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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.

The growth and metastasis of malignant solid tumors depend closely on new blood vessels. Vasculogenic mimicry provides a special pathway of blood supply during the early growth of malignant tumors, and real-time monitoring of its occurrence and development in vivo is important to clinical applications. However, there are few labels with sufficient brightness and stability in vivo to achieve high spatiotemporal resolution imaging of deep tissue for noninvasive optical detection of vasculogenic mimicry in tumor tissues. In this study, we constructed a high-brightness fluorescent label with fluorescence in the near-infrared-II region, which can be used not only for in vivo tumor imaging but also for tissue section imaging. Real-time high-resolution imaging of tumor vessels has been achieved with PbS quantum dots (QDs) surface-coupled with horseradish peroxidase (HRP) (HRP-QDs) by taking advantage of the low background autofluorescence of tissue at the near-infrared-II wavelength for in vivo and tissue section imaging. Qualitative and quantitative analysis of early blood supply patterns of tumor growth enables monitoring neovascularization to accurate noninvasive identification of benign and malignant solid tumors.

Superoxide dismutase (SOD) nanozymes show mitigating effect on oxidative stress-related diseases, but are limited by their modest activity. Here, the authors report a carbon dot SOD nanozyme with catalytic activity matching natural enzymes and unveil its catalytic mechanism.

Abstract Extracellular vesicles (EVs) are cell‐derived membrane‐enclosed structures that deliver biomolecules for intercellular communication. Developing visualization methods to elucidate the spatiotemporal dynamics of EVs’ behaviors will facilitate their understanding and translation. With a quantum dot (QD) labeling strategy, a single particle tracking (SPT) platform is proposed here for dissecting the dynamic behaviors of EVs. The interplays between tumor cell‐derived small EVs (T‐sEVs) and endothelial cells (ECs) are specifically investigated based on this platform. It is revealed that, following a clathrin‐mediated endocytosis by ECs, T‐sEVs are transported to the perinuclear region in a typical three‐stage pattern. Importantly, T‐sEVs frequently interact with and finally enter lysosomes, followed by quick release of their carried miRNAs. This study, for the first time, reports the entire process and detailed dynamics of T‐sEV transportation and cargo‐release in ECs, leading to better understanding of their proangiogenic functions. Additionally, the QD‐based SPT technique will help uncover more secrets of sEV‐mediated cell–cell communication.

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.

Abstract Background Quantum dot (QD)-based single virus tracking has become a powerful tool for dissecting virus infection mechanism. However, only virus behaviors at the early stage of retrograde trafficking have been dynamically tracked so far. Monitoring of comprehensive virus retrograde transportation remains a challenge. Results Based on the superior fluorescence properties of QDs and their labeling of virus internal component, the dynamic interactions between baculoviruses and all key transportation-related cellular structures, including vesicles, acidic endosomes, actins, nuclear pores and nuclei, were visualized at the single-virus level. Detailed scenarios and dynamic information were provided for these critical interaction processes. Conclusions A comprehensive model of baculovirus retrograde trafficking involving virus endocytosis, fusion with acidic endosome, translocation to nuclear periphery, internalization into nucleus, and arriving at the destination in nucleus was proposed. Thus the whole retrograde transportation of baculovirus in live host cells was elucidated at the single-virus level for the first time.

