Objective:To analyze the changes and clinical significance of homocysteine (Hcy), folate, and cardiac enzyme levels in patients with alcohol dependence and depression.Methods:A total of 102 patients with alcohol dependence and depression, who received treatment at Wenzhou Seventh People's Hospital from January 2022 to June 2023, were included in the observation group. The degree of alcohol dependence in patients in the observation group was assessed using the Michigan Alcoholism Screening Test (MAST). According to the assessment results, the patients in the observation group were divided into the following subgroups: mild alcohol dependence ( n = 33), moderate alcohol dependence ( n = 37), heavy alcohol dependence ( n = 15), and severe alcohol dependence ( n = 17). The severity of depression among patients in the observation group was assessed with the Hamilton Depression Scale (HAMD). Based on the assessment results, the patients in the observation group were divided into the following subgroups: mild depression ( n = 43), moderate depression ( n = 34), and severe depression ( n = 25). The cognitive function of patients in the observation group was assessed using the Montreal Cognitive Assessment Scale (MoCA). According to the assessment results, the patients in the observation group were divided into normal cognitive function ( n = 73) and cognitive impairment ( n = 29) subgroups. Thirty healthy volunteers from our hospital during the same period were included in the control group. The levels of Hcy, folate, and cardiac enzymes were compared among all groups. The correlations between Hcy, folate, and cardiac enzyme levels with HAMD, MoCA, and MAST scores were analyzed using the Pearson method. Results:The Hcy level in the observation group was (15.21 ± 1.99) μg/L, which was significantly higher than that in the control group [(11.38 ± 1.46) μg/L, t = -9.80, P < 0.001]. The levels of folate, lactate dehydrogenase (LDH), and creatine kinase (CK) in the observation group were (4.82 ± 1.77) μg/L, (122.69 ± 33.98) IU/L, and (87.83 ± 16.52) IU/L, respectively, which were significantly lower than those in the control group [(6.27 ± 1.35) μg/L, (150.56 ± 38.78) IU/L, (98.67 ± 20.29) IU/L, t = 4.16, 3.82, 2.99, all P < 0.05]. The Hcy levels in the mild , moderate, heavy, and severe alcohol dependence subgroups [(13.16 ± 1.23) μg/L, (15.35 ± 0.82) μg/L, (16.79 ± 1.38) μg/L, (17.63 ± 1.22) μg/L] increased sequentially, while the folate levels [(6.11 ± 1.51) μg/L, (4.95 ± 1.40) μg/L, (4.04 ± 0.99) μg/L, (2.70 ± 0.99) μg/L], LDH levels [(153.35 ± 27.47) IU/L, (123.29 ± 16.59) IU/L, (109.83 ± 14.41) IU/L, (73.24 ± 16.86) IU/L], and CK levels [(104.14 ± 12.78) IU/L, (86.48 ± 9.15) IU/L, (78.11 ± 7.85) IU/L, (67.71 ± 9.00) IU/L] decreased sequentially. These differences in Hcy, folate, LDH, and CK levels among the mild, moderate, heavy, and severe alcohol dependence subgroups were statistically significant ( F = 73.24, 26.53, 59.08, 53.86, all P < 0.001). The Hcy levels in the mild, moderate, and severe depression subgroups [(13.75 ± 1.54) μg/L, (15.46 ± 1.17) μg/L, (17.39 ± 1.31) μg/L] increased progressively, while the folate levels [(5.83 ± 1.77) μg/L, (4.67 ± 1.12) μg/L, (3.28 ± 1.26) μg/L], LDH levels [(138.09 ± 33.67) IU/L, (119.73 ± 26.39) IU/L, (100.24 ± 30.88) IU/L], and CK levels [(96.35 ± 15.24) IU/L, (86.73 ± 15.62) IU/L, (74.69 ± 9.71) IU/L] decreased progressively. The differenes in Hcy, folate , LDH, and CK levels among the four depression subgroups were statistically significant ( F = 56.57, 24.36, 12.23, 18.44, all P < 0.001). The Hcy levels in the cognitive impairment group [(17.01 ± 1.63) μg/L] was significantly higher than that in the normal cognitive function group [(14.50 ± 1.64) μg/L, t = -6.97, P < 0.001), and the folate, LDH, and CK levels in the cognitive impairment group were (3.76 ± 1.78) μg/L, (102.71 ± 31.08) IU/L, and (76.00 ± 13.37) IU/L respectively, which were significantly lower than those in the normal cognitive function group [(5.24 ± 1.58) μg/L, (130.63 ± 31.92) IU/L, (92.52 ± 15.31) IU/L, t = 4.11, 4.01, 5.09, all P < 0.001]. Hcy levels were positively correlated with HAMD and MAST scores ( r = 0.854, 0.846, both P < 0.05) and negatively correlated with MoCA scores ( r = -0.648, P < 0.001). Folate, LDH, and CK levels were negatively correlated with HAMD and MAST scores ( r = -0.644, -0.701; r = -0.551, -0.696; r = -0.505, -0.673; all P < 0.001), and they were positively correlated with MoCA scores ( r = 0.514, 0.436, 0.448, all P < 0.001). Conclusion:In patients with alcohol dependence and depression, abnormal levels of Hcy, folate, and cardiac enzymes were observed. These indicators were found to be associated with the severity of alcohol dependence, the level of depression, and cognitive function.

