[Functional analysis of virus-specific CD4(+)T cells and CD8(+)T cells in patients with liver injury caused by Epstein-Barr virus infection].

Objective: To analyze the functional differences between virus-specific CD4(+)T cells and CD8(+)T cells in patients infected with Epstein-Barr virus (EBV) who develop liver injury and those who do not. Methods: 45 cases of EBV infections were enrolled, including 28 cases developing liver injuries and 17 that did not. Mononuclear cells from peripheral blood were isolated. CD4(+)T cells and CD8(+)T cells were purified and cultured using recombinant EBV core antigen 2 (EBNA2) for 96 h with stimulation. The CCK-8 method was used to detect cell proliferation. Flow cytometry was used to detect the proportion of CD4(+)T cells and CD8(+)T cells. An enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of CD4(+)T cells secreting cytokines and CD8(+)T cells secreting molecular toxicity. Real-time quantitative PCR was used to detect the mRNA levels of transcription factors and molecular toxicity in CD4(+)T cell subsets. Flow cytometry was used to detect the immune checkpoints at molecular levels in CD8(+)T cells. The inter-group comparison was performed using a t-test or Mann-Whitney test. Results: There was no statistically significant difference (P > 0.05) in the proliferation proportion of peripheral blood mononuclear cells, CD4(+)T cells, and CD8(+)T cells after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group (P > 0.05). There was no statistically significant difference in the proportion of CD4(+)T cells secreting related cytokines and the mRNA levels of transcription factors after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group (P > 0.05).The levels of perforin secreted by CD8(+)T cells and granzyme B after stimulation with recombinant EBNA2 were higher in the EBV infection-induced liver injury group than those in the non-liver injury group [(75.51±23.33) pg/ml vs. (58.99±18.39) pg/ml, P = 0.017] [(117.8±44.55) pg/ml vs. (90.22±34.21) pg/ml, P = 0.034]. The mRNA levels of Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand in CD8(+)T cells in the liver injury group caused by EBV infection were approximately 1.5 and 1.2 times higher than those in the non-liver injury group, respectively, and the difference was statistically significant (P < 0.001), but there was no statistically significant difference in the proportional expression of programmed cell death-1 and cytotoxic T lymphocyte-associated antigen-4 in CD8(+)T cells between the EBV-infected non-liver injury group and infected liver injury group (P > 0.05) Conclusion: Patients with liver injury caused by EBV infection have strong virus-specific CD8(+) T cell toxic effects, which may mediate EBV-induced liver injury.

[Y-shaped osteotomy in the apical vertebra for treating congenital complex rigid scoliosis:at least 2-year follow-up].

Objective: To investigate the clinical outcome of the coronal Y-shaped osteotomy in the apical vertebra for treating congenital complex rigid scoliosis. Methods: A retrospective analysis was conducted on 66 cases who underwent Y-shaped osteotomy treatment for congenital complex rigid scoliosis in the uppermost vertebra at the Department of Orthopedics,the Second Hospital of Shanxi Medical University from June 2007 to August 2020. There were 19 males and 47 females,with an age of (13.1±5.3) years(range:2 to 30 years).Classification of congenital scoliosis:25 cases (37.9%) were incomplete,13 cases (19.7%) were dysarthritic,and 28 cases (42.4%) were mixed. There were 25 cases (37.9%) with thoracic or rib malformations. 45 cases (68.2%) were complicated with spinal cord malformation.The main radiological indicators included Cobb angle of the curvature,Cobb angle of the local bend,apical vertebral translation (AVT),trunk shift (TS),thoracic trunk shift (TTS),radiographic shoulder height (RSH),coronal balance and sagittal vertebral axis. The preoperative,postoperative immediate,and last follow-up radiological indicators were collected and the operation time,blood loss,hospitalization time,and operation-related complications were recorded. Data were compared by repeated measure ANOVA and paired-t test. Results: All patients underwent surgery successfully. The duration of the first surgery was (221.4±52.8) minutes,and the blood loss during the first surgery was (273.2±41.8) ml. The length of the first hospital stay was (8.8±1.7) days.Unilateral fixation was performed in 19 cases (28.8%),while bilateral fixation was performed in 47 cases (71.2%). The fused segments were 7.5±2.9,and the vertebral pedicle screw density was (68.5±20.6)%. The follow-up time for the 66 patients was (36.7±17.0) months(range:24 to 102 months).The main curve Cobb Angle was improved from (58.5±18.9)°before surgery to (21.1±11.8)°after surgery,and was (23.6±15.3) ° at the last follow-up(F=273.957,P<0.01),with a correction rate of 66.2%. Segmental curve Cobb Angle was improved from (47.9±18.0)° to (16.0±11.3)° after surgery,and was (16.8±12.8) °at the last follow-up (F=270.483,P<0.01)with a correction rate of 69.2%. The AVT,TS,TTS and RSH values improved significantly at the final follow-up (all P<0.05),while coronal balance and sagittal vertical axis were maintained without significant differences between pre-operation and post-operation(both P>0.05). A total of 5 patients underwent staged operation,all of which were residual scoliosis aggravated after the first stage of orthosis operation and had good prognosis after the second stage of operation. Conclusions: Y-shaped osteotomy for the treatment of congenital rigid scoliosis results in good clinical and radiological outcomes without serious complications. This procedure can be considered as an option for the treatment of congenital complex rigid scoliosis.

