Spectral library search for improved TMTpro labelled peptide assignment in human plasma proteomics.

Clinical biomarker discovery is often based on the analysis of human plasma samples. However, the high dynamic range and complexity of plasma pose significant challenges to mass spectrometry-based proteomics. Current methods for improving protein identifications require laborious pre-analytical sample preparation. In this study, we developed and evaluated a TMTpro-specific spectral library for improved protein identification in human plasma proteomics. The library was constructed by LC-MS/MS analysis of highly fractionated TMTpro-tagged human plasma, human cell lysates, and relevant arterial tissues. The library was curated using several quality filters to ensure reliable peptide identifications. Our results show that spectral library searching using the TMTpro spectral library improves the identification of proteins in plasma samples compared to conventional sequence database searching. Protein identifications made by the spectral library search engine demonstrated a high degree of complementarity with the sequence database search engine, indicating the feasibility of increasing the number of protein identifications without additional pre-analytical sample preparation. The TMTpro-specific spectral library provides a resource for future plasma proteomics research and optimization of search algorithms for greater accuracy and speed in protein identifications in human plasma proteomics, and is made publicly available to the research community via ProteomeXchange with identifier PXD042546.

Perampanel's forgiveness factor in a variable medication adherence paradigm in a rat model of chronic epilepsy.

BACKGROUND: Poor medication adherence contributes to increased morbidity and mortality in patients with epilepsy and may be under-addressed in clinical practice. Ethical concerns make it impossible to study the impact of medication nonadherence in clinical trials, but our previous work emphasizes the importance of using preclinical approaches to address these questions. With over 30 clinically available antiseizure medicines (ASM's), it remains an important question to understand the relationship between poor adherence and seizure incidence across mechanistically distinct ASM's, including the broad-spectrum ASM, perampanel (PER). METHODS: We formulated PER into chow pellets to deliver to rats in a 100% fully adherent or 50% variable nonadherent paradigm via our novel automated medication-in-food delivery system. Chronic oral dosing was initiated in male rats with chronic epilepsy while monitoring 24/7 for videoEEG evidence of seizures during a 4-week placebo baseline and 4-week treatment phase. PER concentrations were monitored in plasma at 1-week intervals and correlated with degree of seizure control. The relationship between missed doses and extended patterns of nonadherence were correlated with breakthrough seizures. RESULTS: Fully adherent rats demonstrated a median reduction in seizure frequency of 50%, whereas nonadherent rats had a median increase of 54%. Plasma concentrations of PER were stable over the 4-week treatment period in both fully adherent and nonadherent groups, with levels being twice as high in fully adherent animals. There was no correlation between a single missed dose or series of missed doses and the incidence of breakthrough seizures. However, those animals in the nonadherent group that received PER for every meal during a 24-h period had a reduced likelihood of seizure incidence. CONCLUSIONS: If our preclinical data is supported in the clinic, PER's favorable pharmacokinetic profile in humans, combined with a lowered risk of breakthrough seizures suggests that it may provide a certain forgiveness factor if a dose is missed within a 24-h window.

Costs and Health Care Utilization Analysis of Medical Group Visits for Adults With Type 2 Diabetes in Community Health Centers.

