Biology subjects

Dong, L.

Publications and source records attributed to Dong, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Dissecting organoid-bacteria interaction highlights decreased contractile force as a key factor for heart infection

Bacterial endocarditis is a fatal cardiovascular disease exacerbated by weakened heart contraction, yet the direct impact of cardiac contractility on bacterial adhesion remains elusive. Here, we present a novel quantitative physics model integrating finite element analysis and live-cell imaging to uncover their strong correlation. Using this model, we quantified the real-time force magnitude generated by organoid-type cardiac microtissue derived from healthy donors and dilated cardiomyopathy patients - mimicking normal and suppressed heart contractility, respectively - to the approaching bacteria in a real fluidic system. The data revealed that weakened cardiac contractility facilitated bacterial invasion of the myocardium. Verifying this finding in a mouse transverse aortic constriction model demonstrated that increasing heart contraction efficiently mitigated bacterial invasion, with a 25% increase in heart contractility reducing endocarditis risk by 80%. Our findings demonstrate that patient-derived cardiac organoids provide a physiologically relevant platform for studying bacterial infections in vitro, offering high clinical fidelity. This platform establishes a valuable tool for drug screening and the development of novel therapeutic strategies.

bioengineering↗

bioRxiv2025

The GSA Family in 2025: A Broadened Sharing Platform for Multi-Omics and Multimodal Data

The Genome Sequence Archive Family (GSA family) provides a suite of database resources for archiving, retrieving, and sharing multi-omics data for the global academic and industrial communities. It currently comprises four distinct database members: the Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/), the Genome Sequence Archive for Human (GSA-Human, https://ngdc.cncb.ac.cn/gsa-human/), the open Archive for Miscellaneous Data (OMIX, https://ngdc.cncb.ac.cn/omix/), and the Open Biomedical Imaging Archive (OBIA, https://ngdc.cncb.ac.cn/obia). Compared to the 2021 version, the GSA family has significantly expanded by introducing a new database member, the OBIA, and through comprehensive upgrades to the existing ones. Notable enhancements to the existing members include broadening the range of accepted data types, enhancing the quality control systems, fine-tuning the data retrieval system, and refining the data-sharing management mechanisms.

genomics↗

bioRxiv2025

A Telomere-to-Telomere Diploid Reference Genome and Centromere Structure of the Chinese Quartet

Recent advances in sequencing technologies have enabled the complete assembly of human genomes from telomere to telomere (T2T), resolving previously inaccessible regions such as centromeres and segmental duplications. Here, we present an updated, higher-quality, haplotype-phased T2T assembly of the Chinese Quartet (T2T-CQ), a family cohort comprising monozygotic twins and their parents, generated using high-coverage ONT ultralong and PacBio HiFi sequencing. The T2T-CQ assembly serves as a crucial reference genome for integrating publicly available multi-omics data and advances the utility of the Quartet reference materials. The T2T-CQ assembly scores highly on multiple metrics of continuity and completeness, with Genome Continuity Inspector (GCI) scores of 77.76 (maternal) and 76.41 (paternal), quality values (QV) > 70, and Clipping Reveals Assembly Quality (CRAQ) scores > 99.6 for both haplotypes, enabling complete annotation of centromeric regions. Within these regions, we identified novel 13-mer higher-order repeat patterns on chromosome 17 which exhibited a monophyletic origin and emerged approximately 230 thousand years ago. Overall, this work establishes an essential genomic resource for the Han Chinese population and advances the development of a T2T pan-Chinese reference genome, which will significantly enable future investigations both into population-specific structural variants and the evolutionary dynamics of centromeres.

genomics↗

bioRxiv2025

Restoration of tumor suppressor protein with enhanced activity using engineered tRNAs to induce tumor regression

