Biology subjects

Yilmaz, E.

Publications and source records attributed to Yilmaz, E..

15 recordsLinked to original sources

bioRxiv2026

Snurportin-1 maintains muscle niche integrity and myogenic progenitor homeostasis

Loss-of-function variants in SNUPN, encoding the nuclear import factor Snurportin-1 (SPN1) required for spliceosomal small nuclear ribonucleoprotein (snRNP) transport, cause a recently described form of limb-girdle muscular dystrophy (LGMD). However, the role of SPN1 in skeletal muscle homeostasis remains poorly understood, in part due to the lack of a suitable in vivo model. Here, we generated a zebrafish snupn loss-of-function model that recapitulates key features of the skeletal muscle phenotype observed in patients. Mutant larvae developed severe locomotor impairment by 6 days post-fertilization (dpf), accompanied by sarcomeric disorganization and impaired muscle fiber integrity. Transcriptomic profiling at 6 dpf revealed widespread alternative splicing and transcriptional dysregulation, with prominent alterations in extracellular matrix and basement membrane components, together with upregulation of stress- and inflammation-associated genes. Notably, these late-stage abnormalities were preceded by disruption of the muscle progenitor population at 2 dpf, with reduced Pax7 progenitor abundance and myogenic gene expression together with altered muscle differentiation and organization. Together, these findings identify SPN1 as a key regulator of skeletal muscle homeostasis linking RNA processing to extracellular niche integrity and myogenic progenitor maintenance. This zebrafish model provides an in vivo platform for dissecting LGMD-associated disease mechanisms and developing therapeutic strategies aimed at restoring muscle function and regenerative capacity.

genetics↗

bioRxiv2026

A mechanically stable neural probe for percutaneous high-resolution, multichannel recordings in peripheral nerves

The development of neural probes has enabled a deeper understanding and improved treatment for neurological disorders. Microneurography is currently the gold standard for assessing the electrophysiological signature of pain mechanisms in the human peripheral nervous system. However, its clinical utility is limited by the low recording yield and signal-to-noise ratio of single-electrode probes. To overcome these limitations, we developed mechanically robust, multi-electrode probes designed for acute percutaneous insertion and recording in peripheral nerves. The electrical and mechanical stability of these probes was confirmed through repeated insertions in artificial human skin and rat peripheral nerves. In addition, ex vivo and in vivo experiments demonstrated enhanced functional performance, with multi-site recordings enabling the isolation of single-fiber activity. Importantly, our probes can be operated analogously to conventional microneurography needles while substantially increasing the information yield, providing enhanced capabilities for minimally invasive peripheral nerve assessment.

neuroscience↗

bioRxiv2026

Multi-omics characterization of vascular, neurodegenerative, and mixed neuropathology in the aging human brain

Late-life cognitive impairment most commonly occurs in the setting of mixed neurodegenerative and cerebrovascular pathology, yet the molecular programs distinguishing vascular, neurodegenerative, and mixed pathology in the aging human brain remain incompletely defined. We performed neuropathology-stratified proteomic and transcriptomic profiling of postmortem brain tissue from participants in the Religious Orders Study and Rush Memory and Aging Project. Dorsolateral prefrontal cortex proteomics (n = 733) were analyzed alongside bulk RNA sequencing from dorsolateral prefrontal cortex (n = 938), posterior cingulate cortex (n = 569), and anterior caudate (n = 632). Participants were classified into vascular, neurodegenerative, and mixed pathology groups based on comprehensive autopsy assessment. Neurodegenerative and mixed pathology, relative to vascular pathology, showed coordinated upregulation of immune and inflammatory pathways and downregulation of mitochondrial and oxidative phosphorylation programs across molecular layers. Although few individual proteins differed between mixed and neurodegenerative groups, pathway-level analyses identified additional remodeling programs in mixed pathology, including extracellular matrix organization and vesicle-mediated transport. Proteomic co-expression network analysis identified immune-stress modules associated with amyloid burden, tau pathology, and cognitive decline, whereas a mitochondrial bioenergetic module showed relative preservation in vascular pathology. Cross-omics concordance was robust at the pathway level but limited at the level of individual genes and proteins. These findings define conserved molecular programs distinguishing vascular, neurodegenerative, and mixed pathology and demonstrate that pathway-level organization provides a stable framework for interpreting molecular heterogeneity in late-life dementia.

