Immune niches for hair follicle development and homeostasis.

The hair follicle is a dynamic mini-organ that has specialized cycles and architectures with diverse cell types to form hairs. Previous studies for several decades have investigated morphogenesis and signaling pathways during embryonic development and adult hair cycles in both mouse and human skin. In particular, hair follicle stem cells and mesenchymal niches received major attention as key players, and their roles and interactions were heavily revealed. Although resident and circulating immune cells affect cellular function and interactions in the skin, research on immune cells has mainly received attention on diseases rather than development or homeostasis. Recently, many studies have suggested the functional roles of diverse immune cells as a niche for hair follicles. Here, we will review recent findings about immune niches for hair follicles and provide insight into mechanisms of hair growth and diseases.

ER stress and lipid imbalance drive diabetic embryonic cardiomyopathy in an organoid model of human heart development.

Congenital heart defects are the most prevalent human birth defects, and their incidence is exacerbated by maternal health conditions, such as diabetes during the first trimester (pregestational diabetes). Our understanding of the pathology of these disorders is hindered by a lack of human models and the inaccessibility of embryonic tissue. Using an advanced human heart organoid system, we simulated embryonic heart development under pregestational diabetes-like conditions. These organoids developed pathophysiological features observed in mouse and human studies before, including ROS-mediated stress and cardiomyocyte hypertrophy. scRNA-seq revealed cardiac cell-type-specific dysfunction affecting epicardial and cardiomyocyte populations and alterations in the endoplasmic reticulum and very-long-chain fatty acid lipid metabolism. Imaging and lipidomics confirmed these findings and showed that dyslipidemia was linked to fatty acid desaturase 2 mRNA decay dependent on IRE1-RIDD signaling. Targeting IRE1 or restoring lipid levels partially reversed the effects of pregestational diabetes, offering potential preventive and therapeutic strategies in humans.

A patterned human primitive heart organoid model generated by pluripotent stem cell self-organization.

Pluripotent stem cell-derived organoids can recapitulate significant features of organ development in vitro. We hypothesized that creating human heart organoids by mimicking aspects of in utero gestation (e.g., addition of metabolic and hormonal factors) would lead to higher physiological and anatomical relevance. We find that heart organoids produced using this self-organization-driven developmental induction strategy are remarkably similar transcriptionally and morphologically to age-matched human embryonic hearts. We also show that they recapitulate several aspects of cardiac development, including large atrial and ventricular chambers, proepicardial organ formation, and retinoic acid-mediated anterior-posterior patterning, mimicking the developmental processes found in the post-heart tube stage primitive heart. Moreover, we provide proof-of-concept demonstration of the value of this system for disease modeling by exploring the effects of ondansetron, a drug administered to pregnant women and associated with congenital heart defects. These findings constitute a significant technical advance for synthetic heart development and provide a powerful tool for cardiac disease modeling.

Tumor-associated macrophages respond to chemotherapy by detrimental transcriptional reprogramming and suppressing stabilin-1 mediated clearance of EGF.

Introduction: Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor-associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs. Methods: The effect of chemotherapeutic platinum agent cisplatin was assessed in the model system of human ex vivo TAMs. Whole-transcriptome sequencing for paired TAMs stimulated and not stimulated by cisplatin was analysed by NGS. Endocytic uptake of EGF was quantified by flow cytometry. Confocal microscopy was used to visualize stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells expressing ectopically recombinant stabilin-1 and in stabilin-1+ TAMs. In cohort of patients with breast cancer, the effect of platinum therapy on the transcriptome of TAMs was validated, and differential expression of regulators of endocytosis was identified. Results: Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated that TAMs' scavenger receptor stabilin-1 is responsible for the clearance of epidermal growth factor (EGF), a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on synaptotagmin-11 (SYT11), confirmed in patients with breast cancer. Conclusion: Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.

Exosomes Released by Influenza-Virus-Infected Cells Carry Factors Capable of Suppressing Immune Defense Genes in Naïve Cells.

