(+)-Borneol, the main component of "Natural Borneol" in the Chinese Pharmacopoeia, is a high-end spice and precious medicine. Plant extraction cannot meet the increasing demand for (+)-borneol, while microbial biosynthesis offers a sustainable supply route. However, its production was extremely low compared with other monoterpenes, even with extensively optimizing the mevalonate pathway. We found that the key challenge is the complex and unusual dephosphorylation reaction of bornyl diphosphate (BPP), which suffers the side-reaction and the competition from the cellular dephosphorylation process, especially lipid metabolism, thus limiting (+)-borneol synthesis. Here, we systematically optimized the dephosphorylation process by identifying, characterizing phosphatases, and balancing cellular dephosphorylation metabolism. For the first time, we identified two endogenous phosphatases and seven heterologous phosphatases, which significantly increased (+)-borneol production by up to 152%. By engineering BPP dephosphorylation and optimizing the MVA pathway, the production of (+)-borneol was increased by 33.8-fold, which enabled the production of 753 mg/L under fed-batch fermentation in shake flasks, so far the highest reported in the literature. This study showed that rewiring dephosphorylation metabolism was essential for high-level production of (+)-borneol in Saccharomyces cerevisiae, and balancing cellular dephosphorylation is also helpful for efficient biosynthesis of other terpenoids since all whose biosynthesis involves the dephosphorylation procedure.

Objective To explore the expression differences of the docking protein 1(DOK1)gene in multiple tissues of mice during the development stage from prediabetes to type 2 diabetes mellitus(T2DM),as well as its correlation with blood lipid levels.Methods The mice were divided into the 0 d group(control),the 20 d group,the 40 d group,the 60 d group,the 80 d group and the T2DM group.The 0 d group was fed with normal diet,while the other four groups were fed with high-fat diet to establish the prediabetes model.On this basis,the T2DM group was given low-dose multiple intraperitoneal injections of streptozotocin(STZ)to establish the T2DM model.The fasting and intraperitoneal glucose tolerance test(IPGTT)and insulin tol-erance test(IPITT)blood glucose of mice in each group were detected,and the expression of the DOK1 gene in nine tissues(heart,liver,kidney,skeletal muscle,pancreas,adipose,spleen,lung and colon tissues)was de-tected by qPCR.The level of DOK1 in serum was determined by ELISA,and the lipid level in serum was measured by an automatic biochemical analyzer.The correlations between serum DOK1 and TG,TC,HDL-C,LDL-C,lipoprotein A[LP(a)],and free fatty acids(FFA)were analyzed by Spearman correlation analysis.Re-sults The prediabetic and T2DM model mice were successfully established.Compared with the 0 d group,the blood glucose and lipid levels in the 80 d group and the T2DM group were significantly increased(P<0.001).Compared with the 0 d group,the expression level of DOK1 mRNA in the T2DM group was significantly de-creased in heart,liver,kidney,skeletal muscle,pancreas and adipose tissue(P<0.05).Compared with the 0 d group,the expression level of DOK1 mRNA in the 80 d group was significantly decreased in heart,liver,kid-ney and skeletal muscle tissue(P<0.05).Compared with the 0 d group,the expression levels of DOK1 mR-NA in the 40 d group and the 60 d group was significantly decreased in the liver tissues(P<0.05).Compared with group 0 d,there was no statistically significant difference in the expression level of DOK1 mRNA in the spleen,lung and colon tissues among each group(P>0.05).DOK1 in the 80 d group and the T2DM group was negatively correlated with TG,TC,LDL-C,LP(a),and FFA(r=-0.787 8,-0.727 2,-0.763 7,-0.764 2,-0.892 5,P<0.001),while positively correlated with HDL-C(r=0.961 3,P<0.001).Conclu-sion The reduction of DOK is related to the mechanism of insulin resistance and may play a key role in lipid metabolism disorders associated with diabetes.

