Biology subjects

Tian, T.

Publications and source records attributed to Tian, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Neuregulin-1 Attenuates Myocardial Ischemia/Reperfusion Injury by Activating the UCP2/PINK1/LC3B-mediated Mitophagy

BACKGROUNDIschemia/reperfusion (I/R) injury may significantly affect the treatment outcomes and prognosis of patients with acute myocardial infarction following coronary artery recanalization. Available evidence suggests that neuregulin-1 (NRG-1) can provide a protection against myocardial I/R injury and is involved in various cardioprotective interventions by potential regulation of mitophagy. However, the molecular mechanisms linking NRG-1 and mitophagy remain to be clarified. This experiment aimed to determine whether NRG-1 postconditioning attenuated myocardial I/R injury through the regulation of mitophagy and to explore the underlying mechanisms. METHODBoth an in vivo myocardial I/R injury model of rats and an in vitro hypoxia/reoxygenation (H/R) model of H9C2 cardiomyocytes were applied. NRG-1 treatment was conducted immediately after I/R or H/R intervention. In the in vivo experiment, cardioprotective effects of NRG-1 were determined by infarct size, cardiac enzyme and histopathologic examinations. The potential downstream pathways and molecular targets of NRG-1 were screened by the RNA sequencing and the Protein-Protein Interaction Networks (PPI). The expression levels of mitochondrial uncoupling protein 2 (UCP2) and mitophagy-related protein in both the I/R myocardium and H/R cardiomyocytes were measured by immunofluorescence staining and Western blots. The activation of mitophagy was observed with the transmission electron microscopy (TEM) and JC-1 staining. RESULTSThe KEGG and GSEA analyses showed that the mitophagy-related pathways were enriched in the I/R myocardium treated with NRG-1, and UCP2 exhibited a significant correlation between mitophagy and interaction with PINK1. Meanwhile, the treatment with mitophagy inhibitor Mdivi-1 significant eliminated the cardioprotective effects of NRG-1 postconditioning in vivo, and the challenge with UCP2 inhibitor genipin could also attenuate the activating effect of NRG-1 postconditioning on mitophagy. Consistently, the in vitro experiment using H9C2 cardiomyocytes showd that NRG-1 treatment significantly up-regulated the expression levels of UCP2 and mitophagy-related proteins, and activated the mitophagy, whereas the challenge with small interfering RNA (siRNA)-mediated UCP2 knockdown abolished the effects of NRG-1. CONCLUSIONSNRG-1 postconditioning can produce a protection against the myocardial I/R injury by activating mitophagy through the UCP2/PINK1/LC3B signaling pathway.

molecular biology↗

bioRxiv2024

A Comprehensive Proteogenomic and Spatial Analysis of Innate and Acquired Resistance of Metastatic Melanoma to Immune Checkpoint Blockade Therapies

While a subset of patients with metastatic melanoma achieves durable responses to immune checkpoint blockade (ICB) therapies, the majority ultimately exhibit either innate or acquired resistance to these treatments. However, the molecular mechanisms underlying resistance to ICB therapies remain elusive and are warranted to elucidate. Here, we comprehensively investigated the tumor and tumor immune microenvironment (TIME) of paired pre- and post-treatment tumor specimens from metastatic melanoma patients who were primary or secondary resistance to anti-CTLA-4 and/or anti-PD-1/PD-L1 therapies. Differentially expressed gene (DEG) analysis and single-sample gene set enrichment analysis (ssGSEA) with transcriptomic data identified cell cycle and c-MYC signaling as pathway-based resistance signatures. And weighted gene co-expression network analysis (WGCNA) revealed the activation of a cross-resistance meta-program involving key signaling pathways related to tumor progression in ICB resistant melanoma. Moreover, spatially-resolved, image-based immune monitoring analysis by using NanoStrings digital spatial profiling (DSP) and Cyclic Immunofluorescence (CyCIF) showed infiltration of suppressive immune cells in the tumor microenvironment of melanoma with resistance to ICB therapies. Our study reveals the molecular mechanisms underlying resistance to ICB therapies in patients with metastatic melanoma by conducting such integrated analyses of multi-dimensional data, and provides rationale for salvage therapies that will potentially overcome resistance to ICB therapies. Statement of translational relevanceThis study paves the way for the creation of innovative therapeutic strategies, aimed at subverting resistance to immune checkpoint blockade (ICB) therapies in metastatic melanoma patients. By unraveling the specific molecular mechanisms underlying resistance, scientists can design effective alternative treatments that target pathways such as pathways associated with cell cycle dysregulation and c-MYC signaling. Furthermore, through the application of advanced immune monitoring techniques such as NanoString Digital Spatial Profiling (DSP) and Cyclic Immunofluorescence (CyCIF), this study has significantly enriched our understanding of the tumor microenvironment. This enhanced characterization facilitates the discovery of potential biomarkers that may forecast a patients response to ICB treatment. Ultimately, these advancements could potentially refine patient outcomes and foster the development of more personalized cancer treatments in the future.

