Biology subjects

Nguyen, N. T.

Publications and source records attributed to Nguyen, N. T..

17 recordsLinked to original sources

bioRxiv2026

Chronotype asymmetry arises from stochastic sleep homeostasis under circadian entrainment

Study ObjectivesChronotype, typically defined by the mid-sleep phase on free days, is often interpreted as a direct proxy for circadian phase. However, a fundamental paradox challenges this assumption: worldwide chronotype distributions show a heavy right skew, whereas circadian period distributions show a slight left skew. We investigated whether fast, noisy cortical processes in homeostatic sleep pressure dynamics could resolve this paradox. MethodsDrawing upon the classical two-process model of sleep regulation, we simulated how interindividual differences in sleep homeostasis (Process S), combined with realistic circadian period distributions (Process C), interact to shape chronotype variability. We introduce stochasticity into sleep pressure build-up to test whether homeostatic variance generates the empirically observed right-skew in chronotype distributions. ResultsExploring the parameter space reveals that stable sleep-wake cycles are constrained within physiological limits of entrainment in an Arnold tongue-like structure. While our simulations confirm a general positive correlation between intrinsic period and chronotype, the heavy right tail of the chronotype distribution arises from interindividual variability in homeostatic sleep dynamics operating near the unstable boundaries of entrainment. This mechanism preserves the individual-level correlation but inverts the population-level skew from left to right. ConclusionsBehavioral chronotype emerges from the nonlinear interaction between fluctuating homeostatic sleep pressure and a hierarchically entrained circadian system. We caution against inferring the intrinsic circadian period solely from chronotype, particularly in clinical contexts where accurate assessment of circadian rhythmicity is critical for targeted therapies.

biophysics↗

bioRxiv2025

VN1K: a genome graph-based and function-driven multi-omics and phenomics resource for the Vietnamese population

Vietnam, the 16th most populated nation, remains profoundly underrepresented in global genomic databases. Here, we present VN1K, a first-ever comprehensive and well-curated resource of multi-omics data with a wide-range of phenotypic information of 1,011 unrelated Vietnamese individuals. High-depth short-read whole-genome sequencing data were generated for all samples along with various - omic data, including microarray, long-read whole-genome sequencing, and RNA sequencing. Using a high-sensitivity variant detection pipeline, which included a pangenome graph reference and a deep-learning framework, we identified nearly 40 million variants of which 8.5 million are novel with nearly 900 thousand short insertions/deletions and 39 thousand structural variants. Specifically, VN1K featured a first-ever whole-genome methylation profile based on long read sequencing. A genotype imputation panel was also created with the highest accuracy on the Vietnamese population. Variants with significantly different allele frequencies in the Vietnamese population compared to others were found to be functionally significant, especially in genes associated with immune diseases (HLA-B, KIR3DL3, KIR2DL1, KIR2DL4) or drug responses (CYP2C19, CYP2D6, VKORC1, CYP2B6). We were also able to map various loci related to hepatitis B virus infection as well as six disease traits, including triglyceride levels, LDL-C, serum glucose levels, HbA1c, and levels of two liver enzymes (ALT and AST). VN1K dataset is accessible via genome.vinbigdata.org, an integrated platform with both linear and graph-based genome browser for facilitating data exploration, research, and applications in precision medicine.

genomics↗

bioRxiv2024

Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical nonhomologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented various approaches to shift the repair pathway preference toward HR by using a dominant-negative KU80 mutant protein (KUDN) to disrupt the initiation of cNHEJ and enhance DSB end resection through nucleases. Our results show from 1.71- to 3.55-fold improvement of the GT efficiency at the callus stage and a more remarkable, up to 9.84-fold, increase in GT efficiency at two specific tomato loci, SlHKT1;2 and SlEPSPS1, when we screened transformants obtained from the KUDN-mediated cNHEJ suppression approach. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated GT at an additional locus, SlCAB13. These findings provide a promising method for more efficient and precise plant breeding in the future.

plant biology↗

bioRxiv2024

Breakthrough in Dicot Prime Editing: Enabling Heritable Desired Edits in Tomato and Arabidopsis