genomics↗

bioRxiv2025

Functional Genomic Screens Reveal RBBP4 as a Key Regulator of Cell Cycle Progression in TMZ-Resistant Glioblastoma

Temozolomide (TMZ) remains the standard of care for glioblastoma; however, its efficacy is frequently influenced by epigenetic mechanisms, notably the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. While MGMT promoter hypermethylation is associated with enhanced responsiveness to TMZ, additional epigenetic determinants of TMZ resistance remain largely undefined. In this study, we established TMZ-resistant glioblastoma cell lines that consistently maintained their resistant phenotype both in vitro and in vivo. Transcriptomic analyses revealed a marked upregulation of MGMT expression in these models. To systematically investigate the epigenetic regulators governing TMZ resistance and cell survival, we conducted CRISPR/Cas9-based functional genomic screens using our focused Epigenetic Knock-Out Library (EPIKOL), which targets 800 chromatin regulators alongside selected positive and negative controls. These unbiased screens validated MGMT as a primary mediator of TMZ resistance, confirming the robustness of our approach. Moreover, dropout screens across multiple resistant cell line models identified Retinoblastoma Binding Protein 4 (RBBP4) as a critical vulnerability. Notably, RBBP4 knockout significantly impaired cell proliferation without affecting MGMT expression, suggesting a distinct mechanism supporting the survival of TMZ-resistant glioblastoma cells. Subsequent transcriptomic profiling following RBBP4 loss demonstrated significant downregulation of cell cycle pathways, particularly the G2/M checkpoint. Live-cell imaging and immunofluorescence analyses further revealed increased cell size and multinucleation in RBBP4-deficient cells, indicative of disrupted mitotic progression. Collectively, our results identify RBBP4 as a key regulator of cell cycle progression and survival in TMZ-resistant glioblastoma and highlight its potential as a novel epigenetic target for therapeutic intervention in recurrent disease.

cancer biology↗

bioRxiv2025

APOE-ϵ4-induced Fibronectin at the blood-brain barrier is a conserved pathological mediator of disrupted astrocyte-endothelia interaction in Alzheimer's disease

