The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H(+) symport, an outward-facing cavity is thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.
Metallic subwavelength apertures can be used in epi-illumination fluorescence to achieve focal volume confinement. Because of the near field components inherent to small metallic structures, observation volumes are formed that are much smaller than the conventional diffraction limited volume attainable by high numerical aperture far field optics (circa a femtoliter). Observation volumes in the range of 10−4fl have been reported previously. Such apertures can be used for single-molecule detection at relatively high concentrations (up to 20μM) of fluorophores. Here, we present a novel fabrication of metallic subwavelength apertures in the visible range. Using a new electron beam lithography process, uniform arrays of such apertures can be manufactured efficiently in large numbers with diameters in the range of 60–100nm. The apertures were characterized by scanning electron microscopy, optical microscopy, focused ion beam cross sections/transmission electron microscopy, and fluorescence correlation spectroscopy measurements, which confirmed their geometry and optical confinement. Process throughput can be further increased using deep ultraviolet photolithography to replace electron beam lithography. This enables the production of aperture arrays in a high volume manufacturing environment.
We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.
Colorectal cancer (CRC) is the third most common cancer in the world in terms of morbidity and mortality, which brings great health hazards and economic burdens to patients and society. A fecal examination is an effective method for clinical examination and the most commonly used method for the census. It is simple, non-invasive, and suitable for large-scale population screening. With the development of molecular biology, lots of efforts have been made to discover new fecal biomarkers for the early screening of colorectal cancer. In this review, we summarize and discuss the recent advances of fecal biomarkers for CRC screening or diagnosis, including DNA biomarkers, RNA biomarkers, protein biomarkers, gut microbes and volatile organic compounds focusing on their diagnostic evaluation for CRC, which can provide a basis for the further development of new and effective CRC fecal screening and early diagnosis techniques.
We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 μs and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Förster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein.
Peptide-coated quantum dot−photosensitizer conjugates were developed using novel covalent conjugation strategies on peptides which overcoat quantum dots (QDs). Rose bengal and chlorin e6, photosensitizers (PSs) that generate singlet oxygen in high yield, were covalently attached to phytochelatin-related peptides. The photosensitizer−peptide conjugates were subsequently used to overcoat green- and red-emitting CdSe/CdS/ZnS nanocrystals. Generation of singlet oxygen could be achieved via indirect excitation through Förster (fluorescence) resonance energy transfer (FRET) from the nanocrystals to PSs, or by direct excitation of the PSs. In the latter case, by using two color excitations, the conjugate could be simultaneously used for fluorescence imaging and singlet oxygen generation. Singlet oxygen quantum yields as high as 0.31 were achieved using 532-nm excitation wavelengths.
The interaction between the upper and lower rotors greatly influences the aeroacoustic characteristics of a counter-rotating nano-coaxial rotor. To study this influence, a numerical investigation was carried out. The unsteady flow field of a single upper rotor was first studied with a large-eddy simulation computational fluid dynamics method coupled with a sliding-mesh technique. The Ffowcs Williams–Hawking equation method was used to investigate the aeroacoustic characteristics of the upper rotor based on the flow field. An experimental setup was established to validate the computational approach. The experimental results matched well with the computational results. Additionally, results show that the peak value of the total sound pressure level appeared near the blade tip, which verified that the tip vortex was one of the most important sources of rotor noise. Then the aeroacoustic noise of the nano-coaxial rotor was studied numerically. It was found that the total sound pressure level of the nano-coaxial rotor was greater than that of the upper rotor. Flow field analysis showed that the shedding vortices of the upper rotor interacted with the lower rotor, resulting in a blade–vortex interaction. It was evident that the aeroacoustic noise was enhanced by the interference between the upper and lower rotors.
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