OBJECTIVE: We evaluated the economic impact of group visits (GVs) in adults with uncontrolled diabetes in community health centers (CHCs) in the United States. RESEARCH DESIGN AND METHODS: In this prospective controlled trial, we implemented 6 monthly GV sessions in 5 CHCs and compared intervention patients (n=49) to control patients (n=72) receiving usual care within the same CHCs. We conducted patient chart reviews to obtain health care utilization data for the prior 6 months at baseline, 6 months (during the GV implementation), and 12 months (after the implementation). We also collected monthly logs of CHC expenses and staff time spent on activities related to GVs. Per-patient total costs included CHCs' expenses and costs associated with staff time and patients' health care use. For group comparison, we used the Wilcoxon rank-sum test and the bootstrapping method that was to bootstrap generalized estimating equation models. RESULTS: The GV group had fewer 6-month hospitalizations (mean: GV: 0.06 vs. control: 0.24, rate: 6.1% vs. 19.4%) ( P ≤ 0.04) and similar emergency department visits at 12 months than the control group. Implementing GV incurred $1770 per-patient. The intervention cost $1597 more than the control at 6 months ($3021 vs. $1424) but saved $1855 at 12 months ($857 vs. $2712) ( P =0.002). CONCLUSIONS: The diabetes GV care model reduced hospitalizations and had cost savings at 12 months, while it improved patients' diabetes-related quality of life and glucose control. Future studies should assess its lifetime cost-effectiveness through a randomized controlled trial.

Plasma calcitonin gene-related peptide and nerve growth factor as headache and pain biomarkers in recently deployed soldiers with and without a recent concussion.

OBJECTIVE: The objective of this study was to characterize the utility of calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) as potential biomarkers for headache and pain disorders in the post-military deployment setting. BACKGROUND: The need to improve recognition, assessment, and prognoses of individuals with posttraumatic headache or other pain has increased interest in the potential of CGRP and NGF as biomarkers. METHODS: The Warrior Strong Study (NCT01847040) is an observational longitudinal study of United States-based soldiers who had recently returned from deployment to Afghanistan or Iraq from 2009 to 2014. The present nested cross-sectional analysis uses baseline data collected from soldiers returning to Fort Bragg, North Carolina. RESULTS: In total, 264 soldiers (mean (standard deviation [SD] age 28.1 [6.4] years, 230/264 [87.1%] men, 171/263 [65.0%] White) were analyzed. Mean (SD) plasma levels of CGRP were 1.3 (1.1) pg/mL and mean levels of NGF were 1.4 (0.4) pg/mL. Age was negatively correlated with NGF (-0.01 pg/mL per year, p = 0.007) but was not associated with CGRP. Men had higher mean (SD) CGRP plasma levels than women (1.4 95% confidence interval [CI; 1.2] vs. 0.9 95% CI [0.5] pg/mL, p < 0.002, Kruskal-Wallis test). CGRP levels were lower in participants who had a headache at the time of the blood draw (1.0 [0.6] pg/mL vs. 1.4 [1.2] pg/mL, p = 0.024). NGF was lower in participants with continuous pain (all types; 1.2 [0.4] vs. 1.4 [0.4] pg/mL, p = 0.027) and was lower in participants with traumatic brain injury (TBI) + posttraumatic headache (PTH) versus TBI without PTH (1.3 [0.3] vs. 1.4 [0.4] pg/mL, p = 0.021). Otherwise, CGRP and NGF were not associated with migraine-like headache, TBI status, or headache burden as measured by the number of medical encounters in crude or adjusted models. CONCLUSION: In this exploratory study, plasma levels of NGF and CGRP showed promise as biomarkers for headache and other types of pain. These findings need to be replicated in other cohorts.

Elements of Integrated Behavioral Health Associated with Primary Care Provider Confidence in Managing Depression at Community Health Centers.

