Decreased Glycocalyx Shedding on Presentation in Hemorrhaging Geriatric Trauma Patients.

INTRODUCTION: Plasma levels of syndecan-1 (Sdc-1), a biomarker of endothelial glycocalyx (EG) damage, correlate with worse outcomes in trauma patients. However, EG injury is not well characterized in injured older adults (OA). The aims of this study were to characterize Sdc-1 shedding in OA trauma patients relative to younger adults (YA) and determine associations with putative regulators of EG sheddases. METHODS: We performed a secondary analysis of data from the Pragmatic, Randomized Optimal Platelet, and Plasma Ratios (PROPPR) trial, stratifying bluntly injured subjects into OA and YA groups based on upper age quartile (57 y). Plasma Sdc-1 levels were compared in OA and YA at hospital arrival through postinjury day 3, and the independent association between age and Sdc-1 level at arrival was determined after adjusting for differences in gender, shock index (SI), and pre-existing comorbidities. In a follow-up analysis, case-control matching was used to create populations of OA and YA with equivalent SI and injury severity score. Levels of Sdc-1 were compared between these matched groups, and the relationships with candidate regulators of EG shedding were assessed. RESULTS: Of 680 subjects in the Pragmatic, Randomized Optimal Platelet, and Plasma Ratios trial, 350 (51%) had blunt injuries, and 92 (26.3%) of these were OA. Plasma Sdc-1 levels at arrival, 2 h, and 6 h were significantly lower in OA compared to YA (all P < 0.05). After adjusting for sex, pre-existing morbidities and SI, age was associated with decreased Sdc-1 levels at arrival. In the matched analyses, Sdc-1, high-mobility group box 1 and tissue inhibitor of metalloproteinase-2 levels were lower in OA compared to YA. Both high-mobility group box-1 and tissue inhibitor of metalloproteinase-2 significantly correlated with arrival Sdc-1 and were inversely associated with age. CONCLUSIONS: This study indicates that increased age is independently associated with decreased Sdc-1 levels among patients with blunt injuries. Suppressed plasma levels of sheddases in relation to diminished Sdc-1 shedding suggest that mechanisms regulating EG cleavage may be impaired in injured older adults. These findings provide novel insight into the age-dependent impact of injury on the vascular endothelium, which could have important implications for the clinical management of older adults following trauma.

Fragmentation dynamics of the doubly charged aniline: the source of kinetically excited CnH3+ ions.

The goals of this work are to attempt to decipher if an aniline dication can isomerize to a picoline dication in a given astrochemical environment and if the dissociation of such dications could be a source of kinetically hot fragment ions, some of which could be of significance in the interstellar medium. Toward this purpose, the VUV-induced dication dissociation was investigated experimentally using ion-ion coincidence and computationally by optimizing various pathways. Contrary to previous reports, we show here that the dication of aniline is structurally too weak to retain its ring structure while following the dissociation pathways. A fragile open ring structure could lead to all the experimentally observed pathways of noticeable intensity. The significance of this, especially in terms of molecular dynamics, can be assessed by the fact that all the transformations were facilitated by specific hydrogen migration. A clear selectivity is seen where the dication of aniline was found to prefer a rearrangement of hydrogen within the ring rather than transferring from nitrogen to the ring, which is conventionally expected and has to do with the charge state and charge localization.

Endothelial Glycocalyx Degradation Patterns in Sepsis-Associated Pediatric Acute Respiratory Distress Syndrome: A Single Center Retrospective Observational Study.

