Effect of radiofrequency therapy on HPV16-E7 lentivirus infection in the reproductive tract of mice and its effect on immune function of splenic lymphocytes

Objective: To investigate the effect of radiofrequency therapy (RFT) on HPV16-E7 lentivirus infection in the reproductive tract of mice and reveal its effect on immune function of splenic lymphocytes. Materials and Methods: The mouse reproductive tract model was established by infection with HPV16-E7 lentivirus. Fluorescence microscope was used to evaluate successful injection. The expression of HPV16-E7 protein was detected by Western blotting test. The levels of CD4+ and CD8+ were determined by flow cytometry, and the ratio was calculated. The proliferation of splenic lymphocytes was detected by MTT assay. Expression of Interleukin (IL)-2 and interferon-γ (IFN-γ) messenger RNA (mRNA) in lymphocyte was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results: Fluorescence microscope determined the successful injection of HPV16-E7 lentivirus. Compared with model group, RFT treatment decreased HPV16-E7 protein, and increased CD4+/CD8+ ratio and the proliferation activity of splenic lymphocytes. Besides, RFT treatment increased the mRNA expression levels of IL-2 and IFN-γ compared to the model group. In particular, the proliferation activity of spleen lymphocytes and the expression levels of IL-2 mRNA and IFN-γ mRNA in RFT were higher at 12 days than at 6 days after treatment. Conclusion: RFT could eliminate HPV16-E7 lentivirus infection in the reproductive tract of mice, and the mechanism was related to the immune system.

Expression, Microencapsulation of Recombinant Human Epidermal Growth Factor, and Release Study in Gastric Ulcer Representing Condition

Recombinant human epidermal growth factor (rhEGF) has been studied and expressed in various expression systems. It has been also commercialized and clinically used, yet limited to topical diseases. However, being naturally expressed in different tissues, the rhEGF is potential to be applied not only for external wound and skin disorders, but also to regenerates internal damaged epidermal cells such found in gastric ulcer. In the recent study, chitosan microparticles were developed to facilitate delivery of the rhEGF and to overcome gastric degradation that majorly interfere protein, particularly rhEGF oral administration. The rhEGF was expressed in E. coli BL21(DE3) and purified using Ni-NTA chromatography. The refolded rhEGF showed proliferation activity on MC7 cells. rhEGF loaded chitosan microparticles were stable in the gastric and specifically released the loaded rhEGF in the high oxidative environment in acidic pH representing gastric ulcer condition.

Proliferation activity in the polyps of Cassiopea xamachana: where the planuloid buds grow

Polyps of the Cassiopeidae family possess a unique type of asexual reproduction by producing free-swimming buds — planuloids. The process of planuloid development and transformation to polyp has been described earlier, however, the source of tissue formation is still poorly studied. Using the method of EdU incorporation we have analyzed DNA synthesis activity during planuloid formation and growth in Cassiopea xamachana. We revealed the active proliferation zone at the early stage of bud formation. This zone continued to function during planuloid growth, providing the formation of polyp structures, and preserved in polyp calyx after metamorphosis. Its proliferation activity varied at different growth stages, whereas the localization remained relatively the same.

Case Report: Primary Diffuse Leptomeningeal Oligodendrogliomatosis in a Young Adult Cat

A 2-year-old cat was presented with progressive ataxia. Despite treatment the animal died. Pathomorphological examination revealed a widespread leptomeningeal mass at all levels of the central nervous system accentuated on the cervical spinal cord and the medulla oblongata without presence of a primary intraaxial tumor. The neoplasm was mainly composed of round, uninucleate cells with hyperchromatic nuclei, which were immunopositive for OLIG2, doublecortin, MAP2, synaptophysin, and vimentin, indicating components of both oligodendroglial and neuronal differentiation. Ki-67 immunohistochemistry indicated a high proliferation activity of the neoplasm. Few GFAP positive and Iba-1 positive cells were interpreted as reactive astrocytes and macrophages or microglia, respectively. The tumor was immunonegative for CD3, CD20, PAX5, MUM1, pan-cytokeratin, S100, NSE, p75NTR, NeuN and periaxin. These findings led to the diagnosis of primary diffuse leptomeningeal oligodendrogliomatosis. This is the first reported case of this entity in a young cat, which should be considered as a differential diagnosis for diffuse subarachnoidal round cell infiltrates.