Prime editing (PE) enables almost all types of precise genome editing in animals and plants. It has been successfully adapted to edit several plants at variable efficiency and versatility. However, this technique is inefficient for dicots for unknown reasons. Here, by employing novel combinations of PE components, including an RNA chaperone and modified epegRNAs driven by a PolII-PolIII composite promoter and a viral replicon system, we obtained up to 9.7% of the desired PE efficiency at the callus stage assessed by targeted deep sequencing. Subsequently, we identified that up to 38.2% of transformants contained desired PE alleles in tomatoes and Arabidopsis, marking the first successful heritable PE transmission in dicots. Our PE tools also showed high accuracy, specificity, and multiplexing capability, which unlocked the potential for practical PE applications in dicots, paving the way for transformative advancements in plant sciences.

synthetic biology↗

bioRxiv2023

Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub

Integrin signaling plays important roles in development and disease. An adhesion signaling network called the integrin adhesome has been principally defined using bioinformatics and proteomics. To date, the adhesome has not been studied using integrated proteomic and genetic approaches. Here, proteomic studies in C. elegans identified physical associations between the RPM-1 ubiquitin ligase signaling hub and numerous adhesome components including Talin, Kindlin and beta-integrin. C. elegans RPM-1 is orthologous to human MYCBP2, a prominent player in nervous system development associated with a neurodevelopmental disorder. Using neuron-specific, CRISPR loss-of-function strategies, we show that core adhesome components affect axon development and interact genetically with RPM-1. Mechanistically, Talin opposes RPM-1 in a functional tug-of-war on growth cones that is required for accurate axon termination. Thus, our findings orthogonally validate the adhesome via multi-component genetic and physical interfaces with a key neuronal signaling hub and identify new links between the adhesome and brain disorders.

neuroscience↗

bioRxiv2023

Generation of Parthenocarpy tomato on multiple commercial cultivars utilizing CRIPR/Cas9 system

Tomato (Solanum lycopersicum L.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming the SlANT1 gene overexpression cassette and then later used to produce the SlIAA9 knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockout sliaa9 were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygous sliaa9 knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials.

plant biology↗

bioRxiv2022

Characterization of yield and fruit quality parameters of Vietnamese elite tomato lines generated through phenotypic selection and conventional breeding methods

Tomato (Solanum lycopersicum L.) is the second most important vegetable crop after potatoes, and global demands have been steadily increasing in recent years. Conventional breeding has been applied to breed and domesticate tomato varieties to meet the need for higher yield or superior agronomical traits that allow to sustain under different climatic conditions. In the current study, we applied bulk population breeding by crossing eight tomato accessions procured from the Asian Vegetable Research and Development Center (AVRDC) with three heat-resistant tomato inbred lines from Vietnam and generated ten elite tomato (ET) lines in the F8 generation. The individual F8 lines exhibited robust vigor and adaptability to Vietnamese climate conditions. Among the ten lines, ET1 and ET3 displayed indeterminate growth. ET2 showed semi-determinate, while all the other lines had determinate growth. The different ET lines showed distinctive superior agronomical traits, including early maturing (ET4, ET7, and ET10), highly efficient fruit set (ET1), higher yield (ET1, ET8, ET10), jointless pedicels (ET2), and partial parthenocarpy (ET9). Molecular analysis revealed that the ET3 line consisted of Ty-1 and Ty-3 loci that positively contribute to Tomato yellow leaf curl virus (TYCLV) resistance in tomato plants. The elite tomato lines developed in this study would contribute significantly to the Vietnamese and Asian gene pool for improved tomato production and may be a valuable resource for various breeding goals.

