Transcriptional Coactivator BOB1 (OBF1, OCA-B) Modulates the Specificity of DNA Recognition by the POU-Domain Factors OCT1 and OCT2 in a Monomeric Configuration.

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.

Preclinical toxicity evaluation of novel antibacterial contezolid acefosamil in rats and dogs.

Contezolid acefosamil (CZA) is an intravenous prodrug of oxazolidinone antibiotic contezolid (CZD). It is being developed to treat infections due to Gram-positive bacteria including multidrug-resistant pathogens, while addressing myelosuppression and neurotoxicity limitations associated with long-term use of this class of antibiotics. In vivo, CZA is rapidly deacylated into its first metabolite MRX-1352, which is then dephosphorylated to release active drug CZD. Four-week repeat-dose toxicity studies of intravenous CZA were conducted in Sprague-Dawley rats (40, 80, and 160/120 mg/kg/dose twice a day [BID]) and beagle dogs (25, 50, and 100/75 mg/kg/dose BID). The high doses administered to both rats and dogs were adjusted due to adverse effects including decreased body weight and food consumption. Additionally, a dose-dependent transient reduction in erythrocyte levels was recorded at the end of dosing phase. Importantly, no myelosuppressive reduction in platelet counts was observed, in contrast to the myelosuppression documented for standard-of-care oxazolidinone linezolid. The no-observed-adverse-effect level (NOAEL) of CZA was 80 and 25 mg/kg/dose BID in rats and dogs, respectively. Separately, 3-month neuropathological evaluation in Long-Evans rats (25, 37.5, and 50 mg/kg/dose, oral CZA, BID) demonstrated no neurotoxicity in the central, peripheral, and optical neurological systems. Toxicokinetic data from these studies revealed that CZD exposures at NOAELs were higher than or comparable with that for the intended clinical dose. These results confirm the favorable safety profile for CZA and support its clinical evaluation for long-term therapy of persistent Gram-positive infections, beyond the application for earlier oxazolidinones.

Patterns of genetic differentiation imply distinct phylogeographic history of the mosquito species Anopheles messeae and Anopheles daciae in Eurasia.

Detailed knowledge of phylogeography is important for control of mosquito species involved in the transmission of human infectious diseases. Anopheles messeae is a geographically widespread and genetically diverse dominant vector of malaria in Eurasia. A closely related species, An. daciae, was originally distinguished from An. messeae based on five nucleotide substitutions in its ribosomal DNA (rDNA). However, the patterns of phylogeographic history of these species in Eurasia remain poorly understood. Here, using internal transcribed spacer 2 (ITS2) of rDNA and karyotyping for the species identification we determined the composition of five Anopheles species in 28 locations in Eurasia. Based on the frequencies of 11 polymorphic chromosomal inversions used as genetic markers, a large-scale population genetics analysis was performed of 1932 mosquitoes identified as An. messeae, An. daciae and their hybrids. The largest genetic differences between the species were detected in the X sex chromosome suggesting a potential involvement of this chromosome in speciation. The frequencies of autosomal inversions in the same locations differed by 13%-45% between the species demonstrating a restricted gene flow between the species. Overall, An. messeae was identified as a diverse species with a more complex population structure than An. daciae. The clinal gradients in frequencies of chromosomal inversions were determined in both species implicating their possible involvement in climate adaptations. The frequencies of hybrids were low ~1% in northern Europe but high up to 50% in south-eastern populations. Thus, our study revealed critical differences in patterns of phylogeographic history between An. messeae and An. daciae in Eurasia. This knowledge will help to predict the potential of the malaria transmission in the northern territories of the continent.

Phylogenomics revealed migration routes and adaptive radiation timing of Holarctic malaria mosquito species of the Maculipennis Group.

