A near-infrared fluorescent probe for the ratiometric detection and living cell imaging of ß-galactosidase.

ß-Galactosidase (ß-gal) has captured the attention of biologists, chemists, and medical researchers as an important biomarker for cell senescence and primary ovarian cancer. Therefore, many fluorescent probes with visible light emission have been developed for the detection and imaging of ß-gal in living cells. However, near-infrared (NIR) ratiometric probes are more suitable for bioimaging because near-infrared light can effectively avoid the interference of autofluorescence and the ratiometric approach can improve sensitivity and accuracy of the detection. In this work, we designed an NIR ratiometric probe (TMG) for the highly sensitive detection of ß-gal. Using a spontaneous degradation mechanism based on the ICT effect, the change in ratio (F650/F580) exhibited a prominent ß-gal-dependent performance and proved a strong linear response to the activity of ß-gal at an enzyme concentration between 0 and 200 U L-1, with a limit of detection as low as 0.86 U L-1, and the response speed is much faster than the same type of probes previously reported. The probe also revealed an excellent biocompatibility and a large Stokes shift. Moreover, fluorescence microscopy imaging experiments confirmed that this probe could be successfully used for the detection of endogenous ß-gal in living cells. Graphical abstract.

Cytochrome P450 CYP6AE70 Confers Resistance to Multiple Insecticides in a Lepidopteran Pest, Spodoptera exigua.

Cytochrome P450 monooxygenases are associated with the detoxification of xenobiotics, and overexpression of P450 genes has been proven to be associated with insecticide resistance in insect species. Our previous study has revealed that multiple CYP6AE genes were significantly overexpressed in a resistant strain of Spodoptera exigua, and among these genes, CYP6AE70 was particularly highly expressed. However, the functional roles of the CYP6AE genes in insecticide resistance remain unknown in this pest. Here, we investigate the relationship between the CYP6AE genes and insecticide resistance by focusing on CYP6AE70. The expression of CYP6AE70 was increased after exposure to chlorpyrifos, cypermethrin, and deltamethrin. Ectopic overexpression of P450 in transgenic flies by the GAL4/UAS system dramatically enhanced the tolerance to these three insecticides. Furthermore, the recombinant CYP6AE70 was functionally expressed in Sf9 cells, and metabolic assays revealed that the recombinant P450 protein could efficiently metabolize chlorpyrifos, cypermethrin, and deltamethrin. Finally, molecular modeling and docking also showed that this P450 protein were tightly bound to the three insecticides. These results determine that the upregulation of CYP6AE genes results in resistance to multiple insecticides in S. exigua. This study improves our understanding of P450-mediated insecticide resistance and will help us to design more effective resistance management for pest control.

Novel ratiometric fluorescent probe for real-time detection of alkaline phosphatase and its application in living cells.

A novel ratiometric fluorescent probe has been developed through a simple synthetic route for the detection of alkaline phosphatase(ALP) in aqueous media and for fluorescence imaging in living cells. The introduction of a spontaneous-degradation spacer in the design of the fluorescent probe is beneficial for the ratio detection method and allows the selection of a fluorophore with an amino group. Under catalysis by ALP, the phosphate monoester bond breaks; this is followed by 1,4-elimination, decomposition of the carbamate moiety, and subsequent formation of the 4-amine-1,8-naphthalimide fluorophore. The probe APN shows a significant fluorescence colour change from blue to green in response to ALP, and the fluorescence intensity ratio of the probe solution (F550/F480) has a good linear relationship with the ALP concentration in the range of 0 to 100 U L-1. Our studies have demonstrated that APN exhibits high accuracy in recognising ALP, with a detection limit as low as 0.16 U L-1. Furthermore, the probe shows very good biocompatibility, which is beneficial for its application in biological systems.

Near-infrared ratiometric probe with a self-immolative spacer for rapid and sensitive detection of alkaline phosphatase activity and imaging in vivo.