Blood-brain barrier (BBB) dysfunction is a key feature of Alzheimers disease (AD), particularly in individuals carrying the APOE-{varepsilon}4 allele. This dysfunction worsens neuroinflammation and hinders the removal of toxic proteins, such as amyloid-beta (A{beta}42), from the brain. In post-mortem brain tissues and in animal models, we previously reported that fibronectin accumulates at the BBB predominantly in APOE-{varepsilon}4 carriers. Furthermore, we found a loss-of-function variant in the fibronectin 1 (FN1) gene significantly reduces aggregated fibronectin levels and decreases AD risk among APOE-{varepsilon}4 carriers. Yet, the molecular mechanisms downstream of fibronectin at the BBB remain unclear. The extracellular matrix (ECM) plays a crucial role in maintaining BBB homeostasis and orchestrating the interactions between BBB cell types, including endothelia and astrocytes. Understanding the mechanisms affecting the ECM and BBB cell types will be critical for developing effective therapies against AD, especially among APOE-{varepsilon}4 carriers. Here, we demonstrate that APOE-{varepsilon}4, A{beta}42, and inflammation drive the induction of FN1 expression in several models including zebrafish, mice, iPSC-derived human 3D astrocyte and 3D cerebrovascular cell cultures, and in human brains. Fibronectin accumulation disrupts astroglial-endothelial interactions and the signalling cascade between vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF) and Insulin-like growth factor 1 (IGF1). This accumulation of fibronectin in APOE-{varepsilon}4- associated AD potentiates BBB dysfunction, which strongly implicates reducing fibronectin deposition as a potential therapeutic target for AD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/634732v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1efff03org.highwire.dtl.DTLVardef@14f40cdorg.highwire.dtl.DTLVardef@27743aorg.highwire.dtl.DTLVardef@3fa9a9_HPS_FORMAT_FIGEXP M_FIG C_FIG Accessibility textThis image illustrates the effects of different APOE isoforms (ApoE-{varepsilon}3 and ApoE-{varepsilon}4) on blood-brain barrier (BBB) integrity, focusing on the molecular interactions between astrocytes and endothelial cells. This figure emphasizes the detrimental effects of ApoE-{varepsilon}4 on BBB integrity via fibronectin accumulation and altered signaling pathways. The top section provides a schematic overview of the blood-brain barrier, highlighting astrocytes, endothelial cells, and their interface. The left panel represents the ApoE-{varepsilon}3 condition: Normal fibronectin (FN1) levels support healthy interactions between astrocytes and endothelial cells. Growth factors, including VEGFA, HBEGF, and IGF1, maintain BBB integrity through their respective receptors (VEGFR and EGFR). Green arrows indicate activation of these signaling pathways. The right panel depicts the ApoE-{varepsilon}4 condition: Elevated fibronectin (FN1) disrupts astrocyte-endothelium interactions. FN1 binds integrins and activates focal adhesion kinase (FAK), inhibiting VEGFA, which is required for endothelial HBEGF that in turn activates IGF1 signaling. Red symbols indicate inhibition of HBEGF, VEGFA, and IGF1 pathways, leading to BBB dysfunction. HighlightsAPOE-{varepsilon}4 drives fibronectin deposition in Alzheimers, disrupting astrocyte-endothelia interactions. APOE-{varepsilon}4 and fibronectin co-localize, forming aggregates at blood-brain barrier (BBB). Fibronectin alters the signaling between VEGF, IGF1, and HBEGF impairing BBB function. Reducing fibronectin restores BBB integrity and offsets APOE-{varepsilon}4 pathology.

cell biology↗

bioRxiv2025

NCBP2-AS2 is a mitochondrial microprotein, regulates energy metabolism and neurogenesis, and is downregulated in Alzheimer's disease

Microproteins, short functional peptides encoded by small genes, are emerging as critical regulators of cellular processes, yet their roles in mitochondrial function and neurodegeneration remain underexplored. In this study, we identify NCBP2-AS2 as an evolutionarily conserved mitochondrial microprotein with significant roles in energy metabolism and neurogenesis. Using a combination of cellular and molecular approaches, including CRISPR/Cas9 knockout models, stoichiometric co- immunoprecipitation, and advanced imaging techniques, we demonstrate that NCBP2-AS2 localizes to the inner mitochondrial space and interacts with translocase of the inner membrane (TIM) chaperones. These interactions suggest a role in ATPase subunit transport, supported by the observed reductions in ATPase subunit levels and impaired glucose metabolism in NCBP2-AS2-deficient cells. In zebrafish, NCBP2-AS2 knockout led to increased astroglial proliferation, microglial abundance, and enhanced neurogenesis, particularly under amyloid pathology. Notably, we show that NCBP2-AS2 expression is consistently downregulated in human Alzheimers disease brains and zebrafish amyloidosis models, suggesting a conserved role in neurodegenerative pathology. These findings reveal a novel link between mitochondrial protein transport, energy metabolism, and neural regeneration, positioning NCBP2-AS2 as a potential therapeutic target for mitigating mitochondrial dysfunction and promoting neurogenesis in neurodegenerative diseases such as Alzheimers disease.

cell biology↗

bioRxiv2024

Ethanol exposure model in zebrafish causes phenotypic, behavioral and gene expression changes that mimic Fetal Alcohol Spectrum Disorders in human birth cohorts