BACKGROUND: Sepsis-associated destruction of the pulmonary microvascular endothelial glycocalyx (EGCX) creates a vulnerable endothelial surface, contributing to the development of acute respiratory distress syndrome (ARDS). Constituents of the EGCX shed into circulation, glycosaminoglycans and proteoglycans, may serve as biomarkers of endothelial dysfunction. We sought to define the patterns of plasma EGCX degradation products in children with sepsis-associated pediatric ARDS (PARDS), and test their association with clinical outcomes. METHODS: We retrospectively analyzed a prospective cohort (2018-2020) of children (≥1 month to <18 years of age) receiving invasive mechanical ventilation for acute respiratory failure for ≥72 h. Children with and without sepsis-associated PARDS were selected from the parent cohort and compared. Blood was collected at time of enrollment. Plasma glycosaminoglycan disaccharide class (heparan sulfate, chondroitin sulfate, and hyaluronan) and sulfation subtypes (heparan sulfate and chondroitin sulfate) were quantified using liquid chromatography tandem mass spectrometry. Plasma proteoglycans (syndecan-1) were measured through an immunoassay. RESULTS: Among the 39 mechanically ventilated children (29 with and 10 without sepsis-associated PARDS), sepsis-associated PARDS patients demonstrated higher levels of heparan sulfate (median 639 ng/mL [interquartile range, IQR 421-902] vs 311 [IQR 228-461]) and syndecan-1 (median 146 ng/mL [IQR 32-315] vs 8 [IQR 8-50]), both p = 0.01. Heparan sulfate subtype analysis demonstrated greater proportions of N-sulfated disaccharide levels among children with sepsis-associated PARDS (p = 0.01). Increasing N-sulfated disaccharide levels by quartile were associated with severe PARDS (n = 9/29) with the highest quartile including >60% of the severe PARDS patients (test for trend, p = 0.04). Higher total heparan sulfate and N-sulfated disaccharide levels were independently associated with fewer 28-day ventilator-free days in children with sepsis-associated PARDS (all p < 0.05). CONCLUSIONS: Children with sepsis-associated PARDS exhibited higher plasma levels of heparan sulfate disaccharides and syndecan-1, suggesting that EGCX degradation biomarkers may provide insights into endothelial dysfunction and PARDS pathobiology.

The x-ray absorption spectrum of the tert-butyl radical: An experimental and computational investigation.

We report the x-ray absorption spectrum (XAS) of the tert-butyl radical, C4H9. The radical was generated pyrolytically from azo-tert-butane, and the XAS of the pure radical was obtained by subtraction of spectra recorded at different temperatures. The bands in the XAS were assigned by ab initio calculations that are in very good agreement with the experimental data. The lowest energy signal in the XAS is assigned to the C1s electron transition from the central carbon atom to the singly occupied molecular orbital (SOMO), while higher transitions correspond to C1s excitations from terminal carbon atoms. Furthermore, we investigated the fragmentation of the radical following resonant C1s excitation by electron-ion-coincidence spectroscopy. Several fragmentation channels were identified. The C1s excitation of the terminal carbons is associated with a stronger fragmentation tendency compared to the lowest C1s excitation of the central carbon into the SOMO. For this core excited state, we still observe an intact parent ion, C4H9+, and a comparatively higher tendency to dissociate into CH3+ + C3H6+.

Facile Energy Release from Substituted Dewar Isomers of 1,2-Dihydro-1,2-Azaborinines Catalyzed by Coinage Metal Lewis Acids.

Molecular solar thermal systems (MOST) represent an auspicious solution for the storage of solar energy. We report silver salts as a unique class of catalysts, capable of releasing the stored energy from the promising 1,2-dihydro-1,2-azaborinine based MOST system. Mechanistic investigations provided insights into the silver catalyzed thermal backreaction, concurrently unveiling the first crystal structure of a 2-aza-3-borabicyclo[2.2.0]hex-5-ene, the Dewar isomer of 1,2-dihydro-1,2-azaborinine. Quantification of activation energies by kinetic experiments has elucidated the advantageous energy change associated with Lewis acid catalysts, a phenomenon corroborated through computational analysis. By means of low temperature NMR spectroscopy, mechanistic insights into the coordination of Ag+ to the 1,2-dihydro-1,2-azaborinine were gained.

