Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules.

The development of new ion-activation/dissociation methods continues to be one of the most active areas of mass spectrometry owing to the broad applications of tandem mass spectrometry in the identification and structural characterization of molecules. This Review will showcase the impact of ultraviolet photodissociation (UVPD) as a frontier strategy for generating informative fragmentation patterns of ions, especially for biological molecules whose complicated structures, subtle modifications, and large sizes often impede molecular characterization. UVPD energizes ions via absorption of high-energy photons, which allows access to new dissociation pathways relative to more conventional ion-activation methods. Applications of UVPD for the analysis of peptides, proteins, lipids, and other classes of biologically relevant molecules are emphasized in this Review.

Photorelaxation and Photorepair Processes in Nucleic and Amino Acid Derivatives.

Understanding the fundamental interaction between electromagnetic radiation and matter is essential for a large number of phenomena, with significance to civilization.[...].

Photoinduced processes in nucleic acids.

Photoinduced processes in nucleic acids are phenomena of fundamental interest in diverse fields, from prebiotic studies, through medical research on carcinogenesis, to the development of bioorganic photodevices. In this contribution we survey many aspects of the research across the boundaries. Starting from a historical background, where the main milestones are identified, we review the main findings of the physical-chemical research of photoinduced processes on several types of nucleic-acid fragments, from monomers to duplexes. We also discuss a number of different issues which are still under debate.

Photosynthesis and photo-stability of nucleic acids in prebiotic extraterrestrial environments.

Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including molecules important for biology such as amino acids, quinones, and amphiphiles. When pyrimidine is introduced into these ices, the products of irradiation include the nucleobases uracil, cytosine, and thymine, the informational sub-units of DNA and RNA, as well as some of their isomers. The formation of these compounds, which has been studied both experimentally and theoretically, requires a succession of additions of OH, NH2, and CH3groups to pyrimidine. Results show that H2O ice plays key roles in the formation of the nucleobases, as an oxidant, as a matrix in which reactions can take place, and as a catalyst that assists proton abstraction from intermediate compounds. As H2O is also the most abundant icy component in most cold astrophysical environments, it probably plays the same roles in space in the formation of biologically relevant compounds. Results also show that although the formation of uracil and cytosine from pyrimidine in ices is fairly straightforward, the formation of thymine is not. This is mostly due to the fact that methylation is a limiting step for its formation, particularly in H2O-rich ices, where methylation must compete with oxidation. The relative inefficiency of the abiotic formation of thymine to that of uracil and cytosine, together with the fact that thymine has not been detected in meteorites, are not inconsistent with the RNA world hypothesis. Indeed, a lack of abiotically produced thymine delivered to the early Earth may have forced the choice for an RNA world, in which only uracil and cytosine are needed, but not thymine.

Investigating the biochemical response of proton minibeam radiation therapy by means of synchrotron-based infrared microspectroscopy.

The biology underlying proton minibeam radiation therapy (pMBRT) is not fully understood. Here we aim to elucidate the biological effects of pMBRT using Fourier Transform Infrared Microspectroscopy (FTIRM). In vitro (CTX-TNA2 astrocytes and F98 glioma rat cell lines) and in vivo (healthy and F98-bearing Fischer rats) irradiations were conducted, with conventional proton radiotherapy and pMBRT. FTIRM measurements were performed at ALBA Synchrotron, and multivariate data analysis methods were employed to assess spectral differences between irradiation configurations and doses. For astrocytes, the spectral regions related to proteins and nucleic acids were highly affected by conventional irradiations and the high-dose regions of pMBRT, suggesting important modifications on these biomolecules. For glioma, pMBRT had a great effect on the nucleic acids and carbohydrates. In animals, conventional radiotherapy had a remarkable impact on the proteins and nucleic acids of healthy rats; analysis of tumour regions in glioma-bearing rats suggested major nucleic acid modifications due to pMBRT.

Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins.

Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photodynamic action but not considered the main cause of cell death. The in vivo effect of the photodynamic process on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein. Two cationic porphyrins (Tetra-Py(+)-Me and Tri-Py(+)-Me-PF) were used to photoinactivate E. coli (5.0µM; 10(8)cellsmL(-1)) and S. warneri (0.5µM; 10(8)cellsmL(-1)) upon white light irradiation at 4.0mWcm(-2) for 270min and 40min, respectively. Total nucleic acids were extracted from photosensitized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double-stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry. E. coli was completely photoinactivated with both porphyrins (5.0µM), whereas S. warneri was only completely inactivated by Tri-Py(+)-Me-PF (0.5µM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA>16S rRNA>genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5min with Tri-Py(+)-Me-PF but almost unchanged with Tetra-Py(+)-Me after 40min of irradiation. The amount of Tri-Py(+)-Me-PF bound to E. coli after washing the cells is higher than Tetra-Py(+)-Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells.

Developing pathogen reduction technologies for RBC suspensions.

