Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.

The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.

Identification and Characterization of GAL-021 as a Novel Breathing Control Modulator.

BACKGROUND: The authors describe the preclinical pharmacological properties of GAL-021, a novel peripheral chemoreceptor modulator. METHODS: The ventilatory effects of GAL-021 were characterized using tracheal pneumotachometry (n = 4 to 6), plethysmography (n = 5 to 6), arterial blood gas analyses (n = 6 to 11), and nasal capnography (n = 3 to 4) in naive animals and those subjected to morphine-induced respiratory depression. Morphine analgesia in rats was evaluated by tail-flick test (n = 6). Carotid body involvement in GAL-021 ventilatory effects was assessed by comparing responses in intact and carotid sinus nerve-transected rats. Hemodynamic effects of GAL-021 were evaluated in urethane-anesthetized rats (n = 7). The pharmacological profile of GAL-021 in vitro was investigated using radioligand binding, enzyme inhibition, and cellular electrophysiology assays. RESULTS: GAL-021 given intravenously stimulated ventilation and/or attenuated opiate-induced respiratory depression in rats, mice, and nonhuman primates, without decreasing morphine analgesia in rats. GAL-021 did not alter mean arterial pressure but produced a modest increase in heart rate. Ventilatory stimulation in rats was attenuated by carotid sinus nerve transection. GAL-021 inhibited KCa1.1 in GH3 cells, and the evoked ventilatory stimulation was attenuated in Slo1 mice lacking the pore-forming α-subunit of the KCa1.1 channel. CONCLUSIONS: GAL-021 behaved as a breathing control modulator in rodents and nonhuman primates and diminished opioid-induced respiratory depression without compromising opioid analgesia. It acted predominantly at the carotid body, in part by inhibiting KCa1.1 channels. Its preclinical profile qualified the compound to enter clinical trials to assess effects on breathing control disorders such as drug (opioid)-induced respiratory depression and sleep apnea.

N-acetylaspartate as a reservoir for glutamate.

N-acetylaspartate (NAA) is an intermediary metabolite that is found in relatively high concentrations in the human brain. More specifically, NAA is so concentrated in the neurons that it generates one of the most visible peaks in nuclear magnetic resonance (NMR) spectra, thus allowing NAA to serve as "a neuronal marker". However, to date there is no generally accepted physiological (primary) role for NAA. Another molecule that is found at similar concentrations in the brain is glutamate. Glutamate is an amino acid and neurotransmitter with numerous functions in the brain. We propose that NAA, a six-carbon amino acid derivative, is converted to glutamate (five carbons) in an energetically favorable set of reactions. This set of reactions starts when aspartoacylase converts the six carbons of NAA to aspartate and acetate, which are subsequently converted to oxaloacetate and acetyl CoA, respectively. Aspartylacylase is found in astrocytes and oligodendrocytes. In the mitochondria, oxaloacetate and acetyl CoA are combined to form citrate. Requiring two steps, the citrate is oxidized in the Kreb's cycle to alpha-ketoglutarate, producing NADH. Finally, alpha-ketoglutarate is readily converted to glutamate by transaminating the alpha-keto to an amine. The resulting glutamate can be used by multiple cells types to provide optimal brain functional and structural needs. Thus, the abundant NAA in neuronal tissue can serve as a large reservoir for replenishing glutamate in times of rapid or dynamic signaling demands and stress. This is beneficial in that proper levels of glutamate serve critical functions for neurons, astrocytes, and oligodendrocytes including their survival. In conclusion, we hypothesize that NAA conversion to glutamate is a logical and favorable use of this highly concentrated metabolite. It is important for normal brain function because of the brain's relatively unique metabolic demands and metabolite fluxes. Knowing that NAA is converted to glutamate will be important for better understanding myriad neurodegenerative diseases such as Canavan's Disease and Multiple Sclerosis, to name a few. Future studies to demonstrate the chemical, metabolic and pathological links between NAA and glutamate will support this hypothesis.

