Diabetic chronic wounds are characterized by difficult healing, recurrent progression, and high rates of disability and mortality, which make their clinical treatment a medical challenge urgent to be addressed. However, the complex local microenvironment conditions of chronic wounds, such as high protease activity and persistent inflammatory responses, result in low bioavailability of exogenous cytokines (e.g., chemokine CXCL12) at the wound site, limiting their clinical application. In this study, we utilized Lactobacillus plantarum WCFS1 as the chassis to develop an efficient CXCL12 delivery system based on synthetic biology. Subsequently, we evaluated the role of the engineered probiotic strain in promoting the chronic wound healing in diabetic mice. Firstly, we fused the endogenous secretion signal peptide lp_3050 (SP_{lp_3050}) of L. plantarum WCFS1 and the commonly used secretion signal peptide usp45 (SP_usp45) of lactic acid bacteria with the reporter gene gusA and inserted them into the pTRK892-P_32(pgm) plasmid by molecular cloning. Then, we prepared the engineered strains and characterized the efficacy of the two signal peptides in driving the secretion of GusA. The results showed that SP_{lp_3050} efficiently drove the secretion of GusA in L. plantarum WCFS1, increasing the activity of GusA in the culture supernatant by nearly five times compared with that of SP_{lp_3050}. Further, we fused SP_{lp_3050} and codon-optimized CXCL12 gene to construct an engineered probiotic strain Lpw-CXCL12 for CXCL12 delivery. The results demonstrated that the content of CXCL12 in the culture supernatant reached (13.40±0.20) μg/mL. Finally, we found that the engineered probiotic strain Lpw-CXCL12 accelerated chronic wound healing in a diabetic mouse model. In conclusion, these results support an engineered probiotic strain in promoting diabetic chronic wound healing, providing a new strategy and technological foundation for the management of diabetic chronic wounds in the future.
Probiotics ; Animals ; Chemokine CXCL12/biosynthesis* ; Mice ; Wound Healing ; Lactobacillus plantarum/metabolism* ; Diabetes Mellitus, Experimental/complications* ; Male

Objective:To investigate the expression, prognosis, and role of G protein-coupled receptor kinase-interacting protein 1 (GIT1) in patients with hepatocellular carcinoma (HCC) tumor micro environments.Methods:Clinical data of 140 cases who underwent complete HCC surgical resection from January 2015 to December 2021 in Subei People's Hospital affiliated to Yangzhou University, Jiangsu Province, were included. Tumor tissue and adjacent tissue samples were collected for immunohistochemical analysis. The patients were divided into a high expression group and a low expression group according to the expression of GIT1. Cox regression was used to analyze the risk factors for prognosis in patients with HCC. Fifteen pairs of cancer tissues and adjacent tissues were randomly matched for quantitative polymerase chain reaction (RT-PCR), western blot (WB), and immunohistochemical analysis. GITI knockout or overexpression cell lines of human hepatoma cell lines HepG2, HuH7 and MHCC97-H, and mouse hepatoma cell line Hepa 1-6 were constructed. The effects on M2 macrophage polarization were analyzed by flow cytometry. A mice tumor model was constructed. The growth curve of tumor tissue overexpressing GIT1 was plotted. Bioinformatics analysis of the Cancer Genome Atlas (TCGA) data was performed using OncoLnc, Kaplan-Meier Plotter, UALCAN, and GEPIA databases to explore the differential expression of GIT1 in HCC patients and its effect on prognosis.Results:Bioinformatics analysis showed that the expression level of GIT1 was significantly higher in HCC tissues than in normal liver tissues ( P<0.05). RT-PCR and WB experiments showed that GIT1 was highly expressed in HCC. The follow-up results showed that high expression of GIT1 was associated with poor prognosis in patients with HCC. The high expression of GIT1 was an independent risk factor for the prognosis in patients with HCC ( HR=2.562, 95% CI: 0.231-0.704, P<0.05). Functional enrichment analysis combined with TIMER database analysis found that GIT1 expression level was associated with multiple immune cell infiltrations in HCC, but the correlation coefficient with macrophage infiltration was the highest ( r=0.545, P<0.001). Mice tumorigenesis experiments showed that the tumor volume of GIT1-overexpressing mice was significantly increased ( P<0.05). Additionally, flow cytometry indicated that after GIT1 overexpression, there was a low degree of M1 infiltration/polarization (wild type: 5.06%±0.11%, overexpression type: 4.09%±0.04%; P<0.05) and a high degree of M2 infiltration/polarization (wild type: 10.20%±0.33%, overexpression type: 14.7%±0.12%; P<0.05). Conclusion:GIT1 serves as a prognostic biomarker in HCC, promoting tumor progression through its high expression and enhances M2 macrophage infiltration.

