Design and Biological Evaluation of the Long-Acting C5-Inhibited Ornithodoros moubata Complement Inhibitor (OmCI) Modified with Fatty Acid.

Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.

High-titer production of staurosporine by heterologous expression and process optimization.

Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5 kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145 mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750 mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568 mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine. KEYPOINTS: • Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine. • Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine. • The highest yield of staurosporine was achieved to 4568 mg/L by stepwise increase strategy.

A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy.

Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2+ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2+ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.

Epidemiological and genomic characteristics of Acinetobacter baumannii from different infection sites using comparative genomics.

BACKGROUND: Acinetobacter baumannii is a common nosocomial pathogen that poses a huge threat to global health. Owing to the severity of A. baumannii infections, it became necessary to investigate the epidemiological characteristics of A. baumannii in Chinese hospitals and find the reasons for the high antibiotic resistance rate and mortality. This study aimed to investigate the epidemiologic and genetic characteristics of A. baumannii isolated from patients with hospital acquired pneumonia (HAP), bloodstream infection (BSI) and urinary tract infection (UTI) in China and uncover potential mechanisms for multi-drug resistance and virulence characteristics of A. baumannii isolates. RESULTS: All isolates were classified into two primary clades in core gene-based phylogenetic relationship. Clonal complex 208 (CC208) mainly consisted of ST195 (32 %) and ST208 (24.6 %). CC208 and non-CC208 isolates had carbapenem resistance rates of 96.2 and 9.1 %, respectively. Core genes were enriched in 'Amino acid transport and metabolism', 'Translation', 'Energy production and conversion', 'Transcription', 'Inorganic ion transport and metabolism' and 'Cell wall/membrane/envelope synthesis'. Most isolates possessed virulence factors related to polysaccharide biosynthesis, capsular polysaccharide synthesis and motility. Eleven isolates belong to ST369 or ST191 (oxford scheme) all had the virulence factor cap8E and it had a higher positive rate in UTI (35.3 %) than in BSI (18.9 %) and HAP (12.9 %). ABGRI1 antibiotic resistance islands were responsible for streptomycin, tetracycline and sulfonate resistance. The blaOXA-23 gene was the most probable cause for carbapenem resistance, although the blaOXA-66 gene with nonsynonymous SNPs (F82L, I129L) was not. CONCLUSIONS: A. baumannii is a genomically variable pathogen that has the potential to cause a range of infectious diseases. There is high proportion of carbapenem resistance in isolates from all three infection sites (HAP, BSI and UTI), which can be attributed to the blaOXA-23 gene. CC208 is the predominant clone in blaOXA-23-carrying A. baumannii that should be monitored. Virulence factors involving bacteria motility and polysaccharide biosynthesis which are widespread in clinical A. baumannii strains deserve our attention.

Long-Acting Human Interleukin 2 Bioconjugate Modified with Fatty Acids by Sortase A.

Human Interleukin 2 (IL-2) has already achieved impressive results as a therapeutic agent for cancer and autoimmune diseases. However, one of the limitations associated with the clinical application of IL-2 is its short half-life owing to rapid clearance by the kidneys. Modification with fatty acids, as an albumin noncovalent ligand with the advantage of deep penetration into tissues and high activity-to-mass ratio, is a commonly used approach to improve the half-life of native peptides and proteins. In this investigation, we attempted to extend the half-life of IL-2 through conjugation with a fatty acid using sortase A (srtA). We initially designed and optimized three IL-2 analogues with different peptide linkers between the C-terminus of IL-2 and srtA recognition sequence (LPETG). Among these, analogue A3 was validated as the optimal IL-2 analogue for further modification. Next, six fatty acid moieties with the same fatty acid and different hydrophilic spacers were conjugated to A3 through srtA. The six bioconjugates generated were screened for in vitro biological activity, among which bioconjugate B6 was identified as near-optimal to IL-2. Additionally, B6 could effectively bind albumin through the conjugated fatty acid, which contributed to a significant improvement in its pharmacokinetic properties in vivo. In summary, we have developed a novel IL-2 bioconjugate, B6, modified with fatty acids using srtA, which may effectively serve as a new-generation long-acting IL-2 immunotherapeutic agent.

