Active Constituents with Tyrosinase Inhibitory Activities from Waste Tobacco Leaves.

One novel compound, (R)-3, 6-diethoxy-4-hydroxycyclohex-3-en-1-one (1) and thirteen known compounds were isolated from the waste tobacco leaves. The structures of two compounds (1-2) were confirmed and attributed firstly by the extensive spectroscopic data, including 1D/2D NMR, IR, HR-ESI-MS, CD, and ECD spectra. Notably, seven compounds (2, 3, 9, 10, 11, 12, and 13) exhibited better tyrosinase inhibitory activity than the positive control kojic acid. The binding modes of these compounds revealed that their structure formed strong hydrogen bonds and van der Waals forces with the active sites of tyrosinase. These results indicated that waste tobacco leaves are good resources for developing tyrosinase inhibitors.

An in vitro and in vivo study on extracorporeal transducer optimization for high-intensity focused ultrasound to improve the safety and efficacy of breast tumor ablation.

OBJECTIVE: To observe the characteristics of a new extracorporeal high intensity focused ultrasound transducer, titled Haifu system JCQ-B, and to compare its safety and efficacy for breast ablation with the standard Haifu system JC transducer. MATERIALS AND METHODS: Ox liver with pig skin and pork ribs were prepared in a semi-sphere shape, served as in vitro acoustic model. The udders of female goats were used as in vivo acoustic model. Both in vitro and in vivo models were ablated by either JCQ-B or JC transducer. The morphology of biological focal region (BFR), the coagulative necrosis volume, and the temperature increase were observed and compared. RESULTS: The BFR morphology of JCQ-B transducer was circular both in vitro and in vivo, with a length-width ratio close to one. Under the same sonication parameters (sonication power, time and depth in tissue), coagulation necrosis volume caused by JCQ-B transducer was larger than that caused by JC transducer both in vitro and in vivo. The increase in temperature in the near and far acoustic pathways with JCQ-B transducer was significantly lower than that of JC transducer in vitro. After receiving high sonication energy during in vivo experimentation, there were no complications observed after the ablation of JCQ-B transducer, while small skin damage was observed after the ablation of JC transducer. CONCLUSIONS: The JCQ-B transducer improved the safety and efficacy of treatment by optimizing BFR morphology and ablation efficiency, which could be applied in the treatment of breast tumor.

Three New Constituents with Anti-Inflammatory and Antimicrobial Activities in Vitro from the Roots of Capsicum annuum L.

Three new compounds, including two new sesquiterpenes (1-2), named Annuumine E-F, and one new natural product, 3-hydroxy-2,6-dimethylbenzenemethanol (3), together with seventeen known compounds (4-20) were isolated from the ethanol extract of the roots of Capsicum annuum L. Among them, five compounds (4, 5, 9, 10 and 20) were isolated from this plant for the first time. The structures of new compounds (1-3) were determined via detailed analysis of the IR, HR-ESI-MS and 1D and 2D NMR spectra. The anti-inflammatory activities of the isolated compounds were evaluated by their ability to reduce NO release by LPS-induced RAW 264.7 cells. Notably, compound 11 exhibited moderate anti-inflammatory activity (IC50 =21.11 µM). Moreover, the antibacterial activities of the isolated compounds were also evaluated.

Cytostatic Activity of Sanguinarine and a Cyanide Derivative in Human Erythroleukemia Cells Is Mediated by Suppression of c-MET/MAPK Signaling.

Sanguinarine (1) is a natural product with significant pharmacological effects. However, the application of sanguinarine has been limited due to its toxic side effects and a lack of clarity regarding its molecular mechanisms. To reduce the toxic side effects of sanguinarine, its cyanide derivative (1a) was first designed and synthesized in our previous research. In this study, we confirmed that 1a presents lower toxicity than sanguinarine but shows comparable anti-leukemia activity. Further biological studies using RNA-seq, lentiviral transfection, Western blotting, and flow cytometry analysis first revealed that both compounds 1 and 1a inhibited the proliferation and induced the apoptosis of leukemic cells by regulating the transcription of c-MET and then suppressing downstream pathways, including the MAPK, PI3K/AKT and JAK/STAT pathways. Collectively, the data indicate that 1a, as a potential anti-leukemia lead compound regulating c-MET transcription, exhibits better safety than 1 while maintaining cytostatic activity through the same mechanism as 1.

