OBJECTIVE To design and synthesize rifamycin S derivatives and load them into milk exosomes to evaluate their in vitro antimicrobial activity.METHODS Rifamycin S derivatives were synthe-sized and characterized by mass spectrometry and NMR.Using the dilution assay method,the inhibitory activity of each rifamycin S derivatives molecule against Staphylococcus aureus and Pseudomonas aerugi-nosa was determined,and the IC50 was calculated.Derivatives molecules with excellent antimicrobial activity were selected and loaded into milk exosomes using the ultrasonication method,resulting in the preparation of milk exosome-loaded rifamycin S derivatives.The antimicrobial activity against Staphylo-coccus aureus was determined using the dilution assay method.The inhibitory effect of the exosome-loaded rifamycin S derivatives on Staphylococcus aureus residing within macrophages was detected using the plate colony counting method.RESULTS Three rifamycin S derivatives were successfully designed and synthesized,which demonstrated superior antimicrobial activity against Staphylococcus aureus(the parent compound's antimicrobial activity is merely from 1/20 to 1/80 of that of the three rifamycin S derivatives)and Pseudomonas aeruginosa(the parent compound's antimicrobial activity is only 1/14 and 1/9 of that of compound 1 and compound 3)compared to the parent compound.The loading of milk exosomes with the rifamycin S derivatives compound 3 was successfully achieved,with a loading efficiency of 10.9％.The antimicrobial activity of the compound after exosome loading was significantly enhanced against Staphylococcus aureus in vitro and against Staphylococcus aureus residing within macrophages(P＜0.01).CONCLUSION The designed and synthesized derivatives of rifamycin S possess stronger anti-microbial activity,and their antibacterial efficacy against both extracellular and intracellular bacteria can be further enhanced after loading into exosomes.

Viruses constitute a significant group of pathogens that have caused numerous fatalities and substantial economic losses in recent years, particularly with the emergence of coronaviruses. While the impact of SARS-CoV-2 appears to be diminishing in daily life, only a limited number of drugs have received approval or emergency use authorization for its treatment. Given the high mutation rate of viral genomes, host-directed agents (HDAs) have emerged as a preferred choice due to their broad applicability and lasting effectiveness. In contrast to direct-acting antivirals (DAAs), HDAs offer several advantages, including broad-spectrum antiviral activities, potential efficacy against future emerging viruses, and a lower likelihood of inducing drug resistance. In our review article, we have synthesized known host-directed antiviral targets that span diverse cellular pathways and mechanisms, shedding light on the intricate interplay between host cells and viruses. Additionally, we have provided a brief overview of the development of HDAs based on these targets. We aim for this comprehensive analysis to offer valuable perspectives and insights that can guide future antiviral research and drug development efforts.

OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

AIM:To compare the degree of disease simulation between the two mouse models of acute exacer-bation of chronic obstructive pulmonary disease(AECOPD)using intranasal instillation of lipopolysaccharide(LPS)and fine particulate matter(PM2.5)for 3 d based on exposure to cigarette smoke(CS)for 90 d.METHODS:Thirty-two male C57BL/6 mice were randomly divided into 4 groups:control group(n=8),CS group(n=8),CS+PM2.5 group(n=8)and CS+LPS group(n=8).The AECOPD models in CS+PM2.5 and CS+LPS groups were established by CS exposure combined with intranasal PM2.5 and LPS instillation.Lung function,lung pathology and airway goblet cell hyperplasia using histologi-cal staining were measured.To evaluate the degree of lung inflammation and mucus secretion in mice,the prorein levels of mucin 5AC(MUC5AC),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the bronchoalveolar lavage fluid(BALF)were detected by ELISA and total white blood cell(WBC)counts and the BALF differential cell counts(neutro-phils,macrophages,lymphocytes)were detected by Giemsa staining.RESULTS:In CS group,lung function decreased(P<0.05),and bronchial inflammation index increased(P<0.01),airway goblet cell hyperplasia and airway collagen de-position were significant(P<0.01),total WBC count and differential cell count in the BALF increased(P<0.05),MUC5AC and inflammatory factor IL-6 and TNF-α levels increased(P<0.05),compared with control group.Compared with CS group,lung function decreased(P<0.05),the bronchial inflammation index increased(P<0.01),airway goblet cell hyperplasia and airway collagen deposition were significant(P<0.01),total WBC count and differential cell count in the BALF increased(P<0.05),and MUC5AC and inflammatory factor IL-6 and TNF-α levels increased(P<0.05)in CS+PM2.5 and CS+LPS groups.Compared with CS+PM2.5 group,lung function decreased(P<0.05),the bronchial inflamma-tion index increased(P<0.01),airway goblet cell hyperplasia and airway collagen deposition were significant(P<0.01),total WBC count and differential cell count in the BALF increased(P<0.05),and MUC5AC and inflammatory factor IL-6 and TNF-α levels increased(P<0.05)in CS+LPS group.CONCLUSION:Exposure to CS combined with both intrana-sal PM2.5 and LPS instillation allowed for establishing AECOPD models in mice,and CS exposure combined with intrana-sal LPS instillation better simulated AECOPD characteristics.