Background: Exosomes are involved in intercellular communication and can transfer regulatory molecules between cells. Consequently, they can participate in host immune response regulation. For the influenza A virus (IAV), there is very limited information on changes in exosome composition during cell infection shedding light on the potential role of these extracellular membrane vesicles. Thus, the aim of our work was to study changes in exosomal composition following IAV infection of cells, as well as to evaluate their effect on uninfected cells. Methods: To characterize changes in the composition of cellular miRNAs and mRNAs of exosomes during IAV infection of A549 cells, NGS was used, as well as PCR to identify viral genes. Naïve A549 cells were stimulated with infected-cell-secreted exosomes for studying their activity. Changes in the expression of genes associated with the cell's immune response were shown using PCR. The effect of exosomes on IAV replication was shown in MDCK cells using In-Cell ELISA and PCR of the supernatants. Results: A change in the miRNA composition (miR-21-3p, miR-26a-5p, miR-23a-5p, miR-548c-5p) and mRNA composition (RPL13A, MKNK2, TRIB3) of exosomes under the influence of the IAV was shown. Many RNAs were involved in the regulation of the immune response of the cell, mainly by suppressing it. After exosome stimulation of naïve cells, a significant decrease in the expression of genes involved in the immune response was shown (RIG1, IFIT1, MDA5, COX2, NFκB, AnxA1, PKR, IL6, IL18). When infecting MDCK cells, a significant decrease in nucleoprotein levels was observed in the presence of exosomes secreted by mock-infected cells. Viral levels in supernatants also decreased. Conclusions: Exosomes secreted by IAV-infected cells could reduce the immune response of neighboring intact cells, leading to more effective IAV replication. This may be associated both with regulatory functions of cellular miRNAs and mRNAs carried by exosomes, or with the presence of viral mRNAs encoding proteins with an immunosuppressive function.

Pathogenicity Reclassification of Genetic Variants Related to Early-Onset Breast Cancer among Women of Mongoloid Origin.

BACKGROUND: Germline alterations in BRCA1, BRCA2, and other genes are responsible for early-onset breast cancer. However, up to 20% of molecular tests report genetic variant of unknown significance (VUS) or novel variants that have never been previously described and their clinical significance are unknown. This study aimed to reclassify variant of unknown significance (VUS) or novel variants by using the ActiveDriveDB database that annotates variants through the lens of sites of post-translational modifications (PTM). METHODS: Our study included thirty-eighth young Buryat BC patients, belonging to the Mongoloid race and anthropologically to the Central Asia. Genomic DNA was extracted from the peripheral blood lymphocytes using the phenol/chloroform method. DNA library were prepared using the Hereditary Cancer SolutionTM kit (Sophia GENETICS, Switzerland) to cover 27 genes, such as ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PMS2, PMS2CL, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2. Paired-end sequencing (2 x 150 bp) was conducted using NextSeq 500 system (Illumina, USA). RESULTS: We re-examined 135 rare variants (41 VUS, 25 conflicting, 64 benign and 5 new variants). We identified 10 out of 135 (7.4%) mutations that affected the sites of post-translational modification in proteins. Of 135 rare mutations, 1 benign variant was reclassified as network-rewiring - motif loss mutation, 3 VUS and 1 new variant were reclassified as distal PTM- mutations, 2 new and 1 benign variant were classified as proximal PTM- mutations and 1 benign and 1 conflicting variant were classified as direct PTM- mutations. CONCLUSIONS: For the first time, 7.4% (10 out of 135) of mutations that affected the sites of post-translational modification in proteins were identified among early-onset breast cancer women of Mongoloid origin.

Characterization of the novel heterozygous SCN5A genetic variant Y739D associated with Brugada syndrome.

