Biology subjects

Tsai, R.

Publications and source records attributed to Tsai, R..

11 recordsLinked to original sources

bioRxiv2026

S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

As a consequence of their sizes, many loss-of-function genetic mutations fall within large genes. A major gene-therapy tool that could be used to solve large swaths of the genetic diseases that result from these inherited mutations is large-fragment knock-in. I.e. instead of attempting to create separate treatments for each and every location that these mutations occur in, large groups of patients could be aided via a single safe-harbor integration of the full-length coding sequence. Towards this goal, we have created a set of early stage gene-editing enzymes that can help mediate large cargo integration at a safe harbor locus in human cells. When expressed in stable lines, our S-SELeCT (Site-Specific Large Cargo Targeting) integrase fusions can facilitate integration of a 10 kb plasmid at frequencies up to 32%, and when delivered transiently via plasmid transfection, we were able to achieve up to 13% knock-in. These are the first serine integrase enzymes that have been evolved fully in human cells, and the first to recognize an endogenous symmetric non-pseudosite - the first true human serine integrase attachment site. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/702954v2_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@1c22676org.highwire.dtl.DTLVardef@16b171corg.highwire.dtl.DTLVardef@1ba4796org.highwire.dtl.DTLVardef@b1d91f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Topoisomerase IIb binding underlies frequently mutated elements in cancer genomes

Type-II topoisomerases resolve topological stress in DNA through controlled double-strand breaks. While TOP2A is a chemotherapy target in proliferating cells, the ubiquitously expressed TOP2B is a potential off-target. Here we explore roles of TOP2B in mutagenesis by generating DNA-binding maps of TOP2B, CTCF, and RAD21 in human cancer samples and analysing these maps for driver mutations and mutational processes in 6500 whole cancer genomes. TOP2B-CTCF-RAD21 and TOP2B-RAD21 sites are enriched in somatic mutations and structural variants (SVs), especially at evolutionary conserved sites displaying high transcription and long-range chromatin interactions. TOP2B binding underlies SVs and hotspot mutations in cancer-driving genes such as TP53, MYC, FOXA1, and VHL, and many cis-regulatory elements. We show that the TOP2B-bound mutational hotspot at RMRP drives tumor initiation and growth in vivo. These data highlight TOP2B as a protector of the genome from topological challenges whose aberrant activity promotes driver and passenger mutations in cancer genomes.

cancer biology↗

bioRxiv2024

Alterations in Lysosomal, Glial and Neurodegenerative Biomarkers in Patients with Sporadic and Genetic Forms of Frontotemporal Dementia

