[Effect of MELK Inhibitor OTSSP167 on Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To investigate the effect of MELK inhibitor OTSSP167 against diffuse large B-cell lymphoma (DLBCL). METHODS: The effect of OTSSP167 on activity, proliferation, and apoptosis of DLBCL cell line (SUDHL2 and HBL1) was detected by CCK-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and Annexin V-FITC/PI double staining, respectively. DLBCL cells were inoculated into nude mice, after 4 weeks of OTSSP167 treatment, the effect of OTSSP167 on DLBCL growth in vivo was detected. Caspase-GloTM 3/7 enzyme activity assay kit was used to detect the effect of OTSSP167 on Caspase 3/7 enzyme activity of DLBCL cells. The expression levels of apoptosis and cycle-related proteins were detected by Western blot. RESULTS: OTSSP167 significantly inhibited the activity of SUDHL2 and HBL1 cells in a dose-dependent manner (r =-0.61, r =-0.52). EdU staining showed that OTSSP167 could significantly inhibit the proliferation of SUDHL2 and HBL1 cells. Annexin V-FITC/PI result showed that OTSSP167 could significantly promote the apoptosis of SUDHL2 and HBL1 cells (P <0.001). The result of in vivo experiment showed that OTSSP167 could inhibit the growth of SUDHL2 cells in nude mice. The result of TUNEL staining of tumor further confirmed that OTSSP167 could promote the apoptosis of SUDHL2 cells. Caspase 3/7 enzyme activity test demonstrated that OTSSP167 could significantly increase caspase activity in SUDHL2 and HBL1 cells (r =0.98, r =0.87). Western blot showed that OTSSP167 could dose-dependently inhibit the expression of PARP, Bcl-xL, and Bcl-2 in apoptosis signaling pathway (r =-0.93, r =-0.66, r =-0.87), while p53 protein was significantly up-regulated (r =0.82). The expression of cell cycle-related proteins cdc2, Cyclin E1, Cyclin A2, and Cyclin B1 also showed a dose-dependent down-regulation (r =-0.89, r =-0.83, r =-0.61, r =-0.93). CONCLUSION: The MELK inhibitor OTSSP167 can inhibit the proliferation and promote the apoptosis of DLBCL cells by inhibiting the expression of cycle-related proteins and anti-apoptosis-related proteins.

Cuproptosis-related long non-coding RNAs model that effectively predicts prognosis in hepatocellular carcinoma.

BACKGROUND: Cuproptosis has recently been considered a novel form of programmed cell death. To date, long-chain non-coding RNAs (lncRNAs) crucial to the regulation of this process remain unelucidated. AIM: To identify lncRNAs linked to cuproptosis in order to estimate patients' prognoses for hepatocellular carcinoma (HCC). METHODS: Using RNA sequence data from The Cancer Genome Atlas Live Hepatocellular Carcinoma (TCGA-LIHC), a co-expression network of cuproptosis-related genes and lncRNAs was constructed. For HCC prognosis, we developed a cuproptosis-related lncRNA signature (CupRLSig) using univariate Cox, lasso, and multivariate Cox regression analyses. Kaplan-Meier analysis was used to compare overall survival among high- and low-risk groups stratified by median CupRLSig risk score. Furthermore, comparisons of functional annotation, immune infiltration, somatic mutation, tumor mutation burden (TMB), and pharmacologic options were made between high- and low-risk groups. RESULTS: Three hundred and forty-three patients with complete follow-up data were recruited in the analysis. Pearson correlation analysis identified 157 cuproptosis-related lncRNAs related to 14 cuproptosis genes. Next, we divided the TCGA-LIHC sample into a training set and a validation set. In univariate Cox regression analysis, 27 LncRNAs with prognostic value were identified in the training set. After lasso regression, the multivariate Cox regression model determined the identified risk equation as follows: Risk score = (0.2659 × PICSAR expression) + (0.4374 × FOXD2-AS1 expression) + (-0.3467 × AP001065.1 expression). The CupRLSig high-risk group was associated with poor overall survival (hazard ratio = 1.162, 95%CI = 1.063-1.270; P < 0.001) after the patients were divided into two groups depending upon their median risk score. Model accuracy was further supported by receiver operating characteristic and principal component analysis as well as the validation set. The area under the curve of 0.741 was found to be a better predictor of HCC prognosis as compared to other clinicopathological variables. Mutation analysis revealed that high-risk combinations with high TMB carried worse prognoses (median survival of 30 mo vs 102 mo of low-risk combinations with low TMB group). The low-risk group had more activated natural killer cells (NK cells, P = 0.032 by Wilcoxon rank sum test) and fewer regulatory T cells (Tregs, P = 0.021) infiltration than the high-risk group. This finding could explain why the low-risk group has a better prognosis. Interestingly, when checkpoint gene expression (CD276, CTLA-4, and PDCD-1) and tumor immune dysfunction and rejection (TIDE) scores are considered, high-risk patients may respond better to immunotherapy. Finally, most drugs commonly used in preclinical and clinical systemic therapy for HCC, such as 5-fluorouracil, gemcitabine, paclitaxel, imatinib, sunitinib, rapamycin, and XL-184 (cabozantinib), were found to be more efficacious in the low-risk group; erlotinib, an exception, was more efficacious in the high-risk group. CONCLUSION: The lncRNA signature, CupRLSig, constructed in this study is valuable in prognostic estimation of HCC. Importantly, CupRLSig also predicts the level of immune infiltration and potential efficacy of tumor immunotherapy.

