Effect of gold-conjugated resveratrol nanoparticles on glioma cells and its underlying mechanism.

BACKGROUND:  Glioblastoma is the most aggressive brain tumor with poor prognosis. Although Resveratrol (Rsv) is known to have therapeutic effects on glioma, the effects of gold-conjugated resveratrol nanoparticles (Rsv-AuNPs) on glioma cells are rarely reported. OBJECTIVE: We aimed to investigate the effects of Rsv-AuNPs on glioma cells and its underlying mechanism. METHOD: Human glioma cell line U87 was treated with different concentrations of Rsv-AuNPs. CCK-8, transwell, and wound healing assay were performed to measure the effects of Rsv-AuNPs on cell proliferation, invasion, and migration ability, respectively. Flow cytometry assay was used to detect the effects of Rsv-AuNPs on apoptosis. Changes of protein expressions related to proliferation, invasion, migration, and apoptosis were measured by Western blot assay. In addition, the inhibitory role of Rsv-AuNPs in the PI3K/AKT/mTOR signaling pathway was verified by using PI3K inhibitor LY294002. RESULTS: Rsv-AuNPs treatment significantly suppressed proliferation, migration, and invasion of U87 cells (all P < 0.05) and increased the apoptosis rate (P < 0.05). The changes of proteins related to proliferation, migration, invasion and apoptosis were consistent (all P < 0.05). Moreover, Rsv-AuNPs treatment significantly inhibited the phosphorylation of PI3K, AKT and mTOR proteins in U87 cells (P < 0.05). CONCLUSION: The present study found that Rsv-AuNPs inhibited the proliferation, migration, and invasion of U87 cells and induced apoptosis by inhibiting the activation of PI3K/AKT/mTOR signaling pathway. In the future, Rsv-AuNPs might be applied to the clinical treatment of glioma through more in-depth animal and clinical research.

Single-Position Complete Retroperitoneoscopic Radical Nephroureterectomy with Bladder Cuff Excision for Upper Urinary Tract Urothelial Carcinoma.

Purpose: We proposed a new technique, single-position complete retroperitoneoscopic radical nephroureterectomy (SCRNU), which proved to be efficient for the treatment of upper urinary tract urothelial carcinoma (UTUC). Materials and Methods: In this study, we retrospectively evaluated 86 patients diagnosed with UTUC at our hospital from June 2013 to June 2021. The patients who underwent traditional retroperitoneoscopic nephroureterectomy (TRNU) (n = 28) and SCRNU (n = 58) were consecutively enrolled. Demographic characteristics, perioperative parameters, and follow-up data were collected and compared between the two groups. Results: Both procedures were performed effectively in 86 patients without converting to open surgery. The mean follow-up time was 45.4 months for the SCRNU group and 39 months for the TRNU group. All follow-up patients survived without incidence of bladder incision tumor. Further, the follow-up results showed that there was no significant difference in the recurrence rate of bladder tumor between the two methods. SCRNU group was superior to TRNU group because of shorter operating time, fewer perioperative complications, less postoperative pain, lower recurrence rate, and cheaper medical expenditure. Conclusions: The SCRNU technique is less invasive, have fewer complications, and has a better cosmetic outcome.

Characterization and acceleration of genome shuffling and ploidy reduction in synthetic allopolyploids by genome sequencing and editing.

Polyploidy and the subsequent ploidy reduction and genome shuffling are the major driving forces of genome evolution. Here, we revealed short-term allopolyploid genome evolution by sequencing a synthetic intergeneric hybrid (Raphanobrassica, RRCC). In this allotetraploid, the genome deletion was quick, while rearrangement was slow. The core and high-frequency genes tended to be retained while the specific and low-frequency genes tended to be deleted in the hybrid. The large-fragment deletions were enriched in the heterochromatin region and probably derived from chromosome breaks. The intergeneric translocations were primarily of short fragments dependent on homoeology, indicating a gene conversion origin. To accelerate genome shuffling, we developed an efficient genome editing platform for Raphanobrassica. By editing Fanconi Anemia Complementation Group M (FANCM) genes, homoeologous recombination, chromosome deletion and secondary meiosis with additional ploidy reduction were accelerated. FANCM was shown to be a checkpoint of meiosis and controller of ploidy stability. By simultaneously editing FLIP genes, gene conversion was precisely introduced, and mosaic genes were produced around the target site. This intergeneric hybrid and genome editing platform not only provides models that facilitate experimental evolution research by speeding up genome shuffling and conversion but also accelerates plant breeding by enhancing intergeneric genetic exchange and creating new genes.

Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference.

Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%-95.26%) and specificity (83.72%-98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.

Elevated Interleukin-6 Levels within 72 Hours Post Admission Are Associated with Disease Progression in Nonseptic Critically Ill Children.

It has been established that the innate and adaptive immune suppression was heterogeneous in septic and nonseptic critically ill patients, while the value of immune function in pediatric patients with nonseptic critical illness is limited. We conducted a single-center retrospective study to explore this issue. A total of 65 children with nonseptic illnesses were studied for lymphocyte subpopulations, immunoglobulin concentrations, complement concentrations, and cytokines in peripheral blood in the next 72 hours after admission to our Pediatric Intensive Care Unit (PICU). When compared to clinically recovered patients, patients with disease progression had a numerically lower but not significantly different median pediatric critical illness score and longer PICU median stays. The analysis of serum immunoglobulin (IgG, IgM, and IgA), serum complement (C3, C4) concentrations, and lymphocyte subpopulations showed no significantly difference between patients with and without relieved clinical symptoms by day 4. For the cytokine analysis, the level of IL-6 was significantly higher in patients with disease progression than that in patients who clinically recovered (p = 0.046). In the univariate Cox regression analysis, plasma IL-6 levels were associated with outcome. Multivariate analysis evidenced that the level of plasma IL-6 was one of the factors determining the length of hospital stays. In conclusions, our results demonstrate that increased IL-6 levels in the initial 72 hours post admission are associated with prolonged stays and disease progression in nonseptic critically ill children in the PICU.

T-cell acute lymphoid leukemia resembling Burkitt leukemia cell morphology: A case report.

Biphenotypic acute leukemia (BAL) is an uncommon type of cancer, which accounts for <5% of all adult ALs. Based upon a previously described scoring system, the European Group for the Immunological Classification of Leukemias (EGIL) proposed a set of diagnostic criteria for BAL. This scoring system is based upon the number and degree of specificity of several markers for myeloid or T/B-lymphoid blasts. The present study describes a case of T-cell acute lymphoblastic leukemia (T-ALL) with Burkitt-like cytology, which according to the French-American-British classification, corresponded to a diagnosis of Burkitt type L3 ALL. Flow cytometry analysis demonstrated that the blasts were positive for T-lymphoid markers, cytoplasmic cluster of differentiation (CD)3, CD7 and CD56, and myeloid markers, CD13, CD33 and CD15. At first, a diagnosis of BAL was suggested by the EGIL score, however, according to the 2008 World Health Organization criteria, a case of T-ALL with aberrant myeloid markers was established. The study also reviewed the literature and discussed the limitations of the EGIL scoring system in clinical decision making, to aid in the selection of an appropriate therapeutic regimen.

Analysis of IgG subclass antibodies and expression of T-Cell receptor signaling molecules in anti-CD4 monoclonal antibody treated mice with autoimmune cardiomyopathy.

T-cell immune abnormality in patients of dilated cardiomyopathy has been intensively studied over the past 10 years. In this study, we aim to focus on the molecular mechanism of T-cells in autoimmune cardiomyopathy mouse model by detecting the expression of three T-cell signaling molecules. Balb/C mice (n = 12) were immunized with the peptides derived from human ADP/ATP carrier on the 1st, 14th, 28th, 49th and 79th days, and half of them were also injected with anti-L3T4 McAb on the - 1st, 0 and 1st days. The sham-immunized mice were taken as the controls (n = 6). The main result shows that the antibody response of IgG subclasses such as IgG1, IgG2b and IgG3 were definitely blocked except IgG2a in CD4+ cell-depleted Balb/C mice. In addition, the average mRNA expression of p56lck, p59fyn and zap-70 were all found to be dramatically higher in the mice immunized with only ADP/ATP carrier peptides than in the control-group. At meantime, reduced levels of the protein kinases p56lck, p59fyn and zap-70 were clearly observed in anti-CD4 McAb immunized group compared with DCM group. We propose that the proliferation of T-cells was significantly inhibited in anti-CD4 treated mice and CD4+ T-cells may play a critical role in ADP/ATP carrier caused mouse DCM.

