Biology subjects

Yao, D.

Publications and source records attributed to Yao, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Rotten to the core - a neurofunctional signature of subjective core disgust generalizes to socio-moral contexts

While disgust originates in the hard-wired mammalian distaste response, the conscious experience of disgust in humans strongly depends on subjective appraisal and may even extend to sociomoral contexts. In a series of studies, we combined functional magnetic resonance imaging (fMRI) with machine-learning based predictive modeling to establish a comprehensive neurobiological model of subjective disgust. The developed neurofunctional signature accurately predicted momentary self-reported subjective disgust across discovery (n=78) and pre-registered validation (n=30) cohorts and generalized across core disgust (n=34 and n=26), gustatory distaste (n=30), and sociomoral (unfair offers; n=43) contexts. Disgust experience was encoded in distributed cortical and subcortical systems, and exhibited distinct and shared neural representations with subjective fear or negative affect in interoceptive-emotional awareness and conscious appraisal systems while the signatures most accurately predicted the respective target experience. We provide an accurate fMRI-signature for disgust with a high potential to resolve ongoing evolutionary debates.

neuroscience↗

bioRxiv2023

Development of compact transcriptional effectors using high-throughput measurements in diverse contexts

Human nuclear proteins contain >1000 transcriptional effector domains that can activate or repress transcription of target genes. We lack a systematic understanding of which effector domains regulate transcription robustly across genomic, cell-type, and DNA-binding domain (DBD) contexts. Here, we developed dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous targets, and tested effector function for a library containing 5092 nuclear protein Pfam domains across varied contexts. We find many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. We then confirm these findings and further map context dependencies of effectors drawn from unannotated protein regions using a larger library containing 114,288 sequences tiling chromatin regulators and transcription factors. To enable efficient perturbations, we select effectors that are potent in diverse contexts, and engineer (1) improved ZNF705 KRAB CRISPRi tools to silence promoters and enhancers, and (2) a compact human activator combination NFZ for better CRISPRa and inducible circuit delivery. Together, this effector-by-context functional map reveals context-dependence across human effectors and guides effector selection for robustly manipulating transcription.

systems biology↗

bioRxiv2023

The central renin angiotensin II system: a genetic pathway, functional decoding and selective target engagement characterization in humans

The brain renin angiotensin II system plays a pivotal role in cognition and neuropathology via the central angiotensin II type 1 receptor (AT1R), yet the lack of a biologically informed framework currently impedes translational and therapeutic progress. We combined imaging transcriptomic and meta-analyses with pharmaco-resting state fMRI employing a selective AT1R antagonist in a discovery-replication design (n=132 individuals). The AT1R was densely expressed in subcortical systems engaged in reward, motivation, stress, and memory. Pharmacological target engagement suppressed spontaneous neural activity in subcortical systems with high AT1R expression and enhanced functional network integration in cortico-basal ganglia-thalamo-cortical circuits. AT1R-regulation on functional network integration was further mediated by dopaminergic, opioid and corticotrophin-releasing hormone pathways. Overall, this work provides the first comprehensive characterization of the architecture and function of the brain renin angiotensin II system indicating that the central AT1R-mediates human cognition and behavior via regulating specific circuits and interacting with classical transmitter systems.

neuroscience↗

bioRxiv2023

A brief real-time fNIRS-informed neurofeedback training of the prefrontal cortex changes brain activity and connectivity during subsequent working memory challenge

