Infectious agents and their physiological correlates in early marine Chinook salmon (Oncorhynchus tshawytscha).

The early marine life of Pacific salmon is believed to be a critical period limiting population-level survival. Recent evidence suggests that some infectious agents are associated with survival but linkages with underlying physiological mechanisms are lacking. While challenge studies can demonstrate cause and effect relationships between infection and pathological change or mortality, in some cases pathological change may only manifest in the presence of environmental stressors; thus, it is important to gain context from field observations. Herein, we examined physiological correlates with infectious agent loads in Chinook salmon during their first ocean year. We measured physiology at the molecular (gene expression), metabolic (plasma chemistry) and cellular (histopathology) levels. Of 46 assayed infectious agents, 27 were detected, including viruses, bacteria and parasites. This exploratory study identified.a strong molecular response to viral disease and pathological change consistent with jaundice/anemia associated with Piscine orthoreovirus,strong molecular signals of gill inflammation and immune response associated with gill agents `Candidatus Branchiomonas cysticola' and Parvicapsula pseudobranchicola,a general downregulation of gill immune response associated with Parvicapsula minibicornis complementary to that of P. pseudobranchicola.Importantly, our study provides the first evidence that the molecular activation of viral disease response and the lesions observed during the development of the PRV-related disease jaundice/anemia in farmed Chinook salmon are also observed in wild juvenile Chinook salmon.

Single molecule infrared spectroscopy in the gas phase.

Spectroscopy is a key analytical tool that provides valuable insight into molecular structure and is widely used to identify chemical samples. Tagging spectroscopy is a form of action spectroscopy in which the absorption of a single photon by a molecular ion is detected via the loss of a weakly attached, inert 'tag' particle (for example, He, Ne, N2)1-3. The absorption spectrum is derived from the tag loss rate as a function of incident radiation frequency. So far, all spectroscopy of gas phase polyatomic molecules has been restricted to large molecular ensembles, thus complicating spectral interpretation by the presence of multiple chemical and isomeric species. Here we present a novel tagging spectroscopic scheme to analyse the purest possible sample: a single gas phase molecule. We demonstrate this technique with the measurement of the infrared spectrum of a single gas phase tropylium (C7H7+) molecular ion. The high sensitivity of our method revealed spectral features not previously observed using traditional tagging methods4. Our approach, in principle, enables analysis of multicomponent mixtures by identifying constituent molecules one at a time. Single molecule sensitivity extends action spectroscopy to rare samples, such as those of extraterrestrial origin5,6, or to reactive reaction intermediates formed at number densities that are too low for traditional action methods.

Low-Temperature Gas-Phase Kinetics of Ethanol-Methanol Heterodimer Formation.

The structures of gas-phase noncovalently bound clusters have long been studied in supersonic expansions. This method of study, while providing a wealth of information about the nature of noncovalent bonds, precludes observation of the formation of the cluster, as the clusters form just after the orifice of the pulsed valve. Here, we directly observe formation of ethanol-methanol dimers via microwave spectroscopy in a controlled cryogenic environment. Time profiles of the concentration of reagents in the cell yielded gas-phase reaction rate constants of kMe-g = (2.8 ± 1.4) × 10-13 cm3 molecule-1 s-1 and kMe-t = (1.6 ± 0.8) × 10-13 cm3 molecule-1 s-1 for the pseudo-second-order ethanol-methanol dimerization reaction at 8 K. The relaxation cross section between the gauche and trans conformers of ethanol was also measured using the same technique. In addition, thermodynamic relaxation between conformers of ethanol over time allowed for selection of conformer stoichiometry in the ethanol-methanol dimerization reaction, but no change in the ratio of dimer conformers was observed with changing ethanol monomer stoichiometry.

Delivery of a Group Hypnosis Protocol for Managing Chronic Pain in Outpatient Integrative Medicine.

