Biology subjects

Gao, H.

Publications and source records attributed to Gao, H..

At least 199 records · Page 11Linked to original sources

bioRxiv2020

Simulation of Ca2+ oscillations in astrocytes mediated by amyloid beta in Alzheimer's disease

Disruptions of astrocyte Ca2+ signaling is important in Alzheimers disease (AD) with the unclear mechanism of amyloid beta peptide (A{beta}). We have modified our previous computational model of spontaneous Ca2+ oscillations in astrocytes to investigate the effects of A{beta} on intracellular Ca2+ dynamics. The simulation results have shown consistence with the previous experiments. A{beta} can increase the resting concentration of intracellular Ca2+ and change the regime of Ca2+ oscillations by activating L-type voltage-gated calcium channels and the metabolic glutamate receptors, or by increasing ryanodine receptors sensitivity and Ca2+ leakage, respectively. This work have provided a toolkit to study the influence of A{beta} on intracellular Ca2+ dynamics in AD. It is helpful for understanding the toxic role of A{beta} during the progression of AD. Statement of SignificanceAlzheimers disease (AD) is the most common neurodegenerative disease with the unclear mechanism of amyloid beta peptide (A{beta}). This work have implemented a computational model to address the Ca2+ dynamics of astrocyte mediated by A{beta} with the four different pathways: voltage-gated calcium channels, metabotropic glutamate receptors 5, ryanodine receptor channels and membrane leak. The Ca2+ oscillations and bifurcation diagram indicate that astrocytes exhibit ionic excitability mediated by A{beta} and become the potential targets of A{beta} neurotoxicity. We expect this shared computational model would advance the understanding of AD.

biophysics↗

bioRxiv2020

Rhesus macaques can be effectively infected with SARS-CoV-2 via ocular conjunctival route

The outbreak of Corona Virus Disease 2019 caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly transmitted. The potential extra-respiratory transmission routes remain uncertain. Five rhesus macaques were inoculated with 1x106 TCID50 of SARS-CoV-2 via conjunctival (CJ), intratracheal (IT), and intragastric (IG) routes, respectively. Remarkably, the CJ inoculated-macaques developed mild interstitial pneumonia and viral load was detectable in the conjunctival swabs at 1 days post-inoculation (dpi). Only via IT inoculation, viral load was detected in the anal swab at 1-7 dpi and macaque showed weight loss. However, viral load was undetectable after IG inoculation. Comparatively, viral load was higher in the nasolacrimal system but lesions of lung were relatively mild and local via CJ inoculation compared with that via IT inoculation, demonstrating distinct characteristics of virus dispersion. Both the two routes affected the alimentary tract. Therefore the clinicians need to protect eye while working with patients.

microbiology↗

bioRxiv2020

Reinfection could not occur in SARS-CoV-2 infected rhesus macaques

A global pandemic of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is ongoing spread. It remains unclear whether the convalescing patients have a risk of reinfection. Rhesus macaques were rechallenged with SARS-CoV-2 during an early recovery phase from initial infection characterized by weight loss, interstitial pneumonia and systemic viral dissemination mainly in respiratory and gastrointestinal tracts. The monkeys rechallenged with the identical SARS-CoV-2 strain have failed to produce detectable viral dissemination, clinical manifestations and histopathological changes. A notably enhanced neutralizing antibody response might contribute the protection of rhesus macaques from the reinfection by SARS-CoV-2. Our results indicated that primary SARS-CoV-2 infection protects from subsequent reinfection. One Sentence SummaryNeutralizing antibodies against SARS-CoV-2 might protect rhesus macaques which have undergone an initial infection from reinfection during early recovery days.

microbiology↗

bioRxiv2020

Meta-analysis of the effects of sleep deprivation on depression in patients and animals

