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.

Realizing the Future We Want: Leveraging Technology to Achieve a Better Health Care Experience for All.

The health care ecosystem is evolving rapidly. Technology must be leveraged to achieve better outcomes and meet the goals of providing more coordinated, patient-centric, and high-quality care. Premier, Inc. is a Charlotte-based health information technology company working to build efficiencies and innovate in this area to help health care practitioners deliver the best results for patients.

Sex-specific vascular responses of the rat aorta: effects of moderate term (intermediate stage) streptozotocin-induced diabetes.

Hyperglycemia affects male and female vascular beds differently. We have previously shown that 1 week after the induction of diabetes with streptozotocin (STZ), male and female rats exhibit differences in aortic endothelial function. To examine this phenomenon further, aortic responses were studied in male and female rats 8 weeks after the induction of diabetes (intermediate stage). Endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) was measured in phenylephrine (PE) pre-contracted rat aortic rings. Concentration response curves to PE were generated before and after L-NAME, a nitric oxide synthase (NOS) inhibitor. Furthermore, mRNA expression of endothelial nitric oxide synthase (eNOS) and NADPH oxidase subunit (Nox1) were determined. At 8 weeks, diabetes impaired EDV to a greater extent in female than male aortae. Furthermore, the responsiveness to PE was significantly enhanced only in female diabetic rats, and basal NO, as indicated by the potentiation of the response to PE after L-NAME, was reduced in female diabetic rat aortae to the same levels as in males. In addition, eNOS mRNA expression was decreased, while the Nox1 expression was significantly enhanced in diabetic female rats. These results suggest that aortic function in female diabetic rats after 8 weeks exhibits a more prominent impairment and that NO may be involved.

Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this "fit-for-purpose" approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

Statistical design for biospecimen cohort size in proteomics-based biomarker discovery and verification studies.

Protein biomarkers are needed to deepen our understanding of cancer biology and to improve our ability to diagnose, monitor, and treat cancers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Although contemporary proteomics technologies support the measurement of large numbers of proteins in individual clinical specimens, sample throughput remains comparatively low. This problem is amplified in typical clinical proteomics research studies, which routinely suffer from a lack of proper experimental design, resulting in analysis of too few biospecimens to achieve adequate statistical power at each stage of a biomarker pipeline. To address this critical shortcoming, a joint workshop was held by the National Cancer Institute (NCI), National Heart, Lung, and Blood Institute (NHLBI), and American Association for Clinical Chemistry (AACC) with participation from the U.S. Food and Drug Administration (FDA). An important output from the workshop was a statistical framework for the design of biomarker discovery and verification studies. Herein, we describe the use of quantitative clinical judgments to set statistical criteria for clinical relevance and the development of an approach to calculate biospecimen sample size for proteomic studies in discovery and verification stages prior to clinical validation stage. This represents a first step toward building a consensus on quantitative criteria for statistical design of proteomics biomarker discovery and verification research.

Women's groups, covariate shocks, and resilience: An evidence synthesis of past shocks to inform a response to COVID-19.

Background: Interventions with women's groups are increasingly seen as an important strategy for advancing women's empowerment, health, and economic outcomes in low- and middle-income countries, with the potential to increase the resiliency of members and their communities during widespread covariate shocks, such as coronavirus disease 2019 (COVID-19). Methods: This evidence synthesis compiles evidence from past shocks on women's group activities and the extent to which women's groups mitigate the effects of shocks on members and communities. We reviewed 90 documents from academic databases, organizational reports, and additional gray literature, and included literature diverse in geography, type of women's group, and shock. Results: The literature suggests that covariate shocks tend to disrupt group activities and reduce group resources, but linkages to formal institutions can mitigate this impact by extending credit beyond the shock-affected resource pool. Evidence was largely supportive of women's groups providing resilience to members and communities, though findings varied according to shock severity, group purpose and structure, and outcome measures. Further, actions to support individual resilience during a shock, such as increased payment flexibility, may run counter to group resilience. The findings of the evidence synthesis are largely consistent with emerging evidence about women's groups and COVID-19 in South Asia and sub-Saharan Africa. Conclusions: We finalize the paper with a discussion on policy implications, including the importance of sustainable access to financial resources for women's group members; equity considerations surrounding the distribution of group benefits and burdens; and the potential for meaningful partnerships between women's groups and local governments and/or non-governmental organizations (NGOs) to enhance community response amidst crises.

