Leukoencephalopathy with vanishing white matter(VWM) is one of the most prevalent inherited white matter disorders in childhood,and it′s the only known hereditary human disease due to the direct defects in protein synthesis process,with the gene defects in EIF2B1-5,encoding the five subunits of eukaryotic translation initiation factor(eIF2B ?,?,?,? and ?) respectively.eIF2B is essential for the protein translation initiation process,and its action is realized via eukaryotic translation initiation factor2(eIF2).Phosphorylation of eIF2? and eIF2B? is an important way to regulate eIF2B function,and thus play a key role in control of the protein translation level under physiological condition.Mutant eIF2B results in functional defects and decrease of the overall protein translation in cells,but in increase the translation of proteins with multiple upstream open reading frames,such as activating transcription factor 4(AFT4),which leads to the susceptibility to un-folded protein response under stress,and the following apoptosis.The exact pathogenic mechanisms of VWM are far from well understood.It′s suggested that level of AFT4 in cells with eIF2B mutations is higher than in wild type cells under physiological condition,which makes the mutant cells more susceptible to endoplasmic reticulum(ER) stress and unfolded protein response(UPR).Under stress,the defect eIF2B leads to a vicious cycle of UPR activation,which may underlie the neurological aggravation in VWM patients after minor stress,a specific cli-nical feature of VWM.Elucidating the pathogenesis of VWM will be helpful to further understand the protein translation process in eukaryotic cells,and provide a clue for possible therapeutic targets and treatment strategies in the future.Abstract:SUMM ARY Leukoencephalopathy with vanishing white matter(VWM) is one of the most prevalent in-herited white matter d isorders in childhood,and i′ts the only known hered itary human d isease due to the d irect defects in protein synthesis process,with the gene defects inEIF2B1-5,encod ing the five sub-units of eukaryotic translation initiation factor(eIF2B?,?,?,?and?) respectively.eIF2B is essential for the protein translation initiation process,and its action is realized via eukaryotic translation initiation factor2(eIF2).Phosphorylation of eIF2?and eIF2B?is an important way to regulate eIF2B function,and thus play a key role in control of the protein translation level under physiological cond ition.Mutant eIF2B results in functional defects and decrease of the overall protein translation in cells,but in increase the translation of proteins with multiple upstream open read ing frames,such as activating transcription factor 4(AFT4),which leads to the susceptibility to un-folded protein response under stress,and the following apoptosis.The exact pathogenic mechanisms ofVWM are far from well understood.I′ts sugges-ted that level ofAFT4 in cells with eIF2B mutations is higher than in wild type cells under physiological cond ition,which makes the mutant cellsmore susceptible to endoplasm ic reticulum(ER) stress and un-folded protein response(UPR).Under stress,the defect eIF2B leads to a vicious cycle ofUPR activa-tion,which may underlie the neurological aggravation in VWM patients afterm inor stress,a specific cli-nical feature ofVWM.E lucidating the pathogenesis ofVWM will be helpful to further understand the pro-tein translation process in eukaryotic cells,and provide a clue for possible therapeutic targets and treat-ment strategies in the future.

Objective To study the method and conditions of determination of niclosamide in water. Methods Niclosamide was extracted by mixed extractent, then striped by NaOH. Niclosamide in NaOH solution was determined with dual-wavelength photometry. Results The recovery of niclosamide was 93%. The linear ranger was 0-4. 0 g/m3 and the detectable limit for niclosamide was 0. 042 7 g/m3. The apparent molar absorptivity was 1. 26 ?105 L/(mol?cm). Conclusion Extraction photometry is suitable for determination of niclosamide in water in the field.

