Biology subjects

Gao, S.

Publications and source records attributed to Gao, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Novel Calcin peptides from Scorpion Venom Exhibit Humidity-Dependent Expression and Ryanodine Receptor Activation

Scorpions are venomous animals of considerable pharmacological potential, traditionally used in East Asia to treat cardiovascular and autoimmune diseases. However, geographical variations in the expression of their venom components remain poorly understood. We performed transcriptome profiling on scorpion samples collected from eight distinct climatic and environmental regions in East Asia, we successfully identified two distinct scorpion species: Uroctonus mordax and Mesobuthus martensii. Furthermore, we identified two novel calcins from the venoms of these species, with their expression levels showing a significant correlation to environmental humidity. Functional assays revealed that these calcins differentially potentiate [3H]ryanodine binding to RyR isoforms and induce a persistent subconductance state, suggesting distinct mechanisms of action. Our findings suggest biogeographical variation in venom composition between species and suggest that environmental humidity as a key factor influencing calcin expression. These results expand the structural diversity of the calcin family and provide a mechanistic basis for the medicinal application and ecological adaptation of scorpion venom.

molecular biology↗

bioRxiv2025

RhoV Promotes Proliferation, Migration, and MAPK Activation in Pancreatic Ductal Adenocarcinoma

Background and AimsPancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by diagnosis at advanced stages, limited therapeutic options, and frequent resistance to therapies. Although oncogenic KRAS mutations are central drivers of PDAC, alternative pathways indisputably contribute to its tumorigenesis and progression. RhoV, a member of the Rho family of small GTPases, has been implicated in tumor development in other cancer types, such as breast cancer and lung adenocarcinoma; however, its role in PDAC remains unclear. MethodsIn this study, we investigated the expression and functional impact of RhoV on PDAC. Analysis of publicly available datasets and immunohistochemical profiling of 114 PDAC patient specimens were used to evaluate the expression of RhoV in PDAC and its prognostic impact. Overexpression and CRISPR-Cas9-mediated knockout of RhoV were established in three pancreatic cancer cell lines. Functional analyses, such as cell proliferation, migration, invasion, colony formation, spheroid growth, and mouse xenograft, were used to evaluate the role of RhoV in PDAC cells. ResultsRhoV overexpression was associated with reduced overall and recurrence-free survival in public datasets and our own patient cohort. Functional assays demonstrated that RhoV overexpression promoted PDAC cell proliferation, colony formation, and spheroid growth, whereas knockout of RhoV suppressed these changes. Moreover, RhoV enhanced PDAC cell migration and invasion in vitro, accompanied by downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating induction of epithelial-mesenchymal transition. Mechanistically, RhoV overexpression activated key MAPK pathway components, including phosphorylation of ERK, JNK, and p38. In vivo, xenograft models confirmed that RhoV drives tumor growth and increases tumor burden. ConclusionThese results establish RhoV as a novel oncogenic factor in PDAC progression and highlight its potential as a biomarker and therapeutic target, warranting further investigation into combinatorial targeting strategies to overcome KRAS inhibitor resistance.

cancer biology↗

bioRxiv2025

Disgust propensity, not disgust sensitivity, shapes the reactivity of a subjective disgust circuit in humans

Disgust constitutes an evolutionary adaptive defensive-avoidance response, yet humans vary markedly in their dispositional tendency to experience disgust (disgust propensity) and in their negative appraisal of such experience (disgust sensitivity). Conceptual frameworks and neuroimaging studies suggest that these traits may differentially modulate neural responses to disgust-eliciting stimuli; however, methodological constraints have left their precise roles unresolved. Our comparably large fMRI study (n = 142) therefore aimed to systematically determine how trait disgust modulates neural responses to carefully selected and validated disgust-specific visual stimuli across varying levels of subjective disgust experience. The whole-brain voxel-wise regression analyses revealed a neural dissociation between the two disgust traits, with disgust propensity, but not disgust sensitivity, modulating disgust-related neural activity in the anterior, middle, and posterior insula, as well as the caudate, putamen, thalamus, hippocampus, and parahippocampal gyrus. Mediation and network-level analyses further supported this dissociation by showing that disgust propensity shapes disgust experience via insula - striatal - hippocampal pathways. Together, these findings provide evidence for a neurofunctional dissociation of disgust propensity and sensitivity and elucidate how trait disgust shapes subjective experiences. They further suggest that disgust propensity and the identified systems may represent promising targets for the regulation of disgust-related pathology.