BACKGROUND: Phylogenetic analyses of closely related species of mosquitoes are important for better understanding the evolution of traits contributing to transmission of vector-borne diseases. Six out of 41 dominant malaria vectors of the genus Anopheles in the world belong to the Maculipennis Group, which is subdivided into two Nearctic subgroups (Freeborni and Quadrimaculatus) and one Palearctic (Maculipennis) subgroup. Although previous studies considered the Nearctic subgroups as ancestral, details about their relationship with the Palearctic subgroup, and their migration times and routes from North America to Eurasia remain controversial. The Palearctic species An. beklemishevi is currently included in the Nearctic Quadrimaculatus subgroup adding to the uncertainties in mosquito systematics. RESULTS: To reconstruct historic relationships in the Maculipennis Group, we conducted a phylogenomic analysis of 11 Palearctic and 2 Nearctic species based on sequences of 1271 orthologous genes. The analysis indicated that the Palearctic species An. beklemishevi clusters together with other Eurasian species and represents a basal lineage among them. Also, An. beklemishevi is related more closely to An. freeborni, which inhabits the Western United States, rather than to An. quadrimaculatus, a species from the Eastern United States. The time-calibrated tree suggests a migration of mosquitoes in the Maculipennis Group from North America to Eurasia about 20-25 million years ago through the Bering Land Bridge. A Hybridcheck analysis demonstrated highly significant signatures of introgression events between allopatric species An. labranchiae and An. beklemishevi. The analysis also identified ancestral introgression events between An. sacharovi and its Nearctic relative An. freeborni despite their current geographic isolation. The reconstructed phylogeny suggests that vector competence and the ability to enter complete diapause during winter evolved independently in different lineages of the Maculipennis Group. CONCLUSIONS: Our phylogenomic analyses reveal migration routes and adaptive radiation timing of Holarctic malaria vectors and strongly support the inclusion of An. beklemishevi into the Maculipennis Subgroup. Detailed knowledge of the evolutionary history of the Maculipennis Subgroup provides a framework for examining the genomic changes related to ecological adaptation and susceptibility to human pathogens. These genomic variations may inform researchers about similar changes in the future providing insights into the patterns of disease transmission in Eurasia.

Surgical treatment of chronic thromboembolic pulmonary hypertension in combination with a left anterior descending artery myocardial bridge: A case report.

Pulmonary thromboendarterectomy is a potentially curative option for most patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, a special group of patients with CTEPH requires simultaneous cardiac procedures. We report a rare case of successful surgical treatment of a CTEPH patient with a left anterior descending artery myocardial bridge. Despite the complexity of performing pulmonary thromboendarterectomy (PTE), the issue concerning the method of revascularization of the artery in the case of the left anterior descending artery myocardial bridge is controversial. PTE and supracoronary myotomy were performed. In our case, the optimal surgery method for the left anterior descending artery myocardial bridge was chosen intraoperatively based on the depth and length of the myocardial bridge. The patient's significant functional improvement after surgery and hemodynamic normalization were confirmed at the follow-up assessment. This case demonstrates rare but potentially dangerous pathologies that can be treated with minimal adverse effects.

Redo aortic valve-sparing root replacement for failing autograft after the Ross procedure: A case report.

INTRODUCTION AND IMPORTANCE: Late pulmonary autograft dilatation is observed in 10-20 % of patients after the Ross procedure, more often during the second decade of follow-up. Composite aortic root replacement with a valved conduit is the most common redo procedure. An aortic valve-sparing root replacement does not require lifelong anticoagulation and may significantly decrease the risk of complications, associated with a valve prosthesis. PRESENTATION OF THE CASE: We report a case of late pulmonary autograft dilatation developed after the Ross procedure. The annual transthoracic echocardiography after 20 years revealed severe dilatation of the pulmonary autograft, measuring 60 mm in diameter. The patient underwent a successful elective redo valve-sparing aortic root replacement (David I procedure). The aortic cross-clamp time was 144 min, and the CPB time was 181 min. The patient had an uneventful midterm postoperative course. CLINICAL DISCUSSION: Late pulmonary autograft failure after the Ross procedure is a relatively rare condition, leading to repeat operation. Late autograft failure can contribute to aortic regurgitation, heart failure, and death due to ascending aortic dissection and rupture. Several research groups reported good early and midterm results of redo valve-sparing root replacement in such cases. In a reoperative valve-sparing root replacement after the Ross procedure, the portion of the native aorta with the adjacent part of the autograft may complicate the aortic root proper sizing. CONCLUSION: Redo valve-sparing root replacement (David I procedure) is a viable option in pulmonary autograft dilatation with unaffected valve leaflets.