Design, Synthesis, and Evaluation of the COX-2 Inhibitory Activities of New 1,3-Dihydro-2H-indolin-2-one Derivatives.

Thirty-three 1,3-dihydro-2H-indolin-2-one derivatives bearing α, ß-unsaturated ketones were designed and synthesized via the Knoevenagel condensation reaction. The cytotoxicity, in vitro anti-inflammatory ability, and in vitro COX-2 inhibitory activity of all the compounds were evaluated. Compounds 4a, 4e, 4i-4j, and 9d exhibited weak cytotoxicity and different degrees of inhibition against NO production in LPS-stimulated RAW 264.7 cells. The IC50 values of compounds 4a, 4i, and 4j were 17.81 ± 1.86 µM, 20.41 ± 1.61 µM, and 16.31 ± 0.35 µM, respectively. Compounds 4e and 9d showed better anti-inflammatory activity with IC50 values of 13.51 ± 0.48 µM and 10.03 ± 0.27 µM, respectively, which were lower than those of the positive control ammonium pyrrolidinedithiocarbamate (PDTC). Compounds 4e, 9h, and 9i showed good COX-2 inhibitory activities with IC50 values of 2.35 ± 0.04 µM, 2.422 ± 0.10 µM and 3.34 ± 0.05 µM, respectively. Moreover, the possible mechanism by which COX-2 recognized 4e, 9h, and 9i was predicted by molecular docking. The results of this research suggested that compounds 4e, 9h, and 9i might be new anti-inflammatory lead compounds for further optimization and evaluation.

Synthesis and Antileukemia Activity Evaluation of Benzophenanthridine Alkaloid Derivatives.

Thirty-three benzophenanthridine alkaloid derivatives (1a-1u and 2a-2l) were synthesized, and their cytotoxic activities against two leukemia cell lines (Jurkat Clone E6-1 and THP-1) were evaluated in vitro using a Cell Counting Kit-8 (CCK-8) assay. Nine of these derivatives (1i-l, 2a, and 2i-l) with IC50 values in the range of 0.18-7.94 µM showed significant inhibitory effects on the proliferation of both cancer cell lines. Analysis of the primary structure-activity relationships revealed that different substituent groups at the C-6 position might have an effect on the antileukemia activity of the corresponding compounds. In addition, the groups at the C-7 and C-8 positions could influence the antileukemia activity. Among these compounds, 2j showed the strongest in vitro antiproliferative activity against Jurkat Clone E6-1 and THP-1 cells with good IC50 values (0.52 ± 0.03 µM and 0.48 ± 0.03 µM, respectively), slightly induced apoptosis, and arrested the cell-cycle, all of which suggests that compound 2j may represent a potentially useful start point to undergo further optimization toward a lead compound.

Pentacyclic Triterpenoids from Sabia discolor Dunn and Their α-Glycosidase Inhibitory Activities.

Four new pentacyclic triterpenoids named Sabiadiscolor A-D (1 and 7-9) together with eleven known ones were isolated by repeated column chromatography. Their structures were identified and characterized by NMR and MS spectral data as 6 oleanane-type pentacyclic triterpenoids (1-6), 7 ursane-type ones (7-13), and 2 lupanane-type ones (14-15). Except for compound 15, all other compounds were isolated from Sabia discolor Dunn for the first time. Their α-glycosidase inhibitory activities were evaluated, which showed that compounds 1, 3, 8, 9, 13, and 15 implied remarkable activities with IC50 values ranging from 0.09 to 0.27 µM, and the preliminary structure-activity relationship was discussed.

Hepatoprotective Constituents of Total Dibenzocyclooctadiene Lignans from Schisandra chinensis Based on the Spectrum-Effect Relationship.

To scientifically clarify the hepatoprotective constituents of Fructus Schizandrae chinensis, eleven batches samples of total dibenzocyclooctadiene lignans (TDL) from Schisandra chinensis were prepared by using the optimum extraction technique. Characteristic high-performance liquid chromatography (HPLC) chromatograms were obtained through HPLC analysis technology, and the hepatoprotective effects of the eleven batches of TDL were evaluated by MTT assay. Based on the chemical and biological activity results, the spectrum-effect relationship between the characteristic HPLC fingerprints and the hepatoprotective effect of TDL was established using Minitab 16.0 data analysis software. On the basis of the spectrum-effect relationship, thirteen compounds (1-13) were obtained from the TDL by chemical natural product chemical separation and purification technology, and their structures were identified on the basis of the spectral data and the literature. Based on these compounds, thirteen common peaks among the thirty-three chromatographic peaks in the above HPLC fingerprints were identified. Our findings showed that some components, including, schisandrin B (2), schisandrin A (3), and schisandrol B (7) had significant roles in promoting hepatoprotective activity. Preliminary verification of the spectrum-effect relationship of TDL from S. chinensis was carried out, and the results confirmed that the activity of a composite of these three key components in optimal ratios was better than that of any individual compound, which potentially confirmed the reliability of the spectrum-effect relationship and the synergistic effects of traditional Chinese medicine.

