Centrifugated lateral flow assay strips based on dual-emission carbon dots modified with europium ions for ratiometric determination and on-site discrimination of tetracyclines in environment.

Due to the serious detrimental impact on human health, antibiotic pollution particularly tetracyclines residues has become a serious problem. Herein, a multiple response fluorescent probe consisted of dual-emission carbon dots and Eu3+ (D-CDs@Eu3+) is designed for the determination and discrimination of tetracyclines (TCs). Specifically, the carboxyl and amidogen group of dual-emission carbon dots (D-CDs) can coordinate with Eu3+ to form the D-CDs@Eu3+. Upon adding TCs, the fluorescence intensities of D-CDs at 405 nm and 495 nm are quenched due to inner filter effect (IFE) and the localization of fluorescence resonance energy transfer (L-FRET) between the D-CDs@Eu3+ and TC. Simultaneously, the D-CDs@Eu3+ may chelate with TCs to enhance the occurrence of antenna effect, while the characteristic peaks of Eu3+ at 590 nm and 615 nm are enhanced. On these bases, the TCs detection is achieved with low detection limits from 46.7 to 72.0 nM. Additionally, through the distinct efficiencies of L-FRET, the discrimination of TCs is achieved. Moreover, a novel centrifugated lateral flow assay strips (CLFASs) device is developed by integrating the D-CDs@Eu3+, lateral flow assay strips and smartphone using RGB variations for TCs detection, achieving remarkable recoveries (98.6-103.7 %) in real samples. Therefore, this CLFASs device provides a reliable approach for the TCs detection, demonstrating potential applications.

A sensitive lateral flow test strip sensor for visual detection of acid red 18 in food using bicentric-emission carbon dots.

Hazardous synthetic colorants have found widespread use in food production, and excessive consumption of these pigments can pose potential risks to human health. In this study, we propose an ultrasensitive fluorescence method for the analysis of Acid Red 18 (AR18) in food products. The method is based on the nitrogen-doped carbon dots (N-CDs) derived from tris and resorcinol through a hydrothermal way. The as-synthesized N-CDs exhibit two emission centers at 425 nm and 541 nm, corresponding to the excitation wavelengths of 377 nm and 465 nm, respectively. Upon the addition of AR18, the fluorescence intensity at 541 nm significantly decreases with a simultaneous, though less pronounced, reduction in the intensity at 425 nm, which is attributed to the localization of fluorescence resonance energy transfer (L-FRET). Specifically, a novel ratiometric fluorescent probe was constructed based on the extracted data from the 3D fluorescence excitation-emission matrix. This probe demonstrates a wide linear range from 0.0539 to 30 µM and a low limit of detection (LOD) of 53.9 nM. For practical applications, a portable fluorescent sensor based on a lateral flow test strip (LFTS) was designed for real-time monitoring of AR18. Color channel values were determined using a smartphone application, resulting in a satisfactory LOD of 75.3 nM. Furthermore, the suitability of the proposed ratiometric fluorescent probe was validated through the detection of AR18 in real food samples, consistently achieving recovery rates in the range of 99.7-101.4%. This research not only expands the scope of CDs in sensing fields, but also provides an effective strategy for the development of an excellent platform for real-time AR18 detection, contributing to public food safety.

Construction of fluorescent sensor array and three-dimensional microfluidic paper based analytical device for specific identification and visual determination of antibiotics in food.

For antibiotics misuse since the global outbreak of COVID 19, a novel strategy for discriminating and detecting antibiotics is proposed based on the graphene quantum dots with multi-doped heteroatoms including F, N and P (M-GQDs), which exhibit blue emission (419.0 nm) under the excitation of 336.0 nm. Specifically, the fluorescence of M-GQDs is quenched by tetracyclines (TCs) owing to inner filter effect (IFE) and enhanced by alkane-modified fluoroquinolones (AFQs), which is attributed to restricted conformational rotation based on π-π stacking, hydrogen-bonding and electrostatic interactions. Meanwhile, the electron-accepting property of oxazine ring in oxazine-modified fluoroquinolones (OFQs) increases emission peak at 498.0 nm and decreases emission peak at 419.0 nm as the color changes from blue to cyan. Moreover, a cascade system integrated with 3D microfluidic paper-based analytical device (3D-µPAD) is applied successfully for visually distinguishing three antibiotics, which shows great potential and versatility of M-GQDs for food safety monitoring.

