Biology subjects

Kozlowski, P.

Publications and source records attributed to Kozlowski, P..

8 recordsLinked to original sources

bioRxiv2025

Modeling Atrial Fibrillation through Intermittent Tachypacing-Induced Remodeling in hiPSC-Derived Atrial Cardiomyocytes and Atrial Fibroblast

BackgroundHuman in vitro models for atrial fibrillation (AF) are limited. Human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) provide a valuable tool to study AF pathophysiology by facilitating in vitro modeling. ObjectivesTo investigate the effects of an intermittent tachypacing protocol (ITPP) in matured hiPSC-aCMs co-cultured with human atrial cardiac fibroblast (haCF) to mimic AF-associated electroanatomical phenotypes. Methods and ResultshiPSC-aCMs were cultured alone or co-cultured with haCFs at 90/10 and 70/30 ratios. ITPP was applied through field stimulation, and optical mapping assessed action potentials (APs) and calcium transients (CaTs). Immunostaining was performed to quantify pro-fibrotic biomarkers (Collagen III and TGF{beta}1). ITPP led to increased spontaneous AP frequency ({Delta}=+31{+/-}7%, P<0.0001) and reduced AP duration at 80% repolarization (APD80%; {Delta}=-15{+/-}4%, P=0.001). Additionally, the upstroke slope ({Delta}=-41{+/-}11%, P=0.001) and amplitude (dF/F0; {Delta}=-51{+/-}13%, P<0.001) of intracellular CaT were significantly reduced. Co-culture at the 70/30 hiPSC-aCM/haCF ratio, showed a >100-fold increase in Collagen III expression (P<0.0001), diminished excitability ({Delta}Hz=-61{+/-}6%, P<0.0001), prolonged {Delta}APD80% ({Delta}=+130{+/-}10%, P<0.0001), prolonged AP triangulation ({Delta}APDTri=+143{+/-}13%, P<0.0001), reduced upstroke slope ({Delta}=-66{+/-}6%, P<0.0001), conduction block ({Delta}=-52{+/-}18%, P=0.0260), and diminished intracellular calcium handling (upstroke slope {Delta}=-50{+/-}8%, P<0.0001; {Delta}dF/F0=-34{+/-}9%, P=0.0003). Finally, the application of ITPP to the 70/30 co-culture model recapitulated an AF-mediated phenotype ({Delta}Hz=+25{+/-}8%, P=0.02; {Delta}APD80%=-16{+/-}6%, P=0.01) while introducing conduction block ({Delta}CV100/0 vs 70/30= -27{+/-}15%; P=0.0005). ConclusionsCo-cultures of matured hiPSC-aCMs and haCFs exhibited structural and electrophysiological remodeling, including conduction abnormalities, mirroring key AF mechanisms. This model holds potential for patient-specific therapies and drug discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/655123v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1c56760org.highwire.dtl.DTLVardef@105d4a2org.highwire.dtl.DTLVardef@1cb18d4org.highwire.dtl.DTLVardef@cb3353_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Human iPSC-Model for Atrial Fibrillation C_FIG

bioengineering↗

bioRxiv2025

Whole-miRNome sequencing (WMS) - a panel for targeted sequencing of all human miRNA genes

There is a growing interest in the genetic variation of noncoding genomic elements, including miRNAs, and several mutations in miRNA genes implicated in human diseases, including cancer, have already been detected. However, the lack of dedicated analytical tools severely hampers progress in this area. In this study, we developed whole-miRNome sequencing (WMS), which enables targeted sequencing of all human miRNA genes (n[~]2000) and 28 miRNA biogenesis genes. Herein, by sequencing almost 600 samples, including [~]300 tumor/normal pairs of samples from different cancer types, we identified [~]2, 000 mutations, including 1, 435 cancer somatic mutations, with 879 occurring in miRNA genes. These mutations were located in all parts of the genes, including seed or cleavage sites essential for the functioning of miRNA genes. The high reliability of the mutations was confirmed through various approaches, including different sequencing methods. The analysis identified several miRNA genes with functional enrichment of cancer mutations, including MIR3928, specifically mutated in basal cell carcinoma (BCC), indicating its potential role in this cancer. WMS also allowed the identification of multiple copy number alterations, hotspots of which often encompassed miRNA genes. WMS provides highly effective, low-cost sequencing of all miRNA genes in different types of samples, including highly degraded FFPE samples.

genomics↗

bioRxiv2023

SARS-CoV-2 and its ORF3a, E and M viroporins activate inflammasome in human macrophages and induce of IL-1α in pulmonary epithelial and endothelial cells

