 patients, median HU are 0.75 (VAS), 0.86 (EQ5D), 0.91 (TTO) and 0.99 (SG). The underlying genotype is not associated with HU measures. VAS and EQ5D are associated with lung function, while TTO and SG values are not sensitive to any of the examined factors. Among the QOL-PCD scales, physical functioning and lower respiratory symptoms explained much of VAS (R2= 0.419) and EQ5D (R2= 0.538) variability.Conclusions: Our study demonstrates that HU elicitation in PCD is feasible using both direct and indirect methods. Overall, HU scores are relatively high among adult patients, with higher scores observed in SG and TTO, followed by EQ5D and VAS. VAS and EQ5D HU values are sensitive to lung function as well as to QOL-PCD physical functioning and lower respiratory symptom scores.

Background Next-generation sequencing (NGS) represents a significant advancement in clinical genetics. However, its use creates several technical, data interpretation and management challenges. It is essential to follow a consistent data analysis pipeline to achieve the highest possible accuracy and avoid false variant calls. Herein, we aimed to compare the performance of twenty-eight combinations of NGS data analysis pipeline compartments, including short-read mapping (BWA-MEM, Bowtie2, Stampy), variant calling (GATK-HaplotypeCaller, GATK-UnifiedGenotyper, SAMtools) and interval padding (null, 50 bp, 100 bp) methods, along with a commercially available pipeline (BWA Enrichment, Illumina®). Fourteen germline DNA samples from breast cancer patients were sequenced using a targeted NGS panel approach and subjected to data analysis. Results We highlight that interval padding is required for the accurate detection of intronic variants including spliceogenic pathogenic variants (PVs). In addition, using nearly default parameters, the BWA Enrichment algorithm, failed to detect these spliceogenic PVs and a missense PV in the TP53 gene. We also recommend the BWA-MEM algorithm for sequence alignment, whereas variant calling should be performed using a combination of variant calling algorithms; GATK-HaplotypeCaller and SAMtools for the accurate detection of insertions/deletions and GATK-UnifiedGenotyper for the efficient detection of single nucleotide variant calls. Conclusions These findings have important implications towards the identification of clinically actionable variants through panel testing in a clinical laboratory setting, when dedicated bioinformatics personnel might not always be available. The results also reveal the necessity of improving the existing tools and/or at the same time developing new pipelines to generate more reliable and more consistent data.

Assessment of dietary intake is fundamental for evaluating the interrelationships between diet and disease. The present study aimed to develop and validate the semiquantitative Cypriot food frequency questionnaire (CyFFQ).A 171-item paper-and-pencil semiquantitative interview-administered FFQ was developed, including local foods and culturally specific meals commonly consumed among Cypriot adults. FFQ reproducibility was assessed by comparing the energy-adjusted daily macro- and micronutrients intake at baseline (FFQ1) and 1 year later (FFQ2) using a Wilcoxon matched pairs signed rank test and intraclass correlation coefficients (ICCs) in a random sample of Cypriot adults. FFQ relative validity was evaluated by comparing the intake as estimated by FFQ2 with that obtained from the average of three 24-h recalls taken over the year between FFQ1 and FFQ2. Associations between nutrient intakes estimated using FFQ2 and the 24-h recalls were assessed using Spearman rank correlation and Bland-Altman plots were used to assess agreement between the FFQ and the 24-h recalls.Among eligible participants, 68 (78%) completed the study (44.1% males, aged 30.5-47.5 years). The energy-adjusted intakes of macro- and micronutrients did not significantly differ between the two FFQs, excluding magnesium. The FFQ2 and the averaged 24-h recalls were significantly correlated for most macro- and micronutrients. The median (interquartile) ICC for all macro- and micronutrients was 0.46 (0.38-0.52) (p 0.05). Agreement was satisfactory (30%) for most micro- and macronutrients. Bland-Altman plots also confirmed good agreement between the two methods.The CyFFQ is a valid and reliable tool for assessing dietary consumption in Cypriot adults.