Working memory (WM) represents a building-block of higher cognitive functions and a wide range of mental disorders are associated with WM impairments. Initial studies have shown that several sessions of functional nearinfrared spectroscopy (fNIRS) informed real-time neurofeedback (NF) allow healthy individuals to volitionally increase activity in the dorsolateral prefrontal cortex (DLPFC), a region critically involved in WM. For the translation to therapeutic or neuroenhancement applications, however, it is critical to assess whether fNIRS-NF success transfers into neural and behavioral WM enhancement in the absence of feedback. We therefore combined single-session fNIRS-NF of the left DLPFC with a randomized sham-controlled design (N = 62 participants) and a subsequent WM challenge with concomitant functional MRI. Over four runs of fNIRS-NF, the left DLPFC NF training group demonstrated enhanced neural activity in this region, reflecting successful acquisition of neural selfregulation. During the subsequent WM challenge, we observed no evidence for performance differences between the training and the sham group. Importantly, however, examination of the fMRI data revealed that - compared to the sham group - the training group exhibited significantly increased regional activity in the bilateral DLPFC and decreased left DLPFC - left anterior insula functional connectivity during the WM challenge. Exploratory analyses revealed a negative association between DLPFC activity and WM reaction times in the NF group. Together, these findings indicate that healthy individuals can learn to volitionally increase left DLPFC activity in a single training session and that the training success translates into WM-related neural activation and connectivity changes in the absence of feedback. This renders fNIRS-NF as a promising and scalable WM intervention approach that could be applied to various mental disorders.

neuroscience↗

bioRxiv2023

Compressed Perturb-seq: highly efficient screens for regulatory circuits using random composite perturbations

Pooled CRISPR screens with single-cell RNA-seq readout (Perturb-seq) have emerged as a key technique in functional genomics, but are limited in scale by cost and combinatorial complexity. Here, we reimagine Perturb-seqs design through the lens of algorithms applied to random, low-dimensional observations. We present compressed Perturb-seq, which measures multiple random perturbations per cell or multiple cells per droplet and computationally decompresses these measurements by leveraging the sparse structure of regulatory circuits. Applied to 598 genes in the immune response to bacterial lipopolysaccharide, compressed Perturb-seq achieves the same accuracy as conventional Perturb-seq at 4 to 20-fold reduced cost, with greater power to learn genetic interactions. We identify known and novel regulators of immune responses and uncover evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing GWAS or trans-eQTL studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.

systems biology↗

bioRxiv2022

Multi-center integrated analysis of non-coding CRISPR screens

The ENCODE Consortiums efforts to annotate non-coding, cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes which play a major role in health and disease. Pooled, non-coding CRISPR screens are a promising approach for systematically investigating gene regulatory mechanisms. Here, the ENCODE Functional Characterization Centers report 109 screens comprising 346,970 individual perturbations across 13.3Mb of the genome, using a variety of methods, readouts, and statistical analyses. Across 332 functionally confirmed CRE-gene links, we identify principles for screening endogenous, non-coding elements for causal regulatory mechanisms. Nearly all CREs show strong evidence of open chromatin, and targeting accessibility peak summits is a critical component of our proposed sgRNA design rules. We provide experimental guidelines to accurately detect CREs with variable, often low, transcriptional effects. We discover a previously undescribed DNA strand-bias for CRISPRi in transcribed regions with implications for screen design and analysis. Benchmarking five screen analysis tools, we find CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity sgRNAs. Together, we provide an accessible data resource, predesigned sgRNAs targeting 3,275,697 ENCODE SCREEN candidate CREs, and screening guidelines to accelerate functional characterization of the non-coding genome.

genomics↗

bioRxiv2022

CRISPR screens in 3D assembloids reveal disease genes associated with human interneuron development

The assembly of cortical circuits involves the generation and migration of cortical interneurons from the ventral to the dorsal forebrain, which has been challenging to study in humans as these processes take place at inaccessible stages of late gestation and early postnatal development. Autism spectrum disorder (ASD) and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development, but which of the hundreds of NDD genes impact interneuron generation and migration into circuits and how they mediate these effects remain unknown. We previously developed a stem cell-based platform to study human cortical interneurons in self-organizing organoids resembling the ventral forebrain and their migration using forebrain assembloids. Here, we integrate assembloid technology with CRISPR screening to systematically investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including the RNA-binding protein CSDE1 and the canonical TGF{beta} signaling activator SMAD4. Then, we ran an interneuron migration screen in [~]1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and, notably, the endoplasmic reticulum (ER)-related gene LNPK. Interestingly, we discovered that, during interneuron migration, the ER is displaced along the leading neuronal branch prior to nuclear translocation. Deletion of LNPK interfered with this ER displacement and resulted in reduced interneuron saltation length, indicating a critical role for the ER in this migratory process. Taken together, these results highlight how this versatile CRISPR-assembloid platform can be used to systematically map disease genes onto early stages of human neural development and to reveal novel mechanisms regulating interneuron development.