Discovery of Antibacterial Contezolid Acefosamil: Innovative O-Acyl Phosphoramidate Prodrug for IV and Oral Therapies.

New oral antibiotic contezolid (CZD) is effective against Gram-positive infections but unsuitable for intravenous (IV) administration due to its modest solubility. To address the medical need for an IV form of CZD, its isoxazol-3-yl phosphoramidate derivatives have been explored, and contezolid acefosamil (CZA, 8), the first representative of a novel O-acyl phosphoramidate prodrug class, has been identified. CZA exhibits high aqueous solubility (>200 mg/mL) and good hydrolytic stability at media pH suitable for IV administration. CZA rapidly converts into the active drug CZD in vivo. In a pharmacokinetic (PK) rat model, the exposure of active drug CZD after IV administration of the prodrug CZA was similar to or higher than that from the IV administration of CZD. The prodrug CZA is bioequivalent to or better than CZD in several preclinical infection models. CZA is likewise active upon its oral administration. To date, CZA has been evaluated in Phase 1 and Phase 2 clinical trials in the USA. It is advancing into further clinical studies including step-down therapy with in-hospital intravenous CZA administration followed by outpatient oral CZD treatment.

Penetrating Cardiac Injury from Crossbow Successfully Treated With Cardiac Autotransplantation.

A middle-aged male patient presented with self-inflicted penetrating cardiac injury from 2 crossbow bolts causing injury to multiple cardiac structures and surrounding great vessels. He was successfully treated with peripheral cannulation for cardiopulmonary bypass, median sternotomy, hypothermic circulatory arrest, autotransplantation of the heart, and repair of all intracardiac injuries.

Effects of ischaemic postconditioning in aortic valve replacement: a multicenter randomized controlled trial.

OBJECTIVES: The effect of ischaemic postconditioning (IPost) on postcardioplegic cardiac function is not known. We hypothesized that IPost was cardioprotective in adult patients undergoing elective aortic valve replacement. METHODS: In a multicentre, prospective, randomized trial, patients (n = 209) were randomized to either a standard operation (controls) or postconditioning. Immediately before the cross-clamp was released, patients in the postconditioning group underwent 3 cycles of flow/non-flow (2 min each) of normothermic blood via the antegrade cardioplegia line. The primary end point was cardiac index. Secondary end points included additional haemodynamic measurements, biomarkers of cardiomyocyte injury, renal function parameters, intra- and postoperative arrhythmias and use of inotropic agents. RESULTS: There was no significant difference between the groups regarding cardiac index [mean between-group difference, 95% confidence interval (CI), 0.11 (-0.1 to 0.3), P = 0.27]. Postconditioning had no effect on other haemodynamic parametres. There was no between-group difference regarding troponin T or creatine kinase MB. Postconditioning reduced the relative risk for arrhythmias by 45% (P = 0.03) when postoperative atrial fibrillation and intraoperative ventricular fibrillation were combined. There were no differences in patients with/without diabetes, patients above/below 70 years of age or between the centres. However, after postconditioning, the cardiac index [95% CI, 0.46 (0.2-0.7), P = 0.001], cardiac output (P < 0.001), mean arterial pressure (P < 0.001) and left ventricular stroke work index (P < 0.001) were higher in males compared to females. CONCLUSIONS: IPost had no overall cardioprotective effects in patients undergoing aortic valve replacement but improved postoperative cardiac performance in men compared to women.

New Cytogenetic Photomap and Molecular Diagnostics for the Cryptic Species of the Malaria Mosquitoes Anopheles messeae and Anopheles daciae from Eurasia.

