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Cells are normally cultured in 2D environment, which is usually inconsistent with the real microenvironment in vivo, and it is rarely reported that an effective cancer cell killing process occurs in a 3D network environment. Herein, a kind of new biomimetic composite hydrogel which can achieve 3D cell culture has been prepared and constructed by assembly of polyisocyanopeptide (PIC) with cationic oligo (p-phenylene vinylene) (OPV). The polymer chains of PIC can be bound and frizzled to form a 3D network when the temperature rises above the gelation temperature, followed by encapsulating the cells into biomimetic composite hydrogel. Cells grow and proliferate well in 3D composite hydrogels with excellent cell viability. When the cells undergo cancerization or microbial infection during the 3D culture, the addition of the luminol luminescence system can cause a strong bioluminescence resonance energy transfer (BRET) process to produce highly active reactive oxygen species (ROS) in 3D culture and kill the ca...

DNA–protein interactions control DNA transcription, recombination, restriction, and replication, which play an important role in regulating life activities. We developed a new strategy to photothermally regulate DNA unzipping and binding to single-stranded binding protein (SSBP) based on the hybrid systems of graphene oxide (GO) and conjugated polymer. GO is applied as the photothermal modulator to activate the unzipping of dsDNA into ssDNA. Upon near-infrared (NIR) laser irradiation, the thermal melting transition of DNA is promoted, which results in much farther distance between poly[(9,9-bis(6′-N,N,N-trimethylammonium)hexyl)-fluorenylene phenylene dibromide] (PFP) and fluorescein labeled at the terminus of the DNA in comparison to that of dsDNA before NIR irradiation in the presence of GO. Therefore, the fluorescence resonance energy transfer (FRET) efficiency for PFP/fluorescein becomes weaker upon irradiation. Moreover, FRET efficiency could readily recover when the unzipped DNA hybridizes with SSBP....

Fluorescent probes have been demonstrated to be promising candidates as biomarkers and biological carriers. Our study focuses on the development of a novel amphiphilic fluorescent probe with good photostability, high water solubility, excellent specificity and promising loading capability for tumor diagnosis and treatment. At first, BODIPY dye and O-carboxymethyl chitosan were prepared via a chemical reaction. Then, the prepared BODIPY dye and cRGD were bonded to O-carboxymethyl chitosan successively via an acylation reaction. Finally, we obtained the desired amphiphilic fluorescent probe: BODIPY–O-CMC–cRGD, which was based on the fluorescence resonance energy transfer (FRET) principle for selective visualization of tumors in vitro. Through a series of experiments, we found that this fluorescent probe possessed better fluorescence characteristics and tumor targeting properties. Simultaneously, by self-assembly, the amphiphilic probe encapsulated the other flexible structure of BODIPY2 and the rigid structure of porphyrin, which formed distinct nanoparticles with different particle sizes. Hence, we could observe different phagocytosis processes of the two nanoparticles in the tumor cells via the fluorescence of dyes by confocal laser scanning microscopy. Therefore, the results suggest that the fluorescent probe has advantages in tumor detection, and the constructed tumor-specific nanoparticles show high clinical potential to be utilized not only in visual and precise diagnosis but also in excellent drug delivery for tumor treatment. Henceforth, we will prepare new targeted and visualized pharmaceuticals by replacing BODIPY2 and porphyrin with antineoplastic drugs for future tumor treatment.

Hybrid nanocomposites consisting of uniform Fe 3 O 4 nanoparticles and boron nitride (BN) nanospheres were synthesized via an ethanol-thermal reaction method. The spherical BN nanoparticles (BNNSs) with average diameter 150 nm have been uniformly coated with dense ultra-small Fe 3 O 4 nanoparticles (with average diameter of 10 nm), forming novel Fe 3 O 4 @BNNS nanocomposites. Magnetic measurement by using vibrating sample magnetometer (VSM) indicates that the Fe 3 O 4 coating is superparamagnetic, and the nanocomposites can be physically manipulated at a low magnetic field. Preliminary biocompatibility study has also been performed to evaluate the toxicity of the nanocomposites. The nanocomposites show cytocompatibility at low concentration and have little effect on cell viability of MCF-7, MCF-10 and Hela cell lines. The Fe 3 O 4 @BNNS nanocomposites may find a wide range of potential applications including water treatment, catalysts, carriers for boron neutron capture therapy and magnetic-targeted drug delivery.

Glutathione (GSH) as a biothiol is an essential peptide related to various diseases. Although multiple strategies for biothiols detection have been developed, there is increasing demand for sensors that can differentiate GSH from cysteine (Cys) and homocysteine (Hcy), owing to the similar structures and thiol groups in these amino acids. Herein, we report a novel Eu3+/LAPONITE (Lap)-based organic/inorganic hybrid material for selective detection of GSH via an “off–on” process. The fluorescence of Eu(DPA)3@Lap-Tris can be quenched by Cu2+ through photoinduced electron transfer (PET). The addition of GSH into the Eu(DPA)3@Lap-Tris/Cu2+ system induces the removal of Cu2+ from Eu(DPA)3@Lap-Tris and blocks PET, resulting in the recovery of fluorescence. This proposed assay demonstrates higher selectivity toward GSH than Cys and Hcy, and showed a detection limit of 162 nM within a linear range of 0.5–30 μM. Unlike other GSH selective sensors, this platform could be formed into a hydrogel while its sensitivity w...