BackgroundFrontotemporal dementia (FTD) is the most common cause of early-onset dementia with 10-20% of cases caused by mutations in one of three genes: GRN, C9orf72, or MAPT. To effectively develop therapeutics for FTD, the identification and characterization of biomarkers to understand disease pathogenesis and evaluate the impact of specific therapeutic strategies on the target biology as well as the underlying disease pathology are essential. Moreover, tracking the longitudinal changes of these biomarkers throughout disease progression is crucial to discern their correlation with clinical manifestations for potential prognostic usage. MethodsWe conducted a comprehensive investigation of biomarkers indicative of lysosomal biology, glial cell activation, synaptic and neuronal health in cerebrospinal fluid (CSF) and plasma from non-carrier controls, sporadic FTD (symptomatic non-carriers) and symptomatic carriers of mutations in GRN, C9orf72, or MAPT, as well as asymptomatic GRN mutation carriers. We also assessed the longitudinal changes of biomarkers in GRN mutation carriers. Furthermore, we examined biomarker levels in disease impacted brain regions including middle temporal gyrus (MTG) and superior frontal gyrus (SFG) and disease-unaffected inferior occipital gyrus (IOG) from sporadic FTD and symptomatic GRN carriers. ResultsWe confirmed glucosylsphingosine (GlcSph), a lysosomal biomarker regulated by progranulin, was elevated in the plasma from GRN mutation carriers, both symptomatic and asymptomatic. GlcSph and other lysosomal biomarkers such as ganglioside GM2 and globoside GB3 were increased in the disease affected SFG and MTG regions from sporadic FTD and symptomatic GRN mutation carriers, but not in the IOG, compared to the same brain regions from controls. The glial biomarkers GFAP in plasma and YKL40 in CSF were elevated in asymptomatic GRN carriers, and all symptomatic groups, except the symptomatic C9orf72 mutation group. YKL40 was also increased in SFG and MTG regions from sporadic FTD and symptomatic GRN mutation carriers. Neuronal injury and degeneration biomarkers NfL in CSF and plasma, and UCHL1 in CSF were elevated in patients with all forms of FTD. Synaptic biomarkers NPTXR, NPTX1/2, and VGF were reduced in CSF from patients with all forms of FTD, with the most pronounced reductions observed in symptomatic MAPT mutation carriers. Furthermore, we demonstrated plasma NfL was significantly positively correlated with disease severity as measured by CDR+NACC FTLD{square}SB in genetic forms of FTD and CSF NPTXR was significantly negatively correlated with CDR+NACC FTLD{square}SB in symptomatic GRN and MAPT mutation carriers. ConclusionsIn conclusion, our comprehensive investigation replicated alterations in biofluid biomarkers indicative of lysosomal function, glial activation, synaptic and neuronal health across sporadic and genetic forms of FTD and unveiled novel insights into the dysregulation of these biomarkers within brain tissues from patients with GRN mutations. The observed correlations between biomarkers and disease severity open promising avenues for prognostic applications and for indicators of drug efficacy in clinical trials. Our data also implicated a complicated relationship between biofluid and tissue biomarker changes and future investigations should delve into the mechanistic underpinnings of these biomarkers, which will serve as a foundation for the development of targeted therapeutics for FTD.

neuroscience↗

bioRxiv2022

In vivo CRISPR screens reveal SCAF1 and USP15 as novel drivers of pancreatic cancer

Functionally characterizing the genetic alterations that drive pancreatic cancer progression is a prerequisite for Precision Medicine. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to assess the transforming potential of 125 recurrently mutated long-tail pancreatic cancer genes, which revealed USP15 and SCAF1 as novel and potent Pancreatic ductal adenocarcinoma PDAC tumor suppressors, with USP15 functioning in a haplo-insufficient manner. Mechanistically, we found that loss of USP15 leads to reduced inflammatory responses associated with TNF, TGF-{beta} and IL6 signaling and sensitizes pancreatic cancer cells to PARP inhibition and gemcitabine. Similarly, genetic ablation of SCAF1 reduced inflammatory responses linked to TNF, TGF-{beta} and mTOR signaling and increased sensitivity to PARP inhibition. Furthermore, we identified that loss of SCAF1 resulted in the formation of a truncated inactive USP15 isoform at the expense of full length USP15, functionally coupling SACF1 and USP15. Notably, USP15 and SCAF1 mutations or copy number losses are observed in 31% of PDAC patients. Together, our results demonstrate the utility of in vivo CRISPR to integrate human cancer genomics with mouse modeling to delineate novel cancer driver genes USP15 and SCAF1 such as with potential prognostic and therapeutic implications.

cancer biology↗

bioRxiv2022

In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer

How the genetic landscape of a tumor governs the tumors response to immunotherapy remains largely elusive. Here, we established a direct in vivo CRISPR/Cas9 gene editing methodology to assess the immune-modulatory capabilities of 573 putative cancer genes associated with altered cytotoxic activity in human cancers. Using KrasG12D- and BrafV600E-driven mouse lung cancer models, we identify Serpinb9 and Adam2 as our top immune suppressive and immune enhancing genes, respectively. Mechanistically, we show that Serpinb9 ablation in KrasG12D- and BrafV600E-mutant lung tumor cells greatly enhances the efficacy of cytotoxic T-cells in vitro and in vivo. ADAM2 is a cancer testis antigen broadly expressed in human cancers such as lung adenocarcinoma (13.9%), renal (74.7%), prostate (72.4%), uterine (28.6%) and invasive breast (9.5%) cancer. In our mouse models, we show that Adam2 expression is induced in KrasG12D- but not BrafV600E-driven murine lung tumors and that its expression is further enhanced by immunotherapy. We show that loss of Adam2 significantly decreases KrasG12D-lung tumor burden but blocks the efficacy of cytotoxic T-cells. Consistently, Adam2 overexpression dramatically increases tumor growth and enhances immunotherapy efficacy. Mechanistically, we find that Adam2s oncogenic function depends on modulating the tumor immune microenvironment by restraining productive type I and type II interferon responses as well as cytokine signaling, reducing the presentation of tumor-associated antigen, and modulating surface expression of several immunoregulatory receptors within Kras-driven lung tumors. Adam2 expression also leads to reduced levels of immune checkpoint inhibitors such as Pd-l1, Lag3, Tigit and Tim3. This reduced exhaustion within the tumor microenvironment may explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy against Adam2 overexpressing lung tumors. Together, our study highlights the power of integrating cancer genomic with in vivo CRISPR/Cas9 screens to uncover how cancer-associated genetic alterations control responses to immunotherapies.

