Fluorescent probe-based detection of outer membrane damage of Gram-negative bacteria.

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

New approaches are needed to discover novel antimicrobials, particularly antibiotics that target the Gram-negative outer membrane. By exploiting bacterial sensing and responses to outer membrane (OM) damage, we used a biosensor approach consisting of polymyxin resistance gene transcriptional reporters to screen natural products and a small drug library for biosensor activity that indicates damage to the OM. The diverse antimicrobial compounds that cause induction of the polymyxin resistance genes, which correlates with outer membrane damage, suggest that these LPS and surface modifications also function in short-term repair to sublethal exposure and are required against broad membrane stress conditions.

Open AccessCCS ChemistryRESEARCH ARTICLES11 Mar 2022A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals Wenjuan Song, Zhi-Ling Song, Qian Li, Chengwen Shang, Qiqi Chao, Xingfu Liu, Rongmei Kong, Gao-Chao Fan and Xiliang Luo Wenjuan Song Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 , Zhi-Ling Song *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 , Qian Li Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 , Chengwen Shang Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 , Qiqi Chao Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 , Xingfu Liu Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 , Rongmei Kong School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165 , Gao-Chao Fan Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 and Xiliang Luo *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 https://doi.org/10.31635/ccschem.022.202101618 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Quantifying the microRNAs (miRNAs) levels in living cells, while essential for the study of fundamental biology and medical diagnostics, has barely been achieved due to insufficient probe delivery and unquantifiable signals. We report a cell-anchored and self-calibrated DNA nanoplatform, a cholesterol-headed DNA nanowire that is capable of efficiently delivering to various cells and simultaneously detecting two target miRNAs. One miRNA target can be utilized as an endogenous control against cell-to-cell variations. Moreover, the photocleavable linkers inserted in the nanostructures allow us to precisely regulate the probe structure and fluorescence signaling at the desired time and location in vivo. As a second control, the maximum fluorescence can be elicited by UV light, which further facilitates the normalization of the absolute fluorescence signal. With two introduced internal controls, the maximum fluorescence and endogenous control gene, this approach displays excellent stability and self-calibration performance, effectively avoiding the interference from operating conditions and cell-to-cell variations, such as the laser powers and intracellular probe concentrations. Importantly, this design is capable of unifying the output signal intensity between in vitro test and cell imaging, making the in vitro linear calibration curve appropriate for the quantification of miRNA expression in living cells. Download figure Download PowerPoint Introduction MicroRNAs (miRNAs) are involved in essential physiological processes, including cellular survival, proliferation, apoptosis, and differentiation.1–3 Aberrant miRNA expression is associated with the occurrence and progression of some diseases such as malignant tumors.4,5 Hence, monitoring intracellular miRNA levels is especially important in medical diagnostics. Alternative in vitro methodologies,6–8 such as quantitative real-time polymerase chain reaction (RT-PCR), northern blotting, and microarray screening, while achieving significant advances in detecting the expression levels of miRNAs in cell lysates, are unable to work in living cells. Fluorescence imaging approaches9–13 have been believed to be powerful in cellular biosensing due to in situ visualization, high resolution, and their noninvasive capability. However, quantifying miRNAs in living cells has been limited by insufficient probe delivery and unquantifiable signals. As for the delivery of nucleic acid probes, alternative strategies, including liposomes, inorganic nanomaterials, organic polymers, and proteins, have been proposed for endogenous miRNA analysis.14–17 While these methods have achieved the goal of delivering cargo into specific cells, there is still a need to improve the performance of the delivery system, so as to reduce costs, simplify the preparation process, improve the biocompatibility, and generalize the system to multiple cells. Given these findings, DNA self-assembly based on the hybridization chain reaction (HCR)18–20 is a good choice, which can be easily synthesized by roughly mixing DNA strands. Moreover, to achieve generalized detection, the DNA nanosystem also needs to be able to recognize multiple cells, whether tumor