[Preparation of interleukin-1ß-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice].

Objective: To prepare interleukin-1ß-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1ß-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1ß-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1ß-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1ß (IL-1ß) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1ß antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 µg/ml and 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

[Clinical characteristics and surgical outcomes of pediatric epiretinal membranes without specific etiologies].

Objective: To describe clinical characteristics and surgical outcomes of pediatric epiretinal membranes (ERMs) without specific etiologies. Methods: Medical data of a cohort of pediatric patients (≤14 years) who had ERMs without specific etiologies, underwent surgical removal from January 2019 to September 2021, and were followed up for at least 12 months were retrospectively reviewed. Age at presentation, chief complaints, color fundus photographs, optical coherence tomographic images, preoperative and postoperative visual acuities, anatomical changes, and postoperative complications were assessed. Results: There were 14 patients (17 eyes), including 5 females (6 eyes) and 9 males (11 eyes). The mean age at surgery was 6.31±2.91 years, and the follow-up duration was 17.3±9.5 months. Eight patients were found to have low vision in the school physical examination. Fifteen eyes had an appearance of cellophane macular reflex on fundus images. On optical coherence tomographic images, 10 eyes had"taco"folds, and 7 eyes had"ripple"folds. Five eyes had ellipsoid zone disruptions, while 12 eyes had ellipsoid zone integrity. The preoperative and postoperative best-corrected visual acuities in logMAR were 0.532±0.302 and 0.340±0.298. One patient suffered traumatic cataract and secondary retinal detachment postoperatively, and after further vitrectomy, the retina became attached. Conclusion: Pediatric ERMs without specific etiologies were mostly found in school-age children with cellophane macular reflex and"taco"folds. Vitrectomy may result in both potential visual acuity and macular anatomical improvements.

The combination of sucrose dilaurate and sucrose laurate suppresses HMGB1: an enhancer of melanocyte dendricity and melanosome transfer to keratinocytes.

BACKGROUND: Hyperpigmented spots are common issues in all ethnicities, involving multiple intrinsic and extrinsic factors such as UVB exposure, hormone balance, inflammatory status and ageing. OBJECTIVES: To determine (i) melanocyte dendricity in multiple facial spot types, (ii) impact of High Mobility Group Box 1 (HMGB1), and the combination of sucrose dilaurate and sucrose laurate (SDL) on melanogenesis and melanocyte dendricity, and (iii) SDL effect on facial spots in a human use test. METHODS: Facial spot and adjacent non-spot skin biopsies were collected from Chinese women (age 20-70). Histological assessment of melanocyte dendricity was performed for 3 spot types (solar lentigo, melasma and postinflammatory hyperpigmentation) by immunofluorescent staining for c-kit/MITF. Keratinocyte, melanocyte and melanocyte-keratinocyte co-culture models were used to assess HMGB1 release by UVB radiation, the effects of HMGB1 and SDL on melanin production, melanocyte dendricity and melanosome transfer. The effect of an SDL-containing moisturizer on appearance of facial hyperpigmented spots was assessed against a vehicle control in an 8-week human use test. RESULTS: Melanocytes in spot areas are more dendritic than melanocytes in adjacent non-spot skin across three investigated spot types. In cell culture models, a moderate UVB-radiation exposure caused release of HMGB1 from keratinocytes. HMGB1 did not alter melanin production in melanocytes, but enhanced melanocyte dendricity and melanosome transfer. SDL reduced HMGB1 release from keratinocytes, inhibited melanin production, reversibly suppressed melanocyte dendricity and reduced melanosome transfer. In the human use test, SDL-containing moisturizer reduced appearance of spots versus vehicle. CONCLUSION: Increased melanocyte dendricity was observed in multiple types of facial spots. Addition of HMGB1 protein increased melanocyte dendricity and melanosome transfer in cell cultures, implicating potential involvement in spot formation. SDL suppressed melanin production, melanocyte dendricity and melanosome transfer in vitro and reduced appearance of spots in the use test, suggesting SDL is an effective solution to address hyperpigmented spot concerns.

[Mechanism of Mycobacterium tuberculosis on interleukin-6 receptor 3'-untranslated region methylation in CD4+T cells].