neuroscience↗

bioRxiv2022

Death-seq identifies regulators of cell death and senolytic therapies

Selectively ablating senescent cells ("senolysis") is an evolving therapeutic approach for age-related diseases. Current senolytics are limited to local administration by potency and side effects. While genetic screens could identify senolytics, current screens are underpowered for identifying genes that regulate cell death due to limitations in screen methodology. Here, we establish Death-seq, a positive selection CRISPR screen optimized to identify enhancers and mechanisms of cell death. Our screens identified synergistic enhancers of cell death induced by the known senolytic ABT-263, a BH3 mimetic. SMAC mimetics, enhancers in our screens, synergize with ABT-199, another BH3 mimetic that is not senolytic alone, clearing senescent cells in models of age-related disease while sparing human platelets, avoiding the thrombocytopenia associated with ABT-263. Death-seq enables the systematic screening of cell death pathways to uncover molecular mechanisms of regulated cell death subroutines and identify drug targets for diverse pathological states such as senescence, cancer, and neurodegeneration.

cell biology↗

bioRxiv2022

Harmonized-Multinational qEEG Norms (HarMNqEEG)

This paper extends our frequency domain quantitative electroencephalography (qEEG) methods pursuing higher sensitivity to detect Brain Developmental Disorders. Prior qEEG work lacked integration of cross-spectral information omitting important functional connectivity descriptors. Lack of geographical diversity precluded accounting for site-specific variance, increasing qEEG nuisance variance. We ameliorate these weaknesses. i) Create lifespan Hermitian Riemannian multinational qEEG norms for cross-spectral tensors. These norms result from the HarMNqEEG project fostered by the Global Brain Consortium. We calculate the norms with data from 9 countries, 12 devices, and 14 studies, including 1564 subjects. Instead of raw data, only anonymized metadata and EEG cross-spectral tensors were shared. After visual and automatic quality control developmental equations for the mean and standard deviation of qEEG traditional and Hermitian Riemannian descriptive parameters were calculated using additive mixed-effects models. We demonstrate qEEG "batch effects" and provide methods to calculate harmonized z-scores. ii) We also show that the multinational harmonized Hermitian Riemannian norms produce z-scores with increased diagnostic accuracy to predict brain dysfunction at school-age produced by malnutrition only in the first year of life. We provide data and software for constructing norms. iii) We offer open code and data to calculate different individual z-scores from the HarMNqEEG dataset. These results contribute to developing bias-free, low-cost neuroimaging technologies applicable in various health settings. HighlightsO_LIWe create lifespan Hermitian Riemannian qEEG norms for cross-spectral tensors. C_LIO_LIThe norms are based on 9 countries, 12 devices, and 14 studies, with 1564 subjects. C_LIO_LIWe demonstrate qEEG "batch effects", providing harmonization methods to remove them. C_LIO_LIMultinational harmonized z-scores increase diagnostic accuracy of brain dysfunction. C_LIO_LIData and software are available for norm and individual z-scores calculation. C_LI

neuroscience↗

bioRxiv2021

The different brain areas occupied for integrating information of hierarchical linguistic units: a study based on EEG and TMS

