As a biocatalyst, laccase has been widely studied and applied in the papermaking industry. However, the low catalytic efficiency and poor stability of natural laccase limit its application in the pulping process. To develop the laccase with high activity and strong tolerance, we carried out directed evolution for modification of the laccase derived from Bacillus pumilus and screened out the mutants F282L/F306L and Q275P from the random mutant library by high-throughput screening. The specific activities of F282L/F306L and Q275P were 280.87 U/mg and 453.94 U/mg, respectively, which were 1.42 times and 2.30 times that of the wild-type laccase. Q275P demonstrated significantly improved thermal stability, with the relative activity 20% higher than that of the wild-type laccase after incubation at 40 ℃, 50 ℃, and 70 ℃ for 4 h. F282L/F306L and Q275P showed greater tolerance to metal ions and organic solvents than the wild-type laccase. The K_m value of the wild-type laccase was 374.97 μmo/L, and those of F282L/F306L and Q275P were reduced to 318.96 μmo/L and 360.71 μmo/L, respectively, which suggested that the substrate affinity of laccase was improved after mutation. The k_cat values of F282L/F306L and Q275P for the substrate ABTS were 574.00 s^-1 and 898.03 s^-1, respectively, which were 1.1 times and 1.7 times that of the wild-type laccase, indicating the improved catalytic efficiency. Q275P demonstrated better performance than the wild-type laccase in pulping, as manifested by the reduction of 0.82 in the Kappa number and the increases of 2.00% ISO, 7.8%, and 7.2% in whiteness, tensile index, and breaking length, respectively. This work lays a foundation for improving the adaptation of laccase to the environment of the papermaking industry.
Laccase/chemistry* ; Directed Molecular Evolution ; Enzyme Stability ; Bacillus pumilus/genetics* ; Mutation ; Biocatalysis ; Catalysis

Ferritin, a ubiquitous protein in living organisms, plays a crucial role in storing and converting iron, as well as maintaining cellular iron metabolism balance. Due to the ability of self-assembling into unique nanocage-like structures in vitro and the special physicochemical properties, ferritin has garnered extensive attention in the biomedical field. This paper provides a brief overview of the structure and cargo loading strategies of ferritin, with a specific focus on its applications in various biological fields such as nanomedicine, bioimaging, and nanoparticle vaccine carriers. The aim is to offer a valuable reference for the future research involving ferritin nanoparticles.
Ferritins/chemistry* ; Nanoparticles/chemistry* ; Humans ; Nanomedicine/methods* ; Animals

The rationale for the design of control groups in tuina clinical trial is the foundation for rigorously validating the effectiveness and safety of this therapy. This article reviewed the current state of the design of tuina placebo in control groups of clinical trials， pointed out the necessity of setting up tuina placebo in clinical trials of tuina， analyzed the challenges in implementing blinding of tuina manipulation， and concluded that tuina placebo is still challenged by the placebo effect， the diversification of tuina manipulation but the lack of standardization， and the difficulty of implementing blinding due to the high level of public awareness of tuina. This article also summarized the design of placebo manipulation in three types of clinical trials， including spinal manipulation， acupressure， and paediatric tuina， and proposed four strategies for designing placebo tuina manipulation-controlling placebo effects， developing operational standards for placebo tuina manipulation， ensuring the rigor of blinding implementation， and applying new technologies to enhance the standardization and blinding capacity of placebo tuina methods. So the article is aimed at improving the methodological quality of tuina clinical trial designs， and promoting the standardization and scientificity of tuina clinical trial design.

Functional dyspepsia(FD)is a common functional gastrointestinal disease in clinical practice,which has the characteristics of high incidence,difficult to cure,and recurrence.FD belongs to the categories of"ruffian"and"stomach pain"in TCM,and the disease is located in the stomach,which is closely related to the liver and spleen,and the syndrome of liver depression and spleen deficiency is the most common.This article summarized the literature related to the TCM treatment for FD with liver depression and spleen deficiency syndrome,and concluded the clinical application,efficacy characteristics and mechanism,so as to provide reference for clinical treatment and basic research.The analysis found that the clinical efficacy of TCM in the treatment of FD is significant,which can not only improve the digestive symptoms of patients,but also improve their anxiety and depression state and daily life quality,and has the characteristics of overall regulation,syndrome differentiation and treatment,and improvement of physical fitness.Its mechanism may involve multiple pathways and levels such as abnormal gastric motility,abnormal brain-intestinal interaction and immune inflammatory response.

