Quantitative UV-C dose validation with photochromic indicators for informed N95 emergency decontamination.

With COVID-19 N95 shortages, frontline medical personnel are forced to reuse this disposable-but sophisticated-multilayer respirator. Widely used to decontaminate nonporous surfaces, UV-C light has demonstrated germicidal efficacy on porous, non-planar N95 respirators when all surfaces receive ≥1.0 J/cm2 dose. Of utmost importance across disciplines, translation of empirical evidence to implementation relies upon UV-C measurements frequently confounded by radiometer complexities. To enable rigorous on-respirator measurements, we introduce a photochromic indicator dose quantification technique for: (1) UV-C treatment design and (2) in-process UV-C dose validation. While addressing outstanding indicator limitations of qualitative readout and insufficient dynamic range, our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%) required for UV-C dose measurements. In a measurement infeasible with radiometers, we observe a striking ~20× dose variation over N95s within one decontamination system. Furthermore, we adapt consumer electronics for accessible quantitative readout and use optical attenuators to extend indicator dynamic range >10× to quantify doses relevant for N95 decontamination. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical considerations for both photochromic indicators themselves and UV-C decontamination processes.

Stability of reagents used for chiral amino acid analysis during spaceflight missions in high-radiation environments.

The search for biosignatures on spaceflight missions requires in situ instrumentation capable of highly selective and sensitive organic analyses. To this end, CE-LIF is a uniquely promising technique, capable of determining the type, abundance, and chirality of amino acids present in environmental samples at nanomolar concentrations. However, this type of assay requires several reagents that have not yet been used on spaceflight missions. A key concern, particularly for future missions to Europa, is the survivability of these critical components for CE separation and LIF detection under high levels of radiation. Here we present an investigation of the chemical stability of the reagents and associated fused silica capillary after a total ionizing dose of 300 krad, exceeding the predicted total ionizing dose for the potential Europa Lander Mission payload by two-fold. Neither the fused silica capillary nor the fluorescent dye (5-carboxyfluorescein succinimidyl ester) showed significant change in performance following irradiation. Following the irradiation of the pre-mixed background electrolyte, both migration time and resolution were affected. However, when the reagents (sodium tetraborate, sodium taurocholate, and γ-cyclodextrin) and the acetonitrile solution were irradiated separately and mixed afterwards, there was no change in the separation performance.

Iron-catalyzed ortho trifluoromethylation of anilines via picolinamide assisted photoinduced C-H functionalization.

A convenient, oxidant-free protocol was developed for the ortho trifluoromethylation of aniline via picolinamide assisted Fe-promoted C-H functionalization under ultraviolet irradiation. In this transformation acetone essentially acted as both a solvent to dissolve reactants and a low-cost radical initiator to efficiently generate a CF3 radical from Langlois' reagent. A broad substrate scope was tolerated and picolinamide bearing strong electron withdrawing groups also could be transformed into the corresponding products with acceptable yields. Furthermore, the value of this method has been highlighted via the efficient synthesis of the nonsteroidal anti-inflammatory drug floctafenine.

Are clusters important in understanding the mechanisms in atmospheric pressure ionization? Part 1: Reagent ion generation and chemical control of ion populations.

It is well documented since the early days of the development of atmospheric pressure ionization methods, which operate in the gas phase, that cluster ions are ubiquitous. This holds true for atmospheric pressure chemical ionization, as well as for more recent techniques, such as atmospheric pressure photoionization, direct analysis in real time, and many more. In fact, it is well established that cluster ions are the primary carriers of the net charge generated. Nevertheless, cluster ion chemistry has only been sporadically included in the numerous proposed ionization mechanisms leading to charged target analytes, which are often protonated molecules. This paper series, consisting of two parts, attempts to highlight the role of cluster ion chemistry with regard to the generation of analyte ions. In addition, the impact of the changing reaction matrix and the non-thermal collisions of ions en route from the atmospheric pressure ion source to the high vacuum analyzer region are discussed. This work addresses such issues as extent of protonation versus deuteration, the extent of analyte fragmentation, as well as highly variable ionization efficiencies, among others. In Part 1, the nature of the reagent ion generation is examined, as well as the extent of thermodynamic versus kinetic control of the resulting ion population entering the analyzer region.

Novel water-resistant UV-activated oxygen indicator for intelligent food packaging.

