Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease.

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

Phenotype-aware prioritisation of rare Mendelian disease variants.

A molecular diagnosis from the analysis of sequencing data in rare Mendelian diseases has a huge impact on the management of patients and their families. Numerous patient phenotype-aware variant prioritisation (VP) tools have been developed to help automate this process, and shorten the diagnostic odyssey, but performance statistics on real patient data are limited. Here we identify, assess, and compare the performance of all up-to-date, freely available, and programmatically accessible tools using a whole-exome, retinal disease dataset from 134 individuals with a molecular diagnosis. All tools were able to identify around two-thirds of the genetic diagnoses as the top-ranked candidate, with LIRICAL performing best overall. Finally, we discuss the challenges to overcome most cases remaining undiagnosed after current, state-of-the-art practices.

Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone-marrow-failure disorder characterized by a triad of mucocutaneous features that include abnormal skin pigmentation, nail dystrophy, and oral leucoplakia. Despite the identification of several genetic variants that cause DC, a significant proportion of probands remain without a molecular diagnosis. In a cohort of eight independent DC-affected families, we have identified a remarkable series of heterozygous germline variants in the gene encoding thymidylate synthase (TYMS). Although the inheritance appeared to be autosomal recessive, one parent in each family had a wild-type TYMS coding sequence. Targeted genomic sequencing identified a specific haplotype and rare variants in the naturally occurring TYMS antisense regulator ENOSF1 (enolase super family 1) inherited from the other parent. Lymphoblastoid cells from affected probands have severe TYMS deficiency, altered cellular deoxyribonucleotide triphosphate pools, and hypersensitivity to the TYMS-specific inhibitor 5-fluorouracil. These defects in the nucleotide metabolism pathway resulted in genotoxic stress, defective transcription, and abnormal telomere maintenance. Gene-rescue studies in cells from affected probands revealed that post-transcriptional epistatic silencing of TYMS is occurring via elevated ENOSF1. These cell and molecular abnormalities generated by the combination of germline digenic variants at the TYMS-ENOSF1 locus represent a unique pathogenetic pathway for DC causation in these affected individuals, whereas the parents who are carriers of either of these variants in a singular fashion remain unaffected.

A comparative study of mirror self-recognition in three corvid species.

Mirror self-recognition (MSR) assessed by the Mark Test has been the staple test for the study of animal self-awareness. When tested in this paradigm, corvid species return discrepant results, with only the Eurasian magpies and the Indian house crow successfully passing the test so far, whereas multiple other corvid species fail. The lack of replicability of these positive results and the large divergence in applied methodologies calls into question whether the observed differences are in fact phylogenetic or methodological, and, if so, which factors facilitate the expression of MSR in some corvids. In this study, we (1) present new results on the self-recognition abilities of common ravens, (2) replicate results of azure-winged magpies, and (3) compare the mirror responses and performances in the mark test of these two corvid species with a third corvid species: carrion crows, previously tested following the same experimental procedure. Our results show interspecies differences in the approach of and the response to the mirror during the mirror exposure phase of the experiment as well as in the subsequent mark test. However, the performances of these species in the Mark Test do not provide any evidence for their ability of self-recognition. Our results add to the ongoing discussion about the convergent evolution of MSR and we advocate for consistent methodologies and procedures in comparing this ability across species to advance this discussion.

Quantifying Multiple Work-Related Psychosocial Risk Factors: Proposal for a Composite Indicator Based on the COPSOQ II.

PURPOSE: To determine national reference values for the Copenhagen Psychosocial Questionnaire (COPSOQ II) across occupational sectors and develop a composite score to estimate the cumulative effect of multiple work-related stressors, in order to facilitate the implementation of occupational health directives on psychosocial risk assessment. METHOD: Cross-sectional data was collected via an online questionnaire. The sample included 13,104 individuals and was representative of the general Hungarian adult working population in terms of gender, age, education, and occupation. Mean scores were calculated for 18 scales on work environment and for 5 outcome scales of the COPSOQ II across 18 occupational sectors. We analyzed the association between a composite psychosocial risk score (CPRS), reflecting severity of exposure to multiple risk factors, and high stress, burnout, sleep troubles, and poor self-rated health. RESULTS: We found occupation-related differences in the mean scores on all COPSOQ II scales. Scores on the "Stress" scale ranged from 47.9 to 56.2, with the highest mean score in accommodation and food services sector. Variability was greatest with respect to emotional demands (range 40.3-67.6) and smallest with respect to role clarity (range 70.3-75.7). The prevalence of negative health outcomes increased with the CPRS. Five risk categories were formed, for which the odds ratio of negative outcomes ranged from 1.6 to 56.5. CONCLUSION: The sector-specific psychosocial risk profiles covering 18 work environmental factors can be used as a reference in organizational surveys and international comparisons. The CPRS proved to be a powerful predictor of self-reported negative health outcomes.

