Biology subjects

Li, M.-H.

Publications and source records attributed to Li, M.-H..

16 recordsLinked to original sources

bioRxiv2025

Targeting STING-ATM axis overcomes radiotherapy-induced immune suppression and restores anti-tumor immunity in nasopharyngeal carcinoma

BackgroundRadiotherapy (RT) is a standard treatment for nasopharyngeal carcinoma (NPC), however, RT-induced immune suppression and tumor radioresistance limit durable cancer control, with approximately 20% of patients developing recurrence or metastasis after treatment. We investigated the molecular effects of RT on the immune landscape of NPC and identified a key role of the STING-ATM-MPO axis in RT-induced immune suppression, which is associated with regulatory T cell (Treg) expansion and radioresistance. MethodsWe analyzed leukocyte counts from 653 NPC patients before treatment and at multiple time points (week 1-2, week 3-4, and week 5-6) after RT. Single-cell RNA sequencing (scRNA-seq) was performed on peripheral blood mononuclear cell (PBMC) samples from stage I NPC patients (T1N0M0) treated with RT alone. Blood samples were collected before and after 18 Gy RT (in 2 Gy fractions). A randomized six-arm syngeneic mouse tumor model and in vitro cell studies were used to investigate interactions among STING-ATM signaling, immune modulation, and tumor response. ResultsIn NPC patients, RT suppressed cGAS-STING signaling in monocytes and dendritic cells but promoted immunosuppressive programs, including PD-1 signaling, checkpoint expression, regulatory T cell expansion, and upregulation of TGF{beta}1, IL-10, VEGF, PDGF, and MDSC-associated pathways. In a syngeneic mouse model, co-treatment with the STING agonist diABZI reversed these effects and improved tumor control. Bulk RNA sequencing confirmed that RT suppressed cGAS-STING and ATM signaling in both spleens and tumors. Combination treatment restored this signaling, enhanced neutrophil degranulation, and inhibited PD-1 signaling and immune checkpoints. Immunohistochemistry showed that diABZI increased ATM activation, MPO expression, and reduced Treg infiltration in spleens and tumors. ATM activation correlated positively with MPO and inversely with FOXP3 immunoreactivity, suggesting that STING-ATM-MPO activation counteracts Treg-mediated immune suppression. In vitro, diABZI activated STING-ATM signaling in PBMCs, THP-1 cells, and Jurkat T cells, while inhibiting NPC cell growth partly via CYLD upregulation. ConclusionsThese findings link RT-induced suppression to inhibition of cGAS-STING signaling. Activation of STING-ATM-MPO axis counteracts Treg-mediated immune suppression, supporting the therapeutic potential of STING agonists as radiosensitizers in NPC.

cancer biology↗

bioRxiv2025

Super-giga and tiny orchid genomes illuminate evolution of Orchidaceae

Orchidaceae (orchids) is commonly known as one of the largest families of seed plants, and grow in an extensive range of habitats worldwide. In the present study, we generated chromosome-level reference genomes for two orchids using a combination of PacBio, Illumina, and Hi-C sequencing, Cypripedium singchii has the largest genome and chromosomes among the sequenced species so far, with a genome size of 43.19 Gb (1C) with ten chromosomes, and Apostasia fujianica has the smallest known genome and chromosomes in Orchidaceae, with a genome size of 340.90 Mb (1C) with 35 chromosomes. We predicted a total of 32,412 and 21,724 protein-coding genes for C. singchii and A. fujianica, respectively. The overall BUSCO score was 85.01% for C. singchii and 91.80% in A. fujianica. Based on protein-coding sequences from 55 conserved single-copy families across 21 plant species, we constructed a high-confidence phylogenetic tree and estimated the divergence times. The high-quality genomes of super-giga and tiny orchids offer key insight for future evolutionary researches.

genomics↗

bioRxiv2025

PANDA: A simple and affordable chamber system for measuring the whole-plant net CO2 flux