Although strong evidence exists for using individual hypnosis to treat pain, evidence regarding group applications is limited. This project evaluated changes in multiple outcome measures in persons with chronic pain treated with 8 weeks of group hypnosis. Eighty-five adults with diverse chronic pain etiologies completed an 8-session, structured group hypnosis treatment. Pain intensity, pain interference, and global health were evaluated at baseline, posttreatment, and 3- and 6-months posttreatment. Linear mixed effects models assessed changes in outcomes over time. In a model testing, all three outcome measures simultaneously, participants improved substantially from pre- to posttreatment and maintained improvement across follow-up. Analyses of individual outcomes showed significant pre- to posttreatment reductions in pain intensity and interference, which were maintained for pain intensity and continued to improve for pain interference across follow-up. The findings provide compelling preliminary evidence that a group format is an effective delivery system for teaching individual skills in using hypnosis for chronic pain management. Larger randomized controlled trials are warranted to demonstrate equivalence of outcomes between treatment modes.

Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution.

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.

Utility of SPECT Functional Neuroimaging of Pain.

Functional neuroimaging modalities vary in spatial and temporal resolution. One major limitation of most functional neuroimaging modalities is that only neural activation taking place inside the scanner can be imaged. This limitation makes functional neuroimaging in many clinical scenarios extremely difficult or impossible. The most commonly used radiopharmaceutical in Single Photon Emission Tomography (SPECT) functional brain imaging is Technetium 99 m-labeled Ethyl Cysteinate Dimer (ECD). ECD is a lipophilic compound with unique pharmacodynamics. It crosses the blood brain barrier and has high first pass extraction by the neurons proportional to regional brain perfusion at the time of injection. It reaches peak activity in the brain 1 min after injection and is then slowly cleared from the brain following a biexponential mode. This allows for a practical imaging window of 1 or 2 h after injection. In other words, it freezes a snapshot of brain perfusion at the time of injection that is kept and can be imaged later. This unique feature allows for designing functional brain imaging studies that do not require the patient to be inside the scanner at the time of brain activation. Functional brain imaging during severe burn wound care is an example that has been extensively studied using this technique. Not only does SPECT allow for imaging of brain activity under extreme pain conditions in clinical settings, but it also allows for imaging of brain activity modulation in response to analgesic maneuvers whether pharmacologic or non-traditional such as using virtual reality analgesia. Together with its utility in extreme situations, SPECTS is also helpful in investigating brain activation under typical pain conditions such as experimental controlled pain and chronic pain syndromes.

Opportunities, barriers, and recommendations in down syndrome research.

BACKGROUND: Recent advances in medical care have increased life expectancy and improved the quality of life for people with Down syndrome (DS). These advances are the result of both pre-clinical and clinical research but much about DS is still poorly understood. In 2020, the NIH announced their plan to update their DS research plan and requested input from the scientific and advocacy community. OBJECTIVE: The National Down Syndrome Society (NDSS) and the LuMind IDSC Foundation worked together with scientific and medical experts to develop recommendations for the NIH research plan. METHODS: NDSS and LuMind IDSC assembled over 50 experts across multiple disciplines and organized them in eleven working groups focused on specific issues for people with DS. RESULTS: This review article summarizes the research gaps and recommendations that have the potential to improve the health and quality of life for people with DS within the next decade. CONCLUSIONS: This review highlights many of the scientific gaps that exist in DS research. Based on these gaps, a multidisciplinary group of DS experts has made recommendations to advance DS research. This paper may also aid policymakers and the DS community to build a comprehensive national DS research strategy.

DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation.

Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, we developed biocompatible immune cell-engaging particles (ICEp) that use synthetic short DNA as scaffolds for efficient and tunable protein loading. To improve the safety of chimeric antigen receptor (CAR) T cell therapies, micrometre-sized ICEp were injected intratumorally to present a priming signal for systemically administered AND-gate CAR-T cells. Locally retained ICEp presenting a high density of priming antigens activated CAR T cells, driving local tumour clearance while sparing uninjected tumours in immunodeficient mice. The ratiometric control of costimulatory ligands (anti-CD3 and anti-CD28 antibodies) and the surface presentation of a cytokine (IL-2) on ICEp were shown to substantially impact human primary T cell activation phenotypes. This modular and versatile biomaterial functionalization platform can provide new opportunities for immunotherapies.

