Biology subjects

Borowiak, M.

Publications and source records attributed to Borowiak, M..

9 recordsLinked to original sources

bioRxiv2025

KuPID: Kmer-based Upstream Preprocessing of Long Reads forIsoform Discovery

Eukaryotic genes can encode multiple protein isoforms based on alternative splicing of their transcribed regions. Most modern novel isoform discovery methods function by identifying and assembling exon splice junctions from an RNAseq sample. However, splice junctions can only be accurately annotated with time-intensive dynamic programming alignment. This manuscript introduces KuPID, a method for preprocessing long RNAseq reads with the goal of better identifying novel isoform transcripts. KuPID utilizes kmer sketching as a pre-filter to quickly pseudo-align reads to known reference isoforms. Full alignment need only then be applied to reads that are most relevant to isoform discovery. Not only does KuPID speed up the discovery pipeline, it also increases downstream accuracy by filtering out extraneous reads. KuPID preprocessing simultaneously increases the f1 accuracy of isoform discovery pipelines by up to 16.7 points while decreasing the runtime by a factor of 2-3x. An optional mode permits a KuPID sample to be paired with both isoform discovery and transcript quantification. Code availability: https://github.com/mboro2000/KuPID.git

bioinformatics↗

bioRxiv2025

ER stress and cell fate: FKBP2 regulates proinsulin folding and α- vs. β-cell differentiation via NFAT and HDAC9

The continuous demand for insulin production places a burden on the endoplasmic reticulum (ER) of pancreatic {beta} cells, making them highly susceptible to protein misfolding and ER stress. While general ER chaperones are known to be essential for {beta} cell homeostasis, the specific mechanisms by which individual chaperones facilitate proinsulin folding and influence cell fate remain unclear. This study identifies FK506-binding protein 2 (FKBP2), an ER-localized cis-trans prolyl isomerase, as a critical regulator of human {beta} cell differentiation and proinsulin processing. Using loss- and gain-of-function FKBP2 models in human pluripotent stem cells (hPSCs), we demonstrate that its deficiency impairs insulin processing, leading to abnormal insulin granule morphology and reduced insulin secretion. Unexpectedly, our findings reveal a crucial and unreported role for FKBP2 in cell fate determination. Single-cell RNA sequencing reveals that FKBP2 loss disrupts the proper endocrine lineage allocation, causing a shift towards the cell formation at the expense of {beta} cells. In KO endocrine cells, we observed sustained ER stress and elevated intracellular calcium levels, along with activation of the NFAT2-HDAC9 axis. Pharmacological inhibition of HDAC class IIa activity partially rescued {beta} cell differentiation, supporting a causal role for this pathway. Collectively, our results provide new mechanistic insights into how ER chaperones can control pancreatic development and contribute to the pathogenesis of diabetes.

cell biology↗

bioRxiv2023

iPSC-based modeling of helicase deficiency reveals impaired cell proliferation and increased apoptosis after NK cell lineage commitment

While monogenic variants in CDC45-MCM-GINS (CMG) replisome proteins cause human natural killer cell deficiencies (NKD), family members with the same inherited variants often have variable clinical and cellular phenotypes. We investigated two siblings with inherited compound heterozygous GINS4 variants but variable disease expressivity. Cell cycle impairment and increased apoptosis were detected following NK cell lineage commitment but not in pluripotent cells. While this effect was detected in both siblings, the efficiency of NK cell differentiation was variable and correlated with differential clinical severity of NKD. Further investigation of allelic expression of inherited GINS4 variants demonstrated expected biallelic expression of GINS4 in pluripotent cells and progenitors. However, allelic bias in lineage-committed NK cells led to over- or under-representation of more damaging GINS4 heterozygous variants associated with differential cellular and clinical severity. This study identifies allelic bias that causes phenotypic variation of monogenic diseases and defines mechanisms underlying immunodeficiency.