Increased population of CD4(+)CD25(high), regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients.

PURPOSE: Regulatory T cells (T-reg) that control harmful autoimmune T cells in the periphery may also suppress the immune response against cancer. In this study we investigated the possible involvement of CD4(+)CD25(high) T-reg in the immune impairment of patients with acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: The frequencies and phenotypes of CD4(+)CD25(high) T cells in the peripheral blood of AML patients were determined by flow cytometry. To assess the functional activity of CD4(+)CD25(high) T cells, CD4(+)CD25(high), and CD4(+)CD25(-) T cells were sorted from peripheral blood mononuclear cells with FACS Vantage. The immunoregulatory properties of CD4(+)CD25(high) and CD4(+)CD25(-) T cells were characterized by proliferation assays and cytokine production assays. In addition, the frequency of apoptotic and proliferating cells in CD4(+)CD25(high) T cells were respectively evaluated by 7AAD and ki67 binding cells using flow cytometry. RESULTS: Compared with healthy controls, AML patients had a higher proportion of CD4(+)CD25(high) T cells in peripheral blood. These cells were CD45-RA(-), CD69(-), CD45-RO(+), CD95(+), and intercellular CTLA-4(+), and secreted low levels of TNF-alpha and IL-10, but no IL-2, IL-4, IL-5, and IFN-gamma. They inhibited the proliferation and cytokine production (IL-2, IFN-gamma) of CD4(+)CD25(-) T cells, but improved IL-10 production under the co-culture of both subsets with stimulation, thus behaving as T-reg. Notably, CD4(+)CD25(high) T cells in AML patients presented significantly higher apoptosis and proliferation than that of healthy individuals. CONCLUSIONS: The frequency of CD4(+)CD25(high) T-reg in peripheral blood in AML patients is significantly higher when compared with healthy individuals, likely due to the increasing proliferation of CD4(+)CD25(high) T cells.

[Correlation of immunophenotype to cytogenetics and clinical features of adult acute myeloid leukemia].

BACKGROUND & OBJECTIVE: New WHO classification has been rapidly used in diagnosis of leukemia. Based on coexpression and correlation of lineage-associated antigens, multiparameter high-resolution flow cytometry has been developed to precisely identify lineage characteristics of leukemia. Some immunophenotypes correlate with cytogenetic abnormality and prognosis. This study was to analyze immunophenotype of naive acute myeloid leukemia (AML), and explore its correlations to cytogenetics, clinical features, and FAB subtype of AML. METHODS: Multiparameter high-resolution flow cytometry with a panel of 25 different monoclonal antibodies was used to analyze the surface and cytoplasmic antigens expressions of 96 adults with AML; G-binding technique was used to analyze karyotype of 73 of the 96 patients. RESULTS: In these AML patients, some antigens were correlated with FAB subtypes:expression of CD2 was enhanced in AML-M3; HLA-DR, CD34, and CD56 were absent in AML-M3; expression of CD19 was increased in AML-M2; expressions of CD14 and CD56 were enhanced in AML-M5; MPO was absent in AML-M0. Karyotype abnormality was detected in 40(54.8%) patients. CD22, CD56, and TdT expressions were correlated with karyotype abnormality. t(8; 21) was only detected in 10 AML-M2 patients with high expressions of CD15, CD19, CD34, and CD56; no lymphoid lineage antigens were detected in 7 AML-M3 patients with t (15; 17). Expressions of CD4 and TdT were positively correlated with patient's age; expressions of CD7 and CD14 were positively correlated with high white blood cell count; expressions of CD4, CD14, and CD56 were positively correlated with high platelet count. CONCLUSIONS: The abnormal antigen expression of AML is tightly linked with karyotype abnormality. Detection of immunophenotype may help to diagnose and classify AML.

[Immunophenotypes in 115 patients with acute myeloid leukemia by multi-color flow cytometry].