It is unknown whether the ssDNA-binding-protein (SSB) possesses the ability to catalyze DNA recombination. We investigated the recombination function of SSB and the recombination kinetics of Escherichia coli using a new transformation method with a modified double-layered plate. We found that SSB catalysed intermolecular recombination in vitro. Its intermolecular recombination rate versus substrate concentration or homologous sequence length fitted the Hill equation, and while the plasmid intramolecular recombination rate versus substrate concentration fitted a positively linear correlation, the dominant intermolecular recombination was a non-homologous recombination in vivo, similar to RecA. However, ssb-dependent recombination occurred later and at a lower recombination rate than the recA-dependent, probably because ssb expression was about two-fold lower than recA during the E. coli earlier growth stage. The affinity to substrate and the recombination efficiency of SSB was lower than RecA, whereas SSB enhanced the catalytic efficiency of RecA. Knocking out both recA and ssb led to loss of recombination. Our results confirmed that as SSB has the recombination function itself as an allosteric enzyme, recA-independent recombination in E. coli should be ssb-dependent. ssb-dependent recombination may be the third DNA double-strand break repair pathway, in addition to recA-dependent recombination and non-homologous end joining.

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.

A novel magnetic/fluorometric bimodal sensor was built from carbon dots (CDs) and MnO2. The resulting sensor was sensitive to glutathione (GSH), leading to apparent enhancement of magnetic resonance (MR) and fluorescence signals along with visual changes. The bimodal detection strategy is based on the decomposition of the CDs-MnO2 through a redox reaction between GSH and MnO2. This process causes the transformation from non-MR-active MnO2 to MR-active Mn(2+), and is accompanied by fluorescence restoration of CDs. Compared with a range of other CDs, the polyethylenimine (PEI) passivated CDs (denoted as pCDs) were suitable for detection due to their positive surface potential. Cross-validation between MR and fluorescence provided detailed information regarding the MnO2 reduction process, and revealed the three distinct stages of the redox process. Thus, the design of a CD-based sensor for the magnetic/fluorometric bimodal detection of GSH was emphasized for the first time. This platform showed a detection limit of 0.6 μM with a linear range of 1-200 μM in the fluorescence mode, while the MR mode exhibited a linear range of 5-200 μM and a GSH detection limit of 2.8 μM with a visible change being observed rapidly at 1 μM in the MR images. Furthermore, the introduction of the MR mode allowed the biothiols to be easily identified. The integration of CD fluorescence with an MR response was demonstrated to be promising for providing detailed information and discriminating power, and therefore extend the application of CDs in sensing and imaging.

An assembly was fabricated and was revealed to be a multiple-stimulus-responsive biomimetic hybrid polymer architecture. It was constructed by the hydrophobic interactions between a conjugated polyfluorene that contained 2,1,3-benzothiadiazole units (PFBT) and a tri(ethylene glycol)-functionalized polyisocyanopeptide (3OEG-PIC). The introduction of PFBT to the polyisocyanopeptide (PIC) network allowed for the incorporation of responsiveness to multiple stimuli including temperature, CO2 , carbonic anhydrase, and nonlinear mechanics, which mimics natural processes and interactions. Furthermore, the light-harvesting and signal amplification characteristics of PFBT endowed the supramolecular assembly with the essential function of fluorescence monitoring for biological processes.

A conjugated polymer centered on fluorene and 2,1,3-benzothia-diazole (PFBT) is prepared for sensing CO2 in situ with high sensitivity and low background. Upon introducing CO2, the weaker electrostatic repulsion and stronger hydrophobic interactions between neighboring PFBT molecules enhance the interchain contacts compared to that without CO2, leading to the energy transfer from fluorene to 2,1,3-benzothia-diazole sites and the emission color shift from blue to green, which is sensitive to sensing CO2 in atmospheric air with a content of ∼400 ppm. Importantly, PFBT is employed to monitor photosynthesis and respiration upon cycling day and night in situ.

Background Most immune reactions related to transfusion and transplantation are caused by IgM ABO antibodies. However, IgG also plays an important role in these reactions. Therefore, a method to measure antibodies, including IgG, is necessary. We investigated ABO antibody titers of healthy individuals using a column agglutination technique (CAT) with or without dithiothreitol (DTT) and compared them with titers obtained using a conventional tube method. Methods Among healthy adults who underwent a medical examination, 180 individuals (60 with blood group A, 60 with group B, and 60 with group O) were selected. Antibody titrations were performed using the immediate spin (IS) tube, anti-human globulin (AHG) tube, and CAT with or without DTT methods. Results Higher median values of anti-B and anti-A titers in groups A and B individuals, respectively, were obtained using the IS method than using the AHG method. Higher values for group O individuals were obtained using the AHG method. Higher median titers of anti-B and anti-A in group O individuals were obtained using CAT without DTT than using the AHG method. Median titers of anti-B and anti-A in all blood groups were higher in CAT without DTT than in CAT with DTT, especially for group O individuals. Conclusions We recommend CAT with and without DTT for titration of anti-A and anti-B, especially in group O individuals, to provide more sensitive results that include IgG data. Adjustment of insurance coverage of fees associated with antibody titration might be necessary, considering the actual cost of reagents and personnel.

