Rational Development of a New Reaction-Based Ratiometric Fluorescent Probe with a Large Stokes Shift for Selective Detection of Bisulfite in Tap Water, Real Food Samples, Onion Tissues, and Zebrafish.

Bisulfite (HSO3-) is usually widely added to tap water and food because it has antibacterial, bleaching, and antioxidant effects. However, its abnormal addition would cause a series of serious diseases related to it. Therefore, development of an effective method for HSO3- detection was of great significance to human health. In this work, a new reaction-based ratiometric fluorescent probe KQ-SO2 was rationally designed, which could be used for the highly selective detection of HSO3- in tap water, real food samples, onion tissues, and zebrafish. Specifically, a positively charged benzo[e]indolium moiety and a carbazole group through a condensation reaction resulted in KQ-SO2, which displayed two well-resolved emission bands separated by 225 nm, fast response (1 min), and high selectivity and sensitivity toward HSO3- upon undergoing the Michael addition reaction, as well as low cytotoxicity in vitro. In addition, KQ-SO2 has been successfully applied for the detection of HSO3- in tap water, real food samples, onion tissues, and zebrafish with satisfactory results. We predict that KQ-SO2 could be used as a powerful tool to reveal the relationship between HSO3- and the human health.

A novel fluorescent probe for ratiometric detection of formaldehyde in real food samples, living tissues and zebrafish.

Herein, a two-photon (TP) ratiometric fluorescent probe (NpFA) was developed for detecting formaldehyde (FA) in real food samples, living onion tissues and zebrafish by fluorescence resonance energy transfer (FRET) strategy. Specifically, a TP fluorophore as the donor and a FA turn-on naphthalimide fluorophore as the acceptor were connected by a non-conjugated linker to construct the TP-FRET-based NpFA, which exhibited a target-modulated ratiometric fluorescence response to FA rapidly with high selectivity and sensitivity during 65 s, and a large ratio ~5-fold enhancement at I550/I410 after addition of FA, displaying ~60-fold enhancement at 550 nm and a quite low DOC of 5.8 ± 0.2 nM. Moreover, NpFA has a good imaging resolution and depth of deep tissue penetration. Therefore, based on the above results, NpFA has the capability to be a useful tool for investigating FA in real samples application, and we also hope NpFA will further study of the physiological and pathological function of FA.

TP-FRET-Based Fluorescent Sensor for Ratiometric Detection of Formaldehyde in Real Food Samples, Living Cells, Tissues, and Zebrafish.

Formaldehyde (FA, HCHO) is a highly reactive carbonyl species, which is very harmful to humans and the environment as a tissue fixative and preservative. Therefore, developing some highly sensitive, selective, and rapid detection methods is significant for human health in food safety and environmental protection. Herein, a two-photon (TP) ratiometric sensor, CmNp-CHO, has been constructed by conjugating a TP donor (Π-push-pull-structure) with a FA off-on acceptor (functioned with hydrazide moiety) via a nonconjugated linker through the fluorescence resonance energy transfer mechanism. Such a scaffold affords CmNp-CHO a reliable and specific probe for detecting FA with two well-resolved emission peaks separated by 124 nm. Also, it responds to FA rapidly with high selectivity and sensitivity during 1.0 min and a large ratio enhancement at I550/I426 with addition of 0-20µM FA, exhibiting ∼4-fold ratio increase and a fairly low LOD of 8.3 ± 0.3 nM. Moreover, CmNp-CHO has been successfully employed for detecting FA in live cells, onion tissues, and zebrafish, exhibiting that CmNp-CHO can serve as a useful tool for investigating FA in real food application and offering strong theoretical support and technical means for investigation of physiological and pathological functions of FA.

An efficient TP-FRET-based lysosome-targetable fluorescent probe for imaging peroxynitrite with two well-resolved emission channels in living cells, tissues and zebrafish.

Peroxynitrite (ONOO-), an important role of reactive oxygen species (ROS) in vivo, and studies showed abnormal of ROS can induce lysosomal membrane permeabilization (LMP) and lead to the death of cells. Thus, it is of great significance for designing an effective method for investigating relationship between physiology and pathology between ONOO- and lysosome. Herein, for the first time, we adopted a Förster resonance energy transfer (FRET) strategy to construct a lysosome-targetable small molecular ratiometric two-photon (TP) fluorescent probe (NpRh-ONOO) for detecting ONOO- in living cells, tissues and zebrafish. Specifically, a TP fluorophore and a rhodamine B fluorophore are directly connected by a flexible piperidine linker to form the TP-FRET-scaffold, a hydrazide as ONOO- reactive set, and a dimethylamino as lysosome targeting-group, which shows a target-modulated ratiometric TP fluorescence response, two well-resolved emission peaks separated by 73 nm, rapid response (<10 s), high selectivity and sensitivity with the detection limit is as low as 3.3 nM for ONOO-. These prominent features of probe were then applied for ratiometric bioimaging both exogenous and endogenous peroxynitrite in living cells, tissues and zebrafish, demonstrating it can be used as a powerful tool for biological research of lysosomal peroxynitrite in biological systems.