Objective To analyze the characteristics,causes,influencing factors of compensation,and appraisal features of second-instance medical malpractice cases involving cardiovascular diseases in Henan Province from 2017 to 2022,and to provide reference for forensic appraisal and judicial trial.Methods Cases were retrieved from China Judgments Online between 2017 and 2022.A total of 1,957 documents were reviewed,including 1,397 medical malpractice cases and 130 cardiovascular disease cases.Results The total compensation awarded in these second-instance cases was 27.04 million yuan,with a median of 158,600 yuan.Cases involving patient death accounted for 80.00％(104/130).Among 107 cases with first-instance appraisals,55.14％(59/107)raised objections,while 74.62％(97/130)of the second-instance trials upheld the original judgment.The most common degree of responsibility borne by medical institutions was secondary responsibility(41.54％,54/130).The top three medical faults were:inadequate observation and management of patient conditions(46.15％),omission of auxiliary examinations(37.50％),and insufficient notification(36.54％).In terms of violations-including medical record documentation,inappropriate treatment,misdiagnosis or missed diagnosis,out-of-scope practice,and improper medication use-the actual proportions in judgments(24/113,34/113,12/113,7/113,14/113)were all significantly lower than the patients' claims(all P＜0.05).Conclusion Cardiovascular medical malpractice cases in second-instance trials involve substantial compensation and a high proportion of death outcomes.Both medical institutions and patients should pay greater attention to first-instance trials.Appraisal organizations should proactively provide explanation and education regarding issues likely to raise patient doubts,thereby reducing unnecessary appeals.They should also avoid hasty revisions of appraisal opinions when faced with objections and instead focus on improving the quality and credibility of appraisals.In determining medical faults,emphasis should be placed on evaluating whether there was inadequate patient monitoring and management,omission of auxiliary examinations,and insufficient notification.

AIM:To compare the degree of disease simulation between the two mouse models of acute exacer-bation of chronic obstructive pulmonary disease(AECOPD)using intranasal instillation of lipopolysaccharide(LPS)and fine particulate matter(PM2.5)for 3 d based on exposure to cigarette smoke(CS)for 90 d.METHODS:Thirty-two male C57BL/6 mice were randomly divided into 4 groups:control group(n=8),CS group(n=8),CS+PM2.5 group(n=8)and CS+LPS group(n=8).The AECOPD models in CS+PM2.5 and CS+LPS groups were established by CS exposure combined with intranasal PM2.5 and LPS instillation.Lung function,lung pathology and airway goblet cell hyperplasia using histologi-cal staining were measured.To evaluate the degree of lung inflammation and mucus secretion in mice,the prorein levels of mucin 5AC(MUC5AC),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the bronchoalveolar lavage fluid(BALF)were detected by ELISA and total white blood cell(WBC)counts and the BALF differential cell counts(neutro-phils,macrophages,lymphocytes)were detected by Giemsa staining.RESULTS:In CS group,lung function decreased(P<0.05),and bronchial inflammation index increased(P<0.01),airway goblet cell hyperplasia and airway collagen de-position were significant(P<0.01),total WBC count and differential cell count in the BALF increased(P<0.05),MUC5AC and inflammatory factor IL-6 and TNF-α levels increased(P<0.05),compared with control group.Compared with CS group,lung function decreased(P<0.05),the bronchial inflammation index increased(P<0.01),airway goblet cell hyperplasia and airway collagen deposition were significant(P<0.01),total WBC count and differential cell count in the BALF increased(P<0.05),and MUC5AC and inflammatory factor IL-6 and TNF-α levels increased(P<0.05)in CS+PM2.5 and CS+LPS groups.Compared with CS+PM2.5 group,lung function decreased(P<0.05),the bronchial inflamma-tion index increased(P<0.01),airway goblet cell hyperplasia and airway collagen deposition were significant(P<0.01),total WBC count and differential cell count in the BALF increased(P<0.05),and MUC5AC and inflammatory factor IL-6 and TNF-α levels increased(P<0.05)in CS+LPS group.CONCLUSION:Exposure to CS combined with both intrana-sal PM2.5 and LPS instillation allowed for establishing AECOPD models in mice,and CS exposure combined with intrana-sal LPS instillation better simulated AECOPD characteristics.