or normal. The hydrophobic DNA label, cholesterol, possesses a strong hydrophobic affinity with the phospholipid bilayer in the cell membrane, and it can bind to different cells without complex chemical modifications, additional genetic manipulation, or metabolic regulation.21–23 Therefore, integrating cholesterol with fluorescent DNA self-assembly will contribute to efficient probe delivery into a variety of cells. Additionally, for further quantitative miRNA analysis in living cells, the most commonly used fluorescent approaches have suffered from the challenges of unequal signal intensity between cell imaging and in vitro test, making the in vitro calibration curve unavailable for intracellular detection. The key factors contributing to these challenges include the inhomogeneity of probe transfection efficiency and the variation of operating parameters, such as the probe concentration and laser power. For the in vitro techniques, such as RT-PCR, a key part in relative quantification of miRNA levels involves the adoption of a control gene, including the endogenous and exogenous control genes.24,25 In particular, miR-16, a stable and highly expressed gene, has been generally utilized as a reference to calibrate the raw experimental data.24 The Miller group26 indicated that the Let-7a and miR-16 genes were suitable endogenous control genes for miRNA analysis in human breast cancer. The Zhang group27 also revealed that miR-16 was one of the most suitable miRNA endogenous control genes across all cell lines. Accordingly, researchers have sought to introduce these control strategies into their in vivo genetic analysis. The Mirkin group28 developed a multiplexed nanoflare labeled with Cy3 and Cy5 fluorophores for simultaneous detection of two mRNA targets (survivin-target and actin-control) inside cells. In other words, these fluorescence probes should carry two independent fluorescence signals, one for the first target, the miRNA of interest, and the other for the second target, the endogenous control gene. However, due to the two distinct excited lasers, the two absolute-fluorescence outputs tend to fluctuate inconsistently with operating parameters, leading to the target-independent cell-to-cell variations. Therefore, quantitative detection of miRNA in living cells remains a major challenge. Motivated by the control strategy26,27 and single laser-excited ratiometric strategy,29,30 we hypothesize that the absolute fluorescence intensity can be normalized by a control fluorescencethat is excited under the same laser parameter. In this regard, the maximum fluorescence of the identical fluorophore is a desirable candidate. The key step toward this goal is to obtain the maximum fluorescence, which requires precise regulation of the probe structure and fluorescence signaling in vivo at the desired time and location. Light tools with low cost, clean energy, and remote controllability have been widely used to manipulate molecular engineering, fluorescence signaling, gene expression, and tumor therapeutics.31–33 The photocleavable (PC) probe is a typical example, whose molecular structure is locked by a PC linker and then cut by light irradiation at the right time and place.31–33 With the advantages of high spatiotemporal resolution, this light-controllable approach allows us to decide when and where to edit the probe structure to trigger the maximum fluorescence and ultimately normalize the initial absolute fluorescence. However, the incorporation of light-controllable technique into the control gene strategies for intracellular quantification assay has not been reported thus far. Herein, we develop a cell-anchored and self-calibrated DNA nanoplatform for in situ imaging and quantification of miRNA in living cells. As shown in Scheme 1, this DNA nanoplatform, a large DNA fluorescence nanowire, is self-assembled by three functional domains (A, H1–P1, and H2–P2). The activator, A strand, induces HCR assembly of hairpin probes H1–P1 and H2–P2 to construct a cholesterol-headed HCR nanowire (HCRW). Cholesterol is known to anchor strongly to the cell membrane. Thus, a strand with the cholesterol label at the head also serves as a guidance for the effective delivery of HCRW nanoprobes to various cells. Two other functional regions are the signaling elements, hairpin probes H1–P1 and H2–P2, which are designed to signal the two targets, the control miRNA and the target miRNA of interest. Furthermore, the H1 and H2 sequences are inserted with the PC-linkers containing an o-nitrobenzyl group, facilitating further spatiotemporal regulation. Scheme 1 | Schematic illustration of the synthesis of HCRW nanoplatform and its application in quantification of endogenous miRNA in living cell. Download figure Download PowerPoint miR-21, a highly expressed tumor marker, is designated as a model target of interest. miR-16 is selected as an endogenous control gene because of its stable expression in all human breast cell lines. In the presence of miR-21 