Thymocyte selection-associated high mobility group box (TOX), a member of the high mobility group protein super-family, plays an important role in T cell development, functional maintenance, and exhaustion. It has been recently found that TOX exerts critical immunoregulatory functions during pathogen infections, and TOX expression is strongly associated with the intensity and tolerance of host immune responses. This review systematically summarizes the structural and functional features of TOX and focuses on its expression dynamics, mechanisms of action, and immunomodulatory effects during viral, bacterial, and parasitic infections, which provides a theoretical support to better understanding of the role of TOX in infectious diseases and provides new insights into development of potential immunotherapeutic strategies targeting TOX.

BACKGROUND:The formation of Lewy bodies due to abnormal α-synuclein aggregation is a characteristic pathological change in Parkinson's disease.In recent years,several studies have revealed that the formation of α-synuclein aggregates is closely related to its post-translational modifications.The modification of α-synuclein such as phosphorylation,nitration,acetylation,and ubiquitination has attracted extensive attention in the pathogenesis and progression of Parkinson's disease. OBJECTIVE:To review the research progress in the effect of modification types and sites of α-synuclein on the characteristic pathological formation and progression of Parkinson's disease. METHODS:PubMed and CNKI databases were searched by the first author with the key words of"α-synuclein,Parkinson's disease,phosphorylation,acetylation,ubiquitination,nitration"in English and Chinese respectively to collect and sort out the literature related to abnormal modification of α-synuclein in recent years.Finally,61 articles were included for further review. RESULTS AND CONCLUSION:Abnormal modification of α-synuclein is closely related to its protein structure and its positive and negative charges.Its amino terminus is positively charged and prone to ubiquitination and acetylation modifications.The central hydrophobic region is prone to forming β-pleated sheet due to its hydrophobic property.The carboxyl terminus is negatively charged,which is the main phosphorylation modification region.Phosphorylation modification sites promote phosphorylation modification and are closely related to α-synuclein aggregation,while protein kinases can target the activation of translational modifications,which may help to promote or inhibit aggregate formation.The degradation pathway of α-synuclein mainly plays a role in removing pathological proteins.Various kinase catalysts contribute to impaired protein ubiquitination modifications that lead to abnormal protein accumulation,thereby exacerbating neurodegeneration.The amino-terminal acetylation of α-synuclein improves the shuttle ability of the protein to the cell membrane and slows down the protein aggregation,which may be the protection target of nerve cells.However,the acetylation modification of the mutant protein produces the opposite effect.The protein nitration modification is mainly related to oxidative stress.The aggregation tendency of the protein modified by nitration is enhanced under the action of reactive oxygen species.Different post-translational modifications have different effects.Therefore,elucidating the main mechanisms of their post-translational modifications and inhibiting the post-translational modifications that contribute to protein aggregation may provide a reference for new targets for early diagnosis and treatment of Parkinson's disease.

Taking the Ligustrum lucidum extract as an example, this study investigated the influence of external mechanical forces under different processing conditions on the crystallographic solid form and the key parameter of drug dissolution rate, so as to provide guidance for the preparation and quality control of traditional Chinese medicine preparations. Under different pulverization and tableting conditions, the change of crystallographic solid form of L. lucidum extract was observed by polarizing light microscope. Meanwhile, the effect of this change on the dissolution rate was investigated, and the mathematical relationship between crystal content and dissolution rate was analyzed. The results showed that the process of ball milling and tableting had a significant impact on the crystallographic solid form of L. lucidum extract. The amorphous extract displayed crystal transformation, which induced a significant decline in dissolution rate. Further studies revealed that there was a negative linear relationship between crystal content and dissolution rate. The results of this study indicated that the crystallographic solid form transformation of traditional Chinese medicine extracts might occur during the preparation process, which may cause potential risks to the quality of traditional Chinese medicine preparations. It is suggested that we should pay attention to and strengthen the investigation of the crystallographic solid form during the preparation process, so as to guarantee the safety, effectiveness, and quality controllability of traditional Chinese medicine preparations.