[The relationship of work-related psychosocial risk factors with depressive symptoms among Hungarian workers: preliminary results of the Hungarian Work Stress Survey]. / Munkahelyi pszichoszociális kockázati tényezok kapcsolata a depressziós tünetegyüttessel a magyar munkavállalók körében. Az Országos Munkahelyi Stresszfelmérés elozetes eredményei.

INTRODUCTION: Research has shown that psychosocial stress acts as a risk factor for mental disorders. AIM: The present study aims at processing the preliminary results of the Hungarian Survey of Work Stress, concerning the relationship between depressive symptoms and work stress. METHODS: Cross-sectional survey among Hungarian workers was carried out (n = 1058, 27.5% man, 72.5% woman, age 37.2 years, SD = 11 years). Psychosocial factors were measured using the COPSOQ II questionnaire, while BDI-9 was used for the assessment of depressive symptoms. Statistical analysis was carried out applying Spearman's correlation and logistic regression. RESULTS: A quarter of the workers reported moderate or severe symptoms of depression (BDI≥19). The study confirmed the association between depressive symptoms and work-family conflict (OR = 2.21, CI: 1.82-2.68), possibilities for development (OR = 0.76, CI: 0.59-0.97) meaning of work (OR = 0.69, CI: 0.59-0.89) and commitment (OR = 0.60, CI: 0.47-0.78). CONCLUSION: The results point toward the need of such organizational measures that allow for the reduction of psychosocial stress.

Autosomal dominant stromal corneal dystrophy associated with a SPARCL1 missense variant.

Corneal dystrophies are phenotypically and genetically heterogeneous, often resulting in visual impairment caused by corneal opacification. We investigated the genetic cause of an autosomal dominant corneal stromal dystrophy in a pedigree with eight affected individuals in three generations. Affected individuals had diffuse central stromal opacity, with reduced visual acuity in older family members. Histopathology of affected cornea tissue removed during surgery revealed mild stromal textural alterations with alcianophilic deposits. Whole genome sequence data were generated for four affected individuals. No rare variants (MAF < 0.001) were identified in established corneal dystrophy genes. However, a novel heterozygous missense variant in exon 4 of SPARCL1, NM_004684: c.334G > A; p.(Glu112Lys), which is predicted to be damaging, segregated with disease. SPARC-like protein 1 (SPARCL1) is a secreted matricellular protein involved in cell migration, cell adhesion, tissue repair, and remodelling. Interestingly, SPARCL1 has been shown to regulate decorin. Heterozygous variants in DCN, encoding decorin, cause autosomal dominant congenital stromal corneal dystrophy, suggesting a common pathogenic pathway. Therefore, we performed immunohistochemistry to compare SPARCL1 and decorin localisation in corneal tissue from an affected family member and an unaffected control. Strikingly, the level of decorin was significantly decreased in the corneal stroma of the affected tissue, and SPARCL1 appeared to be retained in the epithelium. In summary, we describe a novel autosomal dominant corneal stromal dystrophy associated with a missense variant in SPARCL1, extending the phenotypic and genetic heterogeneity of inherited corneal disease.

Tissue-specific TCF4 triplet repeat instability revealed by optical genome mapping.

BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is the most common repeat-mediated disease in humans. It exclusively affects corneal endothelial cells (CECs), with ≤81% of cases associated with an intronic TCF4 triplet repeat (CTG18.1). Here, we utilise optical genome mapping (OGM) to investigate CTG18.1 tissue-specific instability to gain mechanistic insights. METHODS: We applied OGM to a diverse range of genomic DNAs (gDNAs) from patients with FECD and controls (n = 43); CECs, leukocytes and fibroblasts. A bioinformatics pipeline was developed to robustly interrogate CTG18.1-spanning DNA molecules. All results were compared with conventional polymerase chain reaction-based fragment analysis. FINDINGS: Analysis of bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin. However, clusters of CTG18.1 molecules, encompassing ∼1800-11,900 repeats, were exclusively detected in diseased CECs from expansion-positive cases. Additionally, both progenitor allele size and age were found to influence the level of leukocyte-specific CTG18.1 instability. INTERPRETATION: OGM is a powerful tool for analysing somatic instability of repeat loci and reveals here the extreme levels of CTG18.1 instability occurring within diseased CECs underpinning FECD pathophysiology, opening up new therapeutic avenues for FECD. Furthermore, these findings highlight the broader translational utility of FECD as a model for developing therapeutic strategies for rarer diseases similarly attributed to somatically unstable repeats. FUNDING: UK Research and Innovation, Moorfields Eye Charity, Fight for Sight, Medical Research Council, NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Grantová Agentura Ceské Republiky, Univerzita Karlova v Praze, the National Brain Appeal's Innovation Fund and Rosetrees Trust.