Aggregation-induced bimodal excitation of nitrogen-doped carbon dots for ratiometric sensing of new coccine and solid-state multicolor lighting.

Trace detection of foodstuff pigments have gained increasing attention because of their close association with biological and environmental processes. Herein, we propose an innovative bimodal excitation nitrogen-doped carbon dots (N-CDs) for ratiometric sensing of new coccine (NC) pigment, which are synthesized by using melamine and o-phenylenediamine as precursors via solvothermal treatment. With the increase of the N-CDs concentration, N-CDs exhibit not only a concentration-dependent tunable color behavior, but also a novel aggregation-induced bimodal excitation phenomenon. Considering this distinctive bimodal excitation behavior, a ratiometric sensor based on N-CDs has been developed for the detection of the NC in different organic solvents due to the inner filter effect and fluorescence resonance energy transfer. The intensity ratio of two excitation signals is linear with the NC concentration in the range of 0.032-100 µM, and the limit of detection is as low as 32.1 nM. Meanwhile, we realize the design of multicolor-emission N-CDs/polymer films. All in all, this work presents a novel kind view of the mechanism of distinctive bimodal excitation of N-CDs, and further proposes an innovative ratiometric method for the screening analysis of NC in food samples and environmental pollutants.

Activated cascade effect for dual-mode ratiometric and smartphone-assisted visual detection of curcumin and F- based on nitrogen-doped carbon dots.

The growing persistence of harmful ion or drug molecular residues has always been considered as a matter of concern due to its importance in biological and environmental processes, which requires taking measures to maintain environmental health sustainably and effectively. Inspired by the multi-system and visual quantitative detection of nitrogen-doped carbon dots (N-CDs), we develop a novel cascade nano-system based on dual emission carbon dots for on-site visual quantitative detection of curcumin and fluoride ion (F-). Herein, tris (hydroxymethyl) aminomethane (Tris) and m-dihydroxybenzene (m-DHB) are elected as reaction precursors to synthesize dual-emission N-CDs by a one-step hydrothermal method. The obtained N-CDs exhibit dual emission peaks at 426 nm (blue) and 528 nm (green) with quantum yields of 53 % and 71 %, respectively. Then, trace curcumin and F- intelligent off-on-off sensing probe is formed by taking advantage of the activated cascade effect. As for the occurrence of inner filter effect (IFE) and fluorescence resonance energy transfer (FRET), the green fluorescence of N-CDs quenches remarkably, called as OFF initial state. Then the curcumin-F- complex leads to the hypochromatic shift of the absorption band from 532 to 430 nm, which activates the green fluorescence of N-CDs, named as ON state. Meanwhile, the blue fluorescence of N-CDs is quenched due to the FRET, called as OFF terminal state. This system shows good linear relationships from 0 to 35 µM and 0 to 40 µM with low detection limits of 29 nM and 42 nM for curcumin and F- ratiometric detection, respectively. Moreover, a smartphone-assisted analyzer is developed for on-site quantitative detection. Furthermore, we design a logic gate for logistics information storage, which proves the possibility of a logic gate based on N-CDs in practical application. Thus, our work will provide an effective strategy for environmental quantitative monitoring and information storage encryption.

NGF accelerates cutaneous wound healing by promoting the migration of dermal fibroblasts via the PI3K/Akt-Rac1-JNK and ERK pathways.

As a well-known neurotrophic factor, nerve growth factor (NGF) has also been extensively recognized for its acceleration of healing in cutaneous wounds in both animal models and randomized clinical trials. However, the underlying mechanisms accounting for the therapeutic effect of NGF on skin wounds are not fully understood. NGF treatment significantly accelerated the rate of wound healing by promoting wound reepithelialization, the formation of granulation tissue, and collagen production. To explore the possible mechanisms of this process, the expression levels of CD68, VEGF, PCNA, and TGF-ß1 in wounds were detected by immunohistochemical staining. The levels of these proteins were all significantly raised in NGF-treated wounds compared to untreated controls. NGF also significantly promoted the migration, but not the proliferation, of dermal fibroblasts. NGF induced a remarkable increase in the activity of PI3K/Akt, JNK, ERK, and Rac1, and blockade with their specific inhibitors significantly impaired the NGF-induced migration. In conclusion, NGF significantly accelerated the healing of skin excisional wounds in rats and the fibroblast migration induced by NGF may contribute to this healing process. The activation of PI3K/Akt, Rac1, JNK, and ERK were all involved in the regulation of NGF-induced fibroblast migration.

bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways.

