Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals ...with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.
Regulatory T cells (Tregs) play crucial role in maintenance of peripheral tolerance. Recent clinical trials confirmed safety and efficacy of Treg treatment of deleterious immune responses. However, ...Tregs lose their characteristic phenotype and suppressive potential during expansion ex vivo. Therefore, multiple research teams have been studding Treg biology in aim to improve their stability in vitro. In the current paper, we demonstrate that mild hypothermia of 33 °C induces robust proliferation of Tregs, preserves expression of FoxP3, CD25 and Helios, and prevents TSDR methylation during culture in vitro. Tregs expanded at 33 °C have stronger immunosuppressive potential and remarkably anti-inflammatory phenotype demonstrated by the whole transcriptome sequencing. These observations shed new light on impact of temperature on regulation of immune response. We show that just a simple change in temperature can preserve Treg stability, function and accelerate their proliferation, responding to unanswered question- how to preserve Treg stability in vitro.
Adipose-derived mesenchymal stromal cells (AD-MSCs) are an essential issue in modern medicine. Extensive preclinical and clinical studies have shown that mesenchymal stromal/stem cells, including ...AD-MSCs, have specific properties (ability to differentiate into other cells, recruitment to the site of injury) of particular importance in the regenerative process. Ongoing research aims to elucidate factors supporting AD-MSC culture and differentiation in vitro. Angiopoietin-like proteins (ANGPTLs), known for their pleiotropic effects in lipid and glucose metabolism, may play a significant role in this context. Regeneration is a complex and dynamic process controlled by many factors. ANGPTL6 (Angiopoietin-related growth factor, AGF), among many activities modulated the biological activity of stem cells. This study examined the influence of synthesized AGF-derived peptides, designated as AGF9 and AGF27, on AD-MSCs. AGF9 and AGF27 enhanced the viability and migration of AD-MSCs and acted as a chemotactic factor for these cells. AGF9 stimulated chondrogenesis and lipid synthesis during AD-MSCs differentiation, influenced AD-MSCs cytokine secretion and modulated transcriptome for such basic cell activities as migration, transport of molecules, and apoptosis. The ability of AGF9 to modulate the biological activity of AD-MSCs warrants the consideration of this peptide a noteworthy therapeutic agent that deserves further investigation for applications in regenerative medicine.
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•Angiopoietin-like growth factor-derived peptides modulate AD-MSCs activities.•AGF9 affects adipogenesis and differentiation into osteocytes (osteogenesis).•AGF9 stimulates the chondrogenesis process.•AD-MSCs transcriptomic response to AGF9 changes some related biological processes.•AGF9 peptide is a potential medicinal product for use in the regeneration process.
Hypoxia-inducible factors (HIF) are heterodimeric transcription factors that allow cells to adapt and survive during hypoxia. Regulation of HIF1A and HIF2A mRNA is well characterized, whereas HIF3A ...mRNA regulation and function are less clear. Using RNA-Seq analysis of primary human umbilical vein endothelial cells, we found two isoforms of HIF3A were expressed, HIF3A2 and HIF3A3. Comparing HIF3A expression profiles to HIF1A mRNA during 48 hours of hypoxia revealed that HIF1A message peaked at 4 hours, whereas HIF3A expression increased while HIF1A was decreasing. Given that HIF1A mRNA is regulated by miR-429, we tested miR-429 effects on both HIF3A isoforms and found that they too were regulated by miR-429. Analysis of a HIF-3 target, DNA-damage-inducible transcript 4, a key survival gene, indicated that DDIT4 mRNA is induced by HIF-3 and negatively regulated by miR-429 through miR-429's actions on HIF3A message. This provides a compelling model for how hypoxia-induced miR-429 regulates the switch between HIF-1 adaptive responses to HIF-3 survival responses by rapidly decreasing HIF1A levels while simultaneously slowing the progression of HIF3A expression until the miR-429 levels drop below normoxic levels. Since HIF-1 drives HIF3A and miR-429 expression, this establishes a regulatory network in which miR-429 plays a pivotal role.
Visium Spatial Gene Expression (ST) is a method combining histological spatial information with transcriptomics profiles directly from tissue sections. The use of spatial information has made it ...possible to discover new modes of gene expression regulations. However, in the ST experiment, the nucleus size of cells may exceed the thickness of a tissue slice. This may, in turn, negatively affect comprehensive capturing the transcriptomics profile in a single slice, especially for tissues having large differences in the size of nuclei.
Here, we defined the effect of Consecutive Slices Data Integration (CSDI) on unveiling accurate spot clustering and deconvolution of spatial transcriptomic spots in human postmortem brains. By considering the histological information as reference, we assessed the improvement of unsupervised clustering and single nuclei RNA-seq and ST data integration before and after CSDI.
Apart from the escalated number of defined clusters representing neuronal layers, the pattern of clusters in consecutive sections was concordant only after CSDI. Besides, the assigned cell labels to spots matches the histological pattern of tissue sections after CSDI.
CSDI can be applied to investigate consecutive sections studied with ST in the human cerebral cortex, avoiding misinterpretation of spot clustering and annotation, increasing accuracy of cell recognition as well as improvement in uncovering the layers of grey matter in the human brain.
