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Viral diseases are among the main threats to public health. Understanding the factors affecting viral invasion is important for antiviral research. Until now, it was known that most viruses have very low plaque-forming unit (PFU)-to-particle ratios. However, further investigation is required to determine the underlying factors. Here, using quantitative single-particle analysis methods, the invasion of Semliki Forest virus (SFV), Japanese encephalitis virus (JEV), and influenza A virus (IAV) containing attachment to the cell surface, entry into the cell, transport towards the cell interior, and fusion with endosomes to release nucleocapsids were quantitatively analysed in parallel. It was found that for SFV with an PFU-to-particle ratio of approximately 1:2, an entry efficiency of approximately 31% limited infection. For JEV, whose PFU-to-particle ratio was approximately 1:310, an attachment efficiency of approximately 27% and an entry efficiency of 10% were the main factors limiting its infection. Meanwhile, for IAV with PFU-to-particle ratios of 1:8100, 5% attachment efficiency, 9% entry efficiency, and 53% fusion efficiency significantly limited its infection. These results suggest that viruses with different infectivities have different limited steps in the invasion process. Moreover, there are significant differences in attachment efficiencies among viruses, emphasizing the pivotal role of attachment in viral invasion. The influence of the virus purification method on virus invasion was also investigated. This study, for the first time, reports the efficiencies of different stages of virus invasion, leading to a better understanding of virus invasion and providing a protocol to quantitatively analyse the virus invasion efficiency.

Lateral intakes are common in rivers. The pump efficiency and sediment deposition are determined by the local hydrodynamic characteristics and mainstream division width. The hydraulic characteristics of lateral withdrawal from inclined river slopes at different intake elevations should be investigated. Meanwhile, the division width exhibits significant vertical non-uniformity at an inclined river slope, which should be clarified. Hence, a three-dimensional (3-D) hydrodynamic and particle-tracking model was developed with the Open Source Field Operation and Manipulation (OpenFOAM), and the model was validated with physical model tests for 90° lateral withdrawal from an inclined side bank. The flow fields, withdrawal sources, and division widths were investigated with different intake bottom elevations, withdrawal discharges, and main channel velocities. This study showed that under inclined side bank conditions, water entered the intake at an oblique angle, causing significant 3-D spiral flows in the intake rather than two-dimensional closed recirculation. A lower withdrawal discharge, a lower bottom elevation of the intake, or a higher main channel velocity could further strengthen this phenomenon. The average division width and turbulent kinetic energy were smaller under inclined side bank conditions than under vertical bank conditions. With a low intake bottom elevation, a low withdrawal discharge, or a high main channel velocity, the sources of lateral withdrawal were in similar ranges near the local inclined bank in the vertical direction. Under inclined slope conditions, sediment deposition near the intake entrance could be reduced, compared to that under vertical slope conditions. The results provide hydrodynamic and sediment references for engineering designs for natural rivers with inclined terrains.