Human linguistic units are hierarchical, and our brain responds differently when processing linguistic units during sentence comprehension, especially when the modality of the received signal is different (auditory, visual, or audio-visual). However, it is unclear how the brain processes and integrates language information at different linguistic units (words, phrases, and sentences) provided simultaneously in audio and visual modalities. To address the issue, we presented participants with sequences of short Chinese sentences through auditory or visual or combined audio- visual modalities, while electroencephalographic responses were recorded. With a frequency tagging approach, we analyzed the neural representations of basic linguistic units (i.e., characters/monosyllabic words) and higher-level linguistic structures (i.e., phrases and sentences) across the three modalities separately. We found that audio-visual integration occurs at all linguistic units, and the brain areas involved in the integration varied across different linguistic levels. In particular, the integration of sentences activated the local left prefrontal area. Therefore, we used continuous theta-burst stimulation (cTBS) to verify that the left prefrontal cortex plays a vital role in the audio-visual integration of sentence information. Our findings suggest the advantage of bimodal language comprehension at hierarchical stages in language-related information processing and provide evidence for the causal role of the left prefrontal regions in processing information of audio-visual sentences.

neuroscience↗

bioRxiv2021

Crystal structure of a constitutive active mutant of adenosine A2A receptor

Previously we reported a common activation pathway of the class A G protein-coupled receptors (GPCRs) in which a series of conserved residues/motifs undergo conformational change during extracellular agonist binding and finally induce the coupling of intracellular G protein, and successfully predicted several novel constitutive active or inactive mutations for A2A adenosine receptor (A2AAR) through this mechanism (Zhou et al., 2019). Here we determined the crystal structure of a typical A2AAR constitutive active mutant I92N in complex with agonist UK-432097 to reveal the molecular mechanism of mutation-induced constitutive activity. The mutated I92N forms a hydrophilic interaction network with nearby residues including W6.48 of the CWxP motif, which is absent in the wild type A2AAR. Although the mutant structure is overall similar to the previously determined intermediate state A2AAR structure (PDB ID: 3QAK), the molecular dynamics simulations suggested that the I92N mutant stabilizes the metastable intermediate state through the hydrophilic interaction network and favors the receptor conformational transition towards the active state. This research provides a structural template toward the special pharmacological outcome triggered by conformational mutation and sheds light on future structural or pharmacological studies among class A GPCRs.

molecular biology↗

bioRxiv2021

Differential Binding Kinetics for Evaluating Immune Checkpoint Inhibitors in Serum

The ability to characterize the binding kinetics of drug-target interactions in a biologically relevant matrix, such as serum or plasma, remains a fundamental challenge in drug discovery. We apply a novel label-based giant magnetoresistance (GMR) biosensor platform to measure protein binding kinetics and affinities of drug-target pairs in buffer and different levels of serum. Specifically, we evaluate three well-established immune checkpoint inhibitors, pembrolizumab, nivolumab and atezolizumab and compare the results with label-free kinetic platforms: surface plasmon resonance (SPR) and bio-layer interferometry (BLI). Labeling of analytes does not affect their association and dissociation rates (on and off rates) from GMR biosensors which enables kinetic measurements in biologically relevant matrices. Only the GMR biossensors is consistently suitable for measuring binding kinetics in up to 80% serum. The faster and different off-rates of the three immune checkpoint inhibitors in the presence of serum should be considered when modeling their pharmacological performance. TeaserWe reveal the effects of serum on binding kinetics of antibody drugs, relevant to the pharmacological performance of immunotherapeutic.

bioengineering↗

bioRxiv2021

Leveraging gene co-regulation to identify gene sets enriched for disease heritability

Identifying gene sets that are associated to disease can provide valuable biological knowledge, but a fundamental challenge of gene set analyses of GWAS data is linking disease-associated SNPs to genes. Transcriptome-wide association studies (TWAS) can be used to detect associations between the genetically predicted expression of a gene and disease risk, thus implicating candidate disease genes. However, causal disease genes at TWAS-associated loci generally remain unknown due to gene co-regulation, which leads to correlations across genes in predicted expression. We developed a new method, gene co-regulation score (GCSC) regression, to identify gene sets that are enriched for disease heritability explained by the predicted expression of causal disease genes in the gene set. GCSC regresses TWAS chi-square statistics on gene co-regulation scores reflecting correlations in predicted gene expression; GCSC determines that a gene set is enriched for disease heritability if genes with high co-regulation to the gene set have higher TWAS chi-square statistics than genes with low co-regulation to the gene set, beyond what is expected based on co-regulation to all genes. We verified via simulations that GCSC is well-calibrated, and well-powered to identify gene sets that are enriched for disease heritability explained by predicted expression. We applied GCSC to gene expression data from GTEx (48 tissues) and GWAS summary statistics for 43 independent diseases and complex traits (average N =344K), analyzing a broad set of biological pathways and specifically expressed gene sets. We identified many enriched gene sets, recapitulating known biology. For Alzheimers disease, we detected evidence of an immune basis, and specifically a role for antigen presentation, in analyses of both biological pathways and specifically expressed gene sets. Our results highlight the advantages of leveraging gene co-regulation within the TWAS framework to identify gene sets associated to disease.