and miR-16, their complementary probe sequences P1 and P2 are released from the HCRW to distance the fluorophores from black hole quenchers (BHQ), leading to the enhancement of the corresponding Cy5 fluorescence (FCy5) and tetramethylrhodamine (TAMRA) fluorescence (FTAMRA). After target-triggered responses, normalization of their absolute signals is performed with reference to the second control, the maximum fluorescence. The UV light irradiation is added to release all BHQ quenchers, thereby triggering the fully recovered maximum fluorescence of Cy5 and TAMRA, namely FCy5-max and FTAMRA-max. Then, the fluorescence recovery efficiencies, called normalized intensities, are calculated by plugging the four absolute fluorescence signals of individual cells into eqs. 1 and 2. Referring to two controls, our designed HCRW adopts the normalized fluorescence ratio (NCy5/NTAMRA) as the final output signal for the quantitative detection of miR-21. Compared with the commonly used signaling modes, it displays excellent stability and self-calibration performance, effectively shielding the influences of various experimental conditions such as the laser power and probe concentration. In this way, the linear calibration curve obtained in vitro allows for the miRNA quantification in living cells. Accordingly, the mean concentrations of endogenous miR-21 in the MCF-7, MDA-MB-231, and MCF-10A cells have been calculated to be 7.09, 2.15, and 0.75 nM, respectively, presenting a higher expression of miR-21 in cancer cells than in normal cells. This approach provides a solution for in situ quantitative detection of miRNA in living cells, and it can also be extended to the analysis of other biomolecules. Experimental Methods Preparation of HCRW Before the synthesis of the nanoprobe, all the DNA sequences were solved in phosphate-buffered saline (PBS) solution. First, hairpin DNA sequences H1 and H2 were heated at 90 °C for 10 min and then slowly cooled to room temperature for the later experiment. Next, equivalent probe sequences P1 and P2 were incubated with H1 and H2 for 30 min at 37 °C, respectively, so as to form the monomers H1–P1 and H2–P2. Finally, a mixture of H1–P1, H2–P2, and A was incubated at 37 °C for 1 h to trigger the HCR self-assembly, and then the HCRW was synthesized. All nucleotide sequences used in this study were shown in Supporting Information Tables S1–S3. Verification of the fluorescence sensing capacity of HCRW HCRW nanoprobes (10 μL) were added to 90 μL PBS solution containing different concentrations of targets (miR-21 and miR-16). After incubation for 40 min at 37 °C, the fluorescence changes were monitored by an F-7000 fluorescence spectrophotometer (Hitach, Japan). The independent fluorescence signals of TAMRA and Cy5 were detected under the excitation wavelengths of 543 and 633 nm, respectively. All experiments were conducted at least three times. In vitro UV irradiation test The PC-linkers inserted in the HCRW nanostructures allowed us to precisely regulate the probe structure and fluorescence signaling at the right time and place. To investigate this, a nanostructure without PC-linkers, HCRW-noPC, was synthesized for comparision. The common UV light (365 nm, 1 W) was used to cleave the PC-linkers and trigger the maximum fluorescence signals. The HCRW and HCRW-noPC solutions were irradiated under UV light for different periods of time, ranging from 0 to 10 min, and then the dynamic fluorescence signals were monitored with the F-7000 fluorescence spectrophotometer. In this way, the optimization of irradiation time was also conducted. Stability and self-calibration test Laser power was used as a model experimental variate to explore the detection performance of the HCRW probe. Typically, 10 μL HCRW nanoprobes were added to 90 μL PBS solution containing fixed miR-16 (5 nM) and different concentrations of miR-21. After incubation for 40 min at 37 °C, the target-triggered fluorescence signals of TAMRA and Cy5 were detected under different laser powers and read as FCy5 and FTAMRA, respectively. Next, the mixed solution was irradiated under UV light for 6 min to elicit the maximum fluorescence signals, FCy5-max and FTAMRA-max, which were detected under different laser powers. Then, the normalized fluorescence intensities, NCy5 and NTAMRA, were calculated by plugging the data into eqs. 1 and 2. Finally, the normalized ratio, NCy5/NTAMRA, was used as the final output signal. A comparison among the absolute intensity FCy5, normalized intensity NCy5, absolute ratio FCy5/FTAMRA, and normalized ratio NCy5/NTAMRA was performed to demonstrate the changes of detection signal under different laser powers. The test under varying probe concentrations was similar to the above steps, except that the laser power was constant throughout the test, while the probe concentration was changed. All the experiments were conducted at least three times. Confirmation of cholesterol-guided intracellular delivery The cholesterol label plays an