Cancer develops through a gradual accumulation of mutations including nonsense mutations in tumor suppressor genes (TSGs), which drive tumor proliferation and progression due to the impairment or complete loss of tumor suppressor protein function. The ability to restore functional tumor suppressor protein from mutant driver TSGs is a longstanding aspiration in cancer therapy, however, current strategies face formidable challenges in recovering proteins harboring heterogeneous mutations and circumventing dominant-negative effects exerted by endogenous mutant proteins. Therefore, the development of therapeutic interventions that are effective and generalizable across diverse nonsense mutations in cancer, to rescue endogenous mutant tumor suppressor proteins remains an urgent yet unmet need. Here we find that engineered suppressor transfer RNA (sup-tRNA) could overcome nonsense mutations in key driver tumor suppressor genes, such as TP53, thereby avoiding the dominant-negative effects through the restoration of endogenous mutant p53 with its original functions. Optimized sup-tRNA that we named super tRNA, enabling high-fidelity incorporation of desired amino acids, can further enhanced the transcriptional activity of p53 to suppress tumor cell proliferation. Moreover, we also establish a computational model to guide super tRNAs design, and demonstrate that super tRNA could effectively inhibit tumor growth by targeted readthrough of nonsense mutations in different tumor suppressor genes. In two xenograft models harboring nonsense mutant TP53, we observed up to a 70% reduction in tumor volume after lipid nanoparticle (LNP)-based sup-tRNA treatment, along with robust recovery of p53 expression and function, and no hematological or histological abnormalities detected. Our findings demonstrate the potential of sup-tRNA as a feasible and effective strategy to rescue and enhance tumor suppressor protein function, thereby preventing cancer development. As a novel therapeutic RNA, sup-tRNA provides a flexible strategy for targeting driver mutations in cancer, thereby expanding the potential of tRNA-based therapy in precision oncology.

bioengineering↗

bioRxiv2024

ALDH3A1-mediated detoxification of reactive aldehydes contributes to distinct muscle responses to denervation and Amyotrophic Lateral Sclerosis progression

Different muscles exhibit varied susceptibility to degeneration in Amyotrophic Lateral Sclerosis (ALS), a fatal neuromuscular disorder. Extraocular muscles (EOMs) are particularly resistant to ALS progression, and exploring the underlying molecular nature may offer significant therapeutic value. Reactive aldehyde 4-hydroxynonenal (HNE) is implicated in ALS pathogenesis, and Aldh3a1 is an inactivation-resistant intracellular aldehyde dehydrogenase that detoxifies 4-HNE to protect eyes against UV-induced oxidative stress. We detected prominently higher levels of Aldh3a1 in mouse EOMs compared to other muscles under normal physiological conditions. In an ALS mouse model (hSOD1G93A) reaching end-stage, Aldh3a1 expression was maintained high in EOMs, substantially elevated in soleus and diaphragm, but only moderately increased in extensor digitorum longus (EDL) muscle, which endured the most severe pathological remodeling, as demonstrated by unparalleled upregulation of a denervation marker Ankrd1. Importantly, sciatic nerve transection in wildtype mice further confirmed induced Aldh3a1 and Ankrd1 expression in an inverse manner across muscle types in response to denervation. Mechanistically, whole-muscle RNA-Seq and pharmacological tests indicate that higher basal levels of lipid oxidation in soleus and diaphragm muscles may predispose them to stronger Nrf2 antioxidant responses under pathological stress compared to EDL, leading to more prominent Aldh3a1 upregulation. Additionally, the identification of the myoblast fusion marker Mymk as an EOM signature gene suggests that the spontaneous activation of satellite cells contributes to high levels of Aldh3a1 in EOMs. Functionally, adeno-associated virus-mediated overexpression of Aldh3a1 protected myotubes from 4-HNE-induced DNA fragmentation and plasma membrane leakage. It also restored MG53-mediated membrane repair, highlighting its potential for clinical applications.

cell biology↗

bioRxiv2024

ApoDock: Ligand-Conditioned Sidechain Packing for Flexible Molecular Docking

Molecular docking is a crucial technique for elucidating protein-ligand interactions. Machine learning-based docking methods offer promising advantages over traditional approaches, with significant potential for further development. However, many current machine learning-based methods face challenges in ensuring the physical plausibility of generated docking poses. Additionally, accommodating protein flexibility remains difficult for existing methods, limiting their effectiveness in real-world scenarios. Diffusion based models has already show a good solution of those problems, such as Alphafold3(AF3). Herein, we present ApoDock, a modular docking paradigm that combines machine learning-driven conditional sidechain packing based on protein backbone and ligand information with traditional sampling methods to ensure physically realistic poses. With accurate sidechain packing and physical based pose sampling, ApoDock demonstrates competitive performance across diverse applications, highlighting its potential as a valuable tool for protein-ligand binding studies and related applications.

