Versatile, do-it-yourself, low-cost spinning disk confocal microscope.

Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (∼400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers.

Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy.

Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid-Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy.

Multiplayer Noncooperative and Cooperative Minimax H∞ Tracking Game Strategies for Linear Mean-Field Stochastic Systems With Applications to Cyber-Social Systems.

The multiplayer stochastic noncooperative tracking game (NTG) with conflicting target strategy and cooperative tracking game (CTG) with a common target strategy of the mean-field stochastic jump-diffusion (MFSJD) system with external disturbance is investigated in this study. Due to the mean (collective) behavior in the system dynamic and cost function, the designs of the NTG strategy and CTG strategy for target tracking of the MFSJD system are more difficult than the conventional stochastic system. By the proposed indirect method, the NTG and CTG strategy design problems are transformed into linear matrix inequalities (LMIs)-constrained multiobjective optimization problem (MOP) and LMIs-constrained single-objective optimization problem (SOP), respectively. The LMIs-constrained MOP could be solved effectively for all Nash equilibrium solutions of NTG at the Pareto front by the proposed LMIs-constrained multiobjective evolutionary algorithm (MOEA). Two simulation examples, including the share market allocation and network security strategies in cyber-social systems, are given to illustrate the design procedure and validate the effectiveness of the proposed LMI-constrained MOEA for all Nash equilibrium solutions of NTG strategies of the MFSJD system.

Robust State/Fault Estimation and Fault-Tolerant Control in Discrete-Time T-S Fuzzy Systems: An Embedded Smoothing Signal Model Approach.

This study investigates a simple design method of the robust state/fault estimation and fault-tolerant control (FTC) of discrete-time Takagi-Sugeno (T-S) fuzzy systems. To avoid the corruption of the fault signal on state estimation, a novel smoothing signal model of fault signal is embedded in the T-S fuzzy model for the robust H∞ state/fault estimation of the discrete-time nonlinear system with external disturbance by the traditional fuzzy observer. When the component and sensor faults are generated from different fault sources, two smoothing signal models for component and sensor faults are both embedded in the T-S fuzzy system for robust state/fault estimation. Since the nonsingular smoothing signal model and T-S fuzzy model are augmented together for signal reconstruction, the traditional fuzzy Luenberger-type observer can be employed to robustly estimate state/fault signal simultaneously from the H∞ estimation perspective. By utilizing the estimated state and fault signal, a traditional H∞ observer-based controller is also introduced for the FTC with powerful disturbance attenuation capability of the effect caused by the smoothing model error and external disturbance. Moreover, the robust H∞ observer-based FTC design is transformed into a linear matrix inequality (LMI) -constrained optimization problem by the proposed two-step design procedure. With the help of LMI TOOLBOX in MATLAB, we can easily design the fuzzy Luenberger-type observer for efficient robust H∞ state/fault estimation and solve the H∞ observer-based FTC design problem of discrete nonlinear systems. Two simulation examples are given to validate the performance of state/fault estimation and FTC of the proposed methods.

Incorporation of sensing modalities into de novo designed fluorescence-activating proteins.

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein-protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

Stochastic Robust Team Tracking Control of Multi-UAV Networked System Under Wiener and Poisson Random Fluctuations.

In this article, a robust leader-follower tracking control scheme is proposed to deal with the stochastic multi-unmanned aerial vehicle (UAV) networked team tracking control problem to achieve a prescribed H∞ robust tracking performance. By taking the leader's desired path and every UAV networked system into account, the leader-follower tracking error networked system is constructed by arranging the UAVs network systems into a leader-follower formation. To effectively reduce the effect of the external disturbance on the team tracking process, a robust H∞ controller is proposed. With the help of the Itô-Lévy formula, the robust team tracking control design of the multi-UAV system is transformed to a Hamilton-Jacobin inequality (HJI)-constrained optimization problem. Since the HJI constraint cannot be easily solved, the Takagi-Sugeno (T-S) fuzzy interpolation method is employed to approximate the nonlinear networked system with a set of local linearized stochastic networked systems to simplify the design procedure. By the proposed T-S fuzzy control method, the H∞ robust leader-follower tracking control design of the stochastic multi-UAV networked system can be transformed to equivalent linear matrix inequalities (LMIs)-constrained optimization problem which can be efficiently solved by using the convex optimization techniques. Finally, a design example is given with simulation to illustrate the design procedure of the robust team tracking control of desired attitude and path and validate the effectiveness of the proposed robust H∞ team tracking control method of the multi-UAV networked system.

Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy.

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

De novo design of a fluorescence-activating ß-barrel.

The regular arrangements of ß-strands around a central axis in ß-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with ß-barrels. Here we show that accurate de novo design of ß-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of ß-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.

A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay.

We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.

Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions.

We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide-protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored. Using the well characterized p53-Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53-Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.

Antihyperlipidemic and antioxidant effects of extracts from Pleurotus citrinopileatus.

Pleurotus citrinopileatus is a popular edible mushroom which is physiologically active in both humans and animals. In the study we investigate the effects of this mushroom on hyperlipidemic hamster rats. Four dietary forms of the mushroom were created as follows. The powdered dry fruiting body, hot-water extract, and two kinds of elutes were obtained, from ethyl acetate extract and methanol extract, respectively, in different mixed proportion solvents over silica gel column chromatography (referred to as EAE and MOE, respectively). They were tested at different dosages as a supplement to a high-fat diet in hyperlipidemic rats. Serum triglycerides and total cholesterol levels were significantly lower in groups supplemented with the highest dosages of EAE and MOE (0.5 g/kg, body weight daily) as compared with the control groups that received no mushroom additive. High-density lipoprotein levels in these same two experimental groups were also significantly higher than those in the negative control group. The tested rats that were fed with EAE had the highest serum glutathione peroxidase and superoxide dismutase activity, and those with the MOE and EAE had the highest DPPH free radical scavenging activities and ferric-reducing abilities, tested in vitro. The major constituents of MOE and EAE were identified as ergosterol and nicotinic acid, respectively. P. citrinopileatus extracts may have a significant antihyperlipidemia effect. Furthermore, antioxidant activities and antihyperlipidemic effects of MOE and EAE seemed to display similar tendencies.

Antihyperglycemic effect of polysaccharide from fermented broth of Pleurotus citrinopileatus.

Pleurotus citrinopileatus is an edible mushroom, which has recently become very popular, with a consequent increase in industrial production. Water-soluble polysaccharides (WSPS), extracted from edible mushrooms, have been found to have antitumor and immunoenhancing effects. In this study, we investigate the effects of WSPS extracted from submerged fermented medium of P. citrinopileatus on hyperglycemia and damaged pancreatic cells in rats with streptozotocin (STZ)-induced diabetes. The diabetic rats fed with water-soluble polysaccharide of P. citrinopileatus (SPPC) lost less body weight than those fed SPPC-free regular diet. Serum total cholesterol and triglyceride levels in the diabetic rats fed with SPPC at a dose of 0.4 g/kg bw daily was lower than in the groups fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The fasting blood glucose levels of diabetic rats fed with SPPC were 44% lower than the negative controls. The degree of damage to the islets of Langerhans of the rats fed with the highest dosage of SPPC was significantly lower than those fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The results showed that STZ-induced diabetic rats fed with SPPC might help alleviate the elevation of the level of that in fasting blood glucose.