Personalized Model to Predict Keratoconus Progression From Demographic, Topographic, and Genetic Data.

PURPOSE: To generate a prognostic model to predict keratoconus progression to corneal crosslinking (CXL). DESIGN: Retrospective cohort study. METHODS: We recruited 5025 patients (9341 eyes) with early keratoconus between January 2011 and November 2020. Genetic data from 926 patients were available. We investigated both keratometry or CXL as end points for progression and used the Royston-Parmar method on the proportional hazards scale to generate a prognostic model. We calculated hazard ratios (HRs) for each significant covariate, with explained variation and discrimination, and performed internal-external cross validation by geographic regions. RESULTS: After exclusions, model fitting comprised 8701 eyes, of which 3232 underwent CXL. For early keratoconus, CXL provided a more robust prognostic model than keratometric progression. The final model explained 33% of the variation in time to event: age HR (95% CI) 0.9 (0.90-0.91), maximum anterior keratometry 1.08 (1.07-1.09), and minimum corneal thickness 0.95 (0.93-0.96) as significant covariates. Single-nucleotide polymorphisms (SNPs) associated with keratoconus (n=28) did not significantly contribute to the model. The predicted time-to-event curves closely followed the observed curves during internal-external validation. Differences in discrimination between geographic regions was low, suggesting the model maintained its predictive ability. CONCLUSIONS: A prognostic model to predict keratoconus progression could aid patient empowerment, triage, and service provision. Age at presentation is the most significant predictor of progression risk. Candidate SNPs associated with keratoconus do not contribute to progression risk.

["The phlebotomist" - venous punction and venous access obtaining in the department of oncology]. / A "vérvételes" ­ vénapunkció és -biztosítás onkológiai centrumunkban.

The protection of the veins in patients with malignancies is of high importance (regarding both blood taking and the drug administration). In order to prevent any unnecessary injury, every phlebotomy needs to have a sophisticated indication and requires skilled hands and adequate mentality. For the administration of cytostatic treatment, insertion of cannula is necessary, in most of the cases through a peripheral vein. Recently with more successful treatment possibilities, patients undergo many subsequent types of treatment. Thus, more examinations and diagnostic procedures (e.g. blood taking, radiologic examinations) are needed. Therefore, the need for central venous catheter or Porth-a-Cath device is increasing. Our report demonstrates general guidelines of blood taking and our everyday practice.

Machine Learning Algorithms to Detect Subclinical Keratoconus: Systematic Review.

BACKGROUND: Keratoconus is a disorder characterized by progressive thinning and distortion of the cornea. If detected at an early stage, corneal collagen cross-linking can prevent disease progression and further visual loss. Although advanced forms are easily detected, reliable identification of subclinical disease can be problematic. Several different machine learning algorithms have been used to improve the detection of subclinical keratoconus based on the analysis of multiple types of clinical measures, such as corneal imaging, aberrometry, or biomechanical measurements. OBJECTIVE: The aim of this study is to survey and critically evaluate the literature on the algorithmic detection of subclinical keratoconus and equivalent definitions. METHODS: For this systematic review, we performed a structured search of the following databases: MEDLINE, Embase, and Web of Science and Cochrane Library from January 1, 2010, to October 31, 2020. We included all full-text studies that have used algorithms for the detection of subclinical keratoconus and excluded studies that did not perform validation. This systematic review followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) recommendations. RESULTS: We compared the measured parameters and the design of the machine learning algorithms reported in 26 papers that met the inclusion criteria. All salient information required for detailed comparison, including diagnostic criteria, demographic data, sample size, acquisition system, validation details, parameter inputs, machine learning algorithm, and key results are reported in this study. CONCLUSIONS: Machine learning has the potential to improve the detection of subclinical keratoconus or early keratoconus in routine ophthalmic practice. Currently, there is no consensus regarding the corneal parameters that should be included for assessment and the optimal design for the machine learning algorithm. We have identified avenues for further research to improve early detection and stratification of patients for early treatment to prevent disease progression.