bioinformatics↗

bioRxiv2024

CXXC-finger protein 1 associates with FOXP3 to stabilize homeostasis and suppressive functions of regulatory T cells

FOXP3-expressing regulatory T (Treg) cells play a pivotal role in maintaining immune homeostasis and tolerance, with their activation being crucial for preventing various inflammatory responses. However, the mechanisms governing the epigenetic program in Treg cells during their dynamic activation remain unclear. In this study, we demonstrate that CXXC finger protein 1 (CXXC1) interacts with the transcription factor FOXP3 and facilitates the regulation of target genes by modulating H3K4me3 deposition. Cxxc1 deletion in Treg cells leads to severe inflammatory disease and spontaneous T-cell activation, with impaired immunosuppressive function. As a transcriptional regulator, CXXC1 promotes the expression of key Treg functional markers under steady-state conditions, which are essential for the maintenance of Treg cell homeostasis and their suppressive functions. Epigenetically, CXXC1 binds to the genomic regulatory regions of Treg program genes in mouse Treg cells, overlapping with FOXP3 binding sites. Given its critical role in Treg cell homeostasis, CXXC1 presents itself as a promising therapeutic target for autoimmune diseases.

immunology↗

bioRxiv2024

Circadian modulation of mosquito host-seeking persistence by Pigment-Dispersing Factor impacts daily biting patterns

Daily rhythms in mosquito attraction to humans are thought to drive biting patterns and contribute to disease transmission dynamics. Behavioral rhythms in many insects are controlled by specialized clock cells in the brain that are coordinated by the neuropeptide Pigment-Dispersing Factor (PDF). We show that female Aedes aegypti mosquitoes with genetically disrupted PDF display altered daily behavioral timing with reduced locomotor activity and biting in the morning. Using an automated behavioral tracking system, we also report that mosquitoes exhibit daily modulation of response persistence to the host cue carbon dioxide and loss of PDF alters this pattern. These findings indicate that PDF regulates temporal features of host-seeking behavior that promote biting success at specific times of day and may underlie blood feeding patterns observed in the field.

neuroscience↗

bioRxiv2024

A wheat tandem kinase and NLR pair confers resistance to multiple fungal pathogens

Recently discovered tandem kinase proteins (TKPs) are pivotal to the innate immune systems of cereal plants, yet how they initiate plant immune responses remains unclear. This report identifies the wheat protein WTN1, a non-canonical NLR receptor featuring tandem NB-ARC domains, as crucial for WTK3-mediated disease resistance. Both WTK3 and its allelic variant Rwt4, known for conferring resistance to wheat powdery mildew and blast respectively, are capable of recognizing the blast effector PWT4, and activate WTN1 to form calcium-permeable channels, akin to ZAR1 and Sr35. This study unveils a unique plant defense mechanism wherein TKPs and associated NLRs operate as "sensor-executor" pairs against fungal pathogens. Additionally, evolutionary analyses reveal a co-evolutionary trajectory of the TKP-NLR module, highlighting their synergistic role in triggering plant immunity. One Sentence SummaryAn ancient synergistic TKP-NLR pair triggers innate immunity for multiple disease resistance in wheat.

plant biology↗

bioRxiv2024

Ultra-resolution Deconvolution of Spatial Transcriptomics Unveils Spatiotemporal Cellular Dynamics in Complex Microenvironments