Aim Alport syndrome (AS) is the second most common hereditary kidney disease caused by mutations in collagen IV genes. Patients present with microhaematuria that progressively leads to proteinuria and end stage renal disease. Currently, no specific treatment exists for AS. Using mass spectrometry based proteomics, we aimed to detect early alterations in molecular pathways implicated in AS before the stage of overt proteinuria, which could be amenable to therapeutic intervention. Methods Kidneys were harvested from male Col4a3 −/− knock out and sex and age‐matched Col4a3 +/+ wild‐type mice at 4 weeks of age. Purified peptides were separated by liquid chromatography and analysed by high resolution mass spectrometry. The Cytoscape bioinformatics tool was used for function enrichment and pathway analysis. PPARα expression levels were evaluated by immunofluorescence and immunoblotting. Results Proteomic analysis identified 415 significantly differentially expressed proteins, which were mainly involved in metabolic and cellular processes, the extracellular matrix, binding and catalytic activity. Pathway enrichment analysis revealed among others, downregulation of the proteasome and PPAR pathways. PPARα protein expression levels were observed to be downregulated in Alport mice, supporting further the results of the discovery proteomics. Conclusion This study provides additional evidence that alterations in proteins which participate in cellular metabolism and mitochondrial homeostasis in kidney cells are early events in the development of chronic kidney disease in AS. Of note is the dysregulation of the PPAR pathway, which is amenable to therapeutic intervention and provides a new potential target for therapy in AS., SUMMARY AT A GLANCE Alterations in proteins which participate in cellular metabolism and mitochondrial homeostasis in kidney cells, are early events in the development of CKD in Alport syndrome. In addition, pathway enrichment analysis revealed among others, dysregulation of the proteasome, protein digestion and absorption pathways as well as PPAR pathway in AS compared to control mice. Targeting the PPAR pathway could be a potential therapeutic approach in AS.

// Lola Koniali 1 , * , Andreas Hadjisavvas 1 , 2 , * , Anastasia Constantinidou 3 , Kyproula Christodoulou 2 , 4 , Yiolanda Christou 5 , Christiana Demetriou 6 , Andreas S. Panayides 7 , Constantinos Pitris 8 , Constantinos S. Pattichis 7 , Eleni Zamba-Papanicolaou 2 , 5 and Kyriacos Kyriacou 1 , 2 1 Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 2 The Cyprus School of Molecular Medicine, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 3 Medical School, University of Cyprus and the Bank of Cyprus Oncology Centre, Nicosia, Cyprus 4 Neurogenetics Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 5 Neurology Clinic D, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 6 Department of Primary Care and Population Health, University of Nicosia Medical School, Nicosia, Cyprus 7 Department of Electrical and Computer Engineering, School of Engineering, University of Cyprus, Nicosia, Cyprus 8 Department of Computer Science, University of Cyprus, Nicosia, Cyprus * These authors contributed equally to this work Correspondence to: Kyriacos Kyriacou, email: kyriacos@cing.ac.cy Keywords: breast cancer; brain metastases; risk factors; biomarkers Received: August 21, 2019 Accepted: January 04, 2020 Published: February 11, 2020 ABSTRACT Background: Brain metastasis (BM) is an increasingly common and devastating complication of breast cancer (BC). Methods: A systematic literature search of EMBASE and MEDLINE was conducted to elucidate the current state of knowledge on known and novel prognostic factors associated with 1) the risk for BCBM and 2) the time to brain metastases (TTBM). Results: A total of 96 studies involving institutional records from 28 countries were identified. Of these, 69 studies reported risk factors of BCBM, 46 factors associated with the TTBM and twenty studies examined variables for both outcomes. Young age, estrogen receptor negativity (ER-), overexpression of human epidermal factor (HER2+), and higher presenting stage, histological grade, tumor size, Ki67 labeling index and nodal involvement were consistently found to be independent risk factors of BCBM. Of these, triple-negative BC (TNBC) subtype, ER-, higher presenting histological grade, tumor size, and nodal involvement were also reported to associate with shorter TTBM. In contrast, young age, hormone receptor negative (HR-) status, higher presenting stage, nodal involvement and development of liver metastasis were the most important risk factors for BM in HER2-positive patients. Conclusions: The study provides a comprehensive and individual evaluation of the risk factors that could support the design of screening tools and interventional trials for early detection of BCBM.