Isoprenoids are all derived from two five-carbon building blocks called isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized either by the mevalonate (MVA) pathway or 2-C-methyld-D-erythritol-4-phosphate (MEP) pathway. In this study, the MVA pathway genes were integrated into the chromosome of LYC101, in which the expression of key genes in the MEP synthesis pathway and lycopene synthesis pathway were optimized by artificial regulatory parts, to further improve the production of isoprenoids in Escherichia coli. The plasmids pALV23 and pALV145 were screened from a plasmid library that constructed by using the RBS library to link the genes of the MVA pathway, which greatly increased the production of β-carotene. The effects of plasmids pALV23 and pALV145 on the lycopene production in low and high lycopene production strain, LYC001 and LYC101, were compared, respectively. The production of lycopene was increased by plasmids pALV23 and pALV145 in both strains. In high lycopene production strain LYC101, pALV23 produced more lycopene than pALV145. Then, the MVA gene together of promoter of pALV23 was integrated into the chromosome of LYC101 at poxB site using method of homologous recombination helped by CRISPR-Cas9 system, resulted in genetically stable strain, LYC102. The yield of lycopene of LYC102 was 40.9 mg/g DCW, 1.19-folds higher than that of LYC101, and 20% more than that of LYC101 with pALV23. Simultaneous expression of MVA pathway and MEP pathway in recombinant E. coli can effectively increase the yield of terpenoids. In this study, a plasmid-free, genetically stable, high-yielding lycopene strain was constructed, which could be used for industrialization. Also, the platform strain can be used for the synthesis of other terpenoids.
Chromosomes, Bacterial ; Escherichia coli ; Lycopene ; Mevalonic Acid ; beta Carotene

The introduction of the mevalonate pathway (MVA pathway) in recombinant Escherichia coli can improve the synthesis of terpenoids. But the imbalance expression of MVA pathway genes and accumulation of intermediates inhibit cell growth and terpenoids production. In this study, each gene of MVA pathway and key genes of lycopene synthesis pathway were cloned in plasmid to express in the recombinant E. coli LYC103 with optimizing the expression of the key genes of the 2-methyl-D-erythritol-4-phosphate pathway (MEP pathway), chromosome recombinant MVA pathway and the lycopene synthesis pathway. The results showed that the overexpression of ispA, crtE, mvaK1, idi and mvaD genes did not affect the cell growth, while lycopene production increased by 13.5%, 16.5%, 17.95%, 33.7% and 61.1% respectively, indicating that these genes may be the rate-limiting steps for the synthesis of lycopene. mvaK1, mvaK2, mvaD of MVA pathway were the rate-limiting steps and were in an operon. The mvaK1, mvaK2, mvaD operon was regulated by mRS (mRNA stabilizing region) library in front of mvaK1, obtaining strain LYC104. Lycopene yield of LYC104 was doubled and cell growth was increased by 32% compared with the control strain LYC103. CRISPR-cas9 technology was used to integrate idi into chromosome at lacZ site to obtain LYC105 strain. Cell growth of LYC105 was increased by 147% and lycopene yield was increased by 2.28 times compared with that of LYC103. In this study, each gene of lycopene synthesis pathway was expressed in plasmid to certify the rate-limiting gene based on the complete MVA pathway on the chromosome. Then the rate-limiting gene was integrated in chromosome with homologous recombination to release the rate-limiting, which providing a new strategy for the construction of high-yield strains for metabolic engineering.

OBJECTIVE： To investigate the effects of Kangfuxin liquid on repairing cartilage defect model of knee osteoarthritis （KOA） in rabbits and its mechanism. METHODS： Totally 72 male New Zealand rabbits were selected and randomly divided into model control group and Kangfuxin low-dose， medium-dose， high-dose groups， with 18 rabbits in each group. A cartilage defect model of the medial femoral condyle of the right knee joint in rabbits was established by drilling after anesthesia surgery. Then the rabbits in each group were given medicine via articular cavity immediately. Kangfuxin low-dose， middle-dose and high-dose groups were given 20％， 40％， 80％ Kangfuxin liquid； model control group was given constant volume of normal saline consecutively， 0.2 mL/kg， once every 3 days. At 4th， 8th， 12th week after medication， the wound repair of cartilage defect in rabbits was observed. Immediately after medication and at 4th， 8th， 12th week after medication， repaired tissue of cartilage defect in rabbits was scored histologically with Wakitani scoring standard under light microscope. At 12th week after medication， pathological changes of repaired tissue of cartilage defect in rabbits were observed by Masson staining. The levels of NO， SOD and LPO in joint fluid and PYD in urine of rabbits were detected by ELISA. RESULTS： At 4th， 8th， 12th week after medication， compared with model control group， cartilage defects in rabbits were repaired well in Kangfuxin low-dose， medium-dose and high-dose groups. At 4th， 8th， 12th week after medication， compared with immediately after medication and model control group at same time point， histomorphological score of repairing cartilage defect of knee joint in rabbits decreased significantly in Kangfuxin low-dose， medium-dose and high-dose groups （P＜0.05）. At 12th week after medication， compared with model control group， the histopathology degree of cartilage defect of knee joint in rabbits was significantly alleviated in Kangfuxin low-dose， medium-dose and high-dose groups. At 4th， 8th， 12th week after medication， compared with model control group， the levels of NO and LPO in joint fluid and PYD level in urine were decreased to different extent in Kangfuxin low-dose， medium-dose and high-dose groups， while SOD level was increased to different extent； at 12th week after medication， the difference of each index has statistical significance （P＜0.05 or P＜0.01）. CONCLUSIONS： Kangangxin liquid can significantly repair cartilage defect of KOA cartilage defect model rabbits， the mechanism of which may be associated with increasing the expression of SOD and mediating NO-inhibited chondrocyte apoptosis.