Boronate-based fluorescent probes for imaging cellular hydrogen peroxide

The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H2O2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H2O2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H2O2.     

Parallel biomolecular computation on surfaces with advanced finite automata

A biomolecular, programmable 3-symbol-3-state finite automaton is reported. This automaton computes autonomously with all of its components, including hardware, software, input, and output being biomolecules mixed together in solution. The hardware consisted of two enzymes: an endonuclease, BbvI, and T4 DNA ligase. The software (transition rules represented by transition molecules) and the input were double-stranded (ds) DNA oligomers. Computation was carried out by autonomous processing of the input molecules via repetitive cycles of restriction, hybridization, and ligation reactions to produce a final-state output in the form of a dsDNA molecule. The 3-symbol-3-state deterministic automaton is an extension of the 2-symbol-2-state automaton previously reported, and theoretically it can be further expanded to a 37-symbol-3-state automaton. The applicability of this design was further amplified by employing surface-anchored input molecules, using the surface plasmon resonance technology to monitor the computation steps in real time. Computation was performed by alternating the feed solutions between endonuclease and a solution containing the ligase, ATP, and appropriate transition molecules. The output detection involved final ligation with one of three soluble detection molecules. Parallel computation and stepwise detection were carried out automatically with a Biacore chip that was loaded with four different inputs.     

Preplay negotiations and the prisoner's dilemma

In order to improve outcomes of one shot noncooperative games a formal procedure for conducting preplay negotiations is proposed. For the prisoners' dilemma game it is shown that all the perfect equilibrium in the induced game (the game with the preplays) yield the cooperative pay-off. For another game it is shown that all perfect equilibrium payoffs converge to be Pareto optimal as the number of preplays increases.     

On economically based quality control decisions

A new approach to the problem of quality control is investigated. It is based on the theories and models developed for optimal maintenance policies for equipment subject to stochastic failure. Four alternative sampling/inspection procedures are examined. They are easy to implement, do not require the process operator to compute his own operating decisions, and are capable of finding optimal intervals between successive samplings/inspections. Both fixed and variable costs are included in the models. Relatively efficient algorithms have been developed for finding the optimal quality control procedures; this is due, partially, to the simple form of the objective function.     

Intrinsic Fluorescence of Metabolite Amyloids Allows Label‐Free Monitoring of Their Formation and Dynamics in Live Cells

The formation of apoptosis‐inducing amyloidal structures by metabolites has significantly extended the “amyloid hypothesis” to include non‐proteinaceous, single metabolite building blocks. However, detection of metabolite assemblies is restricted compared to their larger protein‐based counterparts owing to the hindrance of external labelling and limited immunohistochemical detection tools. Herein, we present the detection of the formation, dynamics, and cellular distribution of metabolite amyloid‐like structures and provide mechanistic insights into the generation of supramolecular chromophores. Moreover, the intrinsic fluorescence properties allow the detection of metabolite assemblies in living cells without the use of external dyes. Altogether, this intrinsic fluorescence of metabolite assemblies further verifies their amyloidal nature, while providing an important tool for further investigation of their pathological role in inborn error of metabolism disorders.     

Photodynamic Treatment of Red Blood Cell Concentrates For Virus Inactivation Enhances Red Blood Cell Aggregation: Protection with Antioxidants

Abstract— Photodynamic treatment (PDT) using phthalocyanines and red light appears to be a promising procedure for decontamination of red blood cell (RBC) concentrates for transfusion. A possible complication of this treatment may be induced aggregation of RBC. The production of RBC aggregates was measured with a novel computerized cell flow properties analyzer (CFA). The PDT of RBC concentrates with sulfonated aluminum phthalocy-anine (AIPcS4) and the silicon phthalocyanine Pc 4 under virucidal conditions markedly enhanced RBC aggregation and higher shear stress was required to disperse these aggregates. The clusters of cells were huge and abnormally shaped, unlike the rouleaux formed by untreated RBC. This aggregation was prevented when a mixture of antioxidants was included during PDT. Addition of the antioxidants after PDT reduced aggregation only partially. It is concluded that inclusion of antioxidants during PDT of RBC concentrates prior to transfusion may reduce or eliminate the hemodynamic risk that the virucidal treatment may present to the recipient.     

Protecting Fibrinogen with Rutin during UVC Irradiation for Viral Inactivation

Abstract— Fibrinogen solutions were irradiated with UVC (254 nm) to inactivate contaminating viruses. In order to protect fibrinogen during UVC irradiation, 0.5 m M rutin was added prior to UVC exposure and subsequently removed during processing. Viral kill by 0.1 J/cm² UVC resulted in the following inactivation values (log 10): non-lipid-enveloped viruses: Parvo 5.5; encephalomyocarditis virus 6.5; hepatitis A virus 6.5: lipid-enveloped viruses: human immunodeficiency virus 5.7; vesicular stomatitis virus 5.7. Fibrinogen irradiated with 0.5 m M rutin did not significantly differ from unirradiated material in terms of clot time and breaking strength. In the absence of rutin, UVC irradiation of fibrinogen at similar fluence led to loss of solubility, increased clot time and the cleavage of fibrino-peptides that reacted with dinitro-phenyl hydrazine as a test for ketonic carbonyl groups. High-performance liquid chromatography and mass spectrometry data showed that rutin exposed to UVC formed numerous breakdown, oxidation and combinational products. Experiments with ³H-rutin showed that after UVC irradiation, subsequent processing by a C18 resin and alcohol precipitation removed >99% rutin, representing <10 ppm rutin in the final fibrinogen preparations. Residual ³H-rutin was not covalently bonded to the fibrinogen. Immunochemical studies with rabbit antisera to UVC irradiated (with rutin) fibrinogen showed the absence of neoimmungens. By all measures, rutin prevents fibrinogen degradation during virucidal UVC irradiation.     

Encoding and Processing of Alphanumeric Information by Chemical Mixtures

A novel infochemical device that is based on ¹H NMR readout of chemical information is presented. This chemical encoding system utilizes two measurable parameters of homogeneous mixtures, chemical shift and peak integration, for three different applications: 1) a text‐encoding device that is based on spectral representation of a sequence of symbols, 2) encoding of 21‐digit binary numbers, each represented by an NMR spectrum, and their algebraic manipulations, such as addition and subtraction, and 3) encoding of 21‐digit decimal numbers. The first application enables molecular information storage and encryption. The relative concentration of each component, as measured by the relevant peak integration, can represent a symbol. The second application of this system, in addition to its obvious memory capability, enables mathematical operations. The NMR spectrum of a given mixture represents a 21‐digit binary number where each of the peaks encodes for a specific digit. In any of the input mixtures (numbers) each compound is either present or absent, representing either 1 or 0, respectively. We used the various binary numbers to carry out addition operations by combining two or more solutions (numbers). Subtraction operations were also preformed by digital processing of the information. The third application is the representation of decimal numbers. As before, each of the peaks encodes for a specific digit. In any of the input mixtures each compound is present in one of 10 different relative concentrations, representing the 10 digits of a decimal number.