Reoviruses are non-enveloped viruses with wide host range, can cause serious infections in animals, plants and microorganism, e.g., aquareovirus, which is capable of causing serious haemorrhagic in aquatic animals. To date, the entry process of aquareovirus infection remains obscure. Real-time single-virus tracking are effective tools for exploring the details in viral infection process, which are crucial for understanding the pathogenic mechanism. Here, we used quantum dots-based single particle tracking technology combined with biochemical assays and ultrastructural observation to reveal unobservable infection steps and map dynamic interactions between a reovirus, Scophthalmus maximus reovirus (SMReV), and its host cell in real time. The results showed that the single membrane-bound reovirus particle can enter into the cell within several seconds through nascent clathrin-caoted pits, and most of the particles could internalize into cytoplasm within 30 min post-infection. The specific inhibitors analysis also showed that entry of SMREV depended on clathrin-mediated endocytosis rather than cavolin-mediated endocytosis. The motion analysis of internalized single particle indicated that the reovirus initially experienced slow and directed motion in the actin-enriched cell periphery, while it underwent relatively faster and directed movement toward the cell interior, suggesting that transport of SMReV was dependent on the cytoskeleton. Further, dual-labeling of virus and cytoskeleton and inhibitor analysis both demonstrated that transport of internalized SMReV was firstly dependent on actin filaments at the cell periphery, and then on microtubules toward the cell interior. Then visualization of SMReV trafficking in the endosomes revealed that the internalized reovirus particles were sorted from early endosomes to late endosomes, then part of them were delivered to lysosome. This study for the first time revealed the entry pathway, intracellular dynamic and the infection fate of fish reovirus in host cell in real time and in situ, which provided new insights into the infection mechanism of non-enveloped viruses.

Cancer is a major threat to public health in the 21st century because it is one of the leading causes of death worldwide. The mechanisms of carcinogenesis, cancer invasion, and metastasis remain unclear. Thus, the development of a novel approach for cancer detection is urgent, and real-time monitoring is crucial in revealing its underlying biological mechanisms. With the optical and chemical advantages of quantum dots (QDs), QD-based nanotechnology is helpful in constructing a biomedical imaging platform for cancer behavior study. This review mainly focuses on the application of QD-based nanotechnology in cancer cell imaging and tumor microenvironment studies both in vivo and in vitro, as well as the remaining issues and future perspectives.

BACKGROUND:As a marker for tumor cell proliferation, Ki67 has important impacts on breast cancer (BC) prognosis. Although immunohistochemical staining is the current standard method, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study was to develop a fluorescent spectrum-based quantitative analysis of Ki67 expression by quantum-dots (QDs) multiple imaging technique. METHODS:A QDs-based in situ multiple fluorescent imaging method was developed, which stained nuclear Ki67 as red signal and cytoplasmic cytokeratin (CK) as green signal. Both Ki67 and CK signals were automatically separated and quantified by professional spectrum analysis software. This technique was applied to tissue microarrays from 240 BC patients. Both Ki67 and CK values, and Ki67/CK ratio were obtained for each patient, and their prognostic value on 5-year disease free survival was assessed. RESULTS:This method simultaneously stains nuclear Ki67 and cytoplasmic CK with clear signal contrast, making it easy for signal separation and quantification. The total fluorescent signal intensities of both Ki67 sum and CK sum were obtained, and Ki67/CK ratio calculated. Ki67 sum and Ki67/CK ratio were each attributed into two grades by X-tile software based on the best P value principle. Multivariate analysis showed Ki67 grade (P = 0.047) and Ki67/CK grade (P = 0.004) were independent prognostic factors. Furthermore, area under curve (AUC) of ROC analysis for Ki67/CK grade (AUC: 0.683, 95%CI: 0.613-0.752) was higher than Ki67 grade (AUC: 0.665, 95%CI: 0.596-0.734) and HER-2 gene (AUC: 0.586, 95%CI: 0.510-0.661), but lower than N stage (AUC: 0.760, 95%CI: 0.696-0.823) and histological grade (AUC: 0.756, 95%CI: 0.692-0.820) on predicting the risk for recurrence. CONCLUSIONS:A QDs-based quantitative and in situ multiple imaging on Ki67 and CK was developed to improve Ki67 assessment in BC, and Ki67/CK grade had better performance than Ki67 grade in predicting prognosis.

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn2-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu(2+) or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu(2+): a moderate-affinity site (10(6) M(-1)) and a high-affinity site (10(8) M(-1)). Furthermore, Cu(2+) binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu(2+) plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.