Fetal Alcohol Spectrum Disorders (FASD) represent a significant global health challenge, characterized by physical and neurodevelopmental abnormalities in offspring resulting from prenatal alcohol exposure. This study aims to utilize the zebrafish to examine the phenotypic, behavioral, and molecular changes associated with embryonic ethanol exposure, providing a model for human FASD conditions. Our study exposed zebrafish embryos to 0.5% ethanol during a critical developmental window (2-24 hours post-fertilization) and documented significant craniofacial and cardiac deformities, which recapitulate what has been observed in human FASD in humans. Notably, exposed zebrafish exhibited reduced skull and eye sizes, thickened jaw size, and enlarged heart chambers. We found reduced burst swim distance following a touch stimulus, a novel behavioral assessment of potential deficits in sensory processing such as processing speed and/or stress/startle response, both of which are affected in human FASD. Whole-organism gene expression was found to be altered by ethanol for orthologs of four of five inflammation-related genes for which placental expression was previously found to be altered in response to alcohol in human placentas (SERPINE1, CRHB, BCL2L1, PSMB4, PTGS2A). We conclude that the zebrafish model effectively mimics several FASD phenotypes observed in humans, confirming gene expression changes we have previously documented in a human observational study and providing a valuable platform for exploring the underlying mechanisms of alcohol-induced embryonic alterations and for developing diagnostic markers and therapeutic targets for early intervention.

pathology↗

bioRxiv2024

Chromatin-focused genetic and chemical screens identify BRPF1 as a targetable vulnerability in Taxol-resistant triple-negative breast cancer

Triple-negative breast cancer (TNBC) stands out as a particularly aggressive and frequently recurring form of breast cancer. Due to the absence of hormone receptors, the available treatment avenues are constrained, making chemotherapy the primary approach. Unfortunately, the development of resistance to chemotherapy poses a significant challenge, further restricting the already limited therapeutic alternatives for recurrent cases. Understanding the molecular basis of chemotherapy resistance in TNBC is pivotal for improving treatment outcomes. Here, we generated two different Taxol-resistant TNBC cell lines with a dose-escalation method to mimic chemotherapy resistance in vitro. These cells exhibited hallmark features of resistance, including reduced cell growth, altered morphology, and evasion of apoptosis. Transcriptome analysis uncovered elevated ABCB1 expression and multidrug-resistant phenotype in the resistant cells. To comprehensively investigate the key epigenetic regulators of Taxol resistance, we conducted chromatin-focused genetic and chemical screens and pinpointed Bromodomain and PHD Finger Containing 1 (BRPF1) as a novel regulator of Taxol resistance in TNBC cells. Knockout of BRPF1, the reader protein in the MOZ/MORF histone acetyl-transferase complex, but not the other complex members, sensitized resistant cells to Taxol. Additionally, BRPF1 inhibitors, PFI-4 and OF-1, in combination with Taxol significantly reduced cell viability. Transcriptome analysis upon BRPF1 loss or inhibition revealed a negative impact on ribosome biogenesis-related gene sets, resulting in a global decrease in protein translation in Taxol-resistant cells. Our ChIP-qPCR analysis demonstrated that active BRPF1 directly interacts with the ABCB1 promoter, enhancing its expression towards inducing a multidrug-resistant phenotype. Conversely, knockout or inhibition of BRPF1 leads to decreased ABCB1 expression. This dual mechanism critically sensitizes Taxol-resistant TNBC cells to chemotherapy. Our findings uncover a comprehensive molecular framework, highlighting the pivotal role of epigenetic reader protein BRPF1 in Taxol resistance and providing potential avenues for therapeutic intervention in TNBC.

