This paper summarizes professor LIN Yi's clinical experience in treating brain metastases of triple-negative breast cancer （TNBC）. It is believed that the pathogenesis of this disease is fundamentally based on spleen and lung qi deficiency， with the binding of phlegm， stasis， and toxins acting as the critical link for disease progression， and qi stagnation serving as a major factor in its protracted course. The core therapeutic principle of "consolidating the root and cultivating the origin， resolving phlegm and dredging collaterals" is proposed. Clinically， the disease is staged into active， stable， and convalescent phases. During the active phase， characterized by spleen and lung qi deficiency alongside hyperactivity of phlegm-toxin pathogens， the treatment focuses on supplementing qi， fortifying the spleen， resolving phlegm， and dredging collaterals， utilizing the Yiqi Jiangtan Tongluo Formula （益气降痰通络方）. During the stable phase， marked by spleen deficiency， weak qi， and residual phlegm， stasis， and toxins lingering in the collaterals， the treatment aims to fortify spleen， cultivate the origin， expel pathogens， and dredge collaterals， using modified Baihua Qiling Formula （白花芪苓方）. During the convalescent phase， characterized by depletion of healthy qi， dysfunction of zang-fu （脏腑） organs， and deep-seated residual phlegm， stasis， and toxins within the brain collaterals， the treatment focuses on regulating qi movement， protecting body fluids， and applying constitution-based treatments， mostly using mild and nourishing medicinals.

This paper summarizes professor LIN Yi's clinical experience in treating brain metastases of triple-negative breast cancer （TNBC）. It is believed that the pathogenesis of this disease is fundamentally based on spleen and lung qi deficiency， with the binding of phlegm， stasis， and toxins acting as the critical link for disease progression， and qi stagnation serving as a major factor in its protracted course. The core therapeutic principle of "consolidating the root and cultivating the origin， resolving phlegm and dredging collaterals" is proposed. Clinically， the disease is staged into active， stable， and convalescent phases. During the active phase， characterized by spleen and lung qi deficiency alongside hyperactivity of phlegm-toxin pathogens， the treatment focuses on supplementing qi， fortifying the spleen， resolving phlegm， and dredging collaterals， utilizing the Yiqi Jiangtan Tongluo Formula （益气降痰通络方）. During the stable phase， marked by spleen deficiency， weak qi， and residual phlegm， stasis， and toxins lingering in the collaterals， the treatment aims to fortify spleen， cultivate the origin， expel pathogens， and dredge collaterals， using modified Baihua Qiling Formula （白花芪苓方）. During the convalescent phase， characterized by depletion of healthy qi， dysfunction of zang-fu （脏腑） organs， and deep-seated residual phlegm， stasis， and toxins within the brain collaterals， the treatment focuses on regulating qi movement， protecting body fluids， and applying constitution-based treatments， mostly using mild and nourishing medicinals.