Objective: To investigate the role and mechanism of DNA methylation in Mycobacterium tuberculosis (MTB lysate) -induced downregulation of interleukin-6 receptor(IL-6R) expression in CD4+T cells. Methods: A prospective study was conducted. Bisulfite sequencing (BSP) was applied to determine the methylation levels of CpG island in IL-6R promoter region and 3'untranslated region (3'UTR) region in CD4+T cells from peripheral blood mononuclear cells (PBMC) of control group (healthy person, n=10) and TB group (tuberculosis patients, n=10) in Shenzhen Third People's Hospital between 2019 and 2020. Quantitative reverse transcription-PCR (RT-qPCR) and Western blotting were used to detect the expression of IL-6R, DNMT1, DNMT3A and DNMT3B in MTB lysate-stimulated CD4+T cells and Jurkat E6-1 cells. Furthermore, PBMC in control group and Jurkat E6-1 cells activated by anti-CD3/CD28 antibody were stimulated by MTB lysates to detect the methylation levels of CpG island and IL-6R and DNMT expression. Transcriptional activity of differently methylation regions of IL-6R 3'UTR was detected by using luciferase reporter gene system. Results: IL-6R expression in TB group was lower than that in control group, but DNMT1 and DNMT3B expressions were higher than those in control group in CD4+T cells isolated from PBMC. There was no significant difference in the methylation rate of IL-6R promoter CpG island of CD4+T cells between control and TB group. However, the methylation rates of CpG island in 3'UTR region were significantly higher (P<0.001) in TB (69.5%±3.4%), compared with control (54.3%±4.7%). Besides, IL-6R expression was lower than unstimulated, while DNMT1 and DNMT3B expression was higher than unstimulated after MTB lysate-stimulation of activated control PBMC in vitro. The methylation rate of CpG island in IL-6R 3'UTR region of CD4+T cells increased from 58.8%±11.6% to 79.4%±10.9% (P<0.001) after MTB lysate-stimulated PBMC of the control. The same results were observed in the MTB lysate-stimulated CD4+T cells isolated from PBMC in control and Jurkat E6-1 cell line. Furthermore, IL-6R expression after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysate was higher than that stimulated by MTB lysate alone. In addition, the methylation levels of CpG islands in the 3' UTR region of IL-6R were lower than those stimulated by MTB lysates alone after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysates. The transcriptional activity of the fully unmethylated IL-6R 3'UTR CpG island reporter gene was higher than that of the fully methylated IL-6R 3'UTR CpG island. Conclusions: MTB lysates stimulation inhibited IL-6R expression transcriptionalely as well as on the protein level by inducing hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells. The hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells induced by MTB may be related to the increased expression of DNMT1 and DNMT3B.

Identification of DNA methylation-driven genes and construction of a nomogram to predict overall survival in pancreatic cancer.

BACKGROUND: The incidence and mortality of pancreatic cancer (PC) has gradually increased. The aim of this study was to identify survival-related DNA methylation (DNAm)-driven genes and establish a nomogram to predict outcomes in patients with PC. METHODS: The gene expression, DNA methylation database, and PC clinical samples were downloaded from TCGA. DNAm-driven genes were identified by integrating analyses of gene expression and DNA methylation data. Survival-related DNAm-driven genes were screened via univariate, least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression analyses to develop a risk score model for prognosis. Based on analyses of clinical parameters and risk score, a nomogram was built and validated. The independent cohort from GEO database were used for external validation. RESULTS: A total of 16 differentially expressed methylation-driven genes were identified. Based on LASSO Cox regression and multivariate Cox regression analysis, six genes (FERMT1, LIPH, LAMA3, PPP1R14D, NQO1, VSIG2) were chosen to develop the risk score model. In the Kaplan-Meier analysis, age, T stage, N stage, AJCC stage, radiation therapy history, tumor size, surgery type performed, pathological type, chemotherapy history, and risk score were potential prognostic factors in PC (P < 0.1). In the multivariate analysis, stage, chemotherapy, and risk score were significantly correlated to overall survival (P < 0.05). The nomogram was constructed with the three variables (stage, chemotherapy, and risk score) for predicting the 1-year, 2-year, and 3-year survival rates of PC patients. Nomogram performance was assessed by receiver operating characteristic (ROC) curves and calibration curves. 1-year, 2-year and 3-year AUC of nomogram model was 0.899, 0.765 and 0.776, respectively. CONCLUSIONS: In our study, we successfully identified the six DNAm-driven genes (FERMT1, LIPH, LAMA3, PPP1R14D, NQO1, VSIG2) with a relationship to the outcomes of PC patients. The nomogram including stage, chemotherapy, and risk score could be used to predict survival in PC patients.