genetics↗

bioRxiv2021

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens

The trafficking of specific protein cohorts to the correct subcellular location at the correct time is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or FACS. To broadly empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identify genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in the mislocalization and destabilization of mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

bioengineering↗

bioRxiv2021

Exploring the dynamic mechanisms of steady-state visual evoked potentials at the whole-brain level: evidence from a computational study

Periodic visual stimulation can induce stable steady-state visual evoked potentials (SSVEPs) distributed in multiple brain regions and has potential applications in both neural engineering and cognitive neuroscience. However, the underlying dynamic mechanisms of SSVEPs at the whole-brain level are still not completely understood. Here, we addressed this issue by simulating the rich dynamics of SSVEPs with a large-scale brain model designed with constraints of neuroimaging data acquired from the human brain. By eliciting activity of the occipital areas using an external periodic stimulus, our model was capable of replicating both the spatial distributions and response features of SSVEPs that were observed in experiments. In particular, we confirmed that alpha-band (8-12 Hz) stimulation could evoke stronger SSVEP responses; this frequency sensitivity was due to nonlinear entrainment and resonance, and could be modulated by endogenous factors in the brain. Interestingly, the stimulus-evoked brain networks also exhibited significant superiority in topological properties near this frequency-sensitivity range, and stronger SSVEP responses were demonstrated to be supported by more efficient functional connectivity at the neural activity level. These findings not only provide insights into the mechanistic understanding of SSVEPs at the whole-brain level but also indicate a bright future for large-scale brain modeling in characterizing the complicated dynamics and functions of the brain.

neuroscience↗

bioRxiv2021

Function-structure Coupling：White matter fMRI hyper-activation associates with structural integrity reductions in schizophrenia

BackgroundWhite matter (WM) microstructure deficit may be an underlying factor in the brain dysconnectivity hypothesis of schizophrenia using diffusion tensor imaging (DTI). However, WM dysfunction is unclear in schizophrenia. This study aimed to investigate the association between structural deficits and functional disturbances in major WM tracts in schizophrenia. MethodsUsing functional magnetic resonance imaging (fMRI) and DTI, we developed the skeleton-based white matter functional analysis, which could achieve voxel-wise function-structure coupling by projecting the fMRI signals onto a skeleton in WM. We measured the fractional anisotropy (FA) and WM low-frequency oscillation activation and their couplings in ninety-three schizophrenia patients and 122 healthy controls (HCs). An independent open database (62 schizophrenia patients and 71 HCs) was used to test the reproducibility. Finally, associations between WM activations and five behaviour assessment categories (cognition, emotion, motor, personality and sensory) were examined. ResultsThis study revealed a reversed pattern of structure and function in frontotemporal tracts, as follows. (1) WM hyper-activation was associated with reduced FA in schizophrenia. (2) The function-structure association was positive in healthy controls but negative in schizophrenia patients. Furthermore, function-structure dissociation was exacerbated by long illness duration and severe negative symptoms. (3) WM activations were significantly related to cognition and emotion. ConclusionsThis study indicated function-structure dys-coupling, with higher functional activation and reduced structural integration in frontotemporal WM, which may reflect a potential mechanism in WM neuropathologic processing of schizophrenia.

