Gene therapy with tumor-specific promoter mediated suicide gene plus IL-12 gene enhanced tumor inhibition and prolonged host survival in a murine model of Lewis lung carcinoma

<p>Abstract</p> <p>Background</p> <p>Gene therapy is a promising therapeutic approach for cancer. Targeted expression of desired therapeutic proteins within the tumor is the best approach to reduce toxicity and improve survival. This study is to establish a more effective and less toxic gene therapy of cancer.</p> <p>Methods</p> <p>Combined gene therapy strategy with recombinant adenovirus expressing horseradish peroxidase (HRP) mediated by human telomerase reverse transcriptase (hTERT) promoter (AdhTERTHRP) and murine interleukin-12 (mIL-12) under the control of Cytomegalovirus (CMV) promoter (AdCMVmIL-12) was developed and evaluated against Lewis lung carcinoma (LLC) both <it>in vivo </it>and <it>in vitro</it>. The mechanism of action and systemic toxicities were also investigated.</p> <p>Results</p> <p>The combination of AdhTERTHRP/indole-3-acetic acid (IAA) treatment and AdCMVmIL-12 resulted in significant tumor growth inhibition and survival improvement compared with AdhTERTHRP/IAA alone (tumor volume, 427.4 ± 48.7 mm³<it>vs </it>581.9 ± 46.9 mm³, <it>p </it>= 0.005 on day 15; median overall survival (OS), 51 d <it>vs </it>33 d) or AdCMVmIL-12 alone (tumor volume, 362.2 ± 33.8 mm³<it>vs </it>494.4 ± 70.2 mm³, <it>p </it>= 0.046 on day 12; median OS, 51 d <it>vs </it>36 d). The combination treatment stimulated more CD4⁺and CD8⁺T lymphocyte infiltration in tumors, compared with either AdCMVmIL-12 alone (1.3-fold increase for CD4⁺T cells and 1.2-fold increase for CD8⁺T cells, <it>P </it>< 0.01) or AdhTERTHRP alone (2.1-fold increase for CD4⁺T cells and 2.2-fold increase for CD8⁺T cells, <it>P </it>< 0.01). The apoptotic cells in combination group were significantly increased in comparison with AdCMVmIL-12 alone group (2.8-fold increase, <it>P </it>< 0.01) or AdhTERTHRP alone group (1.6-fold increase, <it>P </it>< 0.01). No significant systematic toxicities were observed.</p> <p>Conclusions</p> <p>Combination gene therapy with AdhTERTHRP/IAA and AdCMVmIL-12 could significantly inhibit tumor growth and improve host survival in LLC model, without significant systemic adverse effects.</p

Recent Development in Reversible Solid Oxide Fuel Cells: Theory, Integration and Prospective

Abstract Reversible solid oxide fuel cell (RSOC) has gained widespread attention due to their potential for high efficiency in implementing multi‐energy distributed systems. When high power demand is required, RSOC can operate in the solid oxide fuel cell (SOFC) mode, directly converting the chemical energy from hydrogen or other renewable fuels into electricity. When excess electricity is available, RSOC can operate in the solid oxide electrolysis cell (SOEC) mode, producing fuels through the electrolysis of water or co‐electrolysis of water and carbon dioxide. The reversible operation of RSOC enables the direct conversion between chemical energy and electricity, offering a promising solution for clean and sustainable energy with low cost and high round‐trip efficiency. This paper introduces the research background and working principles of RSOC, provides a detailed overview of the current research status of electrolyte, fuel electrode, and oxygen electrode materials, discusses the optimization design of RSOC stacks and energy utilization strategies in the system. In addition, the future development directions of RSOC were also explored, which is of significant importance for the commercialization of RSOC

Telomere-binding protein TPP1 modulates telomere homeostasis and confers radioresistance to human colorectal cancer cells.

Radiotherapy is one of the major therapeutic strategies in cancer treatment. The telomere-binding protein TPP1 is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1 expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1 on radiosensitivity is unclear.In this study, we found that elevated TPP1 expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Moreover, TPP1 overexpression showed lengthened telomere length and a significant decrease of radiosensitivity to X-rays. TPP1 mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. Furthermore, TPP1 overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1 signal pathway after IR exposure. Moreover, TPP1 overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation.We demonstrated that elevated expressions of TPP1 in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1 may be a potential target in the radiotherapy of colorectal cancer

Inhibition of UBE2D3 expression attenuates radiosensitivity of MCF-7 human breast cancer cells by increasing hTERT expression and activity.