Immune cells infiltrate tissues in response to exogenous pathogens or spontaneous tumors, generating protective immunity to safeguard tissues. Despite advancements in spatial transcriptomics (ST), the precise spatial distribution and functional specialization of immune cells within tissue microenvironments remain elusive. Here, we introduce an ultra-precision ST deconvolution algorithm (UCASpatial) that enhances the mapping of cell subpopulations to spatial locations by leveraging the contribution of genes indicative of cell identity through entropy-based weighting. Using both in silico and real ST datasets, we demonstrate that UCASpatial improves the robustness and accuracy in identifying low-abundant cell subpopulations and distinguishing transcriptionally heterogeneous cell subpopulations. Applying UCASpatial to human colorectal cancer (CRC), we link genomic alterations in individual cancer clones to multi-cellular characteristics of the tumor immune microenvironment (TIME) and reveal the co-evolution of tumor cells and TIME at a clonal resolution. We show that the copy number gain on chromosome 20q (chr20q-gain) in tumor cells orchestrates a T cell-excluded TIME, indicative of resistance to immunotherapy in CRC, and is associated with tumor-intrinsic human endogenous retrovirus subfamily H (HERV-H) silencing and impaired type I interferon response. In murine wound healing models, we illuminate the spatiotemporal dynamics of individual cell subsets across various stages of the healing process. We discovered that the scarring-healing mice (C57BL/6) exhibited a replacement of Prg4+ chondrogenic progenitors to Igfbp5+ chondrocytes in the wound bed at the regeneration stage, a change not observed in the regenerative strain (MRL/MpJ). The Igfbp5+ chondrocyte, spatially coordinating with Cd36+ Gpnmb+ Il1b- macrophage and Fmod+ fibroblast, forms a pro-fibrotic community associated with regeneration failure. The cell-cell interactions within this three-cell cluster, mediated by the IL11-IL11RA axis, drive the pro-fibrotic community formation and limit regeneration in C57BL/6 mice. Our findings present UCASpatial as a versatile tool for deciphering fine-grained cellular landscapes in ST and exploring intercellular mechanisms in complex and dynamic microenvironments.

bioinformatics↗

bioRxiv2024

MIPDB: A maize image-phenotype database with multi-angle and multi-time characteristics

Plant phenomics has become one of the most significant scientific fields in recent years. However, typical phenotyping procedures have low accuracy, low throughput, and are labor-intensive and time-consuming. Large-scale phenotypic collection equipment, on the other hand, is pricy, rigid, and inconvenient. The advancement of phenomics has been hampered by these restrictions. Lightweight picture collection equipment can now be used to capture plant phenotypic data thanks to the development of deep learning-based image identification. For the purpose of training the model, this approach needs high-quality annotated datasets. In this study, we used a handheld camera to gather multi-angle, multi-time series images and an unmanned aerial vehicle (UAV) to create a maize image phenotyping database (MIPDB). Over 30,000 high-resolution photos are available in the MIPDB, with 17,631 of those images having been carefully tagged with point-line method. The MIPDB can be accessed by the general public at http://phenomics.agis.org.cn. We anticipate that the availability of this superior dataset will stimulate a new revolution in crop breeding and advance deep learning-based phenomics research.

plant biology↗

bioRxiv2024

Temperature and photoperiod differentially impact maternal phenotypes in diapause egg-laying Aedes albopictus mosquitoes

