Utilization of feather keratin waste to antioxidant and migration-enhancer peptides by Bacillus licheniformis 8-4.

AIMS: Feathers are keratin-rich byproducts of poultry processing, but those are often frequently abandoned as garbage and thus polluting the environment. Therefore, the study focused on the efficient biodegradation, bioactivity, and high-value application of feather keratin. METHODS AND RESULTS: Feather-degrading bacteria were identified, and the degradation properties were characterized. DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) and ABTS (2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid))radical scavenging assays, cytotoxicity assays, intracellular reactive oxygen scavenging assays, and cell migration assays were used to examine the biological activities of the feather keratin hydrolysis peptides (FKHPs). The results showed that we screened a feather-degrading strain of Bacillus licheniformis 8-4, which achieved complete degradation of 2% (w/v) feathers within 48 h. Notably, the feather fermentation broth was particularly high in FKHPs, which exhibited good DPPH and ABTS radical scavenging ability. Further studies revealed that FKHPs had both the ability to scavenge H2O2-induced ROS from HaCat cells and the ability to promote HaCat cell migration, while remaining non-toxic. CONCLUSIONS: The effective feather-degrading ability of B. licheniformis 8-4 allowed for the fermentation of feather medium to yield active peptides that were both antioxidants and cell-migration enhancers.

Computer-assisted in vitro reconstitution of purine degradation pathway to lower the purine content in food.

BACKGROUND: With the increasing prevalence of gout and its etiological hyperuricemia, dietary control of gout based on low-purine food according to patients' eating habits is becoming a better choice compared to the existing drug treatment such as allopurinol with notorious side effects. Reconstructing the purine metabolic pathway in vitro to degrade purine substances in food into natural functional allantoin appears to be an innovative method for preparing nutritious and healthy food of low purine content. The present study reports a computer-assisted in vitro reconstruction of four purinolytic enzymes metabolizing adenosine into allantoin to reduce purine content in food for personalized dietary control of hyperuricemia and gout. RESULTS: Under the optimum reaction conditions of 40 °C and pH 7, 0.1 U of enzymes and 0.5 mmol L-1 adenosine determined by an orthogonal test design, 16 different enzyme complexes were experimentally tested. The tested enzyme composition and allantoin production values were used as input and output to build a three-layer back propagation artificial neural network (BP-ANN) model, which was further optimized by a genetic algorithm (GA). The optimum enzyme complex predicted by the GA-BP-ANN model produced 248.08±7.832 µmol L-1 allantoin, which was 19.9% higher than equimolar mixture of enzymes, and also more efficiently lowered purine contents in beer, as well as beef and yeast extracts. CONCLUSION: This is the first in vitro reconstitution of complete purine metabolic pathway by combining ANN and GA technologies, with successful application with respect to lowering the purine content in food, indicating a promising application of computer-assisted in vitro reconstitution of purinolytic pathway in low-purine food to prevent hyperuricemia and gout. © 2022 Society of Chemical Industry.

Mechanisms Affecting the Biosynthesis and Incorporation Rate of Selenocysteine.

Selenocysteine (Sec) is the 21st non-standard proteinogenic amino acid. Due to the particularity of the codon encoding Sec, the selenoprotein synthesis needs to be completed by unique mechanisms in specific biological systems. In this paper, the underlying mechanisms for the biosynthesis and incorporation of Sec into selenoprotein were comprehensively reviewed on five aspects: (i) the specific biosynthesis mechanism of Sec and the role of its internal influencing factors (SelA, SelB, SelC, SelD, SPS2 and PSTK); (ii) the elements (SECIS, PSL, SPUR and RF) on mRNA and their functional mechanisms; (iii) the specificity (either translation termination or translation into Sec) of UGA; (iv) the structure-activity relationship and action mechanism of SelA, SelB, SelC and SelD; and (v) the operating mechanism of two key enzyme systems for inorganic selenium source flow before Sec synthesis. Lastly, the size of the translation initiation interval, other action modes of SECIS and effects of REPS (Repetitive Extragenic Palindromic Sequences) that affect the incorporation efficiency of Sec was also discussed to provide scientific basis for the large-scale industrial fermentation for the production of selenoprotein.

Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site-directed mutagenesis.

BACKGROUND: Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis. RESULTS: Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s-1 and 568.93 L mmol-1 s-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol-1 s-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference. CONCLUSIONS: The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.

Characterization of a novel Acinetobacter baumannii xanthine dehydrogenase expressed in Escherichia coli.