Sex differences in mesenteric endothelial function of streptozotocin-induced diabetic rats: a shift in the relative importance of EDRFs.

Several studies suggest that diabetes affects male and female vascular beds differently. However, the mechanisms underlying the interaction of sex and diabetes remain to be investigated. This study investigates whether there are 1) sex differences in the development of abnormal vascular responses and 2) changes in the relative contributions of endothelium-derived relaxing factors in modulating vascular reactivity of mesenteric arteries taken from streptozotocin (STZ)-induced diabetic rats at early and intermediate stages of the disease (1 and 8 wk, respectively). We also investigated the mesenteric expression of the mRNAs for endothelial nitric oxide (NO) synthase (eNOS) and NADPH oxidase (Nox) in STZ-induced diabetes in both sexes. Vascular responses to acetylcholine (ACh) in mesenteric arterial rings precontracted with phenylephrine were measured before and after pretreatment with indomethacin (cyclooxygenase inhibitor), N(ω)-nitro-L-arginine methyl ester (NOS inhibitor), or barium chloride (K(ir) blocker) plus ouabain (Na(+)-K(+)-ATPase inhibitor). We demonstrated that ACh-induced relaxations were significantly impaired in mesenteric arteries from both male and female diabetic rats at 1 and 8 wk. However, at 8 wk the extent of impairment was significantly greater in diabetic females than diabetic males. Our data also showed that in females, the levels of eNOS, Nox2, and Nox4 mRNA expression and the relative importance of NO to the regulation of vascular reactivity were substantially enhanced, whereas the importance of endothelium-derived hyperpolarizing factor (EDHF) was significantly reduced at both 1 and 8 wk after the induction of diabetes. This study reveals the predisposition of female rat mesenteric arteries to vascular injury after the induction of diabetes may be due to a shift away from a putative EDHF, initially the major vasodilatory factor, toward a greater reliance on NO.

The role of nonsteroidal anti-inflammatory drugs in pediatric patients.

Like in the adult population, nonsteroidal anti-inflammatory drugs (NSAIDS) are commonly used agents for their anti-inflammatory, anti-pyretic and analgesic effects in pediatrics. They are also used for some distinct indications in pediatrics such as Kawasaki disease, patent ductus arteriosus (PDA) closure, and Juvenile Idiopathic Arthritis (JIA). The primary mechanism thought to cause their therapeutic effects is the inhibition of prostaglandin synthesis. NSAIDs inhibit cyclooxygenase (COX) which is an enzyme that is necessary for the formation of prostaglandins. Unfortunately, this same mechanism, the inhibition of prostaglandins, is thought to be the most likely cause of gastrointestinal (GI) mucosal damage, because prostaglandins through multiple mechanisms assist in the preparation and maintenance of the protective barrier of the mucosal lining of the stomach. Similarly, prostaglandins in the kidney promote intrarenal plasma flow and electrolyte balance. The efficacy and safety of NSAIDs must be considered in prescribing these agents. The real conundrum of these therapies is determining the role of newer agents such as intravenous ibuprofen compared to existing alternatives. Available data for intravenous ibuprofen in adults is promising, but further studies are needed in pediatric patients to determine efficacy, place in therapy, and safety.

An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers.

There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern "-omics" experiments. Stable isotope standards with capture by anti-peptide antibodies (SISCAPA) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this study, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform, we demonstrate detection limits in the physiologically relevant ng/ml range (from 10 microl of plasma) with sufficient precision (median coefficient of variation, 12.6%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 ml) can extend the limits of detection to the low pg/ml range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates.

Inpatient utilization of blood cultures drawn in an urban ED.

Bloodstream infections are now ranked as the 10th leading cause of death in the United States. Given the severity of bacteremia, physicians routinely order multiple sets of blood cultures in the emergency department. This is a retrospective chart review on 1124 patients admitted to the hospital for suspected bacteremia during calendar year 2004. The aims of the present investigation were to investigate the overall utility of blood cultures by the admitting services and to identify patient factors that might influence culture yield. Data were collected regarding patient demographics, comorbidities, vital signs, laboratory results, antibiotic use, blood culture results, and notation of blood culture results by admitting physicians. Increased age, elevated heart rate, use of chemotherapy, decreased sodium, and increased blood urea nitrogen significantly increased the likelihood of yielding a positive blood culture in our patient population. Culture results were noted in 517 patient charts by the primary medical team (46.0%) and were adjusted in 223 patients (43.3%). Of 1124 cultures, 10.3% were positive in at least 1 bottle for a pathogenic organism (true positive), and 6.3% were contaminants (false positive). In conclusion, cultures must be followed closely by the admitting physician after being obtained. Our data emphasize that blood cultures are currently not well used by the admitting physicians and that measures need to be taken to improve the overall utility of blood culture data by the admitting physician.