To verify whether chaperones can promote the soluble expression of PlpE in Escherichia coli and whether the expressed protein is active,prokaryotic expression and Western blot detection were performed.The results showed that:The PlpE prokaryotic expression vector pET-32a(+)-plpE was expressed as inclusion body,and the expression form was not changed by changing the concentration of inducer,induction time and temperature.The companion proteins pG-KJE8,pGro7,pKJE7 and pG-Tf2 were co-expressed with pET-32a(+)-plpE in Eschierichia coli expres-sion system,respectively.When the final concentration of IPTG of 0.5 mmol/L,L-arabinose of 0.5 g/L or tetracycline of 5.0 μg/L were added as inducers and induced at 37 ℃ for 8 h,the results showed that the molecular companion pGro7 could change the expression of rp-PlpE from inclu-sion body to soluble expression.pG-KJE8,pKJE7 and pG-Tf2 had no effect on the expression of rp-PlpE.The soluble rp-PlpE can react specifically with the positive serum of Mannheimia haemolyti-ca.Therefore,the study showed that the co-expression of the chaperone protein pGro7 can make the rp-PlpE protein express in a soluble form,and the purified protein exhibits reactogenicity.These findings lay the foundation for the establishment of a subunit vaccine and serological diagno-sis methods for Mannheimia haemolytica.

To verify whether chaperones can promote the soluble expression of PlpE in Escherichia coli and whether the expressed protein is active,prokaryotic expression and Western blot detection were performed.The results showed that:The PlpE prokaryotic expression vector pET-32a(+)-plpE was expressed as inclusion body,and the expression form was not changed by changing the concentration of inducer,induction time and temperature.The companion proteins pG-KJE8,pGro7,pKJE7 and pG-Tf2 were co-expressed with pET-32a(+)-plpE in Eschierichia coli expres-sion system,respectively.When the final concentration of IPTG of 0.5 mmol/L,L-arabinose of 0.5 g/L or tetracycline of 5.0 μg/L were added as inducers and induced at 37 ℃ for 8 h,the results showed that the molecular companion pGro7 could change the expression of rp-PlpE from inclu-sion body to soluble expression.pG-KJE8,pKJE7 and pG-Tf2 had no effect on the expression of rp-PlpE.The soluble rp-PlpE can react specifically with the positive serum of Mannheimia haemolyti-ca.Therefore,the study showed that the co-expression of the chaperone protein pGro7 can make the rp-PlpE protein express in a soluble form,and the purified protein exhibits reactogenicity.These findings lay the foundation for the establishment of a subunit vaccine and serological diagno-sis methods for Mannheimia haemolytica.