Inflammasome assembly is a potent mechanism responsible for the host protection against pathogens, including viruses. When compromised, it can allow viral replication, while when disrupted, it can perpetuate pathological responses by IL-1 signaling and pyroptotic cell death. SARS-CoV-2 infection was shown to activate inflammasome in the lungs of COVID-19 patients, however, potential mechanisms responsible for this response are not fully elucidated. In this study, we investigated the effects of ORF3a, E and M SARS-CoV-2 viroporins in the inflammasome activation in major populations of alveolar sentinel cells: macrophages, epithelial and endothelial cells. We demonstrated that each viroporin is capable of activation of the inflammasome in macrophages to trigger cell death and IL-1 release from epithelial and endothelial cells. Small molecule NLRP3 inflammasome inhibitors reduced IL-1 release but weakly affected the pyroptosis. Importantly, we discovered that while SARS-CoV-2 could not infect the pulmonary microvascular endothelial cells it induced IL-1 and IL-33 release. Together, these findings highlight the essential role of macrophages as the major inflammasome-activating cell population in the lungs and point to endothelial cell expressed IL-1 as a potential novel component driving the pulmonary immunothromobosis in COVID-19.

immunology↗

bioRxiv2021

Profile of basal cell carcinoma mutations and copy number alterations - focus on gene-associated noncoding variants

Basal cell carcinoma (BCC) of the skin is the most common cancer in humans, characterized by the highest mutation rate among cancers, and is mostly driven by mutations in genes involved in the hedgehog pathway. To date, almost all BCC genetic studies have focused exclusively on protein-coding sequences; therefore, the impact of noncoding variants on the BCC genome is unrecognized. In this study, with the use of whole-exome sequencing of 27 tumor/normal pairs of BCC samples, we performed an analysis of somatic mutations in both protein-coding sequences and gene-associated noncoding regions, including 5UTRs, 3UTRs, and exon-adjacent intron sequences. Separately, in each region, we performed hotspot identification, mutation enrichment analysis, and cancer driver identification with OncodriveFML. Additionally, we performed a whole-genome copy number alteration analysis with GISTIC2. Of the >80,000 identified mutations, [~]50% were localized in noncoding regions. The results of the analysis generally corroborated the previous findings regarding genes mutated in coding sequences, including PTCH1, TP53, and MYCN, but more importantly showed that mutations were also clustered in specific noncoding regions, including hotspots. Some of the genes specifically mutated in noncoding regions were identified as highly potent cancer drivers, of which BAD had a mutation hotspot in the 3UTR, DHODH had a mutation hotspot in the Kozak sequence in the 5UTR, and CHCHD2 frequently showed mutations in the 5UTR. All of these genes are functionally implicated in cancer-related processes (e.g., apoptosis, mitochondrial metabolism, and de novo pyrimidine synthesis) or the pathogenesis of UV radiation-induced cancers. We also found that the identified BAD and CHCHD2 mutations frequently occur in melanoma but not in other cancers via The Cancer Genome Atlas analysis. Finally, we identified frequent deletion of chr9q, encompassing PTCH1, and unreported frequent copy number gain of chr9p, encompassing the genes encoding the immune checkpoint ligands PD-L1 and PD-L2. In conclusion, this study is the first systematic analysis of coding and noncoding mutations in BCC and provides a strong basis for further analyses of the variants in BCC and cancer in general. Author summaryThe study is the first systematic analysis of both coding and noncoding mutations in basal cell carcinoma (BCC), the most common and the most highly mutated human cancer. Noncoding mutations accounted for [~]50% ([~]40K mutations) of all mutations detected by the standard WES approach. Among the genes frequently mutated in noncoding regions are: BAD with a hotspot in the 3UTR, DHODH with a hotspot in the Kozak sequence, and CHCHD2 with mutations in 5UTR, all genes functionally related to cell metabolism and apoptosis. Analysis of copy number alterations showed frequent chr9q deletion, encompassing PTCH1 (the key BCC tumor suppressor), and frequent copy number gain of chr9p, encompassing the genes of the immune checkpoint proteins PD-L1 and PD-L2.

