Variants in the circadian clock genes PER2 and PER3 associate with familial sleep phase disorders.

Delayed sleep phase disorder and advanced sleep phase disorder cause disruption of the circadian clock and present with extreme morning/evening chronotype with unclear role of the genetic etiology, especially for delayed sleep phase disorder. To assess if genotyping can aid in clinical diagnosis, we examined the presence of genetic variants in circadian clock genes previously linked to both sleep disorders in Slovenian patient cohort. Based on Morning-evening questionnaire, we found 15 patients with extreme chronotypes, 13 evening and 2 morning, and 28 controls. Sanger sequencing was used to determine the presence of carefully selected candidate SNPs in regions of the CSNK1D, PER2/3 and CRY1 genes. In a patient with an extreme morning chronotype and a family history of circadian sleep disorder we identified two heterozygous missense variants in PER3 gene, c.1243C>G (NM_001377275.1 (p.Pro415Ala)) and c.1250A>G (NM_001377275.1 (p.His417Arg)). The variants were significantly linked to Advanced sleep phase disorder and were also found in proband's father with extreme morningness. Additionally, a rare SNP was found in PER2 gene in a patient with clinical picture of Delayed sleep phase disorder. The novel variant in PER2 (NM_022817.3):c.1901-218 G>T was found in proband's parent with eveningness, indicating an autosomal dominant inheritance. We identified a family with autosomal dominant inheritance of two PER3 heterozygous variants that can be linked to Advanced sleep phase disorder. We revealed also a rare hereditary form of Delayed sleep phase disorder with a new PER2 variant with autosomal dominant inheritance, shedding the light into the genetic causality.

Mechanisms of pathogenicity and the quest for genetic modifiers of kidney disease in branchiootorenal syndrome.

Backgound: Branchiootorenal (BOR) syndrome is an autosomal dominant disorder caused by pathogenic EYA1 variants and clinically characterized by auricular malformations with hearing loss, branchial arch anomalies, and congenital anomalies of the kidney and urinary tract. BOR phenotypes are highly variable and heterogenous. While random monoallelic expression is assumed to explain this phenotypic heterogeneity, the potential role of modifier genes has not yet been explored. Methods: Through thorough phenotyping and exome sequencing, we studied one family with disease presentation in at least four generations in both clinical and genetic terms. Functional investigation of the single associated EYA1 variant c.1698+1G>A included splice site analysis and assessment of EYA1 distribution in patient-derived fibroblasts. The candidate modifier gene CYP51A1 was evaluated by histopathological analysis of murine Cyp51+/- and Cyp51-/- kidneys. As the gene encodes the enzyme lanosterol 14α-demethylase, we assessed sterol intermediates in patient blood samples as well. Results: The EYA1 variant c.1698+1G>A resulted in functional deletion of the EYA domain by exon skipping. The EYA domain mediates protein-protein interactions between EYA1 and co-regulators of transcription. EYA1 abundance was reduced in the nuclear compartment of patient-derived fibroblasts, suggesting impaired nuclear translocation of these protein complexes. Within the affected family, renal phenotypes spanned from normal kidney function in adulthood to chronic kidney failure in infancy. By analyzing exome sequencing data for variants that potentially play roles as genetic modifiers, we identified a canonical splice site alteration in CYP51A1 as the strongest candidate variant. Conclusion: In this study, we demonstrate pathogenicity of EYA1 c.1698+1G>A, propose a mechanism for dysfunction of mutant EYA1, and conjecture CYP51A1 as a potential genetic modifier of renal involvement in BOR syndrome.

COVID-19 and cholesterol biosynthesis: Towards innovative decision support systems.

With COVID-19 becoming endemic, there is a continuing need to find biomarkers characterizing the disease and aiding in patient stratification. We studied the relation between COVID-19 and cholesterol biosynthesis by comparing 10 intermediates of cholesterol biosynthesis during the hospitalization of 164 patients (admission, disease deterioration, discharge) admitted to the University Medical Center of Ljubljana. The concentrations of zymosterol, 24-dehydrolathosterol, desmosterol, and zymostenol were significantly altered in COVID-19 patients. We further developed a predictive model for disease severity based on clinical parameters alone and their combination with a subset of sterols. Our machine learning models applying 8 clinical parameters predicted disease severity with excellent accuracy (AUC = 0.96), showing substantial improvement over current clinical risk scores. After including sterols, model performance remained better than COVID-GRAM. This is the first study to examine cholesterol biosynthesis during COVID-19 and shows that a subset of cholesterol-related sterols is associated with the severity of COVID-19.