Neurological disorders are closely linked to the alterations in cell membrane permeability (CMP) and mitochondrial membrane potential (MMP). Changes in CMP and MMP may lead to damage and death of nerve cells, thus triggering the onset and progression of neurological diseases. Therefore, monitoring the changes of these two physiological parameters not only benefits the accurate assessment of nerve cell health status, but also enables providing key information for the diagnosis and treatment of neurological diseases. However, the simultaneous monitoring of these two cellular physiological parameters is still challenging. Herein, we design and synthesize two quinolinium-carbazole-derivated fluorescent probes (OQ and PQ). As isomers, the only difference in their chemical structures is the linking position of the carbazole unit in quinoline rings. Strikingly, such a subtle difference endows OQ and PQ with significantly different organelle-staining behaviors. PQ mainly targets at the nucleus, OQ can simultaneously stain cell membranes and mitochondria in normal cells, and performs CMP and MMP-dependent translocation from the cell membrane to mitochondria then to the nucleus, thus holding great promise as an intracellular translocation probe to image the changes of CMP and MMP. After unraveling the intrinsic mechanism of their different translocation abilities by combining experiments with molecular dynamics simulations and density functional theory calculations, we successfully used OQ to monitor the continuous changes of CMP and MMP in three neurological disease-related cell models, including oxidative stress-damaged, Parkinson's disease, and virus-infected ones. Besides providing a validated imaging tool for monitoring cellular physiological parameters, this work paves a promising route for designing intracellular translocation probes to analyze cellular physiological parameters associated with various diseases. [ABSTRACT FROM AUTHOR]

A hydrophilic ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]B[F.sub.4]) is used as a model for examining the electrostatic interaction between ILs and calf thymus DNA (ctDNA) by using a surface electrochemical micromethod. The quantitative thermodynamic and kinetic parameters about the interaction of [bmim]B[F.sub.4] and ctDNA are obtained.

A simple method is described for integrating minisolenoids into microfluidic devices for process controlled and low-volume magneto immunoassays. Several pairs of minisolenoids are integrated in the same chip in order to manipulate superparamagnetic beads with different surface chemistries for simultaneous analysis of sample components.

A study is conducted on a strategy for the fabrication of quantum dot (QD) photosensitized nano-TiO(sub 2) films through the use of monodisperse core/shell QD as photosensitizes. The result of bactericidal and DNA damage experiments demonstrate that photosensitized nano-TiO(sub 2) films have excellent photoactivity.

Development of efficient targeting nanomaterials is extremely challenging due to the nonspecific accumulation in immune tissues, such as the liver and the spleen. Ultrasmall nanoparticles (USNPs) could possess small molecule-like in vivo pharmacokinetic profiles, coupled with integrated functions capacity, improving the molecular imaging efficiency, particularly in oncology. For nuclear imaging, radiometals are often incorporated into the structures of USNPs using chelator and chelator-free strategies. However, the incorporated chelator may change the surface properties and in vivo behavior of UNSPs, while chelator-free labeling strategies either involve complicated resynthesis or rely on the active properties of the metal ions. Herein, a novel chelator-free and postsynthetic strategy for broad-spectrum metal ion attachment is reported. The ultrasmall Ag2Se quantum dots (QDs) are developed with an active oxygen layer on the surface, allowing for facile incorporation of both active and inert metals with high labeling efficiency. The particles enable fluorescence, magnetic resonance imaging, and positron emission tomography (PET) trimodality imaging. After conjugation with targeting peptide, the probe yields a high tumor-to-muscle ratio of nine in PET imaging. Importantly, the QDs are predominantly excreted from body through the renal route within 12 h. This chelator-free strategy opens an avenue for exploring broad-spectrum radiometal isotope labeling and USNP-based renal-excreting imaging probes. [ABSTRACT FROM AUTHOR]

Double-stranded DNA(poly(dA)30.poly(dT)30) modified gold electrodes, prepared by air-drying/adsorption method, is investigated by various techniques, including cyclic voltammetry (CV), quartz crystal microbalance (QCM), electrochemical scanning tunneling microscopy (EC-STM), and surface-enhanced Raman scattering spectroscopy (SERS). CV, QCM, and EC-STM techniques are useful tools for profound insight into the nature of DNA attached to surfaces.

