Objective:To explore the feasibility of modifying hemerythrin molecules with natural cross-linker genipin,and evaluate its efficacy and safety.Methods:Hemerythrin was isolated and purified from sipunculid worms using tangential flow ultrafiltration.Subsequently,genipin cross-linked hemerythrin nanoparticles(GHrNPs)were constructed by adding 20％w/w genipin under mildly acidic conditions,and glutaraldehyde cross-linked hemerythrin nanoparticles(GAHrNPs)were constructed by adding 10％w/w glutaraldehyde under mildly alkaline conditions.The diameter,dispersity index,zeta potential,functional group structure,P50,and Hill coefficient of the two nanoparticle groups were measured.The two nanoparticle groups at different concentrations were co-cultured with vascular endothelial cells for 24 hours,then the cell viability and NO concentration in the culture medium were measured.Results:After glutaraldehyde/genipin molecular cross-linking,infrared spectra showed the continuous presence of amide bands Ⅰ and Ⅱ.The hydrated particle sizes of hemerythrin,GHrNP and GAHrNP were(93.14±2.11),(109.53±3.54),and(115.65±2.65)nm,dispersity indexes were 0.30±0.06,0.27±0.05,and 0.25±0.03,zeta potentials were(-24.00±1.54),(-19.52±1.31),and(-18.90±1.25)mV,P50 values were(9.28±0.22),(8.50±0.54),and(5.75±0.90)mmHg,and Hill coefficients were 1.61±0.14,1.58±0.17,and 1.41±0.22,respectively.The average hydrated particle size increased after cross-linking with hemerythrin,the negative value of the zeta potential decreased(both P＜0.05).The P50 value of GAHrNP was significantly decreased than that of hemerythrin and GHrNP(P＜0.05).The viability of vascular endothelial cells in the GHrNP group was higher than that in the GAHrNP group at different mass concentrations(P＜0.05).The NO concentration in the culture medium of vascular endothelial cells in the GHrNP group was higher than that in the GAHrNP group only at 2.0 mg/ml(P＜0.05).Conclusion:Hemerythrin molecules cross-linked by genipin can form stable nanoparticles with good oxygen-carrying activity and lower cytotoxicity compared to glutaraldehyde.

Objectives:Comparative analysis of the beagles acute-phase electrocardiographic,hemodynamic,left ventricular flow field status,and energy consumption characteristics of pacing at different sites of conduction system may help to elucidate the scientific mechanism of left bundle branch pacing(LBBP)as a option of physiological pacing therapy.Methods:Eight healthy adult beagles were used in this study.Initially,an active fixation lead was implanted in the right atrial appendage,followed by implantation of another active fixation lead at the right ventricular apex,distal His bundle,and left bundle septal branch,respectively.After connecting a dual-chamber pacemaker,electrocardiographic and acute phase hemodynamic parameters under sinus rhythm,right ventricular apex pacing(RVAP),distal His bundle pacing(DHBP),and LBBP states were collected and analyzed.Three complete cardiac cycles of standard apical three-chamber color Doppler dynamic images were acquired under vector flow mapping(VFM)mode.Offline analysis was performed on obtained parameters including isovolumic contraction period,rapid ejection period,isovolumic relaxation period,rapid filling period,atrial contraction period,and left ventricular intracavitary energy consumption.These parameters were compared under pacing at different sites and the linear correlations of major parameters were analyzed.Results:The QRS duration of baseline intrinsic sinus rhythm,RVAP,DHBP and LBBP were(45.0±4.0)ms,(98.4±6.2)ms,(50.0±4.5)ms and(62.0±4.7)ms,respectively.The LBBP-QRS duration was significantly wider than intrinsic sinus rhythm and DHBP,but significantly narrower than RVAP(all P<0.01).Compared with baseline AOO mode(the pacing rate was performed at 10 beats/min above the intrinsic heart rate),the change of acute phase maximum left ventricular pressure rise rate(LVdP/dtmax)in RVAP,DHBP and LBBP was([-7.89±5.67]% ),([0.74±2.05]% )and([-0.14±3.59]% ),respectively.There was no significant difference in LVdP/dtmax changes between DHBP and LBBP(P=0.667),but both pacing modalities were significantly better than RVAP(all P<0.01).The average energy consumption of the left ventricle under RVAP was significantly higher than that of intrinsic sinus rhythm,DHBP,and LBBP in isovolumic contraction period,rapid ejection period,isovolumic relaxation period,rapid filling period,and atrial contraction period(all P<0.01).However,there was no statistically significant difference in energy consumption among intrinsic sinus rhythm,DHBP,and LBBP during the above five phases(all P>0.05).DHBP and LBBP did not show a significant increase in the number of left ventricular vortices,vortex area,and circulation intensity compared to intrinsic sinus rhythm,and LBBP did not show a significant increase in vortex area and circulation intensity compared to DHBP.Conclusions:Although LBBP canines significantly prolonged the paced QRS duration,it showed no significant differences in acute phase left ventricular hemodynamics,left ventricular flow field status,and energy consumption compared to intrinsic sinus rhythm and DHBP.Performance of LBBP was superior to RVAP.This study may contribute to revealing the theoretical basis of LBBP as a feasible physiological pacing therapy.