Genetic variants in SCN5A gene were identified in patients with various arrhythmogenic conditions including Brugada syndrome. Despite significant progress of last decades in studying the molecular mechanism of arrhythmia-associated SCN5A mutations, the understanding of relationship between genetics, electrophysiological consequences and clinical phenotype is lacking. We have found a novel genetic variant Y739D in the SCN5A-encoded sodium channel Nav1.5 of a male patient with Brugada syndrome (BrS). The objective of the study was to characterize the biophysical properties of Nav1.5-Y739D and provide possible explanation of the phenotype observed in the patient. The WT and Y739D channels were heterologously expressed in the HEK-293T cells and the whole-cell sodium currents were recorded. Substitution Y739D reduced the sodium current density by 47 ± 2% at -20 mV, positively shifted voltage-dependent activation, accelerated both fast and slow inactivation, and decelerated recovery from the slow inactivation. The Y739D loss-of-function phenotype likely causes the BrS manifestation. In the hNav1.5 homology models, which are based on the cryo-EM structure of rat Nav1.5 channel, Y739 in the extracellular loop IIS1-S2 forms H-bonds with K1381 and E1435 and pi-cation contacts with K1397 (all in loop IIIS5-P1). In contrast, Y739D accepts H-bonds from K1397 and Y1434. Substantially different contacts of Y739 and Y739D with loop IIIS5-P1 would differently transmit allosteric signals from VSD-II to the fast-inactivation gate at the N-end of helix IIIS5 and slow-inactivation gate at the C-end of helix IIIP1. This may underlie the atomic mechanism of the Y739D channel dysfunction.

PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse.

Introduction: Circulating monocytes are main source for tumor-associated macrophages (TAMs) that control tumor growth, angiogenesis, metastasis and therapy resistance. We raised the questions how monocyte programming is affected by growing tumors localized in colon and rectal sections, and how treatment onsets affect monocyte programming in the circulation. Methods: Patients with rectal cancer and colon cancer were enrolled in the study. Peripheral blood monocytes were characterized by phenotypic analysis using flow cytometry, by transcriptomic analysis using RNA sequencing and by gene expression analysis using real-time RT-PCR. Phenotypic analysis was performed with IF/confocal microscopy. Spatial transcriptomic analysis was applied using GeoMX DSP-NGS. Results: In patients with rectal cancer, increased amount of CCR2+ monocytes was indicative for the absence of both lymphatic and hematogenous metastasis. In contrast, in patients with colon cancer CD163+ monocytes were indicative for LN metastasis. NGS analysis identified tumor-specific transcriptional programming of monocytes in all CRC patients compared to healthy individuals. The key transcriptional difference between monocytes of patients with colon and rectal cancer was increased expression of PFKFB3, activator of glycolysis that is currently considered as therapy target for major solid cancers. PFKFB3-expressing monocyte-derived macrophages massively infiltrated tumor in colon. Nanostring technology identified correlation of PFKFB3 with amount and tumor-promoting properties of TAMs in colon but not in rectal cancer. PFKFB3 was indicative for tumor relapse specifically in colon cancer. Discussion: Our findings provide essential argument towards CRC definition to cover two clinically distinct cancers - colon cancer and rectal cancer, that differentially interact with innate immunity.

Context-Specific Osteogenic Potential of Mesenchymal Stem Cells.

Despite the great progress in the field of bone tissue regeneration, the early initiating mechanisms of osteogenic differentiation are not well understood. Cells capable of osteogenic transformation vary from mesenchymal stem cells of various origins to mural cells of vessels. The mechanisms of pathological calcification are thought to be similar to those of bone formation. Notch signaling has been shown to play an important role in osteogenic differentiation, as well as in pathological calcification. Nevertheless, despite its known tissue- and context-specificity, the information about its role in the osteogenic differentiation of different cells is still limited. We compared mesenchymal stem cells from adipogenic tissue (MSCs) and interstitial cells from the aortic valve (VICs) by their ability to undergo Notch-dependent osteogenic differentiation. We showed differences between the two types of cells in their ability to activate the expression of proosteogenic genes RUNX2, BMP2, BMP4, DLX2, BGLAP, SPRY, IBSP, and SPP1 in response to Notch activation. Untargeted metabolomic profiling also confirms differences between MSCs and VICs in their osteogenic state. Analysis of the activity of RUNX2 and SPP1 promoters shows fine-tuned dose-dependency in response to Notch induction and suggests a direct link between the level of Notch activation, and the proostogenic gene expression and corresponding osteogenic induction. Our data suggest that osteogenic differentiation is a context-dependent process and the outcome of it could be cell-type dependent.

Suppression of IgE-mediated anaphylaxis and food allergy with monovalent anti-FcεRIα mAbs.