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2′O-Methyl (2′OMe)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2′OMe AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2′OMe AON and LNA/2′OMe chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2′OMe AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.

Simple Summary Breast cancer is the most commonly diagnosed type of cancer in women worldwide. Stratification of women based on their individual breast cancer risk could guide targeted preventative strategies and population screening. Integrated models that combine the effects of a polygenic risk score (PRS) with classical breast cancer risk factors could provide an individualized breast-cancer risk estimation. Although various studies have extensively evaluated the performance of such integrated models in populations of European ancestry, no previous studies have included individuals of Greek-Cypriot origin. To this end, we have assessed the predictive performance of a 15-SNP PRS (PRS15), in combination with classical breast-cancer risk factors, in women of Greek-Cypriot origin. This proof-of-concept study suggests that models combining genetic data with classical risk factors may be used in the future for the prediction of breast-cancer risk and, therefore, supports their potential clinical utility for targeted preventative strategies in Cypriot women. Abstract The PRS combines multiplicatively the effects of common low-risk single nucleotide polymorphisms (SNPs) and has the potential to be used for the estimation of an individual’s risk for a trait or disease. PRS has been successfully implemented for the prediction of breast cancer risk. The combination of PRS with classical breast cancer risk factors provides a more comprehensive risk estimation and could, thus, improve risk stratification and personalized preventative strategies. In this study, we assessed the predictive performance of the combined effect of PRS15 with classical breast-cancer risk factors in Cypriot women using 1109 cases and 1177 controls from the MASTOS study. The PRS15 was significantly associated with an increased breast cancer risk in Cypriot women OR (95% CI) 1.66 (1.25–2.19). The integrated risk model obtained an AUC (95% CI) 0.70 (0.67–0.72) and had the ability to stratify women according to their disease status at the extreme deciles. These results provide evidence that the combination of PRS with classical risk factors may be used in the future for the stratification of Cypriot women based on their disease risk, and support its potential clinical utility for targeted preventative actions and population screening.

The purpose of this study was to evaluate the neuroprotective effects of omega-3 polyunsaturated fatty acid (ω3-PUFA) supplementation in a mouse model of OPA1-associated autosomal dominant optic atrophy (ADOA). The blood level of arachidonic acid (AA) and eicosapentaenoic acid (EPA) served to adjust the treatment dosage (AA/EPA = 1.0-1.5). Eight-month-old mice were allocated to four groups (n = 20/group): the ω3-PUFA-treated Opa1

Objective To examine whether the employees of an oil and gas company in Shaanxi Province are more vulnerable to hypertension than the general residents, aged 18-60, living in the same geographical region. Design Cross-sectional analysis of the hypertension prevalence of two different population by using propensity score matching (PSM). Participants Employees of Changqing Oil Field Filiale took part in Health Risk Factors Survey (2013) and residents took part in the National Health Service Survey (2013) in Shaanxi Province, China. Main outcome measures The primary outcome was hypertention which was defined as systolic blood pressure of at least 140 mm Hg, or diastolic blood pressure of at least 90 mm Hg, or self-reported antihypertensive medication use in the previous 2 weeks, or self-reported history of hypertention. Results Changqing employees were much younger, with a higher proportion of men, and their lifestyle was less healthy, with higher BMI, more drinkers and smokers. Before PSM, hypertension prevalence of Changqing employees was slightly lower than Shaanxi residents (5.8% vs 7.6%). After PSM, the results of the adjusted logistic model showed that Changqing employees were more likely to be hypertensive (OR =1.10, 95% CI =1.02-1.19, P =0.01). Among the male samples, Changqing employees were more likely to be hypertensive (OR =1.43, 95% CI =1.31-1.58, P Conclusion Changqing’s employees, as a whole, and male employees in particular, were more vulnerable to hypertension than the general residents of the region. However, the reverse was found for female employees.