SrB4O7:Sm2+ fluorescence improves the accuracy of temperature measurements in externally heated diamond anvil cells.

The sample temperature in an externally heated diamond anvil cell (EHDAC) is generally measured by a thermocouple fixed to the pavilions of diamond anvils, ignoring the temperature difference between the thermocouple and the sample. However, the measured temperature depends strongly on the placement of the thermocouple, thus seriously reducing the accuracy of the temperature measurement and hindering the use of EHDAC in experiments requiring precise temperature measurements, such as high-pressure melting and phase-diagram investigations. In this study, the full width at half maximum (FWHM) of the 0-0 fluorescence line of strontium borate doped with bivalent samarium ions (SrBO4:Sm2+, SBO) is found to be highly sensitive to temperature and responds extremely rapidly to small temperature fluctuations, which makes it an excellent temperature indicator. We propose herein a precise method to measure temperature that involves measuring the FWHM of the 0-0 fluorescence line of SBO. This method is used to correct the temperature discrepancy between the thermocouple and the sample in an EHDAC. These corrections significantly improve the accuracy of temperature measurements in EHDACs. The accuracy of this method is verified by measuring the melting point of tin at ambient pressure. We also use this method to produce a tentative elementary phase diagram of tin up to 109 GPa and 495 K. This method facilitates high-pressure, high-temperature experiments demanding accurate temperature measurements in various disciplines. The study also discusses, in general, the experimental approach to measuring temperature.

[Interaction of notch signaling pathway with toll-like receptor 4 on the function of CD14+ monocytes in chronic hepatitis C patients].

Objective: To observe the expressional changes in Notch signaling pathway and toll-like receptor 4 (TLR4) and their interactions on the functions of CD14(+) monocytes in chronic hepatitis C patients. Methods: A total of 24 treatment-naïve chronic hepatitis C cases and 10 healthy individuals, who visited Shaanxi Provincial People's Hospital from August to October 2017, were enrolled. Selected CD14(+) monocytes were stimulated by the Notch signaling pathway inhibitor DAPT or transfected with TLR4 siRNA, and the levels of Notch1, Notch2, Hes1 and Hes5 mRNA were detected by real-time quantitative PCR. TLR4 protein levels and phosphorylation of NF-κB was detected by Western blot. ELISA was used to detect the level of cytokines secreted from CD14(+) monocytes. A t-test or paired t-test was used for comparison between groups. Results: The relative expression of Notch1 mRNA (3.97 ± 2.03 vs. 0.91 ± 0.76, P < 0.01) and downstream of Notch signaling pathway (5.96 ± 2.31 vs. 0.99 ± 0.45, P < 0.01), Hes1 mRNA and Hes5 mRNA (4.31 ± 1.05 vs. 0.84 ± 0.20, P < 0.01) in CD14+ monocytes of chronic hepatitis C patients was significantly higher than that of healthy individuals. The relative expression of TLR4 mRNA (5.14 ± 1.09 vs. 1.27 ± 0.39) and protein level in CD14(+) monocytes of chronic hepatitis C patients were significantly higher than those of healthy individuals (P < 0.01). An inhibition of Notch signaling pathway with DAPT had reduced the relative expression level of TLR4 mRNA (2.58 ± 1.36 vs. 4.34 ± 1.88, P < 0.05), protein expression and phosphorylation of NF-B in CD14(+) monocytes of chronic hepatitis C patients. Furthermore, the secretion level of MCP-1 [(94.32 ± 23.59) pg/ml vs. (64.07 ± 9.39) pg/ml, P < 0.01] and IL-8 [(12.54 ± 4.89) pg/ml vs. (7.92 ± 3.01) pg/ml, P < 0.05] was significantly reduced. TLR4 siRNA transfection reduced the expressions of Notch1 mRNA (2.09 ± 1.72 vs. 3.73 ± 1.75, P < 0.05), Hes1 (2.87 ± 0.84 vs. 5.54 ± 0.97, P < 0.01), and Hes5 (2.89 ± 0.93 vs. 4.51 ± 1.54, P < 0.01) in CD14(+) monocytes of chronic hepatitis C patients. Conclusion: Interaction of Notch signaling pathway with TLR4 can promote the function of CD14(+) monocytes in chronic hepatitis C patients.