ABSTRACT Quantum dots (QDs) possess optical properties of superbright fluorescence, excellent photostability, narrow emission spectra, and optional colors. Labeled with QDs, single molecules/viruses can be rapidly and continuously imaged for a long time, providing more detailed information than when labeled with other fluorophores. While they are widely used to label proteins in single-molecule-tracking studies, QDs have rarely been used to study virus infection, mainly due to a lack of accepted labeling strategies. Here, we report a general method to mildly and readily label enveloped viruses with QDs. Lipid-biotin conjugates were used to recognize and mark viral lipid membranes, and streptavidin-QD conjugates were used to light them up. Such a method allowed enveloped viruses to be labeled in 2 h with specificity and efficiency up to 99% and 98%, respectively. The intact morphology and the native infectivity of viruses were preserved. With the aid of this QD labeling method, we lit wild-type and mutant Japanese encephalitis viruses up, tracked their infection in living Vero cells, and found that H144A and Q258A substitutions in the envelope protein did not affect the virus intracellular trafficking. The lipid-specific QD labeling method described in this study provides a handy and practical tool to readily “see” the viruses and follow their infection, facilitating the widespread use of single-virus tracking and the uncovering of complex infection mechanisms. IMPORTANCE Virus infection in host cells is a complex process comprising a large number of dynamic molecular events. Single-virus tracking is a versatile technique to study these events. To perform this technique, viruses must be fluorescently labeled to be visible to fluorescence microscopes. The quantum dot is a kind of fluorescent tag that has many unique optical properties. It has been widely used to label proteins in single-molecule-tracking studies but rarely used to study virus infection, mainly due to the lack of an accepted labeling method. In this study, we developed a lipid-specific method to readily, mildly, specifically, and efficiently label enveloped viruses with quantum dots by recognizing viral envelope lipids with lipid-biotin conjugates and recognizing these lipid-biotin conjugates with streptavidin-quantum dot conjugates. It is not only applicable to normal viruses, but also competent to label the key protein-mutated viruses and the inactivated highly virulent viruses, providing a powerful tool for single-virus tracking.

The presence of submerged vegetation on river beds can change the water flow structure and alter the state of sediment motion. In this study, the incipient motion of sediment in the presence of submerged flexible vegetation in open channels was investigated in a laboratory experiment. The vegetation was simulated with flexible rubber cylinders arranged in parallel arrays. The effect of the vegetation density, water depth, and sediment grain size on the incipient motion was investigated. The experimental results indicate that the incipient motion velocity of sediment increases as the vegetation density decreases and the water depth and sediment grain size increase. With flexible plants, the incipient motion velocity of sediment is lower than it is without vegetation, and is larger than it is with rigid vegetation. A general incipient motion velocity equation was derived, which can be applied to both flexible and rigid vegetation conditions.

The velocity dip phenomenon may occur in a part of or in the whole flow field of open channel flows due to the secondary flow effect. Based on rectangular flume experiments and the laser Doppler velocimetry, the influence of the distance to the sidewall and the aspect ratio on the velocity dip is investigated. Through application of statistical methods to the experimental results, it is proposed that the flow field may be divided into two regions, the relatively strong sidewall region and the relatively weak sidewall region. In the former region, the distance to the sidewall greatly affects the location of maximum velocity, and, in the latter region, both the distance to the sidewall and the aspect ratio influence the location of the maximum velocity.

When subway tunnels are routed underneath rivers, riverbed scour may expose the structure, with potentially severe consequences. Thus, it is important to identify the maximum scour depth to ensure that the designed buried depth is adequate. There are a range of methods that may be applied to this problem, including the fluvial process analysis method, geological structure analysis method, scour formula method, scour model experiment method, and numerical simulation method. However, the application ranges and forecasting precision of these methods vary considerably. In order to quantitatively analyze the characteristics of the different methods, a subway tunnel passing underneath a river was selected, and the aforementioned five methods were used to forecast the maximum scour depth. The fluvial process analysis method was used to characterize the river regime and evolution trend, which were the baseline for examination of the scour depth of the riverbed. The results obtained from the scour model experiment and the numerical simulation methods are reliable; these two methods are suitable for application to tunnel projects passing underneath rivers. The scour formula method was less accurate than the scour model experiment method; it is suitable for application to lower risk projects such as pipelines. The results of the geological structure analysis had low precision; the method is suitable for use as a secondary method to assist other research methods. To forecast the maximum scour depth of the riverbed above the subway tunnel, a combination of methods is suggested, and the appropriate analysis method should be chosen with respect to the local conditions.