important role in effectively guiding the delivery of nanoprobe to various cells. A cholesterol-headed HCR flare system (Cho-HCRF) was labeled with Cy5 and TAMRA to light up the transfection trace along with its intracellular location. Meanwhile, an HCR flare system without cholesterol label, HCRF, was also synthesized for comparison. Before the addition of probes, MCF-7 cells were preinoculated in 96-well plates at 37 °C for 24 h and then washed with PBS solution three times. Next, 150 μL 250 nM Cho-HCRF or HCRF probes were added to each well and incubated at 37 °C for 50 min. During this time period, real-time confocal imaging was performed on the cells at 10, 20, 30, 40, and 50 min. Fluorescence images were captured under a 10× objective lens (Leica, Wetzlar, Germany). The laser with a wavelength of 543 nm/633 nm was adopted as the excitation source of Cy5 (red)/TAMRA (green) fluorescence, and the bandpass filter range was set at 570–610 nm/650–690 nm. Optimization of UV irradiation time of HCRW in living cells MCF-7 cells were preinoculated in 96-well plates at 37 °C for 24 h and then washed three times with PBS. Then, 150 μL 250 nM HCRW was added into each well and incubated at room temperature under dark conditions for 40 min. After washing three times with PBS buffer, the MCF-7 cells were exposed to UV irradiation for different times (0, 1, 5, 10, and 15 min). Then, the temporal changes of Cy5 and TAMRA fluorescence in the living cells were measured with confocal microscopy. Fluorescence imaging in live cells For the in situ imaging of living cells, the HCRW nanoprobes were incubated with MCF-7 MDA-MB-231 and MCF-10A cells for 40 min under dark conditions in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Then the cell imaging was analyzed with confocal microscopy under the lasers of 543 and 633 nm, and the emissions of TAMRA and Cy5 were collected in the green and red channels, respectively. Next, the cells were exposed to UV irradiation for 10 min. Finally, the maximum fluorescence of Cy5 and TAMRA in the cells was measured with confocal microscopy. Results and Discussion Construction and characterization As shown in Scheme 1, the HCRW nanoplatform was synthesized starting from the HCR self-assembly of five DNA oligonucleotides (H1, H2, P1, P2, and A). miR-21 was designated as a target gene in this work because of its high relevance in tumor progression.34–37 miR-16 was chosen as another target and an endogenous control gene, which was stably and highly expressed across human breast cells and frequently used as a control in conventional biological methodologies. The probing sequence P1 was partially hybridized with PC-linker-embedded hairpin strand 1 (termed H1), and then the hairpin probe H1–P1 capable of binding miR-21 was prepared. Another hairpin probe H2–P2 for miR-16 was also formed in this way. Different dyes (Cy5 for miR-21 and TAMRA for miR-16) were labeled into the 5′ ends of the probe sequences of HCRW to make the independent determination of each target. The synthetic hairpin probes were then mixed with cholesterol-guided activator (termed A) to induce HCR self-assembly, thus constructing the large nanowire HCRW. A polyacrylamide gel electrophoresis (PAGE) assay was used to characterize the synthesis of HCRW. In Figure 1a, the gel migration rate slowed with the sequential addition of five DNA oligonucleotides, indicating successful synthesis of HCRW (Figure 1a, lanes 1–6). Moreover, the target recognition performance of P1 and P2 in this large nanowire remained good, as evidenced by an extra accelerated band in lane 7 compared with lane 6. Figure 1 | Characterization and verification of HCRW. (a) Native PAGE analysis. From lanes 0 to 8: lane 0: marker; lane 1: H1; lane 2: P1; lane 3: H1 + P1; lane 4: H1 + P1 + H2 + P2; lane 5: A; lane 6: lane 4 + A; lane 7: lane 6 + DNA21 + DNA16; lane 8: DNA21 + DNA16. Kinetic curve responses of HCRW platform for 20 nM miR-21 (b) and 5 nM miR-16 (c), where FCy5 represents the absolute intensity of Cy5 fluorescence and FTAMRA represents the absolute intensity of TAMRA fluorescence. (d) Original Cy5 fluorescence spectra of HCRW platform (50 nM) in response to different concentrations of miR-21 (0, 0.5, 2, 5, 10, 20, 30, 40, 50, and 100 nM). (e) Original TAMRA fluorescence spectra of HCRW platform (50 nM) in response to different concentrations of miR-16 (0, 0.5, 2, 5, 10, 20, 30, 40, 50, and 100 nM). Download figure Download PowerPoint Next, the fluorescence-sensing capacity of the HCRW was investigated. As designed in Supporting Information Figure S1a, in the initial HCRW nanostructure, the fluorescence of TAMRA and Cy5 was greatly quenched by the proximal BHQ quenchers. In the presence of miR-21 target, it hybridized with the P1 strand to form a completely complementary duplex, while Cy5 fluorophore was far away from the BHQ quencher, generating an enhanced Cy5 fluorescence. As shown in Figure 1b, the Cy5 fluorescence at 663 nm showed significant increase with the