ObjectiveResearch on the antidepressant effects of sleep deprivation (SD) is lagging and has not produced completely uniform results in humans and animals. The present study aimed to reassess the effect of SD on patients and animals by meta-analysis based on updated research. MethodsWe searched PubMed, Embase and Cochrane Library for articles since the first relevant literature published up to June 10th, 2019. Data on sample characteristics, features of SD, and tests for depression were extracted. ResultsFourteen articles were included, eight on humans and six on animals. We found that when the duration of SD in patients was 7-14 days, it reflected antidepression [-1.52 (-2.07, -0.97); I2=19.6%]. In animals, the results of sucrose consumption experiments showed that SD has depressogenic effects [-1.06 (-1.63, -0.49); I2=81.1%], while the results of forced swimming experiments showed that SD treated depression [-1.17 (-2.19, -0.16); I2=80.1%], regardless of the duration of sleep deprivation. ConclusionSD can be an effective antidepressant measure when the duration is 7-14 days in patients. In animal studies, SD has shown more antidepressant effects when measured by forced swimming experiments, whereas using sucrose consumption tests had the effect of worsening depression.

neuroscience↗

bioRxiv2020

Protection of Rhesus Macaque from SARS-Coronavirus challenge by recombinant adenovirus vaccine

A recombinant adenovirus vaccine against the SARS Coronavirus (SARS-CoV) was constructed, which contains fragments from the S, N, and Orf8 genes. Rhesus Macaques immunized with the recombinant adenovirus generated antigen-specific humoral and cellular response. Furthermore, the vaccine provided significant protection against subsequent live SARS-CoV challenge. In contrast, three out of four monkeys immunized with placebo suffered severe alveolar damage and pulmonary destruction.

immunology↗

bioRxiv2020

The Pathogenicity of 2019 Novel Coronavirus in hACE2 Transgenic Mice

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) caused the Corona Virus Disease 2019 (COVID-19) cases in China has become a public health emergency of international concern (PHEIC). Based on angiotensin converting enzyme 2 (ACE2) as cell entry receptor of SARS-CoV, we used the hACE2 transgenic mice infected with SARS-CoV-2 to study the pathogenicity of the virus. Weight loss and virus replication in lung were observed in hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes and monocytes in alveolar interstitium, and accumulation of macrophages in alveolar cavities. Viral antigens were observed in the bronchial epithelial cells, alveolar macrophages and alveolar epithelia. The phenomenon was not found in wild type mice with SARS-CoV-2 infection. The pathogenicity of SARS-CoV-2 in hACE2 mice was clarified and the Kochs postulates were fulfilled as well, and the mouse model may facilitate the development of therapeutics and vaccines against SARS-CoV-2.

microbiology↗

bioRxiv2020

DPHL: A pan-human protein mass spectrometry library for robust biomarker discovery using Data-Independent Acquisition and Parallel Reaction Monitoring

To answer the increasing need for detecting and validating protein biomarkers in clinical specimens, proteomic techniques are required that support the fast, reproducible and quantitative analysis of large clinical sample cohorts. Targeted mass spectrometry techniques, specifically SRM, PRM and the massively parallel SWATH/DIA technique have emerged as a powerful method for biomarker research. For optimal performance, they require prior knowledge about the fragment ion spectra of targeted peptides. In this report, we describe a mass spectrometric (MS) pipeline and spectral resource to support data-independent acquisition (DIA) and parallel reaction monitoring (PRM) based biomarker studies. To build the spectral resource we integrated common open-source MS computational tools to assemble an open source computational workflow based on Docker. It was then applied to generate a comprehensive DIA pan-human library (DPHL) from 1,096 data dependent acquisition (DDA) MS raw files, and it comprises 242,476 unique peptide sequences from 14,782 protein groups and 10,943 SwissProt-annotated proteins expressed in 16 types of cancer samples. In particular, tissue specimens from patients with prostate cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, squamous cell lung carcinoma, diseased thyroid, glioblastoma multiforme, sarcoma and diffuse large B-cell lymphoma (DLBCL), as well as plasma samples from a range of hematologic malignancies were collected from multiple clinics in China, the Netherlands and Singapore and included in the resource. This extensive spectral resource was then applied to a prostate cancer cohort of 17 patients, consisting of 8 patients with prostate cancer (PCa) and 9 with benign prostate hyperplasia (BPH), respectively. Data analysis of DIA data from these samples identified differential expressions of FASN, TPP1 and SPON2 in prostate tumors. Thereafter, PRM validation was applied to a larger PCa cohort of 57 patients and the differential expressions of FASN, TPP1 and SPON2 in prostate tumors were validated. As a second application, the DPHL spectral resource was applied to a patient cohort consisting of samples from 19 DLBCL patients and 18 healthy individuals. Differential expressions of CRP, CD44 and SAA1 between DLBCL cases and healthy controls were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supported that DIA-PRM MS pipeline enables robust protein biomarker discoveries.