COVID-19 bacteremic co-infection is a major risk factor for mortality, ICU admission, and mechanical ventilation.

BACKGROUND: Recent single-center reports have suggested that community-acquired bacteremic co-infection in the context of Coronavirus disease 2019 (COVID-19) may be an important driver of mortality; however, these reports have not been validated with a multicenter, demographically diverse, cohort study with data spanning the pandemic. METHODS: In this multicenter, retrospective cohort study, inpatient encounters were assessed for COVID-19 with community-acquired bacteremic co-infection using 48-h post-admission blood cultures and grouped by: (1) confirmed co-infection [recovery of bacterial pathogen], (2) suspected co-infection [negative culture with ≥ 2 antimicrobials administered], and (3) no evidence of co-infection [no culture]. The primary outcomes were in-hospital mortality, ICU admission, and mechanical ventilation. COVID-19 bacterial co-infection risk factors and impact on primary outcomes were determined using multivariate logistic regressions and expressed as adjusted odds ratios with 95% confidence intervals (Cohort, OR 95% CI, Wald test p value). RESULTS: The studied cohorts included 13,781 COVID-19 inpatient encounters from 2020 to 2022 in the University of Alabama at Birmingham (UAB, n = 4075) and Ochsner Louisiana State University Health-Shreveport (OLHS, n = 9706) cohorts with confirmed (2.5%), suspected (46%), or no community-acquired bacterial co-infection (51.5%) and a comparison cohort consisting of 99,170 inpatient encounters from 2010 to 2019 (UAB pre-COVID-19 pandemic cohort). Significantly increased likelihood of COVID-19 bacterial co-infection was observed in patients with elevated ≥ 15 neutrophil-to-lymphocyte ratio (UAB: 1.95 [1.21-3.07]; OLHS: 3.65 [2.66-5.05], p < 0.001 for both) within 48-h of hospital admission. Bacterial co-infection was found to confer the greatest increased risk for in-hospital mortality (UAB: 3.07 [2.42-5.46]; OLHS: 4.05 [2.29-6.97], p < 0.001 for both), ICU admission (UAB: 4.47 [2.87-7.09], OLHS: 2.65 [2.00-3.48], p < 0.001 for both), and mechanical ventilation (UAB: 3.84 [2.21-6.12]; OLHS: 2.75 [1.87-3.92], p < 0.001 for both) across both cohorts, as compared to other risk factors for severe disease. Observed mortality in COVID-19 bacterial co-infection (24%) dramatically exceeds the mortality rate associated with community-acquired bacteremia in pre-COVID-19 pandemic inpatients (5.9%) and was consistent across alpha, delta, and omicron SARS-CoV-2 variants. CONCLUSIONS: Elevated neutrophil-to-lymphocyte ratio is a prognostic indicator of COVID-19 bacterial co-infection within 48-h of admission. Community-acquired bacterial co-infection, as defined by blood culture-positive results, confers greater increased risk of in-hospital mortality, ICU admission, and mechanical ventilation than previously described risk factors (advanced age, select comorbidities, male sex) for COVID-19 mortality, and is independent of SARS-CoV-2 variant.

Two-body dissociation of isoxazole following double photoionization - an experimental PEPIPICO and theoretical DFT and MP2 study.