genetics↗

bioRxiv2024

Responses of root architectural and anatomical traits to low nitrogen stress in rice

Improving nitrogen use efficiency in rice would provide economic and environmental benefits, but little is known about root morphological and anatomical responses to low nitrogen. In this study, two sets of rice genotypes, one set from the RDP1 panel, and one set of recombinant inbred lines, were used to characterize responses to gradual nitrogen depletion by plant uptake and movement of nitrogen to deeper soil strata as a result of leaching, so that more nitrogen was available at depth in aerobic soil mesocosms in a greenhouse. There was significant genetic variation in shoot biomass reductions in response to low nitrogen. The root to shoot biomass ratio was increased by low nitrogen in both sets of genotypes. Relative investment in nodal root number was accentuated with low nitrogen, and shoot biomass was correlated with numbers and lengths of nodal and large lateral roots. There was genetic variation for nodal root number and length in both sets of genotypes. Anatomical responses to low nitrogen were assessed in nodal roots of the RILs, where root cross-sectional area, stele area, and metaxylem vessel number were reduced by low nitrogen, and root diameter was reduced in the RDP1 genotypes. There were significant interactions of nitrogen with genotype for stele area and percent aerenchyma in the RILs. Genetic variation for low nitrogen responses may be useful for selection of rice lines with greater nitrogen acquisition under nitrogen-leaching conditions.

plant biology↗

bioRxiv2024

The clinical antiprotozoal drug halofuginone promotes weight loss by elevating GDF15 and FGF21

Obesity is a debilitating disease with increasing worldwide prevalence. Despite its high prevalence, specific pharmacologic intervention for obesity is challenging. Here, we report that halofuginone, an FDA-approved anti-scleroderma and anti-protozoal drug, is a promising anti-obesity agent in rodent models. Halofuginone suppressed food intake, increased energy expenditure, and resulted in weight loss in preclinical diet-induced obese mouse models, while also decreasing insulin resistance and hepatic steatosis. By combining genetic and pharmacological tools with transcriptomics, we identified that halofuginone increases FGF21 and GDF15 levels via ATF4. Using knockout mice, we show these hormones are both necessary for its anti-obesity effects. Thus, our study first reports the beneficial metabolic effects of halofuginone and underscores its utility to treat obesity and its associated metabolic complications.

pharmacology and toxicology↗

bioRxiv2023

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge, while incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to 7 genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expressions of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/L, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased FAD biosynthesis, enhanced flux entering the TCA cycle and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories. Impact StatementIn this study, yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and successfully incorporating modules for large-scale disulfide bond formation. KmYACs were stably propagated in K. marxianus without compromising the normal growth of the host, irrespective of the selection of telomeres (either Tetrahymena or K. marxianus) and configuration (either linear or circular). KmYACs notably enhanced the expressions of various heterologous proteins, with further yield improvement by supplementing lysine or arginine in the medium. Our findings affirm KmYAC as a robust and versatile platform for transferring large-scale function modules, demonstrating immense potential for both industrial applications and fundamental research.

synthetic biology↗

bioRxiv2023

Resident memory T cell development is associated with AP-1 transcription factor upregulation across anatomical niches.