BackgroundAedes albopictus (Skuse 1894) mosquitoes can transmit deadly arboviruses and are globally invasive due to their ability to survive in both tropical and temperate climates. Although adults cannot survive harsh winters, females are capable of anticipating seasonal change and producing overwintering diapause (DP) eggs that remain in a state of arrested development over the winter and hatch when favorable conditions return in the spring. Previous work has shown that shortening photoperiod (day length) alone is sufficient for DP induction. While decreasing temperatures can facilitate DP entry, temperature signals alone are not sufficient to induce DP. Methodology/Principal FindingsTo identify maternal phenotypes predictive of DP egg production, we characterized aspects of maternal physiology and behavior to identify those that correlate with DP egg production and changes in photoperiod, versus changes in temperature. Neither changes in temperature nor photoperiod impacted protein preference, blood meal consumption, or total number of eggs produced per female. Egg retention and oviposition timing were influenced by temperature, independent of DP egg production. However, females housed under short photoperiod conditions showed increased starvation resistance, despite showing similar levels of locomotor activity and internal stores of triacylglycerols, glucose, glycogen, and trehalose compared to females housed in long photoperiods. Conclusions/SignificanceThese results suggest that temperature and photoperiod differentially affect maternal phenotypes and identify starvation resistance as a maternal phenotype that is influenced by photoperiod and can be used to predict DP egg status. AUTHOR SUMMARYAedes albopictus mosquitoes can survive in temperate climates because females are able to detect changes in temperature and day length in the autumn to produce "diapause" eggs that are provisioned with extra nutrients to survive harsh winters by remaining in a state of arrest and hatching in the spring. Although temperature and day length normally change in concert with each other throughout the year, studies have shown that day length is the most important cue used by female mosquitoes to initiate diapause egg production. In this study, the authors examined aspects of feeding and reproduction to determine which of these correlated with day length. Many aspects of reproduction were influenced by temperature; however, housing female mosquitoes under autumn-like short day conditions led to increased starvation resistance. Surprisingly these females survive longer even though they do not appear to expend less energy compared to females housed in spring-like long day conditions at the same temperature. This suggests that the mosquito mothers undergo changes in their metabolism that can be used predict whether she will produce eggs that can survive the winter. This new knowledge may lead to new targets to disrupt seasonal reproduction in mosquitoes and limit the parts of the world where they can survive.

physiology↗

bioRxiv2024

Worldwide soundscape ecology patterns across realms

Abstract and keywordsO_ST_ABSAimC_ST_ABSThe urgency for remote, reliable, and scalable biodiversity monitoring amidst mounting human pressures on ecosystems has sparked worldwide interest in Passive Acoustic Monitoring (PAM), which can track life underwater and on land. However, we lack a unified methodology to report this sampling effort and a comprehensive overview of PAM coverage to gauge its potential as a global research and monitoring tool. To remediate this, we created the Worldwide Soundscapes project, a collaborative network and growing database comprising metadata from 409 datasets across all realms (terrestrial, marine, freshwater, and subterranean). LocationWorldwide, 12 200 sites, all ecosystems. Time period1991 to present. Major taxa studiedAll soniferous taxa. MethodsWe synthesise sampling coverage across spatial, temporal, and ecological scales using metadata describing sampling locations, deployment schedules, focal taxa, and recording parameters. We explore global trends in biological, anthropogenic, and geophysical sounds based on 168 recordings from twelve ecosystems across all realms. ResultsTerrestrial sampling is spatially denser (45 sites/Mkm2) than aquatic sampling (0.3 and 1.8 sites/Mkm2 in oceans and fresh water) with only two subterranean datasets. Although diel and lunar cycles are well-covered in all realms, only marine datasets (56%) comprehensively sample all seasons. Across twelve ecosystems, biological sounds show contrasting diel patterns, decline with distance from the equator and negatively correlate with anthropogenic sounds. Main conclusionsPAM can inform macroecology studies as well as global conservation and phenology syntheses, but representation can be improved by expanding terrestrial taxonomic scope, sampling coverage in the high seas and subterranean ecosystems, and spatio-temporal replication in freshwater habitats. Overall, this global PAM network holds promise to support global biodiversity research and monitoring efforts.