cancer biology↗

bioRxiv2022

Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings

Accurate, high-resolution environmental monitoring of SARS-CoV-2 traces indoors through sentinel cards is a promising approach to help students safely return to in-person learning. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface types, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution, but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, wet wipes, RNase Away) at three different viral loads: "high" (4 x 104 GE/mL), "medium" (1 x 104 GE/mL), and "low" (2.5 x 103 GE/mL). RNAse Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. ImportanceBecause SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are persistent signal or new infectious cases, and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

bioinformatics↗

bioRxiv2021

Loss of epigenetic regulation disrupts lineage integrity, reactivatesmultipotency and promotes breast cancer

Systematically investigating the scores of genes mutated in cancer and discerning real drivers from inconsequential bystanders is a prerequisite for Precision Medicine, but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent long-tail breast cancer genes, which revealed epigenetic regulation as major tumor suppressive mechanism. We report that core or accessory components of the COMPASS histone methylase complex including KMT2C, KDM6A, BAP1 and ASXL2 ("EpiDrivers") cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that Cre-mediated activation of PIK3CAH1047R elicited an aberrant alveolar lactation program in luminal cells, which was exacerbated upon loss of EpiDrivers. Remarkably, EpiDriver loss in basal cells also triggered an alveolar-like lineage conversion and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. As EpiDrivers are mutated in 39% of human breast cancers, lineage infidelity and lactation mimicry may significantly contribute to early steps of breast cancer progression. Statement of significanceInfrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In-vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of lactation genes as potential early events in tumorigenesis.

cancer biology↗

bioRxiv2021

Comparison of heat-inactivated and infectious SARS-CoV-2 across indoor surface materials shows comparable RT-qPCR viral signal intensity and persistence

Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with COVID-19 and inform appropriate infection mitigation responses. Research groups have reported detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2 positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over seven days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle (Cq)) can be correlated to surface viral load using only one linear regression model per material category. The same experiment was performed with infectious viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and time points tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods. ImportanceEnvironmental monitoring is an important tool for public health surveillance, particularly in settings with low rates of diagnostic testing. Time between sampling public environments, such as hospitals or schools, and notifying stakeholders of the results should be minimal, allowing decisions to be made towards containing outbreaks of coronavirus disease 2019 (COVID-19). The Safer At School Early Alert program (SASEA) [1], a large-scale environmental monitoring effort in elementary school and child care settings, has processed > 13,000 surface samples for SARS-CoV-2, detecting viral signals from 574 samples. However, consecutive detection events necessitated the present study to establish appropriate response practices around persistent viral signals on classroom surfaces. Other research groups and clinical labs developing environmental monitoring methods may need to establish their own correlation between RT - qPCR results and viral load, but this work provides evidence justifying simplified experimental designs, like reduced testing materials and the use of heat-inactivated viral particles.

microbiology↗

bioRxiv2019

Comprehensive analysis of lncRNAs reveals candidate prognostic biomarkers in multiple cancer types