addition of miR-21. This effect was also observed in the fluorescence behavior of TAMRA at 582 nm after the addition of miR-16 (Figure 1c). In addition, HCRW demonstrated gradual enhancement in Cy5 fluorescence (FCy5) with increasing miR-21 concentration (Figure 1d and Supporting Information Figure S1). Moreover, a linear relationship (FCy5 = 112.2 + 45.7x) for target miR-21 in the range of 0.5–40 nM has been found, and the limit of detection was 127 pM (S/N = 3). FCy5 and x represent the Cy5 fluorescence intensity and miR-21 concentration, respectively. Meanwhile, the HCRW nanoprobe showed good specificity ( Supporting Information Figure S1) for miR-21 detection. with miR-16 concentration P2 to from H1 leading to enhanced TAMRA fluorescence (Figure and Supporting Information Figure Therefore, an in the absolute intensity of TAMRA fluorescence was observed with increasing miR-16 concentration ( Supporting Information Figure In addition, the specificity assay that enhanced TAMRA fluorescence was by miR-16 ( Supporting Information Figure To further that the probe recognition was and fluorescence detection was the of miR-16 on the detection of miR-21 was ( Supporting Information Figure The fluorescence response for miR-21 was by miR-16, and the two corresponding calibration for target miR-21 with and without the addition of miR-16 with each other ( Supporting Information all the indicated that HCRW allowed for the response to and miR-16 targets with two independent fluorescence signals in a in Stability and self-calibration In addition to the of its stability and self-calibration were also important to the of intracellular miRNA To the of unequal signals in different experimental this HCRW probe was used to detection in two (Figure First, under the trigger of miR-21 and miR-16 targets, the absolute fluorescence signals FCy5 and FTAMRA were The second step this target-triggered response was the of maximum fluorescence signals and under UV which was by the fluorescence changes of HCRW ( Supporting Information Figure After these fluorescence data and were Accordingly, the two absolute fluorescence signals be easily normalized by the maximum fluorescence signals to 1, the normalized signals were 1 and 2. Next, the ratio of normalized fluorescence as the final output signal for the miR-21 detection. We then the stability of fluorophores under UV irradiation ( Supporting Information Figure further The fluorescence changes of Cy5 and TAMRA indicated that the UV light the of the fluorophores ( Supporting Information 5 = 5 5 TAMRA = TAMRA TAMRA 5 TAMRA = ( 5 TAMRA ( 5 TAMRA Figure | of the response of fluorescence methods and this for the detection of target miR-21 under different laser powers. (a) Schematic illustration of HCRW for the detection of target miR-21 under different laser powers. (b) The fluorescence responses of HCRW platform (50 nM) for the 10 nM target miR-21 under different laser when the miR-16 was fixed at 5 The maximum fluorescence spectra of HCRW platform after UV light (d) The responses of the absolute fluorescence intensity FCy5 of HCRW platform for the 10 nM target miR-21 under different laser powers. (e) The corresponding (NCy5/NTAMRA) of normalized fluorescence intensity NCy5 to under different laser powers. The linear of absolute fluorescence FCy5 the concentration of miR-21 under different laser powers. The linear of NCy5/NTAMRA the concentration of miR-21 under different laser powers. Download figure Download PowerPoint As a the probe should be able to calibrate the of different operating parameters, a stable and detection signal to the same target. The laser power was used as a model operating variate to explore the detection behavior of the HCRW probe (Figure The fluorescence changes of the Cy5 and TAMRA were in solution. First, the concentrations of miR-21 and miR-16 in solution were fixed at 10 and 5 nM, respectively. Next, the target miR-21 signal was read as the ratio of NCy5 to NTAMRA, namely NCy5/NTAMRA, while miR-16 was utilized as the first control and maximum fluorescence as the second control for normalization in fluorescence For the fluorescent methods based on other signaling output modes, such as absolute intensity FCy5, normalized intensity NCy5, and absolute ratio FCy5/FTAMRA, were also Figure and Supporting Information Figure the signal changes of HCRW to the same targets under different laser powers. the concentration of target miR-21 was a large variation was observed in target-triggered Cy5 fluorescence (Figure and the maximum Cy5 fluorescence also the same (Figure and Supporting Information Figure During this process, the TAMRA fluorescence signals were also monitored for the further of output signals ( Supporting Information Figure The absolute signal FCy5 was (Figure The absolute ratio obtained by the absolute fluorescence response of miR-21 to that of miR-16, was also greatly ( Supporting Information Figure The normalized approach a in the of the detection signal when the absolute