plant biology↗

bioRxiv2022

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control

There remains an urgent need for new therapies for drug-resistant epilepsy (DRE). Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bi-directional control of sodium channel expression are needed. MicroRNAs (miRNA) are small non-coding RNAs which negatively regulate gene expression. Here, we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the novel anti-seizure medicine cannabidiol (CBD) and plasma from patients with DRE, converge on a single target, miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking of miR-335-5p also increased voltage-gated sodium currents in human iPSC-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazole seizure model, whereas AAV9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect brain excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide new biomarker or therapeutic strategies for different types of drug-resistant epilepsy. Significance StatementDespite the clinical availability of over 30 anti-seizure medications (ASMs), around 30% of people with epilepsy do not achieve seizure freedom. MicroRNAs are small non-coding RNAs which negatively regulate protein expression by binding to target mRNAs. Here, we identified the brain-enriched miR-335-5p to be commonly altered in three heterogenous miRNA profiling datasets. Bi-directional modulation of miR-335-5p identified a potential homeostatic role of miR-335-5p in brain excitability involving voltage-gated sodium channels. Electrophysiological and in vivo approaches revealed pro-epileptic activity of miR-335-5p inhibition whereas overexpression of miR-335-5p resulted in anti-epileptic activity. Overall, targeting miR-335-5p could provide a new approach in the modulation of brain excitability, with possible therapeutic applications in drug-resistant epilepsies and other neurological diseases.

neuroscience↗

bioRxiv2022

CRISPR/Cas-based precision gene replacement in plants via homologous recombination-independent approaches

Precise gene or allele replacement is a desirable technology, but implementing it in plants remains challenging. CRISPR-Cas-based approaches, such as gene targeting (GT) and prime editing (PE), have opened up new possibilities for precise gene replacement in plants. However, their editing size and efficiency still need improvement. Recently, strategies using canonical nonhomologous end-joining (cNHEJ) and microhomology-mediated end joining (MMEJ) have been considered promising alternatives for precise gene replacement in yeast and mammals. However, these approaches have not been extensively explored and applied to plants. Here, we proposed and tested a CRISPR-Cas-based tool, termed PREMJ (precision gene replacement via microhomology-mediated end joining), for precision gene replacement in plants. The PREMJ strategy employing 20-bp microhomology MMEJ donors ([~]100 bp lengths) and a canonical nonhomologous end-joining (cNHEJ) inhibitor, NU7441, produced high targeted gene replacement efficiencies, up to 1.60 {+/-} 0.14, 4.47 {+/-} 1.98, and 8.98 {+/-} 4.73 % in protoplasts of tomato, lettuce, and cabbage, respectively. Our data also revealed the critical impacts of the microhomology length and NU7441 concentration on PREMJ-based precision gene editing in plants. Although obtaining edited plants remains challenging due to inefficient protoplast regenerations and Agrobacterium-mediated delivery, PREMJ may significantly contribute to precision gene editing in plants competent to PREMJ complex delivery and plant regeneration. Key MessageWe designed and tested a method, termed PREMJ, for precise gene replacement using the CRISPR-Cas-mediated double-stranded break (DSB) formation and repairing the DSBs via microhomology-mediated end joining (MMEJ) with MMEJ donor template carrying desired base changes and microhomologies flanking the DSB ends. PREMJ showed feasibility in precise gene replacement in protoplasts of tomatoes, lettuce, and cabbage, albeit its efficacy in Agrobacterium-mediated plant transformation requires further optimization.

synthetic biology↗

bioRxiv2022

Mucosal microbiomes and Fusobacterium genomics in Vietnamese colorectal cancer patients

Perturbations in the gut microbiome have been linked to the promotion and prognosis of colorectal cancer (CRC), with the colonic overabundance of Fusobacterium nucleatum shown as the most consistent marker. Despite the increasing health burden inflicted by CRC in low- and middle-income countries like Vietnam, the CRC-specific microbiome in these populations remains underexplored. Here we conducted a study in Vietnam to enrol 43 CRC patients (cases) and 25 patients with non-cancerous colorectal polyps (controls) between December 2018 and January 2020. Our study investigated the mucosal microbiome signature and genomic diversity of Fusobacterium in Vietnamese CRC patients, using a combination of 16S rRNA gene profiling, anaerobic microbiology, and whole genome sequencing. We found that several oral bacteria, including F. nucleatum and Leptotrichia, were significantly more abundant in the tumour mucosa, and these two bacteria were also more enriched in tumours of advanced CRC stages (III-IV). We obtained 53 Fusobacterium genomes from the saliva, tumour and non-tumour mucosa of six CRC patients. Isolates from the gut mucosa belonged to diverse F. nucleatum subspecies (nucleatum, animalis, vincentii, polymorphum) and a potential new subspecies of F. periodonticum. The Fusobacterium population within each individual was distinct and in many cases diverse, with minimal intra-clonal variation. Phylogenetic analyses showed that within each individual, tumour-associated Fusobacterium were clonal to those isolated from non-tumour mucosa, but distantly related to those isolated from saliva. Genes encoding major virulence factors (Fap2 and RadD) showed variability in length and evidence of horizontal gene transfer. Our work provides a framework to understand the genomic diversity of Fusobacterium within the CRC patients, which can be exploited for the development of CRC diagnostic and therapeutic options targeting this oncobacterium.