Numerous studies have evaluated a wide variety of photosensitizers and alkylating agents as candidates for a pathogen reduction process to be used in RBC suspensions. The methodologies that produce robust inactivation of pathogens with maintenance of RBC properties during storage involve those that specifically target nucleic acids. This has been demonstrated through in vitro studies by flexible photosensitizers, which specifically target nucleic acid but do not engage in photochemistry when free in solution and nucleic acid alkylating agents in conjunction with extracellular quencher(s) to protect against RBC membrane alkylation. The flexible photosensitizer method must be scaled up to entire units, and toxicology studies would need to be performed for further development. Clinical trials will ultimately be necessary to further develop either flexible photosensitizers or nucleic acid alkylating methods with quenchers for use in Transfusion Medicine.

Research progress in the effects of terahertz waves on biomacromolecules.

With the rapid development of terahertz technologies, basic research and applications of terahertz waves in biomedicine have attracted increasing attention. The rotation and vibrational energy levels of biomacromolecules fall in the energy range of terahertz waves; thus, terahertz waves might interact with biomacromolecules. Therefore, terahertz waves have been widely applied to explore features of the terahertz spectrum of biomacromolecules. However, the effects of terahertz waves on biomacromolecules are largely unexplored. Although some progress has been reported, there are still numerous technical barriers to clarifying the relation between terahertz waves and biomacromolecules and to realizing the accurate regulation of biological macromolecules by terahertz waves. Therefore, further investigations should be conducted in the future. In this paper, we reviewed terahertz waves and their biomedical research advantages, applications of terahertz waves on biomacromolecules and the effects of terahertz waves on biomacromolecules. These findings will provide novel ideas and methods for the research and application of terahertz waves in the biomedical field.

ps-TRIR covers all the bases--recent advances in the use of transient IR for the detection of short-lived species in nucleic acids.

Recent developments of the picosecond transient absorption infrared technique and its ability to elucidate the nature and kinetic behaviour of transient species formed upon pulsed laser excitation of nucleic acids are described.

Destruction of Bacillus licheniformis spores by microwave irradiation.

AIMS: To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores. METHODS AND RESULTS: We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0.5 kW) was modified to output power at 2.0 kW, which allowed a shorter sterilization cycle. A 2.0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0.5 kW microwave oven. In contrast to boiling and 0.5 kW microwave treatments, the 2.0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2.0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane. CONCLUSIONS: These results suggest that 2.0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.

Multi-level patterning nucleic acid photolithography.

The versatile and tunable self-assembly properties of nucleic acids and engineered nucleic acid constructs make them invaluable in constructing microscale and nanoscale devices, structures and circuits. Increasing the complexity, functionality and ease of assembly of such constructs, as well as interfacing them to the macroscopic world requires a multifaceted and programmable fabrication approach that combines efficient and spatially resolved nucleic acid synthesis with multiple post-synthetic chemical and enzymatic modifications. Here we demonstrate a multi-level photolithographic patterning approach that starts with large-scale in situ surface synthesis of natural, modified or chimeric nucleic acid molecular structures and is followed by chemical and enzymatic nucleic acid modifications and processing. The resulting high-complexity, micrometer-resolution nucleic acid surface patterns include linear and branched structures, multi-color fluorophore labeling and programmable targeted oligonucleotide immobilization and cleavage.

The effect of the short wavelength ultraviolet radiation. An extension of biological dosimetry to the UV-C range.

Polycrystalline uracil thin layer can be used as biological dosimeter for assessing exposure to UV radiation. The dimerization and reversion efficiency of the ultraviolet radiation in the UV-B and the UV-C range were quantified on polycrystalline uracil thin layers irradiated with quasi-monochromatic radiation using interference filters of 10nm bandwidth. The dimer formation and monomerization (reversion) dose-effect relations were determined by optical spectroscopy. The decrease of the OD value of the uracil thin layer at 288 nm was taken as a measure of the dimer formation, while the increase of the OD of a completely irradiated (until reaching the saturation level) uracil layer was taken as the sign of the monomerization. The two processes in the UV-B and the UV-C range take place simultaneously, the individual characterization of the dimerization efficiency was performed from the initial slope of the dimerization dose-effect function and an action spectrum for dimerization was constructed in the UV-C range too. The reversion efficiency was found to be practically the same with all of the investigated wavelengths: 200 nm, 210 nm, 220 nm, 230 nm, 240 nm The possible biological relevance of the reversion of dimers are discussed.

Practical Radiation Damage-Induced Phasing.

Although crystallographers typically seek to mitigate radiation damage in macromolecular crystals, in some cases, radiation damage to specific atoms can be used to determine phases de novo. This process is called radiation damage-induced phasing or "RIP." Here, we provide a general overview of the method and a practical set of data collection and processing strategies for phasing macromolecular structures using RIP.

Effects of electromagnetic fields on molecules and cells.