Extensive research has focused on finding effective strategies to prevent or improve recovery from brain ischemia and reperfusion (I/R) injury. The basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some central nervous system (CNS) disorders, including ischemic injury. In this study, we demonstrate that bFGF administration can improve locomotor activity and inhibit the ER stress induced in the CA1 region of the hippocampus in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response proteins CHOP, XBP-1, ATF-6 and caspase-12 that are induced by H(2)O(2) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signaling pathways, PI3K/Akt and ERK1/2. Inhibition of the PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and U0126, respectively, partially reduce the protective effect of bFGF. Taken together, our results indicate that the neuroprotective role of bFGF involves the suppression of ER stress in the ischemic oxidative damage models and oxidative stress-induced PC12 cell injury, and these effects is underlying the activation of the PI3K/Akt and ERK1/2 signal pathway.

bFGF expression mediated by a hypoxia-regulated adenoviral vector protects PC12 cell death induced by serum deprivation.

Basic fibroblast growth factor (bFGF) is a known neuroprotectant against a number of brain injury conditions such as cerebral ischemia. However, bFGF also regulates a plethora of brain developmental processes and functions as a strong mitogen. Therefore, unregulated long-term expression of bFGF in brain may potentially be tumorigenic, limiting its utility in brain therapy. Here, we report the successful construction of an adenoviral vector (Ad-5HRE-bFGF) expressing bFGF under the regulation of five hypoxia-responsive elements (5HRE) and a minimal cytomegalovirus promoter (CMVmp). Following hypoxia treatment in a hypoxic chamber with less than 1% of oxygen, Ad-5HRE-bFGF induced a significant and time-dependent expression of bFGF protein and the fluorescent tag, humanized GFP (hrGFP) protein, in infected PC12 cells. In contrast, normoxia treatment evoked extremely low level of bFGF and hrGFP expression, demonstrating that the 5HRE-CMVmp cassette was effective in regulating the expression of bFGF gene in response to hypoxia. More importantly, bFGF expressed by the Ad-5HRE-bFGF viral vector under the regulation of hypoxia was significantly neuroprotective against PC12 cell death evoked by serum deprivation. Taken together, these studies demonstrated the feasibility to express bFGF in a hypoxia-regulated fashion to provide neuroprotection. The Ad-5HRE-bFGF can be further developed as an effective tool to provide neuroprotection against hypoxia-induced brain diseases, such as cerebral ischemia.

CaMKII phosphorylates collapsin response mediator protein 2 and modulates axonal damage during glutamate excitotoxicity.

Intracellular calcium influx through NMDA receptors triggers a cascade of deleterious signaling events which lead to neuronal death in neurological conditions such as stroke. However, it is not clear as to the molecular mechanism underlying early damage response from axons and dendrites which are important in maintaining a network essential for the survival of neurons. Here, we examined changes of axons treated with glutamate and showed the appearance of betaIII-tubulin positive varicosities on axons before the appearance of neuronal death. Dizocilpine blocked the occurrence of varicosities on axons suggesting that these microstructures were mediated by NMDA receptor activities. Despite early increased expression of pCaMKII and pMAPK after just 10 min of glutamate treatment, only inhibitors to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and calpain prevented the occurrence of axonal varicosities. In contrast, inhibitors to Rho kinase, mitogen-activated protein kinase and phosphoinositide 3-kinase were not effective, nor were they able to rescue neurons from death, suggesting CaMKII and calpain are important in axon survival. Activated CaMKII directly phosphorylates collapsin response mediator protein (CRMP) 2 which is independent of calpain-mediated cleavage of CRMP2. Over-expression of CRMP2, but not the phosphorylation-resistant mutant CRMP2-T555A, increased axonal resistance to glutamate toxicity with reduced numbers of varicosities. The levels of both pCRMP2 and pCaMKII were also increased robustly within early time points in ischemic brains and which correlated with the appearance of axonal varicosities in the ischemic neurons. Collectively, these studies demonstrated an important role for CaMKII in modulating the integrity of axons through CRMP2 during excitotoxicity-induced neuronal death.

