BLA-involved circuits in neuropsychiatric disorders.

The basolateral amygdala (BLA) is the subregion of the amygdala located in the medial of the temporal lobe, which is connected with a wide range of brain regions to achieve diverse functions. Recently, an increasing number of studies have focused on the participation of the BLA in many neuropsychiatric disorders from the neural circuit perspective, aided by the rapid development of viral tracing methods and increasingly specific neural modulation technologies. However, how to translate this circuit-level preclinical intervention into clinical treatment using noninvasive or minor invasive manipulations to benefit patients struggling with neuropsychiatric disorders is still an inevitable question to be considered. In this review, we summarized the role of BLA-involved circuits in neuropsychiatric disorders including Alzheimer's disease, perioperative neurocognitive disorders, schizophrenia, anxiety disorders, depressive disorders, posttraumatic stress disorders, autism spectrum disorders, and pain-associative affective states and cognitive dysfunctions. Additionally, we provide insights into future directions and challenges for clinical translation.

Distribution of mercury and methylmercury in aquacultured fish in special waters formed by coal mining subsidence.

In China, fence net aquaculture practices have been established in some subsidence waters that have been formed in coal mining subsidence areas. Within this dynamic ecological context, diverse fish species grow continuously until being harvested at the culmination of their production cycle. The purpose of this study was to investigate diverse factors influencing the bioavailability and distribution of mercury (Hg) and methylmercury (MeHg), which have high physiological toxicity in fish, in the Guqiao coal mining subsidence area in Huainan, China. Mercury and MeHg were analyzed in 38 fish samples of eight species using direct mercury analysis (DMA-80) and gas chromatography-cold vapor atomic fluorescence spectrometry (GC-CVAFAS). The analysis results show that the ranges of Hg and MeHg content and methylation rate in the fish were 7.84-85.18 ng/g, 0.52-3.52 ng/g, and 0.81-42.68 %, respectively. Meanwhile, conclusions are also summarized as following: (1) Monophagous herbivorous fish that were fed continuously in fence net aquaculture areas had higher MeHg levels and mercury methylation rates than carnivorous fish. Hg and MeHg contents were affected by different feeding habits of fish. (2) Bottom-dwelling fish show higher MeHg levels, and habitat selection in terms of water depth also partially affected the MeHg content of fish. (3) The effect of fence net aquaculture on methylation of fish in subsidence water is mainly from feed and mercury-containing bottom sediments. However, a time-lag is observed in the physiological response of benthic fishes to the release of Hg from sediments. Our findings provides baseline reference data for the ecological impact of fence net aquaculture in waters affected by soil subsidence induced by coal mining in China. Prevalent environmental contaminants within coal mining locales, notably Hg, may infiltrate rain-induced subsidence waters through various pathways.

Ultra-robust informational metasurfaces based on spatial coherence structures engineering.

Optical information transmission is vital in modern optics and photonics due to its concurrent and multi-dimensional nature, leading to tremendous applications such as optical microscopy, holography, and optical sensing. Conventional optical information transmission technologies suffer from bulky optical setup and information loss/crosstalk when meeting scatterers or obstacles in the light path. Here, we theoretically propose and experimentally realize the simultaneous manipulation of the coherence lengths and coherence structures of the light beams with the disordered metasurfaces. The ultra-robust optical information transmission and self-reconstruction can be realized by the generated partially coherent beam with modulated coherence structure even 93% of light is recklessly obstructed during light transmission, which brings new light to robust optical information transmission with a single metasurface. Our method provides a generic principle for the generalized coherence manipulation on the photonic platform and displays a variety of functionalities advancing capabilities in optical information transmission such as meta-holography and imaging in disordered and perturbative media.

Mirror Symmetry Broken of Sound Vortex Transmission in a Single Passive Metasurface via Phase Coupling.