[Objective]To explore the effect of Baoyuan Jiedu Decoction(BJD)on liver lipids and liver mitochondrial function in cancer cachexia mice.[Methods]The mice model of cancer cachexia was established by subcutaneous inoculation of Lewis lung cancer cells,and the mice were randomly divided into normal group,model group,BJD group and medroxyprogesterone acetate(MPA)group,which were given continuously for 21 days,and the food intake,body weight and tumor volume of the mice were recorded.Multi-dimensional mass spectrometry-based shotgun lipidomics(MDMS-SL)was used to detect liver lipid content.Changes of liver mitochondria were observed by transmission electron microscope.Enzyme-linked immunosorbent assay(ELISA)was used to detect adenosine triphosphate(ATP)content in liver.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression levels of liver mitochondrial respiratory chain complexes NADH:ubiquinone oxidoreductase subunit B8(NDUFB8),succinate dehydrogenase complex iron sulfur subunit B(SDHB)and ubiquinol-cytochrome-c reductase complex core protein 2(UQCRC2).The expression level of liver mitochondrial respiratory chain complex NDUFB8 protein was detected by Western blot.[Results]Compared with model group,the food intake and body weight of BJD group increased,and the tumor growth was slow(P<0.05).The results of lipidomics showed that the liver lipids in model group changed significantly compared with those in normal group,and BJD could obviously regulate 33 lipid molecules after intervention(P<0.05).The transmission electron microscope showed that the morphology of mitochondria in the model group was distorted,some mitochondrial contents were degraded,the outer membrane of mitochondria was broken,and the crista of mitochondria was obviously shortened and broken.Compared with the model group,the morphology of mitochondria in Baoyuan Jiedu Decoction group was obviously relieved,and the damage of crista structure was alleviated.The results of ELISA showed that the ATP content in the liver of BJD group was significantly higher than that of model group(P<0.05).RT-qPCR results showed that BJD could significantly increase the mRNA expression levels of mitochondrial respiratory chain complexes NDUFB8,SDHB and UQCRC2 after intervention(P<0.05).Western blot results showed that BJD group could significantly increase the protein expression level of mitochondrial respiratory chain complex NDUFB8 after intervention(P<0.05).[Conclusion]BJD can obviously increase the body weight and food intake of cancer cachexia mice,regulate the abnormal changes of liver lipid metabolism,restore the activity of liver mitochondrial respiratory chain complex,and improve mitochondrial respiratory function and energy metabolism.

Objective:To express,purify,prepare antibodies,and analyze the subcellular localization of Toxoplasma gondii thioredoxin 20(Trx20),and to provide the reference for the development of Toxoplasma gondii vaccine.Methods:Bioinformatics-related websites and software were used to perform bioinformatics analysis of the Trx20 protein;specific primers were designed to amplify the target fragment and construct the prokaryotic expression vector;the protein was expressed in vitro and purified;experimental animals were immunized to prepare antibodies;enzyme-linked immunosorbent assay(ELISA)method was used to detect the titer of the polyclonal antibodies;Western blotting method was used to verify the specificity and sensitivity of the antibodies and to determine the natural expression of the protein;immunofluorescence assay(IFA)was used to analyze the subcellular localization of the protein.Results:The bioinformatics analysis results showed that Trx20 protein was a relatively stable hydrophilic protein with a molecular formula of C2172H3412N548O616S20,containing 424 amino acids,a predicted relative molecular mass of 47 700,and a theoretical isoelectric point of 8.55;it was predicted that the protein had one signal peptide,no transmembrane region,contained one domain named"Thioredoxin like Superfamily",and had 35 phosphorylation sites,one N-glycosylation site,and 17 antigenic determinants;in the secondary structure,alpha-helices accounted for 41.51％of the total amino acids,and random coils accounted for 39.86％;the recombinant plasmids pET-28a-Trx20 and pGEX-4T-1-Trx20 were successfully constructed,and the soluble recombinant protein was expressed and purified;polyclonal antibodies were successfully prepared with a titer as high as 1:64 000,and they specifically recognized the endogenous Trx20 protein in Toxoplasma gondii;the subcellular localization results showed that Trx20 protein was widely distributed in the cytoplasm of the parasite.Conclusion:Toxoplasma gondii Trx20 protein is a secretory protein containing phosphorylation/glycosylation modification sites and a thioredoxin domain,and it is localized in the cytoplasm of the parasite.