The Eurasian malaria vector Anopheles messeae is a widely spread and genetically diverse species. Five widespread polymorphic chromosomal inversions were found in natural populations of this mosquito. A cryptic species, Anopheles daciae, was differentiated from An. messeae by the presence of several nucleotide substitutions in the Internal Transcribed Spacer 2 (ITS2) region of ribosomal DNA. However, because of the absence of a high-quality reference cytogenetic map, the inversion polymorphisms in An. daciae and An. messeae remain poorly understood. Moreover, a recently determined heterogeneity in ITS2 in An. daciae questioned the accuracy of the previously used Restriction Fragment Length Polymorphism (RFLP) assay for species diagnostics. In this study, a standard-universal cytogenetic map was constructed based on orcein stained images of chromosomes from salivary glands for population studies of the chromosomal inversions that can be used for both An. messeae and An. daciae. In addition, a new ITS2-RFLP approach for species diagnostics was developed. Both methods were applied to characterize inversion polymorphism in populations of An. messeae and An. daciae from a single location in Western Siberia in Russia. The analysis demonstrates that cryptic species are remarkably different in their frequencies of chromosomal inversion variants. Our study supports previous observations that An. messeae has higher inversion polymorphism in all autosomes than the cryptic species An. daciae.

Pharmacokinetics and Disposition of Contezolid in Humans: Resolution of a Disproportionate Human Metabolite for Clinical Development.

Contezolid (MRX-I), a novel oxazolidinone antibiotic, was recently approved for the treatment of serious Gram-positive infections. The pharmacokinetics and disposition of [14C]contezolid were investigated in a single-dose human mass balance study. Cross-species comparison of plasma exposure for contezolid and metabolites was performed, and the safety of the disproportionate metabolite in human was evaluated with additional nonclinical studies. After an oral administration of 99.1 µCi/602-mg dose of [14C]contezolid, approximately 91.5% of the radioactivity was recovered in 0 to 168 h postdose, mainly in urine followed by that in feces. The principal metabolic pathway of contezolid in human comprised an oxidative ring opening of the 2,3-dihydropyridin-4-one fragment into polar metabolites MRX445-1 and MRX459, with recovery of approximately 48% and 15% of the dose, respectively, in urine and feces. Contezolid, MRX445-1, and MRX459 accounted for 68.0%, 19.5%, and 4.84% of the plasma exposure of the total radioactivity, respectively. Metabolites MRX445-1 and MRX459 were observed in disproportionately larger amounts in human plasma than in samples from rat or dog, the rodent and nonrodent species, respectively, used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies, wherein the primary metabolite, MRX445-1, was further characterized. The no-observed-adverse-effect level (NOAEL) of MRX445-1 was determined as 360 mg/kg body weight/day in a 14-day repeat-dose test in pregnant and nonpregnant Sprague Dawley rats. Furthermore, MRX445-1 exhibited no antibacterial activity in vitro. Thus, MRX445-1 is not expected to exert clinically relevant pharmacology and toxicity.

Nonclinical Evaluation of Antibacterial Oxazolidinones Contezolid and Contezolid Acefosamil with Low Serotonergic Neurotoxicity.

Linezolid, the principal oxazolidinone antibiotic for therapy of Gram-positive infections, is limited by its myelosuppression and monoamine oxidase (MAO) inhibition, with the latter manifested as serotonergic neurotoxicity. The oral oxazolidinone contezolid and its injectable prodrug contezolid acefosamil are developed to overcome the above limitations. Serotonergic profiles for contezolid in vitro and for orally administered contezolid acefosamil in rodents are reported. Contezolid exhibited 2- and 148-fold reduction over linezolid reversible inhibition of MAO-A and MAO-B human enzyme isoforms. In the mouse head-twitch model, contezolid acefosamil was devoid of neurotoxicity at supratherapeutic oral doses of 40, 80, and 120 mg/kg. In the rat tyramine challenge model, no significant increase in arterial blood pressure was observed for contezolid acefosamil up to 120 mg/kg oral dosing. In these tests, the comparator linezolid has elicited serotonergic responses. Thus, contezolid and contezolid acefosamil exhibited an attenuated propensity to induce MAO-related serotonergic neurotoxicity. The data support a continued clinical evaluation of these agents, with potential to expand oxazolidinone therapies to patient populations on concurrent selective serotonin reuptake inhibitor medications or where MAO inhibitors are contraindicated.

