Biology subjects

Karakulah, G.

Publications and source records attributed to Karakulah, G..

11 recordsLinked to original sources

bioRxiv2025

BrAIn: A comprehensive artificial intelligence-based morphology analysis system for brain organoids and neuroscience

Human-induced pluripotent stem cells (iPSCs) offer transformative potential for biomedical research, with iPSC-derived organoids providing more physiologically relevant models than traditional 2D cell cultures. Among these, brain organoids are particularly valuable for drug screening, disease modeling, and investigations into molecular pathways. Accurate representation of brain morphology is critical, as more complex organoid structures better mimic the human brain. Deep learning (DL) and machine learning (ML) approaches have become integral to analyzing organoid morphology, yet tools for comprehensive, time-resolved assessments are scarce. Here, we introduce BrAIn, a DL-based application for analyzing the developmental progression of brain organoids. BrAIn tracks their evolution from embryoid bodies and quantifies parameters including area, Feret diameter, perimeter, roundness, and circularity. It also classifies budding and abnormal morphologies of 3D organoids and detects monolayer neural rosette structures, key features of neuronal differentiation. Designed with accessibility in mind, BrAIn provides a no-code interface, enabling researchers of all technical backgrounds to conduct advanced morphological analyses with ease. Our study demonstrates the application of BrAIn to evaluate the effects of different growth conditions - static, orbital shaker, and microfluidic chip-based - on brain organoid development. Orbital shaker cultures resulted in the largest organoids, while chip-based systems achieved more homogeneous growth. Both conditions produced organoids with greater morphological complexity compared to static culture. BrAIn emerges as a robust, user-friendly tool to quantify brain organoid development and explore how versatile growth conditions influence their morphology and maturation.

bioengineering↗

bioRxiv2024

Characterization of a novel neurodevelopmental rare disease caused by a mutation within the autophagy gene ATG9B

Autophagy is a highly conserved eukaryotic cellular process whose dysfunction results in human pathologies including cancer and neurodegenerative disease. First identified in yeast, ATG genes are central players in autophagy. Although their roles in cancer and neurodegenerative disease are well known, Mendelian diseases associated with ATG genes are rare. Mutations in core autophagy genes ATG5 and ATG7 have been previously reported to cause rare genetic disorders with autosomal recessive inheritance pattern. Here we report, for the first time, a rare genetic disorder that results from a deletion/frameshift mutation in human ATG9B, the placenta specific homologue of yeast ATG9 in humans. The 11-nucleotide deletion causes a frameshift and addition of a premature stop codon, truncating the C-terminal cytosolic domain of the ATG9B protein. The pediatric patients carrying the mutant allele homozygous were children of a consanguineous marriage and displayed neurodevelopmental anomalies including mental retardation. We hypothesized that this phenotype originates during placental development. To characterize the effects of the mutation and gain insight on the specific functions of ATG9B in a physiological setting, we used mammalian cells and generated a knock-in mouse model. Truncated ATG9B was not stable when expressed in cells. It was localized to perinuclear vesicles like the WT protein, but not to peripheral vesicles. Homozygous knock-in mice were viable, fertile and displayed no gross phenotypical abnormalities. Histomorphometry analysis of the placenta layers did not reveal a significant difference between mutant and control embryos. The assessments of neurobehavioral tests were similar in wild-type and homozygous knock-in mice. However, knock-in mice had a reduced fear memory trend, which is an amygdala-involved response.

genetics↗

bioRxiv2023

TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype and is associated with increased survival in cancer patients with high tumor macrophage content

BackgroundTNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an inbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known. MethodsPrimary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients. ResultsTRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content. ConclusionsTRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.

immunology↗

bioRxiv2023

Retinoids and EZH2 inhibitors cooperate to orchestrate cytotoxic effects on bladder cancer cells

Emerging evidence has highlighted the importance of targeting EZH2 in bladder cancer owing to the highly mutated nature of bladder cancers harboring mutations in chromatin regulatory genes opposing Polycomb-mediated repression. Besides, enhanced expression of EZH2 contributes to pathogenesis. Furthermore, the critical role of the retinoic acid signaling pathway in the development and homeostasis of the urothelium is well established. Here we report that coordinated targeting of EZH2 and the retinoic acid signaling pathway caused cytotoxic effects on bladder cancer cells by inducing a synergistic reduction in proliferative potential that was associated with increased apoptosis and cell cycle arrest in a cooperative and orchestrated manner. Moreover, combined treatment caused the modulation of the expression of genes associated with an anti-oncogenic profile, as reflected by the stimulation of marker genes associated with apoptosis and differentiation. We further portrayed a molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of certain genes associated with unfolded protein response and some metabolic processes. This work also characterized an apoptotic program centered on the master transcriptional regulators C/EBP{beta} and CHOP. These findings highlight the importance of co-targeting the EZH2/retinoic acid pathway in bladder cancers and encourage the design of novel treatments employing retinoids coupled with EZH2 inhibitors in bladder carcinoma.