microbiology↗

bioRxiv2020

A novel method for measuring phenotypic colistin resistance in Escherichia coli populations from chicken flocks

Colistin is extensively used in animal production in many low- and middle-income countries. There is a need to develop methodologies to benchmark and monitor changes in resistance in commensal bacterial populations in farms. We aimed to evaluate the performance of a broth microdilution method based on culturing a pooled Escherichia coli suspension (30-50 organisms) from each sample. In order to confirm the biological basis and sensitivity of the method, we prepared 16 standard suspensions containing variable ratios of colistin-susceptible and mcr-1 encoded colistin-resistant E. coli which were grown in 2mg/L colistin. The optical density (OD600nm) readings over time were used to generate a growth curve, and were adjusted to the values obtained in the absence of colistin. The median limit of detection of the method was 1 colistin-resistant in 104 susceptible colonies [1st - 3rd quartile, 1:102 - 1:105]. We applied this method to 108 pooled faecal samples from 36 chicken flocks in the Mekong Delta (Vietnam) over the production cycle. The correlation between this method and the prevalence of colistin resistance in individual colonies harvested from field samples, determined by the Minimum Inhibitory Concentration (MIC), was established. The overall prevalence of colistin resistance at sample and isolate level was 38.9% and 19.4%, respectively. Increased colistin resistance was associated with recent (2 weeks) use of colistin and other, non-colistin antimicrobials (OR=3.67 and OR=1.84, respectively). Our method is a sensitive and affordable approach to monitor changes in colistin resistance in pooled E. coli populations from faecal samples over time. IMPORTANCEColistin (polymyxin E) is an antimicrobial with poor solubility properties, and therefore broth microdilution is the only appropriate method for testing colistin resistance. However, estimating colistin resistance in commensal mixed Escherichia coli populations is laborious since it requires individual colony isolation, identification and susceptibility testing. We developed a growth-based microdilution method suitable for pooled faecal samples. We validated the method by comparing it with results from individual MIC testing of 909 E. coli isolates. We used the method to investigate phenotypic colistin resistance in 108 pooled faecal samples from 36 healthy chicken flocks, each sampled three times over the production cycle. A higher level of resistance was seen in flocks recently supplemented with colistin in drinking water, although the observed generated resistance was short-lived. Our method is affordable, and may potentially be integrated into surveillance systems aiming at estimating the prevalence of resistance at colony level in flocks/herds. Furthermore, it may also be adapted to other complex biological systems, such as farms and abattoirs.

microbiology↗

bioRxiv2020

Glypican4 mediates Wnt transport between germ layers via signaling filopodia

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescues C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2, and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound Wnt5b and regulated formation of the filopodia that transport Wnt5b to neighboring cells. Blocking signaling filopodia that extend from endodermal cells suppressed this rescue. Thus, endodermal signaling filopodia that expressed GFP-Gpc4 transported Wnt5b, and likely Wnt11f2, to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

developmental biology↗

bioRxiv2020

Synergistic Effects of the DRD2/3 Antagonist ONC201 and Radiation in Glioblastoma