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli

Chitosan kills bacteria through cell membrane damage

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.

Chitosan kills bacteria through cell membrane damage

Antimicrobial action of novel chitin derivative

Sensitivities to polycationic peptides and EDTA were compared in Yersinia enterocolitica pathogenic and environmental biogroups. As shown by changes in permeability to the fluorescent hydrophobic probe N-phenylnaphthylamine (NPN), the outer membranes (OMs) of pathogenic and environmental strains grown at 26 degrees C in standard broth were more resistant to poly-L-lysine, poly-L-ornithine, melittin, cecropin P1, polymyxin B, and EDTA than Escherichia coli OMs. At 37 degrees C, OMs of pathogenic biogroups were resistant to EDTA and polycations and OMs of environmental strains were resistant to EDTA whereas E. coli OMs were sensitive to both EDTA and polycations. Similar results were found when testing deoxycholate sensitivity after polycation exposure or when isogenic pairs with or without virulence plasmid pYV were compared. With bacteria grown without Ca++ available, OM permeability to NPN was drastically increased in pathogenic but not in environmental strains or E. coli. Under these conditions, OMs of pYV+ and pYV- cells showed small differences in NPN permeability but differences in polycation sensitivity could not be detected by fluorimetry. O:1,6 (environmental type) lipopolysaccharide (LPS), but not O:3 or O:8 LPS, was markedly rough at 37 degrees C, and this could explain the differences in polycation sensitivity. LPSs from serotypes O:3 and O:8 grown at 37 degrees C were more permeable to NPN than O:1,6 LPS, and O:8 LPS was resistant to polycation-induced permeabilization. These data suggest that LPSs relate to some but not all the OM differences described. It is hypothesized that the different OM properties of environmental and pathogenic biogroups reflect the adaptation of the latter biogroups to pathogenicity.

