Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eµ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

Recent Advances in Cardiovascular Diseases Research Using Animal Models and PET Radioisotope Tracers.

Cardiovascular diseases (CVD) is a collective term describing a range of conditions that affect the heart and blood vessels. Due to the varied nature of the disorders, distinguishing between their causes and monitoring their progress is crucial for finding an effective treatment. Molecular imaging enables non-invasive visualisation and quantification of biological pathways, even at the molecular and subcellular levels, what is essential for understanding the causes and development of CVD. Positron emission tomography imaging is so far recognized as the best method for in vivo studies of the CVD related phenomena. The imaging is based on the use of radioisotope-labelled markers, which have been successfully used in both pre-clinical research and clinical studies. Current research on CVD with the use of such radioconjugates constantly increases our knowledge and understanding of the causes, and brings us closer to effective monitoring and treatment. This review outlines recent advances in the use of the so-far available radioisotope markers in the research on cardiovascular diseases in rodent models, points out the problems and provides a perspective for future applications of PET imaging in CVD studies.

Hypoxia-Induced FAM13A Regulates the Proliferation and Metastasis of Non-Small Cell Lung Cancer Cells.

Hypoxia in non-small cell lung cancer (NSCLC) affects cancer progression, metastasis and metabolism. We previously showed that FAM13A was induced by hypoxia in NSCLC but the biological function of this gene has not been fully elucidated. This study aimed to investigate the role of hypoxia-induced FAM13A in NSCLC progression and metastasis. Lentiviral shRNAs were used for FAM13A gene silencing in NSCLC cell lines (A549, CORL-105). MTS assay, cell tracking VPD540 dye, wound healing assay, invasion assay, BrdU assay and APC Annexin V staining assays were performed to examine cell proliferation ability, migration, invasion and apoptosis rate in NSCLC cells. The results of VPD540 dye and MTS assays showed a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured under normal and hypoxia (1% O2) conditions (p < 0.05), while the effect of FAM13A downregulation on CORL-105 cells was observed after 96 h exposition to hypoxia. Moreover, FAM13A inhibition induced S phase cell cycle arrest in A549 cells under hypoxia conditions. Silencing of FAM13A significantly suppressed migration of A549 and CORL-105 cells in both oxygen conditions, especially after 72 and 96 h (p < 0.001 in normoxia, p < 0.01 after hypoxia). It was showed that FAM13A reduction resulted in disruption of the F-actin cytoskeleton altering A549 cell migration. Cell invasion rates were significantly decreased in A549 FAM13A depleted cells compared to controls (p < 0.05), mostly under hypoxia. FAM13A silencing had no effect on apoptosis induction in NSCLC cells. In the present study, we found that FAM13A silencing has a negative effect on proliferation, migration and invasion activity in NSCLC cells in normal and hypoxic conditions. Our data demonstrated that FAM13A depleted post-hypoxic cells have a decreased cell proliferation ability and metastatic potential, which indicates FAM13A as a potential therapeutic target in lung cancer.

Multiparametric Evaluation of Post-MI Small Animal Models Using Metabolic ([18F]FDG) and Perfusion-Based (SYN1) Heart Viability Tracers.

Cardiovascular diseases (CVD), with myocardial infarction (MI) being one of the crucial components, wreak havoc in developed countries. Advanced imaging technologies are required to obtain quick and widely available diagnostic data. This paper describes a multimodal approach to in vivo perfusion imaging using the novel SYN1 tracer based on the fluorine-18 isotope. The NOD-SCID mice were injected intravenously with SYN1 or [18F] fluorodeoxyglucose ([18F]-FDG) radiotracers after induction of the MI. In all studies, the positron emission tomography-computed tomography (PET/CT) technique was used. To obtain hemodynamic data, mice were subjected to magnetic resonance imaging (MRI). Finally, the biodistribution of the SYN1 compound was performed using Wistar rat model. SYN1 showed normal accumulation in mouse and rat hearts, and MI hearts correctly indicated impaired cardiac segments when compared to [18F]-FDG uptake. In vivo PET/CT and MRI studies showed statistical convergence in terms of the size of the necrotic zone and cardiac function. This was further supported with RNAseq molecular analyses to correlate the candidate function genes' expression, with Serpinb1c, Tnc and Nupr1, with Trem2 and Aldolase B functional correlations showing statistical significance in both SYN1 and [18F]-FDG. Our manuscript presents a new fluorine-18-based perfusion radiotracer for PET/CT imaging that may have importance in clinical applications. Future research should focus on confirmation of the data elucidated here to prepare SYN1 for first-in-human trials.