Reversal of Multidrug Resistance by Apolipoprotein A1-Modified Doxorubicin Liposome for Breast Cancer Treatment.

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.

Cloning, expression, and purification of porcine adrenocorticotropic hormone in Escherichia coli.

Adrenocorticotropic hormone (ACTH) is an old medicine derived from porcine pituitary gland that has been marketed for more than 60 years. In this study, we present a recombinant approach to produce ACTH in Escherichia coli (E. coli). The SUMO-tagged fusion protein was cloned and expressed after induction with isopropyl-ß-d-thiogalactopyranoside (IPTG) at 25 °C for 8 h. The fusion protein was extracted and purified by anion exchange chromatography, and the SUMO tag was subsequently removed by digestion with ubiquitin-like protease 1 (ULP1). Approximately 95.3 mg of recombinant ACTH with 94.2% purity was obtained after cation exchange purification performed on a 5 mL column, from 286 mL fermentation broth based on the amount of pellets homogenized. The molecular mass of the recombinant ACTH was confirmed by mass spectrometry to equal 4567.32 Da.

Chemically synthesized LYRM03 could inhibit the metastasis of human breast cancer MDA-MB-231 cells in vitro and in vivo.

Aminopeptidase N (APN) belongs to the aminopeptidase family, which is widely distributed throughout the animal and plant kingdoms. APN is thought to be a very important target for cancer therapy as it is linked to cancer progression and metastasis. However, bestatin (Ubenimex) is the only approved drug that targets various aminopeptidases for the treatment of acute myelocytic leukemia and lymphedema. A compound 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine (also known as LYRM03), isolated from a Streptomyces strain HCCB10043, exhibited more potent inhibitory activity than bestatin. In this work, we applied a chemical synthesis strategy to generate LYRM03 to overcome the low yields typically achieved from fermentation. Finally, we explored a suite of experiments to determine the bioactivity of LYRM03 and revealed that the metastasis of MDA-MB-231 cells was significantly restrained with LYRM03 treatment or injection both in vitro and in vivo. Because of its anti-metastasis capacity, further structure modifications of LYRM03 will be of interest for its use alone or in combination as a therapy in cancer.

C2-ceramide enhances sorafenib-induced caspase-dependent apoptosis via PI3K/AKT/mTOR and Erk signaling pathways in HCC cells.

Sorafenib as an effective multikinase inhibitor has been approved for the clinical treatment against advanced hepatocellular carcinoma (HCC). HCC treatment requires usually combined therapy because of its complex pathogenesis. Ceramide has been confirmed to induce remarkable apoptosis in human tumor cells and has attracted increasing attention in investigations on combination therapy. In this paper, the anti-HCC effect of sorafenib combined with C2-ceramide was investigated on cell vitality, apoptosis, and migration, and the underlying mechanism was examined using flow cytometry and western blot. Bel7402 cells coincubated with sorafenib and C2-ceramide exhibited lower cell vitality and more irregular cellular morphology and cell cycle arrest. Sorafenib plus C2-ceramide stimulated significantly the production of reactive oxygen species (ROS) and mitochondrial depolarization, which promoted caspases-dependent cell apoptosis as illustrated by related protein expression including caspase 3, caspase 9, Bax, Bcl-2, and cytochrome c. Combination treatment of sorafenib and C2-ceramide inhibited obviously cell growth and proliferation via PI3K/AKT/mTOR and Erk signaling pathways. Furthermore, the combination treatment was proved to inhibit cell migration and epithelial-mesenchymal transition (EMT). These findings indicated that the combination of C2-ceramide and sorafenib provided synergistic inhibitory effects on HCC cells.

Human apolipoprotein A-I exerts a prophylactic effect on high-fat diet-induced atherosclerosis via inflammation inhibition in a rabbit model.