OBJECTIVE: To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications. RESULTS: The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40-45 °C, was stable under alkaline conditions (pH 7-11) and the half-inactivation temperature was 60 °C. The K m, turnover number and catalytic efficiency for xanthine were 25 µM, 69 s(-1) and 2.7 µM(-1) s(-1), respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized. CONCLUSION: The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

Biomimetic Wearable Sensors: Emerging Combination of Intelligence and Electronics.

Owing to the advancement of interdisciplinary concepts, for example, wearable electronics, bioelectronics, and intelligent sensing, during the microelectronics industrial revolution, nowadays, extensively mature wearable sensing devices have become new favorites in the noninvasive human healthcare industry. The combination of wearable sensing devices with bionics is driving frontier developments in various fields, such as personalized medical monitoring and flexible electronics, due to the superior biocompatibilities and diverse sensing mechanisms. It is noticed that the integration of desired functions into wearable device materials can be realized by grafting biomimetic intelligence. Therefore, herein, the mechanism by which biomimetic materials satisfy and further enhance system functionality is reviewed. Next, wearable artificial sensory systems that integrate biomimetic sensing into portable sensing devices are introduced, which have received significant attention from the industry owing to their novel sensing approaches and portabilities. To address the limitations encountered by important signal and data units in biomimetic wearable sensing systems, two paths forward are identified and current challenges and opportunities are presented in this field. In summary, this review provides a further comprehensive understanding of the development of biomimetic wearable sensing devices from both breadth and depth perspectives, offering valuable guidance for future research and application expansion of these devices.

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.

[Advances in research on the MAPK signal transduction pathway of Echinococcus].

Mitogen-activated protein kinase (MAPK) is an important signaling transduction molecules, which can enter the nucleus and activate target gene when it was stimulated and become phosphorylation. MAPK signaling pathway is closely associated with various diseases. Recent studies have indicated that MAPK signaling transduction pathway is also involved in the growth and development of Echinococcus. This review summarizes the progress on the relationship between MAPK signal pathway and Echinococcus.

Improving Catalytic Activity, Acid-Tolerance, and Thermal Stability of Glutathione Peroxidase by Systematic Site-Directed Selenocysteine Incorporation.

Glutathione peroxidase (GPx) is an important antioxidant enzyme. Selenocysteine (Sec)-containing GPxs (Sec-GPxs) are usually superior to their conventional cysteine-containing counterparts (Cys-GPxs), which make up the majority of the natural GPxs but display unsuitable activity and stability for industrial applications. This study first heterologously expressed and characterized a Cys-GPx from Lactococcus lactis (LlGPx), systematically exchanged all the three Cys to Sec and introduced an extra Sec. The results showed that the insertion of Sec at the active site could effectively increase the enzyme activity and confer a lower optimal pH value on the mutants. The double mutant C36U/L157U increased by 2.65 times (5.12 U/mg). The thermal stability of the C81U mutant was significantly improved. These results suggest that site-directed Sec incorporation can effectively improve the enzymatic properties of LlGPx, which may be also used for the protein engineering of other industrial enzymes containing catalytic or other functional cysteine residues.

Production and characterization of novel thermo- and organic solvent-stable keratinase and aminopeptidase from Pseudomonas aeruginosa 4-3 for effective poultry feather degradation.

Feather biodegradation is an important premise for efficient resource development and utilization, in which keratinase plays an important role. However, there are few keratinases that combine the high activity, thermal stability, and organic solvent tolerance required for industrialization. This paper reported an efficient feather-degrading Pseudomonas aeruginosa 4-3 isolated from slaughterhouses. After 48 h of fermentation by P. aeruginosa 4-3 in a feather medium at 40 °C, pH 8.0, keratinase was efficiently produced (295.28 ± 5.42 U/mL) with complete feather degradation (95.3 ± 1.5%). Moreover, the keratinase from P. aeruginosa 4-3 showed high optimal temperature (55 °C), good thermal stability, wide pH tolerance, and excellent organic solvent resistance. In addition, P. aeruginosa 4-3-derived aminopeptidases also exhibit excellent thermal stability and organic solvent tolerance. Encouragingly, the reaction of crude keratinase and aminopeptidase with feathers for 8 h resulted in a 78% degradation rate of feathers. These properties make P. aeruginosa 4-3 keratinase and aminopeptidase ideal proteases for potential applications in keratin degradation, as well as provide ideas for the synergistic degradation of keratin by multiple enzymes.