neuroscience↗

bioRxiv2025

Cost-effective genomic prediction for fertility traits: A comparison of machine learning and conventional models using low-coverage sequencing in Holstein heifers

Fertility is one of the major factors affecting the efficiency of dairy herd, and genomic selection (GS) on milk yield, while ignoring fertility, has resulted in a decline in heifer fertility. Consider the cost of breeding, it is important to enhance the accuracy of GS with cost-effective way for fertility traits. This study investigates the genomic prediction (GP) of fertility traits in Holstein heifers using both machine learning (ML) and conventional methods, based on data from SNP arrays and low-coverage sequencing data. In this study, we collected 45,320 Holstein heifers with phenotype and pedigree records, from which we generated genomic data for 3,683 Holstein heifers using lcGWS. We first estimated the heritability for age at first service (AFS), gestation length (GLh) and age at first calving (AFC). We then compared the prediction performance of ML methods, kernel ridge regression (KRR), support vector regression, and random forest regression, with GBLUP, ssGBLUP and BayesR3 regarding GP accuracy and unbiasedness. Inputs for ML includes genomic relationship matrices (GRM), principal components, and SNPs. The results revealed that the heritability for the three fertility traits ranged from 0.09 to 0.48. Prediction accuracy from imputed low-coverage sequencing data was comparable to that from standard SNP chips. When both pedigree and genotypic data were used for GS, ssGBLUP yielded the highest prediction accuracy for AFS and GLh. Crucially, using only genomic data, KRR_GRM improved GP accuracy by up to 28.57% compared to GBLUP and by up to 9.46% compared to BayesR3. Our results highlight the effectiveness of low-coverage sequencing data in breeding applications and the MLs potential to enhance GP accuracy for fertility traits, offering practical insights for dairy breeding programs. ImplicationsTo improve the accuracy of GS with cost-effective way for fertility traits, this study confirms that low-coverage sequencing offers both accuracy and cost-effectiveness for fertility in Holstein heifers. The research provides a decision-making framework for breeding workers: the ssGBLUP model is optimal when combining pedigree data, while machine learning methods are superior with only genomic data. This study offers a practical tool for achieving efficient and economical genetic improvement in dairy cattle.

genomics↗

bioRxiv2025

Mitochondrial Response to Psychological Stress and Its Medial Prefrontal Biomarker Correlates

BackgroundStress response obligates increased mitochondrial activities to meet stress-induced high energy requirement. This stress-mitochondrial response process involves glucocorticoid but also multiple alternative pathways that are top-down regulated by the medial prefrontal cortex (mPFC). These pathways are important for many neuropsychiatric conditions that are sensitive to stress. However, the field lacks a reliable, clinically accessible stress-mitochondrial response paradigm to study the process in humans. MethodWe used an established psychological stress challenge combined with assaying salivary cell-free mitochondrial DNA (cf-mtDNA), thought to reflect heightened mitochondrial changes or disruptions, in 35 healthy individuals (21 males). We also explored if these stress-induced cf-mtDNA marker elevations were associated brain metabolites as measured by magnetic resonance spectroscopy (MRS), as well as high-resolution brain imaging based cortical thickness focusing on the mPFC. ResultsWe found that salivary cf-mtDNA was significant elevated immediately after the stress challenge (p=2.0x10-7) and gradually declined after. Exploratory causal analysis showed that this cf-mtDNA response was not primarily driven by cortisol response. Instead, individuals with higher baseline dACC lactate+ levels, thought to in part reflect mitochondrial dysfunctions, was significantly associated with the cf-mtDNA response (r=0.80, p<0.001). Higher mtDNA response was also significantly associated with thinner dorsomedial prefrontal cortex (r=-0.52, p=0.01). Age had a U-shape effect such that cf-mtDNA response trended lower in earlier adulthood but higher in older people, explaining 33.8% of the ct-mtDNA response variance (p=0.003). ConclusionThis stress challenge-salivary cf-mtDNA assay paradigm may offer a new, non-invasive approach to evaluate the stress-mitochondrial pathway functioning in aging, psychopharmacology, and neuropsychiatric conditions where psychological stress plays a role.