Objective To examine the role of p38MAPK in high glucose-induced apoptosis of osteoblast MC3T3-E1 cell line, and to investigate its effect on the expressions of apoptosis-related molecules including caspase3, bax, and bcl-2. Methods The lentiviral vector containing short hairpin RNA targeting p38MAPK was constructed. The cultured osteoblast MC3T3-E1 cell were divided into 5 groups:normal control group(A group), high glucose group(B group), p38MAPK-shRNA transfection group(C group), signal transduction inhibitor group(D group), and transfection with negative control siRNA group(E group). RT-PCR was used to determine the p38MAPK mRNA expression levels in MC3T3-E1 cells. Flow cytometry(FCM)was employed to detect the cell apoptotic percentage. The protein levels of apoptosis-related molecules p38MAPK, p-p38MAPK, caspase-3, bax, and bcl-2 were assayed by Western blot. Ultrastructural alternation of MC3T3-E1 cell was observed under transmission electron microscopy(TEM). Results The lentiviral vector containing short hairp in RNA targeting p38MAPK was successfully constructed and transfected into MC3T3-E1 cells. RT-PCR result suggested that the siRNA targeting p38MAPK could effectively reduce the p38MAPK mRNA expression level induced by high glucose in MC3T3-E1 cell line. FCM showed siRNA significantly decreased high glucose-induced apoptosis percentage of MC3T3-E1 cells(P＜0.01). Meanwhile, we also found the siRNA significantly attenuated the proteins levels of p38MAPK, p-p38MAPK, caspase-3, and gene bax induced by high glucose in MC3T3-E1 cells, whereas the protein level of gene bcl-2 was enhanced remarkably when compared with high glucose group and negative control siRNA group(P＜0.01, P＜0.05).Conclusion The iRNA targeting p38MAPK suppressed high glucose-induced MC3T3-E1 cell apoptosis via inhibiting the activation of p38MAPK signaling pathway, thereby reducing the expressions levels of p-p38MAPK, caspase-3 and gene bax, and up-regulating the level of gene bcl-2.

Objective To study the effects of decoy strategy targeted to NF-?B on IL-6 in rat's liver after traumatic inflammatin. Methods Ninty six Wistar rats were randomly divided into 4 groups: control group,traumatic inflammation group, decoy ODN group, and mutant decoy ODN group. Rats were killed on 3 , 6, 12, 24, 48 , and 72 h respectively, for the determination of plasma ALT. Hepatocytes were isolated and nuclear protein was extracted, DNA binding activity of NF-?B was measured by EMSA. Decoy ODN's competition inhibition effect was assayed by EMSA. IL-6 gene expression in liver tissue was assessed by RT-PCR and IL-6 protein level was determined by ELISA. Results After traumatic inflammation, DNA binding activity of NF-?B in the liver increased. IL-6 mRNA and protein level also significantly increased and was in positive correlation with the activity of NF-?B. Decoy ODN effectively inhibited the activity of NF-?B ex vivo. After using decoy ODN, IL-6 mRNA and protein levels of liver tissue significantly decreased, plasma ALT levels were also significantly decreased. Conclusions Decoy strategy targeted to NF-?B could effectively inhibit rat's liver IL-6 release by inhibiting specifically the activity of NF-?B.

Astract:Objective To investigate the ultrastructural changes and HSP70 expression in liver of mice after microwave irradiation with lethal dose and to explore the application of these indexes as the basis of medical identification in microwave irradiation induced death. Methods The mice were divided into the control group and the irradiation group. Mice of the irradiation group were induced death by whole body exposure to 129 W/cm2 microwave irradiation for 30 minutes. The ultrastructure of liver was observed by transmission electron micro-scope;changes of the HSP70 mRNA and protein expression in liver were detected by reverse transcription polymerase chain reaction ( RT-PCR) and Western blotting respectively. Results Liver cytoplasm was observed dissolved with points and sheets and there were mitochondri-al crest and membrane solution in the irradiation group. And the HSP70 mRNA and protein expression level increases significantly compared with the control group with statistically significant difference (P<0. 01). Conclusion Death induced by microwave irradiation could lead to liver cytoplasm dissolution, mitochondria damage, mRNA and protein expression of HSP70 up-regulation, which may be used as important diagnostic indicators of microwave irradiation induced death.

Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Full length gene synthesis is an efficient measure to enhance the expression level of the gene. In order to reduce the non-specific binding between oligonucleotides and bases mutation caused by the complicate secondary structure of DNA and excessive PCR amplification, a frequently phenomenon in one-step gene synthesis, we used a two-step method including assembly PCR (A-PCR) and digestion-ligation step to synthesis Aspergillus niger lipase gene lipA. Assisted by DNA2.0 and Gene2Oliga software, we optimized the codon usage and secondary structure of RNA and induced enzyme sites Cla I (237 site) and Pst I (475 site) into the gene. In the first step, fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) were separately synthesized by assembly PCR. In the second step, fragments F1, F2 and F3 were separately digested by Cla I and Pst I, and then ligated into a full length lipA gene. Two-step method efficiently enhanced successful ratio for full-length gene synthesis and dispersed the risk for gene redesign. The synthesized gene was cloned into pPIC9K vector and transferred into Pichia pastoris. After methanol inducement, the expression level of the codon optimized lipA-syn gene reached 176.0 U/mL, 10.8-fold of the original lipA gene (16.3 U/mL) in Pichia pastoris GS1115. The recombinant offers the possibility for lipase large-scale production.
Base Sequence ; Carboxylic Ester Hydrolases ; biosynthesis ; genetics ; Cloning, Molecular ; Genes, Synthetic ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Mitochondria are the main places of cellular respiration as well as the citric acid cycle and oxidative phospho‐rylation .It plays an important role in controlling the life and death of cells .Mitochondrial dysfunction leads to a series of human diseases such as ischemia‐reperfusion injury ,sepsis and diabetes .Mitochondrial become an attractive target for drug transporters strategy and therapeutic targets for neurodegeneration .Although the molecular mechanisms responsible for mitochondria media‐ted disease processes are not fully elucidated yet ,the oxidative stress appears to be critical .Accordingly ,strategies are being de‐veloped for the targeted delivery of antioxidants to mitochondria .The prospect of development of mitochondrial targeted drugs with anti‐oxidative stress protection is tempting .Mitochondrial targeting antioxidants were the antioxidant drugs which took mi‐tochondria as the target site .In this review ,weintroduced the conception and classification of mitochondrial targeted antioxidants and the research progress of disease treatment by mitochondrial targeted antioxidants .

Objective:To test and evaluate the reliability and clinical effectiveness of osteoporotic thoracolumbar fracture (OTLF) scoring and classification system.Methods:A multicenter retrospective case series study was conducted to analyze the clinical data of 530 OTLF patients admitted to 8 hospitals including Honghui Hospital Affiliated to Xi'an Jiaotong University from January 2021 to June 2022. There were 212 males and 318 females, aged 55-90 years [(72.6±10.8)years]. There were 4 patients with grade C and 18 with grade D according to American Spinal Injury Association (ASIA) classification. According to the osteoporotic thoracolumbar injury classification and severity (OTLICS) score, all patients had an OTLICS score over 4 points and required surgical treatment. Among them, 410 patients had acute symptomatic OTLF (ASOTLF), including 24 patients with type I, 159 type IIA, 47 type IIB, 31 type IIC, 136 type IIIA, 8 type IIIB, 2 type IV (absence of neurological symptoms) and 3 type IV (presence of neurological symptoms), and 120 patients had chronic symptomatic OTLF (CSOTLF), including 62 patients with type I, 21 type II, 17 type III, 3 type IV (reducible under general anesthesia), 9 type IV (not reducible under general anesthesia), 1 type V (reducible under general anesthesia), 5 type V (presence of neurological symptoms), and 2 type V (not reducible under general anesthesia). Surgical procedures included percutaneous vertebroplasty (PVP), positional repositioning plus PVP, percutaneous kyphoplasty (PKP), posterior open reduction combined with bone graft fusion and bone cement augmented screw internal fixation, posterior open reduction combined with decompression, bone graft fusion and bone cement augmented screw internal fixation, and posterior open reduction combined with osteotomy and orthopedics, bone graft fusion and bone cement augmented screw internal fixation. A weighted Kappa was used to test the interobserver and intraobserver reliability of the OTLICS score, the ASOTLF classification, and the CSOTLF classification. The visual analog scale (VAS), Oswestry disability index (ODI), ASIA classification were compared before, at 1 month after surgery and at the last follow-up. Incidence of postoperative complications was observed.Results:The percentage of mean interobserver agreement for OTLICS staging was 93.4%, with a mean confidence Kappa value of 0.86, and the percentage of mean intraobserver agreement was 93.0%, with a mean confidence kappa value of 0.86. The percentage of mean interobserver agreement for ASOTLF staging was 94.2%, with a mean confidence Kappa value of 0.84, and the percentage of mean intraobserver agreement was 92.5%, with a mean confidence Kappa value of 0.83. The percentage of mean interobserver agreement for CSOTLF subtyping was 91.9%, with a mean confidence Kappa value of 0.80, and the percentage of mean intraobserver agreement was 91.3%, with a mean confidence Kappa value of 0.81. All the patients were followed up for 6-12 months [(9.0±2.1)months]. The VAS and ODI scores were significantly lower in patients with ASOTLF and CSOTLF classifications at 1 month after surgery and at the last follow-up than those before surgery (all P<0.05). The VAS scores in patients with ASOTLF types IIA, IIB, IIC, IIIA, and IV were significantly lower at the last follow-up than that at 1 month after surgery; the ODI scores in patients with ASOTLF types I, IIA, IIB, IIIA, IIIB and IV were significantly lower at the last follow-up than those at 1 month after surgery. The VAS scores in patients with CSOTLF types II, III, IV, and V were significantly lower at the last follow-up than those at 1 month after surgery, and the ODI scores in patients with all CSOTLF types were significantly lower at the last follow-up than those at 1 month after surgery (all P<0.05). Two patients with ASIA grade C recovered to grade D, and the rest recovered to grade E at the last follow-up ( P<0.01). No major vessel or nerve injury or internal fixation failure was found during follow-up. There were 18 patients with cement leakage, none of whom showed relevant clinical symptoms. There were 35 patients with new vertebral fractures, all of whom recovered well after symptomatic treatment. Conclusions:The OTLICS score, ASOTLF classification and CSOTLF classification have a high degree of reliability. Application of stepwise treatment for patients with different levels of injury according to the scoring and classification system can reduce pain, promote recovery of the spinal function, and reduce complications, which is of some significance in guiding the selection of clinical treatment.