cancer biology↗

bioRxiv2022

Truncated KDM6A exhibits differential chromatin occupancy and regulatory interactions

Epigenetic deregulation is a critical theme which needs further investigation for bladder cancer research. One of the highly mutated genes in bladder cancer is KDM6A, functioning as a H3K27 demethylase and is part of the MLL3/4 complexes. To decipher the role of KDM6A in normal versus tumor setting, we identified the genomic landscape of KDM6A in normal, immortalized and cancer bladder cells. Our results showed differential KDM6A occupancy at the genes involved in cell differentiation, chromatin organization and Notch signaling depending on the cell type and the mutation status of KDM6A. Transcription factor motif analysis revealed HES1 to be enriched at KDM6A peaks identified for T24 bladder cancer cell line, which has a truncating mutation in KDM6A, lacking demethylase domain. Our co-immunoprecipitation experiments reveal TLE co-repressors and HES1 as potential truncated and wild type KDM6A interactors. With the aid of structural modeling, we explored how the truncated KDM6A could interact with TLE, HES1, as well RUNX, HHEX transcription factors. These structures provide a solid mean to study the functions of KDM6A independent of its demethylase activity. Collectively, our work provides important contributions to the understanding of KDM6A malfunction in bladder cancer.

cancer biology↗

bioRxiv2022

Benchmarking the Widely Used Structure-based Binding Affinity Predictors on the Spike-ACE2 Deep Mutational Interaction Set

Since the start of COVID-19 pandemic, a huge effort has been devoted to understanding the Spike (SARS-CoV-2)-ACE2 recognition mechanism. To this end, two deep mutational scanning studies traced the impact of all possible mutations across Receptor Binding Domain (RBD) of Spike and catalytic domain of human ACE2. By concentrating on the interface mutations of these experimental data, we benchmarked six commonly used structure-based binding affinity predictors (FoldX, EvoEF1, MutaBind2, SSIPe, HADDOCK, and UEP). These predictors were selected based on their user-friendliness, accessibility, and speed. As a result of our benchmarking efforts, we observed that none of the methods could generate a meaningful correlation with the experimental binding data. The best correlation is achieved by FoldX (R = -0.51). Also, when we simplified the prediction problem to a binary classification, i.e., whether a mutation is enriching or depleting the binding, we showed that the highest accuracy is achieved by FoldX with 64% success rate. Surprisingly, on this set, simple energetic scoring functions performed significantly better than the ones using extra evolutionary-based terms, as in Mutabind and SSIPe. Furthermore, we also demonstrated that recent AI approaches, mmCSM-PPI and TopNetTree, yielded comparable performances to the force field-based techniques. These observations suggest plenty of room to improve the binding affinity predictors in guessing the variant-induced binding profile changes of a host-pathogen system, such as Spike-ACE2. To aid such improvements we provide our benchmarking data at https://github.com/CSB-KaracaLab/RBD-ACE2-MutBench with the option to visualize our mutant models at https://rbd-ace2-mutbench.github.io/

bioinformatics↗

bioRxiv2021

FLI1 and FRA1 transcription factors drive the transcriptional regulatory networks characterizing muscle invasive bladder cancer

Bladder cancer is mostly present in the form of urothelium carcinoma, causing over 150,000 deaths each year. Its histopathological classification as muscle invasive (MIBC) and non-muscle invasive (NMIBC) is the most prominent aspect, affecting the prognosis and progression of this disease. In this study, we defined the active regulatory landscape of MIBC and NMIBC cell lines using H3K27ac ChIP-seq and used an integrative approach to combine our findings with existing data. Our analysis revealed FRA1 and FLI1 as two critical transcription factors differentially regulating MIBC regulatory landscape. We show that FRA1 and FLI1 regulate the genes involved in epithelial cell migration and cell junction organization. Knock-down of FRA1 and FLI1 in MIBC revealed the downregulation of several EMT-related genes such as MAP4K4 and FLOT1. Further, ChIP-SICAP performed for FRA1 and FLI1 enabled us to infer chromatin binding partners of these transcription factors and link this information with their target genes. Finally, for the first time we show that knock-down of FRA1 and FLI1 result in significant reduction of invasion capacity of MIBC cells towards muscle microenvironment using IC-CHIP assays. Our results collectively highlight the role of these transcription factors in selection and design of targeted options for treatment of MIBC.