neuroscience↗

bioRxiv2025

Blood and Neuronal Extracellular Vesicle Mitochondrial Disruptions in Schizophrenia

The high energy demand of the human brain obligates robust mitochondrial energy metabolism, while mitochondrial dysfunctions have been linked to neuropsychiatric disorders including schizophrenia spectrum disorders (SSD). However, in vivo assessments that can directly inform brain mitochondrial functioning and its etiopathophysiological path to SSD remain difficult to obtain. We hypothesized that system and brain mitochondrial dysfunctions in SSD may be indexed by elevated cell-free mitochondrial DNA (cf-mtDNA) levels in the blood and in neuronal extracellular vesicles (nEVs). We also explored if these mtDNA marker elevations were associated brain metabolites as measured by magnetic resonance spectroscopy (MRS). We examined blood cf-mtDNA in 58 SSD patients and 33 healthy controls, followed by assessing nEV mtDNA and metabolite levels using MRS in a subgroup of patients and controls. We found that people with SSD had significantly elevated cf-mtDNA levels in both the blood (p=0.0002) and neuronal EVs (p=0.003) compared to controls. These mtDNA abnormalities can be linked back to brain lactate+ levels such that higher blood and nEV mtDNA levels were significantly associated with higher lactate+ levels measured at the anterior cingulate cortex (r=0.53, 0.53; p=0.008, 0.03, respectively) in SSD patients. Furthermore, higher developmental stress and trauma were significantly associated with higher cf-mtDNA levels in both the blood and neuronal EVs in SSD patients (r=0.29, 0.49; p=0.01, 0.03, respectively). In conclusion, if replicated and fully developed, blood and neuronal EV-based cell free mtDNA may provide a clinically accessible biomarker to more directly evaluate the mitochondrial hypothesis and the abnormal bioenergetics pathways in schizophrenia.

neuroscience↗

bioRxiv2025

Genome-Wide Investigation of Transcription Factor Occupancy and Dynamics Using cFOOT-seq

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences. Notably, cFOOT-seq, combined with FootTrack analysis, predicts TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.

molecular biology↗

bioRxiv2025

Structural basis of DNA N6-adenine methylation in eukaryotes

As a transcription-associated epigenetic mark abundantly present in many unicellular eukaryotes, DNA N6-adenine methylation (6mA) is maintained by the AMT1 complex. However, the structure-function relationship of the AMT1 complex remains to be fully elucidated. Here, we employ AlphaFold3 (AF3) for de novo structural modeling of the AMT1 complex, complemented by molecular dynamics simulations. Functional relevance of key structural features, especially those of the AMT1 ternary complex with the double-stranded DNA (dsDNA) substrate and the cofactor, are validated by extensive mutagenesis, both in vivo and in vitro. Our analysis reveals the structural basis for DNA substrate recognition, base flipping, and catalysis in this prototypical eukaryotic DNA 6mA-methyltransferase. It also allows us to delineate the reaction pathway for processive methylation involving translocation of the AMT1 complex along the dsDNA. As the active site is conserved across the MT-A70 family of eukaryotic N6-adenine methyltransferases, the structural insight will facilitate rational design of specific inhibitors.

molecular biology↗

bioRxiv2025

Bromodomain protein IBD1 bridges histone acetylation and H2A.Z deposition to fine-tune transcription and prevent hyperactivation in Tetrahymena thermophila