The known functions of telomerase in tumor cells include replenishing telomeric DNA and maintaining cell immortality. We have previously shown the existence of a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Here we set out to elucidate the molecular mechanisms underlying regulation by telomerase of radiosensitivity in MCF-7 cells. Toward this aim, yeast two-hybrid (Y2H) screening of a human laryngeal squamous cell carcinoma radioresistant (Hep2R) cDNA library was first performed to search for potential hTERT interacting proteins. We identified ubiquitin-conjugating enzyme E2D3 (UBE2D3) as a principle hTERT-interacting protein and validated this association biochemically. ShRNA-mediated inhibition of UBE2D3 expression attenuated MCF-7 radiosensitivity, and induced the accumulation of hTERT and cyclin D1 in these cells. Moreover, down-regulation of UBE2D3 increased hTERT activity and cell proliferation, accelerating G1 to S phase transition in MCF-7 cells. Collectively these findings suggest that UBE2D3 participates in the process of hTERT-mediated radiosensitivity in human breast cancer MCF-7 cells by regulating hTERT and cyclin D1

Microsporidia dressing up: the spore polaroplast transport through the polar tube and transformation into the sporoplasm membrane

ABSTRACTMicrosporidia are obligate intracellular parasites that infect a wide variety of hosts including humans. Microsporidian spores possess a unique, highly specialized invasion apparatus involving the polar filament, polaroplast, and posterior vacuole. During spore germination, the polar filament is discharged out of the spore forming a hollow polar tube that transports the sporoplasm components including the nucleus into the host cell. Due to the complicated topological changes occurring in this process, the details of sporoplasm formation are not clear. Our data suggest that the limiting membrane of the nascent sporoplasm is formed by the polaroplast after microsporidian germination. Using electron microscopy and 1,1′-dioctadecyl-3,3,3′,3′ tetramethyl indocarbocyanine perchlorate staining, we describe that a large number of vesicles, nucleus, and other cytoplasm contents were transported out via the polar tube during spore germination, while the posterior vacuole and plasma membrane finally remained in the empty spore coat. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were also found to localize in the region of the polaroplast and posterior vacuole in mature spores and in the discharged polar tube, which suggested that the polaroplast during transport through the polar tube became the limiting membrane of the sporoplasm. The analysis results of Golgi-tracker green and Golgi marker protein syntaxin 6 were also consistent with the model of the transported polaroplast derived from Golgi transformed into the nascent sporoplasm membrane.IMPORTANCEMicrosporidia, which are obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection by the microsporidia is their unique invasion apparatus which includes the polar filament, polaroplast, and posterior vacuole. When the mature spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that transports the sporoplasm components including the microsporidian nucleus into host cells. Details of the structural difference between the polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting the nucleus and other sporoplasm components. Furthermore, our results indicate that the transported polaroplast transforms into the sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of the polar tube and origin of the sporoplasm membrane, which provide important clarification of the microsporidian infection mechanism

The MCF-7 cells proliferation were illustrated.

<p>After MCF-7 cells were transfected with pshRNA-UBE2D3, cell proliferation was examined by CCK-8 assay. The results were presented as the Means±SD of three independent experiments. *<i>p</i><0.05.</p

TPP1 production, radiosensitivity (SF2) and telomere length (TRF) in human colorectal cancer cell lines.

<div><p>(A) TPP1 production was detected by western blotting.. </p> <p>(B) Telomere length was examined by Southern blot analysis. </p> <p>(C) Relative TPP1 production, radiosensitivity (SF2) and telomere length (TRF) in human colorectal cancer cell lines.</p> <p>(D) Correlation between TPP1 production and radiosensitivity (SF2) in colorectal cancer cells was examined.</p> <p>(E) Correlation between TPP1 production and the TRF length in colorectal cancer cells was examined.</p></div

Effects of TPP1 overexpression on localization of TRF2 with telomeres, telomere length and telomerase activity.

<div><p>(A) Mean TRF lengths at different PDs were detected by southern blot. PD, population doubling. The position of MWs (kb) is indicated to the left.</p> <p>(B) TRAP PCR ELISA assay was used in the analysis of telomerase activity at different PDs.</p> <p>(C) Western blot analysis revealed that TPP1 overexpression had no significant influence on the expression of hTERT.</p> <p>(D) Telomere-ChIP assays were performed using a TRF2 antibody to examine the telomeric DNA bound to by TRF2. Input, supernatant before immunoprecipitation; ppt, protein-DNA immunoprecipitate complex. Specific (telomeric) and nonspecific (Alu) probes were used.</p> <p>Telomeric DNA in ChIP (%) =Telomeric DNA signals of ppt / Telomeric DNA signals of input* 100%.</p></div

The MCF-7 cells radiosensitivity detection were illustrated when exposed to irradiation, depending on doses in GY, MCF-7 cells transfected with pshRNA-UBE2D3 showed reductions of clonogenic survival compared to negative control.

<p>Each group of cells were irradiated at the dose point of 0, 1, 2, 4, 6, 8, 10 GY respectively. After 14 days of incubation, the colonies were fixed and stained. Those colonies containing >50 cells were scored as viable colonies. The data were fit into the linear-quadratic model, and survival curve of each group were demonstrated by Graphpad prism 5.0 software. Each experiment was done at least three times in triplicate wells.</p

hTERT interactors identified in Y2H library screen.

<p>hTERT interactors identified in Y2H library screen.</p