High-throughput measurement of 25-hydroxyvitamin D by LC-MS/MS with separation of the C3-epimer interference for pediatric populations.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for the measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in blood. However, the presence of 25OHD3 C3-epimer (3-epi-25OHD3) may cause interference and overestimation of the 25OHD3 level. We developed a rapid and simple assay for measurement of 25OHD that separates this interference and to investigate its impact on 25OHD3 measurement in adult and pediatric populations. METHODS: Sample preparation consisted of protein precipitation followed by solid-phase extraction with an LC run time of 4.8 min. Method comparison with another LC-MS/MS method of a major reference laboratory that does not separate the C3-epimer interference was performed using adult (n=52) and pediatric (n=40) samples. RESULTS: This method is free from significant ion suppression, carryover and interference. The assay can separate 25OHD3 from the 3-epi-25OHD3, and can measure 25OHD2 and 25OHD3 from 4.2 to 310.7 ng/ml and 5.2 to 311.1 ng/ml, respectively. Method comparison with a LC-MS method that does not separate the interference revealed biases of -0.15 and 4.54 for 25OHD3 measurement in adult and pediatric samples, respectively. CONCLUSION: A fast and simple LC-MS/MS method for quantification of 25OHD3 and 25OHD2 without 3-epi-25OHD3 interference was developed. This assay is required for accurate quantitation of 25OHD3 in pediatric samples and suitable for routine use in a high volume clinical laboratory.

The extracellular domain of myelin oligodendrocyte glycoprotein elicits atypical experimental autoimmune encephalomyelitis in rat and Macaque species.

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund's adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund's adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6-7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction.

Effects of acute psychosocial stress in a nonhuman primate model of allergic asthma.

Current husbandry and care guidelines for laboratory animals recommend social housing for nonhuman primates and all other social species. However, not all individuals of a social species are compatible, which can lead to psychosocial stress on certain members. Because stress affects immune responses, we undertook the present study to determine whether psychosocial stress associated with changes in the group housing of nonhuman primates affected allergic responses in a nonhuman primate model of allergic asthma. Historic records from 35 cynomolgus macaques (Macaca fascicularis) sensitive to house dust mites (HDM) and enrolled in asthma studies from 2007 to 2011 were reviewed for variations in response to aerosolized HDM that could not be explained by clinical or experimental interventions. We then compared these variations with husbandry and clinical records to determine whether the unexplained variations in responses were associated with events known to induce psychosocial stress in this species, including restructuring of social groups, temporary isolation of group members, and changes in cage or room configurations. Adult macaques in stable social groups exhibited little variation in responses to aerosolized antigen. Changes in group membership (conspecifics), cage configurations, and temporary isolation of a group member were associated with decreased responses to HDM. This attenuation lasted 2 to 3 mo on average, although some macaques showed prolonged responses. No evidence for a stress-induced increase in allergic responses was noted. These results demonstrate that acute stress in HDM-sensitive cynomolgus macaques diminishes the physiologic response to inhaled allergen.

Biomarkers of disease and treatment in murine and cynomolgus models of chronic asthma.

BACKGROUND: Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. OBJECTIVE: Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. METHODS: The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. RESULTS: Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. CONCLUSIONS AND CLINICAL RELEVANCE: These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination.

Nocturnal thoracoabdominal asynchrony in house dust mite-sensitive nonhuman primates.

Nocturnal bronchoconstriction is a common symptom of asthma in humans, but is poorly documented in animal models. Thoracoabdominal asynchrony (TAA) is a noninvasive clinical indication of airway obstruction. In this study, respiratory inductive plethysmography (RIP) was used to document nocturnal TAA in house dust mite (HDM)-sensitive Cynomolgus macaques. Dynamic compliance (C(dyn)) and lung resistance (R(L)) measured in anesthetized animals at rest and following exposure to HDM allergen, methacholine, and albuterol were highly correlated with three RIP parameters associated with TAA, ie, phase angle of the rib cage and abdomen waveforms (PhAng), baseline effort phase relation (eBPRL) and effort phase relation (ePhRL). Twenty-one allergic subjects were challenged with HDM early in the morning, and eBPRL and ePhRL were monitored for 20 hours after provocation. Fifteen of the allergic subjects exhibited gradual increases in eBPRL and ePhRL between midnight and 6 am, with peak activity at 4 am. However, as in humans, this nocturnal response was highly variable both between subjects and within subjects over time. The results document that TAA in this nonhuman primate model of asthma is highly correlated with C(dyn) and R(L), and demonstrate that animals exhibiting acute responses to allergen exposure during the day also exhibit nocturnal TAA.

Rho kinase is an effector underlying Ca2+-desensitizing hypoxic relaxation in porcine coronary artery.