Calcium-activated chloride channels (CaCCs) play vital roles in a variety of physiological processes. Dysfunction of the CaCCs is implicated in many diseases. Drug discovery targeting at CaCCs has recently become possible with the determination that TMEM16A is the molecular identity of CaCCs. In this study, we demonstrated that resveratrol (RES), a Chinese traditional medicine compound, is a novel activator of TMEM16A. The yellow fluorescence protein quenching assay and measurement of intracellular calcium fluorescence intensity show that RES activates TMEM16A channels in an intracellular Ca2+-independent way. The data of inside-out patch clamp revealed that RES dose-dependently activates TMEM16A (EC50 = 47.92 ± 9.35 μM). Furthermore, RES enhanced the contractions of the ileum of guinea pigs by activating the TMEM16A channel, which indicated that RES might be a promising drug for the treatment of gastrointestinal hypomotility. As RES was able to induce TMEM16A channel activation, TMEM16A can be added to the list of RES drug targets.

Aim: To investigate the activation of the nuclear factor of activated T cells (NFAT) and its function in the corticosterone (CORT)-induced apoptosis of rat Leydig cells. Methods: NFAT in rat Leydig cells was detected by Western blotting and immunohistochemical staining. Cyclosporin A (CsA) was used to evaluate potential involvement of NFAT in the CORT-induced apoptosis of Leydig cells. Intracellular Ca 2+ was monitored in CORT-treated Leydig cells using Fluo-3/AM. After the Leydig cells were incubated with either CORT or CORT plus CsA for 12 h, the levels of NFAT2 in the nuclei and in the cytoplasm were measured by semi-quantitative Western blotting. The role of NFAT2 in CORTinduced Leydig cell apoptosis was further evaluated by observing the effects of NFAT2 overexpression and the inhibition of NFAT2 activation by CsA on FasL expression and apoptosis. Results: We found that NFAT2 was the predominant isoform in Leydig cells. CsA blocked the CORT-induced apoptosis of the Leydig cells. The intracellular Ca 2+ level in the Leydig cells was significantly increased after the CORT treatment. The CORT increased the level of NFAT2 in the nuclei and decreased its level in the cytoplasm. CsA blocked the CORT-induced nuclear translocation of NFAT2 in the Leydig cells. Both CORT-induced apoptosis and FasL expression in the rat Leydig cells were enhanced by the overexpression of NFAT2 and antagonized by CsA. Conclusion: NFAT2 was activated in CORT-induced Leydig cell apoptosis. The effects of NFAT2 overexpression and the inhibition of NFAT2 activation suggest that NFAT2 may potentially play a pro-apoptotic role in CORT-induced Leydig cell apoptosis through the up-regulation of FasL. (Asian J Androl 2007 Sep; 9: 623–633)

The Leydig cell is the primary source of testosterone in males. Levels of testosterone in circulation are determined by the steroidogenic capacities of individual Leydig cells and the total numbers of Leydig cells per testis. Stress-induced increases in serum glucocorticoid concentrations inhibit testosterone-biosynthetic enzyme activity, leading to decreased rates of testosterone secretion. It is unclear, however, whether the excessive glucocorticoid stimulation also affects total Leydig cell numbers through induction of apoptosis and thereby contributes to the stress-induced suppression of androgen levels. Exposure of Leydig cells to high concentrations of corticosterone (CORT, the endogenously secreted glucocorticoid in rodents) increases their frequency of apoptosis. Studies of immobilization stress indicate that stress-induced increases in CORT are directly responsible for Leydig cell apoptosis. Access to glucocorticoid receptors in Leydig cells is modulated by oxidative inactivation of glucocorticoid by 11 beta-hydroxysteroid dehydrogenase (11 betaHSD). Under basal levels of glucocorticoid, sufficient levels of glucocorticoid metabolism occur and there is likely to be minimal binding of the glucocorticoid receptor. We have established that Leydig cells express type 1 11 betaHSD, an oxidoreductase, and type 2, a unidirectional oxidase. Generation of redox potential through synthesis of the enzyme cofactor NADPH, a byproduct of glucocorticoid metabolism by 11 betaHSD-1, may potentiate testosterone biosynthesis, as NADPH is the cofactor used by steroidogenic enzymes such as type 3 17beta-hydroxysteroid dehydrogenase. In this scenario, inhibition of steroidogenesis will only occur under stressful conditions when high input amounts of CORT exceed the capacity of oxidative inaction by 11 betaHSD. Changes in autonomic catecholaminergic activity may contribute to suppressed Leydig cell function during stress, and may explain the rapid onset of inhibition. However, recent analysis of glucocorticoid action in Leydig cells indicates the presence of a fast, non-genomic pathway that will merit further investigation.