Plasmalogen oxidation induces the generation of excited molecules and electrophilic lipid species.

Plasmalogens are glycerophospholipids with a vinyl ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high reactivity of their vinyl ether groups with reactive oxygen species, our study reveals the generation of subsequent reactive oxygen and electrophilic lipid species from oxidized plasmalogen intermediates. By conducting a comprehensive analysis of the oxidation products by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we demonstrate that singlet molecular oxygen [O2 (1Δg)] reacts with the vinyl ether bond, producing hydroperoxyacetal as a major primary product (97%) together with minor quantities of dioxetane (3%). Furthermore, we show that these primary oxidized intermediates are capable of further generating reactive species including excited triplet carbonyls and O2 (1Δg) as well as electrophilic phospholipid and fatty aldehyde species as secondary reaction products. The generation of excited triplet carbonyls from dioxetane thermal decomposition was confirmed by light emission measurements in the visible region using dibromoanthracene as a triplet enhancer. Moreover, O2 (1Δg) generation from dioxetane and hydroperoxyacetal was evidenced by detection of near-infrared light emission at 1,270 nm and chemical trapping experiments. Additionally, we have thoroughly characterized alpha-beta unsaturated phospholipid and fatty aldehydes by LC-HRMS analysis using two probes that specifically react with aldehydes and alpha-beta unsaturated carbonyls. Overall, our findings demonstrate the generation of excited molecules and electrophilic lipid species from oxidized plasmalogen species unveiling the potential prooxidant nature of plasmalogen-oxidized products.

Colloidal Quantum Dots as an Emerging Vast Platform and Versatile Sensitizer for Singlet Molecular Oxygen Generation.

Singlet molecular oxygen (1O2) has been reported in wide arrays of applications ranging from optoelectronic to photooxygenation reactions and therapy in biomedical proposals. It is also considered a major determinant of photodynamic therapy (PDT) efficacy. Since the direct excitation from the triplet ground state (3O2) of oxygen to the singlet excited state 1O2 is spin forbidden; therefore, a rational design and development of heterogeneous sensitizers is remarkably important for the efficient production of 1O2. For this purpose, quantum dots (QDs) have emerged as versatile candidates either by acting individually as sensitizers for 1O2 generation or by working in conjunction with other inorganic materials or organic sensitizers by providing them a vast platform. Thus, conjoining the photophysical properties of QDs with other materials, e.g., coupling/combining with other inorganic materials, doping with the transition metal ions or lanthanide ions, and conjugation with a molecular sensitizer provide the opportunity to achieve high-efficiency quantum yields of 1O2 which is not possible with either component separately. Hence, the current review has been focused on the recent advances made in the semiconductor QDs, perovskite QDs, and transition metal dichalcogenide QD-sensitized 1O2 generation in the context of ongoing and previously published research work (over the past eight years, from 2015 to 2023).

Nitric Oxide Detection Using a Chemical Trap Method for Applications in Bacterial Systems.

Plant growth-promoting bacteria (PGPB) can be incorporated in biofertilizer formulations, which promote plant growth in different ways, such as fixing nitrogen and producing phytohormones and nitric oxide (NO). NO is a free radical involved in the growth and defense responses of plants and bacteria. NO detection is vital for further investigation in different agronomically important bacteria. NO production in the presence of KNO3 was evaluated over 1-3 days using eight bacterial strains, quantified by the usual Griess reaction, and monitored by 2,3-diaminonaphthalene (DAN), yielding 2,3-naphthotriazole (NAT), as analyzed by fluorescence spectroscopy, gas chromatography-mass spectrometry, and high-performance liquid chromatography. The Greiss and trapping reaction results showed that Azospirillum brasilense (HM053 and FP2), Rhizobium tropici (Br322), and Gluconacetobacter diazotrophicus (Pal 5) produced the highest NO levels 24 h after inoculation, whereas Nitrospirillum amazonense (Y2) and Herbaspirillum seropedicae (SmR1) showed no NO production. In contrast to the literature, in NFbHP-NH4Cl-lactate culture medium with KNO3, NO trapping led to the recovery of a product with a molecular mass ion of 182 Da, namely, 1,2,3,4-naphthotetrazole (NTT), which contained one more nitrogen atom than the usual NAT product with 169 Da. This strategy allows monitoring and tracking NO production in potential biofertilizing bacteria, providing future opportunities to better understand the mechanisms of bacteria-plant interaction and also to manipulate the amount of NO that will sustain the PGPB.

Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis.

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.

Chemiexcited Neurotransmitters and Hormones Create DNA Photoproducts in the Dark.