BACKGROUND:C2 ceramide reduces the formation of Alpha-Synuclein(α-Syn)oligomers as the protein phosphatase 2A agonist,which has an important regulatory effect on cell aging in the central nervous system. OBJECTIVE:To investigate the protective mechanism of C2 ceramide on dopaminergic neurons. METHODS:Twenty-five C57BL/6 mice were randomly divided into control group,model group,C2 ceramide low-,medium-and high-dose groups(n=5 per group).Except for the control group,a mouse model of Parkinson's disease was established by injecting mutant A53T α-Syn oligomers into the left striatum in the other groups.On the 30th day after the striatal injection,three C2 ceramide groups were intragastrically administered with C2 ceramide(1,5,10 μg/g)dissolved in saline at one time,while the control and model groups were administered with the same amount of saline within 30-90 days after modeling,for a total of 60 days.Behavioral changes in each group of mice were observed during this period.On the 90th day after striatal injection,mouse brain tissue was extracted by perfusion under anesthesia,and the changes of dopaminergic neurons in the midbrain substantia nigra were analyzed by immunohistochemical staining.The levels of α-Syn oligomerization and phosphorylation in the midbrain of mice were detected by ELISA,and the changes of enzyme activities related to α-Syn phosphorylation were analyzed. RESULTS AND CONCLUSION:C2 ceramide had an ameliorating effect on Parkinson's disease-like dyskinesia in mice caused by the striatal injection of mutant A53T α-Syn oligomers.High-dose C2 ceramide showed better effects on dyskinesia in mice with Parkinson's disease(P<0.01).The mutant A53T α-Syn oligomers significantly reduced the number of dopaminergic neurons in the substantia nigra of mice(P<0.01),while the number of dopaminergic neurons in the substantia nigra increased significantly in the C2 ceramide high-dose group(P<0.01).The levels of α-Syn oligomers and phosphorylated α-Syn in the brain were significantly reduced in the C2 ceramide high-dose group compared with the model group(P<0.01),while the level of ceramide was increased(P<0.05)and the activity of protein phosphatase 2A was significantly upregulated(P<0.01).To conclude,C2 ceramide can attenuate the neurotoxic effects induced by oligomerized α-Syn by the phosphorylation modification environment of α-Syn in mouse midbrain tissue and protect against the reduction in the number of nigrostriatal dopaminergic neurons in mice,thereby reducing the degree of dyskinesia in Parkinson's disease.

Objective To explore prokaryotic expression,purification of hemolysin coregulatory protein(Hcp)of Vibrio cholerae,and preparation of its polyclonal antibodies.Methods PCR was used to amplify Vibrio cholerae Hcp gene and clone it into pET28a vector to construct recombinant expression vector.The recombinant vector pET28a-hcp was transformed into E.coil BL21(DE3)for expression condition optimization and expression form identification.The soluble Hcp protein was purified by Ni-NTA column.The purified Hcp protein was used to immunize BALB/c mice to prepare polyclonal antibodies.The antibody titer was detected by indirect enzyme-linked immunosorbent assay(ELISA)to evaluate its immunogenicity.Western blot was used to analyze the specific recognition of antibodies to Hcp protein in Vibrio cholerae.Results The enzyme fragment digested by recombinant vector pET28a-hcp was consistent with the expected,the sequencing results were consistent with the Hcp gene sequence in the GenBank database,and the pET28a-hcp recombinant plasmid was successfully constructed.The recombinant plasmid was induced by isopropyl-β-D-thiogalactopyranoside(IPTG)to express the target protein with a relative molecular weight of 28 kD.The pure Hcp protein was obtained after purification by Ni-NTA column,and then Hcp polyclonal antibody(anti-Hcp)with a titer of 1∶512 000 could be obtained from immunized mice.Western blot results showed that anti-Hcp had specificity in recognizing Hcp protein in Vibrio cholerae.Conclusion The soluble expression of Hcp protein is successfully obtained,and high-titer polyclonal antibodies against Hcp are obtained after immunization of mice,which may lay a foundation for subsequent studies on the role of Hcp protein in the pathogenesis of T6SS in non-O1/non-O139 V.cholerae.