Myocardial fibrosis is a serious cause of heart failure and even sudden cardiac death. However, the mechanisms underlying myocardial ischemia-induced cardiac fibrosis remain unclear. Here, we identified that the expression of sterile alpha and TIR motif containing 1 (SARM1), was increased significantly in the ischemic cardiomyopathy patients, dilated cardiomyopathy patients (GSE116250) and fibrotic heart tissues of mice. Additionally, inhibition or knockdown of SARM1 can improve myocardial fibrosis and cardiac function of myocardial infarction (MI) mice. Moreover, SARM1 fibroblasts-specific knock-in mice had increased deposition of extracellular matrix and impaired cardiac function. Mechanically, elevated expression of SARM1 promotes the deposition of extracellular matrix by directly modulating P4HA1. Notably, by using the Click-iT reaction, we identified that the increased expression of ZDHHC17 promotes the palmitoylation levels of SARM1, thereby accelerating the fibrosis process. Based on the fibrosis-promoting effect of SARM1, we screened several drugs with anti-myocardial fibrosis activity. In conclusion, we have unveiled that palmitoylated SARM1 targeting P4HA1 promotes collagen deposition and myocardial fibrosis. Inhibition of SARM1 is a potential strategy for the treatment of myocardial fibrosis. The sites where SARM1 interacts with P4HA1 and the palmitoylation modification sites of SARM1 may be the active targets for anti-fibrosis drugs.

Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease Ⅰ for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease Ⅰ-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.
Genome, Viral/genetics* ; Polymerase Chain Reaction/methods* ; DNA, Viral/genetics* ; Bacteriophage T7/enzymology* ; Synthetic Biology/methods*

Arenavirus infection in humans can lead to viral hemorrhagic fever,which is often fatal and poses a sig-nificant threat to public health.Currently,no effective therapeutic drugs or licensed vaccines are available for arenavirus infections,underscoring the urgency to strengthen the basic research on these viruses and develop novel antiviral therapies.This article summarizes the advancements of research on the structure,life cycle,pathogen-ic mechanisms,epidemiology,detection methods and newly discovered mammalian arenaviruses.Focusing on Las-sa virus,multiple candidate strategies for treatment of arenaviruses are explored,with expectations to provide ref-erences for the development of novel anti-arenaviral drugs.

Nipah virus is a highly pathogenic zoonotic virus.The viral characteristics,epidemiological features,viral life cycle and pathogenic mechanisms of Nipah virus were summarized in the article,and the alternative drugs tar-geting various stages of the viral life cycle,including antibody drugs,peptide drugs,and small molecule com-pounds,were systematically reviewed.The current challenges confronting the process of research and development of alternative drugs for Nipah virus were analyzed,and an outlook was also made on future research direction of candidate drugs for Nipah virus,aiming to provide comprehensive and in-depth theoretical reference for prevention and control of Nipah virus as well as the development of drugs and to facilitate the further development of related fields.

[Objective]To explore the effect of Baoyuan Jiedu Decoction(BJD)on liver lipids and liver mitochondrial function in cancer cachexia mice.[Methods]The mice model of cancer cachexia was established by subcutaneous inoculation of Lewis lung cancer cells,and the mice were randomly divided into normal group,model group,BJD group and medroxyprogesterone acetate(MPA)group,which were given continuously for 21 days,and the food intake,body weight and tumor volume of the mice were recorded.Multi-dimensional mass spectrometry-based shotgun lipidomics(MDMS-SL)was used to detect liver lipid content.Changes of liver mitochondria were observed by transmission electron microscope.Enzyme-linked immunosorbent assay(ELISA)was used to detect adenosine triphosphate(ATP)content in liver.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression levels of liver mitochondrial respiratory chain complexes NADH:ubiquinone oxidoreductase subunit B8(NDUFB8),succinate dehydrogenase complex iron sulfur subunit B(SDHB)and ubiquinol-cytochrome-c reductase complex core protein 2(UQCRC2).The expression level of liver mitochondrial respiratory chain complex NDUFB8 protein was detected by Western blot.[Results]Compared with model group,the food intake and body weight of BJD group increased,and the tumor growth was slow(P<0.05).The results of lipidomics showed that the liver lipids in model group changed significantly compared with those in normal group,and BJD could obviously regulate 33 lipid molecules after intervention(P<0.05).The transmission electron microscope showed that the morphology of mitochondria in the model group was distorted,some mitochondrial contents were degraded,the outer membrane of mitochondria was broken,and the crista of mitochondria was obviously shortened and broken.Compared with the model group,the morphology of mitochondria in Baoyuan Jiedu Decoction group was obviously relieved,and the damage of crista structure was alleviated.The results of ELISA showed that the ATP content in the liver of BJD group was significantly higher than that of model group(P<0.05).RT-qPCR results showed that BJD could significantly increase the mRNA expression levels of mitochondrial respiratory chain complexes NDUFB8,SDHB and UQCRC2 after intervention(P<0.05).Western blot results showed that BJD group could significantly increase the protein expression level of mitochondrial respiratory chain complex NDUFB8 after intervention(P<0.05).[Conclusion]BJD can obviously increase the body weight and food intake of cancer cachexia mice,regulate the abnormal changes of liver lipid metabolism,restore the activity of liver mitochondrial respiratory chain complex,and improve mitochondrial respiratory function and energy metabolism.