The dissociative double photoionization of isoxazole molecules has been investigated experimentally and theoretically. The experiment has been carried out in the 27.5-36 eV photon energy range using vacuum ultraviolet (VUV) synchrotron radiation excitation combined with ion time-of-flight (TOF) spectrometry and photoelectron-photoion-photoion coincidence (PEPIPICO) technique. Five well-resolved two-body dissociation channels have been identified in the isoxazole's coincidence maps, and their appearance energies have been determined. The coincidence yield curves of these dissociation channels have been obtained in the photon energy ranges from their appearance energies up to 36 eV. The double photoionization of isoxazole produces a C3H3NO2+ transient dication, which decomposes into fragments differing from previously reported photofragmentation products of isoxazole. We have found no evidence of pathways leading to the C3H2NO+, HCN+, C2H2O+, C3HN+, or C2H2+ fragments or their neutral counterparts that have been observed in previous neutral photodissociation and single photoionization studies. Instead, the dissociation of isoxazole after the ejection of two electrons is bond-selective and is governed by two reactions, HCO+ + H2CCN+ and H2CO+ + HCCN+, whose appearance energies are 28.6 (±0.3) and 29.4 (±0.3) eV, respectively. A third dissociation channel turns out to be a variant of the most intense channel (HCO+ + H2CCN+), where one of the fragment ions contains a heavy isotope. Two minor dissociation channels occurring at higher energies, CO+ + CH3CN+ and CN+ + H3CCO+, are also identified. The density functional and ab initio quantum chemical calculations have been performed to elucidate the dissociative charge-separating mechanisms and determine the energies of the observed photoproducts. The present work unravels hitherto unexplored photodissociation mechanisms of isoxazole and thus provides deeper insight into the photophysics of five-membered heterocyclic molecules containing two heteroatoms.

A photoelectron spectroscopic investigation of aspirin, paracetamol and ibuprofen in the gas phase.

We have investigated the electronic structure of isolated molecules of paracetamol, aspirin and ibuprofen using computational methods and benchmarked the results against valence and core photoelectron spectra. Paracetamol, aspirin and ibuprofen exist as multiple conformers, and we have calculated the free energies and populations of the lowest energy conformers. We find generally good agreement with previous experimental and theoretical structural results. The valence band spectrum of gas phase aspirin has not been reported previously, and we report it and assign the features based on calculations. The effect of acetylation on the frontier orbitals of the parent molecule, salicylic acid, is to increase the ionization potential of the highest occupied molecular orbital (HOMO), and to exchange the energetic ordering of the following two orbitals. The acetyl π bond contributes to the next orbital, which is hybridised with ring π orbitals. The core level spectra of all three molecules are reported and compared with calculations and with the spectra of parent molecules (salicylic acid for aspirin, 4-aminophenol for paracetamol). Observed core ionization energies are in agreement with theory. Although all compounds share a benzene ring, and they also have a number of other chromophores in common, the spectroscopic data indicate chemical diversity, suggesting that their modes of bonding under physiological conditions are likely to be diverse.

Photofragmentation specificity of photoionized cyclic amino acids (diketopiperazines) as precursors of peptide building blocks.

The photoionisation and photofragmentation of the two cyclic dipetides cyclo(alanyl-glycine) cGA and cyclo(glycyl-glycine) cGG, have been studied combining experiments and simulations. State selected fragments from the ionized molecules are detected using photo-electron photo-ion coincidence (PEPICO) measurements and specific fragmentation paths are identified and characterized via the use of ion-neutral coincidence maps. The simulations, performed using Quantum Chemistry methods, allow us to infer the fragmentation mechanisms of the ionized and excited molecules. We show that ring opening is followed by emission of the neutral fragments CO and HNCO. In the case of cGG the emission of neutral CO leads to a metastable structure that breaks producing small cationic fragments. The studied cyclic dipeptides evolve under ionizing radiation generating different small aziridin moieties and oxazolidinones. These two species are key reactants to elongate producing peptide chains. The corresponding mechanisms have been computed and show that the reaction requires very low energy and may occur in the presence of ionizing radiation.

Autoionization from the plasmon resonance in isolated 1-cyanonaphthalene.