bioinformatics↗

bioRxiv2020

Biomarker-guided treatment strategies for ovarian cancer identified from a heterogeneous panel of patient-derived tumor xenografts

Advanced ovarian cancers are a leading cause of cancer-related death in women. Such cancers are currently treated with surgery and chemotherapy which is often temporarily successful but exhibits a high rate of relapse after which treatment options are few. Here we assess the responses of a panel of patient-derived ovarian cancer xenografts (PDXs) to 19 mono and combination therapies, including small molecules and antibody-drug conjugates. The PDX panel aimed to mimic the heterogeneity of disease observed in patients, and exhibited a distribution of responsiveness to standard of care chemotherapy similar to human clinical data. Three monotherapies and one drug combination were found to be active in different subsets of PDXs. By analyzing gene expression data we identified gene expression biomarkers predictive of responsiveness to each of three novel targeted therapy regimens. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. Biomarker frequency was similar in human patients, suggesting the possibility of a new therapeutic approach to ovarian cancer and demonstrating the potential power of PDX-based trials in broadening the reach of precision cancer medicine.

cancer biology↗

bioRxiv2020

Integrated Omics Analyses Reveal Differential Gene Expression and Potential for Cooperation Between Denitrifying Polyphosphate and Glycogen Accumulating Organisms

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N2O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The 6 transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA, and IC, and a competing glycogen accumulating organism (GAO) affiliated with Candidatus Competibacter. Strongly elevated expression of nitrite reductase compared to nitrous oxide reductase was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N2O accumulation. Surprisingly, while [~]90% of nitrite reductase gene transcripts mapped to the three co-occurring PAO populations, [~]93% of nitric oxide reductase gene transcripts were expressed by the GAO population. This suggests the potential for cooperation between GAOs and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide. Originality-Significance StatementPolyphosphate accumulating organisms (PAOs) affiliated with as-yet-uncultivated Ca. Accumulibacter phosphatis are increasingly employed in enhanced biological phosphorus removal (EBPR) processes, a common environmental biotechnology for removing phosphorus from wastewater and thereby preventing detrimental impacts of nutrient pollution. Under anoxic conditions, PAOs have been associated with unusually high emissions of the potent greenhouse gas and denitrification intermediate nitrous oxide. However, the underlying mechanisms and biological controls on incomplete denitrification by denitrifying Accumulibacter, their ecological interactions with understudied glycogen accumulating organisms (GAOs), and patterns of gene expression under anoxic conditions are all poorly understood. Here, we describe genomic features of a previously unrecognized clade of Accumulibacter that is putatively adapted to high rate P uptake under nitrite-driven denitrification and provide evidence that differential gene expression (namely elevated expression of nitrite reductase compared to nitrous oxide reductase) by Accumulibacter is a key control on nitrous oxide production. Moreover, we document genomic and transcriptional potential for cooperation and crossfeeding of the denitrification intermediate nitric oxide between GAOs and PAOs. This is surprising because GAOs are conventionally considered to be competitors to PAOs, and because nitric oxide is toxic to most microorganisms at low concentrations. Taken together, our work provides significant new understanding of metabolic and ecological interactions in EBPR processes that are critical to environmental protection; demonstrates the potential of previously unrecognized crossfeeding of the denitrification intermediate nitric oxide; and expands our understanding of genomic features and clade level diversity of Accumulibacter.