Integrative computational modeling to unravel novel potential biomarkers in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major health problem around the world. The management of this disease is complicated by the lack of noninvasive diagnostic tools and the few treatment options available. Better clinical outcomes can be achieved if HCC is detected early, but unfortunately, clinical signs appear when the disease is in its late stages. We aim to identify novel genes that can be targeted for the diagnosis and therapy of HCC. We performed a meta-analysis of transcriptomics data to identify differentially expressed genes and applied network analysis to identify hub genes. Fatty acid metabolism, complement and coagulation cascade, chemical carcinogenesis and retinol metabolism were identified as key pathways in HCC. Furthermore, we integrated transcriptomics data into a reference human genome-scale metabolic model to identify key reactions and subsystems relevant in HCC. We conclude that fatty acid activation, purine metabolism, vitamin D, and E metabolism are key processes in the development of HCC and therefore need to be further explored for the development of new therapies. We provide the first evidence that GABRP, HBG1 and DAK (TKFC) genes are important in HCC in humans and warrant further studies.

Bacterial DNA Recognition by SERS Active Plasma-Coupled Nanogold.

It is shown that surface-enhanced Raman spectroscopy (SERS) can identify bacteria based on their genomic DNA composition, acting as a "sample-distinguishing marker". Successful spectral differentiation of bacterial species was accomplished with nanogold aggregates synthesized through single-step plasma reduction of the ionic gold-containing vapored precursor. A high enhancement factor (EF = 107) in truncated coupled plasmonic particulates allowed SERS-probing at nanogram sample quantities. Simulations confirmed the occurrence of the strongest electric field confinement within nanometric gaps between gold dimers/chains from where the molecular fingerprints of bacterial DNA fragments gained photon scattering enhancement. The most prominent Raman modes linked to fundamental base-pair molecular vibrations were deconvoluted and used to proceed with nitrogenous base content estimation. The genomic composition (percentage of guanine-cytosine and adenine-thymine) was successfully validated by third-generation sequencing using nanopore technology, further proving that the SERS technique can be employed to swiftly specify bioentities by the discriminative principal-component statistical approach.

Pyridylethanol(phenylethyl)amines are non-azole, highly selective Candida albicans sterol 14α-demethylase inhibitors.

Sterol 14α-demethylase (CYP51) is the main drug target for the treatment of fungal infections. The worldwide increase in the incidence of opportunistic fungal infections and the emerging resistance to available azole-based antifungal drugs, raise the need to develop structurally distinct and selective fungal CYP51 inhibitors. In this work we have, for the first time, investigated the binding of pyridylethanol(phenylethyl)amines to any fungal CYP51. The comparison of the binding to Candida albicans and human CYP51 studied by spectroscopic and modeling methods revealed moieties decisive for selectivity and potency and resulted in the development of highly selective derivatives with significantly increased inhibitory potency. The structure-based insight into the selectivity requirements of this new chemical class of fungal CYP51 inhibitors, their unique binding properties and the low molecular weight of lead derivatives offer novel directions for the targeted development of antifungal clinical candidates.

Chronic Disruption of the Late Cholesterol Synthesis Leads to Female-Prevalent Liver Cancer.

While the role of cholesterol in liver carcinogenesis remains controversial, hepatocellular carcinoma generally prevails in males. Herein, we uncover pathways of female-prevalent progression to hepatocellular carcinoma due to chronic repression of cholesterogenic lanosterol 14α-demethylase (CYP51) in hepatocytes. Tumors develop in knock-out mice after year one, with 2:1 prevalence in females. Metabolic and transcription factor networks were deduced from the liver transcriptome data, combined by sterol metabolite and blood parameter analyses, and interpreted with relevance to humans. Female knock-outs show increased plasma cholesterol and HDL, dampened lipid-related transcription factors FXR, LXRα:RXRα, and importantly, crosstalk between reduced LXRα and activated TGF-ß signalling, indicating a higher susceptibility to HCC in aging females. PI3K/Akt signalling and ECM-receptor interaction are common pathways that are disturbed by sex-specific altered genes. Additionally, transcription factors (SOX9)2 and PPARα were recognized as important for female hepatocarcinogenesis, while overexpressed Cd36, a target of nuclear receptor RORC, is a new male-related regulator of ECM-receptor signalling in hepatocarcinogenesis. In conclusion, we uncover the sex-dependent metabolic reprogramming of cholesterol-related pathways that predispose for hepatocarcinogenesis in aging females. This is important in light of increased incidence of liver cancers in post-menopausal women.

