A replicative recombinant HPV16 E7 expression virus upregulates CD36 in C33A cells.

Objective: In past decades, the role of high-risk HPV (HR-HPV) infection in cancer pathogenesis has been extensively studied. The viral E7 protein expressed in pre-malignant cells has been identified as an ideal target for immunological intervention. However, the cultivation of HPV in vitro remains a significant challenge, as well as the lack of methods for expressing the HPV E7 protein and generating replication-competent recombinant viral particles, which posed a major obstacle to further exploration of the function and carcinogenic mechanisms of the E7 oncoprotein. Therefore, it is imperative to investigate novel methodologies to construct replication-competent recombinant viral particles that express the HPV E7 protein to facilitate the study of its function. Methods: We initiated the construction of recombinant viral particles by utilizing the ccdB-Kan forward/reverse screening system in conjunction with the Red/ExoCET recombinant system. We followed the infection of C33A cells with the obtained recombinant virus to enable the continuous expression of HPV16 E7. Afterwards, the total RNA was extracted and performed transcriptome sequencing using RNA-Seq technology to identify differentially expressed genes associated with HPV-induced oncogenicity. Results: We successfully established replicative recombinant viral particles expressing HPV16 E7 stably and continuously. The C33A cells were infected with recombinant viral particles to achieve overexpression of the E7 protein. Subsequently, RNA-Seq analysis was conducted to assess the changes in host cell gene expression. The results revealed an upregulation of the CD36 gene, which is associated with the HPV-induced oncogenic pathways, including PI3K-Akt and p53 signaling pathway. qRT-PCR analysis further identified that the upregulation of the CD36 gene due to the expression of HPV16 E7. Conclusion: The successful expression of HPV16 E7 in cells demonstrates that the replicated recombinant virus retains the replication and infection abilities of Ad4, while also upregulating the CD36 gene involved in the PI3K-Akt signaling and p53 pathways, thereby promoting cell proliferation. The outcome of this study provides a novel perspective and serves as a solid foundation for further exploration of HPV-related carcinogenesis and the development of replicative HPV recombinant vaccines capable of inducing protective immunity against HPV.

Quantifying the Fast Dynamics of HClO in Living Cells by a Fluorescence Probe Capable of Responding to Oxidation and Reduction Events within the Time Scale of Milliseconds.

The biological roles of reactive oxygen species (ROS) depend highly on their dynamics. However, it has been challenging for measuring the dynamics of ROS in cells. In this study, we address a core challenge in developing fluorescence probes for monitoring ROS dynamics by designing a redox couple that can respond rapidly to both oxidation and reduction events. We show that such molecules can be designed by taking advantage of the steric effects of electron-donating groups at the ortho position relative to the selenium center. We demonstrate this design in a new fluorescence probe named Fl-Se. Results reveal that Fl-Se and its oxidized product Fl-SeO rapidly respond to HClO, an important member of the ROS family, and glutathione (GSH), with t1/2 = 2.7 ms at [HClO] = 1 µM; t1/2 = 61 ms at [GSH] = 1 mM. When applied in cells, Fl-Se satisfactorily tracks the dynamics of intracellular HClO in H2O2-stimulated HL-60 cells, as well as the different dynamic behaviors of HClO fluctuations involved in the phorbol 12-myristate-13-acetate-activated immune response of RAW264.7 cells and the 3-deazaneplanocin A-induced apoptosis of HL 60 cells.

Theoretical Insights into the Excited State Decays of a Donor-Acceptor Dyad: Is the Twisted and Rehybridized Intramolecular Charge-Transfer State Involved?