The new Internal Transcribed Spacer 2 diagnostic tool clarifies the taxonomic position and geographic distribution of the North American malaria vector Anopheles punctipennis.

BACKGROUND: The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. METHODS: In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. RESULTS: The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. CONCLUSIONS: This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

Modified technique for coarctation of aorta with hypoplastic distal aortic arch.

BACKGROUND: A combination of coarctation of aorta with various severity of distal arch hypoplasia frequently occurs in newborns. Traditional techniques in the neonatal period such as extended end-to-end anastomosis or inner curve patch are controversial. Arch geometry has a marked role in long-term outcomes. We introduce a modified Amato technique of distal aortic arch enlargement with native tissue-to-tissue reconstruction. METHODS: Neonatal patients with coarctation of aorta and distal aortic arch hypoplasia who underwent surgical reconstruction using this technique between January 2016 and December 2019 in our center were included. Patients with concomitant complex heart defects were excluded. Data were obtained from echo protocols, CT scans before and after repair. The dimensions of the arch were assessed using Z-score, arch geometry was evaluated with height/width ratio. RESULTS: Thirty-two patients (22 males, 10 females) were included. Median age and weight were 7 days (5; 18) and 3.5 kg (3.1; 4.0), respectively. The Z-score of distal part of the arch before and after procedure was significantly different (<0.01). No mortality, recoarctation, or bronchial compression was found during 18 (6-38) months of follow-up. CONCLUSION: Modified technique for coarctation of aorta with hypoplastic distal aortic arch provides favorable geometry of the aorta with a low risk of morbidity. The proper selection and accurate technique could minimize potential risks. This method is relatively safe and might improve long-term outcomes associated with the geometry of aorta.

A 300-kb microduplication of 7q36.3 in a patient with triphalangeal thumb-polysyndactyly syndrome combined with congenital heart disease and optic disc coloboma: a case report.

BACKGROUND: Triphalangeal thumb-polysyndactyly syndrome (TPT-PS) is a rare well-defined autosomal dominant disorder characterized by long thumbs with three phalanges combined with pre- and postaxial polydactyly/syndactyly of limbs. By now, the syndrome has been reported in several large families from different ethnic backgrounds, with a high degree of inter- and intrafamilial variability. The genome locus responsible for TPT-PS has been mapped to the 7q36.3 region harboring a long-range sonic hedgehog (SHH) regulatory sequence (ZRS). Both single-nucleotide variants and complete duplications of ZRS were shown to cause TPT-PS and similar limb phenotypes. TPT-PS usually forms as isolated limb pathology not associated with additional malformations, in particular, with cardiovascular abnormalities. CASE PRESENTATION: Here we report on a rare Russian neonatal case of TPT-PS combined with severe congenital heart disease, namely double outlet right ventricle, and microphthalmia with optic disc coloboma. Pedigree analysis revealed TPT-PS of various expressivity in 10 family members throughout five generations, while the cardiac defect and the eye pathology were detected only in the proband. To extend the knowledge on genotype-phenotype spectrum of TPT-PS, the careful clinical and genomic analysis of the family was performed. High-resolution array-based comparative genomic hybridization (array-CGH) revealed a ~ 300 kb microduplication of 7q36.3 locus (arr[GRCh37] 7q36.3(156385810_156684811) × 3) that co-segregated with TPT-PS in the proband and her mother. The duplication encompassed three genes including LMBR1, the intron 5 of which is known to harbor ZRS. Based on whole-exome sequencing data, no additional pathogenic mutations or variants of uncertain clinical significance were found in morbid cardiac genes or genes associated with a microphthalmia/anophthalmia/coloboma spectrum of ocular malformations. CONCLUSIONS: The results support the previous data, indicating that complete ZRS duplication underlies TPT-PS, and suggest a broader phenotypic impact of the 7q36.3 microduplication. Potential involvement of the 7q36.3 microduplication in the patient's cardiac and eye malformations is discussed. However, the contribution of some additional genetic/epigenetic factors to the complex patient`s phenotype cannot be excluded entirely. Further comprehensive functional studies are needed to prove the possible involvement of the 7q36.3 locus in congenital heart disease and eye pathology.