cancer biology↗

bioRxiv2020

A pan-cancer atlas of somatic mutations in miRNA biogenesis genes

It is a well-known and intensively studied phenomenon that the levels of many miRNAs are differentiated in cancer. miRNA biogenesis and functional expression are complex processes orchestrated by many proteins cumulatively called miRNA biogenesis proteins. To characterize cancer somatic mutations in the miRNA biogenesis genes and investigate their potential impact on the levels of miRNAs, we analyzed whole-exome sequencing datasets of over 10,000 cancer/normal sample pairs deposited within the TCGA repository. We identified and characterized over 3,600 somatic mutations in 29 miRNA biogenesis genes and showed that some of the genes are overmutated in specific cancers and/or have recurrent hotspot mutations (e.g., SMAD4 in PAAD, COAD, and READ; DICER1 in UCEC; PRKRA in OV; and LIN28B in SKCM). We identified a list of miRNAs whose level is affected by particular types of mutations in either SMAD4, SMAD2, or DICER1 and showed that hotspot mutations in the RNase domains in DICER1 not only decrease the level of 5p-miRNAs but also increase the level of 3p-miRNAs, including many well-known cancer-related miRNAs. We also showed an association of the mutations with patient survival. Eventually, we created an atlas/compendium of miRNA biogenesis alterations providing a useful resource for different aspects of biomedical research.

genomics↗

bioRxiv2020

Pan-Cancer analysis of somatic mutations in miRNA genes

miRNAs are considered important players in oncogenesis, serving either as oncomiRs or suppressormiRs. Although the accumulation of somatic alterations is an intrinsic aspect of cancer development and many important cancer-driving mutations have been identified in protein-coding genes, the area of functional somatic mutations in miRNA genes is heavily understudied. Here, based on analysis of the whole-exome sequencing of over 10,000 cancer/normal sample pairs deposited within the TCGA repository, we identified and characterized over 10,000 somatic mutations in miRNA genes and showed that some of the genes are overmutated in Pan-Cancer and/or specific cancers. Nonrandom occurrence of the identified mutations was confirmed by a strong association of overmutated miRNA genes with KEGG pathways, most of which were related to specific cancer types or cancer-related processes. Additionally, we showed that mutations in some of the overmutated genes correlate with miRNA expression, cancer staging, and patient survival. Our results may also be the first step (form the basis and provide the resources) in the development of computational and/or statistical approaches/tools dedicated to the identification of cancer-driver miRNA genes.

genomics↗

bioRxiv2019

Somatic mutations in miRNA genes in lung cancer - potential functional consequences of non-coding sequence variants

A growing body of evidence indicates that miRNAs may either drive or suppress oncogenesis. However, little is known about somatic mutations in miRNA genes. To determine the frequency and potential consequences of miRNA gene mutations, we analyzed whole exome sequencing datasets of [~]500 lung adenocarcinoma (LUAD) and [~]500 lung squamous cell carcinoma (LUSC) samples generated in the TCGA. Altogether, we identified >1000 mutations affecting [~]500 different miRNA genes and showed that half of all cancers had at least one such mutation. Mutations occurred in most crucial parts of miRNA precursors, including mature miRNA and seed sequences. We showed that seed mutations strongly affected miRNA:target interactions, drastically changing the pool of predicted targets. Mutations may also affect miRNA biogenesis by changing the structure of miRNA precursors, DROSHA and DICER cleavage sites, and regulatory sequence/structure motifs. We identified 10 significantly overmutated hotspot miRNA genes, including the miR-379 gene in LUAD enriched in mutations in the mature miRNA and regulatory sequences. The occurrence of mutations in the hotspot miRNA genes was also shown experimentally. We present a comprehensive analysis of somatic mutations in miRNA genes and show that some of these genes are mutational hotspots, suggesting their potential role in cancer.

genomics↗

bioRxiv2018

Global increase in circRNA levels in myotonic dystrophy

Splicing aberrations induced as a consequence of the sequestration of MBNL splicing factors on the DMPK transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As MBNLs may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected twenty well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1 and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity. Author SummaryRecently, a great deal of interest has been focused on a new class of RNA molecules called circular RNAs (circRNAs). To date, thousands of circRNAs have been found in different human cells/tissues. Although the function of circRNAs remains mostly unknown, circRNAs have emerged as an important component of the RNA-RNA and RNA-protein interactome. Thus, intensive efforts are being made to fully understand the biology and function of circRNAs, especially their role in human diseases. As an important role in the biogenesis of circRNA may be played by MBNL splicing factors, in this study we used DM1 (to a lesser extent, DM2) as a natural model in which the level of MBNLs is decreased. In contrast to the expected effect, our results consistently showed a global increase in circRNA levels in DM1. As a consequence, whole genome transcriptome analysis revealed dozens of circRNAs with significantly altered (mostly increased) levels in DM1. Furthermore, we observed that the circRNA levels were in many cases strongly associated with DM1 severity.

molecular biology↗