An effective FRET-based two-photon ratiometric fluorescent probe with double well-resolved emission bands for lysosomal pH changes in living cells and zebrafish.

In cells, lysosome is an acidic organelle (approximately pH 4.5-5.5), whose pH changes plays a key role in mediating various biological processes. To address this issue, a lot of fluorescent probes have been developed and prepared for tracking lysosomal pH changes. However, few of these probes can realize the imaging of lysosomal pH changes in biosystems. Herein, a new two-photon (TP) ratiometric fluorescent probe (NpRhLys-pH) by adopting the fluorescence resonance energy transfer (FRET) strategy has been developed for imaging of lysosomal pH changes in living cells and zebrafish. In this probe NpRhLys-pH, constructed by conjugating a TP fluorophore (D-Π-A-structured naphthalimide derivative) with a rhodamine B fluorophore via a non-conjugated flexible linker, the morpholine moiety serves as a targeting unit for anchoring lysosomes, and the xanthane derivative shows a pH-modulated open/close form of the spirocycle. Such a scaffold affords the NpRhLys-pH is a reliable and specific probe for anchoring lysosomes in living cells and zebrafish with dual-channel emission peaks separated by 85 nm, and responds to lysosomal pH rapidly and reversibly with high selectivity and sensitivity, demonstrating it can be used as a powerful tool for the biological research of the relationship between physiology and pathology and lysosomal pH changes in biological systems.

A TP-FRET-based fluorescent sensor for ratiometric visualization of selenocysteine derivatives in living cells, tissues and zebrafish.

Selenium is a biologically essential micronutrient element serving as an essential building block for selenoproteins (SePs), which is playing a key role in various cellular functions. Hence, it is of great significance to developing a reliable and rapid method for detection of Sec in biosystems. Compared with the previously reported probes that have been developed for selective detection of Sec, two-photon (TP) ratiometric Sec-specific probes would be advantageous for the NIR excitation and built-in correction of the dual emission bands. To quantitatively and selectively detect Sec over biothiols with rapid and sensitive response, we for the first time report a new fluorescence resonance energy transfer (FRET)-based TP ratiometric fluorescence probe CmNp-Sec, which was constructed by conjugating a TP fluorophore 6 (coumarin derivative with a D-π-A-structure) with a naphthalimide fluorophore 9 via a non-conjugated linker, and employed a 4-dinitrobenzene-ether (DNB) with a strong ICT effect as Sec responsive moiety. It exhibits quantitatively detect Sec in a wide range (0-50 µM) with a limit of detection of 7.88 nM within 10 min. More impressively, this probe can be conveniently used to detect Sec in living cells, tissues and zebrafish, demonstrating it has the latent capability in further biological applications.

Machine learning-powered antibiotics phenotypic drug discovery.

Identification of novel antibiotics remains a major challenge for drug discovery. The present study explores use of phenotypic readouts beyond classical antibacterial growth inhibition adopting a combined multiparametric high content screening and genomic approach. Deployment of the semi-automated bacterial phenotypic fingerprint (BPF) profiling platform in conjunction with a machine learning-powered dataset analysis, effectively allowed us to narrow down, compare and predict compound mode of action (MoA). The method identifies weak antibacterial hits allowing full exploitation of low potency hits frequently discovered by routine antibacterial screening. We demonstrate that BPF classification tool can be successfully used to guide chemical structure activity relationship optimization, enabling antibiotic development and that this approach can be fruitfully applied across species. The BPF classification tool could be potentially applied in primary screening, effectively enabling identification of novel antibacterial compound hits and differentiating their MoA, hence widening the known antibacterial chemical space of existing pharmaceutical compound libraries. More generally, beyond the specific objective of the present work, the proposed approach could be profitably applied to a broader range of diseases amenable to phenotypic drug discovery.

A mitochondria targetable two-photon excited near-infrared fluorescent probe for imaging of H2O2 in live cells and tissues.