Sportomics method to assess acute phase proteins in Olympic level athletes using dried blood spots and multiplex assay.

Sportomics is a subject-centered holistic method similar to metabolomics focusing on sports as the metabolic challenge. Dried blood spot is emerging as a technique due to its simplicity and reproducibility. In addition, mass spectrometry and integrative computational biology enhance our ability to understand exercise-induced modifications. We studied inflammatory blood proteins (Alpha-1-acid glycoprotein-A1AG1; Albumin; Cystatin C; C-reactive protein-CRP; Hemoglobin-HBA; Haptoglobin-HPT; Insulin-like growth factor 1; Lipopolysaccharide binding protein-LBP; Mannose-binding lectin-MBL2; Myeloperoxidase-PERM and Serum amyloid A1-SAA1), in 687 samples from 97 World-class and Olympic athletes across 16 sports in nine states. Data were analyzed with Spearman's rank-order correlation. Major correlations with CRP, LBP; MBL2; A1AG1, and SAA1 were found. The pairs CRP-SAA1 and CRP-LBP appeared with a robust positive correlation. Other pairs, LBP-SAA1; A1AG1-CRP; A1AG1-SAA1; A1AG1-MBL, and A1AG1-LBP, showed a broader correlation across the sports. The protein-protein interaction map revealed 1500 interactions with 44 core proteins, 30 of them linked to immune system processing. We propose that the inflammation follow-up in exercise can provide knowledge for internal cargo management in training, competition, recovery, doping control, and a deeper understanding of health and disease.

Women's groups and COVID-19: An evidence review on savings groups in Africa.

The coronavirus disease 2019 (COVID-19) pandemic and some of the associated policy responses have resulted in significant gendered impacts that may reverse recent progress in gender equality, including in sub-Saharan Africa. This paper presents emerging evidence from studies in diverse contexts in sub-Saharan Africa -with a deep dive into Nigeria and Uganda-on how COVID-19 has affected women's groups, especially savings groups, and how these groups have helped mitigate the gendered effects of the pandemic's and the associated policy responses' consequences up until April 2021. The synthesis presents evidence that savings groups found ways to continue operating, provided leadership opportunities for women during the pandemic, and mitigated some of the negative economic consequences of COVID-19 on individual savings group members. Savings, credit, and group support from other members all likely contributed to the ability of groups to positively affect the resilience of women's group member during COVID-19. Households with a female member in a savings group in Nigeria and Uganda have coped with the crisis better than those not in savings groups. While savings groups have shown the potential for resilience during the pandemic, they often faced financial challenges because of decreased savings, which sometimes resulted in the depletion of group assets. Savings groups also contributed to community responses and provided women a platform for leadership. These findings are consistent with a recent evidence synthesis on how past covariate shocks affected women's groups and their members. We conclude the paper by presenting various policy recommendations to enable savings groups to achieve improvements in women's empowerment and economic outcomes, and research recommendations to address some of the current evidence gaps on how COVID-19 is affecting women's groups and their members.

Precision multiparameter tracking of inflammation on timescales of hours to years using serial dried blood spots.

Aim: High-frequency longitudinal tracking of inflammation using dried blood microsamples provides a new window for personalized monitoring of infections, chronic inflammatory disease and clinical trials of anti-inflammatory drugs. Results/methodology: Using 1662 dried blood spot samples collected by 16 subjects over periods of weeks to years, we studied the behavior of 12 acute phase response and related proteins in inflammation events correlated with infection, vaccination, surgery, intense exercise and Crohn's disease. Proteins were measured using SISCAPA mass spectrometry and normalized to constant plasma volume using low-variance proteins, generating high precision within-person biomarker trajectories with well-characterized personal baselines. Discussion/conclusion: The results shed new light on the dynamic regulation of APR responses, offering a new approach to visualization of multidimensional inflammation trajectories.

Gender difference in rat aorta vasodilation after acute exposure to high glucose: involvement of protein kinase C beta and superoxide but not of Rho kinase.