Numeric sex chromosome abnormalities are commonly associated with an increased cancer risk. Here, we report a 14-year-old boy with a rare mosaic 45, X/48, XYYY karyotype presenting with subtle ...dysmorphic features and relative height deficiency, requiring growth hormone therapy. As only 12 postnatal cases have been described so far with very limited follow-up data, to assess the proband's long-term prognosis, including cancer risk, we performed high-throughput single-cell RNA sequencing (scRNA-seq) analysis. Although comprehensive cytogenetic analysis showed seemingly near perfect balance between 45, X and 48, XYYY cell populations, scRNA-seq revealed widespread differences in genotype distribution among immune cell fractions, specifically in monocytes, B- and T-cells. These results were confirmed at DNA level by digital-droplet PCR on flow-sorted immune cell types. Furthermore, deregulation of predominantly autosomal genes was observed, including TCL1A overexpression in 45, X B-lymphocytes and other known genes associated with hematological malignancies. Together with the standard hematological results, showing increased fractions of monocytes and CD4+/CD8+T lymphocytes ratio, long-term personalized hemato-oncological surveillance was recommended in the reported patient.
Adipose-derived stem cells (ASCs) have become an important research model in regenerative medicine. However, there are controversies regarding the impact of prolonged cell culture on the ASCs ...phenotype and their differentiation potential. Hence, we studied 10 clinical ASCs replicates from plastic and oncological surgery patients, in six-passage FBS supplemented cultures. We quantified basic mesenchymal cell surface marker transcripts and the encoded proteins after each passage. In parallel, we investigated the differentiation potential of ASCs into chondrocytes, osteocytes and adipocytes. We further determined the effects of FBS supplementation and subsequent deprivation on the whole transcriptome by comprehensive mRNA and miRNA sequencing. Our results show that ASCs maintain differentiation potential and consistent profile of key mesenchymal markers, with apparent expression of distinct isoforms, in long-term cultures. No significant differences were observed between plastic and oncological surgery cohorts. ASCs in FBS supplemented primary cultures are almost committed to mesenchymal lineages as they express key epithelial-mesenchymal transition genes including early mesenchymal markers. Furthermore, combined mRNA/miRNA expression profiling strongly supports a modulatory role for the miR-30 family in the commitment process to mesenchymal lineages. Finally, we propose improvements to existing qPCR based assays that address alternative isoform expression of mesenchymal markers.
Two major types of genetic variation are known: single nucleotide polymorphisms (SNPs), and a more recently discovered structural variation, involving changes in copy number (CNVs) of kilobase- to ...megabase-sized chromosomal segments. It is unknown whether CNVs arise in somatic cells, but it is, however, generally assumed that normal cells are genetically identical. We tested 34 tissue samples from three subjects and, having analyzed for each tissue <=10⁻⁶ of all cells expected in an adult human, we observed at least six CNVs, affecting a single organ or one or more tissues of the same subject. The CNVs ranged from 82 to 176 kb, often encompassing known genes, potentially affecting gene function. Our results indicate that humans are commonly affected by somatic mosaicism for stochastic CNVs, which occur in a substantial fraction of cells. The majority of described CNVs were previously shown to be polymorphic between unrelated subjects, suggesting that some CNVs previously reported as germline might represent somatic events, since in most studies of this kind, only one tissue is typically examined and analysis of parents for the studied subjects is not routinely performed. A considerable number of human phenotypes are a consequence of a somatic process. Thus, our conclusions will be important for the delineation of genetic factors behind these phenotypes. Consequently, biobanks should consider sampling multiple tissues to better address mosaicism in the studies of somatic disorders. Hum Mutat 0,1-7, 2008.
Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural ...cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49–57)-NH2 (R49KKRRQRRR57-amide) and its less basic analogue, PTD4 (Y47ARAAARQARA57-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 μm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49–57)-NH2 adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49–57)-NH2) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide’s ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.
High-throughput technologies generate considerable amount of data which often requires bioinformatic expertise to analyze. Here we present High-Throughput Tabular Data Processor (HTDP), a platform ...independent Java program. HTDP works on any character-delimited column data (e.g. BED, GFF, GTF, PSL, WIG, VCF) from multiple text files and supports merging, filtering and converting of data that is produced in the course of high-throughput experiments. HTDP can also utilize itemized sets of conditions from external files for complex or repetitive filtering/merging tasks. The program is intended to aid global, real-time processing of large data sets using a graphical user interface (GUI). Therefore, no prior expertise in programming, regular expression, or command line usage is required of the user. Additionally, no a priori assumptions are imposed on the internal file composition. We demonstrate the flexibility and potential of HTDP in real-life research tasks including microarray and massively parallel sequencing, i.e. identification of disease predisposing variants in the next generation sequencing data as well as comprehensive concurrent analysis of microarray and sequencing results. We also show the utility of HTDP in technical tasks including data merge, reduction and filtering with external criteria files. HTDP was developed to address functionality that is missing or rudimentary in other GUI software for processing character-delimited column data from high-throughput technologies. Flexibility, in terms of input file handling, provides long term potential functionality in high-throughput analysis pipelines, as the program is not limited by the currently existing applications and data formats. HTDP is available as the Open Source software (https://github.com/pmadanecki/htdp).
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