Effects of early rehabilitation in improvement of paediatric burnt hands function.

BACKGROUND: Hands are one of the most common burn sites in children. Hypertrophic scar contractures in hands after wound healing result in further reductions in their range of motion (ROM), motility, and fine motor activities. Rehabilitation can improve the function of hands. But the optimal time of rehabilitation intervention is still unclear. Therefore, this study was designed to investigate the effects of early rehabilitation management of paediatric burnt hands and to compare the efficacy between early and later rehabilitation intervention. AIM: To investigate the effects of early rehabilitation management of paediatric burnt hands. METHODS: A total of 52 children with burnt hands were allocated into the early intervention group (≤ 1 mo from onset) and a late intervention group (> 1 mo from onset) between January 2016 and December 2017. The children received the same rehabilitation programme including skin care, scar massage, passive ROM exercises, active ROM exercises, compression therapy, orthotic devices wearing and game or music therapy. Rehabilitation assessments were performed before and after the rehabilitation treatment. RESULTS: In the early intervention group, the ROM of the hands was significantly improved after rehabilitation (P = 0.001). But in the late group the effect was not significant statistically (P = 0.142). In the early group, 38.5% of the patients showed significant improvement, while in the late group, 69.2% of the patients showed no significant improvement. The time from onset to posttraumatic rehabilitation (P = 0.0007) and length of hospital stay (P = 0.003) were negatively correlated with the hand function improvement. The length of rehabilitation stay was positively correlated with the hand function improvement (P = 0.005). CONCLUSION: These findings suggest that early rehabilitation might show better results in terms of ROM.

[Effects of P 311 on the migration of epidermal stem cells in mice with superficial partial-thickness burn and injured cell model in vitro].