BACKGROUND: Depression is most often treated by primary care providers (PCPs), but low self-efficacy in caring for depression may impede adequate management. We aimed to identify which elements of integrated behavioral health (BH) were associated with greater confidence among PCPs in identifying and managing depression. DESIGN: Mailed cross-sectional surveys in 2016. PARTICIPANTS: BH leaders and PCPs caring for adult patients at community health centers (CHCs) in 10 midwestern states. MAIN MEASURES: Survey items asked about depression screening, systems to support care, availability and integration of BH, and PCP attitudes and experiences. PCPs rated their confidence in diagnosing, assessing severity, providing counseling, and prescribing medication for depression on a 5-point scale. An overall confidence score was calculated (range 4 (low) to 20 (high)). Multilevel linear mixed models were used to identify factors associated with confidence. KEY RESULTS: Response rates were 60% (N=77/128) and 52% (N=538/1039) for BH leaders and PCPs, respectively. Mean overall confidence score was 15.25±2.36. Confidence was higher among PCPs who were satisfied with the accuracy of depression screening (0.38, p=0.01), worked at CHCs with depression tracking systems (0.48, p=0.045), had access to patients' BH treatment plans (1.59, p=0.002), and cared for more patients with depression (0.29, p=0.003). PCPs who reported their CHC had a sufficient number of psychiatrists were more confident diagnosing depression (0.20, p=0.02) and assessing severity (0.24, p=0.03). Confidence in prescribing was lower at CHCs with more patients living below poverty (-0.66, p<0.001). Confidence in diagnosing was lower at CHCs with more Black/African American patients (-0.20, p=0.03). CONCLUSIONS: PCPs who had access to BH treatment plans, a system for tracking patients with depression, screening protocols, and a sufficient number of psychiatrists were more confident identifying and managing depression. Efforts are needed to address disparities and support PCPs caring for vulnerable patients with depression.

The autoimmunity-associated gene PTPN22 potentiates toll-like receptor-driven, type 1 interferon-dependent immunity.

Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity "risk" gene in the regulation of host defense and inflammation.

Impact of diabetes group visits on patient clinical and self-reported outcomes in community health centers.

BACKGROUND: Diabetes group visits (GVs) are a promising way to deliver high quality care but have been understudied in community health centers (CHCs), across multiple sites, or with a focus on patient-centered outcomes. METHODS: We trained staff and healthcare providers from six CHCs across five Midwestern states to implement a 6-month GV program at their sites. We assessed the impact of diabetes GVs on patient clinical and self-reported outcomes and processes of care compared to patients receiving usual care at these sites during the same period using a prospective controlled study design. RESULTS: CHCs enrolled 51 adult patients with diabetes with glycosylated hemoglobin (A1C) ≥ 8% for the GV intervention and conducted chart review of 72 patients receiving usual care. We analyzed A1C at baseline, 6, and 12 months, low-density lipoproteins (LDL), blood pressure, and patient-reported outcomes. GV patients had a larger decrease in A1C from baseline to 6 months (-1.04%, 95% CI: -1.64, -0.44) and 12 months (-1.76, 95% CI: -2.44, -1.07) compared to usual care; there was no change in blood pressure or LDL. GV patients had higher odds of receiving a flu vaccination, foot exam, eye exam, and lipid panel in the past year compared to usual care but not a dental exam, urine microalbumin test, or blood pressure check. For GV patients, diabetes distress decreased, diabetes-related quality of life improved, and self-reported frequency of healthy eating and checking blood sugar increased from baseline to 6 months, but there was no change in exercise or medication adherence. CONCLUSIONS: A diabetes GV intervention improved blood glucose levels, self-care behaviors, diabetes distress, and processes of care among adults with elevated A1Cs compared to patients receiving usual care. Future studies are needed to assess the sustainability of clinical improvements and costs of the GV model in CHCs.

Assessing the effectiveness of a diabetes group visit training for health center staff: a pilot study of five Midwestern community health centers.

BACKGROUND: Diabetes group visits are shared appointments that include diabetes education in a group setting and individual visits with a medical provider. An 18-month pilot study was designed to evaluate organizational capacity and staff preparedness in implementing and sustaining diabetes group visits. RESULTS: Data were collected and analyzed from pre-post assessments and key informant interviews with community health center (CHC) staff (N = 26) from teams across five Midwestern states. Overall, participants demonstrated high baseline knowledge and awareness about diabetes group visit implementation. Changes in attitudes and practices did occur pertaining to familiarity with billing and increased awareness about potential barriers to diabetes group visit implementation. Key assets to diabetes group visit implementation were access to pre-designed resources and materials, a highly motivated team, and supportive leadership. Key obstacles were socioeconomic challenges experienced by patients, constraints on staff time dedicated to group visit implementation, and staff turnover. CONCLUSIONS: Results of the study provide a framework for implementation of diabetes group visit trainings for CHC staff. Future research is needed to assess the training program in a larger sample of CHCs.

