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Lubomir Skladany,1 Jana Vnencakova,1 Lukas Laffers,2 Beata Skvarkova,1 Eva Hrubá,1 Pavol Molcan,1 Tomas Koller3 1Hepatology, Gastroenterology and Transplantation (HEGITO) Department of the 2nd Department of Medicine, Slovak Medical University, FD Roosevelt Hospital, Banska Bystrica, Slovakia; 2Department of Mathematics, Faculty of Natural Sciences, Matej Bel University, Banska Bystrica, Slovakia; 3Gastroenterology and Hepatology Subdiv. of the 5th Department of Medicine, Comenius University Faculty of Medicine, University Hospital Bratislava, Bratislava, SlovakiaCorrespondence: Tomas KollerGastroenterology and Hepatology Subdiv. of the 5th Department of Medicine, Comenius University Faculty of Medicine, University Hospital Bratislava, Ruzinovska 6, Bratislava 82606, SlovakiaTel +42190518692Email koller.tomas@gmail.comPurpose: Patients with advanced chronic liver disease (ACLD) often have a poor nutritional status. In the management, current guidelines recommend dietary counseling and oral nutritional supplements (ONS). Nutritional goals and adherence to ONS are difficult to achieve while studies addressing adherence are scarce. We aimed to evaluate adherence to ONS, the associated factors, and its impact on outcome among ALCD patients who are discharged from the hospital.Patients and Methods: We identified consecutive hospitalized patients with ACLD from the cirrhosis registry and ONS prescription at discharge. Baseline demographics, anthropometrics, hand-grip strength (HGS), nutritional, and laboratory parameters were recorded. Adherence was assessed at 30, 90, and 180 days, but not in patients who did not survive or in those who underwent liver transplantation (LT) before the time-point.Results: From the registry containing 1004 patients, we included 450 cases, the median age was 56.3 (IQR 47– 62), 60% were males, 63.8% had alcoholic etiology, and the median model for end-stage liver disease score (MELD) was 16 (11– 21). During follow-up, 13.6%, 23.6%, and 31.1% of patients have died within 30, 90, and 180 days, respectively, and 21 underwent LT. Adherence to ONS in surviving patients was observed in 46%, 26.1%, and 16.9% within 30, 90, and 180 days, respectively. Baseline refractory ascites (HR=0.43, 0.24– 0.76), HGS (HR=1.03, 1.01– 1.06), and mid-arm circumference (HR=0.93, 0.88– 0.99) were independently associated with 30-day adherence. Among patients who survived beyond 30 days, adherents for > 30 days had improved synthetic liver function, HGS, a higher probability of LT (HR=1.7, 1.03– 2.8) and lower risk of death (HR=0.65, 0.45– 0.89), particularly those with MELD> 16 (OR=0.55, 0.36– 0.85) and low HGS (OR=0.61, 0.39– 0.93).Conclusion: In ACLD patients after discharge, adherence to ONS steeply declined and was associated with baseline refractory ascites and low muscle strength. Adherence to ONS also improved liver function, muscle strength, and survival.Keywords: adherence, nutritional supplements, malnutrition, cirrhosis, outcome, prognosis

Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel-Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina., (© 2023 Dubaic et al.)

The Sprouty family is a highly conserved group of intracellular modulators of receptor tyrosine kinase (RTK)-signaling pathways, which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders called ciliopathies. We aimed to evaluate Sprouty2 and Sprouty4 gene-dependent alterations of ciliary structure and to focus on the determination of its association with Hedgehog signaling defects in chondrocytes. Analysis of the transgenic mice phenotype with Sprouty2 and Sprouty4 deficiency revealed several defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, reduced bone thickness and trabecular bone mass, skull deformities, or chondroma-like lesions were revealed. All these pathologies might be attributed to ciliopathies. Elongation of the ciliary axonemes in embryonic and postnatal growth plate chondrocytes was observed in Sprouty2 -/- and Sprouty2 +/- /Sprouty4 -/- mutants compared with corresponding littermate controls. Also, cilia-dependent Hedgehog signaling was upregulated in Sprouty2/4 mutant animals. Ptch1 and Ihh expression were upregulated in the autopodium and the proximal tibia of Sprouty2 -/- /Sprouty4 -/- mutants. Increased levels of the GLI3 repressor (GLI3R) form were detected in Sprouty2/4 mutant primary fibroblast embryonic cell cultures and tissues. These findings demonstrate that mouse lines deficient in Sprouty proteins manifest phenotypic features resembling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of RTK and dysfunction of primary cilia during skeletogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR)., (© 2021 American Society for Bone and Mineral Research (ASBMR).)