Asymmetric transmission in a passive vortex system is highly desirable, as it enables the development of compact vortex-based devices. However, breaking the mirror symmetry of transmission via a single metasurface poses challenges due to the inherent symmetric transmission properties in reciprocity. Here, we theoretically propose and experimentally demonstrate a novel transmission-reflection phase coupling mechanism to achieve the broken mirror symmetry of sound vortex transmission. This mechanism establishes a special coupling link between transmission and reflection waves, superimposing asymmetric reflection phases on the transmission phases. By utilizing a single passive phase gradient metasurface with asymmetric reflection phase twists, distinct transmission phase twists for mirror-symmetric incident vortices can be achieved within a cylindrical waveguide. This is typically difficult to imple-ment in a reciprocal system. Numerical and experimental results both demonstrate the broken mirror symmetry of vortex transmission and reflection. Our findings offer a new strategy for controlling vortex wave propagation, which may inspire new directional applications and extend to the field of photonics.

Cross-Linking CdSO4-Type Nets with Hexafluorosilicate Anions to Form an Ultramicroporous Material for Efficient C2H2/CO2 and C2H2/C2H4 Separation.

A 44.610.8 topology hybrid ultramicroporous material (HUM), {[Cu1.5F(SiF6)(L)2.5]·G}n, (L = 4,4'-bisimidazolylbiphenyl, G = guest molecules), 1, formed by cross-linking interpenetrated 3D four-connected CdSO4-type nets with hexafluorosilicate anions is synthesized and evaluated in the context of gas sorption and separation herein. 1 is the first HUM functionalized with two different types of fluorinated sites (SiF6 2- and F- anions) lining along the pore surface. The optimal pore size (≈5 Å) combining mixed and high-density electronegative fluorinated sites enable 1 to preferentially adsorb C2H2 over CO2 and C2H4 by hydrogen bonding interactions with a high C2H2 isosteric heat of adsorption (Qst) of ≈42.3 kJ mol-1 at zero loading. The pronounced discriminatory sorption behaviors lead to excellent separation performance for C2H2/CO2 and C2H2/C2H4 that surpasses many well-known sorbents. Dynamic breakthrough experiments are conducted to confirm the practical separation capability of 1, which reveal an impressive separation factor of 6.1 for equimolar C2H2/CO2 mixture. Furthermore, molecular simulation and density functional theory (DFT) calculations validate the strong binding of C2H2 stems from the chelating fix of C2H2 between SiF6 2- anion and coordinated F- anion.

Spatial Information Lasing Enabled by Full-k-Space Bound States in the Continuum.

Optical amplification and massive information transfer in modern physics depend on stimulated radiation. However, regardless of traditional macroscopic lasers or emerging micro- and nanolasers, the information modulations are generally outside the lasing cavities. On the other hand, bound states in the continuum (BICs) with inherently enormous Q factors are limited to zero-dimensional singularities in momentum space. Here, we propose the concept of spatial information lasing, whose lasing information entropy can be correspondingly controlled by near-field Bragg coupling of guided modes. This concept is verified in gain-loss metamaterials supporting full-k-space BICs with both flexible manipulations and strong confinement of light fields. The counterintuitive high-dimensional BICs exist in a continuous energy band, which provide a versatile platform to precisely control each lasing Fourier component and, thus, can directly convey rich spatial information on the compact size. Single-mode operation achieved in our scheme ensures consistent and stable lasing information. Our findings can be expanded to different wave systems and open new scenarios in informational coherent amplification and high-Q physical frameworks for both classical and quantum applications.

The application of Global Trigger Tool in monitoring antineoplastic adverse drug events: a retrospective study.