Polycyclic aromatic hydrocarbons have widely been conjectured to be ubiquitous in space, as supported by the recent discovery of two isomers of cyanonaphthalene, indene, and 2-cyanoindene in the Taurus molecular cloud-1 using radioastronomy. Here, the photoionization dynamics of 1-cyanonaphthalene (1-CNN) are investigated using synchrotron radiation over the hν = 9.0-19.5 eV range, revealing that prompt autoionization from the plasmon resonance dominates the photophysics for hν = 11.5-16.0 eV. Minimal photo-induced dissociation, whether originating from an excited state impulsive bond rupture or through internal conversion followed by a statistical bond cleavage process, occurs over the microsecond timescale (as limited by the experimental setup). The direct photoionization cross section and photoelectron angular distributions are simulated using an ezDyson model combining Dyson orbitals with Coulomb wave photoejection. When considering these data in conjunction with recent radiative cooling measurements on 1-CNN+, which showed that cations formed with up to 5 eV of internal energy efficiently stabilize through recurrent fluorescence, we conclude that the organic backbone of 1-CNN is resilient to photodestruction by VUV and soft XUV radiation. These dynamics may prove to be a common feature for the survival of small polycyclic aromatic hydrocarbons in space, provided that the cations have a suitable electronic structure to support recurrent fluorescence.

In search of universalities in the dissociative photoionization of PANHs via isomerizations.

In search of the cause behind the similarities often seen in the fragmentation of PANHs, vacuum ultraviolet (VUV) photodissociation of two pairs of isomers quinoline-isoquinoline and 2-naphthylamine-3-methyl-quinoline are studied using the velocity map imaging technique. The internal energy dependence of all primary fragmentation channels is obtained for all four target molecules. The decay dynamics of the four molecules is studied by comparing their various experimental signatures. The dominant channel for the first pair of isomers is found to be hydrogen cyanide (HCN) neutral loss, while the second pair of isomers lose HCNH neutral as its dominant channel. Despite this difference in their primary decay products and the differences in the structures of the four targets, various similarities in their experimental signatures are found, which could be explained by isomerization mechanisms to common structures. The fundamental role of these isomerization in controlling different dissociative channels is explored via a detailed analysis of the experimental photoelectron-photoion coincidences and the investigation of the theoretical potential energy surface. These results add to the notion of a universal PANH fragmentation mechanism and suggests the seven member isomerization as a key candidate for this universal mechanism. The balance between isomerization, dissociation, and other key mechanistic processes in the reaction pathways, such as hydrogen migrations, is also highlighted for the four molecules.

Double Photoionization of Nitrosyl Chloride by Synchrotron Radiation in the 24-70 eV Photon Energy Range.

The behavior of nitrosyl chloride (ClNO) exposed to ionizing radiation was studied by direct probing valence-shell electrons in temporal coincidence with ions originating from the fragmentation process of the transient ClNO2+. Such a molecular dication was produced by double photoionization with synchrotron radiation in the 24-70 eV photon energy range. The experiment has been conducted at the Elettra Synchrotron Facility of Basovizza (Trieste, Italy) using a light beam linearly polarized with the direction of the polarization vector parallel to the ClNO molecular beam axis. ClNO molecules crossing the photon beam at right angles in the scattering region are generated by effusive expansion and randomly oriented. The threshold energy for the double ionization of ClNO (30.1 ± 0.1 eV) and six dissociation channels producing NO+/Cl+, N+/Cl+, N+/O+, O+/Cl+, ClN+/O+, NO+/Cl2+ ion pairs, with their relative abundance and threshold energies, have been measured.

Photochemical Ring-Opening Reaction of 1,3-Cyclohexadiene: Identifying the True Reactive State.

The photochemically induced ring-opening isomerization reaction of 1,3-cyclohexadiene to 1,3,5-hexatriene is a textbook example of a pericyclic reaction and has been amply investigated with advanced spectroscopic techniques. The main open question has been the identification of the single reactive state which drives the process. The generally accepted description of the isomerization pathway starts with a valence excitation to the lowest lying bright state, followed by a passage through a conical intersection to the lowest lying doubly excited state, and finally a branching between either the return to the ground state of the cyclic molecule or the actual ring-opening reaction leading to the open-chain isomer. Here, in a joint experimental and computational effort, we demonstrate that the evolution of the excitation-deexcitation process is much more complex than that usually described. In particular, we show that an initially high-lying electronic state smoothly decreasing in energy along the reaction path plays a key role in the ring-opening reaction.