Achondroplasia is the most prevalent genetic form of dwarfism in humans and is caused by activating mutations in FGFR3 tyrosine kinase. The clinical need for a safe and effective inhibitor of FGFR3 is unmet, leaving achondroplasia currently incurable. Here, we evaluated RBM-007, an RNA aptamer previously developed to neutralize the FGFR3 ligand FGF2, for its activity against FGFR3. In cultured rat chondrocytes or mouse embryonal tibia organ culture, RBM-007 rescued the proliferation arrest, degradation of cartilaginous extracellular matrix, premature senescence, and impaired hypertrophic differentiation induced by FGFR3 signaling. In cartilage xenografts derived from induced pluripotent stem cells from individuals with achondroplasia, RBM-007 rescued impaired chondrocyte differentiation and maturation. When delivered by subcutaneous injection, RBM-007 restored defective skeletal growth in a mouse model of achondroplasia. We thus demonstrate a ligand-trap concept of targeting the cartilage FGFR3 and delineate a potential therapeutic approach for achondroplasia and other FGFR3-related skeletal dysplasias., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mutations in genes affecting primary cilia cause ciliopathies, a diverse group of disorders often affecting skeletal development. This includes Jeune syndrome or asphyxiating thoracic dystrophy (ATD), an autosomal recessive skeletal disorder. Unraveling the responsible molecular pathology helps illuminate mechanisms responsible for functional primary cilia. We identified two families with ATD caused by loss-of-function mutations in the gene encoding adrenergic receptor kinase 1 (ADRBK1 or GRK2). GRK2 cells from an affected individual homozygous for the p.R158* mutation resulted in loss of GRK2, and disrupted chondrocyte growth and differentiation in the cartilage growth plate. GRK2 null cells displayed normal cilia morphology, yet loss of GRK2 compromised cilia-based signaling of Hedgehog (Hh) pathway. Canonical Wnt signaling was also impaired, manifested as a failure to respond to Wnt ligand due to impaired phosphorylation of the Wnt co-receptor LRP6. We have identified GRK2 as an essential regulator of skeletogenesis and demonstrate how both Hh and Wnt signaling mechanistically contribute to skeletal ciliopathies., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disorder categorized into 3 phenotypic variants: infantile, juvenile, and adult. Four recent reports have linked NIID to CGG expansions in the NOTCH2NLC gene in adult NIID (aNIID) and several juvenile patients. Infantile NIID (iNIID) is an extremely rare neuropediatric condition. We present a 7-year-old male patient with severe progressive neurodegenerative disease that included cerebellar symptoms with cerebellar atrophy on brain MRI, psychomotor developmental regression, pseudobulbar syndrome, and polyneuropathy. The diagnosis of iNIID was established through a postmortem neuropathology work-up. We performed long-read sequencing of the critical NOTCH2NLC repeat motif and found no expansion in the patient. We also re-evaluated an antemortem skin biopsy that was collected when the patient was 2 years and 8 months old and did not identify the intranuclear inclusions. In our report, we highlight that the 2 methods (skin biopsy and CGG expansion testing in NOTCH2NLC) used to identify aNIID patients may provide negative results in iNIID patients., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Occupational exposure to diesel exhaust may cause lung cancer in humans. Mechanisms include DNA-damage and inflammatory responses. Here, the potential of NIST SRM2975 diesel exhaust particles (DEP) to transform human bronchial epithelial cells (HBEC3) in vitro was investigated. Long-term exposure of HBEC3 to DEP led to increased colony growth in soft agar. Several DEP-transformed cell lines were established and based on the expression of epithelial-to-mesenchymal-transition (EMT) marker genes, one of them (T2-HBEC3) was further characterized. T2-HBEC3 showed a mesenchymal/fibroblast-like morphology, reduced expression of CDH1, and induction of CDH2 and VIM. T2-HBEC3 had reduced migration potential compared with HBEC3 and little invasion capacity. Gene expression profiling showed baseline differences between HBEC3 and T2-HBEC3 linked to lung carcinogenesis. Next, to assess differences in sensitivity to DEP between parental HBEC3 and T2-HBEC3, gene expression profiling was carried out after DEP short-term exposure. Results revealed changes in genes involved in metabolism of xenobiotics and lipids, as well as inflammation. HBEC3 displayed a higher steady state of IL1B gene expression and release of IL-1β compared with T2-HBEC3. HBEC3 and T2-HBEC3 showed similar susceptibility towards DEP-induced genotoxic effects. Liquid-chromatography-tandem-mass-spectrometry was used to measure secretion of eicosanoids. Generally, major prostaglandin species were released in higher concentrations from T2-HBEC3 than from HBEC3 and several analytes were altered after DEP-exposure. In conclusion, long-term exposure to DEP-transformed human bronchial epithelial cells in vitro. Differences between HBEC3 and T2-HBEC3 regarding baseline levels and DEP-induced changes of particularly CYP1A1, IL-1β, PGE2, and PGF2α may have implications for acute inflammation and carcinogenesis.

In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The EGR1 promoter was engineered to enhance trans -activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies.