Molecular Imaging of Human Skeletal Myoblasts (huSKM) in Mouse Post-Infarction Myocardium.

Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone (p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.

Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and their Molecular Characteristics.

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3, TM4SF18, CFI, GPX8, and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process.

Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus.

Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenine-adenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAA-induced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN. Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus.

Excision of Expanded GAA Repeats Alleviates the Molecular Phenotype of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurological disease caused by expansions of guanine-adenine-adenine (GAA) repeats in intron 1 of the frataxin (FXN) gene. The expansion results in significantly decreased frataxin expression. We report that human FRDA cells can be corrected by zinc finger nuclease-mediated excision of the expanded GAA repeats. Editing of a single expanded GAA allele created heterozygous, FRDA carrier-like cells and significantly increased frataxin expression. This correction persisted during reprogramming of zinc finger nuclease-edited fibroblasts to induced pluripotent stem cells and subsequent differentiation into neurons. The expression of FRDA biomarkers was normalized in corrected patient cells and disease-associated phenotypes, such as decreases in aconitase activity and intracellular ATP levels, were reversed in zinc finger nuclease corrected neuronal cells. Genetically and phenotypically corrected patient cells represent not only a preferred disease-relevant model system to study pathogenic mechanisms, but also a critical step towards development of cell replacement therapy.

Semen Quality, Hormonal Levels, and Androgen Receptor Gene Polymorphisms in a Population of Young Male Volunteers from Two Different Regions of Poland.

BACKGROUND: The population of healthy Polish men has not been frequently and systematically investigated for fertility status. The aim of this study was to assess the quality of semen in a randomly recruited population of young males. The most important task was to find a relationship between semen parameters, sex hormones, and AR gene polymorphism. MATERIAL AND METHODS: Semen and blood samples from young men from the Poznan (n=113) and Lublin regions (n=89) were collected for semen analysis, assessment of hormonal concentrations, and calculation of the CAG and GGN repeats of the AR gene. RESULTS: Statistical comparisons of the hormones and circulating proteins and the seminological parameters revealed significant differences between the regional groups of males studied. Among the correlations found, we emphasize the positive relationship between inhibin B levels and both the number of spermatozoa per ml (R=0.37; p=0.0001) and the total sperm concentration (R=0.40; p=0.00003). Positive correlations between IGF1 and sperm morphology was also found (R=0.40; p=0.000004). The mean number of CAG repeats in our tested groups was 21.93±2.79, in a range from 16 to 31. The mean number of GGN repeats was 23.2±1.66 and ranged from 16 to 29. Numerous significant correlations were found between CAG or GGN repeats and blood hormones or circulating proteins and semen parameters; however, Spearman's rank correlations revealed rather weak coefficients. CONCLUSIONS: This report attempted to determine the quality of semen samples and sex hormones in a population of Polish young men. The results were found to be similar to data obtained in Scandinavia. The calculated means and range of CAG or GGN repeats of the AR gene in Polish males were similar to West European epidemiological data.

Inhibition of the glutamate-cysteine ligase catalytic subunit with buthionine sulfoximine enhances the cytotoxic effect of doxorubicin and cyclophosphamide in Burkitt lymphoma cells.

Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in adults is less satisfactory. Therefore, there is a need to enhance the cytotoxic effect of drugs used in BL treatment. Glutathione (GSH) is an important antioxidant involved in processes such as regulation of oxidative stress and drug detoxification. Elevated GSH levels have been observed in many cancers and were associated with chemoresistance. We previously identified GCLC, encoding an enzyme involved in GSH biosynthesis, as an essential gene in BL. We now confirm that knockout of GCLC decreases viability of BL cells and that the GCLC protein is overexpressed in BL tissues. Moreover, we demonstrate that buthionine sulfoximine (BSO), a known inhibitor of GCLC, decreases growth of BL cells but does not affect control B cells. Furthermore, we show for the first time that BSO enhances the cytotoxicity of compounds commonly used in BL treatment, doxorubicin, and cyclophosphamide. Given the fact that BSO itself was not toxic to control cells and well-tolerated in clinical trials, combination of chemotherapy with BSO may allow reduction of the doses of cytotoxic drugs required to obtain effective responses in BL patients.

Identification of ATM-dependent long non-coding RNAs induced in response to DNA damage.