Long non-coding RNAs (lncRNAs) are increasingly recognized as functional units in cancer pathways and powerful molecular biomarkers, however most lncRNAs remain uncharacterized. Here we performed a systematic discovery of prognostic lncRNAs in 9,326 patient tumors of 29 types using a proportional-hazards elastic net machine-learning framework. lncRNAs showed highly tissue-specific transcript abundance patterns. We identified 179 prognostic lncRNAs whose abundance correlated with patient risk and improved the performance of common clinical variables and molecular tumor subtypes. Pathway analysis revealed a large diversity of the high-risk tumors stratified by lncRNAs and suggested their functional associations. In lower-grade gliomas, discrete activation of HOXA10-AS indicated poor patient prognosis, neurodevelopmental pathway activation and a transcriptomic similarity to glioblastomas. HOXA10-AS knockdown in patient-derived glioblastoma cells caused decreased cell proliferation and deregulation of glioma driver genes and proliferation pathways. Our study underlines the pan-cancer potential of the non-coding transcriptome for developing molecular biomarkers and innovative therapeutic strategies.

cancer biology↗

bioRxiv2019

Oxidation shuts down an auto-inhibitory mechanism of von Willebrand factor

The blood protein von Willebrand factor (VWF) is a key link between inflammation and pathological thrombus formation. In particular, oxidation of methionine residues in specific domains of VWF due to the release of oxidants in inflammatory conditions has been linked to an increased platelet-binding activity. However, the atomistic details how methionine oxidation activates VWF have not been elucidated to date. Yet understanding the activation mechanism of VWF under oxidizing conditions can lead to the development of novel therapeutics that target VWF selectively under inflammatory conditions in order to reduce its thrombotic activity while maintaining its haemostatic function. In this manuscript, we used a combination of a dynamic flow assay and molecular dynamics (MD) simulations to investigate how methionine oxidation removes an auto-inhibitory mechanism of VWF. Results from the dynamic flow assay revealed that oxidation does not directly activate the A1 domain, which is the domain in VWF that contains the binding site to the platelet surface receptor glycoprotein Ib (GpIb), but rather removes the inhibitory function of the neighboring A2 and A3 domains. Furthermore, the MD simulations combined with free energy perturbation calculations suggested that methionine oxidation may destabilize the binding interface between the A1 and A2 domains leading to unmasking of the GpIb-binding site in the A1 domain.

bioengineering↗

bioRxiv2019

The Secretome of Liver X Receptor Agonist Treated Early Outgrowth Cells Decreases Atherosclerosis in Ldlr-/- Mice

ObjectiveEndothelial progenitor cells (EPCs) promote the maintenance of the endothelium by the secretion of vasoreparative factors. A population of EPCs known as early outgrowth cells (EOCs) are currently being investigated as novel cell-based therapies for the treatment of cardiovascular disease. We previously demonstrated that the absence of liver x receptors (LXRs) is detrimental to the formation and function of EOCs under hypercholesterolemic conditions. Here, we investigate whether LXR gain-of-function in EOCs is beneficial for the treatment of atherosclerosis.\n\nApproach and ResultsEOCs were differentiated from the bone marrow of wildtype (WT) and LXR-knockout (Lxr{beta}-/-) mice in the presence of vehicle or LXR agonist (GW3965). WT EOCs treated with GW3965 throughout differentiation showed reduced expression of endothelial lineage markers (Cd144, Vegfr2) compared to WT vehicle and Lxr{beta}-/- cells. GW3965-treated EOCs produced secreted factors that reduced monocyte adhesion to activated endothelial cells in culture. When injected into atherosclerosis-prone Ldlr-/- mice, GW3965-treated EOCs and concentrated conditioned media (CM) from GW3965-treated EOCs, reduced plaque burden within the aortic sinus. Furthermore, when CM from human EOCs (obtained from patients with established CAD) were treated with GW3965, monocyte to endothelial adhesion was decreased suggesting the translatability of the results.\n\nConclusionsEx vivo LXR agonist treatment of EOCs produces a secretome that decreases early atherosclerosis in Ldlr-/- mice. CM from human EOCs significantly inhibits monocyte to endothelial adhesion. Thus, active factor(s) within the GW3965-treated EOC secretome have the potential to be useful for the treatment of atherosclerosis.

cell biology↗