Apolipoprotein A-I (apoA-I) is the major functional protein fraction of high-density lipoprotein. The prophylactic effect and mechanism of human apoA-I on atherosclerosis (AS) were investigated in a high-fat diet-induced AS rabbit model. The rabbits were injected with apoA-I once a week while fed high-fat diet for 20 weeks. Our results showed that apoA-I could raise the serum level of high-density lipoprotein-cholesterol and reduce those of lipid total cholesterol, triglyceride, and low-density lipoprotein-cholesterol in AS rabbits. Decreased aortic plaque area and aortic injury degree were also observed by Oil Red O staining and HE staining in apoA-I-treated high-fat diet-induced AS rabbits. Further study elucidated that apoA-I could down-regulate the expression of some inflammatory mediators including intercellular adhesion molecule type 1, vascular adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-6 (IL-6), and C-reactive protein in serum and aorta of AS rabbits. In addition, real-time quantitative RT-PCR analyses showed that the apoA-I infusions decreased the mRNA levels of two pro-inflammatory molecules, i.e. nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX-2), in aorta of AS rabbits, which was associated with a concomitant reduction in endothelial VCAM-1 and IL-6 mRNA transcription. Together, our results support the atheroprotective and prophylactic role of apoA-I in vivo, and this activity may be correlated with its anti-inflammatory effect.

Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin ß-subunit in Pichia pastoris.

OBJECTIVE: To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin ß-subunit in Pichia pastoris. RESULTS: Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC50 of 7 µg ml(-1), and 30 % of cells were annexin V-positive after treatment with 20 µg endo-CTP ml(-1) for 48 h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague-Dawley rats was much longer than that of its commercial counterpart (Endostar). CONCLUSION: A long-acting endostatin can be produced using CTP technology.

Cationic poly(amidoamine) dendrimers induced cyto-protective autophagy in hepatocellular carcinoma cells.

Poly(amidoamine) (PAMAM) dendrimers are proposed as one of the most promising nanomaterials for biomedical applications because of their unique tree-like structure, monodispersity and tunable properties. In this study, we found that PAMAM dendrimers could induce the formation of autophagosomes and the conversion of microtubule-associated protein 1 light chain 3 (LC3) in hepatocellular carcinoma HepG2 cells, while the inhibition of the Akt/mTOR and activation of the Erk 1/2 signaling pathways were involved in autophagy-induced by PAMAM dendrimers. We also investigated the suppression of autophagy with the obviously enhanced cytotoxicity of PAMAM dendrimers. Moreover, the blockage of a reactive oxygen species (ROS) could enhance the growth inhibition and apoptosis of hepatocellular carcinoma cells, induced by PAMAM dendrimers through reducing autophagic effects. Taken together, these findings explored the role and mechanism of autophagy induced by PAMAM dendrimers in HepG2 cells, provided new insight into the effect of autophagy on drug delivery nanomaterials and tumor cells and contributed to the use of a drug delivery vehicle for hepatocellular carcinoma treatment.

Tumor necrosis therapy antibody interleukin-2 fusion protein elicits prolonged and targeted antitumor effects in vivo.

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) associated with the high-dose IL-2 treatment regimen has limited its use in tumor immunotherapy. To reverse this situation, a tumor-targeted fusion protein, recombinant human TNT-IL2 (rhTNT-IL2), was generated with both the cytokine activity of IL-2 and the tumor-targeting ability of TNT antibody. TNT is a human tumor necrosis therapy monoclonal antibody capable of binding intracellular antigens which are accessible and abundant in necrotic regions of tumors. The immunotherapeutic potential of this fusion protein was tested in murine melanoma and lung cancer models, and tumor-bearing mice showed satisfied tumor regressions after rhTNT-IL2 immunotherapy. Immunohistochemical study showed a distinct penetration of IL-2 in tumors in mice treated with rhTNT-IL2, indicating its evident tumor-targeting activity. Moreover, the rhTNT-IL2 was well tolerated in cynomolgus monkeys in a 12-week long-term repeated toxicity study. These studies indicate that the targeting of IL-2 to necrotic areas of tumors might be a new approach for the immunotherapy of solid tumors.

Enhancement of microbial hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Metarhizium anisopliae using nano-liposome technique.