Modeling the Effect of Hydrophobicity on the Passive Permeation of Solutes across a Bacterial Model Membrane.

Passive diffusion across biomembranes is an important mechanism of permeation for multiple drugs, including antibiotics. However, this process is frequently neglected while studying drug uptake and, in our view, warrants further investigation. Here, we apply molecular dynamics simulations to investigate the impact of changes in molecular hydrophobicity on the permeability of a series of inhibitors of the quorum sensing of Pseudomonas aeruginosa, previously discovered by us, across a membrane model. Overall, we show that permeation across this membrane model does not correlate with the molecule's hydrophobicity. We demonstrate that using a simple model for permeation, based on the difference between the maximum and minimum of the free energy profile, outperforms the inhomogeneous solubility-diffusion model, yielding a permeability ranking that better agrees with the experimental results, especially for hydrophobic permeants. The calculated differences in permeability could not explain differences in in bacterio activity. Nevertheless, substantial differences in molecular orientation along the permeation pathway correlate with the in bacterio activity, emphasizing the importance of analyzing, at an atomistic level, the permeation pathway of these solutes.

Electron and ion spectroscopy of the cyclo-alanine-alanine dipeptide.

The VUV photoionisation and photofragmentation of cyclo-alanine-alanine (cAA) has been studied in a joint experimental and theoretical work. The photoelectron spectrum and the photoelectron-photoion coincidence (PEPICO) measurements, which enable control of the energy being deposited, combined with quantum chemistry calculations, provide direct insight into the cAA molecular stability after photoionisation. The analysis of the ion-neutral coincidence experiments with the molecular dynamics simulations and the exploration of the potential energy surface allows a complete identification of the fragmentation pathways. It has been found that the fragmentation always starts with the ring opening through the C-C bond cleavage, followed by release of neutral CO or HNCO moieties.

Accurate property prediction by second order perturbation theory: The REMP and OO-REMP hybrids.

The prediction of molecular properties such as equilibrium structures or vibrational wavenumbers is a routine task in computational chemistry. If very high accuracy is required, however, the use of computationally demanding ab initio wavefunction methods is mandatory. We present property calculations utilizing Retaining the Excitation Degree - Møller-Plesset (REMP) and Orbital Optimized REMP (OO-REMP) hybrid perturbation theories, showing that with the latter approach, very accurate results are obtained at second order in perturbation theory. Specifically, equilibrium structures and harmonic vibrational wavenumbers and dipole moments of closed and open shell molecules were calculated and compared to the best available experimental results or very accurate calculations. OO-REMP is capable of predicting bond lengths of small closed and open shell molecules with an accuracy of 0.2 and 0.5 pm, respectively, often within the range of experimental uncertainty. Equilibrium harmonic vibrational wavenumbers are predicted with an accuracy better than 20 cm-1. Dipole moments of small closed and open shell molecules are reproduced with a relative error of less than 3%. Across all investigated properties, it turns out that a 20%:80% Møller-Plesset:Retaining the Excitation Degree mixing ratio consistently provides the best results. This is in line with our previous findings, featuring closed and open shell reaction energies.

Electronic state influence on selective bond breaking of core-excited nitrosyl chloride (ClNO).