Preparation, Purification, and Identification of Novel Feather Keratin-Derived Peptides with Antioxidative and Xanthine Oxidase Inhibitory Activities.

Feather keratin is an underappreciated protein resource of high quality, with limited bioavailability, and it urgently requires eco-friendly methods to enhance its value. Here, we report on the preparation, purification, and identification of novel peptides with antioxidant and xanthine oxidase (XOD) inhibitory activities from fermented feather broth, using Bacillus licheniformis 8-4. Two peptides, namely, DLCRPCGPTPLA (DA-12) and ANSCNEPCVR (AR-10), displayed remarkable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging abilities with half-maximal inhibitory concentrations (IC50) values of 0.048, 0.034, and 0.95, 0.84 mg/mL, respectively. These values exceed those of the previously reported feather keratin-derived antioxidant peptides. Another peptide, GNQQVHLQSQDM (GM-12), demonstrated XOD activity inhibition, with an IC50 value of 12.15 mg/mL, and it quenched the fluorescence of XOD. Furthermore, after simulating gastrointestinal digestion, DA-12, AR-10, and GM-12 retained their biological activities. Meanwhile, DA-12 and GM-12 showed an unexpected synergistic inhibition on XOD activity accompanied by fluorescence quenching. This study provides new insights into the potential applications of feather keratin, including functionalized feed with antioxidative and antigout (anti-hyperuricemia) activities.

Novel Antioxidant Peptides Derived from Feather Keratin Alleviate H2O2-Induced Oxidative Damage in HepG2 Cells via Keap1/Nrf2 Pathway.

Reactive oxygen species (ROS) are crucial for signal transduction and the maintenance of cellular homeostasis. However, superfluous ROS may engender chronic pathologies. Feather keratin is a promising new source of antioxidant peptides that can eliminate excess ROS and potentially treat oxidative stress-related diseases, but the underlying mechanisms have remained elusive. This study investigated the antioxidant effects and mechanisms against H2O2-induced oxidative damage in HepG2 cells of the two latest discovered antioxidant peptides, CRPCGPTP (CP-8) and ANSCNEPCVR (AR-10), first decrypted from feather keratin. The results revealed that CP-8 and AR-10 did not exhibit cytotoxicity to HepG2 cells while reducing intracellular ROS accumulation. Simultaneously, they enhanced the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), thus alleviating H2O2-induced cell apoptosis. Molecular docking analysis demonstrated that CP-8, AR-10 interacted well with the key amino acids in the Kelch domain of Keap1, thereby directly disrupting the Keap1-Nrf2 interaction. The peptides' biosafety and antioxidant activity via Keap1/Nrf2 signaling lay the groundwork for further animal studies and applications as functional food additives.

Novel Salt-Tolerant Leucine Dehydrogenase from Marine Pseudoalteromonas rubra DSM 6842.

This study reported the cloning, expression, and characterization of a new salt-tolerant leucine dehydrogenase (PrLeuDH) from Pseudoalteromonas rubra DSM 6842. A codon-optimized 1038 bp gene encoding PrLeuDH was successfully expressed on pET-22b( +) in E. coli BL21(DE3). The purified recombinant PrLeuDH showed a single band of about 38.7 kDa on SDS-PAGE. It exhibited the maximum activity at 40 °C and pH 10.5, while kept high activities in the range of 25-45 °C and pH 9.5-12. The Km value and turnover number kcat for leucine of PrLeuDH were 2.23 ± 0.12 mM and 35.39 ± 0.05 s-1, respectively, resulting in a catalytic efficiency kcat/Km of 15.87 s-1/mM. Importantly, PrLeuDH remained 92.1 ± 2.67% active in the presence of 4.0 M NaCl. The study provides the first in-depth understanding of LeuDH from marine Pseudoalteromonas rubra, meanwhile the unique properties of high activity at low temperature and high salt tolerance make it a promising biocatalyst for the synthesis of non-protein amino acids and α-ketoacids under special conditions in pharmaceutical industry.

Characterization of a Novel Shewanella algae Arginine Decarboxylase Expressed in Escherichia coli.