Objective: This study aimed to establish an antineoplastic drugs trigger tool based on Global Trigger Tool (GTT), to examine the performance by detecting adverse drug events (ADEs) in patients with cancer in a Chinese hospital (a retrospective review), and to investigate the factors associating with the occurrence of antineoplastic ADEs. Methods: Based on the triggers recommended by the GTT and those used in domestic and foreign studies and taking into account the scope of biochemical indexes in our hospital, some of them were adjusted. A total of 37 triggers were finally developed. Five hundred medical records of oncology patients discharged in our hospital from 1 June 2020 to 31 May 2021 were randomly selected according to the inclusion and exclusion criteria. These records were reviewed retrospectively by antineoplastic drugs trigger tool. The sensitivity and specificity of the triggers were analyzed, as well as the characteristics and risk factors for the occurrence of ADEs. Results: Thirty-three of the 37 triggers had positive trigger, and the sensitivity rate was 91.8% (459/500). For the specificity, the positive predictive value of overall ADEs was 46.0% (715/1556), the detection rate of ADEs was 63.0% (315/500), the rate of ADEs per 100 admissions was 136.0 (95% CI, 124.1-147.9), and the rate of ADEs per 1,000 patient days was 208.33 (95% CI, 201.2-215.5). The top three antineoplastic drugs related to ADEs were antimetabolic drugs (29.1%), plant sources and derivatives (27.1%), and metal platinum drugs (26.3%). The hematologic system was most frequently involved (507 cases, 74.6%), followed by gastrointestinal system (89 cases, 13.1%). Multivariate logistic regression analysis showed that the number of combined drugs (OR = 1.14; 95% CI, 1.07-1.22; P < 0.001) and the previous history of adverse drug reaction (ADR) (OR = 0.38; 95% CI, 0.23-0.60; P < 0.001) were the risk factors for ADEs. The length of hospital stay (OR = 0.40; 95% CI, 0.14-1.12; P < 0.05) and the previous history of ADR (OR = 2.18; 95% CI, 1.07-4.45; P < 0.05) were the risk factors for serious adverse drug events (SAE). Conclusion: The established trigger tool could be used to monitor antineoplastic drugs adverse events in patients with tumor effectively but still needs to be optimized. This study may provide some references for further research in order to improve the rationality and safety of antineoplastic medications.

Identification and functional characterization of ABC transporters for selenium accumulation and tolerance in soybean.

ATP-binding cassette (ABC) transporters were crucial for various physiological processes like nutrition, development, and environmental interactions. Selenium (Se) is an essential micronutrient for humans, and its role in plants depends on applied dosage. ABC transporters are considered to participate in Se translocation in plants, but detailed studies in soybean are still lacking. We identified 196 ABC genes in soybean transcriptome under Se exposure using next-generation sequencing and single-molecule real-time sequencing technology. These proteins fell into eight subfamilies: 8 GmABCA, 51 GmABCB, 39 GmABCC, 5 GmABCD, 1 GmABCE, 10 GmABCF, 74 GmABCG, and 8 GmABCI, with amino acid length 121-3022 aa, molecular weight 13.50-341.04 kDa, and isoelectric point 4.06-9.82. We predicted a total of 15 motifs, some of which were specific to certain subfamilies (especially GmABCB, GmABCC, and GmABCG). We also found predicted alternative splicing in GmABCs: 60 events in selenium nanoparticles (SeNPs)-treated, 37 in sodium selenite (Na2SeO3)-treated samples. The GmABC genes showed differential expression in leaves and roots under different application of Se species and Se levels, most of which are belonged to GmABCB, GmABCC, and GmABCG subfamilies with functions in auxin transport, barrier formation, and detoxification. Protein-protein interaction and weighted gene co-expression network analysis suggested functional gene networks with hub ABC genes, contributing to our understanding of their biological functions. Our results illuminate the contributions of GmABC genes to Se accumulation and tolerance in soybean and provide insight for a better understanding of their roles in soybean as well as in other plants.