The CRISPR-Cas9 crATIC HeLa transcriptome: Characterization of a novel cellular model of ATIC deficiency and ZMP accumulation.

In de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (EC 2.1.2.3)/inosine monophosphate cyclohydrolase (EC 3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z-nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). Mutations in ATIC cause an untreatable and devastating inborn error of metabolism in humans. ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimer's disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGFß and other cellular processes.

ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Spatial transcriptomics seeks to integrate single cell transcriptomic data within the three-dimensional space of multicellular biology. Current methods to correlate a cell's position with its transcriptome in living tissues have various limitations. We developed an approach, called 'ZipSeq', that uses patterned illumination and photocaged oligonucleotides to serially print barcodes ('zipcodes') onto live cells in intact tissues, in real time and with an on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in vitro wound healing, live lymph node sections and a live tumor microenvironment. In all cases, we discovered new gene expression patterns associated with histological structures. In the tumor microenvironment, this demonstrated a trajectory of myeloid and T cell differentiation from the periphery inward. A combinatorial variation of ZipSeq efficiently scales in the number of regions defined, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

The Role of the Cerebellum in Pain Perception: A Brain SPECT Perfusion Study in Patients with Burn Injuries.

BACKGROUND AND PURPOSE: Virtual reality (VR) is a promising tool for distraction analgesia. This study aims to compare brain perfusion patterns while patients were undergoing burn wound care in two conditions-VR distraction and control (NoVR). METHODS: With IRB approval, four patients hospitalized for acute burn care (three males and one female) participated in the study. All patients underwent wound care on two consecutive days; 1 day with standard analgesia and adjunctive VR, and the other day with standard analgesia alone, otherwise the wound care was very similar. Tc-99m ethyl cysteinate dimer was injected during wound care at the time of peak pain. Subjective patient reports on a 0-10 scale of pain intensity, time spent thinking about pain, and "fun" as well as opioid equivalent usage were analyzed. Voxel by voxel subtraction analysis of brain perfusion Single Photon Emission Computed Tomography (SPECT) images was performed at the group level. Statistical significance threshold was defined as P < .05. RESULTS: Mean group subjective scores (VR, NoVR, statistical significance, and P-value) were observed for maximal pain intensity (9.0, 8.8, insignificant, and P = .809), time spent thinking about pain (5.2, 10.0, significant, and P = .015), and fun (6.0, 2.5, significant, and P = .012). Subtraction group analysis demonstrated VR-induced modulation of brain activity with statistically significant relative suppression of cerebellar activation in the VR compared to intense cerebellar activation in the NoVR environments. CONCLUSION: Relative decrease in cerebellar perfusion based on stringent statistical threshold in the VR environment combined with improved subjective pain experience supports the hypotheses on the role of cerebellum in perception of noxious stimuli.

Protocatechuic Acid Extends Survival, Improves Motor Function, Diminishes Gliosis, and Sustains Neuromuscular Junctions in the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by motor neuron apoptosis and subsequent skeletal muscle atrophy caused by oxidative and nitrosative stress, mitochondrial dysfunction, and neuroinflammation. Anthocyanins are polyphenolic compounds found in berries that possess neuroprotective and anti-inflammatory properties. Protocatechuic acid (PCA) is a phenolic acid metabolite of the parent anthocyanin, kuromanin, found in blackberries and bilberries. We explored the therapeutic effects of PCA in a transgenic mouse model of ALS that expresses mutant human Cu, Zn-superoxide dismutase 1 with a glycine to alanine substitution at position 93. These mice display skeletal muscle atrophy, hindlimb weakness, and weight loss. Disease onset occurs at approximately 90 days old and end stage is reached at approximately 120 days old. Daily treatment with PCA (100 mg/kg) by oral gavage beginning at disease onset significantly extended survival (121 days old in untreated vs. 133 days old in PCA-treated) and preserved skeletal muscle strength and endurance as assessed by grip strength testing and rotarod performance. Furthermore, PCA reduced astrogliosis and microgliosis in spinal cord, protected spinal motor neurons from apoptosis, and maintained neuromuscular junction integrity in transgenic mice. PCA lengthens survival, lessens the severity of pathological symptoms, and slows disease progression in this mouse model of ALS. Given its significant preclinical therapeutic effects, PCA should be further investigated as a treatment option for patients with ALS.