Galactose-Modified PH-Sensitive Niosomes for Controlled Release and Hepatocellular Carcinoma Target Delivery of Tanshinone IIA.

Increasing the drug tumor-specific accumulation and controlling their release is considered one of the most effective ways to increase the efficacy of drugs. Here, we developed a vesicle system that can target hepatoma and release drugs rapidly within tumor cells. This non-ionic surfactant vesicle is biodegradable. Galactosylated stearate has been used to glycosylate the vesicles to achieve liver targeting; replacement of a portion (Chol:CHEMS = 1:1) of cholesterol by cholesteryl hemisuccinate (CHEMS) allows for a rapid release of drugs in an acidic environment. In vitro release experiments confirmed that galactose-modified pH-sensitive niosomes loaded with tanshinone IIA had excellent drug release performance in acid medium. In vitro experiments using ovarian cancer cells (A2780), colon cancer cells (HCT8), and hepatoma cell (Huh7, HepG2) confirmed that the preparation had specific targeting ability to hepatoma cells compared with free drugs, and this ability was dependent on the galactose content. Furthermore, the preparation also had a more substantial inhibitory effect on tumor cells, and subsequent apoptosis assays and cell cycle analyses further confirmed its enhanced anti-tumor effect. Results of pharmacokinetic experiments confirmed that the vesicle system could significantly extend the blood circulation time of tanshinone IIA, and the larger area under the curve indicated that the preparation had a better drug effect. Thus, the results of biodistribution experiments confirmed the in vivo liver targeting ability of this preparation. Niosomes designed in this manner are expected to be a safe and effective drug delivery system for liver cancer therapy.

Diverse effects of different Akkermansia muciniphila genotypes on Brown adipose tissue inflammation and whitening in a high-fat-diet murine model.

BACKGROUND: The purpose of this study was to investigate the differences in the metabolic protective effects of Akkermansia muciniphila (A.muciniphila) genotypes on high-fat diet mice and explore possible mechanisms. METHODS: Male C57BL/6 mice were randomly divided into 6 groups, including high-fat diet (HFD)+ A. muciniphila I/II/PBS group, normal control diet (NCD)+ A. muciniphila I/II/PBS group, respectively. Dietary intervention and A. muciniphila gavage were performed simultaneously. Blood glucose and lipid metabolism, brown adipose morphology and activities, and intestinal barrier function were examined after the mice were sacrificed. RESULTS: A.muciniphila gavage improved the impaired glucose tolerance, hyperlipidemia and liver steatosis in HFD mice, and that A. muciniphila II (Amuc_GP25) was not as effective as A. muciniphila I (Amuc_GP01). This phenomenon might be because Amuc_GP01 intervention significantly inhibited brown adipose tissue whitening and inflammation induced by HFD, by repairing the intestinal barrier and relieving endotoxemia. Amuc_GP25 did not display the same results as Amuc_GP01 in HFD mice but had stronger effects in the NCD mice. CONCLUSIONS: This study reveals the distinct functions of different A. muciniphila genotypes on diet-induced obesity, suggesting that different A. muciniphila genotypes may affect pathological conditions differently through distinct action pathways.

An Akkermansia muciniphila subtype alleviates high-fat diet-induced metabolic disorders and inhibits the neurodegenerative process in mice.