Objective To investigate the effects of different concentrations of PPF on oxidative stress and apoptosis of PD model cells induced by MPP+.Methods The human neuroblastoma cell SH-SY5Y was induced by 1 mM MPP+ to establish PD cell model.In PPF treatment group,SH-SY5Y cells were stimulated with 10,20,40 and 80 μM PPF for 4 h before MPP+ induction.Cell counting kit-8(CCK-8)was performed to evaluate cell proliferation activity.H2DCF-DA fluorescent probe was used to detect ROS in cells.The levels of MDA and NADPH oxidase were analyzed by the kit.Western blot examined the protein expression of cytochrome c in mitochondria and cytoplasm,as well as the relative expression of Bcl-2,Bax and cleaved caspase-3 in SH-SY5Y cells.Apoptosis rate was analyzed by flow cytometry.Results MPP+ significantly inhibited the proliferation of SH-SY5Y cells(P<0.001),promoted the level of ROS(P<0.001),MDA(P<0.001),NADPH oxidase(P<0.01),cytochrome c in cytoplasm(P<0.01)and induced apoptosis(P<0.001)and the relative expression of pro-apoptosis protein Bax and cleaved caspase-3(P<0.01),reduced cytochrome c protein in mitochondria(P<0.01)and the relative expression of anti-apoptosis protein(P<0.01).PPF pretreatment alleviated the proliferation inhibition,oxidative stress and apoptosis promotion of SH-SY5Y cells induced by MPP+(P<0.001),and the effects of 40 μM and 80 μM on cells were more significant.Conclusion PPF pretreatment can alleviate the oxidative stress of SH-SY5Y cells induced by MMP+ and reduce apoptosis rate.

BACKGROUND:Ankylosing spondylitis is a chronic inflammatory disease with chronic rheumatic immunity.Soft tissue ossification and fusion and spinal stiffness can cause biomechanical changes. OBJECTIVE:To reconstruct the lumbar-sacral intervertebral disc in ankylosing spondylitis patients with lumbar kyphosis by finite element analysis,and to study the range of motion of each segment of T11-S1 and the biomechanical characteristics of annulus fibrosus and nucleus pulposus. METHODS:The imaging data were obtained from an ankylosing spondylitis patient with lumbar kyphosis.The original CT image data of continuously scanned spine were imported into Mimics 21.0 in DICOM format,and T11-S1 was reconstructed respectively.The established model was imported into 3-Matic software in the format of"Stl"to reconstruct the intervertebral disc,and the fibrous intervertebral disc model was obtained.The improved model was further imported into Hypermesh software,and the vertebra,nucleus pulposus,annulus fibrosus and ligament were mesh-divided.After the material properties were given,the model was imported into ABAQUS software to observe the range of motion of each vertebral body in seven different working conditions of T11-S1,and analyze the biomechanical characteristics of each segment of annulus fibrosus and nucleus pulposus. RESULTS AND CONCLUSION:(1)The range of motion of L1 vertebrae was higher than that of other vertebrae under six different working conditions:extension,forward flexion,rotation(left and right),and lateral flexion(left and right).The maximum range of motion was 2.18° during L1 vertebral flexion,and the minimum range of motion was 0.12° during L5 vertebral extension.(2)The annular fiber flexion at L2-L3 segments was greater than the extension(P<0.05),and the annular fiber flexion at L3-L4 and L4-L5 segments was less than the extension(P<0.05).The left rotation of L1-L2 annular fibers was greater than the right rotation(P<0.05).The left flexion of the annulus was greater than the right flexion in L1-L2,L2-L3,L3-L4,L4-L5 and L5-S1 segments(P<0.05).(3)The nucleus pulposus stresses of T11-L12,L1-L2,L2-L3,L3-L4 and L4-L5 segments in forward flexion were greater than in extension(P<0.05).The left rotation of T12-L1 and L3-L4 segments was smaller than the right rotation(P<0.05),and that of T11-T12,L1-L2,and L2-L3 segments was larger than the right rotation(P<0.05).The left flexion was larger than the right flexion in the T11-S1 segment.(4)It is concluded that in ankylosing spondylitis patients with lumbar kyphosis,the minimum range of motion of the vertebral body is located at the L5 vertebral body in extension.To prevent fractures,it is recommended to avoid exercise in the extension position.During the onset of lumbar kyphosis in patients with ankylosing spondylitis,the maximum stress of the annulus fibrosus and nucleus pulposus is located in the L1-L2 segment,which is fixed and will not alter with the change of body position.The late surgical treatment and correction of deformity should focus on releasing the pressure of the annulus fibrosus and nucleus pulposus in this segment to avoid the rupture of the annulus fibrosus and the injury of the nucleus pulposus.