Impaired synaptic plasticity and decreased glutamatergic neuron excitability induced by SIRT1/BDNF downregulation in the hippocampal CA1 region are involved in postoperative cognitive dysfunction.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a common complication after anesthesia/surgery, especially among elderly patients, and poses a significant threat to their postoperative quality of life and overall well-being. While it is widely accepted that elderly patients may experience POCD following anesthesia/surgery, the exact mechanism behind this phenomenon remains unclear. Several studies have indicated that the interaction between silent mating type information regulation 2 homologue 1 (SIRT1) and brain-derived neurotrophic factor (BDNF) is crucial in controlling cognitive function and is strongly linked to neurodegenerative disorders. Hence, this research aims to explore how SIRT1/BDNF impacts cognitive decline caused by anesthesia/surgery in aged mice. METHODS: Open field test (OFT) was used to determine whether anesthesia/surgery affected the motor ability of mice, while the postoperative cognitive function of 18 months old mice was evaluated with Novel object recognition test (NORT), Object location test (OLT) and Fear condition test (FC). The expressions of SIRT1 and other molecules were analyzed by western blot and immunofluorescence staining. The hippocampal synaptic plasticity was detected by Golgi staining and Long-term potentiation (LTP). The effects of SIRT1 and BDNF overexpression as well as chemogenetic activation of glutamatergic neurons in hippocampal CA1 region of 18 months old vesicular glutamate transporter 1 (VGLUT1) mice on POCD were further investigated. RESULTS: The research results revealed that older mice exhibited cognitive impairment following intramedullary fixation of tibial fracture. Additionally, a notable decrease in the expression of SIRT1/BDNF and neuronal excitability in hippocampal CA1 glutamatergic neurons was observed. By increasing levels of SIRT1/BDNF or enhancing glutamatergic neuron excitability in the CA1 region, it was possible to effectively mitigate synaptic plasticity impairment and ameliorate postoperative cognitive dysfunction. CONCLUSIONS: The decline in SIRT1/BDNF levels leading to changes in synaptic plasticity and neuronal excitability in older mice could be a significant factor contributing to cognitive impairment after anesthesia/surgery.

Polarization-insensitive high-numerical-aperture metalens for wide-field super-resolution imaging.

The development of super-oscillatory lens (SOL) offers opportunities to realize far-field label-free super-resolution microscopy. Most microscopes based on a high numerical aperture (NA) SOL operate in the point-by-point scanning mode, resulting in a slow imaging speed. Here, we propose a high-NA metalens operating in the single-shot wide-field mode to achieve real-time super-resolution imaging. An optimization model based on the exhaustion algorithm and angular spectrum (AS) theory is developed for metalens design. We numerically demonstrate that the optimized metalens with an NA of 0.8 realizes the imaging resolution (imaging pixel size) about 0.85 times the Rayleigh criterion. The metalens can achieve super-resolution imaging of an object with over 200 pixels, which is one order of magnitude higher than the unoptimized metalens. Our method provides an avenue toward single-shot far-field label-free super-resolution imaging for applications such as real-time imaging of living cells and temporally moving particles.

Two B-box proteins orchestrate vegetative and reproductive growth in summer chrysanthemum.

Floral transition, the switch from vegetative to reproductive growth, is extremely important for the growth and development of flowering plants. In the summer chrysanthemum, CmBBX8, a member of the subgroup II B-box (BBX) family, positively regulates the transition by physically interacting with CmERF3 to inhibit CmFTL1 expression. In this study, we show that CmBBX5, a B-box subgroup I member comprising two B-boxes and a CCT domain, interacts with CmBBX8. This interaction suppresses the recruitment of CmBBX8 to the CmFTL1 locus without affecting its transcriptional activation activity. CmBBX5 overexpression led to delayed flowering under both LD (long-day) and SD (short-day) conditions, while lines expressing the chimeric repressor gene-silencing (CmBBX5-SRDX) exhibited the opposite phenotype. Subsequent genetic evidence indicated that in regulating flowering, CmBBX5 is partially dependent on CmBBX8. Moreover, during the vegetative growth period, levels of CmBBX5 expression were found to exceed those of CmBBX8. Collectively, our findings indicate that both CmERF3 and CmBBX5 interact with CmBBX8 to dampen the regulation of CmFTL1 via distinct mechanisms, which contribute to preventing the premature flowering of summer chrysanthemum.