The prevalence of obesity and diabetes, and their complicating mental disorders, severely affect public health. This study aimed to investigate the long-term effects of an Akkermansia muciniphila subtype (A. muciniphilasub) on high-fat diet-induced obesity and diabetes, and to evaluate whether this subtype can alleviate their complicated mental disorders. Whole genome sequencing and short chain fatty acid production analysis in supernatant of pure culture were performed. Female adult C57BL/6 mice were fed a high-fat diet or a normal chow diet and were gavaged with A. muciniphilasub or phosphate-buffered saline daily for 10 months. Body weight, food consumption and blood glucose were measured. At the end of the treatment period, all mice were subjected to the Y-maze test, sucrose preference test, analyses of serum, fecal microbiota analysis and histological examination. This A. muciniphilasub had 278 unique genes compared to the type strain (A. muciniphila ATCC BAA-835) and produced short chain fatty acids both. A. muciniphilasub administration significantly reduced body weight gain and improved the spatial memory of high-fat diet-fed mice. A. muciniphilasub increased Nissl bodies in neurons of the hippocampus, and restored the high-fat diet-inhibited tryptophan metabolism. The high-fat diet led to decreased serum 5-hydroxytryptamine and induced depression, which were not alleviated by A. muciniphilasub. A. muciniphilasub increased the relative fecal abundance of Bifidobacterium, and was negatively correlated with the fecal abundance of Bacteroides. The present study demonstrated the beneficial effects of this A. muciniphilasub on body weight, blood glucose control and the alleviation of the memory decay caused by a high-fat diet in mice.

miRNA-1290 Promotes Aggressiveness in Pancreatic Ductal Adenocarcinoma by Targeting IKK1.

BACKGROUND/AIMS: MicroRNAs (miRNAs) are a group of non-coding RNAs that play diverse roles in pancreatic carcinogenesis. In pancreatic ductal adenocarcinoma (PDAC), NF-kB is constitutively activated in most patients and is linked to a mutation in KRAS via IkB kinase complex 1 (IKK1, also known as IKKa). We investigated the link between PDAC aggressiveness and miR-1290. METHODS: We used miRCURYTM LNA Array and in situ hybridization to investigate candidate miRNAs and validated the findings with PCR. The malignant behavior of cell lines was assessed with Cell Counting Kit-8, colony formation, and Transwell assays. A dual-luciferase reporter assay was used to evaluate the interaction between miR-1290 and IKK1. Protein expression was observed by western blotting. RESULTS: In this study, 36 miRNAs were dysregulated in high-grade pancreatic intraepithelial neoplasia (PanIN) and PDAC tissues compared with low-grade PanIN tissues. The area under the curve values of miR-1290 and miR-31-5p were 0.829 and 0.848, respectively (95% confidence interval, 0.722-0.936 and 0.749-0.948, both P < 0.001). There was a significant correlation between miR-1290 and histological differentiation (P = 0.029), pT stage (P = 0.006), and lymph node metastasis (P = 0.001). In addition, the in vitro work showed that miR-1290 promoted PDAC cell proliferation, invasion, and migration. Western blotting and the dual-luciferase reporter assay showed that miR-1290 promoted cancer aggressiveness by directly targeting IKK1. The synergist effect of miR-1290 on the proliferation and metastasis of PDAC cells was attenuated and enhanced by IKK1 overexpression and knockdown, respectively. Consistent with the in vitro results, a subcutaneous tumor mouse model showed that miR-1290 functioned as a potent promoter of PDAC in vivo. CONCLUSION: MiR-1290 may act as an oncogene by directly targeting the 3'-untranslated region of IKK1, and the miR-1290/IKK1 pathway may prove to be a novel diagnostic and therapeutic target for PDAC.

Design and synthesis of novel monoterpenoid indole alkaloid-like analogues and their antitumour activities in vitro.

A biomimetic synthetic strategy and combinatorial chemistry were used to synthesize 34 novel monoterpenoid indole alkaloid (MIA) analogues, and their cytotoxic activities against five cancer cell lines (SW-480, A-549, HL-60, SMMC-7721, and MCF-7) were determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Fourteen of these analogues (7, 16-18, and 23-32) showed significantly greater inhibition of tumour cell proliferation than cisplatin. Compounds 17 and 18 showed the highest cytotoxic activity against the HL-60 cell line with IC50 values of 0.90 µM and 0.43 µM, respectively. Compound 18 slightly induced apoptosis and arrested the cell cycle in SW-480, A-549, HL-60, SMMC-7721, and MCF-7 cells. Analysis of the primary structure-activity relationships reveals that the introduction of different substituent groups at the C-3, C-5, and C-6 positions of the indole moiety and the C-10 position of the genipin moiety might have an effect on the antitumour activity of the resulting compounds.

Hernsubanine E, a new hasubanan alkaloid from Stephania hernandifolia.