Objective:To analyze the clinical features of anti-neurofascin-155 (NF155) antibody positive autoimmune nodopathy in children.Methods:This was a case series study. A total of 6 children who were diagnosed accurately as anti-NF155 antibody positive autoimmune nodopathy by cell immunofluorescence assay at the Children′s Hospital of Fudan University from January 2020 to December 2023 were collected. This study retrospectively analyzed 6 pediatric children′s clinical manifestations, laboratory and electrophysiological examination results, and treatment outcomes.Results:Among 6 children with anti-NF155 antibody positive autoimmune nodopathy, there were 4 boys and 2 girls. The onset age of 6 children ranged from 3 years and 8 months to 12 years. All 6 children had extremity weakness (more severe in the distal and the lower extremities than in the upper extremities), 5 children had sensory deficits such as numbness or pain in the extremities, 4 children had tremors and ataxia, 3 children had cranial nerve involvement. Among the 6 children, 4 children had protein-cell separation in cerebrospinal fluid examinations. Among the 6 children, 1 child had central nervous system demyelination, the brain magnetic resonance imaging showed multiple abnormal signals in the bilateral cerebral hemispheres. Four children showed motor and sensory nerve damage in electrophysiological examination, and 2 children only showed motor nerve damage. Three children showed myelin and axonal damage, and 3 children only showed axonal damage. Among the 6 children, 5 children were treated with intravenous immunoglobulin and steroids. Among them, 2 children underwent plasma exchange due to poor efficacy, and subsequently, rituximab was added. There was 1 child changed the treatment with olfatomumab since the symptoms did not significantly improve after using rituximab. After treatment for 4-15 months, 2 children had no clinical symptoms, 1 child had improvement in clinical symptoms, 2 children had no significant improvement in clinical symptoms, and 1 child who did not receive the immunotherapy had no significant change in clinical symptoms.Conclusions:Anti-NF155 antibody positive autoimmune nodopathy in children presents with varying degrees of clinical manifestations. It is mainly characterized by extremity weakness, numbness and pain, often accompanied bytremorsand ataxia. Some pediatric patients may also have central nervous system demyelination. Cerebrospinal fluid and electrophysiological examination are important auxiliary examination methods. If steroid therapy is not effective, plasma exchange and rituximab treatment should be used as soon as possible.

Objective To establish and evaluate a gas chromatography-tandem mass spectrometry(GC-MS/MS)method for the detection of six common drugs(methamphetamine,meperidine,caffeine,codeine,cocaine and ketamine)in blood,and to improve the determination basis of results.Methods The above six drugs were added into the blank blood,and GC-MS/MS was used for detection after ether extraction.The collection,quantification and confirmation were carried out under the mode of multi-reaction monitoring(MRM).The qualitative results of the above six drugs were evaluated based on the maximum allowable deviation of the retention time and relative ion abundance ratio in the qualitative results of GC-MS/MS.Results There was a good linear relationship between the six common drugs,among which ketamine and caffeine had the lowest detection limit(0.01 μg/mL),methamphetamine had the highest detection limit(0.5 μg/mL).The retention time(RT)and relative retention time(RRT)of the target substance were stable under the six supplemental levels,and the absolute deviation(ΔRTabsolute)of RT was within±0.025 min.The absolute deviation of RRT(ΔRRTabsolute)was within±0.004.The relative ion abundance ratio absolute deviation(ΔIabsolute)is±20%,and the relative ion abundance ratio relative deviation(ΔIrelative)is±50%.Conclusion This study clarified the reference range for qualitative determination of six common drugs in blood matrix detected by GC-MS/MS,and effectively supplemented the qualitative determination indicators of existing instrumental analysis methods.