BACKGROUND: Mast cell and basophil activation by antigen cross-linking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVES: We sought to determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin variable regions and huIgG1 or huIgG4 C regions and were used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα; mice that additionally have an allergy-promoting IL-4Rα mutation; and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a ß-adrenergic receptor antagonist. RESULTS: mv anti-hu FcεRIα mAbs are considerably less able than divalent mAbs are to induce anaphylaxis and deplete mast cell and basophil IgE, but mv mAbs still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant is, apparently because reduced interactions with FcεRs decrease ability to indirectly cross-link FcεRI. CONCLUSIONS: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs do. These mv mAbs may be useful for suppression of huIgE-mediated disease.

Comparative Experimental and Theoretical Study of Mg, Al and Zn Aryloxy Complexes in Copolymerization of Cyclic Esters: The Role of the Metal Coordination in Formation of Random Copolymers.

Homogeneity of copolymers is a general problem of catalytic coordination polymerization. In ring-opening polymerization of cyclic esters, the rational design of the catalyst is generally applied to solve this problem by the equalization of the reactivities of comonomers-however, it often leads to a reduction of catalytic activity. In the present paper, we studied the catalytic behavior of BnOH-activated complexes (ВНТ)Mg(THF)2nBu (1), (ВНТ)2AlMe (2) and [(ВНТ)ZnEt]2 (3), based on 2,6-di-tert-butyl-4-methylphenol (BHT-H) in homo- and copolymerization of L-lactide (lLA) and ε-caprolactone (εCL). Even at 1:5 lLA/εCL ratio Mg complex 1 catalyzed homopolymerization of lLA without involving εCL to the formation of the polymer backbone. On the contrary, Zn complex 3 efficiently catalyzed random lLA/εCL copolymerization; the presence of mono-lactate subunits in the copolymer chain clearly pointed to the transesterification mechanism of copolymer formation. Both epimerization and transesterification side processes were analyzed using the density functional theory (DFT) modeling that confirmed the qualitative difference in catalytic behavior of 1 and 3: Mg and Zn complexes demonstrated different types of preferable coordination on the PLA chain (k2 and k3, respectively) with the result that complex 3 catalyzed controlled εCL ROP/PLA transesterification, providing the formation of lLA/εCL copolymers that contain mono-lactate fragments separated by short oligo(εCL) chains. The best results in the synthesis of random lLA/εCL copolymers were obtained during experiments on transesterification of commercially available PLLA, the applicability of 3/BnOH catalyst in the synthesis of random copolymers of εCL with methyl glycolide, ethyl ethylene phosphonate and ethyl ethylene phosphate was also demonstrated.

Two New Cases of Hypertrophic Cardiomyopathy and Skeletal Muscle Features Associated with ALPK3 Homozygous and Compound Heterozygous Variants.

Hypertrophic cardiomyopathy associated with damaging variants in the ALPK3 gene is a fairly recent discovery, and only a small number of patients have been described thus far. Here we present two additional patients with hypertrophic cardiomyopathy caused by biallelic variants in ALPK3. Genetic investigation was performed using a targeted gene panel consisting of known cardiomyopathy-associated genes and whole exome sequencing. The patients showed a large difference in the age of onset, and both presented with extracardiac features that are often seen in ALPK3 patients. The patient with the later onset showed milder extracardiac symptoms, such as decreased muscle tone and distal muscular dystrophy, but had fast progression of cardiac complications leading to the need of heart transplantation. This study further elucidates the variability of both symptoms and age of onset among these patients.

Genetic Spectrum of Left Ventricular Non-Compaction in Paediatric Patients.