Objective To study the current status of the randomized controlled trial(RCT)outcome indicators of acupuncture and moxibustion in the treatment of chronic fatigue syndrome(CFS);To provide guidance and references for clinical research and protocol design.Methods RCT of acupuncture and moxibustion for CFS were retrieved from CNKI,Wanfang Data,VIP,SinoMed,PubMed,Embase,Web of Science databases from January 1,2015 to December 9,2024.Through literature screening and data extraction,Excel 2019 was used to make a descriptive analysis of the basic characteristics,diagnostic criteria,intervention measures,classification of outcome indicators,use frequency and evaluation time points of the included studies.Origin2021 software was used to draw bubble charts for visual display.Results Totally 119 RCT were included,involving 8 445 subjects,5 diagnostic criteria of Western medicine,5 diagnostic criteria of TCM,and 6 outcome indicators,namely clinical symptoms/signs,symptoms/syndrome of TCM,physical and chemical examination,quality of life,safety evaluation,and patient satisfaction.A total of 58 outcome indicators were reported,with a cumulative frequency of 430 times.Conclusion The RCT of acupuncture and moxibustion treatment of CFS have some problems,such as incomplete baseline assessment,lack of sample size calculation basis,high risk of bias,ignorance of primary and secondary indicators in the selection of outcome indicators,numerous indicators selection and measurement points,lack of long-term follow-up,inconsistent evaluation criteria for TCM syndrome efficacy,and insufficient attention to health economics evaluation and safety outcome indicators.

Objective To study the current status of the randomized controlled trial(RCT)outcome indicators of acupuncture and moxibustion in the treatment of chronic fatigue syndrome(CFS);To provide guidance and references for clinical research and protocol design.Methods RCT of acupuncture and moxibustion for CFS were retrieved from CNKI,Wanfang Data,VIP,SinoMed,PubMed,Embase,Web of Science databases from January 1,2015 to December 9,2024.Through literature screening and data extraction,Excel 2019 was used to make a descriptive analysis of the basic characteristics,diagnostic criteria,intervention measures,classification of outcome indicators,use frequency and evaluation time points of the included studies.Origin2021 software was used to draw bubble charts for visual display.Results Totally 119 RCT were included,involving 8 445 subjects,5 diagnostic criteria of Western medicine,5 diagnostic criteria of TCM,and 6 outcome indicators,namely clinical symptoms/signs,symptoms/syndrome of TCM,physical and chemical examination,quality of life,safety evaluation,and patient satisfaction.A total of 58 outcome indicators were reported,with a cumulative frequency of 430 times.Conclusion The RCT of acupuncture and moxibustion treatment of CFS have some problems,such as incomplete baseline assessment,lack of sample size calculation basis,high risk of bias,ignorance of primary and secondary indicators in the selection of outcome indicators,numerous indicators selection and measurement points,lack of long-term follow-up,inconsistent evaluation criteria for TCM syndrome efficacy,and insufficient attention to health economics evaluation and safety outcome indicators.

Functional dyspepsia(FD)is a common functional gastrointestinal disease in clinical practice,which has the characteristics of high incidence,difficult to cure,and recurrence.FD belongs to the categories of"ruffian"and"stomach pain"in TCM,and the disease is located in the stomach,which is closely related to the liver and spleen,and the syndrome of liver depression and spleen deficiency is the most common.This article summarized the literature related to the TCM treatment for FD with liver depression and spleen deficiency syndrome,and concluded the clinical application,efficacy characteristics and mechanism,so as to provide reference for clinical treatment and basic research.The analysis found that the clinical efficacy of TCM in the treatment of FD is significant,which can not only improve the digestive symptoms of patients,but also improve their anxiety and depression state and daily life quality,and has the characteristics of overall regulation,syndrome differentiation and treatment,and improvement of physical fitness.Its mechanism may involve multiple pathways and levels such as abnormal gastric motility,abnormal brain-intestinal interaction and immune inflammatory response.