For the first time, alginate polymer has been applied to prevent dyes from leaching out of colorimetric oxygen indicator films, which enable people to notice the presence of oxygen in the package in an economic and simple manner. The dye-based oxygen indicator film suffers from dye leaching upon contact with water. In this work, UV-activated visual oxygen indicator films were fabricated using thionine, glycerol, P25 TiO2, and zein as a redox dye, a sacrificial electron donor, UV-absorbing semiconducting photocatalyst, and an encapsulation polymer, respectively. When this zein-coated film was immersed in water for 24h, the dye leakage was as high as 80.80±0.45%. However, introduction of alginate (1.25%) as the coating polymer considerably diminished the dye leaching to only 5.80±0.06%. This is because the ion-binding ability of alginate could prevent the cation dye from leaching into water. This novel water-resistant UV-activated oxygen indicator was also successfully photo-bleached and regained colour fast in the presence of oxygen.

The use of chromophore and fluorophore degradation to quantitate UV dose: FD&C dyes as chemical identicators for UV sterilization.

The accurate measurement of ultraviolet (UV) irradiation, especially within a container or vessel is one of the challenges facing the broad implementation of UV sterilization. Currently, biological indicators are the best method to determine whether an applied UV dose has the necessary efficacy to achieve sterilization. To overcome some of the challenges of using a biological indicator, chemical indicators based upon the degradation of food, drug and cosmetic (FD&C) dyes were developed. In this work, the relationship between UV dose and dye degradation was elucidated and used to create standard curves which could be used as a quantitative measurement system. The use of dye degradation as a measurement of UV dose is especially useful when the levels of UV irradiation within a container cannot be measured directly. Additionally, due to the highly colored nature of the FD&C dyes, the visual changes present upon dye irradiation can be used as a qualitative visual indicator of the UV dose.

An environmentally friendly procedure for the reductive alkylation of amines with ammonium formate using supported reagent.

Reductive alkylation of amines with carbonyl compounds is a useful method for the production of N-alkylated amines, a rapid, mild, efficient, inexpensive and environmentally friendly procedure is reported for the reductive alkylation of amines with aromatic aldehydes using ammonium formate and NiCl2/SiO2 with microwave heating.

An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications.

BACKGROUND: PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. METHODOLOGY/PRINCIPAL FINDINGS: Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. CONCLUSIONS/SIGNIFICANCE: There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.

Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR.

Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.

Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the green fluorescent protein.

The mutant F99S/M153T/V163A of the Green Fluorescent Protein (c3-GFP) has spectral characteristics similar to the wild-type GFP, but it is 42-fold more fluorescent in vivo. Here, we report the crystal structure and the refolding properties of c3-GFP and compare them with those of the less fluorescent wt-GFP and S65T mutant. The topology and the overall structure of c3-GFP is similar to the wild-type GFP. The three mutated residues, Ser99, Thr153, and Ala163, lie on the surface of the protein in three different beta-strands. The side chains of Ser99 and Thr153 are exposed to the solvent, whereas that of Ala163 points toward the interior of the protein. No significant deviation from the structure of the wild-type molecule is found around these positions, and there is not clear evidence of any distortion in the position of the chromophore or of the surrounding residues induced by the mutated amino acids. In vitro refolding experiments on urea-denatured c3-GFP reveal a renaturation behavior similar to that of the S65T molecule, with kinetic constants of the same order of magnitude. We conclude that the higher fluorescence activity of c3-GFP can be attributed neither to particular structural features nor to a faster folding process, as previously proposed.

Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.

Inactivation and stability of viral diagnostic reagents treated by gamma radiation.

The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 (Atomic Energy of Canada, Ltd., Ottawa, Canada) was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0.42 to 3.7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1.7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with greater than 2.4 Mrad radiation at temperatures above 4 degrees C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is reliable and effective replacement for BPL in preparing diagnostic reagents.

Rotational diffusion of rhodopsin-digitonin micelles studied by transient photodichroism.

The transient photodichroism induced by a 0.80 sec plane-polarized light flash in a rhodopsin-digitonin mixture at about -70 degrees C was compared with a theoretical description of the effect. It was concluded that the transient dichroism is entirely due to rotational diffusion of the pigment molecules. When the rhodopsin-digitonin micelles are assumed to be rotationally symmetric it was found from the observed relaxation time that the axial ratio is probably less than 2. The initial photodichroism after each flash as a function of the number of flashes was shown to obey an equation derived for the photochemical equilibrium reaction between rhodopsin, lumirhodopsin, and isorhodopsin. The absolute quantum efficiency of the transition of rhodopsin to lumirhodopsin was found to be equal, within experimental error, to the quantum efficiency of bleaching rhodopsin at room temperature.