We report the development of Hadamard transform fluorescence spectral imaging microscopy by using a movable one-dimensional Hadamard mask to encode the image signals and a linear charge-coupled device (CCD) to detect the encoded spectral image signal. The technique has the ability of multi-spectral imaging and images with four dimensions: location coordinate (Xand Y), fluorescence intensity (Z) and wavelength (λ) (or time (t)) can be obtained. The results show that the system has high spectral resolving power (0.3 nm) and imaging capability, where image resolution up to 511 × 512 can be easily obtained for analysis of tiny samples. Multicolor imaging of acridine orange/ethidium bromide double-stained fibroblast treated with glyoxal shows that 1.5 mM glyoxal obviously causes apoptosis, thus demonstrating that identification of apoptotic cells can be realized using the Hadamard system not only through morphologic changes but also by the spectral evidences of the fluorescently stained cells. In addition, based on immunostaining with quantum dots (QDs) emitting at 550 and 610 nm to tag and trace two breast cancer biomarkers human epidermal growth factor receptor 2 (HER-2) and estrogen receptor (ER) in human breast cancer tissue with in situdual-color fluorescence imaging, sensitive spectra and images were obtained and show that the system can be applied to visualize and quantitatively measure the subcellular proteins inside the tumor tissues, especially when a single laser line is used as the illuminating source for multi-components with different emission peaks. The preliminary biomedical applications indicate the great potential of the Hadamard imaging system as a useful tool in biomedicine. [ABSTRACT FROM AUTHOR]

By using near-infrared surface-enhanced Raman scattering (SERS) with 60 nm gold nanoparticles (Au-NPs) to probe the chemical composition inside single human osteosarcoma cells we have shown that the SERS intensity may increase by a factor of 3–6 times in different parts of the cells depending on the density of gold nanoaggregates within the probed volume after the cell is dehydrated. The cellular points of low-density gold nanoaggregates exhibit more significant increase of SERS signal levels, the cellular macrochemicals such as nucleic acids show conformational changes, and new components can be probed after the cell is completely dried. A comparative study between viable and apoptotic cells indicates that most of the Au-NPs that enter the living cell reside in the cytoplasm and around the nucleus, whereas glyoxal-induced apoptotic cells show relatively uniform distribution of Au-NPs and, interestingly, the presence of DNA fragments is detected throughout the cell, including the cell surface. [ABSTRACT FROM AUTHOR]

We report on the capabilities of near-infrared surface-enhanced Raman scattering (SERS) using gold nanoparticles to obtain detailed chemical information with high spatial resolution from within single cancer cells, living or fixed. Colloidal gold particles, 60 nm in size, were introduced into live human osteosarcoma cells by endocytosis by adding them to the growth medium. Rapid SERS mapping of cells indicated that not only could rich vibrational spectra be obtained from intrinsic cellular constituents both in the cytoplasm and nucleus and but also the distribution of extrinsic molecules introduced into the cells, in this case, rhodamine 6G could be characterized, suggesting that the intracellular distribution of chemotherapeutic agents could potentially be measured by this technique. We show that the SERS signal intensity from the cellular components increases and more spectral detail is acquired from dried cells when compared with hydrated cells in buffer. The data also show spectral fluctuations, mainly in intensity but also in peak position, which are dependent upon the intensity of the excitation light and are probably due to diffusion of molecules on the surface of the gold nanoparticles. A detailed understanding of the origins of these effects is still not complete, but the ability to acquire very sensitive SERS inside living cancer cells indicates the potential of this technique as a useful tool in biomedicine. [ABSTRACT FROM AUTHOR]