The carbon (C) balance of plants is the sum of all source and sink processes. However, due to methodological limitations, most studies focus predominantly on measurements of leaf-level assimilation and respiration, with less attention given to these processes in heterotrophic organs or the whole-plant level. As a result, knowledge of the whole-plant net C balance is scarce, limiting our understanding of the dynamics between C source and sink activities. Therefore, we developed an easily reproducible chamber system for continuous measurements of whole-plant net CO2 fluxes. We present the obtained dynamics of net CO2 fluxes of several C3 and CAM species, including germinating Quercus robur, over several days, as well as the whole-plant net CO2 flux temperature response of Q. robur seedlings, identifying the temperature thresholds at which they shift from a net CO2 sink to source. We show distinct diel patterns of net CO2 fluxes in C3 plants, likely driven by a dynamic diurnal up- and downregulation of sink activities in woody C3 plants. These patterns appear temperature-driven, suggesting a dynamic response of plants sink and source activity to environmental drivers. Our results highlight the importance of whole-plant C balance measurements for understanding plant responses to environmental conditions.

plant biology↗

bioRxiv2025

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

biochemistry↗

bioRxiv2025

Shifting the PPARγ conformational ensemble towards a transcriptionally repressive state improves covalent inhibitor efficacy

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates transcription in response to ligand binding at an orthosteric pocket within the ligand-binding domain (LBD). We previously showed that two covalent ligands, T0070907 and GW9662--extensively used as PPAR{gamma} inhibitors to assess off-target activity--weaken but do not completely block ligand binding via an allosteric mechanism associated with pharmacological inverse agonism (Shang et al., 2024). These covalent inhibitors shift the LBD towards a repressive conformation, where the activation function-2 (AF-2) helix 12 occupies the orthosteric pocket, competing with orthosteric ligand binding. Here, we provide additional support for this allosteric mechanism using two covalent inverse agonists, SR33065 and SR36708, which better stabilize the repressive LBD conformation and are more effective inhibitors of--but also do not completely inhibit--ligand cobinding. Furthermore, we show that ligand cobinding can occur with a previously reported PPAR{gamma} dual-site covalent inhibitor, SR16832, which appears to weaken ligand binding through a direct mechanism independent of the allosteric mechanism. These findings underscore the complex nature of the PPAR{gamma} LBD conformational ensemble and highlight the need to develop alternative methods for designing more effective covalent inhibitors.

biochemistry↗

bioRxiv2025

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of -synuclein (S) are hallmarks of synucleinopathies, including Parkinsons Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that S lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. We used non-canonical amino acid (ncAA) mutagenesis to express and purify all 12 currently known disease-relevant variants of S and studied their binding to membranes as well as their aggregation propensities. We found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric S found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K80 could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K80 acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K80 and other inhibitory sites for therapeutic benefit.

biochemistry↗

bioRxiv2024

Telomere-to-telomere sheep genome assembly reveals new variants associated with wool fineness trait

Ongoing efforts to improve sheep reference genome assemblies still leave many gaps and incomplete regions, resulting in a few common failures and errors in sheep genomic studies. Here, we report a complete, gap-free telomere-to-telomere (T2T) genome of a ram (T2T-sheep1.0) with a size of 2.85 Gb, including all autosomes and chromosomes X and Y. It adds 220.05 Mb of previously unresolved regions (PURs) and 754 new genes to the most updated reference assembly, ARS-UI_Ramb_v3.0, and contains four types of repeat units (SatI, SatII, SatIII, and CenY) in the centromeric regions. T2T-sheep1.0 exhibits a base accuracy of >99.999%, corrects several structural errors in previous reference assemblies, and improves structural variant (SV) detection in repetitive sequences. We identified 192,265 SVs, including 16,885 new SVs in the PURs, from the PacBio long-read sequences of 18 global representative sheep. With the whole-genome short-read sequences of 810 wild and domestic sheep representing 158 global populations and seven wild species, the use of T2T-sheep1.0 as the reference genome has improved population genetic analysis based on [~]133.31 million SNPs and 1,265,266 SVs, including 2,664,979 novel SNPs and 196,471 novel SVs. T2T-sheep1.0 improves selective tests by detecting several novel genes and variants, including those associated with domestication (e.g., ABCC4) and selection for the wool fineness trait (e.g., FOXQ1) in tandemly duplicated regions.

genomics↗

bioRxiv2024

Telomere-to-telomere genome assembly of a male goat reveals novel variants associated with cashmere traits