Eukaryotic gene expression is dynamically regulated through the interplay between histone modifications and chromatin remodeling, yet how these processes are coordinated remains incompletely understood. Here, we identify IBD1 as a critical adaptor that bridges histone acetylation and SWR-mediated H2A.Z deposition. We demonstrate that the bromodomain of IBD1 recognizes histone acetylation, specifically H3K9/14 di-acetylation to recruit the SWR complex subunit ARP6, ensuring precise H2A.Z incorporation into chromatin. Genetic disruption of IBD1, either by deletion or bromodomain mutation, attenuates H2A.Z occupancy at target loci, confirming its essential role in H2A.Z deposition. Strikingly, perturbation of the histone acetylation-IBD1-H2A.Z axis triggers transcriptional hyperactivation, revealing a dual function of H2A.Z in sustaining basal transcription while preventing aberrant gene induction. This defines a novel regulatory paradigm in which IBD1 couples acetyl-mark decoding to SWR-dependent H2A.Z deposition, establishing transcriptional homeostasis. Our findings resolve a central ambiguity by demonstrating that H2A.Z acts as a repressive buffer in highly acetylated regions, counterbalancing the inherent activation potential of acetylation. This study provides a framework for investigating H2A.Z functions and reveals new perspectives on how chromatin modifications and histone variants interplay in transcriptional regulation.

genetics↗

bioRxiv2025

Modeling Post-Gastrula Development via Bidirectional Pluripotent Stem Cells

The absence of stem cells capable of efficiently generating both trophoblast and epiblast lineages has hindered precise recapitulation of embryonic development. Through high-content chemical screening, we established an AL medium to generate mouse and human bidirectional pluripotent stem cells (BPSCs) characterized by concurrent OCT4/CDX2 expression. Mouse BPSCs demonstrated high plastic differentiation into trophoblast, epiblast and primitive endoderm lineages in vitro within 48 hours without exogenous induction factors and efficiently contributed to the embryo and extraembryonic tissues in vivo. Mechanistically, hyperactivation of the Wnt signaling pathway breaks the early lineage differentiation barrier by initiating a Lef1-dependent bypass. Remarkably, BPSCs can efficiently generated E8.5 embryoids that completed gastrulation and displayed advanced features such as brain development, a closed neural tube, a beating heart, somite formation, and primordial germ cells. These findings highlight BPSCs as a powerful tool for investigating early lineage specification and post-gastrulation embryonic development, with potential applications across multiple species.

developmental biology↗

bioRxiv2025

Structures of a lipin/Pah phosphatidic acid phosphatase in distinct catalytic states reveal a signature motif for substrate recognition

Lipin/Pah phosphatidic acid phosphatases (PAPs) are Mg2+-dependent enzymes that catalyze the dephosphorylation of phosphatidic acid (PA) to produce diacylglycerol. Deficiency of lipin PAP activity in humans results in inflammatory disorders such as rhabdomyolysis and Majeed syndrome. Previously, we reported the first PAP enzyme structures of Tetrahymena thermophila (Tt) Pah2 at 3.0[A] resolution. Here, we present five new higher resolution (1.95-2.40[A]) structures of Tt Pah2, in distinct catalytic states, which represent active states of catalysis, including a phospho-Asp transition state mimic, and an inactive state with a distorted active site. The structures, in conjunction with flexible docking simulations and biochemical analysis, connect two highly conserved aspartate and arginine residues in magnesium coordination and recognition of the substrate PA. Overall, this provides a structural basis for catalysis and defines a signature Asp-Arg motif in lipin/Pah PAPs that enables recognition of their lipid substrate PA, providing insight into how the HAD domain of lipin/Pah PAPs evolved to act on a membrane embedded substrate.

biochemistry↗

bioRxiv2025

A distinct class of eukaryotic demethylases safeguards the positional fidelity of N6-methyladenine (6mA)