BackgroundGlioblastoma (GBM) is the deadliest of all brain cancers in adults. The current standard-of-care is surgery followed by radiotherapy and temozolomide, leading to a median survival time of only 15 months. GBM are organized hierarchically with a small number of glioma-initiating cells, responsible for therapy resistance and tumor recurrence, suggesting that targeting glioma-initiating cells could improve treatment response. ONC201 is a first-in-class anti-tumor agent with clinical efficacy in some forms of high-grade gliomas. Here we test its efficacy against GBM in combination with radiation. MethodsUsing patient-derived GBM lines and mouse models of GBM we test the effects of radiation and ONC201 on GBM self - renewal in vitro and survival in vivo. A possible resistance mechanism is investigated using RNA-Sequencing. ResultsTreatment of GBM cells with ONC201 reduced self-renewal, clonogenicity and cell viability in vitro. ONC201 exhibited anti-tumor effects on radioresistant GBM cells indicated by reduced self-renewal in secondary and tertiary glioma spheres. Combined treatment with ONC201 and radiation prolonged survival in syngeneic and patient-derived orthotopic xenograft mouse models of GBM. Subsequent transcriptome analyses after combined treatment revealed shifts in gene expression signatures related to quiescent GBM populations, GBM plasticity, and GBM stem cells. ConclusionsOur findings suggest that combined treatment with the DRD2/3 antagonist ONC201 and radiation improves the efficacy of radiation against GBM in vitro and in vivo through suppression of GICs without increasing toxicity in mouse models of GBM. A clinical assessment of this novel combination therapy against GBM is further warranted. Key points- Combined treatment of ONC201 and radiation exhibit anti-tumor effects on cells from primary and recurrent GBM - Combined treatment significantly prolongs survival in vivo - Combined treatment potentially targets the quiescent GBM cell population Importance of the StudyThe survival rates for patients with GBM are unacceptably low and novel treatment approaches are needed. This study provides evidence that a combination of radiation and the dopamine receptor antagonist ONC201 significantly prolongs survival in mouse models of glioma.

neuroscience↗

bioRxiv2020

Tumor Necrosis Factor Receptor Signaling Modulates Carcinogenesis in a Mouse Model of Breast Cancer

Proinflammatory conditions have long been associated with mammary carcinogenesis and breast cancer progression. The underlying mechanisms are incompletely understood but signaling of TNF through its receptors TNFR1 and TNFR2 is a major mediator of inflammation in both, obesity and in the response of tissues to radiation, two known risk factors for the development of breast cancer. Using the MMTV-Wnt1 mouse model for spontaneous breast cancer and knockout mice for TNFR1 and TNFR2 we report that loss of a TNFR2 allele leads to ductal hyperplasia in the mammary gland with increased numbers of mammary epithelial stem cell and terminal endbuds. Furthermore, that loss of one TNFR2 allele increases the incidence of breast cancers in MMTV-Wnt1 mice and results in tumors with a more aggressive phenotype and metastatic potential. The underlying mechanisms include a preferential activation of canonical NF-{kappa}B signaling pathways and autocrine production of TNF. Analysis of the TCGA dataset indicated inferior overall survival for patients with down-regulated TNFR2 expression. We conclude, that imbalances in TNFR signaling promote the development and progression of breast cancer, indicating that selective agonists of TNFR2 could potentially modulate the risk for breast cancer in high-risk populations. Significance StatementOver the past four decades the treatment results for patients suffering from breast cancer have constantly improved, leaving breast cancer prevention as an important frontier against the second leading cause of cancer death in the United States. Obesity has become a national health crisis and is a known proinflammatory risk factor for breast cancer. Our study describes a previously unknown aspect of proinflammatory signaling on breast cancer development and progression, thus suggesting novel potential targets to modulate the incidence of the disease.

cancer biology↗

bioRxiv2020

1--4-phenylpiperazine Treatment After Brain Irradiation Preserves Cognitive Function in Mice