With the aid of the single-virus tracking technique and the quantum dot-labeling strategy, the internalization of the pseudorabies virus (PrV) has been visualized in real time. The results demonstrate that macropinocytosis can be considered as a major pathway for PrV entering HeLa cells, which facilitates the development of therapeutics for virus-triggered diseases. [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]

We report a non-iterative localization algorithm that utilizes the scaling of a three-dimensional (3D) image in the axial direction and focuses on evaluating the radial symmetry center of the scaled image to achieve the desired single-particle localization. Using this approach, we analyzed simulated 3D particle images by wide-field microscopy and confocal microscopy respectively, and the 3D trajectory of quantum dots (QDs)-labeled influenza virus in live cells. Both applications indicate that the method can achieve 3D single-particle localization with a sub-pixel precision and sub-millisecond computation time. The precision is almost the same as that of the iterative nonlinear least-squares 3D Gaussian fitting method, but with two orders of magnitude higher computation speed. This approach can reduce considerably the time and costs for processing the large volume data of 3D images for 3D single-particle tracking, which is especially suited for 3D high-precision single-particle tracking, 3D single-molecule imaging and even new microscopy techniques [ABSTRACT FROM AUTHOR]

Background: Given the complexity of tumor microenvironment, no single marker from cancer cells could adequately predict the clinical outcomes of gastric cancer (GC). The objective of this study was to evaluate the prognostic role of combined features including conventional pathology, proteinase and immune data in GC. Methods: In addition to pathological studies, immunohistochemistry was used to assess membrane-type 1 matrix metalloproteinase (MT1-MMP) expression and CD11b + immunocytes density in three independent GC tissue microarrays containing 184 GC tissues. Separate and combined features were evaluated for their impact on overall survival (OS). Results: We found that traditional factors including tumor size, histological grade, lymph node status, serosa invasion and TNM stage were associated with OS (P < 0.05 for all). Moreover, statistically significant differences in OS were found among lymph node ratio (LNR) subgroups (P < 0.001), MT1-MMP subgroups (P = 0.015), and CD11b + immunocytes density subgroups (P = 0.031). Most importantly, combined feature (MT1-MMP positive, low CD11b + immunocytes density and high LNR) was found by multivariate analysis to be an independent prognostic factors for OS after excluding other confounding factors (HR = 3.818 [95%CI: 2.223-6.557], P < 0.001). In addition, this combined feature had better performance in predicting clinical outcomes after surgery long before recurrence had occurred (Area under the curve: 0.689 [95%CI: 0.609-0.768], P < 0.001). Conclusions: These findings indicate that better information on GC prognosis could be obtained from combined clinico-pathological factors, tumor cells and the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Bead-based optical encoding or magnetic encoding techniques are promising in high-throughput multiplexed detection and separation of numerous species under complicated conditions. Therefore, a self-assembly strategy implemented in an organic solvent is put forward to fabricate fluorescent-magnetic dual-encoded nanospheres. Briefly, hydrophobic trioctylphosphine oxide-capped CdSe/ZnS quantum dots (QDs) and oleic acid-capped nano-&ggr; -Fe2O3 magnetic particles are directly, selectively and controllably assembled on branched poly(ethylene imine)-coated nanospheres without any pretreatment, which is crucial to keep the high quantum yield of QDs and good dispersibility of &ggr; -Fe2O3. Owing to the tunability of coating amounts of QDs and &ggr; -Fe2O3 as well as controllable fluorescent emissions of deposited-QDs, dual-encoded nanospheres with different photoluminescent emissions and gradient magnetic susceptibility are constructed. Using this improved layer-by-layer self-assembly approach, deposition of hydrophobic nanoparticles onto hydrophilic carriers in organic media can be easily realized; meanwhile, fluorescent-magnetic dual-functional nanospheres can be further equipped with readable optical and magnetic addresses. The resultant fluorescent-magnetic dual-encoded nanospheres possess both the unique optical properties of QDs and the superparamagnetic properties of &ggr; -Fe2O3, exhibiting good monodispersibility, huge encoding capacity and nanoscale particle size. Compared with the encoded microbeads reported by others, the nanometre scale of the dual-encoded nanospheres gives them minimum steric hindrance and higher flexibility. [ABSTRACT FROM AUTHOR]