cell biology↗

bioRxiv2022

FK506-binding protein 2 participates in proinsulin folding

Apart from chaperoning, disulphide bond formation and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94, and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high molecular weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic {beta}-cells increased apoptosis without detectable upregulation of ER-stress response genes. Interestingly, FKBP2 mRNA was overexpressed in {beta}-cells from pancreatic islets of T2D patients. Based on molecular modelling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B chain (P28) is the substrate of FKBP2s isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

molecular biology↗

bioRxiv2022

Next Generation Opto-Jasplakinolides Enable Local Remodeling of Actin Networks

The natural product jasplakinolide is a widely used tool compound to stabilize F-actin and influence actin dynamics. We have previously introduced photoswitchable jasplakinolides (optojasps) that are activated with violet light and deactivated with blue light. Based on insights from cryo-electron microscopy and structure-activity relationship (SAR) studies, we now developed a new generation of functionally superior optojasps that are better suited for biological investigations. These compounds are procured through chemical total synthesis and feature rationally designed red-shifted azobenzene photoswitches. Our new optojasps can be activated with longer wavelengths in the visible range (e.g. 440-477 nm) and rapidly return to their inactive state through thermal relaxation. This has enabled the reversible control of F-actin dynamics, as shown through live-cell imaging and cell migration, as well as cell proliferation assays. Brief sub-cellular activation with blue-green light resulted in highly localized F-actin clusters that gradually dissolved in the dark. Our light-responsive tools can be useful in diverse fields to study actin dynamics with outstanding spatiotemporal precision. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/480923v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1883e3corg.highwire.dtl.DTLVardef@1ae0c4borg.highwire.dtl.DTLVardef@1a92f8forg.highwire.dtl.DTLVardef@718d87_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

SMER28 attenuates PI3K/mTOR signaling by direct inhibition of PI3K p110 delta

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and consequently growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction of phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3K{delta} and to a lesser extent p110{gamma}. The biological relevance of our observations was underscored by interference of SMER28 with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor typrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited.

cell biology↗

bioRxiv2020

German-wide interlaboratory study compares consistency, accuracy and reproducibility of whole-genome short read sequencing

We compared the consistency, accuracy and reproducibility of next-generation short read sequencing between ten laboratories involved in food safety (research institutes, state laboratories, universities and companies) from Germany and Austria. Participants were asked to sequence six DNA samples of three bacterial species (Campylobacter jejuni, Listeria monocytogenes and Salmonella enterica) in duplicate, according to their routine in-house sequencing protocol. Four different types of Illumina sequencing platforms (MiSeq, NextSeq, iSeq, NovaSeq) and one Ion Torrent sequencing instrument (S5) were involved in the study. Sequence quality parameters were determined for all data sets and centrally compared between laboratories. SNP / and cgMLST calling were performed to assess the reproducibility of sequence data collected for individual samples. Overall, we found Illumina short read data to be more accurate and consistent than Ion Torrent sequence data, with little variation between the different Illumina instruments. Two laboratories with Illumina instruments submitted sequence data with lower quality, probably due to the use of a library preparation kit, which shows difficulty in sequencing low GC genome regions. Differences in data quality were more evident after assembling short reads into genome assemblies, with Ion Torrent assemblies featuring a great number of allele differences to Illumina assemblies. Clonality of samples was confirmed through SNP calling, which proved to be a more suitable method for an integrated data analysis of Illumina and Ion Torrent data sets, than cgMLST calling.

genomics↗

bioRxiv2019

NFIA regulates granule recruitment and exocytosis in the adult pancreas

After food ingestion, pancreatic cells secrete zymogen and hormone-containing granules to precisely control digestion and blood glucose levels. Identifying regulators of this process is paramount to combatting multiple pancreatic diseases. Here we show that pancreatic deletion of the transcription factor nuclear factor IA (NFIA) leads to hyperglycemia, hypoinsulinemia, and hypolipidemia. Surprisingly, insulin and digestive enzymes are produced in the absence of NFIA, however, they are not secreted properly and instead accumulate inside pancreatic cells. In NFIA-deficient mice we saw a reduction of insulin granules in the ready releasable pool and the first-phase insulin response was impaired. We found that NFIA binds to and activates Rab39b, a Rab GTPase critical for exocytosis. Re-expression of Rab39b in NFIA knockout islets restored glucose-stimulated insulin secretion. In sum, the NFIA-Rab39b axis regulates pancreatic physiology through granule recruitment and docking, linking NFIA to a new process with potential effects in diabetes, pancreatitis, and lipid disorders.

cell biology↗

bioRxiv2019

Human NK cell deficiency as a result of biallelic mutations in MCM10

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here we report a novel cause of NKD resulting from compound heterozygous mutations in MCM10 that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. By modeling MCM10 deficiency in human NK cell lines and primary NK cell precursors, we demonstrate that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function.

immunology↗