ObjectiveFunctional near-infrared spectroscopy (fNIRS), a novel non-invasive technique for monitoring cerebral activity, can be integrated with upper limb rehabilitation robots to facilitate the real-time assessment of neurological rehabilitation outcomes. The rehabilitation robot is designed with 3 training modes: passive, active, and resistance. Among these, the resistance mode has been demonstrated to yield superior rehabilitative outcomes for patients with a certain level of muscle strength. The control modes in the resistance mode can be categorized into dynamic and static control. However, the effects of different control modes in the resistance mode on the motor function of patients with upper limb hemiplegia in stroke remain unclear. Furthermore, the effects of force, an important parameter of different control modes, on the activation of brain regions have rarely been reported. This study investigates the effects of dynamic and static resistance modes under varying resistance levels on cerebral functional alterations during motor rehabilitation in post-stroke patients. MethodsA cohort of 20 stroke patients with upper limb dysfunction was enrolled in the study, completing preparatory adaptive training followed by 3 intensity-level tasks across 2 motor paradigms. The bilateral prefrontal cortices (PFC), bilateral primary motor cortices (M1), bilateral primary somatosensory cortices (S1), and bilateral premotor and supplementary motor cortices (PM) were examined in both the resting and motor training states. The lateralization index (LI), phase locking value (PLV), network metrics were employed to examine cortical activation patterns and topological properties of brain connectivity. ResultsThe data indicated that both dynamic and static modes resulted in significantly greater activation of the contralateral M1 area and the ipsilateral PM area when compared to the resting state. The static patterns demonstrated a more pronounced activation in the contralateral M1 in comparison to the dynamic patterns. The results of brain network analysis revealed significant differences between the dynamic and resting states in the contralateral PFC area and contralateral M1 area (F=4.709, P=0.038), as well as in the contralateral PM area and ipsilateral M1 area (F=4.218, P=0.049). Moreover, the findings indicated a positive correlation between the activation of the M1 region and the increase in force in the dynamic mode, which was reversed in the static mode. ConclusionBoth dynamic and static resistance training modes have been demonstrated to activate the corresponding brain functional regions. Dynamic resistance modes elicit greater oxygen changes and connectivity to the region of interest (ROI) than static resistance modes. Furthermore, the effects of increasing force differ between the two modes. In patients who have suffered a stroke, dynamic modes may have a more pronounced effect on the activation of exercise-related functional brain regions.

The study aimed to investigate the effect of the CD200R1 gene deletion on microglia activation and nigrostriatal dopamine neuron loss in the Parkinson's disease (PD) process. The CRISPR-Cas9 technology was applied to construct the CD200R1^-/- mice. The primary microglia cells of wild-type and CD200R1^-/- mice were cultured and treated with bacterial lipopolysaccharide (LPS). Microglia phagocytosis level was assessed by a fluorescent microsphere phagocytosis assay. PD mouse model was prepared by nigral stereotaxic injection of recombinant adeno-associated virus vector carrying human α-synuclein (α-syn). The changes in the motor behavior of the mice with both genotypes were evaluated by cylinder test, open field test, and rotarod test. Immunohistochemical staining was used to assess the loss of dopamine neurons in substantia nigra. Immunofluorescence staining was used to detect the expression level of CD68 (a key molecule involved in phagocytosis) in microglia. The results showed that CD200R1 deletion markedly enhanced LPS-induced phagocytosis in vitro by the microglial cells. In the mouse model of PD, CD200R1 deletion exacerbated motor behavior impairment and dopamine neuron loss in substantia nigra. Fluorescence intensity analysis results revealed a significant increase in CD68 expression in microglia located in the substantia nigra of CD200R1^-/- mice. The above results suggest that CD200R1 deletion may further activates microglia by promoting microglial phagocytosis, leading to increased loss of the nigrostriatal dopamine neurons in the PD model mice. Therefore, targeting CD200R1 could potentially serve as a novel therapeutic target for the treatment of early-stage PD.
Animals ; Microglia/physiology* ; Mice ; Phagocytosis ; Parkinson Disease/genetics* ; Disease Models, Animal ; Receptors, Cell Surface/physiology* ; Dopaminergic Neurons/pathology* ; Antigens, CD/metabolism* ; Gene Deletion ; Substantia Nigra ; Mice, Inbred C57BL ; Mice, Knockout ; Cells, Cultured ; Male ; alpha-Synuclein ; CD68 Molecule ; Orexin Receptors

Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes

This study aims to explore the scientific connotation of sinking Rehmanniae Radix has the best quality and compare the quality between floating and sinking fresh Rehmanniae Radix samples. Ultra-performance liquid chromatography tandem quadrupole electrostatic field Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS) was employed to detect the chemical components in floating and sinking fresh Rehmanniae Radix samples. The fingerprint of fresh Rehmanniae Radix was established by high performance liquid chromatography(HPLC), and four index components were determined simultaneously. The cluster analysis, principal component analysis(PCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were conducted to compare the quality of floating and sinking fresh Rehmanniae Radix samples. An evaporative light-scattering detector was used to compare the content of five sugars. The extract yield and drying rate were determined, and the quality connotation of sinking Rehmanniae Radix has the best quality was explained by multiple indicators. A total of 41 components were preliminarily identified from fresh Rehmanniae Radix by UHPLC-Q-Orbitrap HRMS, including 7 iridoid glycosides, 9 phenylethanol glycosides, 6 amino acids, 4 sugars, 3 phenolic acids, 5 nucleosides, 3 organic acids, 1 ionone, 1 furan, 1 coumarin, and 1 phenylpropanoid. The results showed that the main chemical components were consistent between floating and sinking fresh Rehmanniae Radix. Nine common peaks were identified in the fingerprints of 15 batches of floating and sinking fresh Rehmanniae Radix samples, and the similarity of fingerprints was greater than 0.9. The cluster analysis, PCA, and OPLS-DA classified floating and sinking fresh Rehmanniae Radix sasmples into two categories, indicating differences in the quality between them. The total content of catalpol, rehmannioside D, ajugol, and verbascoside in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was catalpol. The total content of fructose, glucose, sucrose, raffinose, and stachyose in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was stachyose. The extract yield and drying rate of the sinking samples were higher than those of floating samples. This study preliminarily showed that floating and sinking fresh Rehmanniae Radix samples had the same components but great differences in the content of medicinal substance basis. The total content of four glycosides and five sugars, extract yield, and drying rate of sinking fresh Rehmanniae Radix samples is higher than that of floating samples of the same batch and specification. These findings, to a certain extent, explains the scientificity of sinking Rehmanniae Radix has the best quality recorded in ancient books and provide a reference for the quality control and clinical application of fresh Rehmanniae Radix.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Rehmannia/chemistry* ; Chemometrics ; Mass Spectrometry/methods* ; Quality Control ; Principal Component Analysis ; Plant Extracts