Objective To investigate the application value of dual-layer spectral computed tomography(DLCT)45 keV single energy image in follow up after transcatheter arterial chemoembolization(TACE)for hepatocellular carcinoma(HCC).Methods The DLCT images of 60 patients with HCC after TACE treatment were analyzed retrospectively.The CT value and standard deviation(SD)value of the lesion region of interest(ROI)and surrounding normal liver parenchyma on two kinds of images with 45 keV energy spectrum CT and 120 kVp conventional CT were measured respectively,then the contrast-to-noise ratio(CNR)between the lesions and surrounding normal liver parenchyma was calculated,and their differences were compared.The image quality of the two kinds of images was scored subjectively with the 3-point method,and the patients were divided into different groups according to the enhance-ment degree and tumor staining.The receiver operating characteristic(ROC)curve was also drawn.Results(1)There was no signif-icant difference in CNR between arterial phase,portal phase,and delayed phase of 120 kVp conventional CT(Hc=1.128,P>0.05).The CNR of 45 keV energy spectrum CT was higher than that of 120 kVp conventional CT,with a statistically significant difference(Z=5.060,P<0.05).(2)The subjective score of 45 keV energy spectrum CT was higher than that of 120 kVp conventional CT,and the difference was statistically significant(Z=5.335,P<0.05).ROC curve analysis showed that 45 keV energy spectrum CT had a larger area under the curve(AUC)than 120 kVp conventional CT,and the difference was statistically significant(Z=4.136,P<0.001).Conclusion 45 keV energy spectrum CT image can significantly improve the CNR between tumor and normal liver parenchyma,and it has the better image quality and higher diagnostic efficiency.

Oxidative stress injury and mitochondrial dysfunction are major obstacles to neurological functional recovery after ischemic stroke. The development of new approaches to simultaneously diminish oxidative stress and resist mitochondrial dysfunction is urgently needed. Inspired by the overproduced reactive oxygen species (ROS) at ischemic neuron mitochondria, multifunctional nanoparticles with ROS-responsiveness and mitochondrial-targeted (SPNPs) were engineered, achieving specific targeting delivery and controllable drug release at ischemic penumbra. Due to the nose-to-brain pathway, SPNPs which were encapsulated in a thermo-sensitive gel by intranasal administration were directly delivered to the ischemic penumbra bypassing the blood‒brain barrier (BBB) and enhancing delivery efficiency. The potential of SPNPs for ischemic stroke treatment was systematically evaluated in vitro and in rat models of middle cerebral artery occlusion (MCAO). Results demonstrated the mitochondrial-targeted and protective effects of SPNPs on H₂O₂-induced oxidative damage in SH-SY5Y cells. In vivo distribution analyzed by fluorescence imaging proved the rapid and enhanced active targeting of SPNPs to the ischemic area in MCAO rats. SPNPs by intranasal administration exhibited superior therapeutic efficacy by alleviating oxidative stress, diminishing inflammation, repairing mitochondrial function, and decreasing apoptosis. This strategy provided a multifunctional delivery system for the effective treatment of ischemic injury, which also implies a potential application prospect for other central nervous diseases.