Objective:To investigate the expression, prognosis, and role of G protein-coupled receptor kinase-interacting protein 1 (GIT1) in patients with hepatocellular carcinoma (HCC) tumor micro environments.Methods:Clinical data of 140 cases who underwent complete HCC surgical resection from January 2015 to December 2021 in Subei People's Hospital affiliated to Yangzhou University, Jiangsu Province, were included. Tumor tissue and adjacent tissue samples were collected for immunohistochemical analysis. The patients were divided into a high expression group and a low expression group according to the expression of GIT1. Cox regression was used to analyze the risk factors for prognosis in patients with HCC. Fifteen pairs of cancer tissues and adjacent tissues were randomly matched for quantitative polymerase chain reaction (RT-PCR), western blot (WB), and immunohistochemical analysis. GITI knockout or overexpression cell lines of human hepatoma cell lines HepG2, HuH7 and MHCC97-H, and mouse hepatoma cell line Hepa 1-6 were constructed. The effects on M2 macrophage polarization were analyzed by flow cytometry. A mice tumor model was constructed. The growth curve of tumor tissue overexpressing GIT1 was plotted. Bioinformatics analysis of the Cancer Genome Atlas (TCGA) data was performed using OncoLnc, Kaplan-Meier Plotter, UALCAN, and GEPIA databases to explore the differential expression of GIT1 in HCC patients and its effect on prognosis.Results:Bioinformatics analysis showed that the expression level of GIT1 was significantly higher in HCC tissues than in normal liver tissues ( P<0.05). RT-PCR and WB experiments showed that GIT1 was highly expressed in HCC. The follow-up results showed that high expression of GIT1 was associated with poor prognosis in patients with HCC. The high expression of GIT1 was an independent risk factor for the prognosis in patients with HCC ( HR=2.562, 95% CI: 0.231-0.704, P<0.05). Functional enrichment analysis combined with TIMER database analysis found that GIT1 expression level was associated with multiple immune cell infiltrations in HCC, but the correlation coefficient with macrophage infiltration was the highest ( r=0.545, P<0.001). Mice tumorigenesis experiments showed that the tumor volume of GIT1-overexpressing mice was significantly increased ( P<0.05). Additionally, flow cytometry indicated that after GIT1 overexpression, there was a low degree of M1 infiltration/polarization (wild type: 5.06%±0.11%, overexpression type: 4.09%±0.04%; P<0.05) and a high degree of M2 infiltration/polarization (wild type: 10.20%±0.33%, overexpression type: 14.7%±0.12%; P<0.05). Conclusion:GIT1 serves as a prognostic biomarker in HCC, promoting tumor progression through its high expression and enhances M2 macrophage infiltration.

Objective:To evaluate the diagnostic efficacy and practicality of the Jaundice color card (JCard) as a screening tool for neonatal jaundice.Methods:Following the standards for reporting of diagnostic accuracy studies (STARD) statement, a multicenter prospective study was conducted in 9 hospitals in China from October 2019 to September 2021. A total of 845 newborns who were admitted to the hospital or outpatient department for liver function testing due to their own diseases. The inclusion criteria were a gestational age of ≥35 weeks, a birth weight of ≥2 000 g, and an age of ≤28 days. The neonate′s parents used the JCard to measure jaundice at the neonate′s cheek. Within 2 hours of the JCard measurement, transcutaneous bilirubin (TcB) was measured with a JH20-1B device and total serum bilirubin (TSB) was detected. The Pearson′s correlation analysis, Bland-Altman plots and the receiver operating characteristic (ROC) curve were used for statistic analysis.Results:Out of the 854 newborns, 445 were male and 409 were female; 46 were born at 35-36 weeks of gestational age and 808 were born at ≥37 weeks of gestational age. Additionally, 432 cases were aged 0-3 days, 236 cases were aged 4-7 days, and 186 cases were aged 8-28 days. The TSB level was (227.4±89.6) μmol/L, with a range of 23.7-717.0 μmol/L. The JCard level was (221.4±77.0) μmol/L and the TcB level was (252.5±76.0) μmol/L. Both the JCard and TcB values showed good correlation ( r=0.77 and 0.80, respectively) and agreements (96.0% (820/854) and 95.2% (813/854) of samples fell within the 95% limits of agreement, respectively) with TSB. The JCard value of 12 had a sensitivity of 0.93 and specificity of 0.75 for identifying a TSB ≥205.2?μmol/L, and a sensitivity of 1.00 and specificity of 0.35 for identifying a TSB ≥342.0?μmol/L. The TcB value of 205.2?μmol/L had a sensitivity of 0.97 and specificity of 0.60 for identifying TSB levels of 205.2 μmol/L, and a sensitivity of 1.00 and specificity of 0.26 for identifying TSB levels of 342.0 μmol/L. The areas under the ROC curve (AUC) of JCard for identifying TSB levels of 153.9, 205.2, 256.5, and 342.0 μmol/L were 0.96, 0.92, 0.83, and 0.83, respectively. The AUC of TcB were 0.94, 0.91, 0.86, and 0.87, respectively. There were both no significant differences between the AUC of JCard and TcB in identifying TSB levels of 153.9 and 205.2 μmol/L (both P>0.05). However, the AUC of JCard were both lower than those of TcB in identifying TSB levels of 256.5 and 342.0 μmol/L (both P<0.05). Conclusions:JCard can be used to classify different levels of bilirubin, but its diagnostic efficacy decreases with increasing bilirubin levels. When TSB level are ≤205.2 μmol/L, its diagnostic efficacy is equivalent to that of the JH20-1B. To prevent the misdiagnosis of severe jaundice, it is recommended that parents use a low JCard score, such as 12, to identify severe hyperbilirubinemia (TSB ≥342.0 μmol/L).