We aimed to determine genetic diagnosis in the national primary ciliary dyskinesia (PCD) cohort of Cyprus, an island with a high disease prevalence. We used targeted next-generation sequencing (NGS) of 39 PCD genes in 48 patients of Greek-Cypriot and other ancestries. We achieved molecular diagnosis in 74% of the unrelated families tested. We identified 24 different mutations in 11 genes, 12 of which are novel. Homozygosity was more common in Greek-Cypriot than non-Greek-Cypriot patients (88% vs 46.2%, p =0.016). Four mutations (DNAH11:c.5095-2A>G, CFAP300:c.95_103delGCCGGCTCC, TTC25:c.716G>A, RSPH9:c.670+2T>C,) were found in 74% of the diagnosed Greek-Cypriot families. Patients with RSPH9 mutations demonstrated higher nasal nitric oxide (57nL/min Vs 15 nL/min, p

Additional file 2: Table S2. EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of NGS applications.

Additional file 1: Table S1. TruSight Cancer (Illumina) target genes in alphabetical order.

Additional file 3: Figure S1. Sequencing and Mapping Evaluation. a. Number of reads and clusters generated per run. b Number of unmapped reads per sample and alignment method.

Additional file 9. Detailed commands and parameters used for data pre-processing, sequence alignment, post-alignment processing and variant discovery.

Additional file 7: Figure S3. Box plot of Matthew Correlation coefficient (MCC) comparisons per alignment, variant calling or padding method. Each dot represents one observation and horizontal bold lines denote median MCC values. Boxes extend from the 25th to the 75th percentile of each group’s distribution of values. Vertical extending lines (whiskers) denote the upper and lower adjacent values. Statistical analyses were performed using the non-parametric Kruskal-Wallis or one-way ANOVA tests.

Additional file 8: Figure S4. Box plot comparisons of False Positive Rates (FPR). Only statistically significant differences are shown. Each dot represents one observation and horizontal bold lines denote median FPR values. Boxes extend from the 25th to the 75th percentile of each group’s distribution of values. Vertical extending lines (whiskers) denote the upper and lower adjacent values. Statistical analysis was performed using the non-parametric Kruskal-–Wallis test.

Additional file 4: Table S3. Number of reads per sample and mapping tool.

Additional file 5: Table S4. Minimum, mean and maximum depth of coverage, per gene and alignment algorithm. Depth of coverage results upon NextSeq 500 High-Output kit 2x75 cycles and 2x150 cycles sequencing.

Additional file 6: Figure S2. Hierarchical clustering of the tools. The d, MCC, p, r and F1 values were used to perform hierarchical clustering analysis based on the Lance–Williams agglomerative hierarchical clustering algorithm, which at each stage recomputes dissimilarities between clusters.