Inhibition of Polo-Like Kinase 1 by BI2536 Reverses the Multidrug Resistance of Human Hepatoma Cells In Vitro and In Vivo.

BACKGROUND: Multi Drug Resistance (MDR) is the phenomenon that cancers develop resistance to majority of chemotherapy drugs and is a serious obstacle to the treatment for Hepatocellular Carcinoma (HCC). Polo-Like Kinase 1 (PLK1) is a serine/threonine kinase associated with tumor growth and clinical prognosis in HCC and BI2536 is its potent inhibitor with IC50 of 0.83nM. AIMS: To test whether the down-regulation of PLK1 by its inhibitor BI2536 would have beneficial effects on the reversal of MDR in HCC cells. METHODS: The CCK-8 assay was used to determine the viability of HepG2/ADM and SMMC7721/ADM cells and their parental cells treated with BI2536. Then animal model studies were performed. Cell invasion assay and wound healing assay were used to determine the invasion ability and motility. Flow cytometric was used to test the apoptosis induced by BI2536. Western blot and quantitative real-time PCR were performed to test the change of expression of MDR and apoptosis-related gene. RESULTS: BI2536 down-regulated the expression of PLK1 protein and mRNA specifically. BI2536 can significantly reduce IC50 for ADM and other drugs in ADM-resistant HCC cells. Meanwhile, it inhibited cell viability, proliferation, and invasion, and induced cell cycle arrest and apoptosis in HCC cells with MDR. CONCLUSION: Our results suggest that PLK1 inhibitor BI2536 can re-sensitize HCC cancer cell with MDR through induction of apoptosis. Thus, PLK1 inhibitor BI2536 may act as an effective chemotherapeutic drug in the clinical treatment of HCC patients with MDR.

A study of FHL1, BAG3, MATR3, PTRF and TCAP in Australian muscular dystrophy patients.

FHL1, BAG3, MATR3 and PTRF are recently identified myopathy genes associated with phenotypes that overlap muscular dystrophy. TCAP is a rare reported cause of muscular dystrophy not routinely screened in most centres. We hypothesised that these genes may account for patients with undiagnosed forms of muscular dystrophy in Australia. We screened a large cohort of muscular dystrophy patients for abnormalities in FHL1 (n=102) and TCAP (n=100) and selected patients whose clinical features overlapped the phenotypes previously described for BAG3 (n=9), MATR3 (n=15) and PTRF (n=7). We found one FHL1 mutation (c.311G>A, p.C104Y) in a boy with rapidly progressive muscle weakness and reducing body myopathy who was initially diagnosed with muscular dystrophy. We identified no pathogenic mutations in BAG3, MATR3, PTRF or TCAP. In conclusion, we have excluded these five genes as common causes of muscular dystrophy in Australia. Patients with reducing body myopathy may be initially diagnosed as muscular dystrophy.

Deficiency of α-actinin-3 is associated with increased susceptibility to contraction-induced damage and skeletal muscle remodeling.