BACKGROUND:Ankylosing spondylitis is a progressive inflammation of spinal stiffness deformity caused by tissue ossification and fibrosis.The posture of ankylosing spondylitis patients is abnormal and their activities are limited that minor injuries can lead to thoracolumbar fractures.Traditional medical image observation limits doctors'preoperative decision planning and postoperative disease prevention for ankylosing spondylitis treatment. OBJECTIVE:Based on the spinal model of ankylosing spondylitis patients before and after posterior spinal cancellous ossification osteotomy("Y"osteotomy for short),to explore the biomechanical changes of"Y"osteotomy and fixation in the treatment of ankylosing spondylitis. METHODS:Based on the preoperative and postoperative CT images of an ankylosing spondylitis patient who went to the Second Affiliated Hospital of Inner Mongolia Medical University,a three-dimensional spine model(T11-S1)before and after"Y"osteotomy(L3 osteotomy)was reconstructed in Mimics 19.0 software.A 7.5 Nm torque was applied to the top of T11 vertebral body to simulate the movement of the spine under six conditions:flexion,extension,left bending,right bending,left rotation and right rotation.Finally,the range of motion of each vertebral body,the stress of each intervertebral disc,and the stress of the screw rod system were simulated. RESULTS AND CONCLUSION:(1)After"Y"type osteotomy and posterior fixation,the range of motion of all vertebrae in the spine decreased,and the loss rate of upper vertebrae was large(L1:77.95%).(2)The maximum stress of the spinal intervertebral disc before operation occurred at the L1-L2 segment(0.55 MPa),and the maximum stress of the spinal intervertebral disc after operation occurred at the T11-T12 segment(0.50 MPa),and the stress of intervertebral disc below T12 was far less than that before operation.(3)The maximum stress of the screw rod system(166.67 MPa)occurred in the upper and middle segments of the rod body and the root of the pedicle screw.(4)In conclusion,the"Y"type posterior fixation operation enhances the stability of the spine and reduces the range of motion of the spine.The vertebral body decompression of the fixed segment is great and the stress-shielding phenomenon of the lower vertebral body is significant.The stiffness of the rod body and the stress concentration area of the pedicle screw should be strengthened to avoid the fracture of the rod caused by stress fatigue.

Objective:To explore the clinical features and genetic variants in three children suspected for β-ketothiolase deficiency (BKTD).Methods:Clinical manifestations, laboratory examination and genetic testing of three children suspected for BKTD at Henan Children′s Hospital between January 2018 and October 2022 were collected, and their clinical and genetic variants were retrospectively analyzed.Results:The children were all males with a age from 7 to 11 months. Their clinical manifestations have included poor spirit, shortness of breath, vomiting, convulsions after traumatic stress and/or infection. All of them had severe metabolic acidosis, elevated ketone bodies in blood and urine, hypoglycemia, with increased isoprenyl-carnitine and 3-hydroxyisovalyl-carnitine in the blood, and 2-methyl-3-hydroxybutyrate and methylprotaroyl glycine in the urine. All of them were found to harbor compound heterozygous variants of the ACAT1 gene, including c. 1183G>T and a large fragment deletion (11q22.3-11q23.1) in child 1, c. 121-3C>G and c. 826+ 5_826+ 9delGTGTT in child 2, and c. 928G>C and c. 1142T>C in child 3. The variants harbored by children 2 and 3 were known to be pathogenic or likely pathogenic. The heterozygous c. 1183G>T variant in child 1 was unreported previously and rated as a variant of unknown significance (PM2_Supporting+ PP3+ PP4) based on guidelines from the American College of Medical Genetics and Genomics. The large segment deletion in 11q22.3-11q23.1 has not been included in the DGV Database and was rated as a pathogenic copy number variation. Conclusion:The variants of the ACAT1 gene probably underlay the pathogenesis of BKTD in these three children.