bioengineering↗

bioRxiv2019

Fibrotic scar after experimental autoimmune encephalomyelitis inhibits oligodendrocyte differentiation

Remyelination failure is a crucial component of disease progression in the autoimmune demyelinating disease Multiple Sclerosis (MS). The regenerative capacity of oligodendrocyte progenitor cells (OPCs) to replace myelinating oligodendrocytes is likely influenced by many aspects of the lesion environment including inflammatory signaling and extracellular matrix (ECM) deposition. These features of MS lesions are typically attributed to infiltrating leukocytes and reactive astrocytes. Here we demonstrate that fibroblasts also contribute to the inhibitory environment in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Using Col11GFP transgenic mice, we show that perivascular fibroblasts are activated in the spinal cord at EAE onset, and infiltrate the parenchyma by the peak of behavioral deficits where they are closely associated with areas of demyelination, myeloid cell accumulation, and ECM deposition. We further show that both fibroblast conditioned media and fibroblast ECM inhibit the differentiation of OPCs into mature oligodendrocytes. Taken together, our results indicate that the fibrotic scar is a major component of EAE pathology that leads to an inhibitory environment for remyelination, thus raising the possibility that anti-fibrotic mechanisms may serve as novel therapeutic targets for MS.

neuroscience↗

bioRxiv2019

Phenotypic Switching of Naïve T cells to Immune-suppressive Treg-like Cells by Mutant KRAS

Oncogenic (mutant) Ras protein Kirsten rat sarcoma viral oncogene homolog (KRAS) promotes uncontrolled proliferation, altered metabolism, and loss of genome integrity in a cell-intrinsic manner. Here, we demonstrate that CD4+ T cells when incubated with tumor-derived exosomes from mutant (MT) KRAS non-small cell lung cancer (NSCLC) cells, patient sera, or a mouse xenograft model, induce phenotypic conversion to FOXP3+ Treg-like cells that are immune-suppressive. Furthermore, transfecting T cells with MT KRAS cDNA alone induced phenotypic switching and mathematical modeling supported this conclusion. Single-cell sequencing identified the interferon pathway as the mechanism underlying the phenotypic switch. These observations highlight a novel cytokine-independent, cell-extrinsic role for KRAS in T cell phenotypic switching. Thus, targeting this new class of Tregs represents a unique therapeutic approach for NSCLC. Since KRAS is the most frequently mutated oncogene in a wide variety of cancers, the findings of this investigation are likely to be of broad interest and have a large scientific impact.

cancer biology↗

bioRxiv2019

Genome sequencing and neurotoxin diversity of a wandering spider Pardosa pseudoannulata (pond wolf spider)

Spiders constitute an extensive and diverse branch of the phylum Arthropoda. Whereas the genomes of four web-weaver spider species and a single cave-living spider have been determined, similar studies have not been reported previously for a wandering spider. The pond wolf spider, Pardosa pseudoannulata, is a wandering hunter that immobilizes prey using venom rather than a web. It is also an important predator against a range of agriculturally important insect pests. The increasing interest in its wandering lifestyle and in the potential of spider venom as a tool for pest control have prompted a detailed study on this wandering spider species. We have generated a high-quality genome sequence of P. pseudoannulata and analysed genes associated with the production of silk and venom toxins. Sequencing reveals that P. pseudoannulata has a large genome of 4.26 Gb. The presence of only 16 spidroin genes and four types of silk glands is consistent with the moderate use of silk and the lack of a prey-catching web. A large number of genes encode neurotoxins and there is evidence that the majority are highly selective for invertebrates. Comparison between spider species reveals a correlation between spider neurotoxin selectivity for target organisms and spider prosoma size, suggesting a possible coevolution of these two features. The genome data provides valuable insights into the biology of P. pseudoannulata and its potential role as a natural enemy in pest control.