A novel series of 7-methoxytacrine (7-MEOTA)-donepezil like compounds was synthesized and tested for their ability to inhibit electric eel acetylcholinesterase (EeAChE), human recombinant AChE (hAChE), equine serum butyrylcholinesterase (eqBChE) and human plasmatic BChE (hBChE). New hybrids consist of a 7-MEOTA unit, representing less toxic tacrine (THA) derivative, connected with analogues of N-benzylpiperazine moieties mimicking N-benzylpiperidine fragment from donepezil. 7-MEOTA-donepezil like compounds exerted mostly non-selective profile in inhibiting cholinesterases of different origin with IC50 ranging from micromolar to sub-micromolar concentration scale. Kinetic analysis confirmed mixed-type inhibition presuming that these inhibitors are capable to simultaneously bind peripheral anionic site (PAS) as well as catalytic anionic site (CAS) of AChE. Molecular modeling studies and QSAR studies were performed to rationalize studies from in vitro. Overall, 7-MEOTA-donepezil like derivatives can be considered as interesting candidates for Alzheimer's disease treatment., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Recent studies suggest that exposure to endocrine-disrupting compounds (EDCs) may play a role in the development of obesity. EDCs such as the flame retardant 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) have been shown to enhance adipocyte differentiation in the murine 3T3-L1 model. The mechanisms by which EDCs direct preadipocytes to form adipocytes are poorly understood. Here, we examined transcriptional and epigenetic mechanisms underlying the induction of in vitro adipocyte differentiation by BDE-47. Quantitative high content microscopy revealed concentration-dependent enhanced adipocyte differentiation following exposure to BDE-47 or the antidiabetic drug troglitazone (TROG). BDE-47 modestly activated the key adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) in COS7 cells, transiently transfected with a GAL4 reporter construct. Increased gene expression was observed for Pparγ2, leptin (Lep), and glucose-6-phophatase catalytic subunit (G6pc) in differentiated 3T3-L1 cells after BDE-47 exposure compared to TROG. Methylation-sensitive high resolution melting (MS-HRM) revealed significant demethylation of three CpG sites in the Pparγ2 promoter after exposure to both BDE-47 and TROG in differentiated 3T3-L1 cells. This study shows the potential of BDE-47 to induce adipocyte differentiation through various mechanisms that include Pparγ2 gene induction and promoter demethylation accompanied by activation of PPARγ, and possible disruption of glucose homeostasis and IGF1 signaling.

Background: Maternal vitamin B(12) (Cbl) deficiency causes nutritional Cbl deficiency in breastfed infants., Aims: To analyse clinical presentation and metabolic consequences in 40 breastfed infants with Cbl deficiency., Methods: Cbl levels in serum and breast milk were determined by an electrochemiluminescence immunoassay, methylmalonic acid level by GC/MS, plasma homocysteine by HPLC and propionylcarnitine by MS/MS. Profound Cbl deficiency was found in 17 children (69 ± 17 ng/l, controls 200-900), and milder Cbl deficiency in 23 children (167 ± 40 ng/l). Maternal Cbl deficiency was mostly caused by insufficient Cbl absorption. Only six mothers were vegetarian., Results: The average age at diagnosis was 4.4 ± 2.5 months. Clinical symptoms included failure to thrive (48% of children), hypotonia (40%), developmental delay (38%) and microcephaly (23%). 63% of children had anaemia (megaloblastic in 28% of all children). All but one patient had methylmalonic aciduria, 80% of patients had hyperhomocysteinemia and 87% had increased aminotransferases. Propionylcarnitine was elevated in two out of 25 infants. Comparing groups with severe and mild Cbl deficiency, a marked difference was found in severity of clinical and laboratory changes., Conclusion: Maternal Cbl status and diagnostic delay are the major factors influencing severity and progression of Cbl deficiency in breastfed infants. In our cohort, propionylcarnitine was not sufficiently sensitive marker of Cbl deficiency. Although symptoms are reversible on Cbl substitution, permanent neurological damage can result. Selective screening for Cbl deficiency is indicated in all breastfed infants with failure to thrive, hypotonia, developmental delay, microcephaly or megaloblastic anaemia. The best prevention in future could be the screening of all pregnant women., (Copyright © 2009 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Ornithine transcarbamylase (OTC) deficiency, transmitted as an X-linked trait, is the most common disorder of the urea cycle. At least 3.5% out of more than 230 mutations consist of large gene deletions, involving one or more exons. Only in 78% of OTC patients the diagnosis was confirmed on DNA level. We analysed OTC intragenic polymorphisms and haplotypes, in an attempt to contribute to the clarification of unresolved cases, in three populations (Czech, Portuguese, and Mozambican) and identified six novel nucleotide changes, all of them occurring with frequency higher than 12.5% in Europeans. Five of these polymorphisms occur with a significant frequency also in Africans. The number and frequency of haplotypes defined with the newly reported markers differ in individual populations.