The potential for selective bond breaking of a small molecule was investigated with electron spectroscopy and electron-ion coincidence experiments on ClNO. The electron spectra were measured upon direct valence photoionization and resonant core excitation at the N 1s- and O 1s-edges, followed by the emission of resonant-Auger (RA) electrons. The RA spectra were analyzed with particular emphasis on the assignment of the participator and spectator states. The states are of special relevance for investigating how distinct electronic configurations influence selective bond breaking. The electron-ion coincidence measurements provided branching fractions of the produced ion fragments as a function of electron binding energy. They explicitly demonstrate how the final electronic states created after photoionization and RA decay influence fragmentation. In particular, we observed a significantly different branching fraction for spectator states compared with participator states. In addition, it was also observed that the bonds broken for the spectator states correlate with the antibonding nature of the spectator-electron orbital.

Comprehensive survey of dissociative photoionization of quinoline by PEPICO experiments.

Dissociative photoionization of quinoline induced by vacuum ultraviolet radiation is investigated using photoelectron-photoion coincidence spectroscopy. Branching ratios of all the detectable fragment ions are measured as a function of internal energy ranging from 2 to 30 eV. A specific generation hierarchy is observed in the breakdown curves of a set of dissociation channels. Moreover, a careful comparison of the breakdown curves of fragments among the successive generations allowed to establish a decay sequence in the fragmentation of quinoline cation. This enabled us to revisit and refine the understanding of the first generation decay and reassign the origin of a few of the higher generation decay products of quinoline cation. With the help of the accompanying computational work (reported concurrently), we have demonstrated the dominance of two different HCN elimination pathways over previously interpreted mechanisms. For the first time, a specific pathway for acetylene elimination is identified in quinoline+ and the role of isomerization in both acetylene as well as hydrogen cyanide loss is also demonstrated. The experiment also established that the acetylene elimination exclusively occurs from the non-nitrogen containing rings of quinoline cation. The formation of a few astronomically important species is also discussed.

Photodissociation of quinoline cation: Mapping the potential energy surface.

A detailed exploration of the potential energy surface of quinoline cation (C9H7N·+) is carried out to extend the present understanding of its fragmentation mechanisms. Density functional theory calculations have been performed to explore new fragmentation schemes, giving special attention to previously unexplored pathways, such as isomerization and elimination of HNC. The isomerization mechanisms producing five- to seven-membered ring intermediates are described and are found to be a dominant channel both energetically and kinetically. Energetically competing pathways are established for the astrochemically important HNC-loss channel, which has hitherto never been considered in the context of the loss of a 27 amu fragment from the parent ions. Elimination of acetylene was also studied in great detail. Overall, the computational results are found to complement the experimental observations from the concurrently conducted PEPICO investigation. These could potentially open the doors for rich and interesting vacuum ultraviolet radiation-driven chemistry on planetary atmospheres, meteorites, and comets.

Mutations in the miR396 binding site of the growth-regulating factor gene VvGRF4 modulate inflorescence architecture in grapevine.

Bunch rot caused by Botrytis cinerea infections is a notorious problem in grapevine cultivation. To produce high quality fruits, grapevine plants are treated with fungicides, which is cost intensive and harmful to the environment. Conversely, loose cluster bunches show a considerably enhanced physical resilience to bunch diseases. With the aim to identify genetic determinants that modulate the development of bunch architecture, we have compared loose and compact 'Pinot noir' clones. Loose cluster architecture was found to be correlated with increased berry size, elongated rachis and elongated pedicels. Using transcriptome analysis in combination with whole genome sequencing, we have identified a growth-regulating factor gene, VvGRF4, upregulated and harbours heterozygous mutations in the loose cluster clones. At late stages of inflorescence development, the mRNA pools of loose cluster clones contain predominantly mRNAs derived from the mutated alleles, which are resistant to miR396 degradation. Expression of the VvGRF4 gene and its mutated variants in Arabidopsis demonstrates that it promotes pedicel elongation. Taken together, VvGRF4 modulates bunch architecture in grapevine 'Pinot noir' clones. This trait can be introduced into other cultivars using marker-assisted breeding or CRISPR-Cas9 technology. Related growth-regulating factors or other genes of the same pathway may have similar functions.