The introduction of 11α-hydroxy to 13-ethyl-gon-4-ene-3,17-dione (GD) by microbial transformation is a key step in the synthesis of oral contraceptive desogestrel, while low substrate solubility and uptake into cells are tough problems influencing biotransformation efficiency greatly. Nano-liposome technique was used in the hydroxylation of GD by Metarhizium anisopliae. The substrate GD was processed to be GD-loaded nano-liposomes (GNLs) with high stability and encapsulation efficiency, and then applied in microbial hydroxylation by M. anisopliae. The results proved that the yield of the main product 11α-hydroxy-13-ethyl-gon-4-ene-3,17-dione (HGD) tripled compared to regular solvent dimethylformamide dispersion method at 2 g/l of substrate feeding concentration, and the HGD conversion rate showed no obvious reduction when the substrate feeding concentration increased from 2 to 6 g/l, which indicated the improvement of GNL addition method on biotransformation. Furthermore, the main byproduct changed from 6ß-hydroxy derivative of GD (with similar polarity to HGD) to 6ß,11α-dihydroxy derivative, which benefits the following purification of HGD from fermentation broth. These advantages suggest a great potential for the application of nano-liposome technique in microbial steroid transformation.

Spatiotemporal characteristics of meteorological drought events in 34 major global river basins during 1901-2021.

Meteorological drought is a crucial driver of various types of droughts; thus, identifying the spatiotemporal characteristics of meteorological drought at the basin scale has implications for ecological and water resource security. However, differences in drought characteristics between river basins have not been clearly elucidated. In this study, we identify and compare meteorological drought events in 34 major river basins worldwide using a three-dimensional drought-clustering algorithm based on the standardised precipitation evapotranspiration index on a 12-month scale from 1901 to 2021. Despite synchronous increases in precipitation and potential evapotranspiration (PET), with precipitation increasing by more than three times the PET, 47 % (16/34) of the basins showed a tendency towards drought in over half their basin areas. Drought events occurred frequently, with more than half identified as short-term droughts (lasting less than or equal to three months). Small basins had a larger drought impact area, with major drought events often originating from the basin boundaries and migrating towards the basin centre. Meteorological droughts were driven by changes in sea surface temperature (SST), especially the El Niño Southern Oscillation (ENSO) or other climate indices. Anomalies in SST and atmospheric circulation caused by ENSO events may have led to altered climate patterns in different basins, resulting in drought events. These results provide important insights into the characteristics and mechanisms of meteorological droughts in different river basins worldwide.

Influence of Lipopolysaccharide-Interacting Peptides Fusion with Endolysin LysECD7 and Fatty Acid Derivatization on the Efficacy against Acinetobacter baumannii Infection In Vitro and In Vivo.

Acinetobacter baumannii has developed multiple drug resistances, posing a significant threat to antibiotic efficacy. LysECD7, an endolysin derived from phages, could be a promising therapeutic agent against multi-drug resistance A. baumannii. In this study, in order to further enhance the antibacterial efficiency of the engineered LysECD7, a few lipopolysaccharide-interacting peptides (Li5, MSI594 and Li5-MSI) were genetically fused with LysECD7. Based on in vitro antibacterial activity, the fusion protein Lys-Li5-MSI was selected for further modifications aimed at extending its half-life. A cysteine residue was introduced into Lys-Li5-MSI through mutation (Lys-Li5-MSIV12C), followed by conjugation with a C16 fatty acid chain via a protonation substitution reaction(V12C-C16). The pharmacokinetic profile of V12C-C16 exhibited a more favorable characteristic in comparison to Lys-Li5-MSI, thereby resulting in enhanced therapeutic efficacy against lethal A. baumannii infection in mice. The study provides valuable insights for the development of novel endolysin therapeutics and proposes an alternative therapeutic strategy for combating A. baumannii infections.

Novel nitroxoline derivative combating resistant bacterial infections through outer membrane disruption and competitive NDM-1 inhibition.