Objective:To investigate the CD163 expression characteristics in intracranial aneurysm (IA) tissue and blood of patients with IA and its feasibility as an early clinical screening indicator for IA.Methods:A total of 28 patients with IA admitted to Department of Neurosurgery, First Affiliated Hospital of Xinjiang Medical University from January 2021 to November 2023 were selected as IA group, and 28 healthy subjects from Health Management Center, First Affiliated Hospital of Xinjiang Medical University at the same time period were selected as control group. Eight saccular IA tissues and 12 superficial temporal artery tissues were collected from patients from IA group accepted IA clipping, and real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the CD163 mRNA expression in these tissues. RT-qPCR was also used to detect the CD163 mRNA expression in the blood of the 2 groups. Seven patients with IA and 7 control subjects from the above 2 groups were randomly selected, respectively; and plasma CD163 protein content was detected by enzyme-linked immunosorbent assay (ELISA). Multivariate Logistic regression was used to analyze the influencing factors for IA. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic values of blood CD163 mRNA expression and plasma CD163 protein content in IA. Results:CD163 mRNA expression in IA tissues was significantly higher than that in superficial temporal artery tissues (41.870±20.355 vs. 6.080±5.444, P<0.05). CD163 mRNA expression in the blood of IA patients was significantly higher than that in the controls (1.969[1.124, 2.318] vs. 1.124[0.933, 1.379], P<0.05). CD163 mRNA expression in the blood of ruptured IA group, unruptured IA group, and control group was gradually decreased, with significant differences ( P<0.05). CD163 mRNA expression in the blood of female IA patients was not statistically different compared with that in male IA patients ( P>0.05). ELISA showed that the CD163 protein content in plasma of the IA group was significantly higher than that in the control group [10.537±1.879] ng/L vs. [8.598±0.885] ng/L, P<0.05). Multivariate Logistic regression analysis showed that age and CD163 mRNA expression in the blood were independent influencing factors for IA occurrence ( OR=0.844, 95% CI: 0.750-0.951, P=0.005; OR=0.111, 95% CI: 0.024-0.506, P=0.004). ROC curve showed that the area under the curve (AUC) of CD163 mRNA expression in blood in diagnosing IA was 0.759 (95% CI: 0.618-0.890, P=0.002), and that of CD163 protein content in plasma in diagnosing IA was 0.864 (95% CI: 0.610-1.000, P=0.035). Conclusion:CD163 mRNA expressions in blood and IA tissues and CD163 protein content in plasma are high in patients with IA; CD163 mRNA expression in blood is an independent risk factor for IA; CD163 protein in plasma can be used as a molecular marker for screening IA.

Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 µg/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 µg/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.

Uncommon epidermal growth factor receptor (EGFR) mutations account for 10%-20% of all EGFR mutations in non-small-cell lung cancer (NSCLC). The uncommon EGFR-mutated NSCLC is associated with poor clinical outcomes and generally achieved unsatisfactory effects to the current therapies using standard EGFR-tyrosine kinase inhibitors (TKIs), including afatinib and osimertinib. Therefore, it is necessary to develop more novel EGFR-TKIs to treat uncommon EGFR-mutated NSCLC. Aumolertinib is a third-generation EGFR-TKI approved in China for treating advanced NSCLC with common EGFR mutations. However, it remains unclear whether aumolertinib is effective in uncommon EGFR-mutated NSCLC. In this work, the in vitro anticancer activity of aumolertinib was investigated in engineered Ba/F3 cells and patient-derived cells bearing diverse uncommon EGFR mutations. Aumolertinib was shown to be more potent in inhibiting the viability of various uncommon EGFR-mutated cell lines than those with wild-type EGFR. And in vivo, aumolertinib could also significantly inhibit tumor growth in two mouse allograft models (V769-D770insASV and L861Q mutations) and a patient-derived xenografts model (H773-V774insNPH mutation). Importantly, aumolertinib exerts responses against tumors in advanced NSCLC patients with uncommon EGFR mutations. These results suggest that aumolertinib has the potential as a promising therapeutic candidate for the treatment of uncommon EGFR-mutated NSCLC.