OBJECTIVES: Several reports suggest that acute hyperglycemia affects male and female vascular beds differently. However, little is known about the interactions between hyperglycemia and gender in the vasculature. The objectives of our study were to investigate if there is a gender-based difference in the relaxation response of rat aorta after acute exposure to high glucose concentration, and the potential role of protein kinase C-beta (PKCbeta), superoxide, and Rho kinase in the gender-specific effect of acute high glucose on the relaxation response. METHODS: Endothelium-dependent dilator responses to acetylcholine (ACh, 10(-8) to 10(-5) M) were obtained before and after 3 h treatment with Krebs' solution containing high glucose (46 mM) in aortic rings pre-contracted with phenylephrine (2 microM) taken from female and male Sprague-Dawley rats. Similar experiments were generated in the presence of 1 microM LY379196, a selective PKCbeta inhibitor, 25 microM MnTMPyP, a superoxide dismutase mimetic, or 1 microM Fasudil, a Rho kinase inhibitor. Furthermore, protein expression of PKCbeta isoforms was measured by Western blotting. RESULTS: We demonstrated that a 3 h incubation with elevated level of glucose impairs ACh responses only in the female rat aortic rings. Inhibition of PKCbeta or superoxide production but not Rho kinase prevents the high glucose-induced impairment of endothelium-dependent relaxation of female rat aorta. In addition, PKCbeta2 expression is significantly higher in the female rat aorta than that in male rat aorta. CONCLUSION: These results suggest that the gender difference in the impairment of endothelium-dependent vasodilation after acute exposure to high glucose in rat aorta is possibly due to differences in PKCbeta2 expression.

Promoting the value and practice of shared decision-making in mental health care.

Active consumer participation is critical in contemporary mental health care and treatment planning and has been a staple of the field of psychiatric rehabilitation for the last three decades. Providing the opportunity for consumers to chose interventions that fit personal preferences and recovery increase the likelihood that these interventions will enhance personal meaning, satisfaction and quality of life (Improving the Quality of Health Care for Mental and Substance Use Conditions, 2006). Similarly, self-determination and shared decision-making are critical components of recovery. As stated in the President's New Freedom Commission on Mental Health Final Report, recovery from mental illnesses should be the expectation in mental health care with services and treatments that are consumer and family-driven. Mental health care should be planned and delivered to ensure that consumers and families with children with mental health problems receive real and meaningful choices about treatment options and providers. The purpose of this paper is to explore the value and use of shared decision-making in health and mental health care, briefly examine the advantages and disadvantages of shared decision making and propose next steps in advancing use of shared decision-making in mental health care.

FDA Regulation of Neurological and Physical Medicine Devices: Access to Safe and Effective Neurotechnologies for All Americans.

The United States Food and Drug Administration (FDA) ensures that patients in the U.S. have access to safe and effective medical devices. The Division of Neurological and Physical Medicine Devices reviews medical technologies that interface with the nervous system. This article addresses how to navigate the FDA's regulatory landscape to successfully bring medical devices to patients.

Pattern recognition scavenger receptor A/CD204 regulates airway inflammatory homeostasis following organic dust extract exposures.

Exposure to agriculture organic dusts, comprised of a diversity of pathogen-associated molecular patterns, results in chronic airway diseases. The multi-functional class A macrophage scavenger receptor (SRA)/CD204 has emerged as an important class of pattern recognition receptors with broad ligand binding ability. The objective was to determine the role of SRA in mediating repetitive and post-inflammatory organic dust extract (ODE)-induced airway inflammation. Wild-type (WT) and SRA knockout (KO) mice were intra-nasally treated with ODE or saline daily for 3 weeks and immediately euthanized or allowed to recover for 1 week. Results show that lung histopathologic changes were increased in SRA KO mice as compared to WT following repetitive ODE exposures marked predominately by increased size and distribution of lymphoid aggregates. After a 1-week recovery from daily ODE treatments, there was significant resolution of lung injury in WT mice, but not SRA KO animals. The increased lung histopathology induced by ODE treatment was associated with decreased accumulation of neutrophils, but greater accumulation of CD4(+) T-cells. The lung cytokine milieu induced by ODE was consistent with a TH1/TH17 polarization in both WT and SRA KO mice. Overall, the data demonstrate that SRA/CD204 plays an important role in the normative inflammatory lung response to ODE, as evidenced by the enhanced dust-mediated injury viewed in the absence of this receptor.