ecology↗

bioRxiv2024

CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification

Infection with human immunodeficiency virus (HIV)-1 leads to acquired immunodeficiency syndrome (AIDS) if left untreated. According to UN figures, approximately 39 million people globally were living with HIV in 2022, with 76% of those individuals accessing antiretroviral therapy. Measurement of plasma viral RNA load using calibrated nucleic acid amplification tests (like reverse transcription quantitative PCR, RT-qPCR) is routinely performed to monitor response to treatment and ultimately prevent viral transmission. RNA quantities measured by commercial tests can vary over many orders of magnitude, from trace single copy levels to, in cases, over 109 /mL of plasma, presenting an analytical challenge for calibrating across a broad measurement range. Interlaboratory study CCQM-P199 "HIV-1 RNA copy number quantification" (April to September 2019) was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting national metrology institutes (NMIs) and designated institutes (DIs) development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements. Thirteen laboratories participated in CCQM-P199 and were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen (gag) gene of in vitro transcribed RNA molecules at low ({approx} 103 /L) and high concentration ({approx} 109 /L) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 were measured by participants using one-step reverse transcription digital PCR (RT-dPCR) (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. Study Material 2 was measured by both RT-dPCR (one-step) (n = 4) and orthogonal methods: single molecule flow cytometric counting (n = 2), high performance liquid chromatograph (HPLC) (n = 1) and isotype dilution-mass spectrometry (ID-MS) (n = 1). Interlaboratory reproducibilities (expressed as %CV) were 21.4 %, 15.3 % and 22.0 % for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties, indicating uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were not statistically significantly different (p = 0.46), the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196 vs. 808 /L; p < 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher molecular weight (MW) impurities constituted 16.6 % of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results. This study demonstrates that reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and precision of contemporary methods for RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to further establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.

molecular biology↗

bioRxiv2024

Plasticity and Dynamics of Hematopoietic Cells within Bone Marrow Microenvironment in Leukemia

Extensive research has been conducted on the plasticity of malignant cells and nonmalignant cells in solid tumor. However, the plasticity of bone marrow hematopoietic cells in leukemia have remained largely unexplored. In this study, we aimed to investigate cell changings in hematopoietic cells through lineage tracing across various types of leukemias. We had compiled a landscape of leukemia and constructed phylogenetic trees of hematopoietic cells through utilizing massively parallel scRNA-seq data, mtDNA mutation and SNP analysis. Based on the observed cell changings, we identified several types of cell changings, including transdifferentiation, dedifferentiation, and state transition, except for differentiation and expansion. In AML and CMML, GMPs and neutrophils showed a higher potential for transferring to other cell types. In BPDCN, pDCs were less prone to switching to other cell types, while T cells demonstrated high plasticity. In B-ALL and B-CLL, B-ALL blast cells and B-CLL blast cells emerged at the most dynamic state. The dynamics of hematopoietic cells in AML, BPDCN and ALL changed along with the clinical process. Extrinsic factors within the leukemia microenvironment may influence the cell changings. Regulons encountered an intermediate cell state during the process of transition to myeloid cells and erythroid cells. We also found a correlation between B-common blast cells and T cells, suggesting a potential transition from B lymphoblastic leukemia to T lymphoblastic leukemia. In conclusion, our study unveiled the distinct plasticity and dynamics of hematopoietic cells in various types of leukemia. This sheds light on the possibility of targeting cell changes as a new strategy for leukemia treatment and improving current immunotherapy.

bioinformatics↗

bioRxiv2024

CCQM-P199b: Interlaboratory comparability study of SARS-CoV-2 RNA copy number quantification

Nucleic acid amplification tests including reverse transcription quantitative PCR (RT-qPCR) are used to detect RNA from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Standardized measurements of RNA can facilitate comparable performance of laboratory tests in the absence of existing reference measurement systems early on in a pandemic. Interlaboratory study CCQM P199b "SARS-CoV-2 RNA copy number quantification" was designed to test the fitness-for-purpose of developed candidate reference measurement procedures (RMPs) for SARS-CoV-2 genomic targets in purified RNA materials, and was conducted under the auspices of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) to evaluate the measurement comparability of national metrology institutes (NMIs) and designated institutes (DIs), thereby supporting international standardization. Twenty-one laboratories participated in CCQM P199b and were requested to report the RNA copy number concentration, expressed in number of copies per microliter, of the SARS-CoV-2 nucleocapsid (N) gene partial region (NC_045512.2: 28274-29239) and envelope (E) gene (NC_045512.2: 26245-26472) (optional measurements) in samples consisting of in vitro transcribed RNA or purified RNA from lentiviral constructs. Materials were provided in two categories: lower concentration ({approx} (101-104) /L in aqueous solution containing human RNA background) and high concentration ({approx} 109 /L in aqueous solution without any other RNA background). For the measurement of N gene concentration in the lower concentration study materials, the majority of laboratories (n = 17) used one-step reverse transcription-digital PCR (RT-dPCR), with three laboratories applying two-step RT-dPCR and one laboratory RT-qPCR. Sixteen laboratories submitted results for E gene concentration. Reproducibility (% CV or equivalent) for RT-dPCR ranged from 19 % to 31 %. Measurements of the high concentration study material by orthogonal methods (isotope dilution-mass spectrometry and single molecule flow cytometry) and a gravimetrically linked lower concentration material were in a good agreement, suggesting a lack of overall bias in RT-dPCR measurements. However methodological factors such as primer and probe (assay) sequences, RT-dPCR reagents and dPCR partition volume were found to be potential sources of interlaboratory variation which need to be controlled when applying this technique. This study demonstrates that the accuracy of RT-dPCR is fit-for-purpose as a RMP for viral RNA target quantification in purified RNA materials and highlights where metrological approaches such as the use of in vitro transcribed controls, orthogonal methods and measurement uncertainty evaluation can support standardization of molecular methods.