Integration of Diabetes and Depression Care Is Associated with Glucose Control in Midwestern Federally Qualified Health Centers.

BACKGROUND: The 2016 American Diabetes Association position statement emphasized that psychosocial and medical care should be integrated and provided to all people with diabetes. OBJECTIVE: To determine whether better integration of diabetes and depression care is associated with better glycemic control. DESIGN: Cross-sectional surveys of Midwestern federally qualified health center (FQHC) leaders and primary care providers (PCPs) in 2016. Responses were linked to FQHC-level data on the percentage of patients with uncontrolled diabetes (glycated hemoglobin ≥ 9%; 75 mmol/mol). PARTICIPANTS: Midwest Clinicians' Network-affiliated FQHC leaders, and PCPs at the FQHCs. MAIN MEASURES: Multilevel models were used to determine associations between the percentage of patients with uncontrolled diabetes and FQHC and PCP characteristics; presence of diabetes and behavioral health care services; and PCPs' perception of the stage of integration between diabetes and depression care services based on the transtheoretical model (i.e., pre-contemplation, contemplation, preparation, action, or maintenance). KEY RESULTS: Response rates were 60% for the FQHC survey (N = 77) and 55% for the PCP survey (N = 538). In adjusted models, FQHCs in which PCPs perceived a higher stage of integration between diabetes and depression care had 3% fewer patients with uncontrolled diabetes per 1-level increase in integration stage (p = 0.01); on-site diabetes self-management education was associated with 7% fewer patients with uncontrolled diabetes (p < 0.01). CONCLUSIONS: At Midwestern FQHCs, a higher stage of perceived integration of diabetes and depression care was associated with better FQHC-level glycemic control. Future studies are needed to elucidate what defines integration of diabetes and depression care services.

Cenobamate (XCOPRI): Can preclinical and clinical evidence provide insight into its mechanism of action?

Approximately one-third of people living with epilepsy are unable to obtain seizure control with the currently marketed antiseizure medications (ASMs), creating a need for novel therapeutics with new mechanisms of action. Cenobamate (CBM) is a tetrazole alkyl carbamate derivative that received US Food and Drug Administration approval in 2019 for the treatment of adult partial onset (focal) seizures. Although CBM displayed impressive seizure reduction in clinical trials across all seizure types, including focal aware motor, focal impaired awareness, and focal to bilateral tonic-clonic seizures, the precise mechanism(s) through which CBM exerts its broad-spectrum antiseizure effects is not known. Experimental evidence suggests that CBM differentiates itself from other ASMs in that it appears to possess dual modes of action (MOAs); that is, it predominately blocks persistent sodium currents and increases both phasic and tonic γ-aminobutyric acid (GABA) inhibition. In this review, we analyze the preclinical efficacy of CBM alongside ASMs with similar MOAs to better understand the mechanism(s) through which CBM achieves such broad-spectrum seizure protection. CBM's preclinical performance in tests, including the mouse 6-Hz model of treatment-resistant seizures, the chemoconvulsant seizure models of generalized epilepsy, and the rat hippocampal kindling model of focal epilepsy, was distinct from other voltage-gated sodium channel blockers and GABAA modulators. This distinction, in light of its proposed mechanism(s) of action, provides insight into the impressive clinical efficacy of CBM in the adult patient with focal onset epilepsy. The results of this comparative reverse translational analysis suggest that CBM is a mechanistically distinct ASM that offers an important advancement in drug development for treatment of therapy-resistant epilepsy.

Acetaminophen and Acetylsalicylic Acid Exposure in a Preterm Infant after Maternal Overdose.