Nomogram to predict survival of patients with advanced and metastatic pancreatic Cancer.

BACKGROUND: Nomograms are rarely employed to estimate the survival of patients with advanced and metastatic pancreatic cancer (PC). Herein, we developed a comprehensive approach to using a nomogram to predict survival probability in patients with advanced and metastatic PC. METHODS: A total of 323 patients with advanced and metastatic PC were identified from the Chinese People's Liberation Army (PLA) General Hospital. A baseline nomogram was constructed using baseline variables of 323 patients. Additionally, 233 patients, whose tumors showed initial responses to first-line chemotherapy, were enrolled in the chemotherapy response-based model. 128 patients and 108 patients with advanced and metastatic PC from January 2019 to April 2021 were selected for external validating baseline model and chemotherapy response-based model. The 1-year and 2-year survival probability was evaluated using multivariate COX regression models. The discrimination and calibration capacity of the nomograms were assessed using C-statistic and calibration plots. The predictive accuracy and net benefit of the nomograms were evaluated using ROC curve and DCA, respectively. RESULTS: In the baseline model, six variables (gender, KPS, baseline TB, baseline N, baseline WBC and baseline CA19-9) were used in the final model. In the chemotherapy response-based model, nine variables (KPS, gender, ascites, baseline N, baseline CA 19-9, baseline CEA, change in CA 19-9 level at week, change in CEA level at week and initial response to chemotherapy) were included in the final model. The C-statistics of the baseline nomogram and the chemotherapy response-based nomogram were 0.67 (95% CI, 0.62-0.71) and 0.74 (95% CI, 0.69-0.77), respectively. CONCLUSION: These nomograms were constructed to predict the survival probability of patients of advanced and metastatic PC. The baseline model and chemotherapy response-based model performed well in survival prediction.

FoxP3 in Treg cell biology: a molecular and structural perspective.

Regulatory T cells (Tregs ) are specialized in immune suppression and play a dominant role in peripheral immune tolerance. Treg  cell lineage development and function maintenance is determined by the forkhead box protein 3 (FoxP3) transcriptional factor, whose activity is fine-tuned by its post-translational modifications (PTMs) and interaction partners. In this review, we summarize current studies in the crystal structures, the PTMs and interaction partners of FoxP3 protein, and discuss how these insights may provide a roadmap for new approaches to modulate Treg  suppression, and new therapies to enhance immune tolerance in autoimmune diseases.

Autophagy activators stimulate the removal of advanced glycation end products in human keratinocytes.

BACKGROUND: The accumulation of advanced glycation end products (AGEs) can impact cellular homoeostasis and protein structure, thus is implicated in numerous skin conditions including yellow, dull appearance. AGE formation is irreversible; thus, understanding of the recycling process of AGEs in the skin is critical for addressing skin appearance conditions. OBJECTIVE: To determine whether (i) accumulation of AGEs occurs in dull appearance group among young population (age 20-29) (ii) in vitro autophagy stimulation results in reduction of AGEs in keratinocytes. METHODS: Facial cheek biopsies were collected from Chinese women (age 20-50) exhibiting either dull or non-dull appearing skin. Histological assessment of glycation was performed for representative subjects among the 20-29 years sub-group by immunofluorescence staining of AGEs. LC-MS methods and keratinocyte cell culture were used to assess impact of autophagy modulators and skin care materials on carboxymethyl lysine (CML) amount, a representative AGE. RESULTS: Notable amounts of AGEs were observed in the epidermal samples among young females. Interestingly, the amount of AGEs was significantly higher among the dull skin appearance group. Treatment of keratinocytes with glyceraldehyde (GLA) enhanced CML in the cells, and postglycation treatment with autophagy activators reduced CML. Two skin care materials, Nymphaea alba flower extract (a.k.a. white water lily extract) and sucrose dilaurate, were identified based from in vitro autophagy activation and found to reduce CML in keratinocytes. CONCLUSION: We found AGEs accumulate in the facial epidermis even among young people, correlating to a yellow and dull appearance. We also demonstrated in vitro activation of autophagy can reduce AGEs in keratinocytes, and autophagy activating skin care materials, N. alba flower extract and sucrose dilaurate, also reduce AGEs in the keratinocyte in vitro model. These data suggest epidermal AGEs contribute to the dull skin appearance, and autophagy activators may provide an effective solution to improve dull appearance by removing and recycling the accumulated glycation in the skin.