Tissue-resident memory T (TRM) cells play a central role in immune responses to pathogens across all barrier tissues after infection. However, the underlying mechanisms that drive TRM differentiation and priming for their recall effector function remains unclear. In this study, we leveraged both newly generated and publicly available single-cell RNA-sequencing (scRNAseq) data generated across 10 developmental time points to define features of CD8 TRM across both skin and small-intestine intraepithelial lymphocytes (siIEL). We employed linear modeling to capture temporally-associated gene programs that increase their expression levels in T cell subsets transitioning from an effector to a memory T cell state. In addition to capturing tissue-specific gene programs, we defined a consensus TRM signature of 60 genes across skin and siIEL that can effectively distinguish TRM from circulating T cell populations, providing a more specific TRM signature than what was previously generated by comparing bulk TRM to naive or non-tissue resident memory populations. This updated TRM signature included the AP-1 transcription factor family members Fos, Fosb and Fosl2. Moreover, ATACseq analysis detected an enrichment of AP-1-specific motifs at open chromatin sites in mature TRM. CyCIF tissue imaging detected nuclear co-localization of AP-1 members Fosb and Junb in resting CD8 TRM >100 days post-infection. Taken together, these results reveal a critical role of AP-1 transcription factor members in TRM biology and suggests a novel mechanism for rapid reactivation of resting TRM in tissue upon antigen encounter.

immunology↗

bioRxiv2023

Long-term stress shapes dynamic reconfiguration of functional brain networks across multi-task demands

Exposure to sustained stress can have a profound impact on the brain, emotion and cognition, with either adaptive or maladaptive effects. Human functional brain networks are dynamically organized to enable rapid and flexible adaptation to meet ever-changing task demands. Yet, little is known about how long-term stress alters the dynamic reconfiguration of functional brain networks across multi-task demands. Here we show prominent changes in the dynamic reconfiguration of large-scale brain networks during resting-state, emotional and working-memory processing under long-term stress. Hidden Markov Model analysis detected several latent brain states and switching processes involving the default mode, emotional salience and executive-control networks that are dominant to rest, emotion and working memory, respectively. Critically, long-term stress increased persistent time on brain states relevant to goal-directed demands and cognitive control, with more frequent transitions to these brain states when compared to controls. Furthermore, long-term stress led to higher correlations of the occupancy and persistency of brain states linked to psychological distress and behavioral performance. Our findings provide a neurocognitive framework whereby long-term stress shapes the way the brain adapts to varying task demands and increases the sensitivity of functional brain networks to psychological and behavioral responses. These changes can be both adaptive and maladaptive, reflecting the complex effects of long-term stress on brain function.

neuroscience↗

bioRxiv2023

Positive coping supports children's emotional wellness: Behavioral evidence and neuroendocrine mechanisms

A positive coping style is recognized as a stable disposition to foster emotional wellness and resilience, enabling an adaptive process of assessing and dealing with environmental challenges. Such an adaptive process is believed to rely on a nuanced interplay of the hippocampal system and the primary stress hormone cortisol activity. As a hallmark of diurnal cortisol rhythm, cortisol awakening response (CAR) is sensitive to upcoming stress and subserves the preparation of the hippocampal system for rapid behavioral adaption. Yet, little is known about how the hippocampal system and CAR contribute to the merit of positive coping on emotional wellness. By two studies, we investigate the effects of positive coping on childrens emotional wellness and CAR, as well as longitudinal changes in hippocampal-neocortical functional systems involved in emotional processing. Behaviorally, positive coping predicted better emotional regulation ability, but lower anxiety and lower response caution in emotional decision-making. At the endocrine and neurocognitive level, positive coping was associated with greater CAR, which further predicted higher connectivity of the hippocampus with ventrolateral prefrontal cortex (vlPFC) and stimulus-sensitive neocortex one year later. Furthermore, CAR mediated an indirect association between positive coping and longitudinal increases in hippocampal-neocortical connectivity. Positive coping and CAR together could account for the maturity of vlPFC through longitudinal changes in hippocampal-neocortical connectivity. Overall, our findings suggest a cognitive-neuroendocrinal framework in which positive coping shapes hippocampal-neocortical maturation via stress hormone response to support emotional wellness. SignificanceThe role of the hippocampal system in regulating stress response is well recognized, but its contribution to emotional well-being is not yet understood. Here we show that the protective effects of positive coping on emotional well-being are contingent on two factors: the cortisol awakening response (CAR), which is sensitive to upcoming stress, and hippocampal development. We found that positive coping practices promoted emotional wellness, enhanced emotional decision-making and increased CAR in young children. Longitudinal neuroimaging analysis revealed that positive coping-related CAR predicted greater hippocampal connectivity with stimulus-sensitive neocortex one year later. Importantly, CAR acted as a mediator of the promotive influence of positive coping on the longitudinal development of hippocampal-neocortical connectivity, which contributed to the maturity of prefrontal control systems. Our findings emphasize the importance of hippocampal-neocortical development in resilient coping and emotional wellness.