Background and purpose:In breast cancer surgery,margin status assessment significantly impacts patient prognosis,with positive margins indicating higher recurrence and metastasis risks.Ensuring complete tumor resection is thus critical for surgical success.Indocyanine green(ICG)has garnered attention for its potential real-time imaging of breast cancer lesions under near-infrared light.This study employed ICG for intraoperative assessment of breast cancer lesion margin status and further explored the possibility of optimizing the safe margin distance surround the lesion in normal breast tissue.Methods:Clinical data of patients admitted to the Department of Thyroid and Breast Surgery,the Fourth Affiliated Hospital of Anhui Medical University(Affiliated Chaohu Hospital),from December 2021 to September 2022 were collected.A retrospective clinical study was conducted on breast cancer patients who were randomly assigned to either the ICG group or the conventional surgery group.Two to three hours before surgery,patients in the ICG group received a peripheral intravenous injection of 0.5 mg/kg ICG.Intraoperative fluorescence imaging was performed on the specimen before and after resection,as well as on the residual cavity.Near-infrared fluorescence imaging equipment was used to quantitatively measure fluorescence intensity of resected lesions at 4 directions(12,3,6,and 9 o'clock)and detect fluorescence in the residual cavity after lesion removal.Specimens were promptly sent to the pathology department for pathological examination,and safety margins of normal breast tissue in the 4 directions were recorded.The Strengthening the Reporting of Observational Studies in Epidemiology(STROBE)checklist was followed for this study.This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Anhui Medical University(Affiliated Chaohu Hospital)(No.KYXM-202310-46).Results:This study included 50 breast cancer patients,with 24 in the ICG group and 26 in the traditional surgery group.In the ICG group,fluorescence signals were detected at all lesion sites.Specifically,fluorescence density values at the lesion center,margin,and surrounding normal breast tissue were measured as 251.08±10.73,208.08±19.74,and 156.76±16.47,respectively,showing a gradual decrease from center outward with statistically significant differences(P＜0.05).Additionally,fluorescence ratios between the lesion center and margin,and center and surrounding normal tissue,were 1.22±0.13 and 1.62±0.19,respectively.After resection,abnormal fluorescence was observed in 2 of 24 cases in the residual cavity,with 1 case being invasive carcinoma with ductal carcinoma in situ and the other normal breast tissue.Ultimately,this study demonstrated that ICG achieved a sensitivity of 95.9%and a specificity of 97.9%in margin assessment.After specimen resection,the safety margins of normal glandular tissue surrounding the lesion were measured.The safety widths for the ICG group and the concurrent breast cancer surgery group were(8.36±6.42)mm and(15.08±4.75)mm,respectively.This difference was statistically significant(P＜0.05).Conclusion:ICG is a real-time,efficient,and cost-effective tracer that can be used to determine breast cancer margins,with excellent sensitivity and specificity.For early-stage breast cancer patients who are eligible for breast-conserving surgery,this tracer helps to reduce the amount of healthy breast tissue that is removed around the lesion.