The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure.

The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.

Comparing the agrochemical properties of compost and vermicomposts produced from municipal sewage sludge digestate.

The aim of this work was to investigate whether the agronomic traits of vermicompost prepared from partially stabilised sewage sludge digestate after thermophilic composting were more favourable than those of conventional compost. The effects of various additives (green waste, spent mushroom compost, wheat straw, biochar) were also tested after 1.5 months precomposting followed by 3 months vermicomposting with Eisenia fetida or by compost maturing. Vermicomposting did not result in significantly more intensive mineralisation than composting; the average organic carbon contents were 21.2 and 22.2% in vermicomposts and composts, respectively. Hence, the average total (N: 2.4%; P: 1.9%; K: 0.9%) and available (N: 160 mg/kg; P: 161 mg/kg; K: 0.8%) macronutrient concentrations were the same in both treatments. The processing method did not influence the organic matter quality (E4/E6) either. However, on average the concentration of the plant growth regulator kinetin was more than twice as high in vermicomposts.

Introducing the National Institute for Sports Medicine in Hungary: a complex sports medical healthcare and screening system.

The Hungarian National Institute for Sports Medicine (NISM) was founded in 1952 to provide medical coverage for national teams, screening and periodic health evaluation (PHE) for all Hungarian athletes. The system of 'all in one and ASAP' evolved by now to a specific state-funded healthcare provider with complex sports medical and sport-related services available for athletes. The NISM created a countrywide network to make health clearance available for all athletes close to their place of residency. This centralised system guarantees the uniformity and financial independence of the network, as it is directly financed by the government and free for every competitive athlete. Thus, it leaves no chance for conflict of interest in evaluating athletes' eligibility. In 2013, NISM established an online registry for preparticipation screening and PHE. This made the registry available for sports physicians and certain data for both sports physicians and athletes themselves. Furthermore, NISM created a nationwide, centrally coordinated, out of turn care with central coordination for elite athletes nationwide. Outpatient and inpatient clinics of NISM provide sports-specific care. Most of the minimally invasive techniques used at the Department of Sports Surgery are applied only here in the country. The medical staff of NISM has special experience in Sports Medicine and sport-related conditions. All tasks are managed within the same system, within institutional frames by professionals at Sports Medicine, which guarantees institutional expertise, competence and responsibility. Our aim is to introduce the complex system, the services and the recent achievements of the Hungarian NISM.

Correlation between pulmonary gas exchange and basal and nitroglycerin (GTN)-induced exhaled nitric oxide (eNO) in patients undergoing cardiac surgery.

The relationship between eNO and events in the alveolar-capillary unit in acute lung injury remains to be established. Since endogenous eNO largely originates from the airway epithelium, but nitroglycerin (GTN)-induced eNO is due to microvascular/alveolar metabolism, we have proposed to use basal and GTN-induced eNO as metabolic markers of the airway--and microvascular/alveolar function, respectively. The current work investigates the relationship between basal and GTN-induced eNO and oxygenation parameters (PaO(2)/FiO(2) ratio) in patients undergoing cardiac surgery utilising cardiopulmonary bypass (CPB). Breath by breath eNO measurements were made in 10 patients before, and 1 and 3 h after CPB either under basal conditions or following intravenous administration of GTN (1, 2 and 3 microg/kg). Basal eNO remained unchanged, whereas GTN-induced eNO was reduced following CPB. Also, there was a transient reduction in PaO(2)/FiO(2) ratio 1 h after CPB (32+/-4 vs. 44+/-3 kPa). A negative correlation was found between oxygenation and basal eNO by Pearson's correlation test and linear regression analysis suggesting that decreased oxygenation was associated with increased basal eNO. In contrast, a decrease in GTN-induced eNO positively correlated with reduced oxygenation index (R=0.533, p=0.002). These data suggest that differential relationships exist between basal and nitrovasodilator-induced eNO and oxygenation indices during subclinical lung injury in patients following CPB and that GTN-induced eNO evolution may reflect better microvascular events and injury.

M-ESLON(R) (retard release morphine sulphate capsules) for Pain Control in Cancer Patients.

The pain of patients who were in the terminal phase of advanced malignant tumors was successfully relieved by M-ESLON(R) capsules of controlled release morphine sulphate (10 30 60 100 mg). The most frequent side effects (sickness, vomiting, obstipation) were effectively controlled. The concentration of the drug in the plasma was stable, therefore the lasting pain relief was ensured.