Sarcomeric α-actinins (α-actinin-2 and -3) are a major component of the Z-disk in skeletal muscle, where they crosslink actin and other structural proteins to maintain an ordered myofibrillar array. Homozygosity for the common null polymorphism (R577X) in ACTN3 results in the absence of fast fiber-specific α-actinin-3 in ∼20% of the general population. α-Actinin-3 deficiency is associated with decreased force generation and is detrimental to sprint and power performance in elite athletes, suggesting that α-actinin-3 is necessary for optimal forceful repetitive muscle contractions. Since Z-disks are the structures most vulnerable to eccentric damage, we sought to examine the effects of α-actinin-3 deficiency on sarcomeric integrity. Actn3 knockout mouse muscle showed significantly increased force deficits following eccentric contraction at 30% stretch, suggesting that α-actinin-3 deficiency results in an increased susceptibility to muscle damage at the extremes of muscle performance. Microarray analyses demonstrated an increase in muscle remodeling genes, which we confirmed at the protein level. The loss of α-actinin-3 and up-regulation of α-actinin-2 resulted in no significant changes to the total pool of sarcomeric α-actinins, suggesting that alterations in fast fiber Z-disk properties may be related to differences in functional protein interactions between α-actinin-2 and α-actinin-3. In support of this, we demonstrated that the Z-disk proteins, ZASP, titin and vinculin preferentially bind to α-actinin-2. Thus, the loss of α-actinin-3 changes the overall protein composition of fast fiber Z-disks and alters their elastic properties, providing a mechanistic explanation for the loss of force generation and increased susceptibility to eccentric damage in α-actinin-3-deficient individuals.

Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.

Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.

Porous polymer films and honeycomb structures based on amphiphilic dendronized block copolymers.

Fabrication of honeycomb patterned films from our synthesized amphiphilic dendronized block copolymer by "on-solid surface spreading" method and "on-water spreading" method was reported for the first time in this paper. The comparison of the two methods indicated honeycomb-patterned films with smaller size, and larger surface density of micropores can be fabricated by spreading on water but with lower regular arrangement. Furthermore, several influencing factors on the formation of the honeycomb structure and the different morphologies, such as the concentration of the copolymer solution and the relative humidity in the atmosphere and the substrates, were investigated. The results showed that comparably high relative humidity from 80% to 95% was needed, and the mica plate as a spreading substrate was suitable to form orderly porous films for such a copolymer. The best ordered pattern could be formed from the copolymer with concentration of 1.00 mg/mL at the relative humidity of 85% using a mica plate. Besides, strong periodicity, regularity, and a large, defect-free area were notable, which made this structure extremely interesting for applications for templated molecular objects formed via intramolecular metal or metal oxide synthesis.

On the antioxidant activity of melatonin.

Melatonin has been widely reported to be an effective antioxidant. Studies of its ability to inhibit the autoxidation of lipids in homogeneous solution and in model heterogeneous systems show that melatonin is not a peroxyl radical trapping antioxidant. In contrast, melatonin can inhibit metal ion-catalyzed oxidation processes.

[Structure-activity relationship studies of chalcones as SRS-A receptor antagonists].

A simple, reliable and highly sensitive bioassay with sensitized longitudinal strips of guinea pig ileum was used for screening the receptor antagonists of slow reacting substance of anaphylaxis (SRS-A). The SRS-A receptor antagonistic activities of 17 chalcones were studied. Most compounds in these chalcones were found to have SRS-A receptor antagonistic action at the concentration of 10(-4) mol.L-1. Among them, compounds 5, 13 and 17 were highly effective with IC50s of 7.5 x 10(-6), 7.5 x 10(-6) and 6.8 x 10(-5) mol.L-1, respectively. Under the same conditions, the IC50 of FPL 55712, a known leukotriene D4 receptor antagonist, was shown to be 3 x 10(-4) mol.L-1. It would appear that compounds 5, 13 and 17 were 40, 40 and 4.4 times more potent, respectively, than FPL 55712. From analysis of structure-activity relationship of chalcones, these results suggest that the following factors may be important for an active antagonist of SRS-A receptors: (a) There is a system of pi, pi conjugation in the molecule; (b) The ester group in the B ring of chalcones is more favorable than the carboxyl group; (c) Antagonism for meta- or para-substituted derivatives of carboxyl or ester group in the B ring are more potent than ortho-substituted compounds; (d) The length of carbon chain of alkyl group in the A ring of chalcones is more effective for 1, 4 or 6 carbon atoms than for 10 or 14 carbon atoms.

[Total synthesis of securinine].

Securinine, an alkaloid having strychnine-like action was synthesized using 1,4-cyclohexanedione as the starting material through the following sequence of reactions: condensation with piperidine, reduction, cyclization catalyzed by mercuric acetate and intramolecular condensation, totally in eleven steps. The IR, 1HNMR and melting point of the synthetic product is identical with those of natural securinine.