ABSTRACTNew Delhi metallo-ß-lactamase-1 (NDM-1) has rapidly disseminated worldwide, leading to multidrug resistance and worse clinical prognosis. Designing and developing effective NDM-1 inhibitors is a critical and urgent challenge. In this study, we constructed a library of long-lasting nitroxoline derivatives and identified ASN-1733 as a promising dual-functional antibiotic. ASN-1733 can effectively compete for Ca2+ on the bacterial surface, causing the detachment of lipopolysaccharides (LPS), thereby compromising the outer membrane integrity and permeability and exhibiting broad-spectrum bactericidal activity. Moreover, ASN-1733 demonstrated wider therapeutic applications than nitroxoline in mouse sepsis, thigh and mild abdominal infections. Furthermore, ASN-1733 can effectively inhibit the hydrolytic capability of NDM-1 and exhibits synergistic killing effects in combination with meropenem against NDM-1 positive bacteria. Mechanistic studies using enzymatic experiments and computer simulations revealed that ASN-1733 can bind to key residues on Loop10 of NDM-1, hindering substrate entry into the enzyme's active site and achieving potent inhibitory activity (Ki = 0.22 µM), even in the presence of excessive Zn2+. These findings elucidate the antibacterial mechanism of nitroxoline and its derivatives, expand their potential application in the field of antibacterial agents and provide new insights into the development of novel NDM-1 inhibitors.

Impact of intra-patient variability of tacrolimus on allograft function and CD4 + /CD8 + ratio in kidney transplant recipients: a retrospective single-center study.

BACKGROUND: Tacrolimus is a critical component of immunosuppressive therapy for kidney transplant recipients. Intra-patient variation (IPV) of tacrolimus levels affects the function of transplanted kidney. AIM: This study aimed to investigate the impact of tacrolimus IPV on kidney function, examine its association with post-transplant duration, and assess its effect on the immune status of transplant recipients. METHOD: This retrospective study was conducted from January 2016 to February 2022. IPV was evaluated using the coefficient of variation (CV) of tacrolimus trough levels from 6 to 48 months after transplantation. Patients were divided into low- and high-IPV groups based on the median CV. Significant differences in kidney function, CD4 + /CD8 + ratio, and post-transplant duration between these groups were analyzed. RESULTS: Among 189 patients, tacrolimus IPV showed a strong correlation with serum creatinine clearance rate (Ccr) and estimated glomerular filtration rate (eGFR) (p < 0.05). Tacrolimus IPV was significantly correlated with post-transplant duration in only two patients (p < 0.05). Using a median CV of 15.4% to categorize patients, the high IPV group, compared to the low IPV group, exhibited significantly higher eGFR at 6-9 months (p < 0.05), lower Ccr at 9-12 months (p < 0.05), and reduced Ccr and eGFR at 15-18 months (p < 0.05). Six months after transplantation, the high IPV group had a significantly lower CD4 + /CD8 + ratio than the low IPV group (p < 0.05). CONCLUSION: This study highlights the significant impact of tacrolimus IPV on transplant kidney function and immune status in transplant patients at various post-transplantation intervals.

Decreasing trends of mean and extreme snowfall in High Mountain Asia.

Intense warming profoundly alters precipitation phase patterns and intensity in High Mountain Asia (HMA). While snowfall climatology and precipitation extremes have been studied, there is a lack of understanding of snowfall extremes within HMA. Here, we investigate the spatial and temporal variability of non-extreme and extreme snowfall in hydrological years 1979-2020 using multi-source meteorological data, compare weather systems during extreme and non-extreme snowfall events, and identify key circulation factors that influence fluctuations in mean annual snowfall and extreme snowfall. The snowfall amount (-0.13 d/mm), days (-0.56 d/a), and fraction (-0.0012) were significantly reduced in HMA, with a shorter snowfall season (-0.52 d/a). Some extreme snowfall metrics (maximum 1-day snowfall and maximum 3-day snowfall) were insensitive to climate change, whereas the maximum consecutive snowfall days (-0.007 d/a), snowfall amount (-0.0023 mm/a), heavy snowfall days (S95pD; 0.0087 d/a), and extremely heavy snowfall days (S99pD; -0.1019 d/a) showed significant decreases. Synthetic analyses show that extreme snowfall events were more likely to occur within a narrow temperature range (-5 °C to 3 °C) with higher relative humidity and precipitation compared to non-extreme events. A stepwise regression method was used to determine that the fluctuation in the average annual snowfall was closely related to the Atlantic Multidecadal Oscillation, whereas the variation in extreme snowfall was mainly influenced by the Southern Oscillation Index. Our research provides a reference for assessing the potential impacts of climate change on a regional scale for risk management and disaster adaptation.