Here, we review the case of a 26 1/7 weeks' gestation premature female infant born to a mother who intentionally ingested a large quantity of Tylenol, aspirin, quetiapine, and prenatal vitamins. The neonate subsequently had markedly elevated levels of both Tylenol and aspirin when checked on the first day of life. While overall clinically stable, the neonate did demonstrate coagulopathy as evidenced by abnormal coagulation studies. Both poison control and a pediatric gastroenterologist/hepatologist were consulted. She successfully tolerated a course of N-acetylcysteine; her subsequent Tylenol level was markedly decreased and the neonate exhibited no further effects of toxicity. The salicylate level decreased on its own accord. To our knowledge, this is the first report of a neonate at 26 weeks' gestation that has been successfully managed for supratherapeutic concentrations of acetaminophen and acetylsalicylic acid secondary to maternal ingestion. While rare, this case may serve as a reference for the effectiveness of N-acetylcysteine in premature infants in such instances.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.

Fluorescent nanodiamonds engage innate immune effector cells: A potential vehicle for targeted anti-tumor immunotherapy.

Fluorescent nanodiamonds (FNDs) are nontoxic, infinitely photostable, and emit fluorescence in the near infrared region. Natural killer (NK) cells and monocytes are part of the innate immune system and are crucial to the control of carcinogenesis. FND-mediated stimulation of these cells may serve as a strategy to enhance anti-tumor activity. FNDs were fabricated with a diameter of 70±28 nm. Innate immune cell FND uptake, viability, surface marker expression, and cytokine production were evaluated in vitro. Evaluation of fluorescence emission from the FNDs was conducted in an animal model. In vitro results demonstrated that treatment of immune cells with FNDs resulted in significant dose-dependent FND uptake, no compromise in cell viability, and immune cell activation. FNDs were visualized in an animal model. Hence, FNDs may serve as novel agents with "track and trace" capabilities to stimulate innate immune cell anti-tumor responses, especially as FNDs are amenable to surface-conjugation with immunomodulatory molecules.

Phosphatase of regenerating liver 2 (PRL2) deficiency impairs Kit signaling and spermatogenesis.

The Phosphatase of Regenerating Liver (PRL) proteins promote cell signaling and are oncogenic when overexpressed. However, our understanding of PRL function came primarily from studies with cultured cell lines aberrantly or ectopically expressing PRLs. To define the physiological roles of the PRLs, we generated PRL2 knock-out mice to study the effects of PRL deletion in a genetically controlled, organismal model. PRL2-deficient male mice exhibit testicular hypotrophy and impaired spermatogenesis, leading to decreased reproductive capacity. Mechanistically, PRL2 deficiency results in elevated PTEN level in the testis, which attenuates the Kit-PI3K-Akt pathway, resulting in increased germ cell apoptosis. Conversely, increased PRL2 expression in GC-1 cells reduces PTEN level and promotes Akt activation. Our analyses of PRL2-deficient animals suggest that PRL2 is required for spermatogenesis during testis development. The study also reveals that PRL2 promotes Kit-mediated PI3K/Akt signaling by reducing the level of PTEN that normally antagonizes the pathway. Given the strong cancer susceptibility to subtle variations in PTEN level, the ability of PRL2 to repress PTEN expression qualifies it as an oncogene and a novel target for developing anti-cancer agents.

A unified lead-oriented synthesis of over fifty molecular scaffolds.

Controlling the properties of lead molecules is critical in drug discovery, but sourcing large numbers of lead-like compounds for screening collections is a major challenge. A unified synthetic approach is described that enabled the synthesis of 52 diverse lead-like molecular scaffolds from a minimal set of 13 precursors. The divergent approach exploited a suite of robust, functional group-tolerant transformations. Crucially, after derivatisation, these scaffolds would target significant lead-like chemical space, and complement commercially-available compounds.