Obesity is a global health problem,and traditional weight loss therapies are often limited in their effectiveness and prone to accompanying adverse reactions In recent years,it has been found that the use of small interfering RNA(siRNA)technology to target regulation of obesity-related genes can effectively reduce body weight,opening up a new path for weight loss treatment.This review summarizes the mechanisms of action,delivery systems,and clinical progress of novel siRNA-based drugs,focusing on targeting genes such as transforming growth factor/cyclooxygenase,inhibin subunit beta E,and activin receptor-like kinase 7,which achieve precise targeting and enhanced therapeutic efficacy through their respective delivery systems.Meanwhile,this review also points out the drawbacks of existing drugs,including low delivery efficiency,risk of immune activation,and off-target effects.

Objective To obtain the recombinant Argonaute protein(TtAgo)from Thermus thermophilus using gene cloning and pro-tein purification techniques and detect its endonuclease activity against the KRAS 12D mutation in vitro,thereby to provide experimental evidence for its subsequent application in the detection of gene mutations.Methods The TtAgo gene sequence was amplified from the genome of Thermus thermophilus by the PCR technology.Then,the amplified sequence was inserted into the prokaryotic expression vec-tor pET-28a to construct the recombinant vector pET-28a-TtAgo.After the recombinant TtAgo protein was induced to be expressed,it was purified sequentially by the nickel ion affinity chromatography(Ni-NTA),heparin affinity chromatography,and size-exclusion chromatography.The high-specificity nuclease activity of the recombinant TtAgo protein mediated by a short DNA guide strand(gDNA)was verified in vitro.Results The prokaryotic expression vector pET-28a-TtAgo was successfully constructed by the gene cloning tech-nique,and the soluble expression of the recombinant TtAgo protein was achieved.With the help of protein purification technology,the high-purity recombinant TtAgo protein was obtained.Under the mediation of gDNA,the recombinant TtAgo protein could specifically cleave wild-type single-stranded DNA(ssDNA),double-stranded DNA(dsDNA),and RNA molecules,while had no cleavage activi-ty for tumor KRAS 12D mutant molecules.Conclusion The high-purity recombinant TtAgo protein with nuclease activity is successful-ly obtained,and its targeted cleavage activity towards the KRAS 12D mutation site is detected in vitro.

Objective To obtain the recombinant Argonaute protein(TtAgo)from Thermus thermophilus using gene cloning and pro-tein purification techniques and detect its endonuclease activity against the KRAS 12D mutation in vitro,thereby to provide experimental evidence for its subsequent application in the detection of gene mutations.Methods The TtAgo gene sequence was amplified from the genome of Thermus thermophilus by the PCR technology.Then,the amplified sequence was inserted into the prokaryotic expression vec-tor pET-28a to construct the recombinant vector pET-28a-TtAgo.After the recombinant TtAgo protein was induced to be expressed,it was purified sequentially by the nickel ion affinity chromatography(Ni-NTA),heparin affinity chromatography,and size-exclusion chromatography.The high-specificity nuclease activity of the recombinant TtAgo protein mediated by a short DNA guide strand(gDNA)was verified in vitro.Results The prokaryotic expression vector pET-28a-TtAgo was successfully constructed by the gene cloning tech-nique,and the soluble expression of the recombinant TtAgo protein was achieved.With the help of protein purification technology,the high-purity recombinant TtAgo protein was obtained.Under the mediation of gDNA,the recombinant TtAgo protein could specifically cleave wild-type single-stranded DNA(ssDNA),double-stranded DNA(dsDNA),and RNA molecules,while had no cleavage activi-ty for tumor KRAS 12D mutant molecules.Conclusion The high-purity recombinant TtAgo protein with nuclease activity is successful-ly obtained,and its targeted cleavage activity towards the KRAS 12D mutation site is detected in vitro.

Obesity is a global health problem,and traditional weight loss therapies are often limited in their effectiveness and prone to accompanying adverse reactions In recent years,it has been found that the use of small interfering RNA(siRNA)technology to target regulation of obesity-related genes can effectively reduce body weight,opening up a new path for weight loss treatment.This review summarizes the mechanisms of action,delivery systems,and clinical progress of novel siRNA-based drugs,focusing on targeting genes such as transforming growth factor/cyclooxygenase,inhibin subunit beta E,and activin receptor-like kinase 7,which achieve precise targeting and enhanced therapeutic efficacy through their respective delivery systems.Meanwhile,this review also points out the drawbacks of existing drugs,including low delivery efficiency,risk of immune activation,and off-target effects.

The ketogenic diet,which has some efficacy in Dravet syndrome,Doose syndrome,West syndrome,Lennox-Gastaut syndrome and other types of refractory epileps,was firstly used in the treatment intractable epilepsy.With the in-depth study in recent years,the researchers found that,the ketogenic diet is the preferred method to treat glucose transporter 1 deficiency syndrome and pyruvate dehydrogenase deficiency,and for a variety of other diseases,such as neurodegenerative diseases,brain(spinal cord)injury,autism may have certain effect,more clinical application is expected.