DNA damage response (DDR) is a complex process, essential for cell survival. Especially deleterious type of DNA damage are DNA double-strand breaks (DSB), which can lead to genomic instability and malignant transformation if not repaired correctly. The central player in DSB detection and repair is the ATM kinase which orchestrates the action of several downstream factors. Recent studies have suggested that long non-coding RNAs (lncRNAs) are involved in DDR. Here, we aimed to identify lncRNAs induced upon DNA damage in an ATM-dependent manner. DNA damage was induced by ionizing radiation (IR) in immortalized lymphoblastoid cell lines derived from 4 patients with ataxia-telangiectasia (AT) and 4 healthy donors. RNA-seq revealed 10 lncRNAs significantly induced 1 h after IR in healthy donors, whereas none in AT patients. 149 lncRNAs were induced 8 h after IR in the control group, while only three in AT patients. Among IR-induced mRNAs, we found several genes with well-known functions in DDR. Gene Set Enrichment Analysis and Gene Ontology revealed delayed induction of key DDR pathways in AT patients compared to controls. The induction and dynamics of selected 9 lncRNAs were confirmed by RT-qPCR. Moreover, using a specific ATM inhibitor we proved that the induction of those lncRNAs is dependent on ATM. Some of the detected lncRNA genes are localized next to protein-coding genes involved in DDR. We observed that induction of lncRNAs after IR preceded changes in expression of adjacent genes. This indicates that IR-induced lncRNAs may regulate the transcription of nearby genes. Subcellular fractionation into chromatin, nuclear, and cytoplasmic fractions revealed that the majority of studied lncRNAs are localized in chromatin. In summary, our study revealed several lncRNAs induced by IR in an ATM-dependent manner. Their genomic co-localization and co-expression with genes involved in DDR suggest that those lncRNAs may be important players in cellular response to DNA damage.

Changes in sub-cellular localisation of trophoblast and inner cell mass specific transcription factors during bovine preimplantation development.

BACKGROUND: Preimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. To gain an insight into these processes we have studied protein and mRNA distribution during the crucial stages of bovine development. Protein distribution of lineage specific markers OCT4, NANOG, CDX2 were analysed in 5-cell, 8-16 cell, morula and blastocyst stage embryos. ICM/TE mRNA levels were compared in hatched blastocysts and included: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, CDX2, KRT-18 and GATA6. RESULTS: At the mRNA level the observed distribution patterns agree with the mouse model. CDX2 and OCT4 proteins were first detected in 5-cell stage embryos. NANOG appeared at the morula stage and was located in the cytoplasm forming characteristic rings around the nuclei. Changes in sub-cellular localisation of OCT4, NANOG and CDX2 were noted from the 8-16 cell onwards. CDX2 initially co-localised with OCT4, but at the blastocyst stage a clear lineage segregation could be observed. Interestingly, we have observed in a small proportion of embryos (2%) that CDX2 immunolabelling overlapped with mitotic chromosomes. CONCLUSIONS: Cell fate specification in cattle become evident earlier than presently anticipated - around the time of bovine embryonic genome activation. There is an intriguing possibility that for proper lineage determination certain transcription factors (such as CDX2) may need to occupy specific regions of chromatin prior to its activation in the interphase nucleus. Our observation suggests a possible role of CDX2 in the process of epigenetic regulation of embryonic cell fate.

The Influence of FAM13A and PPAR-γ2 Gene Polymorphisms on the Metabolic State of Postmenopausal Women.

Recently, we have observed two significant pandemics caused by communicable (COVID-19) and non-communicable factors (obesity). Obesity is related to a specific genetic background and characterized by immunogenetic features, such as low-grade systemic inflammation. The specific genetic variants include the presence of polymorphism of the Peroxisome Proliferator-Activated Receptors gene (PPAR-γ2; Pro12Ala, rs1801282, and C1431T, rs3856806 polymorphisms), ß-adrenergic receptor gene (3ß-AR; Trp64Arg, rs4994), and Family With Sequence Similarity 13 Member A gene (FAM13A; rs1903003, rs7671167, rs2869967). This study aimed to analyze the genetic background, body fat distribution, and hypertension risk in obese metabolically healthy postmenopausal women (n = 229, including 105 lean and 124 obese subjects). Each patient underwent anthropometric and genetic evaluations. The study has shown that the highest value of BMI was associated with visceral fat distribution. The analysis of particular genotypes has revealed no differences between lean and obese women except for FAM13A rs1903003 (CC), which was more prevalent in lean patients. The co-existence of the PPAR-γ2 C1431C variant with other FAM13A gene polymorphisms [rs1903003(TT) or rs7671167(TT), or rs2869967(CC)] was related to higher BMI values and visceral fat distribution (WHR > 0.85). The co-association of FAM13A rs1903003 (CC) and 3ß-AR Trp64Arg was associated with higher values of systolic (SBP) and diastolic blood pressure (DBP). We conclude that the co-existence of FAM13A variants with C1413C polymorphism of the PPAR-γ2 gene is responsible for body fat amount and distribution.