Case Report: Virtual Reality Analgesia in an Opioid Sparing Orthopedic Outpatient Clinic Setting: A Case Study.

Immersive virtual reality is proving effective as a non-pharmacologic analgesic for a growing number of painful medical procedures. External fixator surgical pins provide adjunctive stability to a broken pelvic bone until the bones heal back together, then pins are removed. The purpose of the present case study was to measure for the first time, whether immersive virtual reality could be used to help reduce pain and anxiety during the orthopedic process of removing external fixator pins from a conscious patient in the orthopedic outpatient clinic, and whether it is feasible to use VR in this context. Using a within-subject within wound care design with treatment order randomized, the patient had his first ex-fix pin unscrewed and removed from his healing pelvic bone while he wore a VR helmet and explored an immersive snowy 3D computer generated world, adjunctive VR. He then had his second pin removed during no VR, standard of care pain medications. The patient reported having 43% less pain intensity, 67% less time spent thinking about pain, and 43% lower anxiety during VR vs. during No VR. In addition, the patient reported that his satisfaction with pain management was improved with the use of VR. Conducting simple orthopedic procedures using oral pain pills in an outpatient setting instead of anesthesia in the operating room greatly reduces the amount of opioids used, lowers medical costs and reduces rare but real risks of expensive complications from anesthesia including oversedation, death, and post-surgical dementia. These preliminary results suggest that immersive VR merits more attention as a potentially viable adjunctive non-pharmacologic form of treatment for acute pain and anxiety during medical procedures in the orthopedic outpatient clinic. Recent multi-billion dollar investments into R and D and mass production have made inexpensive immersive virtual reality products commercially available and cost effective for medical applications. We speculate that in the future, patients may be more willing to have minor surgery procedures in the outpatient clinic, with much lower opioid doses, while fully awake, if offered adjunctive virtual reality as a non-pharmacologic analgesic during the procedure. Additional research and development is recommended.

The CRISPR-Cas9 crADSL HeLa transcriptome: A first step in establishing a model for ADSL deficiency and SAICAR accumulation.

Adenylosuccinate lyase (ADSL) catalyzes two steps in de novo purine synthesis (DNPS). Mutations in ADSL can result in inborn errors of metabolism characterized by developmental delay and disorder phenotypes, with no effective treatment options. Recently, SAICAR, a metabolic substrate of ADSL, has been found to have alternative roles in the cell, complicating the role of ADSL. crADSL, a CRISPR KO of ADSL in HeLa cells, was constructed to investigate DNPS and ADSL in a human cell line. Here we employ this cell line in an RNA-seq analysis to initially investigate the effect of DNPS and ADSL deficiency on the transcriptome as a first step in establishing a cellular model of ADSL deficiency. We report transcriptome changes in genes relevant to development, vascular development, muscle, and cancer biology, which provide interesting avenues for future research.

Comparison of infectious agents detected from hatchery and wild juvenile Coho salmon in British Columbia, 2008-2018.