In DNA, electron excitation allows adjacent pyrimidine bases to dimerize by [2 + 2] cycloaddition, creating chemically stable but lethal and mutagenic cyclobutane pyrimidine dimers (CPDs). The usual cause is ultraviolet radiation. Alternatively, CPDs can be made in the dark (dCPDs) via chemically mediated electron excitation of the skin pigment melanin, after it is oxidized by peroxynitrite formed from the stress-induced radicals superoxide and nitric oxide. We now show that the dark process is not limited to the unusual structural molecule melanin: signaling biomolecules such as indolamine and catecholamine neurotransmitters and hormones can also be chemiexcited to energy levels high enough to form dCPDs. Oxidation of serotonin, dopamine, melatonin, and related biogenic amines by peroxynitrite created triplet-excited species, evidenced by chemiluminescence, energy transfer to a triplet-state reporter, or transfer to O2 resulting in singlet molecular oxygen. For a subset of these signaling molecules, triplet states created by peroxynitrite or peroxidase generated dCPDs at levels comparable to ultraviolet (UV). Neurotransmitter catabolism by monoamine oxidase also generated dCPDs. These results reveal a large class of signaling molecules as electronically excitable by biochemical reactions and thus potential players in deviant mammalian metabolism in the absence of light.

Singlet Molecular Oxygen Generation in the Reaction of Biological Haloamines of Amino Acids and Polyamines with Hydrogen Peroxide†.

Leucocytes generate hypohalous acids (HOCl and HOBr) to defend the host against pathogens. In cells, hypohalous acids react with amine-containing molecules, such as amino acids and polyamines, producing chloramines and bromamines, reservoirs of oxidizing power that can potentially damage host tissues at sites of inflammation. Hypohalous acids also react with H2 O2 to produce stoichiometric amounts of singlet molecular oxygen ( 1 O 2 ), but its generation in leucocytes is still under debate. Additionally, it is unclear whether haloamines generate 1 O 2 following a reaction with H2 O2 . Herein, we provide evidence of the generation of 1 O 2 in the reactions between amino acid-derived (taurine, N-α-acetyl-Lysine and glycine) and polyamine-derived (spermine and spermidine) haloamines and H2 O2 in an aqueous solution. The unequivocal formation of 1 O 2 was detected by monitoring its characteristic monomol light emission at 1270 nm in the near-infrared region. For amino acid-derived haloamines, the presence of 1 O 2 was further confirmed by chemical trapping with anthracene-9,10-divinylsulfonate and HPLC-MS/MS detection. Altogether, photoemission and chemical trapping studies demonstrated that chloramines were less effective at producing 1 O 2 than bromamines of amino acids and polyamines. Thus, 1 O 2 formation via bromamines and H2 O2 may be a potential source of 1 O 2 in nonilluminated biological systems.

Micropropagation of Seed-Derived Clonal Lines of the Endangered Agave marmorata Roezl and Their Compatibility with Endophytes.

A. marmorata is the raw material used for tepextate mescal production but is classified as an endangered species. In the present study, we obtain and multiply clonal lines of Agave marmorata Roezl by selecting seedlings derived from seeds. Ten seedlings from two lots of 400 germinated seeds were selected for axillary bud proliferation induced by BAP 5 mg/L in vitamin-free Murashige and Skoog's medium. Differences in shoot numbers, heights and senescent tissue formation were observed. Notably, the AM32 line formed 84 shoots and presented low senescent tissue after 60 d of culture. We also selected the AM31 and AM33 clonal lines. Four-month shoots were extracted with 80% methanol in water to determine the total content of saponins, flavonoids, and phenolic acids and compare the three clonal lines. Some bioactive molecules were identified using HPLC techniques and MALDI-TOF mass spectrometry none showed significant differences in content. Additionally, plants derived from the clonal lines were inoculated with four endophytic bacteria. Among these, Achromobacter xylosoxidans supported plant growth of AM32. A notable effect of plant death was observed after inoculation with Enterobacter cloacae, an endophyte of A. tequilana. Additionally, Pseudomonas aeruginosa, an endophyte from A. marmorata, reduced biomass. Our results demonstrate the incompatibility of A. marmorata to E. cloacae and specialization between the host plant and its endophytes. The compatibility of the plant-endophyte could be exploited to boost the establishment and stability of mutualisms to benefit plant development, stress tolerance and pathogen resistance. The differences in multiplication capacity, stable tissue formation, and endophyte biotization responses may indicate genetic variability. Clonal selection and micropropagation from seed-derived plants could contribute to conserving the endangered A. marmorata plant for reforestation in their natural habitats, thus, assuring mass propagation for sustainable industrial production of mescal, bioactive compounds, and prebiotics.