Objective:To explore the feasibility of modifying hemerythrin molecules with natural cross-linker genipin,and evaluate its efficacy and safety.Methods:Hemerythrin was isolated and purified from sipunculid worms using tangential flow ultrafiltration.Subsequently,genipin cross-linked hemerythrin nanoparticles(GHrNPs)were constructed by adding 20％w/w genipin under mildly acidic conditions,and glutaraldehyde cross-linked hemerythrin nanoparticles(GAHrNPs)were constructed by adding 10％w/w glutaraldehyde under mildly alkaline conditions.The diameter,dispersity index,zeta potential,functional group structure,P50,and Hill coefficient of the two nanoparticle groups were measured.The two nanoparticle groups at different concentrations were co-cultured with vascular endothelial cells for 24 hours,then the cell viability and NO concentration in the culture medium were measured.Results:After glutaraldehyde/genipin molecular cross-linking,infrared spectra showed the continuous presence of amide bands Ⅰ and Ⅱ.The hydrated particle sizes of hemerythrin,GHrNP and GAHrNP were(93.14±2.11),(109.53±3.54),and(115.65±2.65)nm,dispersity indexes were 0.30±0.06,0.27±0.05,and 0.25±0.03,zeta potentials were(-24.00±1.54),(-19.52±1.31),and(-18.90±1.25)mV,P50 values were(9.28±0.22),(8.50±0.54),and(5.75±0.90)mmHg,and Hill coefficients were 1.61±0.14,1.58±0.17,and 1.41±0.22,respectively.The average hydrated particle size increased after cross-linking with hemerythrin,the negative value of the zeta potential decreased(both P＜0.05).The P50 value of GAHrNP was significantly decreased than that of hemerythrin and GHrNP(P＜0.05).The viability of vascular endothelial cells in the GHrNP group was higher than that in the GAHrNP group at different mass concentrations(P＜0.05).The NO concentration in the culture medium of vascular endothelial cells in the GHrNP group was higher than that in the GAHrNP group only at 2.0 mg/ml(P＜0.05).Conclusion:Hemerythrin molecules cross-linked by genipin can form stable nanoparticles with good oxygen-carrying activity and lower cytotoxicity compared to glutaraldehyde.

High mobility group box protein 1(HMGB1)is one of the most widely expressed protein member in the HMGs family,which is well known for its involvement in the body inflammatory response.Previous researches have found that it plays a significant role in cell migration,immune identification and neuroprotection.Spinal cord injury is a disease that causes severe damage to the nervous system,and neural circuits are disrupted after a spinal cord injury,which leads to many conditions including ischemia and hypoxia,inflammatory responses,demyelinating lesions,and glial scar formation that are detrimental to nerve regeneration and repair,making it one of the most difficult diseases to treat in the modern spinal surgery field.HMGB1 is upregulated after spinal cord injury,thereby regulating neuroinflam-matory responses,and participating in the neuronal apoptosis,promoting neuronal regeneration,and inducing neural stem cell differentiation and migration,which plays an important role in the process of neural function recovery.This paper summarizes the structure and function of HMGB1,as well as its role in spinal cord injury,in order to provide direction for founding therapeutic target for neurological function recovery after spinal cord injury.