Alkaline phosphatase (ALP), an enzyme that catalyzes the hydrolysis of phosphate groups, is closely associated with many diseases, including bone disease, prostate cancer, and diabetes. Thus, new assays for ALP detection in live cells are needed to better understand its role in related biological processes. In this study, we constructed a novel near-infrared ratiometric fluorescent probe for detecting ALP activity with high sensitivity. The probe uses a new self-immolative mechanism that can achieve a rapid response (within 10 min) to ALP, detected as a spectral shift (from 580 to 650 nm). This method effectively avoids issues related to instrument variability, and the near-infrared fluorescence emission (650 nm) makes it more suitable for biological detection. Moreover, the high sensitivity (14-fold enhancement of the fluorescence ratio F650/F580) and low detection limit (0.89 U L-1) for ALP allows the probe to be adapted to complex biological environments. The assay was successfully performed using serum samples with a linear range of ALP of up to 150 U L-1. We used the developed probe to detect and image endogenous ALP in cells with satisfactory results, and we successfully used the probes to detect changes in endogenous ALP levels in zebrafish caused by drug-induced organ damage.

Novel fluorescent probe for rapid and ratiometric detection of ß-galactosidase and live cell imaging.

ß-Galactosidase (ß-gal) is an important biomarker for primary ovarian cancers and cell senescence; however, a fast response fluorescent probe for ratiometric monitoring is still rare. A novel, ratiometric water-soluble fluorescent probe (FLM) for ß-gal was developed. The emission ratio F550/F450 reached the maxima at about 5 min and can be used for real-time detection of ß-gal; the ratio gained an ultimate enhancement of about 260-fold. The ratio (F550/F450) displayed brilliant ß-gal-dependent performance and responded linearly with ß-gal activity. The probe showed wonderful biocompatibility and was successfully used for the bioimaging of endogenous ß-gal in the human ovarian cancer cell line OVCAR-3.

A highly selective and ultrafast near-infrared fluorescent turn-on and colorimetric probe for hypochlorite in living cells.

Hypochlorous acid (HOCl)/hypochlorite (OCl-), important reactive oxygen species, play essential roles in many physiological and pathological progresses. Accordingly, we developed a novel dicyanomethylene-4H-pyran (DCM)-based probe DCM-OCl for colorimetric and near-infrared fluorescent turn-on detection of OCl-. The probe exhibited excellent selectivity and sensitivity for OCl- over other bio-related analytes with a detection limit of 80 nM. The excellent selectivity of DCM-OCl for OCl- was ascribed to specific oxidative cleavage of the dimethylthiocarbamate (DMTC) recognition unit by OCl-. Moreover, DCM-OCl exhibited an ultrafast turn-on response (<3 s) to OCl-, potentially allowing real-time detection of OCl-. Furthermore, DCM-OCl was successfully used to image endogenous/exogenous OCl- in living cells.

Ratiometric fluorescent probes with a self-immolative spacer for real-time detection of ß-galactosidase and imaging in living cells.

Ratiometric fluorescent probes with a self-immolative spacer for ß-galactosidase (ß-gal) were developed. They function by ß-gal-cleaving the ß-galactoside bond of fluorescent substrates, followed by self-immolation to liberate the amino group of fluorophore. Thus, a remarkable variation in the photophysical properties was observed and the corresponding ratiometric detection of ß-gal was realized. Our studies demonstrated that the GNPN exhibited high sensitivity for recognition of ß-gal, with a detection limit as low as 0.17 U L-1. GNPN can rapidly quantify ß-gal enzyme activity; the emission ratio F545/F475 for the GNPN reached maxima after approximately 4 min, which was one of the shortest response time ever reported. Furthermore, we demonstrated that these probes possess excellent biocompatibility and can be used to visualize the endogenous ß-gal in ovarian cancer cells.

Moxibustion for pain relief in patients with primary dysmenorrhea: A randomized controlled trial.