Oxalisxishuiensis (Oxalidaceae), a new species from Danxia landforms in Guizhou, China.

Oxalisxishuiensis, a new species of Oxalidaceae from Danxia landforms of Xishui County, Guizhou, China, is described and illustrated. It is morphologically similar to O.wulingensis by the two lateral leaflets arranged at about 180° angle and oblong pink petals with lilac veins, but clearly differs from the latter by leaflets almost as long as wide, obliquely obcordate lateral leaflets, shorter peduncles, longer capsule (1.2-1.5 cm vs. 0.5-0.7 cm) and alveolate seeds.

Application of 18ß-glycyrrhetinic acid in the structural modification of natural products: a review.

18ß-Glycyrrhetinic acid (GA) is an oleane-type pentacyclic triterpene saponin obtained from glycyrrhizic acid by removing 2 glucuronic acid groups. GA and its analogues are active substances of glycyrrhiza aicd, with similar structure and important pharmacological effects such as anti-inflammatory, anti-diabetes, anti-tumor and anti-fibrosis. Although GA combined compounds are in the clinical trial stages, its application potential is severely restricted by its low bioavailability, water solubility and membrane permeability. In this article, synthetic methods and structure-activity relationships (SARs) of GA derivatives from 2018 to present are reviewed based on pharmacological activity. It is hoped that this review can provide reference for the future development of potential GA preclinical candidate compounds, and furnish ideas for the development of pentacyclic triterpenoid lead compounds.

In Situ Fabrication of Silver Nanoparticle-Decorated Polymeric Vesicles for Antibacterial Applications.

Silver/polymeric vesicle composite nanoparticles with good antibacterial properties were fabricated in this study. Silver nanoparticles (AgNPs) were prepared in situ on cross-linked vesicle membranes through the reduction of silver nitrate (AgNO3) using polyvinylpyrrolidone (PVP) via coordination bonding between the Ag+ ions and the nitrogen atoms on the vesicles. X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-vis), and transmission electron microscopy (TEM) analyses confirmed the formation of AgNPs on the vesicles. The antibacterial test demonstrated good antibacterial activity against both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) for the produced AgNP-decorated vesicles. The minimum inhibitory concentration (MIC) values of the AgNP-decorated vesicles for E. coli and S. aureus were 8.4 and 9.6 µg/mL, respectively. Cell viability analysis on the A549 cells indicated that the toxicity was low when the AgNP concentrations did not exceed the MIC values, and the wound healing test confirmed the good antibacterial properties of the AgNP-decorated vesicles.

Extracting Stress-Related EEG Patterns From Pre-Sleep EEG for Forecasting Slow-Wave Sleep Deficiency.

Sleep is vital to our daily activity. Lack of proper sleep can impair functionality and overall health. While stress is known for its detrimental impact on sleep quality, the precise effect of pre-sleep stress on subsequent sleep structure remains unknown. This study introduced a novel approach to study the pre-sleep stress effect on sleep structure, specifically slow-wave sleep (SWS) deficiency. To achieve this, we selected forehead resting EEG immediately before and upon sleep onset to extract stress-related neurological markers through power spectra and entropy analysis. These markers include beta/delta correlation, alpha asymmetry, fuzzy entropy (FuzzEn) and spectral entropy (SpEn). Fifteen subjects were included in this study. Our results showed that subjects lacking SWS often exhibited signs of stress in EEG, such as an increased beta/delta correlation, higher alpha asymmetry, and increased FuzzEn in frontal EEG. Conversely, individuals with ample SWS displayed a weak beta/delta correlation and reduced FuzzEn. Finally, we employed several supervised learning models and found that the selected neurological markers can predict subsequent SWS deficiency. Our investigation demonstrated that the classifiers could effectively predict varying levels of slow-wave sleep (SWS) from pre-sleep EEG segments, achieving a mean balanced accuracy surpassing 0.75. The SMOTE-Tomek resampling method could improve the performance to 0.77. This study suggests that stress-related neurological markers derived from pre-sleep EEG can effectively predict SWS deficiency. Such information can be integrated with existing sleep-improving techniques to provide a personalized sleep forecasting and improvement solution.