cancer biology↗

bioRxiv2024

MU-BRAIN: MUltiethnic Brain Rna-seq for Alzheimer INitiative

Alzheimers Disease (AD) exhibits a complex molecular and phenotypic profile. Investigating gene expression plays a crucial role in unraveling the diseases etiology and progression. Transcriptome data across ethnic groups lack, negatively impacting equity in intervention and research. We employed 565 brains across six U.S. brain banks (n=399 non-Hispanic Whites, n=113 Hispanics, n=12 African Americans) to generated bulk RNA sequencing from prefrontal cortex. We sought to identify cross-ancestry and ancestry-specific differentially expressed genes (DEG) across Braak stages, adjusting for sex, age at death, and RNA quality metrics. We further validated our findings using the Religious Orders Study/Memory Aging Project brains (ROS/MAP; n=1,095) and performed metanalysis (n=1,660). We conducted Gene Set and Variation and Enrichment analysis (GSVA; GSEA). We employed a machine-learning approach for phenotype prediction and gene prioritization to construct a polytranscriptomics risk score (PTRS) splitting our sample into training and testing sub-samples, either randomly or by ethnicity ("ancestry-agnostic" and "ancestry-aware", respectively). Lastly, we validated top DEG using single-nucleus RNA sequencing (snRNAseq) data. We identified several DEG associated with Braak staging: AD-known genes VGF (Padj =3.78E- 07) and ADAMTS2 (Pad j=1.21E-04) were consistently differentially expressed across statistical models, ethnicities, and replicated in ROS/MAP. Genes from the heat shock protein (HSP) family, e.g. HSPB7 (Padj =3.78E-07), were the top differentially expressed genes and replicated in ROS/MAP. Ethnic-stratified analyses prioritized TNFSF14 and SPOCD1 as top Hispanics DEG. GSEA highlighted "Alzheimer disease" (Padj =4.24E-06) and "TYROBP causal network in microglia" (Padj =1.68E-08) pathways. Up- and down-regulated genes were enriched in several pathways (e.g. "Immune response activation signal pathways", "Vesicle-mediated transport in synapse", "cognition"). Ancestry-agnostic and ancestry-aware PTRS effectively classified brains (AUC=0.77 and 0.73 respectively) and replicated in ROS/MAP. snRNAseq validated prioritized genes, including VGF (downregulated in neurons; Padj=1.1 E-07). This is the largest diverse AD transcriptome in post-mortem brain tissue, to our knowledge. We identified perturbated genes, pathways and network expressions in AD brains resulting in cross- ethnic and ethnic-specific findings, ultimately highlighting the diversity within AD pathogenesis. The latter underscores the need for an integrative and personalized approach in AD studies.

genetics↗

bioRxiv2024

FABP4 as a Therapeutic Host Target Controlling SARS-CoV2 Infection

Host metabolic fitness is a critical determinant of infectious disease outcomes. Obesity, aging, and other related metabolic disorders are recognized as high-risk disease modifiers for respiratory infections, including coronavirus infections, though the underlying mechanisms remain unknown. Our study highlights fatty acid-binding protein 4 (FABP4), a key regulator of metabolic dysfunction and inflammation, as a modulator of SARS-CoV-2 pathogenesis, correlating strongly with disease severity in COVID-19 patients. We demonstrate that loss of FABP4 function, by genetic or pharmacological means, reduces SARS-CoV2 replication and disrupts the formation of viral replication organelles in adipocytes and airway epithelial cells. Importantly, FABP4 inhibitor treatment of infected hamsters diminished lung viral titers, alleviated lung damage and reduced collagen deposition. These findings highlight the therapeutic potential of targeting host metabolism in limiting coronavirus replication and mitigating the pathogenesis of infection.

microbiology↗

bioRxiv2024

ABCA7-dependent Neuropeptide-Y signalling is a resilience mechanism required for synaptic integrity in Alzheimer's disease