A complete goat (Capra hircus) reference genome enhances analyses of genetic variation, thus providing novel insights into domestication and selection in goats and related species. Here, we assembled a telomere-to-telomere (T2T) gap-free genome (2.86 Gb) from a cashmere goat (T2T-goat1.0), which included a Y chromosome of 20.9 Mb. With a base accuracy of >99.999%, T2T-goat1.0 corrected numerous genome-wide structural and base errors in previous assemblies and added 288.5 Mb of previously unresolved regions (PURs) and 446 newly assembled genes to the reference genome. We sequenced the genomes of five representative goat breeds for PacBio reads, and used T2T-goat1.0 as a reference to identify a total of 63,417 structural variations (SVs) with up to 4711 SVs (7.41%) in the PURs and 507 overlapping genes significantly enriched (Padj. < 0.05) in the keratin filament pathway. T2T-goat1.0 was applied in population analyses of global wild and domestic goats, which revealed 32,419 SVs and 25,397,794 SNPs, including 870 SVs and 545,026 SNPs in the PURs. Also, our analyses revealed a set of novel selective variants and genes associated with domestication (e.g., NKG2D and ABCC4) and cashmere traits (e.g., ABCC4 and ASIP).

genomics↗

bioRxiv2024

Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

BackgroundCoronavirus disease 2019 (COVID-19) is an immune-related disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The complete pathogenesis of the virus remains to be determined. Unraveling the molecular mechanisms governing SARS-CoV-2 interactions with host cells is crucial for the formulation of effective prophylactic measures and the advancement of COVID-19 therapeutics. MethodsWe analyzed human lung single-cell RNA sequencing dataset to discern the association of butyrophilin subfamily 3 member A2 (BTN3A2) expression with COVID-19. The BTN3A2 gene edited cell lines and transgenic mice were infected by live SARS-CoV-2 in a biosafety level 3 (BSL-3) laboratory. Immunoprecipitation, flow cytometry, biolayer interferometry and competition ELISA assays were performed in BTN3A2 gene edited cells. We performed quantitative real-time PCR, histological and/or immunohistochemical analyses for tissue samples from mice with or without SARS-CoV-2 infection. FindingsThe BTN3A2 mRNA level was correlated with COVID-19 severity. BTN3A2 expression was predominantly identified in epithelial cells, elevated in pathological epithelial cells from COVID-19 patients and co-occurred with ACE2 expression in the same lung cell subtypes. BTN3A2 targeted the early stage of the viral life cycle by inhibiting SARS-CoV-2 attachment through interactions with the receptor-binding domain (RBD) of the Spike protein and ACE2. BTN3A2 inhibited ACE2-mediated SARS-CoV-2 infection by reducing ACE2 in vitro and in vivo. InterpretationThese results reveal a key role of BTN3A2 in the fight against COVID-19. Identifying potential monoclonal antibodies which mimic BTN3A2 may facilitate disruption of SARS-CoV-2 infection, providing a therapeutic avenue for COVID-19. FundingThis study was supported by the National Natural Science Foundation of China (32070569, U1902215, and 32371017), the CAS "Light of West China" Program, and Yunnan Province (202305AH340006). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSOur understanding of the pathogenesis of COVID-19, especially key molecular events in the early stage of viral infection, remains incompletely albeit we witnessed many progresses. This knowledge gap hinders the finding for effective and specific antiviral agents against SARS-CoV-2. The entry of SARS-CoV-2 is mediated by the entry receptor angiotensin-converting enzyme 2 (ACE2) and is affected by host antiviral defenses. Targeting these universal host factors required for virus replication is the most promising approach for effective prevention and treatment of COVID-19. Added value of this studyOur study revealed that BTN3A2, a primate-specific gene, acts as a potent inhibitor of SARS-CoV-2 infection in vitro and in vivo. The up-regulation of BTN3A2 upon SARS-CoV-2 infection competed with the ACE2 receptor for binding to the Spike protein, subsequently reducing ACE2 expression and ACE2-mediated SARS-CoV-2 entry. Implications of all the available evidenceThese data highlighted that BTN3A2 as a novel host factor with protective effects against SARS-CoV-2 infection. The BTN3A2 holds considerable potential as a therapeutic drug for mitigating the impact of SARS-CoV-2 and its variants of concern (VOCs).

cell biology↗

bioRxiv2023

A time-resolved multi-omics atlas of transcriptional regulation in response to high-altitude hypoxia across whole-body tissues