Generation of two iPSC lines (FAMRCi006-A and FAMRCi006-B) from patient with dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy associated with genetic variant LMNAp.Arg527Pro.

Mutations in LMNA gene are known to cause a broad range of diseases called laminopathies. We have generated two induced pluripotent stem cell lines FAMRCi006-A and FAMRCi006-B from a patient carrying LMNA p. p.Arg527Pro mutation associated with Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. Patient-specific peripheral blood mononuclear cells were reprogrammed to iPSCs using Sendai virus reprogramming system. Characterization of iPSCs had revealed pluripotency marker expression, normal karyotype, ability to differentiate into three embryonic germ layers. The reported iPSC lines could be a useful tool for in vitro modeling of laminopathies associated with LMNA genetic variants.

Chromosome and Genome Divergence between the Cryptic Eurasian Malaria Vector-Species Anopheles messeae and Anopheles daciae.

Chromosomal inversions are important drivers of genome evolution. The Eurasian malaria vector Anophelesmesseae has five polymorphic inversions. A cryptic species, An. daciae, has been discriminated from An. messeae based on five fixed nucleotide substitutions in the internal transcribed spacer 2 (ITS2) of ribosomal DNA. However, the inversion polymorphism in An. daciae and the genome divergence between these species remain unexplored. In this study, we sequenced the ITS2 region and analyzed the inversion frequencies of 289 Anopheles larvae specimens collected from three locations in the Moscow region. Five individual genomes for each of the two species were sequenced. We determined that An. messeae and An. daciae differ from each other by the frequency of polymorphic inversions. Inversion X1 was fixed in An. messeae but polymorphic in An. daciae populations. The genome sequence comparison demonstrated genome-wide divergence between the species, especially pronounced on the inversion-rich X chromosome (mean Fst = 0.331). The frequency of polymorphic autosomal inversions was higher in An. messeae than in An. daciae. We conclude that the X chromosome inversions play an important role in the genomic differentiation between the species. Our study determined that An. messeae and An. daciae are closely related species with incomplete reproductive isolation.

Dysregulation of Notch signaling in cardiac mesenchymal cells of patients with tetralogy of Fallot.

BACKGROUND: Tetralogy of Fallot (TF) is a severe congenital defect of heart development. Fine-tuned sequential activation of Notch signaling genes is responsible for proper heart chamber development. Mutations in Notch genes have been associated with TF. The aim of this study was to analyze the activity of the Notch pathway in cardiac mesenchymal cells derived from ventricular tissue of TF patients. METHODS: Cardiac mesenchymal cells were isolated from 42 TF patients and from 14 patients with ventricular septal defects (VSDs), used as a comparison group. The Notch pathway was analyzed by estimating the expression of Notch-related genes by qPCR. Differentiation and proliferation capacity of the cells was estimated. RESULTS: The TF-derived cells demonstrated a dysregulated pattern of Notch-related gene expression comparing to VSD-derived cells. Correlation of Notch signaling activation level by HEY1/HES1 expression level with proliferation and cardiogenic-like differentiation of cardiac mesenchymal cells was observed but not with clinical parameters nor with the age of the patients. CONCLUSIONS: The data suggest a contribution of dysregulated Notch signaling to the pathogenesis of tetralogy of Fallot and importance of Notch signaling level for the functional state of cardiac mesenchymal cells, which could be critical considering these cells for potential cell therapy approaches.