Arginine decarboxylase (ADC) catalyzes the decarboxylation of arginine to form agmatine, an important physiological and pharmacological amine, and attracts attention to the enzymatic production of agmatine. In this study, we for the first time overexpressed and characterized the marine Shewanella algae ADC (SaADC) in Escherichia coli. The recombinant SaADC showed the maximum activity at pH 7.5 and 40 °C. The SaADC displayed previously unreported substrate inhibition when the substrate concentration was higher than 50 mM, which was the upper limit of testing condition in other reports. In the range of 1-80 mM L-arginine, the SaADC showed the Km, kcat, Ki, and kcat/Km values of 72.99 ± 6.45 mM, 42.88 ± 2.63 s-1, 20.56 ± 2.18 mM, and 0.59 s/mM, respectively, which were much higher than the Km (14.55 ± 1.45 mM) and kcat (12.62 ± 0.68 s-1) value obtained by assaying at 1-50 mM L-arginine without considering substrate inhibition. Both the kcat values of SaADC with and without substrate inhibition are the highest ones to the best of our knowledge. This provides a reference for the study of substrate inhibition of ADCs.

A reliable method of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry for determining selenoamino acids in selenoproteins from Lactococcus lactis.

A reliable method for simultaneous determination of four organic selenium species by HPLC-ICP-MS was developed and implemented in determining organic selenoamino acids (Se-AAs) in selenoproteins from Lactococcus lactis (L. lactis) NZ9000. The method consisted of liberating Se-AAs from selenoproteins using ultrasound-assisted protease hydrolysis, and quantitatively detecting Se-AA speciations by HPLC-ICP-MS. After optimizations of proteolysis conditions, chromatographic conditions and determination conditions, the established method could efficiently separate the four Se-AAs, including SeCys, SeCys2, SeMeCys and SeMet within 10 min. It presented high sensitivity with the limits of detection and quantitation in the range of 0.197∼0.240 µg∙L-1 and 0.788∼0.960 µg∙L-1, respectively, good repeatability with a relative standard deviation (RSD) of less than 5%, and good recovery in the desired floating range of 90%∼105%, verifying the good accuracy. The method successfully detected four selenium species in the purified glutathione peroxidase (LlGPx) overexpressed in L. lactis NZ9000, SeCys (0.9716∼1.6784 µg∙g-1), SeCys2 (1.0695∼1.2124 µg∙g-1), SeMeCys (0.7288∼0.7984 µg∙g-1) and SeMet (1.0058∼1.9571 µg∙g-1), accounting for up to 80.14% of total selenium. There was no difference of order of magnitude in the four Se-AAs, indirectly indicating the random incorporation of selenium into selenoprotein LlGPx in L. lactis NZ9000. This work throws new light on the identification and biosynthesis of organic selenium species in selenoproteins and selenium-riched organisms like L. lactis.

Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel.

This study reported simultaneously improved thermostability and hydrolytic pattern of α-amylase from Bacillus subtilis CN7 by rationally engineering the mostly conserved central beta strands in TIM barrel fold. Nine single point mutations and a double mutation were introduced at the 2nd site of the ß7 strand and 3rd site of the ß5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of α-amylase. All the five active mutants changed the compositions and percentages of maltooligosaccharides in final hydrolytic products compared to the product spectrum of the wild-type. A mutant Y204V produced only maltose, maltotriose, and maltopentaose without any glucose and maltotetraose, indicating a conversion from typical endo-amylase to novel maltooligosaccharide-producing amylase. A mutant V260I enhanced the thermal stability by 7.1 °C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of α-amylase by engineering central beta strands of TIM barrel and the novel "beta strands" strategy proposed here may be useful for the protein engineering of other TIM barrel proteins.

Energetic analysis of the two controversial drug binding sites of the M2 proton channel in influenza A virus.

Understanding the mechanism of the M2 proton channel of influenza A is crucially important to both basic research and drug discovery. Recently, the structure was determined independently by high-resolution NMR and X-ray crystallography. However, the two studies lead to completely different drug-binding mechanisms: the X-ray structure shows the drug blocking the pore from inside; whereas the NMR structure shows the drug inhibiting the channel from outside by an allosteric mechanism. Which one of the two is correct? To address this problem, we conducted an in-depth computational analysis. The conclusions drawn from various aspects, such as energetics, the channel-gating dynamic process, the pK(a) shift and its impact on the channel, and the consistency with the previous functional studies, among others, are all in favour to the allosteric mechanism revealed by the NMR structure. The findings reported here may stimulate and encourage new strategies for developing effective drugs against influenza A, particularly in dealing with the drug-resistant problems.

[Diagnosis of chronic enteritis of alpine musk deer (Moschus chrysogaster) based on visible-near infrared reflectance spectra of feces].