genomics↗

bioRxiv2019

RSVdb: A comprehensive database of in vivo mRNA structure

RNA fulfills a crucial regulatory role in cells by folding into a complex RNA structure. To date, a chemical compound, dimethyl sulfate (DMS), has been developed to effectively probe the RNA structure at the transcriptome level. We proposed a database, RSVdb (https://taolab.nwafu.edu.cn/rsvdb/), for the browsing and visualization of transcriptome RNA structures. RSVdb, including 626,225 RNAs with validated DMS reactivity from 178 samples in 8 species, supports four main functions: information retrieval, research overview, structure prediction, and resource download. Users can search for species, studies, transcripts and genes of interest; browse the quality control of sequencing data and statistical charts of RNA structure information; preview and perform online prediction of RNA structures in silico and under DMS restraint of different experimental treatments; and download RNA structure data for species and studies. Together, RSVdb provides a reference for RNA structure and will support future research on the function of RNA structure at the transcriptome level.

bioinformatics↗

bioRxiv2019

RNA Polymerase II CTD phosphatase Rtr1 prevents premature transcription termination

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). Using disruption-compensation (DisCo) protein-protein interaction network analysis, interaction changes were observed within the termination machinery as a consequence of deletion of the serine 5 RNAPII CTD phosphatase Rtr1. Interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1{Delta}, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. These changes could be the result of altered interactions between the termination machinery and/or increased levels of premature termination of RNAPII. Transcriptome analysis in rtr1{Delta} cells found decreased pervasive transcription and a shift in balance of expression of sense and antisense transcripts. Globally, rtr1{Delta} leads to decreases in noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS)-dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes and increased efficiency of snRNA termination. Additionally, premature termination increases globally at protein-coding genes where NNS is recruited during early elongation. The effects of rtr1{Delta} on RNA expression levels were erased following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade terminated noncoding RNAs. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature RNAPII termination of mRNAs and ncRNAs. Additionally, Rtr1 phosphatase activity facilitates low-level elongation of noncoding transcripts that impact the transcriptome through RNAPII interference.\n\nAUTHOR SUMMARYMany cellular RNAs including those that encode for proteins are produced by the enzyme RNA Polymerase II. In this work, we have defined a new role for the phosphatase Rtr1 in the regulation of RNA Polymerase II progression from the start of transcription to the 3 end of the gene where the nascent RNA from protein-coding genes is typically cleaved and polyadenylated. Deletion of the gene that encodes RTR1 leads to changes in the interactions between RNA polymerase II and the termination machinery. Rtr1 loss also causes early termination of RNA Polymerase II at many of its target gene types including protein coding genes and noncoding RNAs. Evidence suggests that the premature termination observed in RTR1 knockout cells occurs through the termination factor and RNA binding protein Nrd1 and its binding partner Nab3. Additionally, many of the prematurely terminated noncoding RNA transcripts are degraded by the Rrp6-containing nuclear exosome, a known component of the Nrd1-Nab3 termination coupled RNA degradation pathway. These findings suggest that Rtr1 normally promotes elongation of RNA Polymerase II transcripts through preventation of Nrd1-directed termination.