The twisted intramolecular charge transfer has been proposed for a number of years and widely accepted to explain the excited-state dynamics of organic molecules. Recently, a new state termed as "twisted and rehybridized intramolecular charge transfer" has been proposed to explain the excited-state dynamics of an aniline-triazine electron donor-acceptor dyad with an alkyne spacer based on ultrafast time-resolved spectroscopy. However, the change of the geometries along the excited-state decay pathway remains unknown. In this study, by optimization of the excited-state geometry of the donor-acceptor dyad and potential energy surface scan along the twisting angle, we successfully reproduce the experimentally observed band in time-resolved infrared absorption spectroscopy. Our calculation results demonstrated that the rehybridization process is not involved and only the twisted intramolecular charge transfer state is formed. Moreover, we located a minimum energy conical intersection between the ground and first excited-state of the donor-acceptor dyad, which is easily reached and corresponding to the primary nonradiative decay pathway of the donor-acceptor dyad. The energy of minimum energy conical intersection is solvent-dependent and consistent with the experimentally observed solvent-dependent lifetime of excited state.

Redox-Responsive Fluorescent Probes with Different Design Strategies.

In an aerobic organism, reactive oxygen species (ROS) are an inevitable metabolic byproduct. Endogenously produced ROS have a significant role in physiological processes, but excess ROS can cause oxidative stress and can damage tissue. Cells possess elaborate mechanisms to regulate their internal redox status. The intracellular redox homeostasis plays an essential role in maintaining cellular function. However, moderate alterations in redox balance can accompany major transitions in a cell's life cycle. Because of the role of ROS in physiology and in pathology, researchers need new tools to study redox chemistry in biological systems.In recent years, researchers have made remarkable progress in developing new, highly sensitive and selective fluorescent probes that respond to redox changes, and in this Account we highlight related research, primarily from our own group. We present an overview of the design, photophysical properties, and fluorescence transduction mechanisms of reported molecules that probe redox changes. We have designed and synthesized a series of fluorescent probes for redox cycles in biological systems relying on the active center of glutathione peroxidase (GPx). We have also constructed probes based on the oxidation and reduction of hydroquinone and of 2,2,6,6-tetramethylpiperidinooxy (TEMPO). Most of these probes exhibit high sensitivity and good selectivity, absorb in the near-infrared, and respond rapidly. Such probes are useful for confocal fluorescence microscopy, a dynamic imaging technique that could allow researchers to observe biologically important ROS and antioxidants in real time. This technique and these probes provide potentially useful tools for exploring the generation, transport, physiological function, and pathogenic mechanisms of ROS and antioxidants.We also describe features that could improve the properties of redox-responsive fluorescent probes: greater photostability; rapid, dynamic, cyclic and ratiometric responses; and broader absorption in the near-IR region. In addition, fluorescent probes that include organochalcogens such as selenium and tellurium show promise for a new class of fluorescent redox probes that are both chemically stable and robustly reversible. However, further investigations of the chemical and fluorescence transduction mechanisms of selenium-based probes in response to ROS are needed.

Selenium as a versatile center in fluorescence probe for the redox cycle between HClO oxidative stress and H2S repair.

Selenium is a biologically important trace element and acts as an active center of glutathione peroxidase (GPx). GPx is the important antioxidant enzyme to protect organisms from oxidative damage via catalyzing the reaction between ROS and glutathione (GSH). Mimicking the oxidation-reduction cycles of the versatile selenium core in GPx, we can develop fluorescence probes to detect oxidation and reduction events in living systems. The cellular redox balance between hypochloric acid (HClO) and hydrogen sulfide (H2S) has broad implications in human health and diseases, such as Alzheimer's disease (AD). Therefore, to further investigate the roles of this redox balance and understand the pathogenesis of neurodegenerative diseases, it is necessary to detect the redox state between HClO and H2S in real time. We have developed a reversible fluorescence probe MPhSe-BOD for imaging of the redox cycle between HClO and H2S based on oxidation and reduction of selenide in living cells.

Switching of the triplet excited state of rhodamine/naphthaleneimide dyads: an experimental and theoretical study.