A new hasubanan alkaloid, hernsubanine E (1), as well as two known compounds p-hydroxybenzaldehyde (2) and (-)-syringaresinol (3) have been isolated from the whole plants of Stephania hernandifolia by various column chromatographic methods. Their structures were identified by physicochemical properties and spectral analyses. Compounds 2 and 3 were isolated from the genus of Stephania for the first time.

Viral strategies to antagonize the host antiviral innate immunity: an indispensable research direction for emerging virus-host interactions.

The public's health is gravely at risk due to the current global outbreak of emerging viruses, specifically SARS-CoV-2 and MPXV. Recent studies have shown that SARS-CoV-2 mutants (such as Omicron) exhibit a higher capability to antagonize the host innate immunity, increasing their human adaptability and transmissibility. Furthermore, current studies on the strategies for MPXV to antagonize the host innate immunity are still in the initial stages. These multiple threats from emerging viruses make it urgent to study emerging virus-host interactions, especially the viral antagonism of host antiviral innate immunity. Given this, we selected several representative viruses that significantly threatened human public health and interpreted the multiple strategies for these viruses to antagonize the host antiviral innate immunity, hoping to provide ideas for molecular mechanism research that emerging viruses antagonize the host antiviral innate immunity and accelerate the research progress. The IAV, SARS-CoV-2, SARS-CoV, MERS-CoV, EBOV, DENV, ZIKV, and HIV are some of the typical viruses. Studies have shown that viruses could antagonize the host antiviral innate immunity by directly or indirectly blocking antiviral innate immune signaling pathways. Proviral host factors, host restriction factors, and ncRNAs (microRNAs, lncRNAs, circRNAs, and vtRNAs) are essential in indirectly blocking antiviral innate immune signaling pathways. Furthermore, via controlling apoptosis, ER stress, stress granule formation, and metabolic pathways, viruses may antagonize it. These regulatory mechanisms include transcriptional regulation, post-translational regulation, preventing complex formation, impeding nuclear translocation, cleavage, degradation, and epigenetic regulation.

Spatial multi-omics at subcellular resolution via high-throughput in situ pairwise sequencing.

Technology for spatial multi-omics aids the discovery of new insights into cellular functions and disease mechanisms. Here we report the development and applicability of multi-omics in situ pairwise sequencing (MiP-seq), a method for the simultaneous detection of DNAs, RNAs, proteins and biomolecules at subcellular resolution. Compared with other in situ sequencing methods, MiP-seq enhances decoding capacity and reduces sequencing and imaging costs while maintaining the efficacy of detection of gene mutations, allele-specific expression and RNA modifications. MiP-seq can be integrated with in vivo calcium imaging and Raman imaging, which enabled us to generate a spatial multi-omics atlas of mouse brain tissues and to correlate gene expression with neuronal activity and cellular biochemical fingerprints. We also report a sequential dilution strategy for resolving optically crowded signals during in situ sequencing. High-throughput in situ pairwise sequencing may facilitate the multidimensional analysis of molecular and functional maps of tissues.

Three-dimensional super resolution ultrasound imaging with a multi-frequency hemispherical phased array.

BACKGROUND: High resolution imaging of the microvasculature plays an important role in both diagnostic and therapeutic applications in the brain. However, ultrasound pulse-echo sonography imaging the brain vasculatures has been limited to narrow acoustic windows and low frequencies due to the distortion of the skull bone, which sacrifices axial resolution since it is pulse length dependent. PURPOSE: To overcome the detect limit, a large aperture 256-module sparse hemispherical transmit/receive array was used to visualize the acoustic emissions of ultrasound-vaporized lipid-coated decafluorobutane nanodroplets flowing through tube phantoms and within rabbit cerebral vasculature in vivo via passive acoustic mapping and super resolution techniques. METHODS: Nanodroplets were vaporized with 55 kHz burst-mode ultrasound (burst length = 145 µs, burst repetition frequency = 9-45 Hz, peak negative acoustic pressure = 0.10-0.22 MPa), which propagates through overlying tissues well without suffering from severe distortions. The resulting emissions were received at a higher frequency (612 or 1224 kHz subarray) to improve the resulting spatial resolution during passive beamforming. Normal resolution three-dimensional images were formed using a delay, sum, and integrate beamforming algorithm, and super-resolved images were extracted via Gaussian fitting of the estimated point-spread-function to the normal resolution data. RESULTS: With super resolution techniques, the mean lateral (axial) full-width-at-half-maximum image intensity was 16 ± 3 (32 ± 6) µm, and 7 ± 1 (15 ± 2) µm corresponding to ∼1/67 of the normal resolution at 612 and 1224 kHz, respectively. The mean positional uncertainties were ∼1/350 (lateral) and ∼1/180 (axial) of the receive wavelength in water. In addition, a temporal correlation between nanodroplet vaporization and the transmit waveform shape was observed, which may provide the opportunity to enhance the signal-to-noise ratio in future studies. CONCLUSIONS: Here, we demonstrate the feasibility of vaporizing nanodroplets via low frequency ultrasound and simultaneously performing spatial mapping via passive beamforming at higher frequencies to improve the resulting spatial resolution of super resolution imaging techniques. This method may enable complete four-dimensional vascular mapping in organs where a hemispherical array could be positioned to surround the target, such as the brain, breast, or testicles.