MiR-29c-3p May Promote the Progression of Alzheimer’s Disease through BACE1

The aim of this study was to explore the specific role of miR-29c-3p in Alzheimer’s disease (AD). Animal models of AD were established by injecting streptozotocin (STZ) into mice through the lateral ventricle, while cell models of AD were induced by 10 μM β-amyloid (Aβ). We detected miR-29c-3p and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) contents and measured AD cell proliferation and apoptosis. A low miR-29c-3p level and a high BACE1 level were detected in the brain tissue of AD animal models and AD cell models. Aβ-processed cells had markedly lower proliferation activity, higher apoptosis, increased phosphorylation of tau protein was over phosphorylated, but the overexpression of miR-29c-3p or the silencing of BACE1 significantly enhanced the cell proliferation activity and reduced cell apoptosis by regulating the contents of related proteins. Inhibition of miR-29c-3p or overexpression of BACE1 aggravated Aβ-induced side effects. We used Targetscan7.2 to predict the downstream target genes of miR-29c-3p. Then, we detected that there were target binding sites between miR-29c-3p and BACE1. The rescue experiment identified BACE1 as a functional target for miR-29c-3p. AD leads to decreased miR-29c-3p level and increased BACE1 level. MiR-29c-3p has specific binding sites with the 3′-untranslated region (3′-UTR) of BACE1 and thus negatively regulates the BACE1 level, thereby affecting the progression of AD.

Efficacy of Er:YAG laser irradiation for decontamination and its effect on biocompatibility of different titanium surfaces

Background Erbium yttrium–aluminum–garnet (Er:YAG) laser have been shown to be suitable for decontamination of titanium surfaces at a wide range of energy settings, however, high intensity of laser irradiation destroy titanium surface and low intensity cannot remove enough microbial biofilm. The aim of this study was to investigate the optimal energy setting of Er:YAG laser for decontamination of sandblasted/acid-etched (SLA) and hydroxyapatite (HA) titanium surfaces. Material and methods After supragingival biofilm construction in vivo, SLA and HA titanium discs were divided into three groups: blank control (BC, clean discs), experimental control (EC, contaminated discs) and experimental groups (EP, contaminated discs irradiated by Er:YAG laser at 40, 70, and 100 mJ/pulse). Scanning electron microscopy (SEM), live/dead bacterial fluorescent detection, and colony counting assay were used to detect the efficacy of laser decontamination. To investigate the effect of laser decontamination on titanium surface biocompatibility, MC3T3-E1 cell adhesion and proliferation activity were examined by SEM and CCK-8 assay. Results Er:YAG laser irradiation at 100 mJ/pulse removed 84.1% of bacteria from SLA titanium surface; laser irradiation at 70 and 100 mJ/pulse removed 76.4% and 77.85% of bacteria from HA titanium surface respectively. Laser irradiation improved MC3T3-E1 cell adhesion on both titanium surfaces. For SLA titanium discs, 100 mJ/pulse group displayed excellent cellular proliferation activity higher than that in BC group (P < 0.01). For HA titanium discs, 70 mJ/pulse group showed the highest activity comparable to BC group (P > 0.05). Conclusions With regards to efficient microbial biofilm decontamination and biocompatibility maintenance, Er:YAG laser at 100 mJ/pulse and 70 mJ/pulse are considered as the optimal energy settings for SLA titanium and HA titanium surface respectively. This study provides theoretical basis for the clinical application of Er:YAG laser in the treatment of peri-implantitis.

Combination of cytostatic chemotherapy with cisplatin and photodynamic therapy with Radachlorin reduced resistance of K562 and Hela cell lines

In this work were study combination effect of photodynamic therapy and cisplatin on the proliferation activity of K562 human leukemia cells and Hela cervical carcinoma cells. A decrease in cell viability and an increase the fraction of apoptotic cells for combination treatment compared with single therapy were observed. It has been shown that the G2/M-phase of cell cycle decreases compared with cisplatin treatment alone, which demonstrates an increase anti-proliferative effect. The combination index of the photodynamic therapy with Radachlorin and cisplatin was calculated and indicates a synergistic effect.

Tissue dermal equivalent – а cellular product based on human dermal keratinocytes and fibroblasts: the properties of equivalent components and perspectives of practical application

The nature and ways of isolation and cultivation in vitro of keratinocytes and fibroblasts, the main cellular components of skin to prepare a new biomedical product, tissue dermal equivalent were considered. The main attention was payed to optimization of upbuilding dermal cell biomass including selection of medium compositions and conditions of cultivation. The information was given on main parameters of cell cultures as proliferation activity, viability and phenotype of the cells. Genotoxicity of fibroblasts and biocompatibility of the cells with organic matrixes to find the optimal carrier for cellular elements of tissue dermal equivalent were studied. The composition, the process of preparation of tissue dermal equivalent and perspectives of its practical application were discussed.