Background::Ainuovirine (ANV) is a new generation of non-nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus (HIV) type 1 infection. This study aimed to evaluate the population pharmacokinetic (PopPK) profile and exposure-response relationship of ANV among people living with HIV.Methods::Plasma concentration-time data from phase 1 and phase 3 clinical trials of ANV were pooled for developing the PopPK model. Exposure estimates obtained from the final model were used in exposure-response analysis for virologic responses and safety responses.Results::ANV exhibited a nonlinear pharmacokinetic profile, which was best described by a two-compartment model with first-order elimination. There were no significant covariates correlated to the pharmacokinetic parameters of ANV. The PopPK parameter estimate (relative standard error [%]) for clearance adjusted for bioavailability (CL/F) was 6.46 (15.00) L/h, and the clearance of ANV increased after multiple doses. The exposure-response model revealed no significant correlation between the virologic response (HIV-RNA <50 copies/mL) at 48 weeks and the exposure, but the incidence of adverse events increased with the increasing exposure ( P value of steady-state trough concentration and area under the steady-state curve were 0.0177 and 0.0141, respectively). Conclusions::Our PopPK model supported ANV 150 mg once daily as the recommended dose for people living with HIV, requiring no dose adjustment for the studied factors. Optimization of ANV dose may be warranted in clinical practice due to an increasing trend in adverse reactions with increasing exposure.Trial registration::Chinese Clinical Trial Registry https://www.chictr.org.cn (Nos. ChiCTR1800018022 and ChiCTR1800019041).

Objective:To establish a nomogram model for predicting the hyper-progression recurrence after hepatectomy in patients with hepatocellular carcinoma (HCC) based on circulating tumor cells (CTC).Methods:Clinical data of 231 HCC patients undergoing hepatectomy at the Department of Hepatobiliary Surgery, Guangxi Medical University Cancer Hospital from January 2013 to December 2022 were retrospectively analyzed, including 200 males and 31 females, aged 46(39, 52) years old. Patients were divided into two groups: the modeling group ( n=154) and the validation group ( n=77). According to the state of postoperative hyper-progression recurrence, patients in the modeling group were subdivided into hyper-progression recurrence ( n=39) and non-hyper-progression recurrence group ( n=115). Patients in the validation group were also subdivided into hyper-progression recurrence ( n=16) and non-hyper-progression recurrence group ( n=61). Clinicopathological data such as the total CTC count, alpha-fetoprotein, and postoperative pathology were collected. Logistic regression analysis was used to analyze the influencing factors of postoperative hyper-progression recurrence. A nomogram model was established based on the results of multivariate logistic regression analysis. The receiver operating characteristic (ROC) curve, calibration curve, decision curve analysis (DCA) and clinical impact curve (CIC) were used to validate the nomogram model. Results:Multivariate logistic regression analysis showed that HCC patients with age ≤45 years old ( OR=6.704, 95% CI: 1.619-27.760, P=0.009), incomplete tumor capsule ( OR=13.292, 95% CI: 3.084-57.295, P=0.001), high total numbers of CTC ( OR=1.101, 95% CI: 1.023-1.186, P=0.011) and high Ki67 index ( OR=52.659, 95% CI: 3.215-862.604, P=0.005) had a high risk of hyper-progression recurrence after hepatectomy. The above three preoperative variables were integrated to construct a nomogram model. The calibration curve showed that the predicted results of the nomogram model were in good agreement with the actual results. The ROC curves of the nomogram model for predicting hyper-progression recurrence after hepatectomy in HCC patients were plotted, and the area under the curve was 0.907 (95% CI: 0.856-0.959) and 0.833 (95% CI: 0.721-0.945) in the modeling group and validation group, respectively. DCA showed that the nomogram model could be used as a valuable predictive tool for the hyper-progression recurrence after hepatectomy. The CIC showed that the population judged by the nomogram model was highly matched with the actual population with hyper-progression recurrence. Conclusions:This study established a nomogram model based on age, tumor capsular integrity and total CTC count, which could accurately predict the postoperative hyper-progression recurrence in HCC patients before hepatectomy. The model is promising in guiding clinical practice after further validation.