Synthesis of amide derivatives containing the imidazole moiety and evaluation of their anti-cardiac fibrosis activity.

Three series of N-{[4-([1,2,4]triazolo[1,5-α]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl]methyl}acetamides (14a-d, 15a-n, and 16a-f) were synthesized and evaluated for activin receptor-like kinase 5 (ALK5) inhibitory activities in an enzymatic assay. The target compounds showed high ALK5 inhibitory activity and selectivity. The half maximal inhibitory concentration (IC50) for phosphorylation of ALK5 of 16f (9.1 nM), the most potent compound, was 2.7 times that of the clinical candidate EW-7197 (vactosertib) and 14 times that of the clinical candidate LY-2157299. The selectivity index of 16f against p38α mitogen-activated protein kinase was >109, which was much higher than that of positive controls (EW-7197: >41, and LY-2157299: 4). Furthermore, a molecular docking study provided the interaction modes between the target compounds and ALK5. Compounds 14c, 14d, and 16f effectively inhibited the protein expression of α-smooth muscle actin (α-SMA), collagen I, and tissue inhibitor of metalloproteinase 1 (TIMP-1)/matrix metalloproteinase 13 (MMP-13) in transforming growth factor-ß-induced human umbilical vein endothelial cells. Compounds 14c and 16f showed especially high activity at low concentrations, which suggests that these compounds could inhibit myocardial cell fibrosis. Compounds 14c, 14d, and 16f are potential preclinical candidates for the treatment of cardiac fibrosis.

DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

Synthesis and Antimicrobial Activity Evaluation of Pyridine Derivatives Containing Imidazo[2,1-b][1,3,4]Thiadiazole Moiety.

Four series of novel pyridine derivatives (17 a-i, 18 a-i, 19 a-e, and 20 a-e) were synthesized and their antimicrobial activities were evaluated. Of all the target compounds, almost half target compounds showed moderate or high antibacterial activity. The 4-F substituted compound 17 d (MIC=0.5 µg/mL) showed the highest antibacterial activity, its activity was twice the positive control compound gatifloxacin (MIC=1.0 µg/mL). For fungus ATCC 9763, the activities of compounds 17 a and 17 d are equivalent to the positive control compound fluconazole (MIC=8 µg/mL). Furthermore, compounds 17 a and 17 d showed little cytotoxicity to human LO2 cells, and did not show hemolysis even at ultra-high concentration (200 µM). The results indicate that these compounds are valuable for further development as antibacterial and antifungal agents.

Synthesis and anti-liver fibrosis activity of imidazole and thiazole compounds containing amino acids.

Four series of imidazoles (15a-g, 20c, and 20d) and thiazoles (18a-g, 22a, and 22b) possessing various amino acids were synthesized and evaluated for activin receptor-like kinase 5 (ALK5) inhibitory activities in an enzymatic assay. Among them, compounds 15g and 18c showed the highest inhibitory activity against ALK5, with IC50 values of 0.017 and 0.025 µM, respectively. Compounds 15g and 18c efficiently inhibited extracellular matrix (ECM) deposition in TGF-ß-induced hepatic stellate cells (HSCs), and eventually suppressed HSC activation. Moreover, compound 15g showed a good pharmacokinetic (PK) profile with a favorable half-life (t1/2 = 9.14 h). The results indicated that these compounds exhibited activity targeting ALK5 and may have potential in the treatment of liver fibrosis; thus they are worthy of further study.

Rational engineering S1' substrate binding pocket to enhance substrate specificity and catalytic activity of thermal-stable keratinase for efficient keratin degradation.

This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.