Objective To investigate the effect of epifriedelanol(Epi)on gene expression of P-glycoprotein(P-gp)in human colorectal adenocarcinoma cell line LS174T and its mechanism.Methods LS174T cells were divided into control group and experimental-L,-M,-H groups.Experimental-L,-M,-H groups were treated with 5,10,20 μmol·L-1 Epi,respectively.Control group was treated with 0.1％dimethyl sulfoxide.Polymerase chain reaction was used to detect the mRNA expression level of P-gp.Theeffect of Epi on multidrug resistance protein 1(MDR1/P-gp)luciferase activity was investigated by pregnane X receptor(PXR)-MDR1/P-gp dual luciferase reporter gene assay.In addition,Western Blot was used to detect the protein expression level of P-gp and the nuclear factor-κB(NF-κB)pathway related proteins.Results The relative expression levels of P-gp mRNA in experimental-M,-H groups and control group were 52.24±5.19,23.00±3.52 and 100.00±9.00;the relative expression levels of P-gp protein were 86.37±9.96,74.85±15.92 and 100.00±12.91;the relative activities P-gp luciferase were 230.19±41.32,203.10±52.84 and 279.67±19.20;the relative expression levels of p65(RelA/p65)in nucleus were 132.36±23.93,145.96±25.15 and 100.00±10.88;the relative expression levels of phosphorylation NF-κB inhibits protein kinase α/β(p-IKKα/β)in cytoplasm were 184.00±54.82,290.10±49.59 and 100.00±15.34;the relative expression levels of phosphorylated NF-κB inhibitory protein α(p-IκBα)in cytoplasm were 125.73±18.77,133.69±20.25 and 100.00±8.12;the relative expression levels of IκBα in cytoplasm were 78.36±14.83,70.44±14.57 and 100.00±22.82,respectively.The above indexes of experimental-M and experimental-H groups were compared with control group,and the differences were statistically significant(P＜0.05,P＜0.01,P＜0.001).Conclusion Epi can down-regulate the gene expression of P-gp in human colorectal adenocarcinoma cell line LS174T,and the mechanism may be related to activation of NF-κB and suppression of PXR.

High mobility group box protein 1(HMGB1)is one of the most widely expressed protein member in the HMGs family,which is well known for its involvement in the body inflammatory response.Previous researches have found that it plays a significant role in cell migration,immune identification and neuroprotection.Spinal cord injury is a disease that causes severe damage to the nervous system,and neural circuits are disrupted after a spinal cord injury,which leads to many conditions including ischemia and hypoxia,inflammatory responses,demyelinating lesions,and glial scar formation that are detrimental to nerve regeneration and repair,making it one of the most difficult diseases to treat in the modern spinal surgery field.HMGB1 is upregulated after spinal cord injury,thereby regulating neuroinflam-matory responses,and participating in the neuronal apoptosis,promoting neuronal regeneration,and inducing neural stem cell differentiation and migration,which plays an important role in the process of neural function recovery.This paper summarizes the structure and function of HMGB1,as well as its role in spinal cord injury,in order to provide direction for founding therapeutic target for neurological function recovery after spinal cord injury.

Objective:To establish a one-pot lyophilized detection system based on recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas13a) technology for the rapid diagnosis of cytomegalovirus (CMV) infection in liver transplantation recipients.Methods:This study is a methodology study. CRISPR RNA (crRNA) and RPA primers were designed targeting the CMV gene sequence. Optimal RPA primer sets were screened to establish the RPA-CRISPR/Cas13a-based CMV detection system. The limit of detection (LOD) was evaluated using gradient-diluted CMV plasmid standards. Cross-reactivity was assessed using genomic DNA from common opportunistic viruses in organ transplant recipients. Lyophilized reagents were validated with CMV-negative and positive samples. P-values were computed using two-sample t-tests for pairwise comparisons and one-way ANOVA for multi-group analyses to assess fluorescence value differences. Subsequently, lyophilized reagents were employed to detect 22 plasma samples from liver transplantation recipients collected at Renji Hospital, Shanghai Jiao Tong University School of Medicine, from June 3, 2024, to May 31, 2025. The test results were then compared with those obtained using quantitative real-time polymerase chain reaction (qPCR). Consistency between the two methods was evaluated using the Kappa coefficient calculated by Kappa test.Result:The established RPA-CRISPR/Cas13a system achieved a detection sensitivity of 1 copy/reaction and exhibited no cross-reactivity with other common opportunistic viruses in organ transplantation. Lyophilized RPA-CRISPR/Cas13a reagents demonstrated performance equivalent to non-lyophilized reagents. Concordance between lyophilized reagent detection and qPCR results for 22 clinical samples was 100% (22/22).Conclusion:A lyophilized CMV detection method based on RPA-CRISPR/Cas13a technology was successfully developed and validated for convenient diagnosing CMV infection in liver transplant recipients.