Cardiac Post-Chest Radiotherapy Complications in a 50-Year-Old Patient with Hodgkin Lymphoma.

Lymphomas are a group of malignant tumors that originate in the lymphatic system. It is the most common type of blood cancer. It affects the lymph nodes, spleen, bone marrow, blood, and other organs. They can be aggressive or chronic. Hodgkin lymphoma survival rate is 2 in 100,000 people. Young adults aged 20-30 and people over 50 are most often affected. The prognosis of Hodgkin's lymphoma is good, with a survival rate of up to 80 percent. Nevertheless, in 20-30 percent of patients who initially respond to treatment, the disease has a tendency to progress. The positive effect of radiotherapy (RT) on patients' survival rates has been proven in many randomized clinical trials. Although the dose of chest RT has significantly reduced over the years, we still struggle with the long-term complications of post-RT repercussions, mainly because there is no established safe dose of RT affecting the heart. Other complications include earlier onset of coronary artery disease, early and late onset of pericarditis, valve degeneration (predominantly of the left heart), calcification of the aorta and its branches, heart failure, and arrhythmias. One patient can manifest each of the abovementioned complications, as in the present case. That is why choosing the right treatment strategy is crucial.

Hybrids of manganese oxide and lipid liquid crystalline nanoparticles (LLCNPs@MnO) as potential magnetic resonance imaging (MRI) contrast agents.

Due to the health risks associated with the use of Gd-chelates and the promising effects of using nanoparticles as T1 contrast agents (CAs) for MRI, Mn-based nanoparticles are considered a highly competitive alternative. The use of hybrid constructs with paramagnetic functionality of Mn-based nanoparticles is an effective approach, in particular, the use of biocompatible lipid liquid crystalline nanoparticles (LLCNPs) as a carrier of MnO nanoparticles. LLCNPs possess a unique internal structure ensuring a payload of different polarity MnO nanoparticles. In view of MRI application, the surface properties including the polarity of MnO are crucial factors determining their relaxation rate and thus the MRI efficiency. Two novel hybrid constructs consisting of LLCNPs loaded with hydrophobic MnO-oleate and hydrophilic MnO-DMSA NPs were prepared. These nanosystems were studied in terms of their physico-chemical properties, positive T1 contrast enhancement properties (in vitro and in vivo) and biological safety. LLCNPs@MnO-oleate and LLCNPs@MnO-DMSA hybrids exhibited a heterogeneous phase composition, however with differences in the inner periodic arrangement and structural parameters, as well as in the preferable localization of MnO NPs within the LLCNPs. Also, these hybrids differed in terms of particle size-related parameters and colloidal stability, which was found to be strongly dependent on the addition of differently functionalized MnO NPs. Embedding both types of MnO NPs into LLCNPs resulted in high relaxivity parameters, in comparison to bare MnO-DMSA NPs and also commercially developed CAs (e.g. Dotarem and Teslascan). Further biosafety studies revealed that cell internalization pathways were dependent on the prepared hybrid type, while viability, effects on the mitochondria membrane potential and cytoskeletal networks were rather related to the susceptibility of the particular cell line. The high relaxation rates achieved with the developed hybrid LLCNPs@MnO enable them to be possibly used as novel and biologically safe MRI T1-enhancing CAs in in vivo imaging.

CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells.

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines-K562, ST486, HepG2, and MCF7-which revealed several essential E-boxes and genes. Among them, we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.

Effect of miR-195 inhibition on human skeletal muscle-derived stem/progenitor cells.