OBJECTIVE To design and synthesize rifamycin S derivatives and load them into milk exosomes to evaluate their in vitro antimicrobial activity.METHODS Rifamycin S derivatives were synthe-sized and characterized by mass spectrometry and NMR.Using the dilution assay method,the inhibitory activity of each rifamycin S derivatives molecule against Staphylococcus aureus and Pseudomonas aerugi-nosa was determined,and the IC50 was calculated.Derivatives molecules with excellent antimicrobial activity were selected and loaded into milk exosomes using the ultrasonication method,resulting in the preparation of milk exosome-loaded rifamycin S derivatives.The antimicrobial activity against Staphylo-coccus aureus was determined using the dilution assay method.The inhibitory effect of the exosome-loaded rifamycin S derivatives on Staphylococcus aureus residing within macrophages was detected using the plate colony counting method.RESULTS Three rifamycin S derivatives were successfully designed and synthesized,which demonstrated superior antimicrobial activity against Staphylococcus aureus(the parent compound's antimicrobial activity is merely from 1/20 to 1/80 of that of the three rifamycin S derivatives)and Pseudomonas aeruginosa(the parent compound's antimicrobial activity is only 1/14 and 1/9 of that of compound 1 and compound 3)compared to the parent compound.The loading of milk exosomes with the rifamycin S derivatives compound 3 was successfully achieved,with a loading efficiency of 10.9％.The antimicrobial activity of the compound after exosome loading was significantly enhanced against Staphylococcus aureus in vitro and against Staphylococcus aureus residing within macrophages(P＜0.01).CONCLUSION The designed and synthesized derivatives of rifamycin S possess stronger anti-microbial activity,and their antibacterial efficacy against both extracellular and intracellular bacteria can be further enhanced after loading into exosomes.

Objective To analyze the characteristics,causes,influencing factors of compensation,and appraisal features of second-instance medical malpractice cases involving cardiovascular diseases in Henan Province from 2017 to 2022,and to provide reference for forensic appraisal and judicial trial.Methods Cases were retrieved from China Judgments Online between 2017 and 2022.A total of 1,957 documents were reviewed,including 1,397 medical malpractice cases and 130 cardiovascular disease cases.Results The total compensation awarded in these second-instance cases was 27.04 million yuan,with a median of 158,600 yuan.Cases involving patient death accounted for 80.00％(104/130).Among 107 cases with first-instance appraisals,55.14％(59/107)raised objections,while 74.62％(97/130)of the second-instance trials upheld the original judgment.The most common degree of responsibility borne by medical institutions was secondary responsibility(41.54％,54/130).The top three medical faults were:inadequate observation and management of patient conditions(46.15％),omission of auxiliary examinations(37.50％),and insufficient notification(36.54％).In terms of violations-including medical record documentation,inappropriate treatment,misdiagnosis or missed diagnosis,out-of-scope practice,and improper medication use-the actual proportions in judgments(24/113,34/113,12/113,7/113,14/113)were all significantly lower than the patients' claims(all P＜0.05).Conclusion Cardiovascular medical malpractice cases in second-instance trials involve substantial compensation and a high proportion of death outcomes.Both medical institutions and patients should pay greater attention to first-instance trials.Appraisal organizations should proactively provide explanation and education regarding issues likely to raise patient doubts,thereby reducing unnecessary appeals.They should also avoid hasty revisions of appraisal opinions when faced with objections and instead focus on improving the quality and credibility of appraisals.In determining medical faults,emphasis should be placed on evaluating whether there was inadequate patient monitoring and management,omission of auxiliary examinations,and insufficient notification.