Objective To investigate the effects of peripheral blood neutrophil infiltration on the polarization regulation of cerebral resident microglia under a permanent ischemic stroke model.Methods Fifty-eight C57BL/6 mice were divided into two groups.One group was sham group,and the other group of mice was subjected to permanent middle cerebral artery occlusion surgery.Mice were euthanized 48 hours,7 days,14 days,and 30 days after surgery for tissue collection.Western blotting was used to detect expression levels of M1 microglia markers CD 16,M2 microglia marker arginase 1(Arg1),inflammatory cytokine interleukin-1 β(IL-1β),and neutrophil marker myeloperoxidase(MPO)in brain tissue.Immunofluorescence histochemical staining was used to assess neutrophil infiltration and M2 microglial distribution around the infarct area in brain sections.In vitro,purified neutrophils were co-cultured with BV2 microglial cells.After lipopolysaccharide stimulation,the phagocytosis of neutrophils by BV2 cells was observed,and the expression levels of CD16 and Arg1 proteins in BV2 cells were detected.Results Western blotting showed that the levels of CD16(P＜0.05),IL-1β(P＜0.001),and MPO(P＜0.05)in brain tissue increased significantly 48 hours and 7 days after surgery,then decreased,with MPO expression returning to normal levels 30 days after surgery.Immunofluorescence showed a significant increase of MPO-positive cells around the infarct area of the mouse cerebral cortex 48 hours after surgery(P＜0.001),followed by a decrease(P＜0.05).The number of ionized calcium binding adapter molecule 1(Iba1)and MPO double-positive cells gradually increased after surgery,and reached their peak at 14 days(P＜0.05).Iba1 and Arg1 double-positive cells also increased significantly 7 days(P＜0.05)and 14 days(P＜0.01)after surgery.In vitro,co-culture experiments showed that after BV2 phagocytosing neutrophils,CD 16(P＜0.05)significantly decreased and Arg1 significantly upregulated(P＜0.05).Conclusion In a permanent ischemic stroke model,microglia transition from M1 to M2 type after phagocytosing neutrophils,and the injured brain area changes from pro-inflammatory state to anti-inflammatory state.

OBJECTIVE: To develop a method for identifying high-risk patients among septic populations requiring mechanical ventilation, and to conduct phenotypic analysis based on this method. METHODS: Data from four sources were utilized: the Medical Information Mart for Intensive Care (MIMIC-IV 2.0, MIMIC-III 1.4), the Philips eICU-Collaborative Research Database 2.0 (eICU-CRD 2.0), and the Anhui Medical University Second Affiliated Hospital dataset. The adult patients in intensive care unit (ICU) who met Sepsis-3 and received invasive mechanical ventilation (IMV) on the first day of first admission were enrolled. The MIMIC-IV dataset with the highest data integrity was divided into a training set and a test set at a 6:1 ratio, while the remaining datasets were served as validation sets. The demographic information, comorbidities, laboratory indicators, commonly used ICU scores, and treatment measures of patients were extracted. Clinical data collected within first day of ICU admission were used to calculate the sequential organ failure assessment (SOFA) score. K-means clustering was applied to cluster SOFA score components, and the sum of squared errors (SSE) and Davies-Bouldin index (DBI) were used to determine the optimal number of disease subtypes. For clustering results, normalized methods were employed to compare baseline characteristics by visualization, and Kaplan-Meier curves were used to analyze clinical outcomes across phenotypes. RESULTS: This study enrolled patients from MIMIC-IV dataset (n = 11 166), MIMIC-III dataset (n = 4 821), eICU-CRD dataset (n = 6 624), and a local dataset (n = 110), with the four datasets showing similar median ages and male proportions exceeding 50%; using 85% of the MIMIC-IV dataset as the training set, 15% as the test set, and the rest dataset as the validation set. K-means clustering based on the six-item SOFA score was performed to determine the optimal number of clusters as 3, and patients were finally classified into three phenotypes. In the training set, compared with the patients with phenotype II and phenotype III, those with phenotype I had the more severe circulatory and respiratory dysfunction, a higher proportion of vasoactive drug usage, more obvious metabolic acidosis and hypoxia, and a higher incidence of congestive heart failure. The patients with phenotype II was dominated by respiratory dysfunction with higher visceral injury. The patients with phenotype III had relatively stable organ function. The above characteristics were consistent in both the test and validation sets. Analysis of infection-related indicators showed that the patients with phenotype I had the highest SOFA score within 7 days after ICU admission, initial decreases and later increases in platelet count (PLT), and higher counts of neutrophils, lymphocytes, and monocytes as compared with those with phenotype II and phenotype III, their blood cultures had a higher positivity rates for Gram-positive bacteria, Gram-negative bacteria and fungi as compared with those with phenotype II and phenotype III. The Kaplan-Meier curve indicated that in the training, test, and validation sets, the 28-day cumulative mortality of patients with phenotype I was significantly higher than that of patients with phenotypes II and phenotype III. CONCLUSIONS Three distinct phenotypes in septic patients receiving IMV based on unsupervised machine learning is derived, among which phenotype I, characterized by cardiorespiratory failure, can be used for the early identification of high-risk patients in this population. Moreover, this population is more prone to bloodstream infections, posing a high risk and having a poor prognosis.
Humans ; Machine Learning ; Sepsis/therapy* ; Respiration, Artificial ; Intensive Care Units ; Organ Dysfunction Scores ; Male ; Female ; Middle Aged ; Adult