neuroscience↗

bioRxiv2023

Conditioning electrical stimulation fails to enhance sympathetic axon regeneration

Peripheral nerve injuries are common, and there is a critical need for the development of novel treatments to complement surgical repair. Conditioning electrical stimulation (CES) is a novel variation of the well-studied perioperative electrical stimulation treatment paradigm. CES is a clinically attractive alternative because of its ability to be performed at the bedside prior to a scheduled nerve repair surgery. Although 60 minutes of CES has been shown to enhance motor and sensory axon regeneration, the effects of CES on sympathetic regeneration are unknown. We investigated how two clinically relevant CES paradigms (10 minutes and 60 minutes) impact sympathetic axon regeneration and distal target reinnervation. Our results indicate that the growth of sympathetic axons is inhibited by CES at acute time points, and at a longer survival time point post-injury, there is no difference between sham CES and the CES groups. We conclude sympathetic axons may retain some regenerative ability, but no enhancement is exhibited after CES, which may be accounted for by the inability of the electrical stimulation paradigm to recruit the small-caliber sympathetic axons into activity. Furthermore, 10-minute CES did not enhance motor and sensory regeneration with a direct repair, and neither 60-minute nor 10-minute CES enhanced motor and sensory regeneration through a graft. Further studies will be needed to optimize electrical stimulation parameters to enhance the regeneration of all neuron types.

neuroscience↗

bioRxiv2023

The ThCre:mTmG mouse has sparse expression in the sympathetic nervous system

The global double-fluorescent cre-dependent reporter mouse mT/mG has been a valuable tool for differential identification of tissues and cells. This line was previously shown to have EGFP expression in a cre-dependent fashion in single neurons. Here, we demonstrate very sparse labeling in a ThCre:mT/mG cross. This is evident in sweat glands, thoracic spinal cord, sciatic nerve, and lumbar sympathetic ganglia. Therefore, this model may be suitable for imaging or electrophysiology studies of postganglionic sympathetic neuron cell bodies but ineffective for visualizing peripheral axons and tissue targets of the sympathetic nervous system.

neuroscience↗

bioRxiv2022

Reducing the Complexity of Fc Glycan Enables the Construction of Antibody Conjugates with Unexpected High Efficiency and Payload Capacity via Glycoengineering

Glycoengineering has been demonstrated to be a powerful tool to construct site-specific antibody conjugates. However, with most glycoengineering strategies, several hours to days are needed to complete the reaction, and the payloads are limited to small molecules. Herein, we show that reducing the complexity of Fc glycan could dramatically boost the enzymatic glycoengineering of IgG molecules to achieve unexpectedly high efficiency and payload capacity. Notably, antibodies with the modified Fc glycan were successfully conjugated with biomacromolecules, e.g., oligonucleotides and nanobodies, in one step by an engineered fucosyltransferase. Moreover, the conversion of this chemoenzymatic engineering could be finished in minutes, or up to hours, affording homogeneous products. The trisaccharide linker of these antibody conjugates exhibits excellent hydrophilicity and stability and reduced ADCC activity. Using this platform, we successfully synthesized an antibody-conjugate-based HER2/CD3 bispecific antibody and applied it to selectively destroy patient-derived cancer organoids by reactivating endogenous T cells inside the organoid. These results suggest that this bispecific antibody format has a high translational value.