Two novel N-(2-mercapto-1,3,4-thiadiazol-5-yl)- N′-(4-substituted-arylacetyl) urea compounds have been synthesized, characterized by NMR and MS, and used as self-assembly reagents to form self-assembled monolayers (SAMs) on Pt electrodes. The modified electrodes were characterized by electrochemical methods. The electrochemical behavior of p-benzenediol at the SAMs electrodes was investigated. It was found that the electrochemical response to p-benzenediol is controlled by diffusion and can be electrocatalyzed to obtain more symmetrical redox peaks and higher voltammetric current response at the SAMs electrodes, with a peak separation of 80 mV. For p-benzenediol the process at the SAMs electrodes is quasi-reversible with a rate constant of 0.6742 s−1. The SAMs electrodes have been used to determine p-benzenediol by differential pulse voltammetry. The peak current was linear for concentrations of p-benzenediol in the range 1×10−7−5×10−4 mol L−1 and the detection limit was 4.0×10−8 mol L−1. The SAMs electrodes were used to determine p-benzenediol in real photographic developer and in a synthetic waste water sample; the standard addition recovery was in the range 96.6–100.4%. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A three-layer core–shell nanostructure consisting of a silver core, a silica spacer, and a fluorescent dye RuBpy-doped outer silica layer was fabricated, and the optimal metal-enhanced fluorescence (MEF) distance was explored through adjusting the thickness of the silica spacer. The results show that the optimal distance is ∼10.4 nm with the maximum fluorescence enhancement factor 2.12. Then a new target-triggered MEF ‘turn-on’ strategy based on the optimized composite nanoparticles was successfully constructed for quantitative detection of prostate specific antigen (PSA), by using RuBpy as the energy donor and BHQ-2 as the acceptor. The hybridization of the complementary DNA of PSA-aptamer immobilized on the surface of the MEF nanoparticles with PSA-aptamer modified with BHQ-2, brought BHQ-2 in close proximity to RuBpy-doped silica shell and resulted in the decrease of fluorescence. In the presence of target PSA molecules, the BHQ-PSA aptamer is dissociated from the surface of the nanoparticles with the fluorescence switched on. Therefore, the assay of PSA was achieved by measuring the varying fluorescence intensity. The results show that PSA can be detected in the range of 1–100 ng ml−1 with a detection limit of 0.20 ng ml−1 (6.1 pM), which is 6.7-fold increase of that using hollow RuBpy-doped silica nanoparticles. Moreover, satisfactory results were obtained when PSA was detected in 1% serum. [ABSTRACT FROM AUTHOR]

We present a simple and sensitive method for multiplexed DNA detection by simultaneously capturing two different DNA sequences with a same silica nanoparticle (NP) through a sandwich mode. This biobarcode assay method was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus (HPV) 16 and 18 L1 genes as model systems. The nonfluorescent carboxyl-modified silica NPs were prepared using water-in-oil (W/O) microemulsion methods. Avidin was immobilized on the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified and the results show that about 8 avidin molecules are bound to one nanoparticle. The silica nano-platforms were prepared through the biotin-avidin interaction and the amounts of capture DNA strands for HPV-16 and HPV-18 (C-16 and C-18, respectively) conjugated to the surface of the same NPs were measured using fluorescent dye hoechst33258. The calculated result shows that the amounts of conjugated C-16 and C-18 on 1 mg of NPs (9.2 pmol) are about 13.5 pmol and 15.5 pmol, respectively. A one-step hybridization reaction was performed by mixing the silica nano-platforms, HPV-16 and HPV-18 target DNA (T-16 and T-18), fluorescein amidite (FAM) or 6-carboxyl-X-rhodamine (Rox) labeled HPV-16 and HPV-18 probes. The hybrid-conjugated NPs were separated by centrifugation, and T-16 and T-18 were detected by measuring fluorescence signals of FAM and Rox respectively. The results show linear dependence of the fluorescence intensity on target DNA concentration in the range from 0.5 to 9 nM, and the detection limit (3σ) of T-16 and T-18 is 0.17 nM and 0.78 nM, respectively. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that sphingomyelin (SM)-sequestered cholesterol, but not accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol independent, whereas the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein that activates actin nucleation, is recruited to the IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of the CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. IMPORTANCE IAV infects cells by harnessing cellular endocytic machineries. A better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in the plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin- coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results provide new insights into IAV infection and the pathway/cargo-specific involvement of the cholesterol pool(s). [ABSTRACT FROM AUTHOR]