BACKGROUND: Though moxibustion is frequently used to treat primary dysmenorrhea in China, relevant evidence supporting its effectiveness is still scanty. METHODS: This study was a pragmatic randomized, conventional drug controlled, open-labeled clinical trial. After initial screen, 152 eligible participants were averagely randomized to receive two different treatment strategies: Moxibustion and conventional drugs. Participants and practitioners were not blinded in this study. The duration of each treatment was 3 months. The primary outcome was pain relief measured by the Visual Analogue Scale. The menstrual pain severity was recorded in a menstrual pain diary. RESULTS: 152 eligible patients were included but only 133 of them eventually completed the whole treatment course. The results showed that the menstrual pain intensity in experimental group and control group was reduced from 6.38±1.28 and 6.41±1.29, respectively, at baseline, to 2.54±1.41 and 2.47±1.29 after treatment. The pain reduction was not significantly different between these two groups (P = 0.76), however; the pain intensity was significantly reduced relative to baseline for each group (P<0.01). Three months after treatment, the effectiveness of moxibustion sustained and started to be superior to the drug's effect (-0.87, 95%CI -1.32 to -0.42, P<0.01). Secondary outcome analyses showed that moxibustion was as effective as drugs in alleviating menstrual pain-related symptoms. The serum levels of pain mediators, such as PGF2α, OT, vWF, ß-EP, PGE2, were significantly improved after treatment in both groups (P<0.05). No adverse events were reported in this trial. CONCLUSIONS: Both moxibustion and conventional drug showed desirable merits in managing menstrual pain, given their treatment effects and economic costs. This study as a pragmatic trial only demonstrates the effectiveness, not the efficacy, of moxibustion for menstrual pain. It can't rule out the effect of psychological factors during treatment process, because no blind procedure or sham control was used due to availability. In clinical practice, moxibustion should be used at the discretion of patients and their physicians. TRIAL REGISTRATION: ClinialTrials.gov NCT01972906.

Use of moxibustion to treat primary dysmenorrhea at two interventional times: study protocol for a randomized controlled trial.

BACKGROUND: Dysmenorrhea is a common menstrual complaint among adolescent girls and women of reproductive age. The treatment of dysmenorrhea is typically selected from multidisciplinary options, including complementary and alternative medicine such as acupuncture and moxibustion. However, there are few published randomized controlled trials concerning moxibustion treatment for dysmenorrhea. This trial aims to investigate the efficacy and safety of moxibustion for primary dysmenorrhea, and to identify the optimal time of moxibustion treatment for primary dysmenorrhea. METHODS/DESIGN: This protocol is for a randomized controlled trial in which the assessor and statistician will be blinded. A total of 222 eligible patients with dysmenorrhea will be randomly assigned to three groups in a 1:1:1 ratio as treatment group A (treated before menstruation onset), treatment group B (treated at the onset of menstruation), or control group C (waiting list group). The participants assigned to the treatment groups will receive suspended moxibustion treatment at Sanyinjiao (SP6) and Guanyuan (CV4), while the waiting list group will not receive moxibustion treatment until the completion of the study. The trial period will consist of three baseline menstrual cycles, three menstrual cycles of treatment, and three menstrual cycles in the follow-up period. The primary outcome will be measured by changes in the Cox Menstrual Symptom Scale and the secondary outcomes will be measured using the Visual Analogue Scale, Cox Retrospective Symptom Scale, diary entries, the Self-rating Depression Scale, and the Self-rating Anxiety Scale. The safety of moxibustion will be assessed at every visit. DISCUSSION: This trial aims to assess the effectiveness and safety of moxibustion for primary dysmenorrhea, as well as to determine whether the optimal time of treatment for primary dysmenorrhea in clinical practice is before or after the onset of menstrual pain. TRIAL REGISTRATION: Chinese Clinical Trial Register: ChiCTR-TRC-14004627 , registered on 9 May 2014.

Acupuncture Deqi Intensity and Propagated Sensation along Channels May, Respectively, Differ due to Different Body Positions of Subjects.

Acupuncture as an essential component of complementary and alternative medicine is gradually recognized and accepted by the mainstream of contemporary medicine. For obtaining preferable clinical effectiveness, Deqi is commonly regarded as efficacy predictor and parameter which is necessary to be achieved. Influential factors for acupuncture efficacy, like Deqi sensation as well as propagated sensation along channels (PSCs), enjoyed a long history in acupuncture basic research. Concerning this study, taking into account different positions on acupuncture Deqi sensation and PSCs, we would like to attest whether different body positions for subjects during needling procedure yield differed acupuncture Deqi sensation, particularly in terms of intensity, and PSCs. Methods. We used self-controlled method and selected 30 healthy subjects to perform needle insertion at Futu point (ST32) bilaterally. Then they were instructed to record the value of intensity of acupuncture sensation and the length and width of PSCs after removing the needle. Results. In regard to intensity of Deqi, kneeling seat position is stronger than supine position, accounting for 90% of the total number of subjects. In length of PSCs, kneeling seat position is greater than supine position, accounting for 56.7%. In width of PSCs, kneeling seat position is greater than supine position, accounting for 66.7%. Conclusion. Our findings show that needle inserting at Futu point (ST32) in kneeling seat position achieve better needle sensation and provide reference for clinical.