Dehydromethionine is a common product of methionine oxidation by singlet molecular oxygen and hypohalous acids.

Methionine is one of the main targets for biological oxidants. Its reaction with the majority of oxidants generates only methionine sulfoxide. However, when N-terminal methionine reacts with hypohalous acids (HOCl and HOBr) or singlet molecular oxygen (1O2), it can also generate a cyclic product called dehydromethionine (DHM). Previously, DHM was suggested as a biomarker of oxidative stress induced by hypohalous acids. However, DHM can also be generated by 1O2 -oxidation of methionine, and the contribution of this pathway of DHM formation in a context of a site-specific redox imbalance in an organism is unknown. In this work, a through comparison of the reactions of hypohalous acids and 1O2 with methionine, either free or inserted in peptides and proteins was undertaken. In addition, we performed methionine photooxidation in heavy water (H218O) to determine the influence of the pH in the mechanism of DHM formation. We showed that for free methionine, or methionine-containing peptides, the yields of DHM formation in the reactions with 1O2 were close to those achieved by HOBr oxidation, but much higher than the yields obtained with HOCl as the oxidant. This was true for all pH tested (5, 7.4, and 9). Interestingly, for the protein ubiquitin, DHM yields after reaction with 1O2 were higher than those obtained with both hypohalous acids. Our results indicate that 1O2 may also be an important source of DHM in biological systems.

Spatial proteomics reveals subcellular reorganization in human keratinocytes exposed to UVA light.

The effects of UV light on the skin have been extensively investigated. However, systematic information about how the exposure to ultraviolet-A (UVA) light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass-spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1,600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins, and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin.

Characterization and Quantification of Tryptophan- and Tyrosine-derived Hydroperoxides.

The reaction of singlet oxygen (1 O2 ) with the amino acids tryptophan and tyrosine, either free or inserted in peptides or proteins, gives rise to hydroperoxides. To understand the impact of these hydroperoxides in complex biological systems, methods allowing their characterization and accurate quantification must be available. In this work, hydroperoxides derived from tryptophan and tyrosine and from peptides containing these amino acids were synthesized by photooxidation, and characterized by high-resolution mass spectrometry. In addition, experiments were carried out to compare two colorimetric methods commonly used for quantification of peroxides, namely the iodometric and the ferric-xylenol orange assays. For the tryptophan hydroperoxide, the quantifications obtained by colorimetric methods were then compared to that obtained by NMR. The results showed that for the ferric-xylenol orange method, the stoichiometry between peroxide and Fe3+ ions varies considerably. On the other hand, for the iodometric assay, the stoichiometry peroxide:I3 - ions is always 1:1. However, the kinetics of the reactions of peroxides with I- vary, and the assay must be performed in anaerobic conditions. Thus, the iodometric method is more appropriate for precise quantification of a given peroxide. The characterization and accurate quantification of biological peroxides is key to understand the mechanisms involved in redox processes.

A single dose of Ultraviolet-A induces proteome remodeling and senescence in primary human keratinocytes.

Epidermal photoaging contributes to skin fragility over time and it is a risk factor for skin cancer. Photoaging has been associated for a long time with exposure to Ultraviolet-A (UVA) light, the predominant component of the solar ultraviolet radiation. While the cellular mechanisms underlying UVA-induced photoaging in the dermis have been well characterized, UVA's action on the epidermis remains elusive. Here, proteomic analysis was conducted to derive the cellular responses induced by an environmentally relevant dose of UVA in primary human keratinocytes. We also investigated the effects of UVA on non-transformed immortalized keratinocytes (HaCaT cells), bearing potentially oncogenic mutations. We showed that UVA induces proteome remodeling and senescence in primary keratinocytes, eliciting potent antioxidant and pro-inflammatory responses. Additionally, we showed that UVA modulates the secretory phenotype of these cells to the extent of inducing paracrine oxidative stress and immune system activation in pre-malignant keratinocytes. These observations offer insights into the cellular mechanisms by which UVA drives photoaging in the skin.

HDL proteome remodeling associates with COVID-19 severity.