BACKGROUND: The application of a circulating miR-195 inhibitor could be a helping factor in the in vitro model of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs). Previously, microRNA-195 (miR-195) expression has been reported to be a negative factor for myogenesis. AIMS: The study aimed to obtain anti-apoptotic and anti-aging effects in in vitro cultured myoblasts and to improve their ability to form myotubes by suppressing miR-195 expression. METHODS: Human wild-type (WT) SkMDS/PC cells incubated with control (nonspecific) miRNA inhibitor and miR-195-inhibited SkMDS/PCs were studied. Functional assays (myotube formation and cell aging), antioxidant, and myogenic gene expression analyses were performed at two time points, at the seventh and eleventh cell passages. RESULTS: Myotube formation was found to be almost 2-fold higher in the miR-195-inhibited SkMDS/PCs population (P < 0.05) compared to WT cells. miR-195 inhibition did not appear to affect cell aging or rejuvenate human SkMDS/PCs. Antioxidant (SOD3 and FOXO) gene expression was augmented in the miR-195-inhibited SkMDS/PCs population, but no positive effect on the remaining antioxidant genes (SOD1, SOD2, and catalase) was observed. A significant increase in MyoD gene expression with a concomitant decrease in MyoG (P < 0.05) was further documented in miR-195- -inhibited SkMDS/PCs compared to WT cells (the eleventh cell passage). CONCLUSIONS: The performed studies may lead to the preconditioning of myogenic stem cells to extend their potential for pro-regenerative activity. The miR-195 inhibitor may serve as a conditioning factor augmenting selective antioxidant gene expression and proliferative potential of SkMDS/PCs, but it does not have an impact on cell aging and/ or apoptosis.

Modeling the human heart ex vivo-current possibilities and strive for future applications.

The high organ specification of the human heart is inversely proportional to its functional recovery after damage. The discovery of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has accelerated research in human heart regeneration and physiology. Nevertheless, due to the immaturity of iPSC-CMs, they are far from being an representative model of the adult heart physiology. Therefore, number of laboratories strive to obtain a heart tissues by engineering methods by structuring iPSC-CMs into complex and advanced platforms. By using the iPSC-CMs and arranging them in 3D cultures it is possible to obtain a human heart muscle with physiological capabilities potentially similar to the adult heart, while remaining in vitro. Here, we attempt to describe existing examples of heart muscle either in vitro or ex vivo models and discuss potential options for the further development of such structures. This will be a crucial step for ultimate derivation of complete heart tissue-mimicking organs and their future use in drug development, therapeutic approaches testing, pre-clinical studies, and clinical applications. This review particularly aims to compile available models of advanced human heart tissue for scientists considering which model would best fit their research needs.

Optimization of human myoblasts culture under different media conditions for application in the in vitro studies.

Human primary in vitro cell cultures are among the most challenging procedures in cellular biology laboratory practice. Myoblasts-progenitor of skeletal muscle origin represent a promising therapeutic cell source since the procedure of their isolation is not technically demanding, and the in vitro culture is relatively straightforward. Myoblasts could be considered as the candidates for clinical applications due to their regenerative potential, and as the carriers of therapeutic proteins introduced through genetic modifications. The main goal of this prospective study was to evaluate different myoblasts isolation strategies based on the pre-plating technique and cells density characteristics. Moreover, testing of different myoblast media formulations-both commercially available and in-house made was performed. Our goal was to establish the in vitro protocol of myoblasts culture allowing for preservation of the proliferative potential and desired phenotype. Our results revealed that in culture of myoblasts of human muscle origin, the pre-plate technique and cell density differences did not correlate with changes in the proliferative potential, however it was observed that low density cells maintained expression of the CD56 marker up to the higher passages. Assessment of different types of culture media confirmed the best performance for DMEM based media without Chicken Embryo Extract (CEE) addition. Cells cultured in DMEM+FBS medium revealed high expression of CD56 and CD90 antigens, absence of the hematopoietic markers and presented stable proliferation profile. This finding is in line with guidelines of regulatory agencies recommending removal of the xeno-derived reagents from the manufacturing process of Advanced Therapy Medicinal Products (ATMP). In this study, human myoblasts culture was optimized in vitro under different media conditions. The next approach in assessment of myoblasts propagation for potential clinical applications will be testing of the clinical grade human platelet lysate (hPL) instead of the FBS.

Whole-genome sequencing identifies new candidate genes for nonobstructive azoospermia.

BACKGROUND: Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established. OBJECTIVE: To identify low-frequency NOA-associated single nucleotide variants (SNVs) using whole-genome sequencing (WGS). MATERIALS AND METHODS: Men with various types of NOA (n = 39), including samples that had been previously tested with whole-exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed. RESULTS: Using WGS, we revealed potential NOA-associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis. DISCUSSION AND CONCLUSION: In this study, we identified novel potential candidate NOA-associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA-associated gene findings.