Simple Summary In Cyprus, approximately 9% of triple-negative (negative in common breast cancer receptors—estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) receptors) breast cancer (TNBC) patients carry inherited mutations in the BRCA1/2 breast cancer (BC) susceptibility genes. These mutations increase the risk of BC. However, the contribution of other BC susceptibility genes has not yet been determined. To this end, we aimed to investigate the prevalence of mutations in BRCA1/2-negative TNBC patients in Cyprus. Ninety-five cancer susceptibility genes were sequenced for 163 TNBC patients. The frequency of non-BRCA mutations and especially PALB2 in TNBC patients in Cyprus appears to be higher compared to other populations, and half of the mutation-positive patients were diagnosed over the age of 60 years. Based on these results, we believe that PALB2 and TP53 along with BRCA1/2 genetic testing could be beneficial for a large proportion of TNBC patients in Cyprus, irrespective of their age of diagnosis. Abstract In Cyprus, approximately 9% of triple-negative (estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative) breast cancer (TNBC) patients are positive for germline pathogenic variants (PVs) in BRCA1/2. However, the contribution of other genes has not yet been determined. To this end, we aimed to investigate the prevalence of germline PVs in BRCA1/2-negative TNBC patients in Cyprus, unselected for family history of cancer or age of diagnosis. A comprehensive 94-cancer-gene panel was implemented for 163 germline DNA samples, extracted from the peripheral blood of TNBC patients. Identified variants of uncertain clinical significance were evaluated, using extensive in silico investigation. Eight PVs (4.9%) were identified in two high-penetrance TNBC susceptibility genes. Of these, seven occurred in PALB2 (87.5%) and one occurred in TP53 (12.5%). Interestingly, 50% of the patients carrying PVs were diagnosed over the age of 60 years. The frequency of non-BRCA PVs (4.9%) and especially PALB2 PVs (4.3%) in TNBC patients in Cyprus appears to be higher compared to other populations. Based on these results, we believe that PALB2 and TP53 along with BRCA1/2 genetic testing could be beneficial for a large proportion of TNBC patients in Cyprus, irrespective of their age of diagnosis.

Background Approximately 50% of systemic lupus erythematosus (SLE) patients develop nephritis, which is among the most severe and frequent complications of the disease and a leading cause of morbidity and mortality. Despite intensive research, there are still no reliable lupus nephritis (LN) markers in clinical use that can assess renal damage and activity with a high sensitivity and specificity. To this end, the aim of this study was to identify new clinically relevant tissue-specific protein biomarkers and possible underlying molecular mechanisms associated with renal involvement in SLE, using mass spectrometry (MS)-based proteomics. Methods Kidneys were harvested from female triple congenic B6.NZMsle1/sle2/sle3 lupus mice model, and the respective sex- and age-matched C57BL/6 control mice at 12, 24 and 36 weeks of age, representing pre-symptomatic, established and end-stage LN, respectively. Proteins were extracted from kidneys, purified, reduced, alkylated and digested by trypsin. Purified peptides were separated by liquid chromatography and analysed by high-resolution MS. Data were processed by the Progenesis QIp software, and functional annotation analysis was performed using DAVID bioinformatics resources. Immunofluorescence and multiple reaction monitoring (MRM) MS methods were used to confirm prospective biomarkers in SLE mouse strains as well as human serum samples. Results Proteomic profiling of kidney tissues from SLE and control mice resulted in the identification of more than 3800 unique proteins. Pathway analysis revealed a number of dysregulated molecular pathways that may be mechanistically involved in renal pathology, including phagosome and proximal tubule bicarbonate reclamation pathways. Proteomic analysis supported by human transcriptomic data and pathway analysis revealed Coronin-1A, Ubiquitin-like protein ISG15, and Rho GDP-dissociation inhibitor 2, as potential LN biomarkers. These results were further validated in other SLE mouse strains using MRM-MS. Most importantly, experiments in humans showed that measurement of Coronin-1A in human sera using MRM-MS can segregate LN patients from SLE patients without nephritis with a high sensitivity (100%) and specificity (100%). Conclusions These preliminary findings suggest that serum Coronin-1A may serve as a promising non-invasive biomarker for LN and, upon validation in larger cohorts, may be employed in the future as a screening test for renal disease in SLE patients.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited, multisystemic disorder characterized clinically by delayed muscle relaxation and weakness. The disease is caused by a CTG repeat expansion in the 3' untranslated region (3' UTR) of the DMPK gene, which leads to the expression of a toxic gain-of-function mRNA. The expanded CUG repeat mRNA sequesters the MBNL1 splicing regulator in nuclear-retained foci structures, resulting in loss of protein function and disruption of alternative splicing homeostasis. In this study, we used CAG repeat antisense oligonucleotides (ASOs), composed of locked nucleic acid (LNA)- and 2'-O-methyl (2'OMe)-modified bases in a chimeric design, to alleviate CUGexpanded-mediated toxicity. Chimeric 14-18mer LNA/2'OMe oligonucleotides, exhibiting an LNA incorporation of ∼33%, significantly ameliorated the misregulated alternative splicing of Mbnl1-dependent exons in primary DM1 mouse myoblasts and tibialis anterior muscles of DM1 mice. Subcutaneous delivery of 14mer and 18mer LNA/2'OMe chimeras in DM1 mice resulted in high levels of accumulation in all tested skeletal muscles, as well as in the diaphragm and heart tissue. Despite the efficient delivery, chimeric LNA/2'OMe oligonucleotides were not able, even at a high-dosage regimen (400 mg/kg/week), to correct the misregulated splicing of Serca1 exon 22 in skeletal muscles. Nevertheless, oligonucleotide doses were well-tolerated as determined by histological and plasma biochemistry analyses. Our results provide proof of concept that inhibition of MBNL1 sequestration by systemic delivery of a steric-blocking ASO is extremely challenging, considering the large number of target sites that need to be occupied per RNA molecule. Although not suitable for DM1 therapy, chimeric LNA/2'OMe oligonucleotides could prove to be highly beneficial for other diseases, such as Duchenne muscular dystrophy, that require inhibition of a single target site per RNA molecule.

Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionarily conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing ER structures. We demonstrate that the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process that does not depend on autophagosome–lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation. This article has an associated First Person interview with the first author of the paper., Highlighted Article: ER stress in mouse myoblasts activates a cell survival mechanism that involves the upregulation of Stbd1 expression that is required for the formation of glycogen-containing structures.

© 2020 The Author(s)., [Introduction]: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. [Areas covered]: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. [Expert commentary]: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine., This paper was funded by the European cooperation in science and technology - COST action No. CA16113 - CliniMARK: ‘good biomarker practice’ to increase the number of clinically validated biomarkers.

Regularly sized platinum electrodes were modified with a layer of thermosensitive hydrogel based on poly(N-isopropylacrylamide) and containing different amounts of crosslinker. The layers were formed by using the electrochemically induced free radical polymerization. The transport of electroactive species through the layers was examined in function of crosslinker content and for two possible forms of the hydrogel. It was found that in the shrunken state, within a specific scan-range, the shape of the voltammograms was typical for a set of independent micro/nanoelectrodes, while in the swollen state of the layers the voltammograms were peak-shaped in the entire scan-rate range. This was a result of either through-film transport (swollen layer) or pinhole/pore (shrunken layer) diffusion of electroactive species. 2% content of crosslinker caused the biggest limitation to the probe transport to the electrode surface.

The evolution of Next Generation Sequencing (NGS) technologies represents a significant advancement in the field of molecular genetics and has set the ground, for the discovery of novel variants which cannot be easily classified as deleterious or neutral. In-vitro and in-vivo characterization of these variants of uncertain clinical significance (VUS) should be followed; however, it is often not feasible to carry out the experimental interpretation for every single VUS. In silico tools have been crucial for the prediction of the impact of VUS on protein structure, stability and function. Our aim was to combine computational approaches to investigate the impact of VUS identified in a cohort of Cypriot Triple-Negative Breast Cancer (TNBC) patients by NGS. Using a combination of structural, functional and network-based bioinformatics approaches for the classification of a nonsense PRF1 mutation in association with BC susceptibility, we propose a possible triggered interaction of the mutant PRF1 protein with the CDKN2A protein, a product of a BC susceptibility gene. Additionally, our results support that the increased probability of interaction of the mutant counterpart of perforin with its top 10 predicted interactors, could play an important role in the obstruction of cellular processes related to carcinogenesis such as cell death, necrosis, DNA damage, immortality, UV stress, DNA repair and cell cycle control. We conclude that probably the nonsense PRF1 mutation could be associated with BC predisposition. However, although in silico tools provide an important tool for the interpretation of VUS, functional studies, co-segregation analyses and/or case-control association studies are needed to draw conclusions on variant classification.