Hydrogen peroxide (H2O2), a member of small-molecule reactive oxygen species (ROS), plays an important role in physiological and/or pathological process within live systems. Herein, to quantitatively investigate the biological role of H2O2 in subcellular level, we constructed of a novel two-photon (TP) in near-infrared (NIR) out fluorescent probe (TP-NIR-H2O2) for visualization of mitochondria H2O2 in living cells and tissues. Specifically, TP-NIR-H2O2 utilized the oxonium ion as the mitochondrial targeting unit and phenylboric acid as the H2O2 reaction moiety. After the phenylboric acid moiety reaction with H2O2, TP-NIR-H2O2 displayed a ~105-fold fluorescence intensity enhancement in 665 nm. Selectivity experiment demonstrated that the probe can detect H2O2 with high selectivity over other ROS. Moreover, TP-NIR-H2O2 could be employed for imaging H2O2 in mice liver tissues with large tissue-image depth (50-170 µm) under two-photon excitation (800 nm).

Evolution of Cancer Registration Combining Online Reporting with Follow-up in the Community: Practices in Guangzhou, China

Background: An efficient registration system with accurate and timely information on cancer incidence and mortality is key to development of policies to prevent and control cancer. A traditional registration system usually needs 3-4 years to collect data and publish a cancer report. However, researchers, policymakers and healthcare professionals need to know the latest cancer registration data quickly. Methods: A computer system has been operating with cases reported online by hospitals and followed up in communities at the Cancer Registry of Guangzhou (CRG) since 2008. The comparability, completeness, accuracy and timeliness of collected data were here evaluated. Results: From 2010 to 2014, 181,194 cancer cases from 1,916,253 medical records of cancer were reported to the CRG online. 53,473 cases were deleted as duplicates (47,906), wrong diagnoses (410) or residents of other places (5,157) during the follow up. Successful final follow-up rates were over 90% for both newly and previously diagnosed cases by general practitioners in community clinics. The CRG coding and classification system follows international standards. The annual trends for all sites by sex were stable from 2010 to 2014. All age-specific incidence rates for childhood cancers were within the limits of the respective international references. The overall M:I ratio for all sites but C44 was 56.7%.,ratios for most sites in Guangzhou being between Hong Kong and Shanghai. A total of 75.7% of the cancer cases reported in 2010­2012 were morphologically verified. Ninety five percent of new cases completed registration within 29.0 months in 2010, reducing to 8.0 months in 2014. Conclusion: The online report system with community follow up at the CRG yields reasonably accurate and close-to-complete data. It takes less time to confirm diagnosis and other information so that reporting annual incidence one year after the close of registration becomes possible.

Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screening Platform.

Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC50 < 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors.

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.

Evaluation of Intravitreal Ranibizumab on the Surgical Outcome for Diabetic Retinopathy With Tractional Retinal Detachment.

This study aims to investigate intravitreal injection of Ranibizumab on the surgical outcome for diabetic patients who had tractional retinal detachment but did not receive any preoperative retinal photocoagulation.Ninety-seven patients (97 eyes) who had diabetic retinopathy with tractional retinal detachment were enrolled to receive 23-G pars plana vitrectomy (PPV). They were assigned to an experimental group (Group I, n = 47 eyes) and a control group (Group II, n = 50 eyes). The patients in Group I were given 1 injection of intravitreal Ranibizumab (Lucentis 0.5 mg/0.05 mL) 1 week before surgery, whereas those in Group II went down to surgery directly. Follow-ups were performed for 6 months to 3 years (16 ±â€Š6 months), and indicators observed included postoperative best-corrected visual acuity, complications, and retinal thickness in the macula measured by optical coherence tomography.In Group I, BCVA improved from logMAR 1.92 ±â€Š0.49 to logMAR 0.81 ±â€Š0.39 following surgery, whereas in Group II, BCVA improved from logMAR 1.91 ±â€Š0.49 to logMAR 0.85 ±â€Š0.41. There was significant postoperative gain in vision, but there was no significant difference between the 2 groups at postoperative follow-up visits. The mean duration of vitrectomy in Group I and Group II was (40 ±â€Š7) minutes and (53 ±â€Š9) minutes, respectively, with significant difference. Iatrogenic breaks were noted in 5 eyes (11%) in the experimental group and 17 eyes (34%) in the control group; the difference was significant. The retinal thickness in the macula measured by OCT was (256 ±â€Š44) µm and (299 ±â€Š84) µm in Group I and Group II respectively with significant difference. Besides, there were significantly more eyes in Group II that required silicone oil tamponade and postoperative retinal photocoagulation.23-G PPV combined with intravitreal tamponade and panretinal photocoagulation still remains an effective regimen for the treatment of diabetic retinopathy complicated with tractional retinal detachment. Preoperative intravitreal injection of Ranibizumab could shorten surgical duration, reduce intraoperative complications, and sometimes spare the need for silicone oil tamponade and postoperative retinal photocoagulation, alleviating patients' suffering from surgery.