Objective To investigate clinical efficacy of transforaminal approach debridement with fusion,thoracolumbar single segment of Brucella spondylitis pedicle screw fixation (TLIF surgery).Methods We analyzed retrospectively the clinical data of 28 patients with Brucella spondylitis treated in our department between January 2009 and January 2014 with TLIF surgery (Group A) and internal fixation with a simple posterior anterior interbody disease debridement,autogenous bone graft (Group B).The two groups were compared in operation time,blood loss,postoperative ambulation time,hospitalization days,erythrocyte sedimentation rate (ESR),Creactive protein (CRP),American Spinal Injury Association (ASIA) classification,visual analogue scale (VAS),Oswestry Disability Index (ODI),Cobb angle of vertebral bone graft healing,and complications.Results All the patients were followed up for an average of 20.2 months (18 to 27 months).They were all cured.Compared with those in Group B,patients in Group A had shorter operation time (164.60±59.19)min,significantly reduced blood loss (346.00±108.90)mL and complications (1 case);significantly shorter postoperative ambulation time (3.36±1.11 days),hospitalization days (17.36 ± 4.19) days and duration (13.16 ± 3.94) months (P ＜ 0.05).The two groups did not significantly differ in VAS scores,ODI,ESR CRP,or Cobb angle (P＞0.05).Conclusion On the basis of norms of anti-drug treatment for brucellosis,TLIF surgery on Brucella spondylitis has the advantages including less trauma,shorter operation time,easier operation,less bleeding,earlier postoperative ambulation,and lower complication rate.

ObjectiveTo investigate the value of serum creatinine(SCr) to monitor the renal function of patients with spinal cord injury(SCI).Methods1298 SCI patients were investigated in Beijing Charity Hospital from January 2004 to May 2009. 249 healthy people were involved as control. The data of SCr were analysed.ResultsThe level of SCr for the SCI patients was (56.81±14.33) μmol/L with normal distribution, 95% CI was 28.73～84.89 μmol/L; The level of SCr for the healthy people was (75.98±11.34) μmol/L, 95% CI was 57.36～101.80 μmol/L. The level of SCr was significantly lower in the SCI patients comparing to the healthy people (P<0.01). Among the SCI patients, the level of SCr was (59.59±13.76) μmol/L for male while (46.41±11.31) μmol/L for female(P<0.05). There was no difference in level of SCr among different ages of the SCI patients(P>0.05). The characteristics were likely in the two groups. There was no difference in level of SCr between paraplegic (55.54±14.96) μmol/L and quadriplegic (57.67±13.83) μmol/L (P>0.05).ConclusionThe characteristics of level of SCr were likely in SCI patients and healthy people with normal distribution. The level of SCr was lower in SCI patients comparing to healthy people, which was higher for male SCI patients comparing to female SCI patients.