biochemistry↗

bioRxiv2022

Chemoenzymatic Measurement of Cell-surface Glycan in Single-cell Multiomics: LacNAc as an Example

Despite the rich information of a cells physiological state encoded in the dynamic changes of cell-surface glycans, methods of capturing glycosylation states at the single-cell level are quite limited. Here we report a chemoenzymatic single-cell N-acetyllactosamine (LacNAc) detection method via tagging the LacNAc with a specific DNA barcode. Compared to the lectin-based glycan detection, the chemoenzymatic labeling does not change the transcriptional status of immune cells and is more compatible with scRNA-seq. Integrated analysis of LacNAc and transcriptome of T cells at a single-cell level reveals that the quantity of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at the basal level in quiescent CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that the LacNAc level is positively correlated to the glycolytic activity of CD8+ T cells at all statues. Taken together, our study demonstrates the feasibility of chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with information of TCR sequence and cell-surface epitopes (i.e., scTCR and CITE-seq) and offers a new way to characterize the biological role of glycan in diversified physiological states.

immunology↗

bioRxiv2022

Sensitive bacterial Vm sensors revealed the excitability of bacterial Vm and its role in antibiotic tolerance

As an important free energy source, the membrane voltage (Vm) regulates many essential physiological processes in bacteria. However, in comparison with eukaryotic cells, knowledge of bacterial electrophysiology is very limited. Here, we developed a set of novel genetically encoded bacterial Vm sensors which allow single-cell recording of bacterial Vm dynamics in live cells with high temporal resolution. Using these new sensors, we reveal the electrically "excitable" and "resting" states of bacterial cells dependent on their metabolic status. In the electrically excitable state, frequent hyperpolarization spikes in bacterial Vm are observed, which facilitates increased antibiotic tolerance. In the electrically resting state, bacterial Vm displays significant cell-to-cell heterogeneity and is linked to the cell fate after antibiotic treatment.

microbiology↗

bioRxiv2022

Single photon kilohertz frame rate imaging of neural activity

Establishing the biological basis of cognition and its disorders will require high precision spatiotemporal measurements of neural activity. Recently developed genetically encoded voltage indicators (GEVIs) report both spiking and subthreshold activity of identified neurons. However, maximally capitalising on the potential of GEVIs will require imaging at the millisecond time scales, which remains challenging with standard camera systems. Here we report application of single photon avalanche diode (SPAD) sensors to imaging neural activity at kilohertz frame rates. SPADs are electronic devices that when activated by a single photon cause an avalanche of electrons and a large electric current. We use an array of SPAD sensors to image individual neurons expressing genetically encoded voltage indicators. We show that subthreshold and spiking activity can be resolved with shot noise limited signals at frame rates of up to 10 kHz. SPAD imaging was able to reveal millisecond scale synchronisation of neural activity in an ex-vivo seizure model. SPAD sensors may have widespread applications for investigation of millisecond timescale neural dynamics. Table of contentsThe high temporal precision of single photon avalanche diodes (SPADs) is leveraged to record neural activity reported by genetically encoded voltage indicators. Sub-threshold and spiking activity of single neurons was resolved with shot noise limited signals at frame rates of up to 10 kHz. SPAD sensors may have widespread applications for neural imaging at high frame rates. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