neuroscience↗

bioRxiv2020

EGCG-modified bone graft to modulate the recruitment of M1 macrophage and alleviate the forming of fibrous capsule

In response to current trends in the modification of guided bone regeneration (GBR) materials, we aimed to build upon our previous studies on epigallocatechin-3-gallate (EGCG) by immersing a commonly used bone graft primarily composed of hydroxyapatite (HA) in EGCG solution, expecting to obtain superior bone-material integration after implantation. Bone grafts are commonly used for bone repair, in which the bone extracellular matrix is stimulated to promote osteogenesis. However, due to its pro-fibrosis effect, this osteoconductive material commonly exhibits implant failure. In addition to providing a basic release profile of EGCG-modified bone graft (E-HA) to clarify the relationship between this material and the environment, we have examined the integration effect via subcutaneous implantation experiments. In this manner, we have assessed the aggregation of pro-inflammatory macrophages, initial angiogenesis, the formation of fibrous capsules, and an enhanced cell viability observed in cultured RAW 264.7 cells. Among these results, we focus on pro-inflammatory macrophages due to their close relationship with fibrosis, which is the most important process in the immune response. Immunofluorescent staining results showed that E-HA substantially compromised the formation of fibrous capsules in hematoxylin-eosin-stained sections, which exhibited less pro-inflammatory macrophage recruitment; meanwhile, the cell viability and primary angiogenesis were improved. This work lays the foundation for future studies on GBR.

immunology↗

bioRxiv2020

Live poultry feeding and trading network and the transmission of avian influenza A(H5N6) virus in a large city in China, 2014-2015

To understand clearly the mechanism of H5N6 transmission, the role of a live poultry feeding and trading network should be explored deeply. However, there is little data to show the network in an area. In this study, we performed a field epidemiological investigation to collect the numbers of farms, wholesale markets, and live poultry retail markets, and the numbers of purchased (from where) and sold (to where) live birds in Changsha City, China in 2014. We also collected samples from the network in the city from January 2014 to March 2015, including the LPMs visited by the patient known to be infected with A(H5N6) virus, and sequenced the genomes of 10 A(H5N6) viral strains isolated from these environmental samples, to determine the source of virus that infected the patient reported in Changsha. Additionally, we collected and analyzed A(H5N6) virus genome sequence isolated from humans, poultry, and LPMs registered in National Center for Biotechnology Information (NCBI; NIH, Bethesda, MD, USA) 14, to determine the source of other human infections with A(H5N6) virus reported in China. Changsha City in China has a large live poultry feeding and trading network (LPFTN) which had 665 farms, 5 wholesale markets, and 223 live poultry retail markets in 2014. The network covered nine provinces and purchased and sold more than 150000 live birds every day. About 840 environmental samples collected from the LPFTN network from January 2014 to March 2015. About 8.45% (71/840) environmental samples were shown to be positive for N6 and 10 full genome sequences of H5N6 virus were analyzed. We performed phylogenetic analyses and virus characterization, which demonstrated that the H5N6 viral strains isolated from Chinese patients were closely related to those isolated from the poultry and environmental samples obtained from the LPFTN network. This indicates that the network with a large volume of live poultry provides a platform for the transmission of H5N6, and provides an infectious pool which makes the people in high risk. ImportanceAvian influenza A(H5N6) virus is an emerging threat to public health. With several human cases reported in recent years, it has become a dominant avian influenza virus (AIV) subtype in China. Live poultry feeding and trading network (LPFTN) contributes to the presence of novel AIV. By a field epidemiological investigation in the complex LPFTN in Changsha, we demonstrated that the H5N6 viral strains isolated from Chinese patients were closely related to those isolated from the poultry and environmental samples obtained from the network, suggesting the LPFTN with a large volume of live poultry provides a platform for the transmission of H5N6 and creates an infectious pool which makes people in high risk. Considering the wide circulation and dynamic reassortment of this highly pathogenic avian influenza H5N6 virus, it should be carefully monitored in poultry and humans due to the pandemic potential.

molecular biology↗