molecular biology↗

bioRxiv2024

MASCARA: coexpression analysis in data from designed experiments

Experiments in plant transcriptomics are usually designed to induce variation in a pathway of interest. Harsh experimental conditions can cause widespread transcriptional changes between groups. Discovering coexpression within a pathway of interest (here the strigolactone pathway) in this context is hampered by the dominant variance induced by the design. Minor changes in experimental conditions not controlled for may affect the plants, leading to small coordinated differences in genes within pathways of interest and related pathways between replicate plants in the same controlled experimental condition. These systematic differences are usually averaged out, but we argue here that they can be used to improve the detection of genes that co-express. We introduce a novel framework "MASCARA" which combines ANOVA simultaneous component analysis and partial least squares to remove the experimentally induced variance and investigate multivariate relationships in the non-designed variance. MASCARA is tested against a selection of competitors on simulated data, created to mimic a designed transcriptome study, where its benefit is demonstrated. In a coexpression analysis of a real dataset MASCARA detects several uncharacterised but relevant transcripts. Our results indicate that there is sufficient structure left in a typical dataset after correcting for experimental variance and that this residual information is useful to investigate coexpression. Author SummaryExperiments in the life sciences usually purposefully induce significant variance between different treatments, in order to activate or repress certain mechanisms of interest. Whilst this is necessary it can make it challenging to detect meaningful relationships within pathways of interest, particularly when the experimental conditions are drastically different. Instead of focusing on the drastic changes in response due to the different treatment, MASCARA uses the systematic synchronous variances between replicates to find related features within the pathway of interest. Through simulation studies and application to a real dataset, we demonstrate the effectiveness of MASCARA in detecting relevant transcripts and extracting coexpression patterns from gene expression data.

bioinformatics↗

bioRxiv2024

Topology-informed regulatory element collections coordinate cell identity gene expression programs

Transcription proteins are concentrated at nuclear transcriptional condensates. These condensates contain cis-regulatory elements (CREs), including enhancers and promoters, that are thought to regulate genes in the same condensate. The roles of condensates are of great current interest, but research into their function is limited by an inability to comprehensively identify their associated CREs. Here, we present a conceptual framework and algorithm, BOUQUET, for integrating genome topology, chromatin occupancy, and graph theory to associate CREs and transcription protein machinery with target genes and identify exceptionally protein-rich communities that interact with condensates. BOUQUET uncovers surprising quantitative correlations between community protein accumulation and gene expression phenotypes by combining accurate CRE-gene assignment with co-activator binding profiles. A small subset of communities, which we call "3D-super-enhancers," is exceptionally protein-rich. BOUQUET-predicted 3D-SEs are comparable in number to co-activator nuclear puncta, and all genes known to interact with co-activator condensates in embryonic stem cells are within 3D-SEs. 3D-SEs are enriched for association with cell identity genes across mammalian tissues. Microscopy analyses show frequent co-localization and co-expression of genes from the same 3D-SE within a single co-activator punctum, suggesting 3D-SE components interact with co-activator condensates. Thus 3D-SEs correspond to co-activator puncta, which nominates additional condensate-associated genes and CREs.

molecular biology↗