Immunophenotyping has become common in the diagnosis and classification of leukemia. To evaluate the immunophenotype of acute myeloid leukemia (AML), multiparameter flow cytometry and CD45/SSC gating were used to analyze the surface and cytoplasmic antigen expressions in 115 cases of AML. The results were compared with the French-American-British (FAB) Cooperative Group classification to help define the best use and role of multiparameter flow cytometry in the diagnosis and proper classification of AML. The results showed that CD38, CD38 and CD13 were the most commonly expressed antigen (94.8%, 91.3% and 89.6%, respectively). CD7 was the most commonly expressed lymphoid antigen (20.2%), followed by CD19 (16.5%) and CD2 (15%). Some immunophenotypes correlated with FAB type, including increased frequency of CD2 in M(3); lack of HLA-DR, CD34 and CD56 expression in M(3); increased frequency of CD19 in M(2), CD14 and CD56 in M(5) and lack of MPO in M(0). In conclusion, multiparameter flow cytometry is a reliable technique in the diagnosis of AML, and some immunophenotypes correlate with FAB type.

[Protein kinase C inhibitor Gö6976 sensitizes arsenic trioxide-induced cell apoptosis in chronic myeloid leukemic cells].

To investigate the As(2)O(3)-chemosensitization of Gö6976 in K562 cells by its abrogation of As(2)O(3)-induced G(2)/M cell cycle arrest, K562 cells were treated with As(2)O(3) (5 micromol/L) and Gö6976 with various concentrations, the distributions of cell cycles were detected by flow cytometry, the cell viability was observed by trypan blue exclusion test and cell proliferation was tested by MTT assay. The results indicated that having treated by As(2)O(3) for 24 h and 48 h, the proportion of K562 cells in G(2)/M phase were (38.02 +/- 7.70)% and (32.58 +/- 7.43)% respectively, and no obvious cell apoptosis appeared. 50 nmol/L Gö6976 combined with As(2)O(3) decrease the proportion of cells in G(2)/M phase to (23.24 +/- 2.93)% and (16.18 +/- 1.60)% respectively and increase the proportion of cells in subG(1) phase to (11.82 +/- 2.31)% and (27.80 +/- 2.89)% respectively. Gö6976 abrogated G(2)/M cell cycle arrest induced by As(2)O(3) and increased cell apoptosis in a concentration- and time-dependent manner. Additionally, comparing to the control group, Gö6976 combined with As(2)O(3) decreased the cell viability and depressed the cell proliferation, but Gö6976 alone showed no same effect on them. In conclusion, the effects of AS(2)O(3)-chemosensitization of Gö6976 on K562 cells is associated with its abrogation of As(2)O(3)-induced G(2)/M cell cycle arrest.

[Effects of PLK1 gene silence on apoptosis of K562 cells].

OBJECTIVE: To investigate the effects of PLK1 gene silence by short hairpin RNA (shRNA) on PLK1 expression and apoptosis in K562 cells, and explore the role of PLK1 in the pathogenesis of leukemia. METHODS: The shRNA fragment targeting at 1416-1436 bp of PLK1 mRNA was synthesized and cloned into pEGFP-H1 vector, named as pEGFP-H1/PLK1. The empty control, pEGFP-H1 and pEGFP-H1/PLK1 were transfected into K562 cells respectively via electroporation. 24 h or 48 h after transfection, gene and protein expression of PLK1 in the cells were assayed by RT-PCR and Western blot analysis respectively, cells viability by MTT assay, caspase-3 activity by colorimetry, cell cycle and apoptosis by FACS. RESULTS: 24 and 48 h after transfection, PLK1 expression in K562 cells was 1.25 +/- 0.07 for control group, 0.52 +/- 0.04 and 0.25 +/- 0.02 for pEGFP-H1/PLK1 group, and 1.24 +/- 0.08 and 1.23 +/- 0.09 for pEGFP-H1 group respectively. The alteration status of PLK1 protein levels were similar to that of PLK mRNA levels. The apoptosis rate was (8.3 +/- 0.6)% in control group, (8.7 +/- 0.7)% in pEGFP-H1 group and (49.7 +/- 3.8)% and (82.3 +/- 6.9)% in pEGFP-H1/PKLK1 group at 24 and 48 h, respectively. In addition, cell fraction at G(2)/M phase was increased obviously compared with control and pEGFP-H1-transfected group. CONCLUSION: The constructed shRNA can remarkably inhibit PLK1 expression and transfected K562 cell proliferation, increase apoptosis and block cell-cycle, suggesting that PLK1 play important roles in apoptosis and cell-cycle control of leukemia cells.

Arsenic trioxide inhibits proliferation in K562 cells by changing cell cycle and survivin expression.