Experimental Study of Rhein Inhibits the Invasion and Migration of HepG2 Cells by ERK Signaling Pathway

Objective: To investigate the effectiveness of Rhein on the proliferation, invasion and migration of human hepatoma cell line HepG2 and its possible mechanism.Methods: Human hepatoma cell line HepG2 was treated with different concentrations of Rhein (Rhein treatment group) and culture in culture medium alone (control group).The proliferation activity of the cells was determined by methyl Thiazolyl Tetrazolium (MTT) colorimetry.Transwell assay detected the invasion and migration of cells in each group.Cell scratch test was used to detect the migration ability of cells in each group.Excella-phospho-excellar signal-regulated kinase (P-ERK) activity was determined by ELISA after treatment with 50μ mol/L Rhein at different times.Western blot was used to detect ERK protein expression in HepG2 cells treated with 50 μmol/L Rhein.Results: Compared with the control group, the proliferation activity, invasion and migration ability of HepG2 cells in the Rhein treatment group were all decreased (P< 0.05), and the p-ERK relative activity of HepG2 cells treated with Rhein was decreased (P < 0.05).Conclusion: Rhein inhibits the invasion and migration of HCC cells, possibly by inhibiting the ERK pathway

Leukemic Cells Expressing ETV6-Frk Identified in a Refractory B-ALL Patient Are Sensitive to Dasatinib in Vitro

[Background] ETV6-FRK is a rare kinase-related fusion gene which was identified only in acute myeloid leukemia (Hosoya N, et al. Genes Chromosomes Cancer. 2005). Herein, we firstly identified ETV6-FRK fusion gene in a patient with high-risk pediatric B cell precursor ALL (B-ALL). Because FRK is Src family tyrosine kinase, we performed functional analysis of ETV6-FRK to establish molecular targeting therapy. [Patient] A 11-year-old boy with B-ALL was refractory to conventional chemotherapy and received allogeneic bone marrow transplantation (allo-BMT) following two courses of blinatumomab. This patient maintains complete remission for three months after allo-BMT. Cytogenetic analysis demonstrated t(6;12)(q21;p13) as a part of complex karyotype. Targeted capture mRNA sequencing identified ETV6-FRK fusion transcript in this patient. [Materials and methods] ETV6-FRK fusion was validated by RT-PCR of the diagnostic leukemic sample from this patient. Full length of ETV6-FRK cDNA was cloned into retroviral vector with Tet-On system. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing ETV6-FRK (Ba/F3-ETV6-FRK) under doxycycline (DOX) dependent manner. Ba/F3-ETV6-FRK was analyzed whether IL-3 independent growth was achieved. To determine whether aberrant activation of FRK-STAT pathway as the downstream effects of ETV6-FRK, activation of FRK-STAT pathway was evaluated by western blot. Finally, in cytotoxic assay, proliferation of Ba/F3-ETV6-FRK was assessed under the media with various concentrations of dasatinib, a tyrosine kinase inhibitor. [Results and discussions] Sequencing of RT-PCR product revealed that ETV6 exon 4 was fused in-frame to FRK exon 3, creating an ETV6-FRK fusion gene. The ETV6-FRK fusion gene produced a chimeric protein consisting of the entire pointed (PNT) oligomerization domain of ETV6 and the kinase domain of FRK (Fig 1). The expression of ETV6-FRK in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-ETV6-FRK proliferated without IL-3 in contrast to Ba/F3 cells not expressing ETV6-FRK (p&lt;0.01), suggesting ETV6-FRK had proliferation activity. Western blot analysis revealed constitutive phosphorylation of tyrosine residues of ETV6-FRK and STAT5/STAT3/STAT1, suggesting constitutive activation of FRK-STATs pathway was associated with IL-3 independent proliferation activity of ETV6-FRK. Considering that dasatinib, which is Src-kinase inhibitor, could block constitutive phosphorylation of ETV6-FRK, we hypothesized that dasatinib might block the IL-3 independent proliferation of Ba/F3-ETV6-FRK. In vitro killing assay showed that dasatinib suppressed efficiently the proliferation of Ba/F3-ETV6-FRK with 50% inhibitory concentration (IC50) 1.64 ± 0.02 nM, although dasatinib didn't show any effect on Ba/F3 cells not expressing ETV6-FRK (Fig. 2). Annexin V assay determined that 35.8 ± 6.9 % of Ba/F3-ETV6-FRK with dasatinib (10nM, 48hrs) were apoptotic than Ba/F3 cells not expressing ETV6-FRK (Fig. 3, p&lt;0.01). These findings suggested that dasatinib abolished the proliferation activity of ETV6-FRK selectively. [Conclusion] We identified the first patient of pediatric high-risk B-ALL harboring ETV6-FRK fusion by targeted capture mRNA sequencing, who was refractory to the conventional chemotherapy. We also provide the first evidence that dasatinib could abrogate proliferation activity of ETV6-FRK in vitro, suggesting that dasatinib might be effective for the patient with B-ALL carrying a ETV6-FRK fusion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.