INTRODUCTION: Left ventricular non-compaction (LVNC) represents a genetically heterogeneous cardiomyopathy which occurs in both children and adults. Its genetic spectrum overlaps with other types of cardiomyopathy. However, LVNC phenotypes in different age groups can have distinct genetic aetiologies. The aim of the study was to decipher the genetic spectrum of LVNC presented in childhood. Patient Group and Methods: Twenty patients under the age of 18 years diagnosed with LVNC were enrolled in the study. Target sequencing and whole-exome sequencing were performed using a panel of 108 cardiomyopathy-associated genes. Pathogenic, likely pathogenic, and variants of unknown significance found in genes highly expressed in cardiomyocytes were considered as variants of interest for further analysis. RESULTS: The median age at presentation was 8.0 (0.1-17) years, with 6 patients presenting before 1 year of age. Twelve (60%) patients demonstrated reduced ejection fraction. Right ventricular (RV) dilation was registered in 6 (30%), often in combination with reduced RV contractility (25%). Almost half (45%) of the patients demonstrated biventricular involvement already at disease presentation. For pathogenic and likely pathogenic variants, the positive genotyping rate was 45%, and these variants were found mainly in non-contractile structural sarcomeric genes (ACTN2, MYPN, and TTN) or in metabolic and signal transduction genes (BRAF and TAZ). Likely pathogenic TAZ variants were detected in all 5 patients suspected of having Barth syndrome. No pathogenic or likely pathogenic variants were found in genes encoding for sarcomeric contractile proteins, but variants of unknown significance were detected in 3 out of 20 patients (MYH6, MYH7, and MYLK2). In 4 patients, variants of unknown significance in ion-channel genes were detected. CONCLUSION: We detected a low burden of contractile sarcomeric variants in LVNC patients presenting below the age of 18 years, with the major number of variants residing in non-contractile structural sarcomeric genes. The identification of the variants in ion-channel and related genes not previously associated with LVNC in paediatric patients requires further examination of their functional role.

New germline mutations in non-BRCA genes among breast cancer women of Mongoloid origin.

In accordance with the Asian BRCA Consortium data, there is a significant difference in incidence rate of breast cancer depending on age, as well as spectrum and prevalence of BRCA1/2 mutations between Mongoloid (East Asian) and Caucasoid (European) people. However, European strategies to identify familial BC are still applied to the Asian population, including Russian Mongoloids (Khakas, Buryats, Tyvans and Yakuts and others). The main purpose of the study was to identify molecular changes associated with hereditary BC in Russian Mongoloid BC patients (Buryats). Thirty-nine patients were included in the study. Genomic DNA extracted from lymphocytes was used to prepare DNA-libraries. Target sequencing was designed to cover 27 genes, such as ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2 and others. Paired-end sequencing (2 × 150 bp) was conducted on a NextSeq 500 system (Illumina, USA). Three pathogenic mutations in non-BRCA genes were found (prevalence of 8%). The pathogenic mutations were found in the RAD51D and PTEN genes. The pathogenic variant in the RAD51D gene (rs137886232, NC_000017.10:g.33428366G>A, p.R141X) was observed in two unrelated individuals aged under 40. One of these patients had a family history of late-onset stomach cancer in second-degree relatives. The pathogenic mutation in the PTEN gene (rs786201044, NC_000010.10:g.89692922T>C, p.C136R) was observed in a 38 years old breast cancer patient with no family history. In our study, we first describe pathogenic mutations in RAD51D and PTEN genes found in young Buryat patients.

The Activity of KIF14, Mieap, and EZR in a New Type of the Invasive Component, Torpedo-Like Structures, Predetermines the Metastatic Potential of Breast Cancer.

Intratumor morphological heterogeneity reflects patterns of invasive growth and is an indicator of the metastatic potential of breast cancer. In this study, we used this heterogeneity to identify molecules associated with breast cancer invasion and metastasis. The gene expression microarray data were used to identify genes differentially expressed between solid, trabecular, and other morphological arrangements of tumor cells. Immunohistochemistry was applied to evaluate the association of the selected proteins with metastasis. RNA-sequencing was performed to analyze the molecular makeup of metastatic tumor cells. High frequency of metastases and decreased metastasis-free survival were detected in patients either with positive expression of KIF14 or Mieap or negative expression of EZR at the tips of the torpedo-like structures in breast cancers. KIF14- and Mieap-positive and EZR-negative cells were mainly detected in the torpedo-like structures of the same breast tumors; however, their transcriptomic features differed. KIF14-positive cells showed a significant upregulation of genes involved in ether lipid metabolism. Mieap-positive cells were enriched in genes involved in mitophagy. EZR-negative cells displayed upregulated genes associated with phagocytosis and the chemokine-mediated signaling pathway. In conclusion, the positive expression of KIF14 and Mieap and negative expression of EZR at the tips of the torpedo-like structures are associated with breast cancer metastasis.

Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage.

Advantages and diagnostic effectiveness of the two most widely used resequencing approaches, whole exome (WES) and whole genome (WGS) sequencing, are often debated. WES dominated large-scale resequencing projects because of lower cost and easier data storage and processing. Rapid development of 3rd generation sequencing methods and novel exome sequencing kits predicate the need for a robust statistical framework allowing informative and easy performance comparison of the emerging methods. In our study we developed a set of statistical tools to systematically assess coverage of coding regions provided by several modern WES platforms, as well as PCR-free WGS. We identified a substantial problem in most previously published comparisons which did not account for mappability limitations of short reads. Using regression analysis and simple machine learning, as well as several novel metrics of coverage evenness, we analyzed the contribution from the major determinants of CDS coverage. Contrary to a common view, most of the observed bias in modern WES stems from mappability limitations of short reads and exome probe design rather than sequence composition. We also identified the ~ 500 kb region of human exome that could not be effectively characterized using short read technology and should receive special attention during variant analysis. Using our novel metrics of sequencing coverage, we identified main determinants of WES and WGS performance. Overall, our study points out avenues for improvement of enrichment-based methods and development of novel approaches that would maximize variant discovery at optimal cost.

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.

Meglumine acridone acetate, the ionic salt of CMA and N-methylglucamine, induces apoptosis in human PBMCs via the mitochondrial pathway.

Meglumine acridone acetate (MA) is used in Russia for the treatment of influenza and other acute respiratory viral infections. It was assumed, until recently, that its antiviral effect was associated with its potential ability to induce type I interferon. Advanced studies, however, have shown the failure of 10-carboxymethyl-9-acridanone (CMA) to activate human STING. As such, MA's antiviral properties are still undergoing clarification. To gain insight into MA's mechanisms of action, we carried out RNA-sequencing analysis of global transcriptomes in MA-treated (MA+) human peripheral blood mononuclear cells (PBMCs). In response to treatment, approximately 1,223 genes were found to be differentially expressed, among which 464 and 759 were identified as either up- or down-regulated, respectively. To clarify the cellular and molecular processes taking place in MA+ cells, we performed a functional analysis of those genes. We have shown that evident MA subcellular localizations are: at the nuclear envelope; inside the nucleus; and diffusely in perinuclear cytoplasm. Postulating that MA may be a nuclear receptor agonist, we carried out docking simulations with PPARα and RORα ligand binding domains including prediction and molecular dynamics-based analysis of potential MA binding poses. Finally, we confirmed that MA treatment enhanced nuclear apoptosis in human PBMCs. The research presented here, in our view, indicates that: (i) MA activity is mediated by nuclear receptors; (ii) MA is a possible PPARα and/or RORα agonist; (iii) MA has an immunosuppressive effect; and (iv) MA induces apoptosis through the mitochondrial signaling pathway.

Truncating Variant in Myof Gene Is Associated With Limb-Girdle Type Muscular Dystrophy and Cardiomyopathy.

Even though genetic studies of individuals with neuromuscular diseases have uncovered the molecular background of many cardiac disorders such as cardiomyopathies and inherited arrhythmic syndromes, the genetic cause of a proportion of cardiomyopathies associated with neuromuscular phenotype still remains unknown. Here, we present an individual with a combination of cardiomyopathy and limb-girdle type muscular dystrophy where whole exome sequencing identified myoferlin (MYOF)-a member of the Ferlin protein family and close homolog of DYSF-as the most likely candidate gene. The disease-causative role of the identified variant c.[2576delG; 2575G>C], p.G859QfsTer8 is supported by functional studies in vitro using the primary patient's skeletal muscle mesenchymal progenitor cells, including both RNA sequencing and morphological studies, as well as recapitulating the muscle phenotype in vivo in zebrafish. We provide the first evidence supporting a role of MYOF in human muscle disease.