systems biology↗

bioRxiv2019

Hair-bearing human skin generated entirely from pluripotent stem cells

The skin is important for regulating bodily fluid retention and temperature, guarding against external stresses, and mediating touch and pain sensation. The skin is also susceptible to damage from burns, diseases, or genetic defects, which affect nearly one billion people worldwide1,2. For the advancement of skin regenerative therapies, it remains challenging to construct new skin with hair follicles and nerves in tissue cultures and in bioengineered skin grafts3-8. Here, we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use step-wise modulation of the TGF{beta} and FGF signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During 4-5 months incubation, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis, and pigmented hair follicles equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking human touch circuitry. Single-cell RNA sequencing and direct comparison to foetal specimens suggest that skin organoids are equivalent to human facial skin in the second-trimester of development. Moreover, we show that skin organoids produce planar hair-bearing skin when grafted on nude mice. Together, our results demonstrate the self-assembly of nearly complete skin tissue in vitro that can be used to reconstitute skin in vivo. We anticipate that our skin organoid model will be foundational to future studies of human skin development, disease modelling, or reconstructive surgery.

developmental biology↗

bioRxiv2019

High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types histological region of small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

biochemistry↗

bioRxiv2019

Mutant p53 Drives Clonal Hematopoiesis through Modulating Epigenetic Pathway

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. We discovered that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Further, genetic and pharmacological inhibition of EZH2 decrease the repopulating potential of p53 mutant HSPCs. Thus, we have uncovered an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.

cancer biology↗

bioRxiv2019

A novel reporter gene assay for pyrogen detection

Fever is a systemic inflammatory response of the body to pyrogens. Nuclear factor {kappa}B (NF-{kappa}B) is a central signalling molecule that causes the excessive secretion of various proinflammatory factors induced by pyrogens. This study explored the feasibility of a novel reporter gene assay (RGA) for pyrogen detection using RAW 264.7 cells stably transfected with the NF-{kappa}B reporter gene as a pyrogenic marker. Pyrogen was incubated with the transgenic cells, and the intensity of the fluorescence signal generated by luciferase secreted by the reporter gene was used to reflect the degree of activation of NF-{kappa}B, so as to quantitatively detect the pyrogens. The RGA could detect different types of pyrogens, including the lipopolysaccharide (LPS) of gram-negative bacteria, the lipoteichoic acid (LTA) of gram-positive bacteria, and the zymosan of fungi, and a good dose-effect relationship was observed in terms of NF-{kappa}B activity. The limits of detection of the RGA to those pyrogens were 0.03 EU/ml, 0.001 g/ml, and 1 g/ml, respectively. The method had good precision and accuracy and could be applied to many biological products (e.g., nivolumab, rituximab, bevacizumab, etanercept, basiliximab, haemophilus influenzae type b conjugate vaccine, 23-valent pneumococcal polysaccharide vaccine, and group A and group C meningococcal conjugate vaccine). The results of this study suggest that the novel RGA has a wide pyrogen detection spectrum and is sufficiently sensitive, stable, and accurate for various applications.\n\nImportancePyrogen testing is mandatory and a critical method to ensure the safety of parenteral products including vaccines.\n\nCurrently, only two pharmacological tests, including the rabbit pyrogen test and the bacterial endotoxins test (BET), are applied to evaluate pyrogenic contamination in parenteral pharmaceuticals by most of state pharmacopoeias. Although generally reliable, both of these assays have shortcomings. The rabbit test is not quantitative but is expensive and involves the use of animals. It can also produce varying responses depending on the strain, age and housing conditions of the rabbits. The BET, however, does not detect pyrogens other than gram-negative bacterial endotoxins and is often problematic when used to test solutions with a high protein content.\n\nTo overcome these shortcomings and satisfy the growing need for new methods prompted by the constantly increasing production of biological compounds, it is necessary to develop the novel assay for pyrogen detection.\n\nHighlightsO_LIThis novel reporter gene assay can detect different types of pyrogens, including the lipopolysaccharide of gram-negative bacteria, the lipoteichoic acid of gram-positive bacteria, and the zymosan of fungi.\nC_LIO_LIThe novel reporter gene assay is sufficiently sensitive, stable, and accurate for various applications.\nC_LI

immunology↗