High-altitude hypoxia acclimatization requires whole-body physiological regulation in highland immigrants, but the underlying genetic mechanism has not been clarified. Here we used sheep as an animal model for plain-to-plateau transplantation. We generated multi-omics data including time-resolved bulk RNA-Seq, ATAC-Seq and single-cell RNA-Seq from multiple tissues as well as phenotypic data from 20 bio-indicators. We characterized transcriptional changes of all genes in each tissue, and examined multi-tissue temporal dynamics and transcriptional interactions among genes. In particular, we identified critical functional genes regulating the short response to hypoxia in each tissue (e.g., PARG in the cerebellum and HMOX1 in the colon). We further identified TAD-constrained cis-regulatory elements, which suppressed the transcriptional activity of most genes under hypoxia. Phenotypic and transcriptional evidence indicated that antenatal hypoxia could improve hypoxia tolerance in offspring. Furthermore, we provided time-series expression data of candidate genes associated with human mountain sickness (e.g., BMPR2) and high-altitude adaptation (e.g., HIF1A). Our study provides valuable resources and insights for future hypoxia-related studies in mammals.

genetics↗

bioRxiv2023

Genomic analyses of Asiatic Mouflon in Iran provide insights into the domestication and evolution of sheep

BackgroundAsiatic mouflon (O. gmelini) consists of several subspecies mainly distributed in Armenia, southern Azerbaijan, Cyprus, northern, southern, and western regions of Iran, and eastern and central regions of Turkey nowadays. Genome analyses of Asiatic mouflon in Iran revealed that they could have diverged from the direct ancestor of domestic sheep, and showed genetic introgression into domestic sheep after domestication. However, the impact of the Asiatic mouflon subspecies in Iran on sheep domestication remains unclear. ResultsHere, we conducted a comprehensive population genomics analysis of Asiatic mouflons in Iran with 780 whole-genome sequences, 767 whole mitogenomes, and 239 Y chromosomes. Whole-genome sequence analyses revealed two subpopulations of the Asiatic mouflons in Iran: O. gmelini_2 limited in Kabudan and O. gmelini_1 over a wide geographic area. Phylogenetic analyses of the Asiatic mouflons in Iran based on uniparental variants revealed a monophyletic lineage with the mitochondrial haplogroups C+E, and clustered into a monophyletic with Y-chromosomal lineage HY2 of sheep. Additionally, introgression tests detected significant signals of genetic introgression from O. gmelini_2 to four sheep populations (e.g., Garut, Garole, Bangladeshi, and Sumatra) in South and Southeast Asia. In the four sheep populations, selective tests and introgression signals revealed that the wild introgression could have contributed to their small body size and local adaptation to the hot and humid environments in the Indian Peninsula. ConclusionsOur results suggested that the maternal haplogroups C+E and paternal lineage HY2 could have originated from the Asiatic mouflon in Iran. Also, our findings provide new insights into sheep domestication and subsequent introgressions from wild ancestors to domestic populations.

evolutionary biology↗

bioRxiv2021

Acute silencing uncovers multiple forms of activity-dependent neuronal survival in the mature entorhinal cortex

Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Many circuits that degenerate in disease are shaped by neural activity during development, raising the possibility that mechanisms governing early cell loss may be misused when activity is compromised in the mature brain. Here we show that electrical activity and synaptic transmission are both required for neuronal survival in the adult entorhinal cortex, but these silencing methods trigger distinct means of degeneration in the same neuronal population. Competition between active and inactive cells drives axonal disintegration caused by synaptic inhibition, but not axon retraction due to electrical suppression. These findings suggest that activity-dependence may persist in some areas of the adult brain long after developmental critical periods have closed. We speculate that lifelong plasticity required to support memory may render entorhinal neurons vulnerable to prolonged activity changes in disease.

neuroscience↗

bioRxiv2021

The Fluorescent Dye 1,6-Diphenyl-1,3,5-Hexatriene Binds to Amyloid Fibrils Formed by Human Amylin and Provides a New Probe of Amylin Amyloid Kinetics

The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (also known as islet amyloid polypeptide) in solution and this results in a 100-fold increase in DPH fluorescence for a sample of 20M human amylin and 0.25 M DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, non-fibrillar human amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined and the time course was independent of the range of DPH concentrations tested (0.25 to 5 M). Maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin pre-fibril oligomers are discussed. Human amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH amylin amyloid interactions. DPH may be especially useful for studies on mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.