Objective To screen the active components of total flavonoid extracts of Sarcandra glabra to promote megakaryocyte differentiation.Methods(1)A model of megakaryocyte differentiation disorder was established by co-culturing human megakaryocytic leukaemia cells(Dami)with human bone marrow stromal cells(HS-5)as an evaluation system,and the experimental groupings were as follows:the Dami group(Dami),the control group(Dami+HS-5),and the PMA group[Dami+HS-5+5 ng·mL-1 foprolol 12-tetradecanoate 13-acetate(PMA)],and model group[Dami+HS-5+1%rabbit anti-rat platelet serum(APS)+5 ng·mL-1 PMA]were cultured for 48 hours.The expressions of megakaryocyte differentiation and maturation surface marker molecules,CD41a and CD61 were detected by flow cytometry.(2)Forty-nine SD male rats were randomly divided into blank plasma group,15-minute group,30-minute group,60-minute group,90-minute group,120-minute group,and 240-minute group,with 7 rats in each group.The rats in each administration group were gavaged with 1.26 g·kg-1 of total flavonoids extracts of Sarcandra glabra,and blood was collected at six set time points(15,30,60,90,120,240 minutes)for the preparation of time-dependent serum-containing plasma of total flavonoids extracts of Sarcandra glabra.(3)Ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)was used to analyze the plasma of the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra,and the peak area was used to construct a matrix(X-matrix)of the amount of chemical composition change over time in the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra.The collected time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra at six different time points was used to intervene in the model of megakaryocyte differentiation and maturation disorder,and the expression of cell surface molecules CD41a and CD61 was detected by flow cytometry to construct the matrix of effect of time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra(Y-matrix).(4)After the data of X and Y matrices were standardized,partial least squares(PLS)was used to calculate and analyze the quantitative and qualitative effect relationship,and variable importance for projection(VIP)>1 was used as the threshold to screen the effect components related to the changes of cell surface molecules CD41a and CD61,and chemical composition identification,as the potential effector components in the total flavonoid extracts of Sarcandra glabra were used to promote the differentiation of megakaryocytes,and finally the regression evaluation system was used to verify the efficacy of its medicinal effect.Results(1)Compared with the Dami group,the expression level of CD41a on the surface of Dami cells in the control group was significantly increased(P＜0.05).Compared with the control group,the expression levels of CD41a and CD61 on the surface of Dami cells in the PMA group were significantly increased(P＜0.01).Compared with the PMA group,the expression levels of CD41a and CD61 on the surface of Dami cells in the model group were significantly reduced(P＜0.01).(2)Compared with the blank plasma group,the expression levels of the molecules CD41a and CD61 on the surface of Dami cells at each time point of 15,30,60,90,120,and 240 minutes were significantly increased(P＜0.01),and the expression levels of CD41a and CD61 were both highest in the 30-minute group.The potential effective components with VIP value greater than 1 were screened out in the positive and negative ion mode,and 540.3638@12.25 and 559.2991@11.53 were selected for pharmacodynamic verification.559.2991@11.53 was identified as daucosterol(Dau),540.3638@12.25 was identified as rosmarinic acid 4-O-β-D-glucoside(Ros).After Ros and Dau intervened in the megakaryocyte differentiation and maturation disorder model respectively,the expression levels of CD41a and CD61 on the surface of Dami cells in the low-,medium-and high-dose groups(40,60 and 80 μg·mL-1)of Ros and Dau were significantly increased compared with the model group(P＜0.05,P＜0.01).Conclusion Ros and Dau may be the active components of the total flavonoids extracts of Sarcandra glabra to promote the differentiation of megakaryocytes.

Objective:To investigate the effect of genetic polymorphism of MTHFR C677T (rs1801133) on methotrexate (MTX) related toxicity in pediatric mature B-cell lymphoma patients. Methods:Fifty-eight intermediate and high risk patients under 18 years of age with mature B-cell lymphoma who received 5 g/m2 MTX (24 h intravenous infusion) in Sun Yat-sen University Cancer Center from August 2014 to December 2021 were included,and their toxicity of high-dose MTX (HD-MTX) were monitored and analyzed. Results:Among the 58 pediatric patients,the number of CC,CT,and TT genotypes for MTHFR C677T was 33,19 and 6,respectively. A total of 101 courses of HD-MTX therapy were counted,of which plasma MTX level>0.2 μmol/L at 48 h post-MTX infusion were observed in 35 courses,≤0.2 μmol/L in 66 courses. Inter-group comparison showed that plasma MTX level>0.2 μmol/L at 48 h post-MTX infusion increased the risk of developing oral mucositis (P<0.05). Compared with wild-type (CC genotype),patients in the mutant group (CT+TT genotype) were more likely to develop myelosuppression,manifested as anemia,leucopenia,neutropenia and thrombocytopenia. However,plasma MTX level at 48 h was not associated with MTHFR C677T gene polymorphism. Conclusion:The risk of developing oral mucositis in children with mature B-cell lymphoma is associated with plasma MTX concentration. Polymorphism of MTHFR C677T gene is not related to plasma MTX concentration in children with mature B-cell lymphoma,but is related to grade Ⅲ to Ⅳ hematological toxicity.