BackgroundNormal tissue toxicity to the CNS is an inevitable consequence of a successful radiotherapy of brain tumors or cancer metastases to the CNS. Cranial irradiation commonly leads to neurocognitive deficits that manifest months or years after treatment. Mechanistically, radiation-induced loss of neural stem/progenitor cells, neuro-inflammation and de-myelinization are contributing factors that lead to progressive cognitive decline. MethodsThe effects of Compound #5 on irradiated murine neurospheres, microglia cells and patients-derived gliomaspheres were assessed in sphere-formation assays, flow cytometry and IL-6 ELISAs, Activation of the Hedgehog pathway was studied by qRT-PCR. The in vivo effects of Compound #5 were analyzed using flow cytometry, sphere-formation assays, immune-histochemistry, behavioral testing and an intracranial mouse model of glioblastoma. ResultsWe report that 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) mitigates radiation-induced normal tissue toxicity in the brains of mice. Compound #5 treatment significantly increased the number of neural stem/progenitor cells after brain irradiation in female animals, inhibited radiation-induced microglia activation and expression of the pro-inflammatory cytokine interleukin-6. Behavioral testing revealed that treatment with Compound #5 after radiotherapy successfully mitigates radiation-induced decline in motor, sensory and memory function of the brain. In mouse models of glioblastoma, Compound #5 showed no toxicity and did not interfere with the growth-delaying effects of radiation. ConclusionsWe conclude that Compound #5 has the potential to mitigate cognitive decline in patients undergoing partial or whole brain irradiation without promoting tumor growth and that the use of this compound as a radiation mitigator of radiation late effects on the CNS warrants further investigation. Importance of the StudySuccessful radiotherapy of CNS malignancies inevitably lead to cognitive decline in cancer survivors and treatment options to mitigate this side effect are limited. We present evidence that a piperazine compound can prevent cognitive decline in mice after total brain irradiation without compromising the antitumor effect of radiation, suggesting that this compound could be used to mitigate radiation side effects in brain tumor patients undergoing radiotherapy.

neuroscience↗

bioRxiv2019

Marker subset selection and decision support range identification for acute myeloid leukemia classification model development with multiparameter flow cytometry

In this study, we developed acute myeloid leukemia (AML) classification model through Wilks lambda-based important marker-identification method and stepwise-forward selection approach, and spotted important decision-support range of flow-cytometry parameter using insights provided by machine-learning algorithm. AML flow-cytometry data released from FlowCAP-II challenge in 2011 was used. In FlowCAP-II challenge, several sample classification algorithms were able to effectively classify AML and non-AML. Most algorithms extracted features from high-dimensional flow-cytometry readout comprised of multiple fluorescent parameters for a large number of antibodies. Multiple parameters with forward scatter and side scatter increase computational complexity in the feature-extraction procedure as well as in the model development. Parameter-subset selection can decrease model complexity, improve model performance, and contribute to a panel design specific for target disease. With this motivation, we estimated importance of each parameter via Wilks lambda and then identified the best subset of parameters using stepwise-forward selection. In the importance-estimation process, histogram matrix of each parameter was used. As a result, parameters, which are associated with blasts gating and identification of immature myeloid cells, were identified as important descriptors in AML classification, and combination of these markers is more effective than an individual marker. A random-forest, supervised-classification machine-learning algorithm was used for the model development. We highlighted decision-support range of the fluorescent signal for the identified important parameters, which significantly contribute to AML classification, through a mean decrease in Gini supported in random forest. These specific ranges could help with establishing diagnosis criteria and elaborate the AML classification model. Because methodology proposed in this study can not only estimate the importance of each parameter but also identify the best subset and the specific ranges, we expect that it would contribute to in silico modeling using flow- and mass-cytometry readout as well as panel design for sample classification.\n\nAuthor summaryFlow cytometry is a widely used technique to analyze multiple physical characteristics of an individual cell and diagnose and monitor human disease as well as response to therapy. Recent developments in hardware (multiple lasers and fluorescence detectors), fluorochromes, and antibodies have facilitated the comprehensive and in-depth analysis of high numbers of cells on a single cell level and led to the creation of various computational analysis methods for cell type identification, rare cell identification, and sample classification. Flow cytometry typically uses panels with a large number of antibodies, leading to high-dimensional multiparameter flow cytometry readout. It increases computational complexity and makes interpretation difficult. In this study, we identified the best subset of the parameters for AML classification model development. The subset would contribute to panel design specific for the target disease and lead to easy interpretation of the results. In addition, we spotted important decision-support range of flow-cytometry parameter via insights provided by machine-learning algorithm. We expect that profiling information of fluorescence expression over the identified decision-support range would complement existing diagnosis criteria.

bioinformatics↗

bioRxiv2018

In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1-5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8-12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

molecular biology↗