The relationship between meteorological factors and mumps incidence in Guangzhou, China, 2005-2012:.

BACKGROUND: Over the past decade, there have been resurgences and large-scale outbreaks of mumps worldwide. Little evidence is available on the relationship between meteorological factors and the incidence of mumps. We aimed to explore the effects of meteorological factors on mumps incidence. METHODS: A Poisson regression model combined with a distributed lag non-linear model (DLNM) was used to evaluate the association between meteorological factors and the mumps incidence in Guangzhou, China, 2005-2012. RESULTS: Nonlinear relationships between meteorological factors, except sunshine hours, and mumps incidence were observed. The relative risks (RRs) of mean temperature, relative humidity and atmospheric pressure were 1.81 (95% confidence interval (CI), 1.41 to 2.32), 1.28 (95% CI, 1.02 to 1.59), and 0.80 (95% CI, 0.67 to 0.95) comparing the 99th percentile to the median of their own, respectively. For wind velocity, the RR was 0.70 (95%CI, 0.54 to 0.91) comparing the 1st percentile to the median. The hot effect and cold effect were larger in females than in males, and the hot effect increased with age. CONCLUSIONS: Mean temperature, relative humidity, wind velocity and atmospheric pressure might be important predictors of the mumps incidence. Tropical cyclone caused a higher increase in mumps cases. Our findings highlight the need to strengthen the awareness of using protective measures during typhoon days and allocating more attention to the susceptible populations during the summer. The two-dose regimen of mumps vaccine should be included in the National Immunization Program schedule, and the catch-up vaccination campaigns should be promoted among adults.

Nucleotide and phospholipid-dependent control of PPXD and C-domain association for SecA ATPase.

The SecA ATPase motor is a central component of the eubacterial protein translocation machinery. It is comprised of N- and C-domain substructures, where the N-domain is comprised of two nucleotide-binding domains that flank a preprotein-binding domain (PPXD), while the C-domain binds phospholipids as well as SecB chaperone. Our recent crystal structure of Bacillus subtilis SecA protomer [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] along with experimental support for the correct dimer structure [Ding, H., Hunt, J. F., Mukerji, I., and Oliver, D. (2003) Biochemistry 42, 8729-8738] have now allowed us to study SecA structural dynamics during interaction with various translocation ligands and to relate these findings to current models of SecA-dependent protein translocation. In this paper, we utilized fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues within the PPXD and C-domains of SecA to investigate the interaction of these two domains and their response to temperature, model membranes, and nucleotide. Consistent with the crystal structure of SecA, we found that the PPXD and C-domains are proximal to one another in the ground state. Increasing temperature or binding to model membranes promoted a loosening of PPXD and C-domain association, while ADP binding promoted a tighter association. A similar pattern of PPXD and C-domain association was obtained also for Escherichia coli SecA protein. Furthermore, a hyperactive Azi-PrlD SecA protein of E. coli had increased PPXD and C-domain separation, consistent with its activation in the ground state. Interestingly, PPXD and C-domain separation occurred prior to the onset of major temperature-induced conformational changes in both the PPXD and C-domains of SecA. Our results support a model in which PPXD and C-domain proximity is important for regulating the initial stages of SecA activation, and they serve also as a template for future structural studies aimed at elucidation of the chemomechanical cycle of SecA-dependent protein translocation.

Bacillus subtilis SecA ATPase exists as an antiparallel dimer in solution.

SecA ATPase promotes the biogenesis of membrane and secretory proteins into and across the cytoplasmic membrane of Eubacteria. SecA binds to translocon component SecYE and substrate proteins and undergoes ATP-dependent conformational cycles that are coupled to the stepwise translocation of proteins. Our recent crystal structure of B. subtilis SecA [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] showed two different dimer interactions in the lattice which both buried significant solvent-accessible surface area in their interface and could potentially be responsible for formation of the physiological dimer in solution. In this paper, we utilize fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues to determine the oligomeric structure of SecA protein in solution. Our results show that of the two dimers interactions observed in the crystal structure, SecA forms an antiparallel dimer in solution that maximizes the buried solvent-accessible surface area and intermolecular contacts. At the submicromolar protein concentrations used in the fluorescence experiments, we saw no evidence for the formation of higher-order oligomers of SecA based on either the alternative dimer or the 3(1) helical fiber observed in the crystal lattice. Our studies are consistent with previous ones demonstrating the existence of a dimerization determinant within the C-domain of SecA as well as those documenting the interaction of N- and C-domains of SecA. Our results also provide a valuable starting point for a determination of whether the subunit status of SecA changes during the protein translocation as well as studies designed to elucidate the conformational dynamics of this multidomain protein during its translocation cycle.