© The Author(s) 2019. This paper examines financial spillovers between the four largest equity markets (by market capitalization) in the GCC region using a VAR-GARCH (1,1) framework that sheds light on interdependence as well as the effects of the 2014 oil crisis. Since the UAE is a federation including two stock exchanges (Abu Dhabi and Dubai), it is pos- sible to test whether being part of a federal union matters more than market size in terms of financial integration. Our results suggest that the latter is more important, since we could not find evidence of stronger linkages between the Abu Dhabi and Dubai markets compared to those between other markets in the region. By contrast, there are significant spillover effects, both in the mean and in the volatility, from the largest market of Saudi Arabia to Qatar and the two markets in the UAE, which confirms that market capitalization is a more important determinant of financial inte- gration than belonging to a federal union. Further, spillovers from the larger markets have become stronger as a result of the 2014 oil crisis. Finally, there is also evidence of spillovers from the smaller to the larger markets. Zayed University, RIF grant R16045.

ObjectiveTo evaluate the therapeutic effects of omega-3 (ω3) fatty acids in the retina of aged mice when the blood arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is maintained between 1.0 and 1.5.Methods and analysisAged (24-month-old) wild-type C57BL/6J mice were allocated to two groups: ω3 treated and untreated. Treatment with ω3 was by daily gavage administration of EPA and docosahexaenoic acid for 60 days. Gas chromatography was used to identify and quantify fatty acids in the blood and retina. To count lipofuscin granules and measure the photoreceptor layer, eyecups were examined histologically using transmission electron microscopy and light microscopy. We also analysed eyecups using mass spectrometry-based proteomics.ResultsAA levels were lower, and EPA levels were higher, in the blood and retinas of the ω3-treated group than in the untreated group, resulting in a lower AA/EPA ratio. The ω3-treated group also showed significantly fewer lipofuscin granules and a thicker outer nuclear layer than the untreated group. Proteomic analysis revealed significantly greater expression of myelin basic protein, myelin regulatory factor-like protein, myelin proteolipid protein and glial fibrillar acidic protein in the ω3-treated group than in the untreated group. Three different pathways were significantly affected by ω3 treatment: fatty acid elongation, biosynthesis of unsaturated fatty acids and metabolic pathways.ConclusionTwo months of ω3 supplementation (when the blood AA/EPA~1.0–1.5) in aged mice reduced lipofuscin granule formation in the retina and protected the photoreceptor layer, suggesting that ω3 supplementation slows normal age-related retinal degeneration.

BESTCILIA Diagnostic Algorithm for PCD. (PDF 188 kb)

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and 19F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENEHD-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine 19F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold 19F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac 19F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with 19F MRI.

Technical Appendix. (DOCX 20 kb)

Secondary Analysis. (DOCX 169 kb)

This paper presents the largest study in Cyprus evaluating the frequency and distribution of BRCA1/2 mutations in a high risk patient cohort. Deleterious mutations in the BRCA1/2 genes were identified in 68 of the 527 patients tested (13%). It is of interest that a quarter of those tested positive, did not have an extensive family history of breast/ovarian cancer but were diagnosed with early onset breast cancer, ovarian cancer under the age of 60 or triple negative breast cancer. The spectrum of mutations identified in our patient cohort is different compared to other Mediterranean countries. Furthermore, several of the mutations detected are novel and have not been identified in other ethnic populations. This highlights the importance of operating a national reference center for cancer genetic diagnosis which offers services tailored to the needs of the Cypriot population.