Alzheimers disease (AD) remains a complex challenge characterized by cognitive decline and memory loss. Genetic variations have emerged as crucial players in the etiology of AD, enabling hope for a better understanding of the disease mechanisms; yet the specific mechanism of action for those genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Using CRISPR/Cas9 gene editing, we generated a knockout model for abca7, orthologous to human ABCA7 - an established AD-risk gene. The abca7+/- zebrafish showed reduced astroglial proliferation, synaptic density, and microglial abundance in response to amyloid beta 42 (A{beta}42). Single-cell transcriptomics revealed abca7-dependent neuronal and glial cellular crosstalk through neuropeptide Y (NPY) signaling. The abca7 knockout reduced the expression of npy, bdnf and ngfra, which are required for synaptic integrity and astroglial proliferation. With clinical data in humans, we showed reduced NPY in AD correlates with elevated Braak stage, predicted regulatory interaction between NPY and BDNF, identified genetic variants in NPY associated with AD, found segregation of variants in ABCA7, BDNF and NGFR in AD families, and discovered epigenetic changes in the promoter regions of NPY, NGFR and BDNF in humans with specific single nucleotide polymorphisms in ABCA7. These results suggest that ABCA7-dependent NPY signaling is required for synaptic integrity, the impairment of which generates a risk factor for AD through compromised brain resilience. O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/573893v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@eecf7forg.highwire.dtl.DTLVardef@78d8e0org.highwire.dtl.DTLVardef@1e65d71org.highwire.dtl.DTLVardef@100db01_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

neuroscience↗

bioRxiv2024

Rare genetic variation in Fibronectin 1 (FN1) protects against APOEe4 in Alzheimer's disease

The risk of developing Alzheimers disease (AD) significantly increases in individuals carrying the APOE{varepsilon}4 allele. Elderly cognitively healthy individuals with APOE{varepsilon}4 also exist, suggesting the presence of cellular mechanisms that counteract the pathological effects of APOE{varepsilon}4; however, these mechanisms are unknown. We hypothesized that APOE{varepsilon}4 carriers without dementia might carry genetic variations that could protect them from developing APOE{varepsilon}4-mediated AD pathology. To test this, we leveraged whole genome sequencing (WGS) data in National Institute on Aging Alzheimers Disease Family Based Study (NIA-AD FBS), Washington Heights/Inwood Columbia Aging Project (WHICAP), and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts and identified potentially protective variants segregating exclusively among unaffected APOE{varepsilon}4 carriers. In homozygous unaffected carriers above 70 years old, we identified 510 rare coding variants. Pathway analysis of the genes harboring these variants showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of functional modifications in ECM proteins. We prioritized two genes that were highly represented in the ECM-related gene ontology terms, (FN1) and collagen type VI alpha 2 chain (COL6A2) and are known to be expressed at the blood-brain barrier (BBB), for postmortem validation and in vivo functional studies. The FN1 and COL6A2 protein levels were increased at the BBB in APOE{varepsilon}4 carriers with AD. Brain expression of cognitively unaffected homozygous APOE{varepsilon}4 carriers had significantly lower FN1 deposition and less reactive gliosis compared to homozygous APOE{varepsilon}4 carriers with AD, suggesting that FN1 might be a downstream driver of APOE{varepsilon}4-mediated AD-related pathology and cognitive decline. To validate our findings, we used zebrafish models with loss-of-function (LOF) mutations in fn1b - the ortholog for human FN1. We found that fibronectin LOF reduced gliosis, enhanced gliovascular remodeling and potentiated the microglial response, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. Our study suggests vascular deposition of FN1 is related to the pathogenicity of APOE{varepsilon}4, LOF variants in FN1 may reduce APOE{varepsilon}4-related AD risk, providing novel clues to potential therapeutic interventions targeting the ECM to mitigate AD risk.

genetics↗

bioRxiv2022

Reliability metrics of outcome measures during the single and double leg drop jump tests