A new method was put forward to diagnose chronic enteritis of alpine musk deer (Moschus chrysogaster) by visible-near infrared reflectance spectra of feces. A total of 125 feces samples, including 70 samples from healthy individuals (healthy samples) and 55 samples from chronic enteritis sufferers (diseased samples), were collected in Xinglongshan musk deer farm, Gansu province. The spectral scan was carried out in the darkroom (temperature 18 degrees C-22 degrees C, humidity 22%-25% and halogen lamp as a sole light source) with an ASD FieldSpec 3 spectrometer. All the samples were divided randomly into two groups, one with 95 samples as the calibration set, and another with 30 samples as the validation set. The samples data were pretreated by the methods of S. Golay smoothing and first derivative. The pretreated spectra were analyzed by principal component analysis (PCA), and the top 6 principal components, which were computed by PCA and accounted for 95.16% variation of the original spectral information, were used for modeling as the new variables. The data of the calibration set were used to build models for diagnosing the chronic enteritis of alpine musk deer by means of back-propagation artificial neural network (ANN-BP), fuzzy pattern recognition, Fisher linear discriminant and Bayes stepwise discriminant, respectively. The predicted outcomes of the 30 unknown samples in validation set showed that the accuracy was 86.7% by themethod of Fisher linear discriminant, 90% by fuzzy pattern recognition and ANN-BP model, and 93.3% by stepwise discrimination. Further analysis found that all misdiagnosed samples were derived from the healthy samples, which were treated as disease samples, and the detection rates of diseased samples were 100% by the four different methods. The results indicated that it was feasible to diagnose the chronic enteritis of alpine musk deer by visible-near infrared reflectance spectra of feces as a rapid and non-contact way, and the PCA combined with Bayes stepwise discriminant was a preferred method.

Bibliometric Analysis of Medication Errors and Adverse Drug Events Studies.

OBJECTIVES: Medication errors and adverse drug events are a key concern of the health-care industry. The objectives of this study were to map the intellectual structure of the studies of medication errors and adverse drug events and to investigate the developing path of this literature and interrelationships among the main topics. METHODS: The Web of Science database was searched for documentation of medication errors and adverse drug events from 1961 to 2013. The most cited articles and references were profiled and analyzed using HistCite software to draw a historiograph and Ucinet software to draw a sociogram. RESULTS: The database search revealed 3343 medication errors and 3342 adverse drug event documents. The most cited articles on medication errors focused on 3 key themes from 1961 to 2013, namely, medication errors in adult inpatients, computerized physician order entry in medication error studies, and medication errors in pediatric inpatients. The developing path for the most cited articles about adverse drug events from 1987 to 2013 was as follows: detection, analysis, effect, and prevention from adult inpatient to pediatric inpatient settings and from hospitalized care to ambulatory care. In addition, social network analysis based on the most cited references revealed a close relationship between medication errors and adverse drug events. CONCLUSIONS: The mapping results provide a valuable tool for researchers to access the literature in this field and can be used to help identify the direction of medication errors and adverse drug events research.

Different treatments for acute myocardial infarction patients via outpatient clinics and emergency department.

To investigate relevant factors and patients with acute myocardial infarction (AMI) were admitted during between weekdays and weekends period.Retrospective population-based study setting: from the 2005 population-based national health insurance underwriting database of millions of people, random sampling (National Health Insurance Research Database [NHIRD]-Longitudinal Health Insurance Database [LHID] 2005).In 2000 to 2009 data of NHIRD, subjects presented with first episode AMI who had received the thrombolytic therapy (TPA), or percutaneous coronary artery intervention (PTCA) or coronary artery bypass graft (CABG) during between weekdays and weekends period.From 2000 to 2009 among patients with first AMI total of 2007 people, the weekday group 1453 people, the weekend group 554. The total mortality within 1 year showed 33.53%, the first-day mortality occupied 8.07% in 1 year of total mortality, increased mortality after admission 3 months later. Cox regression analysis showed that AMI has presented significant risk of death, there are 4 items: weekends, age, Charlson comorbidity index (CCI), thrombolytic therapy; in the other variables including emergency, hospital level, hospital ownership, and urban-rural gap are not significant differences. Further using hierarchical logistic regression analysis for Stratification of AMI mortality risk, it has significant that showed the hospital level, age, CCI, thrombolytic therapy; but emergency, PTCA and 3 CABG treatment are not significant differences.It was approved by the hierarchical logistic regression analysis after stratified correction that the present study will have a direct impact on weekdays and weekends death in the hospital level. It will also affect the individual level.