[Analysis of the effect of percutaneous vertebroplasty combined with (125)I seed implantation in the treatment of spinal metastatic epidural spinal cord compression].

Objective: To investigate the clinical efficacy of percutaneous vertebroplasty (PVP) combined with iodine-125 ((125)I) seed brachytherapy in the treatment of spinal metastatic epidural spinal cord compression (MESCC) and toassess the changes inthe grade of epidural spinal cord compression (ESCC) by magnetic resonance imaging (MRI). Methods: A total of 37 MESCC patients treated with PVP combined with (125)I seed brachytherapy in the interventional and vascular surgery department of Zhongda Hospital affiliated to Southeast University from January 2014 to June 2019 were retrospectively analyzed, including 23 cases of bilateral lower limbs paralysis. Total diseased vertebrae are 39 segments. Visual analogue scale (VAS) and paralysis of lower extremities were evaluated regularly before and after treatment, and VAS values at different follow-up time points were compared. At the same time, MRI was used to evaluate the changes of ESCC grade in the spinal canal and calculate the local lesion efficiency after operation. The postoperative local lesion efficiency at different follow-up times was compared. Results: PVP combined with (125)I seed implantation in all diseased vertebral bodies was successful. The average injection volume of polymethylmethacrylate (PMMA) was (3.2±1.3) ml/segment, the average number of (125)I seed implanted was (25.0±8.6) seeds/segment and the average radiation dose was (15.0±5.1) mCi/segment. The VAS before operation was 8.5, and postoperative VAS were respectively 3.6±1.3, 3.8±1.5, 3.4±1.4, 5.5±1.0, 5.9±1.4 at 5 days, 1 month, 3 months, 6 months, and 1 year after operation. The differences between all follow-up time points and preoperative VAS values were statistically significant (all P<0.001). Compared with 5 days, 1 month and 3 months after operation, VAS increased significantly at 6 months and 1 year after operation, and the difference was statistically significant (all P<0.001); there was no significant difference between the VAS value at 6 months after operation and 1 year after operation (P=0.405). At a follow-up of 3 months, 22 of 23 patients with paralysis of bilateral lower limbs regained the functions of autonomous walking and voiding; the effective rates of MESCC local lesions evaluated by MRI at 1 month, 3 months, 6 months, and>1 year were 89.7%, 91.9%, 90.6%, and 94.7%, respectively, and there was no statistically significant differences among those follow-up time points (all P>0.05). Conclusions: PVP combined with (125)I seed brachytherapy in the treatment of MESCC has significant improvement in immediate pain relief and spinal cord function. After combined treatment, MRI showed that the tumors around the spinal cord regressed dramatically, which could considerably reduce the MESCC grade and remain stable for a long time.

[Advances in the pathogenesis of pre-hepatic failure].

Domestic scholars recognize that patients have a "pre-hepatic failure" before they progress to sub-acute and acute-on-chronic liver failure stage, which is also the golden window for effective clinical intervention, so early identification and intervention during this period can reduce the incidence and mortality of liver failure. The Guidelines for the Diagnosis and Treatment of Liver Failure (2018 Edition) issued by the Chinese Medical Association clearly defines the "pre-stage" of liver failure. And from the perspective of pathophysiological mechanism, the pre-hepatic failure corresponds to the stage of acute liver injury/acute decompensation, inflammation factor/ immunologic derangement. This article briefly introduces the research progress on substantive connotation and pathogenesis of pre-hepatic failure, and puts forward some problems to be explored in the future.