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As2O3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As2O3 induced cell apoptosis, K562 cells were cultured with As2O3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As2O3(2-10 micromol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G2/M phase increased in proportion to As2O3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As2O3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As2O3-induced apoptosis.

[Experimental study of inhibiting CTLL-2 cell apoptosis and enhancing cytotoxicity to Yac-1 cell by hammerhead ribozyme].

OBJECTIVE: To investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in mouse cytotoxic T lymphocyte (CTL) cell line--CTLL-2 cells, and explore a novel approach to enhance the ability of T cells against leukemia in donor lymphocytes infusion (DLI). METHODS: A hammerhead ribozyme targeting the Fas mRNA was synthesized and transfected into CTLL-2 cells by electroporation. Fas expression in CTLL-2 cells was detected by using RT-PCR, Western blot and flow cytometry, CTLL-2 cells viability was measured by MTT assay, caspase-3 proteolytic activity by caspase-3 detection kit, and cell apoptosis by flow cytometry. Killing activity of CTLL-2 was detected by LDH releasing assay in vitro. RESULTS: Expression of Fas mRNA and protein in CTLL-2 cells was reduced to 50% after transfection with anti-Fas ribozyme. Being treated with anti-Fas antibody (JO(2)), compared with control and mock-transfected cells, viability of CTLL-2 cells transfected with anti-Fas ribozyme increased by 1-fold, caspase-3 activity and apoptosis rate of ribozyme-transfected cells decreased to 50% and 37%, respectively, and cell killing activity was enhanced by 2-fold. CONCLUSION: Anti-Fas ribozyme can cleave Fas efficiently and inhibit Fas-mediated apoptosis of CTLL-2 cells, resulting in improvement of their viability.

[Experimental study on activating antileukemic T cells by vaccination with dendritic cells pulsed with survivin].

The objective of this study is to investigate the effect of vaccination with dendritic cells pulsed with survivin antigen on activation of antileukemic T cells, and inhibiting proliferation of leukemic cells. The expression of survivin on acute leukemic cells were detected by cofocal microscopy and immunoprecipitation-Western blot. DCs collected from peripheral blood mononuclear cells were pulsed with survivin purified proteins. Stimulation index (SI) and antileukemia CTL induction were analyzed with (3)H-TdR incorporation and (51)Cr releasing assay, respectively. The phenotype of T cells and DCs were identified by flow cytometry. By immunofluorescence of bone marrow and peripheral blood mononuclear cells, survivin expression was detected in 16 out of 19 AML cases (84.2%). The results showed that survivin fluorescence distribution was in cytoplasm. DCs from peripheral blood mononuclear cells were successfully induced, with typical DC morphologic characteristic. The vaccination with dendritic cells pulsed with survivin antigen dramatically stimulated the proliferation of T cells. The DCs loading survivin activated T cells with higher CD4(+) T(H) ratio as compared with DCs group, T cells activated with DCs expressed CD8 and CD56. Survivin DCs significantly inhibited the growth of leukemic cells in vitro. In conclusion, survivin antigen expressed in the cytoplasm of leukemic cells, leukemic vaccination with DCs pulsed with survivin antigen in vitro inhibited the proliferation of leukemic cells, that may be a pathway for therapy of leukemia.

[Cloning of expression vector of human tissue factor gene and its expression in human ovarian cancer cell line].

The aim was to construct the expressive vector of human tissue factor (TF), and determine its expressive level in stable-transfected human ovarian cancer cell line. The human TFcDNA was obtained from human placenta by RT-PCR and then inserted into eukaryotic expressive vector pcDNA3 to obtain the TF-pcDNA3 recombinant. This recombinant gene was introduced into human ovarian cell line A2780 through transfection mediated by lipofectamine. Stable-transfected cells were screened by G418. The TF expressive levels were detected by RT-PCR and flow cytometry. The results showed that: (1) the constructed product was identified as TF-pcDNA3 recombinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780 cell (transfected cell 3.99 +/- 0.15, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. It was concluded that TF gene was successfully cloned, and was introduced into human ovarian cancer cell, and the subline A2780/TF which stably expresses TF at high level was obtained. It will provide good experimental basis for investigating new mechanisms of tumor growth, invasion, metastasis, hypercoagulability, and for exploring a new strategy of gene therapy.