Infectious diseases are potential contributors to decline in Coho salmon (Oncorhynchus kisutch) populations. Although pathogens are theoretically considered to pose higher risk in high-density rearing environments like hatcheries, there is no direct evidence that hatchery-origin Coho salmon increase the transmission of infectious agents to sympatric wild populations. This study was undertaken to compare prevalence, burden, and diversity of infectious agents between hatchery-reared and wild juvenile Coho salmon in British Columbia (BC), Canada. In total, 2,655 juvenile Coho salmon were collected between 2008 and 2018 from four regions of freshwater and saltwater in BC. High-throughput microfluidics qPCR was employed for simultaneous detection of 36 infectious agents from mixed-tissue samples (gill, brain, heart, liver, and kidney). Thirty-one agents were detected at least once, including ten with prevalence >5%. Candidatus Brachiomonas cysticola, Paraneuclospora theridion, and Parvicapsula pseudobranchiocola were the most prevalent agents. Diversity and burden of infectious agents were substantially higher in marine environment than in freshwater. In Mainland BC, infectious burden and diversity were significantly lower in hatchery smolts than in wild counterparts, whereas in other regions, there were no significant differences. Observed differences in freshwater were predominantly driven by three parasites, Loma salmonae, Myxobolus arcticus, and Parvicapsula kabatai. In saltwater, there were no consistent differences in agent prevalence between hatchery and wild fish shared among the west and east coasts of Vancouver Island. Although some agents showed differential infectious patterns between regions, annual variations likely contributed to this signal. Our findings do not support the hypothesis that hatchery smolts carry higher burdens of infectious agents than conspecific wild fish, reducing the potential risk of transfer to wild smolts at this life stage. Moreover, we provide a baseline of infectious agents in juvenile Coho salmon that will be used in future research and modeling potential correlations between infectious profiles and marine survival.

Cumulative Effects of Thermal and Fisheries Stressors Reveal Sex-Specific Effects on Infection Development and Early Mortality of Adult Coho Salmon (Oncorhynchus kisutch).

Multiple stressors are commonly encountered by wild animals, but their cumulative effects are poorly understood, especially regarding infection development. We conducted a holding study with repeated gill and blood sampling to characterize the effects of cumulative stressors on infection development in adult coho salmon. Treatments included chronic thermal stress (15°C vs. 10°C) and acute gill net entanglement with an air exposure (simulating fisheries bycatch release). The potential loadings of 35 infectious agents and the expression of 17 host immune genes were quantified using high-throughput quantitative polymerase chain reaction, while host physiology was characterized with chemical analysis of blood. Temporal increases in infectious agent richness and loads were concurrent with decreased expression of immune genes in fish sampled in the river. In the laboratory, mortality was minimal in cool water regardless of fishery treatment (<15%). Elevated water temperature under laboratory conditions increased mortality of males and females (8% and 28% mortality, respectively, delayed by >1 wk) and enhanced mortality associated with handling and biopsy (∼40% both sexes). Experimental gillnetting at high temperature further enhanced female mortality (73%). Fish held at high temperature demonstrated heavier infectious agent loads, osmoregulatory impairment, suppressed female maturation, and upregulation of inflammatory and extracellular immune genes. At high temperature, heavy Parvicapsula minibicornis loads were associated with premature mortality. Females exhibited physiological impairment from both stressors after 1 wk, and infection burdens correlated poorly with immune gene regulation compared with males. Cumulative effects of multiple stressors on female mortality are likely a function of physiological impairment and enhanced infections at high temperature.

Assessing the Availability of Pharmacotherapy Options for Tobacco Cessation in Tennessee's Substance Use Facilities.

OBJECTIVES: Smoking is deadly and exacerbates comorbid health conditions, especially among those with co-occurring substance use disorders. Tennessee does not require substance abuse treatment facilities to concurrently provide tobacco-cessation treatment options or limit smoking on facility property as a standard for licensure. This study examines the relation between Tennessee facility region and availability of pharmacotherapeutic options for tobacco cessation, and the relation between the facilities' region and facility smoking policy. METHODS: This study is descriptive, cross-sectional, and exploratory in its design. Study findings are from a secondary analysis of data acquired from the 2016 edition of the National Directory of Drug and Alcohol Abuse Treatment Facilities published by the Substance Abuse and Mental Health Services Administration. RESULTS: The number of substance abuse treatment facilities providing pharmacotherapy options in west Tennessee (7.8%) are statistically significantly lower as compared with substance abuse treatment facilities in both middle (24.6%) and east (39.2%) Tennessee. A statistically significantly higher percentage of facilities in west Tennessee (23.5%) with policies that permit smoking without restriction as compared with the number of facilities in east Tennessee (6.8%) also was found, whereas the facilities in west Tennessee (60.8%) have a policy that permits smoking in designated areas that is significantly lower than the number of facilities in east Tennessee (81.1%). CONCLUSIONS: Tobacco-cessation options are a preventive intervention that can be easily incorporated into a treatment facility's standard of practice and should be made available within the context of all substance abuse treatment facilities. Failure to provide concurrent tobacco-cessation options during substance abuse treatment contradicts the purpose of medical treatment. Medically oriented substance abuse facilities are exemplary settings for offering pharmacotherapeutic options to concurrently treat tobacco use. It is suggested that interdisciplinary clinicians in the field of substance abuse advocate for the implementation of concurrent tobacco cessation treatment in their organization.