biophysics↗

bioRxiv2021

Preparation of Asymmetric Vesicles with Trapped CsCl Avoids Osmotic Imbalance, Non-Physiological External Solutions, and Minimizes Leakage

The natural asymmetry of cellular membranes influences their properties. In recent years, methodologies for preparing asymmetric vesicles have been developed that rely on the methyl--cyclodextrin catalyzed exchange of lipids between donor lipid multilamellar vesicles and acceptor lipid unilamellar vesicles, and the subsequent separation of the, now asymmetric, acceptor vesicles from the donors. Isolation is accomplished by pre-loading acceptor vesicles with a high concentration of sucrose, typically 25% (w/w), and separating from donor and cyclodextrin by sucrose gradient centrifugation. We found that when the asymmetric vesicles were dispersed under hypotonic conditions using physiological salt solutions, there was enhanced leakage of an entrapped probe, 6-carboxyfluorescein. Studies with symmetric vesicles showed this was due to osmotic pressure and was specific to hypotonic solutions. Inclusion of cholesterol partly reduced leakage but did not completely eliminate it. To avoid having to use hypotonic conditions or to suspend vesicles at non-physiological solute concentrations to minimize leakage, a method for preparing asymmetric vesicles using acceptor vesicle-entrapped CsCl at a physiological salt concentration (100 mM) was developed. Asymmetric vesicles prepared with the entrapped CsCl protocol were highly resistant to 6-carboxyfluorescein.

biophysics↗

bioRxiv2020

Soil nutrient availability alters tree carbon allocation dynamics during drought

Drought alters allocation patterns of carbon (C) and nutrients in trees and eventually impairs tree functioning. Elevated soil nutrient availability might alter the response of trees to drought. We hypothesize that increased soil nutrient availability stimulates root metabolism and carbon allocation to belowground tissues under drought stress. To test this hypothesis, we subjected three-year-old Pinus sylvestris saplings in open-top cambers during two subsequent years to drought using three different water treatments (100%, 20% and 0% plant available water in the soil) and two soil nutrient regimes (ambient and nitrogen-phosphorus-potassium (N-P-K) fertilization corresponding to 5 g N/m2/yr) and released drought thereafter. We conducted a 15N and 13C labelling experiment during the peak of the first-year drought by injecting 15N labelled fertilizer in the soil and exposing the tree canopies to 13C labelled CO2. The abundance of the N and C isotopes in the roots, stem and needles was assessed during the following year. C uptake was slightly lower in drought stressed trees, and extreme drought inhibited largely the N uptake and transport. Carbon allocation to belowground tissues was decreased under drought, but not in combination with fertilization. Our results indicate a potential positive feedback loop, where fertilization improved the metabolism and functioning of the roots, stimulating the source activity and hence C allocation to belowground tissues. This way, soil nutrients compensated for drought-induced loss of root functioning, mitigating drought stress of trees.

plant biology↗

bioRxiv2019

Transcriptome analysis reveals the importance of the immune system during early pregnancy in sheep (Ovis aries)

The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. While ovulation rate is the major determinant of litter size in sheep, interactions among the conceptus, corpus luteum and endometrium are essential for pregnancy success. Here, we performed a comparative transcriptome study by sequencing total mRNA from corpus luteum (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses, and mapping the RNA-Seq reads to the latest Rambouillet reference genome. A total of 21,287 genes were expressed in our dataset. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (STAR, CYP11A1, and HSD3B1) and embryo implantation (eg. TIMP1, TIMP2 and TCTP). Among the list of differentially expressed genes, a group of sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs), solute carriers (SLC13A5, SLC15A2, SLC44A5) and chemokines (CCL5, CXCL13, CXCL9) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 genes and two non-coding RNAs (ncRNA) were differentially expressed in breed-wise comparisons owing to flushing diet effect. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including SIGLEC13, SIGLEC14, SIGLEC6, MRP4, and CA5A). Importantly, functional analysis of differentially expressed genes suggested that Finnsheep have a better immune system than Texel and that high prolificacy in Finnsheep might be governed by immune system regulation.

genomics↗