The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 micrograms/ml concanavalin A (Con A) for 30 min at 37 degrees C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by 3H-thymidine assay. Increased fluorescence was not observed in the presence of 10 mM alpha-methyl mannoside, 5 mM sodium azide, 10(-5) M cytochalasin B, or Ca2+-free culture medium. However, incubation with 10(-5) M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response. Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweek mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.

On-line Fluorescence Determination of Pressure Mediated Outer Membrane Damage in Escherichia coli

We have studied the action of human complement (C) on E. coli membranes. We find, as have others, that C disrupts the outer membrane (OM), allowing the release of periplasmic proteins. In addition, we have found 1) that in the complete absence of lysozyme, C damages the inner membrane (IM), 2) IM damage is different from OM damage in that only small molecules traverse a damaged IM whereas macromolecules traverse damaged OM, 3) IM damage and OM damage occur with identical kinetics and dose response, suggesting that IM and OM damage are closely coupled events, and 4) upon the addition of purified C8 and C9 to the washed cellular intermediate, E. coli C 1-7, both IM and OM are damaged coordinately. These results, taken together, suggest that C damages E. coli membranes by acting at a site contiguous with both membranes. We speculate that C may simultaneously gain access to both membranes by acting at the junctions between IM and OM.

Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.

Fluorescence methods have proven to be extremely useful tools for quantitative studies of the equilibria and kinetics of protein-DNA interactions. If the protein contains tryptophan (Trp), as is often the case, and there is a change in intrinsic Trp fluorescence of the protein, one can use this change in signal (quenching/enhancement) to monitor binding. One can also attach an extrinsic fluorophore to either the protein or the DNA and monitor binding due to a change in fluorescence intensity or a change in fluorescence anisotropy. Such equilibrium studies can provide important quantitative information on stoichiometries (occluded site size, number of binding sites) and energetics (affinities and cooperativities) of the interactions. This information is needed to understand the mechanisms of protein-DNA interactions. A critical aspect of such approaches for systems that have non-unity stoichiometries (e.g., a protein that binds multiple ligands) is knowledge of the relationship between the change in fluorescence signal (intensity or anisotropy) and the average extent of binding. Here we describe procedures for using fluorescence approaches to examine the stoichiometries and equilibrium binding affinities of Escherichia coli single-stranded DNA-binding protein (SSB) and Deinococcus radiodurans SSB with long polymeric ssDNA to determine an occluded site size. We also provide examples of studies of SSB binding to shorter oligonucleotides to demonstrate analysis and fitting of the data to an appropriate model (monitoring fluorescence intensity or anisotropy) to obtain quantitative estimates of equilibrium binding parameters. We emphasize that the solution conditions (especially salt concentration and type) can influence not only the binding affinity, but also the mode by which an SSB oligomer binds ssDNA.

Development of fluorescent probes for selectively detecting two or more events simultaneously is imperative for further application in bio-detecting area. Recently, an intelligent fluorescent probe based on Changsha ( CS NIR ) dye and arylboronic acid ( CSBOH ) that can selectively detect H 2 O 2 in alkaline environment was reported. The probe can detect both H 2 O 2 and pH change simultaneously with different fluorescent signal changes, which will be more useful in the H 2 O 2 detection in living organisms. CSBOH exhibited remarkably different fluorescence changes at 650 nm and 720 nm in the presence of H 2 O 2 in different pH buffers when excited at 560 and 670 nm, respectively. The cell experiments demonstrated that CSBOH can image endogenously generated H 2 O 2 in Macrophages and A431 cells. CSBOH was also used to image H 2 O 2 in living animals successfully.