Evidence suggests that cell processes can be influenced by weak electromagnetic fields (EMFs). EMFs appear to represent a global interference or stress to which a cell can adapt without catastrophic consequences. There may be exceptions to this observation, however, such as the putative role of EMFs as promoters in the presence of a primary tumor initiator. The nature of the response suggests that the cell is viewing EMFs as it would another subtle environmental change. The age and state of the cell can profoundly affect the EMF bioresponse. There is no evidence that direct posttranscription effects occur as a result of EMF exposure. Although transcription alterations occur, no apparent disruption in routine physiological processes such as growth and division is immediately evident. What is usually observed is a transient perturbation followed by an adjustment by the normal homeostatic machinery of the cells. DNA does not appear to be significantly altered by EMF. If EMF exposure is associated with an increased risk of cancer, the paucity of genotoxic effects would support the suggestion that the fields act in tumor promotion rather than initiation. The site(s) and mechanisms of interaction remain to be elaborated. Although there are numerous studies and hypotheses that suggest the membrane represents the primary site of interaction, there are also several different studies showing that in vitro systems, including cell-free systems, are responsive to EMFs. The debate about potential hazards or therapeutic value of weak electromagnetic fields will continue until the mechanism of interaction has been clarified.

[Effect of impulse-intermittent ultrahigh frequency irradiation on synthesis of nucleic acids in tumor cells].

In this work is shown that the repetitive high power microwaves is able to exert an inhibitory influence on the process of DNA and RNA syntheses in tumor cells of P-815 mastocytoma. This effect depends on pulse repetition rate. High power microwave pulses inhibit the process of transcription in tumor cells. No activation of DNA reparation system due to the irradiation of non-proliferating mononuclear blood cells was found. This indicates that the repetitive high power microwaves are not able to initiate single-filament rupture in DNA of tumor cells. The conformation of transcription enzymes is assumed to be changed under the influence of the microwave irradiation that makes for significant inhibition of RNA synthesis.

Oxidative damage to nucleic acids photosensitized by titanium dioxide.

The semiconductor TiO2 is known to have photobiological activity in prokaryotic and eukaryotic cells. Applications of this photobiological activity have been suggested including sterilization of waste water and phototherapy of malignant cells. Here, several model and cellular systems were used to study the mechanism of photocatalysis by TiO2. Treatment of TiO2 (anatase, 0.45 microns), suspended in water containing a spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), with UV radiation (320 nm) resulted in an electron spin resonance (ESR) signal characteristic of the hydroxyl radical. Irradiation of solutions containing calf thymus DNA and TiO2 with UVA (320-400 nm) radiation resulted in hydroxylation of guanine bases. The degree of hydroxylation was dependent on both UVA fluence and amount of TiO2 in suspension. Human skin fibroblasts, preincubated 18 h with 10 micrograms/cm2 TiO2 and then UVA-irradiated (0-58 KJ/m2), showed dose dependent photocytoxicity. RNA, isolated from similarly treated fibroblasts, contained significant levels of photooxidation, measured as hydroxylation of guanine bases. However, no oxidative damage was detectable in cellular DNA. These results suggest that nucleic acids are a potential target for photooxidative damage sensitized by TiO2, and support the view that TiO2 photocatalyzes free radical formation.

New concepts in phototherapy: photoisomerization of bilirubin IX alpha and potential toxic effects of light.

New information is summarized, indicating that configurational photoisomerization of bilirubin at the 5 and 15 carbon bridges is the major mechanism of bilirubin photocatabolism in vivo, and that singlet oxygen photooxidation plays only a minor role. The literature is reviewed concerning potentially damaging photodynamic reactions that are observed in vitro with vitamins, proteins, lipids, and nucleic acids, and their possible relationships to the limited number of toxic side-effects that have been detected with clinical phototherapy of neonatal jaundice. Secondary toxic effects, mediated by bilirubin photoderivatives or by retina-neuroendocrine pathways are also considered. Areas requiring further investigations are delineated.

UV light-induced cross-linking of nucleosides, nucleotides and a dinucleotide to the carboxy-terminal heptad repeat peptide of RNA polymerase II as studied by mass spectrometry.

We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross-linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5-bromo-2'-deoxyuridine), two nucleotides (thymidine-5'-monophosphate and 5-bromo-2'-deoxyuridine-5'-monophosphate) and a dinucleotide (thymidylyl-[3'-->5']-2'-deoxyadenosine). The peptide picked was SPSYSPT (L-seryl-L-prolyl-L-seryl-L-tyrosyl-L-seryl-L-prolyl-L-threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed-phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High-energy tandem collision-induced dissociation mass spectrometry pinpointed the location of cross-linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross-linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross-linked DNA-protein complexes.

Testicular proteins, nucleic acids and their synthesis following gamma irradiation.

The effects of two doses (250 and 1000 rads) of local gamma irradiation on testes of adult rats are reported after 1, 2, 4 and 16 weeks. There was a significant increase in DNA content per gm testes at 1 week; a gradual decrease at 2 and 4 week intervals was followed by a trend towards recovery at 16 weeks post-irradiation. The rate of synthesis of both DNA and RNA as studied by the incorporation of (3H)-thymidine and (3H)-uridine, showed similar results. Total protein content per gm testis declined with both doses and at all post-irradiation intervals. Histological observation showed loss of spermatogenic cells suggestive of DNA loss.