Active constituents with α-glucosidase inhibitory activities from Sabia parviflora.

To identify the active constituents with α-glucosidase inhibitory activities in Sabia parviflora, three new compounds, namely, sabiaparviflora A-C (1, 2 and 8), and seven known compounds were isolated from the plant by repeated column chromatography. The structures of the new compounds were identified by extensive application of spectroscopic methods, including 1H NMR, 13C NMR, IR and HR-ESI-MS. All compounds, except for compounds 3-5, 9 and 10 were isolated for the first time from S. parviflora. Their α-glucosidase inhibitory activities were evaluated for the first time by the PNPG method. Three compounds (1, 7 and 10) exhibited marked activities, with IC50 values ranging from 104 to 324 µM, and their structure-activity relationship is preliminarily discussed herein.

Transforming acidic coiled-coil containing protein 3 suppresses influenza A virus replication by impeding viral endosomal trafficking and nuclear import.

Transforming acidic coiled-coil containing protein 3 (TACC3) is a motor spindle protein that plays an essential role in stabilization of the mitotic spindle. In this study, we show that the overexpression of TACC3 reduces the viral titers of multiple influenza A viruses (IAVs). In contrast, the downregulation of TACC3 increases IAVs propagation. Next, we map the target steps of TACC3 requirement to the early stages of viral replication. By confocal microscopy and nuclear plasma separation experiment, we reveal that overexpression of TACC3 results in a substantial decrease of IAV NP accumulation in the nuclei of infected cells. We further show that viral attachment and internalization are not affected by TACC3 overexpression and detect that the early and late endosomal trafficking of IAV in TACC3 overexpression cells is slower than negative control cells. These results suggest that TACC3 exerts an impaired effect on the endosomal trafficking and nuclear import of vRNP, thereby negatively regulating IAV replication. Moreover, the infection of different IAV subtypes decreases the expression level of TACC3 in turn. Consequently, we speculate that IAV ensures the generation of offspring virions by antagonizing the expression of inhibitory factor TACC3. Collectively, our results establish TACC3 as an important inhibitory factor for replication of the IAV, suggesting that TACC3 could be a potential target for the development of future antiviral compounds.

Comprehensive analysis of the key amino acid substitutions in the polymerase and NP of avian influenza virus that enhance polymerase activity and affect adaptation to mammalian hosts.

Accumulation of adaptive mutations in the polymerase and NP genes is crucial for the adaptation of avian influenza A viruses (IAV) to a new host. Here, we identified residues in the polymerase and NP proteins for which the percentages were substantially different between avian and human influenza viruses, to screen for key mammalian adaptive markers. The top 10 human virus-like residues in each gene segment were then selected for analysis of polymerase activity. Our research revealed that the PA-M311I and PA-A343S mutations increased the polymerase activity among the 40 individual mutations that augmented viral transcription and genomic replication, leading to increased virus yields, pro-inflammatory cytokine/chemokine levels and pathogenicity in mice. We also investigated the accumulative mutations in multiple polymerase genes and discovered that a combination of PB2-E120D/V227I, PB1-K52R/L212V/R486K/V709I, PA-R204K/M311I, and NP-E18D/R65K (hereafter referred to as the ten-sites joint mutations) has been identified to generate the highest polymerase activity, which can to some extent make up for the highest polymerase activity caused by the PB2-627 K mutation. When the ten-sites joint mutations co-occur with 627 K, the polymerase activity was further enhanced, potentially resulting in a virus with an improved phenotype that can infect a broader range of hosts, including mammals. This could lead to a greater public health concern than the current epidemic, highlighting that continuous surveillance of the variations of these sites is utmost important.