Objective To explore the trajectory and influencing factors of joint awareness after total knee arthroplasty.Methods With the method for convenience sampling,patients who met the standards for total knee arthroplasty from July 2021 to March 2022 were selected.General information,severity of osteoarthritis,anxiety and depression,and self-efficacy were investigated before surgery(T0).Joint awareness was investigated at 1 month(T1),3 months(T2),and 6 months(T3)after surgery,respectively.Latent growth curve model was used to describe the overall trend of joint awareness;latent class growth model was used to analyze latent subgroups;logistic regression analysis was used to determine the impact of related variables on the trajectory of joint awareness.Results The scores of joint awareness were(32.70±5.80),(47.67±4.67)and(61.53±7.81)respectively at T1 to T3,and joint awareness showed an increasing trend(P＜0.001).The trajectory of joint awareness was divided into 2 potential subgroups:the rapid growth group(61.83％)and the slow growth group(38.17％).Age,BMI,other chronic diseases or not,years of osteoarthritis,severity of osteoarthritis,anxiety,depression,and self-efficacy affected the trend of joint awareness(all P＜0.05).Conclusion Joint awareness showed a linear growth trend with the postoperative time,and there were 2 potential subgroups in the trajectory.Medical workers could develop corresponding interventions based on the influencing factors to improve patients'postoperative awareness of artificial joints.

Objective:To investigate the relationship between dyslipidemia and the risk of non-alcoholic fatty liver disease (NAFLD) in adults.Methods:A total of 13 449 individuals with an average age of 37 (31,48) years including 4 815(35.80%) males, who underwent physical examination and had no NAFLD in Tianjin Medical University General Hospital in 2017 were recruited in this prospective study. Participants were followed up till December 31, 2021, and the newly onset NAFLD was defined as endpoint of the study. Cox regression model was used to analyze the correlation between the dyslipidemia and the risk of NAFLD.Results:The mean follow-up period was 2.80 years with total 37 707 person-years of follow-up. Among 13 449 participants, 2 711 subjects developed NAFLD during the follow-up period with a cumulative incidence rate of 20.2%. After adjusting for confounding factors, compared with the non-dyslipidemia group, the hazard ratio (HR) (95% CI) of the 1, 2, and 3 types of dyslipidemia groups were 1.388 (1.253-1.538), 1.200 (1.143-1.261) and 1.282 (1.226-1.342), respectively (trend test: P<0.001); and the HR (95% CI) of the hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia and low high-density lipoprotein cholesterolemia groups were 1.165 (1.058-1.283), 1.436 (1.345-1.533), 1.291 (1.240-1.344) and 1.068 (1.014-1.124) respectively (all P<0.05). After adjusting for confounding factors, compared with the normal triglycerides group, HR (95% CI) of moderately elevated triglycerides and elevated triglycerides group were 1.774 (1.590-1.980) and 1.443 (1.345-1.549) respectively (trend test: P<0.001). Conclusion:Dyslipidemia can increase the risk of NAFLD, especially for individuals with hypertriglyceridemia, which indicates that appropriate management of blood lipid levels, especially triglyceride level may effectively prevent NAFLD.