Rhodamine-bromonaphthaleneimide (RB-NI) and rhodamine-bromonaphthalenediimide (RB-NDI) dyads were prepared for switching of the triplet excited states. Bromo-NI or bromo-NDI parts in the dyads are the spin converters, i.e., the triplet state producing modules, whereas the RB unit is the acid-activatable electron donor/energy acceptor. NI and NDI absorb at 359 and 541 nm, and the T1 state energy levels are 2.25 and 1.64 eV, respectively. RB undertakes the reversible spirolactam (RB-c) ↔ opened amide (RB-o) transformation. RB-c shows no visible light absorption, and the triplet-state energy level is ET1 = 3.36 eV. Conversely RB-o shows strong absorption at 557 nm, and ET1 is 1.73 eV. Thus, the acid-activated fluorescence-resonance-energy-transfer (FRET) competes with the ISC of NI or NDI. No triplet state was observed for the dyads with nanosecond time-resolved transient absorption spectroscopy. Upon addition of acid, strong fluorescence and long-living triplet excited states were observed. Thus, the producing of triplet state is acid-activatable. The triplet state of RB-NI is localized on RB-o part, whereas in RB-NDI the triplet state is delocalized on both the NDI and RB-o units. The ISC of spin converter was not outcompeted by RET. These studies are useful for switching of triplet excited state.

Switching of the triplet excited state of rhodamine-C60 dyads.

Acid-switching of the triplet excited state in rhodamine-C60 dyads was achieved. The rhodamine moiety acts as an acid-activated visible light-harvesting antenna and C60 as the singlet energy acceptor and the spin converter, and production of the triplet state was enhanced in the presence of acid.

Experimental and theoretical study on the sensing mechanism of a fluorescence probe for hypochloric acid: a Se···N nonbonding interaction modulated twisting process.

In this article, the sensing mechanism of a fluorescence probe for hypochloric acid, NI-Se, has been investigated using experimental and theoretical methods. Based on the results of the steady-state and time-resolved emission spectra of NI-Se and its oxidized form NI-SeO, we suggested that there was twist internal charge transfer (TICT) state with faint fluorescence in NI-Se. Subsequently, the ground and excited state minimum geometries of NI-Se and NI-SeO were optimized with DFT/TD-DFT methods. The results demonstrated there was a twisting process in the excited state of NI-Se and that this twist process was induced by the nonbonding interaction between the Se and N atoms. In addition, the calculated spectra and molecular orbitals confirmed the charge transfer character of the TICT state in NI-Se. To further investigate the driving force behind the twist in NI-Se, we synthesized NI-O, which has no Se···N nonbonding interaction, as a control sample. Herein, we also present the characterization, fluorescence properties and the optimized geometries of NI-O. Moreover, the results showed that Se···N nonbonding interaction plays a significant role in the twisting process of NI-Se.

Ratiometric fluorescence imaging of cellular hypochlorous acid based on heptamethine cyanine dyes.

A series of ratiometric probes based on heptamethine cyanine dyes for detecting hypochlorous acid have been developed. Here we present the synthesis, characterization and fluorescence properties of these probes. And it turns out that the probes are highly sensitive and selective toward hypochlorous acid. More importantly, the application in living cells for ratiometric imaging of hypochlorous acid has been achieved successfully.

A reversible fluorescent probe for detecting hypochloric acid in living cells and animals: utilizing a novel strategy for effectively modulating the fluorescence of selenide and selenoxide.

Based on a novel strategy for modulating the fluorescence of selenide and selenoxide, we have designed and developed a reversible fluorescent probe for hypochloric acid. And the synthesis, characterization, fluorescence properties, as well as the biological applications in living cells and animals, have all been described.

A reversible fluorescence probe based on Se-BODIPY for the redox cycle between HClO oxidative stress and H2S repair in living cells.

We have developed a new reversible fluorescence probe MPhSe-BOD for the redox cycle process between hypochlorous acid and hydrogen sulfide in solution and in living cells. Confocal microscopy imaging using RAW264.7 cell lines shows that the probe has good cell membrane permeability, and can monitor intracellular HClO/H(2)S redox cycles continuously.

A fluorescent probe for rapid detection of thiols and imaging of thiols reducing repair and H2O2 oxidative stress cycles in living cells.

A diselenide containing fluorescent probe based on a fluorescein scaffold for thiols was developed. The fluorescent probe exhibited rapid response, high selectivity and reversibility. Confocal fluorescence microscopy was used to visualize the redox changes mediated by thiols and reactive oxygen species in living HeLa cells.