OBJECTIVE To construct an insulin-resistant(IR)small intestinal organoid model of mice and study the protective effect of flavanomarein(FM)on the intestinal mucosal barrier in the model.METHODS ①Small intestinal organoid models of C57BL/6J and db/db of mice were constructed.The expressions of Ki-67,E-cadherin(E-cad),lysozyme(Lyz)and mucin-2(Muc-2)in small intestinal organ-oids were detected by 3D immunofluorescence.RT-qPCR was used to detect the expressions of fibro-nectin(Fn),glucagon-like peptide-1(GLP-1)and peotide YY(PYY)mRNA while Western blotting was used to detect the expressions of Fn,GLP-1 and PYY protein.The Lyz secretion level was detected by ELISA.② Small intestinal organoids were divided into five groups:C57BL/6J mice 'small intestinal organ-oids as the normal control group,db/db mice' intestinal organoids as the IR model group,db/db mice small intestinal organoids with flavanomarein 25,50 and 100 μmol·L-1 intervention for 48 h as IR model+ FM groups.RT-qPCR was used to detect the expression of Lyz mRNA while Western blotting was used to detect the expression of Lyz protein.RESULTS ① On the 6th day of small intestinal organoid culture,a ring structure with a clear luminal structure was formed and an IR mouse small intestinal organoid model was established.3D Immunofluorescence detection showed that the established small intestinal organoids all expressed Ki-67,E-cad,Lyz and MUC-2.Compared with the normal control group,the expres-sion of Fn mRNA in the IR model group was significantly increased(P<0.05)while the expressions of GLP-1 and PYY mRNA were significantly decreased(P<0.05).Compared with the normal control group,the expression of Fn protein in the IR model group was significantly decreased(P<0.05)while the expressions of GLP-1 and PYY protein were significantly increased(P<0.05).ELISA results showed that compared with the normal control group,the secretion levels of Lyz in the IR model group were signifi-cantly decreased(P<0.01).② RT-qPCR results showed that compared with the normal control group,the expression of Lyz mRNA in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz mRNA in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).Western blotting results showed that compared with the normal control group,the expression of Lyz protein in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz protein in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).CONCLUSION The constructed IR mouse small intestinal organoid model provides a more complete in vitro research model for exploring the pathophysiological mechanism by which drug interventions help repair the intestinal mucosal barrier.FM may maintain the intestinal mucosal barrier by reversing the decrease in Lyz expression levels in IR mice,thereby improving IR.

ObjectiveTo investigate the effects of flavanomarein on the transcriptome of small intestinal organoids in insulin-resistant mice. MethodFirstly， small intestinal organoids of C57BL/6J and db/db mice were established. Ki-67 and E-cadherin expression was determined by immunofluorescence. Small intestinal organoids were divided into the following three groups： C57BL/6J mouse small intestinal organoids as the normal control group， db/db mouse small intestinal organoids as the model group （IR group）， and db/db mouse small intestinal organoids treated with flavanomarein as the administration group （FM group）. Western blot was used to detect the expression of glucagon-like peptide-1（GLP-1） protein on the small intestinal organoids of the three groups. Finally， transcriptome sequencing was performed on samples from the three groups. ResultOn the 6^th day of small intestine organoids culture， a cyclic structure was formed around the lumen， and a small intestine organoids culture model was preliminarily established. Immunofluorescence detection showed that ki-67 and E-cadherin were expressed in small intestinal organoids. Western blot results showed that the expression of GLP-1 protein was increased by flavanomarein. In the results of differential expressed gene （DEG） screening， there were 1 862 DEGs in the IR group as compared with the normal control group， and 2 282 DEGs in the FM group as compared with the IR group. Through protein-protein interaction（PPI） network analysis of the DEGs of the two groups， 10 Hub genes， including Nr1i3， Cyp2c44， Ugt2b1， Gsta1， Gstm2， Ptgs1， Gstm4， Cyp2c38， Cyp4a32， and Gpx3， were obtained. These genes were highly expressed in the normal control group， and their expression was reduced in the IR group. After the intervention of flavanomarein， the expression of the above genes was reversed. ConclusionFlavanomarein may play its role in improving insulin resistance by reversing the expression levels of 10 Hub genes， including Nr1i3， Cyp2c44， Ugt2b1， Gsta1， Gstm2， Ptgs1， Gstm4， Cyp2c38， Cyp4a32， and Gpx3.