BACKGROUND: Besides the well-accepted role in lipid metabolism, high-density lipoprotein (HDL) also seems to participate in host immune response against infectious diseases. OBJECTIVE: We used a quantitative proteomic approach to test the hypothesis that alterations in HDL proteome associate with severity of Coronavirus disease 2019 (COVID-19). METHODS: Based on clinical criteria, subjects (n=41) diagnosed with COVID-19 were divided into two groups: a group of subjects presenting mild symptoms and a second group displaying severe symptoms and requiring hospitalization. Using a proteomic approach, we quantified the levels of 29 proteins in HDL particles derived from these subjects. RESULTS: We showed that the levels of serum amyloid A 1 and 2 (SAA1 and SAA2, respectively), pulmonary surfactant-associated protein B (SFTPB), apolipoprotein F (APOF), and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) were increased by more than 50% in hospitalized patients, independently of sex, HDL-C or triglycerides when comparing with subjects presenting only mild symptoms. Altered HDL proteins were able to classify COVID-19 subjects according to the severity of the disease (error rate 4.9%). Moreover, apolipoprotein M (APOM) in HDL was inversely associated with odds of death due to COVID-19 complications (odds ratio [OR] per 1-SD increase in APOM was 0.27, with 95% confidence interval [CI] of 0.07 to 0.72, P=0.007). CONCLUSION: Our results point to a profound inflammatory remodeling of HDL proteome tracking with severity of COVID-19 infection. They also raise the possibility that HDL particles could play an important role in infectious diseases.

Probiotic Endophytes for More Sustainable Banana Production.

Climatic factors and pathogenic fungi threaten global banana production. Moreover, bananas are being cultivated using excessive amendments of nitrogen and pesticides, which shift the microbial diversity in plants and soil. Advances in high-throughput sequencing (HTS) technologies and culture-dependent methods have provided valuable information about microbial diversity and functionality of plant-associated endophytic communities. Under stressful (biotic or abiotic) conditions, plants can recruit sets of microorganisms to alleviate specific potentially detrimental effects, a phenomenon known as "cry for help". This mechanism is likely initiated in banana plants infected by Fusarium wilt pathogen. Recently, reports demonstrated the synergistic and cumulative effects of synthetic microbial communities (SynComs) on naturally occurring plant microbiomes. Indeed, probiotic SynComs have been shown to increase plant resilience against biotic and abiotic stresses and promote growth. This review focuses on endophytic bacterial diversity and keystone taxa of banana plants. We also discuss the prospects of creating SynComs composed of endophytic bacteria that could enhance the production and sustainability of Cavendish bananas (Musa acuminata AAA), the fourth most important crop for maintaining global food security.

Detection of DNA Adduct Formation in Rat Lungs by a Micro-HPLC/MS/MS Approach.

Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These molecules can react with DNA bases forming mutagenic exocyclic adducts, which have been used as biomarkers of aldehyde exposure and as potential tools for the study of inflammation, metal storage diseases, neurodegenerative disorders, and cancer. High-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) provides a highly precise, specific and ultrasensitive method for the detection of exocyclic DNA adducts. Here we present and describe a validated micro-HPLC-Electro Spray Ionization (ESI)-MS/MS method for the quantification of 1,N2-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.

l-Tryptophan Interactions with the Horseradish Peroxidase-Catalyzed Generation of Triplet Acetone.

Triplet carbonyls generated by chemiexcitation are involved in typical photobiochemical processes in the absence of light. Due to their biradical nature, ultraweak light emission and long lifetime, electronically excited triplet species display typical radical reactions such as isomerization, fragmentation, cycloaddition and hydrogen abstraction. In this paper, we report chemical reactions in a set of amino acid residues induced by the isobutanal/horseradish peroxidase (IBAL/HRP) system, a well-known source of excited triplet acetone (Ac3* ). Accordingly, quenching of Ac3* by tryptophan (Trp) unveiled parallel enzyme damage and inactivation, likely explained by scavenging of IBAL tertiary radical reaction intermediate and Ac3* -derived 2-hydroxy-i-propyl radical. Quenching constants were calculated from Stern-Volmer plots, and the structure of radical adducts was revealed by mass spectrometry. As expected, a concurrent Schiff-type adduct was found to be one of the reaction by-products. These findings draw attention to potential structural and functional changes in enzymes involved in the electronic chemiexcitation of their products.

Heck reaction synthesis of anthracene and naphthalene derivatives as traps and clean chemical sources of singlet molecular oxygen in biological systems.

Studies have previously shown that anthracene and naphthalene derivatives serve as compounds for trapping and chemically generating singlet molecular oxygen [O2(1Δg)], respectively. Simple and efficient synthetic routes to anthracene and naphthalene derivatives are needed, for improved capture and release of O2(1Δg) in cellular environments. Because of this need, we have synthesized a dihydroxypropyl amide naphthlene endoperoxide as a O2(1Δg) donor, as well as five anthracene derivatives as O2(1Δg) acceptor. The anthracene derivatives bear dihydroxypropyl amide, ester, and sulfonate ion end groups connected to 9,10-positions by way of unsaturated (vinyl) and saturated (ethyl) bridging groups. Heck reactions were found to yield these six compounds in easy-to-carry out 3-step reactions in yields of 50-76%. Preliminary results point to the potential of the anthracene compounds to serve as O2(1Δg) acceptors and would be amenable for future use in biological systems to expand the understanding of O2(1Δg) in biochemistry.