[Construction of intrakine mutant SDF-1alpha/54/KDEL and its inhibiting effects upon CXCR4 expression on cell membrane].

To investigate the impact of phenotypic knockout of CXCR4 on Molt-4 cells via intrakine technology,the C-terminal alpha-helix gene SDF-1alpha/54/KDEL of human stromal cell-derived Faceor-1 deletion is fused to a retention signal 4-peptide -KDEL that retains the newly synthesized receptor within the Molt-4 cells endoplasimc reticulum. Subsequently, PCR is used to amplify the target gene SDF-1alpha/54/ KDEL from the constructed plasmid SDF-WT-Gly x 4-Dec/PET-30a(+) at its C-terminal and subclone it into eukaryotic expression vectors pEGFP-C3 for generating recombinant vector cells by lipEGFP-C3/SDF-1alpha/54/KDEL, and then have it sequenced. After the transfection of recombinant plasmids into COS-7 posome, SDF-1alpha/54/KDEL protein is confirmed with Western blot. The recombinant plasmids pEGFP-C3/SDF-1alpha/54/KDEL are isolated and transiently transfected in Molt-4 cells by electroporation. Flow cytometric analysis shows a dramatic reduction of CXCR4 expression on Molt-4 cells. The conclusion is that SDF-1alpha/54/KDEL could assume a role in the phenotypic knockout of CXCR4, and the findings suggest that the inhibiting effect of SDF-1alpha/54 against CXCR4 is not influenced by the deletion of SDF-1alpha helix at the C terminal.

SDF-1/54-DCN: a novel recombinant chimera with dual inhibitory effects on proliferation and chemotaxis of tumor cells.

Several studies have shown that the interaction of CXC chemokine receptor 4 (CXCR4) with its ligand, stromal cell-derived factor-1alpha (SDF-1alpha) is closely involved in the directional migration of some tumors toward specific organs, which provides a new pathway against cancer metastasis. We previously developed an alpha-helix-defective mutant of SDF-1alpha, SDF-1/54 that displays obvious antagonistic activity to CXCR4. But it is necessary to ensure the targeting of SDF-1/54 to tumors in vivo since many normal tissues also express CXCR4. It is known that most tumor cells highly express epidermal growth factor receptor (EGFR). Meanwhile, decorin (DCN), a specific antagonist of EGFR, can target the tumor cells enriched in EGFR and cause a significant downregulation of EGFR. Hereby, we further generated a fusion construct of SDF-1/54 and DCN to expect to enhance the targeting of SDF-1/54 to tumors by dual blocking effects on CXCR4 and EGFR. This study focused on expression of recombinant chimera SDF-1/54-DCN in Escherichia coli, purification and bioactivity to inhibit the physiological functions mediated by CXCR4 and EGFR respectively in various tumor cell lines in vitro. Results indicated that SDF-1/54-DCN could inhibit both chemotaxis and proliferation of the tumor cells we used, which may be attributed to its blocking to CXCR4 and EGFR. These findings suggest that this strategy to link SDF-1/54 with DCN may be a promising approach to increase the targeting of SDF-1/54 to the tumors coexpressing CXCR4 and EGFR.

The synergistic effects of CXCR4 and EGFR on promoting EGF-mediated metastasis in ovarian cancer cells.

CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been reported to mediate the metastasis of many solid tumors including ovarian, breast, lung and prostate. The over-expression of the epidermal growth factor receptor (EGFR) is associated with the majority of ovarian cancer and has been implicated in the process of malignant transformation by promoting cell proliferation, survival, and motility. In this research, the result first showed that epidermis growth factor (EGF) enhanced the expression of CXCR4 and the migration of ovarian cancer cells, moreover, both stromal cell derived factor-1alpha (SDF-1alpha) and EGF-induced high matrix metallopeptidase 9 (MMP9) expressions. Molecular analysis indicated that augmented CXCR4 and MMP9 expression was regulated by phosphatidylinositol-3-kinase(PI3K)/Akt signal transduction pathway. These results suggested a possible important "cross-talk" between CXCR4 and EGFR intracellular pathways that might link signals of tumor deteriorated and provided a plausible explanation for the poor overall survival rate of patients whose co-expression of CXCR4 and EGFR was detected in their tissue sections. It enlightened that, compared to the respective inhibition of the EGFR or CXCR4 signaling, the simultaneous inhibition of them might be a more useful therapeutic strategy of cancer.