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We have previously shown that at high-stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in intracellular [Ca(2+)] (27). This Ca(2+)-desensitizing hypoxic relaxation (CDHR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca(2+)] (10). Rho kinase-dependent phosphorylation of myosin phosphatase-targeting subunit 1 (p-MYPT1) is associated with decreased MRLC phosphatase activity and increased Ca(2+) sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR (33). In the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used alpha-toxin to permeabilize deendothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (MLCK, in the presence of the Rho kinase inhibitor Y-27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPT1 and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the Ca(2+)-desensitizing hypoxic relaxation.

Ca2+-desensitizing hypoxic vasorelaxation: pivotal role for the myosin binding subunit of myosin phosphatase (MYPT1) in porcine coronary artery.

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We showed that at high stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in [Ca(2+)](i). This 'Ca(2+)-desensitizing hypoxic relaxation' was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca(2+)]. Rho kinase-dependent phosphorylation of MYPT1 (p-MYPT1) is associated with decreased MRLC phosphatase (MLCP) activity, and increased Ca(2+) sensitivity of both p-MRLC and force. We tested the hypothesis that hypoxia induces Ca(2+)-desensitizing hypoxic relaxation via dephosphorylation of p-MYPT1, consequently increasing MLCP activity and thus decreasing p-MRLC. alpha-Toxin-permeabilized PCASM pretreated with ATPgammaS did not relax in response to hypoxia. Moreover, when MRLC but not MYPT1 was protected from ATPgammaS thiophosphorylation by the MRLC kinase inhibitor ML7 (300 mum), hypoxia remained ineffective. In contrast, hypoxic relaxation was preserved with further addition of the Rho kinase inhibitor Y27632 (1 mum), to attenuate thiophosphorylation of MYPT1. Importantly, measurements of p-MRLC, and p-MYPT1 at T696 and T853 (human sequence) paralleled that of force. We conclude that Ca(2+)-desensitizing hypoxic relaxation requires dephosphorylation of p-MYPT1. Moreover, no kinases, other then those inhibited by ML7 and Y27632, nor their associated phosphoproteins can be involved in Ca(2+)-desensitizing hypoxic relaxation.

Ca2+-independent hypoxic vasorelaxation in porcine coronary artery.

To demonstrate a Ca(2+)-independent component of hypoxic vasorelaxation and to investigate its mechanism, we utilized permeabilized porcine coronary arteries, in which [Ca(2+)] could be clamped. Arteries permeabilized with beta-escin developed maximum force in response to free Ca(2+) (6.6 microm), concomitant with a parallel increase in myosin regulatory light chain phosphorylation (MRLC-P(i)), from 0.183 +/- 0.023 to 0.353 +/- 0.019 MRLC-P(i) (total light chain)(-1). Hypoxia resulted in a significant decrease in both force (-31.9 +/- 4.1% prior developed force) and MRLC-P(i) (from 0.353 to 0.280 +/- 0.023), despite constant [Ca(2+)] buffered by EGTA (4 mm). Forces developed in response to Ca(2+) (6.6 microm), Ca(2+) (0.2 microm) + GTPgammaS (1 mM), or in the absence of Ca(2+) after treatment with ATPgammaS (1 mM), were of similar magnitude. Hypoxia also relaxed GTPgammaS contractures but importantly, arteries could not be relaxed after treatment with ATPgammaS. Permeabilization with Triton X-100 for 60 min also abolished hypoxic relaxation. The blocking of hypoxic relaxation after ATPgammaS suggests that this Ca(2+)-independent mechanism(s) may operate through alteration of MRLC-P(i) or of phosphorylation of the myosin binding subunit of myosin light chain phosphatase. Treatment with the Rho kinase inhibitor Y27632 (1 microm) relaxed GTPgammaS and Ca(2+) contractures; but the latter required a higher concentration (10 microm) for consistent relaxation. Relaxations to N(2) and/or Y27632 averaged 35% and were not additive or dependent on order. Our data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca(2+)-independent O(2)-sensing mechanism. Importantly, these results unambiguously show that hypoxia-induced vasorelaxation can occur in permeabilized arteries where the Ca(2+) is clamped at a constant value.

Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation.

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of approximately 0.15 mol P(i)/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser(19)-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser(19)-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.