cancer biology↗

bioRxiv2020

Mutation density changes in SARS-CoV-2 are related to the pandemic stage but to a lesser extent in the dominant strain with mutations in spike and RdRp

Since its emergence in Wuhan, China in late 2019, the origin and evolution of SARS-CoV-2 have been among the most debated issues related to COVID-19. Throughout its spread around the world, the viral genome continued acquiring new mutations and some of them became widespread. Among them, 14408 C>T and 23403 A>G mutations in RdRp and S, respectively, became dominant in Europe and the US, which led to debates regarding their effects on the mutability and transmissibility of the virus. In this study, we aimed to investigate possible differences between time-dependent variation of mutation densities (MDe) of viral strains that carry these two mutations and those that do not. Our analyses at the genome and gene level led to two important findings: First, time-dependent changes in the average MDe of circulating SARS-CoV-2 genomes showed different characteristics before and after the beginning of April, when daily new case numbers started levelling off. Second, this pattern was much delayed or even non-existent for the "mutant" (MT) strain that harbored both 14408 C>T and 23403 A>G mutations. Although these differences were not limited to a few hotspots, it is intriguing that the MDe increase is most evident in two critical genes, S and Orf1ab, which are also the genes that harbor the defining mutations of the MT genotype. The nature of these unexpected relationships warrant further research.

genetics↗

bioRxiv2017

Barley Long Non-Coding RNAs and Their Tissue-Specific Co-expression Pattern with Coding-Transcripts

Long non-coding RNAs (lncRNA) with non-protein or small peptide-coding potential transcripts are emerging regulatory molecules. With the advent of next-generation sequencing technologies and novel bioinformatics tools, a tremendous number of lncRNAs has been identified in several plant species. Recent reports demonstrated roles of plant lncRNAs such as development and environmental response. Here, we reported a genome-wide discovery of ~8,000 barley lncRNAs and measured their expression pattern upon excessive boron (B) treatment. According to the tissue-based comparison, leaves have a greater number of B-responsive differentially expressed lncRNAs than the root. Functional annotation of the coding transcripts, which were co-expressed with lncRNAs, revealed that molecular function of the ion transport, establishment of localization, and response to stimulus significantly enriched only in the leaf. On the other hand, 32 barley endogenous target mimics (eTM) as lncRNAs, which potentially decoy the transcriptional suppression activity of 18 miRNAs, were obtained. Presented data including identification, expression measurement, and functional characterization of barley lncRNAs suggest that B-stress response might also be regulated by lncRNA expression via cooperative interaction of miRNA-eTM-coding target transcript modules.

bioinformatics↗

bioRxiv2017

Discovery Of Drought-Responsive Transposable Element-Related Peach miRNAs

MicroRNAs (miRNA) are small non-coding regulatory RNAs that suppress their specific target transcripts either by cleavage or inhibition of translation. Transposable element related miRNAs (TE-miRNA) have been subjected to various studies so far, and some of them were found to be involved in stress response in plants. Here, small RNA (sRNA) sequencing libraries generated from drought stress treated peach tissues were utilized to identify TE-miRNAs. Our computational analysis led to the identification of 63 TE-miRNAs, which were either locating nearby of any TE (TE-related), or overlapping with a TE (TE-derived). Furthermore, 13 out of 63 TE-miRNAs were designated as drought-responsive. Expression pattern of the identified drought-responsive TE-miRNAs are observed as tissue-specific manner, and their specific target transcripts are mostly related to transcription factors and growth-associated genes. Our findings suggest that miRNAs in relation with transposable elements might be key molecular players in the regulation of drought response.

plant biology↗

bioRxiv2016

MicroRNA exocytosis by large dense-core vesicle fusion

Neurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.\n\nOne Sentence SummaryUsing next-generation sequencing (NGS) for microRNA (miRNA) and synthetic neurotransmission, we observed that large dense-core vesicles (LDCVs) contain a variety of miRNA together with classical neurotransmitters, and that miRNA can be released by vesicle fusion mediated by SNARE.

neuroscience↗