Regulation of cytochrome P450 enzyme activity and expression by nitric oxide in the context of inflammatory disease.

Many hepatic cytochrome P450 enzymes and their associated drug metabolizing activities are down-regulated in disease states, and much of this has been associated with inflammatory cytokines and their signaling pathways. One such pathway is the induction of inducible nitric oxide synthase (NOS2) and generation of nitric oxide (NO) in many tissues and cells including the liver and hepatocytes. Experiments in the 1990s demonstrated that NO could bind to and inhibit P450 enzymes, and suggested that inhibition of NOS could attenuate, and NO generation could mimic, the down-regulation by inflammatory stimuli of not only P450 catalytic activities but also of mRNA expression and protein levels of certain P450 enzymes. This review will summarize and examine the evidence that NO functionally inhibits and down-regulates P450 enzymes in vivo and in vitro, with a particular focus on the mechanisms by which these effects are achieved.

Simplified LC-MS Method for Analysis of Sterols in Biological Samples.

We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.

Oxysterols and Gastrointestinal Cancers Around the Clock.

This review focuses on the role of oxidized sterols in three major gastrointestinal cancers (hepatocellular carcinoma, pancreatic, and colon cancer) and how the circadian clock affects the carcinogenesis by regulating the lipid metabolism and beyond. While each field of research (cancer, oxysterols, and circadian clock) is well-studied within their specialty, little is known about the intertwining mechanisms and how these influence the disease etiology in each cancer type. Oxysterols are involved in pathology of these cancers, but final conclusions about their protective or damaging effects are elusive, since the effect depends on the type of oxysterol, concentration, and the cell type. Oxysterol concentrations, the expression of key regulators liver X receptors (LXR), farnesoid X receptor (FXR), and oxysterol-binding proteins (OSBP) family are modulated in tumors and plasma of cancer patients, exposing these proteins and selected oxysterols as new potential biomarkers and drug targets. Evidence about how cholesterol/oxysterol pathways are intertwined with circadian clock is building. Identified key contact points are different forms of retinoic acid receptor related orphan receptors (ROR) and LXRs. RORs and LXRs are both regulated by sterols/oxysterols and the circadian clock and in return also regulate the same pathways, representing a complex interplay between sterol metabolism and the clock. With this in mind, in addition to classical therapies to modulate cholesterol in gastrointestinal cancers, such as the statin therapy, the time is ripe also for therapies where time and duration of the drug application is taken as an important factor for successful therapies. The final goal is the personalized approach with chronotherapy for disease management and treatment in order to increase the positive drug effects.

Personalized therapy when tackling nonalcoholic fatty liver disease: a focus on sex, genes, and drugs.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the most frequent liver disease in the world. It describes a term for a group of hepatic diseases including steatosis, fibrosis, and cirrhosis that can finally lead to hepatocellular carcinoma. There are many factors influencing NAFLD initiation and progression, such as obesity, dyslipidemia, insulin resistance, genetic factors, and hormonal changes. However, there is also lean-NAFLD which is not associated with obesity. NAFLD is considered to be a sexually dimorphic disease. In most cases, men have a higher prevalence for the disease compared to premenopausal women. Areas covered: In this review, we first summarize the NAFLD disease epidemiology, pathology, and diagnosis. We describe NAFLD progression with the focus on sexual and genetic differences for disease development and pharmacological treatment. Personalized treatment for multifactorial NAFLD is discussed in consideration of different factors, including genetics, gender and sex. Expert opinion: The livers of female and male NAFLD patients have different metabolic capacities which influence the metabolism of all drugs applied to such patients. This aspect is not yet sufficiently taken into account. The liver computational models might quicken the pace toward assessing personalized disease progression and treatment options.