Humans ; Child ; Lymphoma, Large-Cell, Anaplastic ; Neoplasm Recurrence, Local ; Prognosis ; Hodgkin Disease

Objective: To explore the diagnostic value of p16/Ki-67 double-stained immunohistochemistry in the diagnosis of human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinoma(opscc) and find out the optimal index to improve the accuracy of HPV detection. Methods: A total of 153 cases, from May 2014 to May 2020, diagnosed OPSCC in Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, were selected. This cohort included 130 males and 23 females, aged (58.6±10.0) years old. HPV RNA in situ hybridization was chosen as the gold standard to detect their HPV status. p16 immunohistochemistry and p16/Ki-67 double-stained immunohistochemistry were performed on all cases, and the p16/Ki-67 double positive index including 20%, 40%, and 60% were used as the thresholds to compare their sensitivity, specificity, and positive prediction value (PPV), negative prediction value (NPV) and prognosis prediction ability. Results: Among the 153 patients with OPSCC, 114 were HPV-negative and 39 were HPV-positive, and the HPV infection rate of OPSCC patients was 25.5% (39/153). Only 58.1% (36/62) of single p16 positive cases were HPV-positive, and the prognosis of patients could not be distinguished using p16 immunohistochemistry only. Using p16/Ki-67 double staining, the accuracy of HPV positive diagnosis has been improved. The HPV diagnostic ability was the highest when the p16/Ki-67 double positive index was 40% (sensitivity=86.8%, specificity=94.8%, PPV=84.6%, NPV=95.6%, area under the curve=0.897), which could distinguish the prognosis of patients (P=0.012). Conclusions: The p16/Ki-67 double-stained immunohistochemistry can improve the accuracy of HPV positive diagnosis rate and diagnosis of HPV-positive oropharyngeal cancer is the most accurate when the double-positive index is 40% as the threshold to judge HPV status and could serve as better surrogate marker for HPV detection.

Objective: To verify the feasibility and accuracy of the transanal multipoint full-layer puncture biopsy (TMFP) technique in determining the residual status of cancer foci after neoadjuvant therapy (nCRT) in rectal cancer. Methods: Between April 2020 and November 2022, a total of 78 patients from the Beijing Chaoyang Hospital of Capital Medical University, the Beijing Friendship Hospital of Capital Medical University, the Qilu Hospital of Shandong University, the Zhongnan Hospital of Wuhan University with advanced rectal cancer received TMFP after nCRT participated in this prospective multicenter trial. There were 53 males and 25 females, aged (M(IQR)) 61 (13) years (range: 35 to 77 years). The tumor distance from the anal verge was 5 (3) cm (range: 2 to 10 cm). The waiting time between nCRT and TMFP was 73 (26) days (range: 33 to 330 days). 13-point transanal puncture was performed with a 16 G tissue biopsy needle with the residual lesion as the center. The specimens were submitted for independent examination and the complications of the puncture were recorded. The consistency of TMFP and radical operation specimen was compared. The consistency of TMPF with clinical remission rates for the diagnosis of complete pathological remission was compared by sensitivity, specificity, negative predictive value, positive predictive value and accuracy. Statistical analysis between groups was performed using the χ² analysis, and a paired χ² test was used to compare diagnostic validity. Results: Before TMFP, clinical complete response (cCR) was evaluated in 27 cases. Thirty-six cases received in vivo puncture, the number of punctures in each patient was 13 (8) (range: 4 to 20), 24 cases of tumor residue were found in the puncture specimens. The sensitivity to judgment (100% vs. 60%, χ²=17.500, P<0.01) and accuracy (88.5% vs. 74.4%, χ²=5.125, P=0.024) of TMFP for the pathologic complete response (pCR) were significantly higher than those of cCR. Implement TMFP based on cCR judgment, the accuracy increased from 74.4% to 92.6% (χ²=4.026, P=0.045). The accuracy of the in vivo puncture was 94.4%, which was 83.3% of the in vitro puncture (χ²=1.382, P=0.240). Overall, the accuracy of TMFP improved gradually with an increasing number of cases (χ²=7.112, P=0.029). Conclusion: TMFP is safe and feasible, which improves the sensitivity and accuracy of rectal cancer pCR determination after nCRT, provides a pathological basis for cCR determination, and contributes to the safe development of the watch and wait policy.

Humans ; Child ; Lymphoma, Large B-Cell, Diffuse/pathology*