OBJECTIVE: To study effects of P311 on the migration of epidermal stem cells (ESCs) in mice with superficial partial-thickness burn and injured cell model in vitro and to explore the mechanism. METHODS: (1) Eighteen male C(57) BL/6 mice were used. Fifteen of them were inflicted with superficial partial-thickness burn on the back. In three injured mice wound tissue and skin of wound edge were obtained at post burn hour (PBH) 6, 12, 24, 48, 72 respectively. The rest three mice were used as normal control, and samples were harvested with the same method as above. The expressions of P311 in harvested samples were assessed with biotin-streptavidin-peroxidase (SP) staining. (2) Six newly born C(57) BL/6 mice were intraperitoneally injected with 50 µg/g BrdU (two times a day) for three days for ESCs-labelling. Seven weeks later, the mice were inflicted with superficial partial-thickness burn on the back. Serial slices of burn wound tissue were prepared at PBH 72 and immunohistochemically stained with SP for observation of the co-localization of BrdU-positive ESCs and P311-positive cells. (3) The empty vector pAdEasy-enhanced green fluorescence protein (EGFP) and the adenovirus P311-expressing vector named pAdEasy-EGFP-P311 were constructed and packed. Human ESCs were isolated by the method of rapid adhesion to collagen IV. After being divided into P311 high-expressing group (n = 3) and EGFP control group (n = 3), the ESCs in two groups were respectively infected by pAdEasy-EGFP-P311 and pAdEasy-EGFP. Scratching assay was performed on ESCs in both groups after they were treated by mitomycin C for 2 hours. The remaining area within the fixed range was measured at post scratching hour (PSH) 0, 24, 48, and 72, and the wound-area healing rate was calculated. Data were processed with independent samples t test. RESULTS: (1) Expression amount of P311 was different in different parts of wound at different time points after burn. Expression amount of P311 in the newly formed epidermis and hair follicle of wound increased along with prolongation of time. Expression amount of P311 in the epidermis and hair follicle of wound edge peaked at PBH 12 and then decreased to normal levels at PBH 72. (2) Co-localization of BrdU-positive ESCs and P311-positive cells was observed in the new epidermal layer of wound tissue of mice, where ESCs were labeled by BrdU. (3) At PSH 48 and 72, wound-area healing rate was obviously higher in P311 high-expressing group [(69 ± 31)%, (89 ± 26)%] than in EGFP control group [(35 ± 12)%, (46 ± 31)%, with t values respectively -2.336, -2.611, P values all below 0.05]. CONCLUSIONS: P311 may promote the migration of ESCs both in rats with superficial partial-thickness burns and in injured cell model in vitro, and it may play an important role in wound healing.

[Biologic effect of nitric oxide on human epidermal stem cells in vitro].

OBJECTIVE: To observe the effect of nitric oxide (NO) on adhesion, proliferation, and migration of human epidermal stem cells (ESC) in vitro. METHODS: ESC were isolated and cultured by the modified method of rapid attachment to type IV collagen. (1) Morphology of cells was observed under inverted phase-contrast microscope. Expression levels of integrin ß(1) and cytokeratin 19 (CK19) of cells were determined by Western blotting and immunofluorescence staining. (2) After being treated with scratching, ESC adhered to the wall was respectively treated with nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in the concentration of 1, 10, 100, 500 µmol/L. ESC without treatment of SNAP was used as control. The migration rate of ESC was detected at post scratching hour (PSH) 12 and 24. The chemotaxis of ESC (treated with SNAP in above-mentioned concentration) was tested by Transwell assay, and the transferred cell number was counted. (3) ESC was respectively treated with SNAP in the concentration of 10, 100, 500 µmol/L for 1 h. ESC without treatment of SNAP was used as control. The adhesion of ESC was detected with adhesion test, and the inhibition rate of adhesion was calculated. The proliferation of ESC (denoted as absorbance value) was determined by microplate reader at post-treatment hour (PTH) 0, 12, 24, 48. Data were processed with one-way analysis of variance and Dunnett t test. RESULTS: (1) Small clone formed on post culture days (PCD) 5 to 9. On PCD 10 to 14, cell proliferation sped up. CK19 and integrin ß(1) were detected to be expressed in the isolated cells. The cells were identified as ESC. (2) Compared with that of ESC without treatment of SNAP [(35.7 ± 0.3)%, (45.7 ± 5.0)%], migration of ESC treated with SNAP in the concentration from 1 to 100 µmol/L was promoted at PSH 12 and 24. Migration rates of ESC treated with 100 µmol/L SNAP were the highest [respectively (48.8 ± 2.7)%, (82.1 ± 15.8)%, with t value respectively 8.34, 5.10, P values both below 0.01]. The number of ESC transferred to membrane after being treated with 100 µmol/L SNAP was significantly larger than that of ESC without treatment of SNAP (t = 9.24, P = 0.00). (3) Absorbance values of ESC treated with 100, 500 µmol/L SNAP were obviously higher than that of ESC without treatment of SNAP (with t value respectively 4.30, 4.67, P values both equal to 0.00). Proliferation of ESC treated with 100, 500 µmol/L SNAP was obviously stronger than that of cells without treatment of SNAP at PTH 24, 48 (with t values from 2.84 to 8.17, P values all below 0.05). CONCLUSIONS: Exogenous NO in suitable concentration can promote the migration of human ESC. Exogenous NO can inhibit the adhesion and promote the proliferation of human ESC in vitro.