Objective To investigate the mechanism of abdominal massage for treating chronic fatigue syndrome(CFS).Methods Thirty clean-grade female Sprague-Dawley rats were randomly divided into the normal control,model control,and abdominal massage groups(n=10 rats per group).The CFS rat model was established through cold water swimming combined with the chronic restraint method in the model control and abdominal massage groups.The rats in the abdominal massage group were treated with the core techniques of Jingu zang-fu massage,namely layer press"Guanyuan"(CV4)for 8 min and Tuanmo"Zhongwan"(CV12)for 12 min as the primary intervention techniques,once a day for 14 consecutive days.The rats in the two control groups did not receive intervention;however,they were bound to the experimental bench when the experimental group was massaged.After the intervention,the indexes of the main organs of the hypothalamic-pituitary-adrenal(HPA)axis and hippocampal cell apoptosis in each group were measured.The morphology of hippocampal cells in each group was observed using Nissl staining of hippocampal tissue and transmission electron microscopy of hippocampal neurons.Results The index of each organ in the model control group was upregulated(P<0.01)compared to that of the normal control group.In contrast,the index of each organ in the abdominal massage group was downregulated(P<0.01)compared with that of the model control group.Compared to the normal control group,the index of each organ in the abdominal massage group was upregulated;however,the difference was not significant.Compared to the normal control group,the cell nuclei in the model control group were significantly consolidated,the nuclear membrane structure was ruptured,and the margins were irregular.Most of the cell morphology in the abdominal massage group was normal compared with that of the model control group,and a small number of nuclear membrane structures were unclear.Compared to the normal control group,the hippocampal neuronal cell in the other two groups was significantly damaged,and the hippocampal neuronal cell in the abdominal massage group was in good condition compared to the model control group.The degree of neuronal apoptosis in the model control group was significantly higher than that in the normal control group(P<0.01).The degree of neuronal apoptosis in the abdominal massage group decreased compared to that of the model control group(P<0.01),which was slightly higher than that in the normal control group but not significant.Conclusion Cold water swimming combined with chronic restraint can simulate CFS in rats,and abdominal massage can reduce the organ index of the hypothalamus,pituitary gland,and adrenal gland,increase hippocampal cell activity,reduce hippocampal tissue damage,inhibit hippocampal cell apoptosis,and maintain the normal physiological function of hippocampal neurons.

BACKGROUND:The pathogenesis of osteoporosis is complex,and its essence is the weakening of bone formation and the enhancement of bone absorption caused by various reasons,resulting in the imbalance of bone metabolism.In recent years,N6-methyladenosine has been found(N6-methyladenosine,m6A)methylation can prevent and treat osteoporosis by regulating bone metabolism. OBJECTIVE:Taking the regulation of bone metabolism by m6A methylation as an entry point,to systematically sort out and summarize the research progress of m6A methylation in osteoporosis,so as to provide certain theoretical reference bases for the search of new therapeutic targets for osteoporosis. METHODS:CNKI,WanFang,VIP,PubMed,MEDLINE,Nature,and Cochrane databases were retrieved for relevant literature published from database inception to 2023.The keywords were"osteoporosis,m6A methylation,bone metabolism,bone marrow mesenchymal stem cells,osteoblasts,osteoclasts"in Chinese and English.Duplicates and obsolete non-referenced documents were excluded,and a total of 73 standard papers were included for further review. RESULTS AND CONCLUSION:m6A methylation can affect the activity and differentiation of bone marrow mesenchymal stem cells,osteoblasts,and osteoclasts through various pathways to regulate bone metabolism and prevent osteoporosis.The regulatory process of m6A methylation is extremely complex,and its related proteins play different roles in different cells.Even in the same kind of cells,the same type of proteins may have radically different roles,regulating different physiological and pathological processes.

Objective:To analyze identification of copper death gene related subtypes,construction of prognosis model and influence of immune infiltration in osteosarcoma(OS)on basis of copper death gene.Methods:Survival and prognosis of OS associated copper death gene were analyzed combining by TARGET and GEO database.OS was divided into different subtypes of copper death by consistent clustering method.SSGSEA was used to analyze difference of immune cells in classification of copper death.Setting P value= 0.05 and q value=0.05,GO and KEGG enrichment analysis were performed on differential genes of copper death typing.Prognosis model was constructed according to results of Lasso regression analysis and cross validation,risk assessment analysis and ROC curve were used to evaluate accuracy of model prediction.Combined with clinical characteristics,nomograms were constructed to predict survival time of patients,and risk differences were analyzed.Immune cell infiltration and tumor microenvironment analysis were performed on OS samples."pRRophetic"package in R software was used to analyze drug sensitivity of OS samples.Results:FDX1,GLS,DLAT and PDHB as high-risk genes for OS prognosis were identified.According to copper death classification of OS samples,OS could be divided into two types:CRGclusterA and CRGclusterB.CRGclusterA was associated with Th2 cells,and CRGclusterB was associated with Th1 cells.Most OS copper death genes were highly expressed in CRGclusterA.Immune cell infiltration analysis results showed that γδ T cells,resting mast cells and resting dendritic cells were positively correlated with risk score,while CD8 T cells were negatively correlated with risk score.Drug sensitivity analysis showed that OS showed higher sensitivity to Elesclomol and GW.441756.Conclusion:Two subtypes of CRGclusterA and CRGclusterB are identified in this study.Four high-risk prognostic genes FDX1,GLS,DLAT and PDHB are identified,providing new insights into prognostic evaluation and immunotherapy target candidates for OS.