MULTI-seq: sample multiplexing for single-cell RNA sequencing using lipid-tagged indices.

Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer.

High sensitivity microwave spectroscopy in a cryogenic buffer gas cell.

We describe an instrument which can be used to analyze complex chemical mixtures at high resolution and high sensitivity. Molecules are collisionally cooled with helium gas at cryogenic temperatures (∼4-7 K) and subsequently detected using chirped pulse microwave spectroscopy. Here, we demonstrate three significant improvements to the apparatus relative to an earlier version: (1) extension of its operating range by more than a factor of two, from 12-18 GHz to 12-26 GHz, which allows a much wider range of species to be characterized; (2) improved detection sensitivity owing to the use of cryogenically cooled low-noise amplifiers and protection switches; and (3) a versatile method of sample input that enables analysis of solids, liquids, gases, and solutions, without the need for chemical separation (as demonstrated with a 12-16 GHz spectrum of lemon oil). This instrument can record broadband microwave spectra at comparable sensitivity to high Q cavity spectrometers which use pulsed supersonic jets, but up to 3000 times faster with a modest increase in the sample consumption rate.

The ozonolysis of isoprene in a cryogenic buffer gas cell by high resolution microwave spectroscopy.

We have developed a method to quantify reaction product ratios using high resolution microwave spectroscopy in a cryogenic buffer gas cell. We demonstrate the power of this method with the study of the ozonolysis of isoprene, CH2[double bond, length as m-dash]C(CH3)-CH[double bond, length as m-dash]CH2, the most abundant, non-methane hydrocarbon emitted into the atmosphere by vegetation. Isoprene is an asymmetric diene, and reacts with O3 at the 1,2 position to produce methyl vinyl ketone (MVK), formaldehyde, and a pair of carbonyl oxides: [CH3CO-CH[double bond, length as m-dash]CH2 + CH2[double bond, length as m-dash]OO] + [CH2[double bond, length as m-dash]O + CH3COO-CH[double bond, length as m-dash]CH2]. Alternatively, O3 could attack at the 3,4 position to produce methacrolein (MACR), formaldehyde, and two carbonyl oxides [CH2[double bond, length as m-dash]C(CH3)-CHO + CH2[double bond, length as m-dash]OO] + [CH2[double bond, length as m-dash]O + CH2[double bond, length as m-dash]C(CH3)-CHOO]. Purified O3 and isoprene were mixed for approximately 10 seconds under dilute (1.5-4% in argon) continuous flow conditions in an alumina tube held at 298 K and 5 Torr. Products exiting the tube were rapidly slowed and cooled within the buffer gas cell by collisions with cryogenic (4-7 K) He. High resolution chirped pulse microwave detection between 12 and 26 GHz was used to achieve highly sensitive (ppb scale), isomer-specific product quantification. We observed a ratio of MACR to MVK of 2.1 ± 0.4 under 1 : 1 ozone to isoprene conditions and 2.1 ± 0.2 under 2 : 1 ozone to isoprene conditions, a finding which is consistent with previous experimental results. Additionally, we discuss relative quantities of formic acid (HCOOH), an isomer of CH2[double bond, length as m-dash]OO, and formaldehyde (CH2[double bond, length as m-dash]O) under varying experimental conditions, and characterize the spectroscopic parameters of the singly-substituted 13C trans-isoprene and 13C anti-periplanar-methacrolein species. This work has the potential to be extended towards a complete branching ratio analysis, as well towards the ability to isolate, identify, and quantify new reactive intermediates in the ozonolysis of alkenes.