neuroscience↗

bioRxiv2022

Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling

Vector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKA), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive. Here we show that TBEV structural proteins, including pre-membrane (prM), envelope, and capsid proteins, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeded the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKA and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKA prM significantly promoted the replication of Sendai virus. Our findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development. Author SummaryAll flaviviruses must overcome the antiviral innate immunity to infect vertebrate host. The non-structural proteins of flaviviruses are mainly responsible for viral replication and host innate immune escape, and the structural proteins are involved in the virus assembly, which are potential targets for prevention and treatment of flaviviral infections. Whether flavivirus structural proteins participate in host innate immune escape remains to be determined. Here, we found that tick-borne encephalitis virus structural proteins precursor membrane (prM), capsid, and envelope proteins can antagonize type I interferon production, in which prM interacts with both MDA5 and MAVS to inhibit RLR antiviral signaling. Additionally, Zika virus and West Nile virus prMs also interact with both MDA5 and MAVS, while dengue virus serotype 2 and yellow fever virus prMs associate only with MDA5 or MAVS to suppress type I interferon production. In contrast, Japanese encephalitis virus prM cannot antagonize type I interferon production. Our findings reveal that flavivirus prM inhibits type I interferon production via interacting with MDA5 and/or MAVS in a species-dependent manner, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention, and vaccine development.

microbiology↗

bioRxiv2021

Angiogenesis is critical for the regenerative effects of exercise.

Summary AbstractEnhancing axon regeneration is a major focus of nerve injury research, and the quality of the surgical nerve repair plays a large role in the aggregate success of nerve regeneration. Additionally, exercise is known to promote successful axon regeneration after surgical nerve repair. In this study, we asked how exercise-induced nerve regeneration is affected when a transected nerve is repaired with or without fibrin glue. Fibrin glue repaired nerves exhibited greater vasculature within the tissue bridge compared to nerves that were intrinsically repaired. Fibrin glue repaired nerves also exhibited more robust axon regeneration after exercise compared to nerves that were not repaired with fibrin glue. When angiogenesis of the tissue bridge was prevented, exercise was unable to enhance regeneration despite the presence of fibrin glue. These findings suggest that the biological properties of fibrin glue enhance angiogenesis within the repair site, and a vascularized bridge is required for enhanced axon elongation with exercise. The combination of fibrin glue repair and exercise resulted in notable differences in vascular growth, axon elongation, neuromuscular junction reinnervation, and functional recovery. Fibrin glue should be considered as an adjuvant for nerve repair to enhance the subsequent efficacy of activity- and physical therapy-based treatment interventions.

cell biology↗

bioRxiv2021

A framework for evaluating edited cell libraries created by massively parallel genome engineering

Genome engineering methodologies are transforming biological research and discovery. Approaches based on CRISPR technology have been broadly adopted and there is growing interest in the generation of massively parallel edited cell libraries. Comparing the libraries generated by these varying approaches is challenging and researchers lack a common framework for defining and assessing the characteristics of these libraries. Here we describe a framework for evaluating massively parallel libraries of edited genomes based on established methods for sampling complex populations. We define specific attributes and metrics that are informative for describing a complex cell library and provide examples for estimating these values. We also connect this analysis to generic phenotyping approaches, using either pooled (typically via a selection assay) or isolate (often referred to as screening) phenotyping approaches. We approach this from the context of creating massively parallel, precisely edited libraries with one edit per cell, though the approach holds for other types of modifications, including libraries containing multiple edits per cell (combinatorial editing). This framework is a critical component for evaluating and comparing new technologies as well as understanding how a massively parallel edited cell library will perform in a given phenotyping approach.

genomics↗

bioRxiv2021

Ultrafast, one-step, and microwave heating-based synthesis of DNA/RNA-AuNP conjugates

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here we discovered that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies showed that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent. Spectroscopy, test strip hybridization, and loading counting experiments indicate that low-affinity poly (T/U) tag mediates the formation of a standing-up conformation, which is distributed in the outer layer of such a SNA structure. In further applications study, CRISPR/Cas9-sgRNA (135 bp), RNA from Nucleocapsid (N) gene of SARS-CoV-2 (1279 bp), and rolling circle amplification (RCA) DNA products (over 1000 bp) could be successfully attached on AuNPs, which overcomes the routine methods in long-chain nucleic acid-AuNP conjugation, exhibiting great promise in novel biosensing and nucleic acids delivery strategy. This novel heating-dry strategy has improved the traditional DNA/RNA-AuNP conjugation methods in simplicity, rapidity, cost, and universality.

biochemistry↗