Although a number of previous studies have studied reliability metrics of outcome measures during single and double leg drop jump tests, none of them presented detailed reliability information for both tests with a group of collegiate athletes of both genders. The aim of this study was therefore to assess the reliability of outcome measures during the single and double leg drop jump tests with proper reliability metrics. Seventeen handball players (11 male and 6 female) participated in the experiments. Each player performed three double leg and three single drop jumps from a 30 cm height box onto a portable force plate in two sessions a week apart. Sixteen outcome measures, including jump height (JH), ground contact time (GCT), reactive strength index (RSI), were calculated. Four groups of reliability metrics like absolute agreement and consistency intraclass correlation coefficients (inter-day and intra-day), standard error of measurement, coefficient of variation, and minimal metrically detectable change were estimated for reliability assessment. The outcome measures as RSI and its individual components JH and GCT together with normalized vertical stiffness (Kvert) yielded high inter-day and intra-day intraclass correlation coefficients and low standard error of measurement and coefficient of variation levels for the double leg drop jump test. The more challenging single leg drop jump test could also be considered reproducible with some other highly reliable outcome measure set by keeping JH, GCT, and normalized Kvert and replacing vertical jump impulse with RSI. The results of the current study therefore suggested the drop jump tests could be deemed reliable to be used for short-term and long-term monitoring needs for a group of collegiate athletes of both genders.

physiology↗

bioRxiv2020

HologLev: Hybrid Magnetic Levitation Platform Integrated with Lensless Holographic Microscopy for Density-Based Cell Analysis

In clinical practice, a variety of diagnostic applications require the identification of target cells. Density has been used as a physical marker to distinguish cell populations since metabolic activities could alter the cell densities. Magnetic levitation offers a great promise for separating cells at single cell level within heterogeneous populations with respect to cell densities. Traditional magnetic levitation platforms need bulky and precise optical microscopes to visualize levitated cells. Moreover, the evaluation process of cell densities is cumbersome, which also requires trained personnel for operation. In this work, we introduce a device (HologLev) as a fusion of magnetic levitation principle and lensless digital inline holographic microscopy (LDIHM). LDIHM provides ease of use by getting rid of bulky and expensive optics. By placing an imaging sensor just beneath the microcapillary channel without any lenses, recorded holograms are processed for determining cell densities through a fully automated digital image processing scheme. The device costs less than $100 and has a compact design that can fit into a pocket. We perform viability tests the device by levitating three different cell lines (MDA-MB-231, U937, D1 ORL UVA) and comparing them against their dead correspondents. We also tested the differentiation of mouse osteoblastic (7F2) cells by monitoring characteristic variations in their density. Lastly, MDA-MB-231 cells exposed to a chemotherapy drug are separated from original cell lines in our platform. HologLev provides cost-effective, label-free, fully automated cell analysis in a compact design which could be highly desirable for laboratory and point-of-care testing applications.

bioengineering↗

bioRxiv2020

Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancer to treat. Due to the asymptomatic nature of the disease and ineffective drug treatment modalities, the survival rate of PDAC patients remains one of the lowest. The recurrent genetic alterations in PDAC are yet to be targeted; therefore, identifying effective therapeutic combinations is desperately needed. Here, we performed an in vivo CRISPR screening in a clinically relevant patient-derived xenograft (PDX) model system to identify synergistic drug combinations for PDAC treatment. Our approach revealed protein arginine methyltransferase gene 5 (PRMT5) as a promising druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to gemcitabine. Genetic and pharmacological inhibition results indicate that of PRMT5 depletion results in synergistic cytotoxicity with Gem due to depleted replication protein A (RPA) levels and an impaired non-homology end joining (NHEJ) DNA repair. Thus, the novel combination creates conditional lethality through the accumulation of excessive DNA damage and cell death, both in vitro and in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is an effective approach in identifying synthetic lethal drug combinations for cancer treatment. STATEMENT of SIGNIFICANCEIdentify synergistic drug combinations for PDAC is a significant unmet need. Through CRISPR screening, we discovered and validated that PRMT5 depletion creates synergistic vulnerability of PDAC cells to gemcitabine. Mechanistically, the combination impairs DNA repair, synergistic accumulation of DNA damage and cell death in vitro and in vivo.

cancer biology↗