DNA N6-methyladenine (6mA) has recently been rediscovered as an epigenetic mark in eukaryotes. Several demethylases responsible for 6mA removal have been identified in multicellular eukaryotes. However, despite the much higher abundance of 6mA in unicellular species, no such enzymes have been reported and the biological significance of 6mA removal in these organisms remain unclear. Here, we identify and functionally characterize DMT3 as a dedicated 6mA demethylase in unicellular eukaryotes. DMT3 targets both fully and hemi-methylated ApT dinucleotides on DNA. It is predominantly localized at transcription start sites (TSS), where it antagonizes the activity of the 6mA methyltransferase AMT1. Deletion or catalytic inactivation of DMT3 leads to genome-wide ectopic 6mA deposition specifically at TSS regions, driven by AMT1, and is accompanied by increased chromatin accessibility and elevated enrichment of transcription-associated histone marks. These epigenetic alterations disrupt gene expression, reduce osmotic stress resistance, and impair the initiation of sexual reproduction. Together, our findings uncover a critical 6mA demethylase in unicellular eukaryotes, and highlight the essential role of active 6mA removal in maintaining transcriptional and developmental integrity.

genetics↗

bioRxiv2025

Stabilized ion selectivity corrects activation drift in kalium channelrhodopsins

Effective optogenetic inhibition of neuronal activity requires tools that can reliably silence neurons across diverse conditions and cell types. Potassium-selective channelrhodopsins (KCRs) have emerged as promising alternatives to chloride-conducting channels for optogenetic inhibition of cellular excitability, but they exhibit dynamic ion selectivity shift under prolonged or intense illumination, causing activation. This inhibition to activation transition limits their utility for silencing circuits. Through behavioral and electrophysiological analyses in Drosophila, and C. elegans, we found that the C29D variant of KCR1 maintains the most stable potassium selectivity among tested variants. While other KCR variants showed excitation defects, KCR1-C29D consistently provided robust in vivo inhibition across cell types, illumination conditions, and species. Electrophysiological recordings revealed that while most KCRs show declining PK/PNa ratios during illumination, KCR1-C29D maintains stable ion selectivity, consistent with its robust silencing capability. This work resolves a key limitation of KCR optogenetics and shows that KCR1-C29D is a superior tool for reliable inhibition. Our findings offer valuable mechanistic and practical insights for the design of next generation optogenetic inhibitors.

neuroscience↗

bioRxiv2025

Computational Assessment of Circularization Impact on Saxitoxin G-quadruplex Aptamer Performance

Although high-affinity and high-specificity aptamers can be obtained by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), and have good activity in vitro, their performance in vivo is poor. This is mainly because of the degradation of linear nucleic acids by nucleases in body fluids and the difficulty of maintaining stable 3D structures in complex body fluid environments, which seriously hinders the clinical application of aptamers. This makes circular aptamers, which can resist degradation and are more stable in complex body fluid environments, an attractive option. However, the cyclization process may have a considerable impact on the structure of the original linear aptamer, and blind cyclization may lead to a decrease in its affinity. Here, we developed a new method to guide the cyclization of saxitoxin (STX) aptamers based on molecular computation, and verified the activity of circular aptamers through bioactivity experiments to further verify the authenticity and reliability of our methods. Consistent with the computational analysis, although circularization disrupts the G4 structure, circular aptamer not only has a higher affinity than the linear one but also provides certain protection to mice and greatly prolongs their survival, which proves the accuracy and reliability of the circular structure designed by our computational method. Our findings provide important ideas for better guidance in the design of circular aptamers and demonstrate their great potential in therapeutic applications.

biochemistry↗

bioRxiv2025

Calpain cleaves the carboxyl terminus of TRPV1 and modulates receptor tachyphylaxis

Transient receptor potential vanilloid-1 (TRPV1) plays a critical role in noxious heat sensation and pain hypersensitivity development in chronic pain. Sustained or repeated exposure to capsaicin, a classic TRPV1 agonist, induces TRPV1 desensitization. This partially accounts for the analgesic effect of capsaicin. However, the regulatory mechanisms of TRPV1 desensitization remain poorly understood. In this study, we found that capsaicin acts on TRPV1 to induce the activation of calpain, a Ca2+-sensitive cysteine protease, in a manner unrelated to cellular injury. Calpain cleaves rTRPV1 at the carboxyl terminus of G819/S820. Lack of the distal carboxyl terminus leads to reduced TRPV1 localization on the plasma membrane, potentially due to increased receptor internalization and impaired subunit assembly. This finding was corroborated by whole-cell patch clamp recordings. Additionally, the {Delta}820-838 mutant of rTRPV1 shows resistance to tachyphylaxis as induced by repetitive capsaicin stimulation. This study reveals a pivotal role for calpain in TRPV1 desensitization where its activation constrains TRPV1 channel function while simultaneously increasing its resistance to tachyphylaxis, thereby acting to maintain TRPV1 activity within an appropriate range.