Human cataractous lenses contain cross-links produced by crystallin-derived tryptophanyl and tyrosyl radicals.

Protein insolubilization, cross-linking and aggregation are considered critical to the development of lens opacity in cataract. However, the information about the presence of cross-links other than disulfides in cataractous lenses is limited. A potential role for cross-links produced from tryptophanyl radicals in cataract development is suggested by the abundance of the UV light-sensitive Trp residues in crystallin proteins. Here we developed a LC-MS/MS approach to examine the presence of Trp-Trp, Trp-Tyr and Tyr-Tyr cross-links and of peptides containing Trp-2H (-2.0156 Da) in the lens of three patients diagnosed with advanced nuclear cataract. In the proteins of two of the lenses, we characterized intermolecular cross-links between ßB2-Tyr153-Tyr104-ßA3 and ßB2-Trp150-Tyr139-ßS. An additional intermolecular cross-link (ßB2-Tyr61-Trp200-ßB3) was present in the lens of the oldest patient. In the proteins of all three lenses, we characterized two intramolecular Trp-Trp cross-links (Trp123-Trp126 in ßB1 and Trp81-Trp84 in ßB2) and six peptides containing Trp -2H residues, which indicate the presence of additional Trp-Trp cross-links. Relevantly, we showed that similar cross-links and peptides with modified Trp-2H residues are produced in a time-dependent manner in bovine ß-crystallin irradiated with a solar simulator. Therefore, different crystallin proteins cross-linked by crystalline-derived tryptophanyl and tyrosyl radicals are present in advanced nuclear cataract lenses and similar protein modifications can be promoted by solar irradiation even in the absence of photosensitizers. Overall, the results indicate that a role for Trp-Tyr and Trp-Trp cross-links in the development of human cataract is possible and deserves further investigation.

Singlet oxygen generation by the reaction of acrolein with peroxynitrite via a 2-hydroxyvinyl radical intermediate.

Acrolein (2-propenal) is an environmental pollutant, food contaminant, and endogenous toxic by-product formed in the thermal decomposition and peroxidation of lipids, proteins, and carbohydrates. Like other α,ß-unsaturated aldehydes, acrolein undergoes Michael addition of nucleophiles such as basic amino acids residues of proteins and nucleobases, triggering aging associated disorders. Here, we show that acrolein is also a potential target of the potent biological oxidant, nitrosating and nitrating agent peroxynitrite. In vitro studies revealed the occurrence of 1,4-addition of peroxynitrite (k2 = 6 × 103 M-1 s-1, pH 7.2, 25 °C) to acrolein in air-equilibrated phosphate buffer. This is attested by acrolein concentration-dependent oxygen uptake, peroxynitrite consumption, and generation of formaldehyde and glyoxal as final products. These products are predicted to be originated from the Russell termination of •OOCH=CH(OH) radical which also includes molecular oxygen at the singlet delta state (O21Δg). Accordingly, EPR spin trapping studies with the 2,6-nitrosobenzene-4-sulfonate ion (DBNBS) revealed a 6-line spectrum attributable to the 2-hydroxyvinyl radical adduct. Singlet oxygen was identified by its characteristic monomolecular IR emission at 1,270 nm in deuterated buffer, which was expectedly quenched upon addition of water and sodium azide. These data represent the first report on singlet oxygen creation from a vinylperoxyl radical, previously reported for alkyl- and formylperoxyl radicals, and may contribute to better understand the adverse acrolein behavior in vivo.

Generation of Singlet Molecular Oxygen by Lipid Hydroperoxides and Nitronium Ion†.

Singlet molecular oxygen is a reactive species involved in biological oxidative processes. The major cellular targets of singlet molecular oxygen are unsaturated fatty acids in the membrane, as well as nucleic acids and proteins. The aim of this study was to investigate whether lipids and commercial hydroperoxides generate singlet molecular oxygen, in presence of nitronium and activated nitronium ion. For this purpose, monomol light emitted in the near-infrared region (λ = 1270 nm) was used to monitor singlet molecular oxygen decay in different solvents, with different hydroperoxides and in the presence of azide. Direct measurements of the singlet molecular oxygen spectrum at 1270 nm recorded during the reaction between lipids and commercial hydroperoxides and nitronium ions unequivocally demonstrated the formation of this excited species.