neuroscience↗

bioRxiv2025

Deciphering complex interactions between LTR retrotransposons and three Papaver species using LTR_Stream

Long terminal repeat retrotransposons (LTR-RTs), a major type of class I transposable elements (TEs), are the most abundant repeat element in plants. The study of the interactions between LTR-RTs and the host genome relies on high-resolution characterization of LTR-RTs. However, for non-model species, this remains a challenge. To address this, we developed LTR_Stream for sub-lineage clustering of LTR-RTs in specific or closely related species, providing higher precision than current database-based lineage-level clustering. Using LTR_Stream, we analyzed Retand LTR-RTs in three Papaver species. Our findings show that high-resolution clustering reveals complex interactions between LTR-RTs and the host genome. For instance, we found autonomous Retand elements could spread among the ancestors of different subgenomes, like retroviruses pandemics, enriching genetic diversity. Additionally, we identified that specific truncated fragments containing transcription factors motifs such as TCP and bZIP may contribute to generation of novel TAD-like boundaries. Notably, our pre- and post-allopolyploidization comparisons show that these effects diminished after allopolyploidization, suggesting that allopolyploidization may be one of the mechanisms by which Papaver species cope with LTR-RTs. We demonstrated the potential application of LTR_Stream and provided a reference case for studying the interactions between LTR-RTs and the host genome in non-model plant species.

bioinformatics↗

bioRxiv2025

Cellular and molecular mechanism for reproductive capacity of male Mongolian cattle basing on single-cell sequencing for testis

Mongolian cattle is an excellent local breed with strong reproductive ability in Inner Mongolia of China. Spermatogenesis is a comprehensive process to decide the reproductive ability. In order to understand the cellular and molecular mechanism of spermatogenesis of Mongolian cattle, scRNA-seq was used to describe the heterogeneity of cells and gene expression profile in testis. The results showed 8 cell clusters. Among somatic cells, Sertoli cells were obviously more in young cattle than that in adult cattle, moreover, there were 4 subpopulations of Sertoli cells (SC1[~]4). In the matured Sertoli cells (SC4), up-regulated genes were mainly enriched in immune system activity, enhancing immune response, and regulating cellular processes. The top 5 marker genes (BTG2, FOSB, EGR1, JUNB, CCNL1) were highly expressed in the testes of Mongolian cattle and water buffalo compared to Holstein cattle, which were highly expressed in the developing SC1. Gain/loss-of-function of EGR1 in Sertoli cells isolated from Mongolian cattle testes positively regulated the expression of FOS and JUN. The results indicated that SC1 acted as the foundation and SC4 as the matured supportive cells and EGR1 would promote the spermatogenesis by regulating AP-1 transcription factor member FOS and JUNto maintain strong reproductive capacity of Mongolian cattle.

developmental biology↗

bioRxiv2025

Establishment of a Reverse Genetics System for the Study of Human Immune Functions in Mice

Reverse genetics approaches in mice are widely utilized to understand gene functions and their aberrations in diseases. However, limitations exist in translating findings from animal models to human physiology. Humanized mice provide a powerful bridge to understanding human physiology and mechanisms of diseases pathogenesis while maintaining the feasibility of working with small animals. Methods for generating humanized mouse models that allow scientists to probe contributions of particular genes have been rudimentary. Here, we established an efficient method for generating genetically modified human cord blood derived CD34+ cells for transplantation, resulting in humanized mice with near-complete loss of specific gene expression by the human immune system. Mice transplanted with Cas9-edited human CD34+ cells recapitulate functional consequences of specific gene losses in the human immune system. This advancement enables the development of humanized mouse models with targeted gene knockouts, offering a valuable research tool for human gene function studies in vivo.

immunology↗