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Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca2+-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only in vitro, i.e., for serum or plasma stability assays, but of lower importance in vivo.

In this study, the levels of circulating mediators of appetite that change after weight loss and promote weight regain did not revert to the values recorded before weight loss. Long-term strategies to counteract these changes may be needed to prevent obesity relapse. Worldwide, there are more than 1.5 billion overweight adults, including 400 million who are obese. 1 Although dietary restriction often results in initial weight loss, the majority of obese dieters fail to maintain their reduced weight. 2 Understanding the barriers to maintenance of weight loss is crucial for the prevention of relapse. Body weight is centrally regulated, with peripheral hormonal signals released from the gastrointestinal tract, pancreas, and adipose tissue integrated, primarily in the hypothalamus, to regulate food intake and energy expenditure. 3 The number of identified peripheral modulators of appetite is expanding rapidly and includes leptin, ghrelin, cholecystokinin, peptide YY, insulin, pancreatic . . .

Measurement of human plasma amyloid-β biomarkers using immunoprecipitation coupled with mass spectrometry reliably predicts individual brain amyloid-β status and has potential clinical utility. Plasma marker predicts amyloid-β pathology in the brain Alzheimer's disease is characterized by the deposition of amyloid-β (Aβ) peptide in the brain. The only available methods to reliably determine the levels of Aβ deposition are Aβ-PET imaging or measurement of Aβ levels in the cerebrospinal fluid. Therefore, identifying a blood-based biomarker that can be assessed in a minimally invasive and cost-effective manner is highly desirable. Katsuhiko Yanagisawa and colleagues use immunoprecipitation and mass spectrometry to measure the levels of several Aβ-related peptide fragments in blood. The APP 669–711 /Aβ 1–42 and Aβ 1–40 /Aβ 1–42 ratios and a composite score reliably predict individual levels of Aβ deposition in the brain. These results highlight the potential clinical utility of plasma biomarkers in predicting brain Aβ burden at an individual level. To facilitate clinical trials of disease-modifying therapies for Alzheimer’s disease, which are expected to be most efficacious at the earliest and mildest stages of the disease 1 , 2 , supportive biomarker information is necessary. The only validated methods for identifying amyloid-β deposition in the brain—the earliest pathological signature of Alzheimer’s disease—are amyloid-β positron-emission tomography (PET) imaging or measurement of amyloid-β in cerebrospinal fluid. Therefore, a minimally invasive, cost-effective blood-based biomarker is desirable 3 , 4 . Despite much effort 3 , 4 , 5 , 6 , 7 , to our knowledge, no study has validated the clinical utility of blood-based amyloid-β markers. Here we demonstrate the measurement of high-performance plasma amyloid-β biomarkers by immunoprecipitation coupled with mass spectrometry. The ability of amyloid-β precursor protein (APP) 669–711 /amyloid-β (Aβ) 1–42 and Aβ 1–40 /Aβ 1–42 ratios, and their composites, to predict individual brain amyloid-β-positive or -negative status was determined by amyloid-β-PET imaging and tested using two independent data sets: a discovery data set (Japan, n  = 121) and a validation data set (Australia, n  = 252 including 111 individuals diagnosed using 11 C-labelled Pittsburgh compound-B (PIB)-PET and 141 using other ligands). Both data sets included cognitively normal individuals, individuals with mild cognitive impairment and individuals with Alzheimer’s disease. All test biomarkers showed high performance when predicting brain amyloid-β burden. In particular, the composite biomarker showed very high areas under the receiver operating characteristic curves (AUCs) in both data sets (discovery, 96.7%, n  = 121 and validation, 94.1%, n  = 111) with an accuracy approximately equal to 90% when using PIB-PET as a standard of truth. Furthermore, test biomarkers were correlated with amyloid-β-PET burden and levels of Aβ 1–42 in cerebrospinal fluid. These results demonstrate the potential clinical utility of plasma biomarkers in predicting brain amyloid-β burden at an individual level. These plasma biomarkers also have cost–benefit and scalability advantages over current techniques, potentially enabling broader clinical access and efficient population screening.

Vitamin D deficiency, characterized by low circulating levels of calcifediol (25-hydroxyvitamin D, 25D) has been linked to increased risk of infections of bacterial and viral origin. Innate immune cells produce hormonal calcitriol (1,25-dihydroxyvitamin D, 1,25D) locally from circulating calcifediol in response to pathogen threat and an immune-specific cytokine network. Calcitriol regulates gene expression through its binding to the vitamin D receptor (VDR), a ligand-regulated transcription factor. The hormone-bound VDR induces the transcription of genes integral to innate immunity including pattern recognition receptors, cytokines, and most importantly antimicrobial peptides (AMPs). Transcription of the human AMP genes β-defensin 2/defensin-β4 (HBD2/DEFB4) and cathelicidin antimicrobial peptide (CAMP) is stimulated by the VDR bound to promoter-proximal vitamin D response elements. HDB2/DEFB4 and the active form of CAMP, the peptide LL-37, which form amphipathic secondary structures, were initially characterized for their antibacterial actively. Notably, calcitriol signaling induces secretion of antibacterial activity in vitro and in vivo, and low circulating levels of calcifediol are associated with diverse indications characterized by impaired antibacterial immunity such as dental caries and urinary tract infections. However, recent work has also provided evidence that the same AMPs are components of 1,25D-induced antiviral responses, including those against the etiological agent of the COVID-19 pandemic, the SARS-CoV2 coronavirus. This review surveys the evidence for 1,25D-induced antimicrobial activity in vitro and in vivo in humans and presents our current understanding of the potential mechanisms by which CAMP and HBD2/DEFB4 contribute to antiviral immunity.

Background Roux-en-Y gastric bypass (RYGB) surgery causes profound changes in secretion of gastrointestinal hormones and glucose metabolism. We present a detailed analysis of the early hormone changes after RYGB in response to three different oral test meals designed to provide this information without causing side effects (such as dumping). Methods We examined eight obese non-diabetic patients before and within 2 weeks after RYGB. On separate days, oral glucose tolerance tests (25 or 50 g glucose dissolved in 200 mL of water) and a liquid mixed meal test (200 mL 300 kcal) were performed. We measured fasting and postprandial glucose, insulin, C-peptide, glucagon, total and intact glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-2 (GLP-2), peptide YY 3-36 (PYY), cholecystokinin (CCK), total and active ghrelin, gastrin, somatostatin, pancreatic polypeptide (PP), amylin, leptin, free fatty acids (FFA), and registered postprandial dumping. Insulin sensitivity was measured by homeostasis model assessment of insulin resistance. Results Fasting glucose, insulin, ghrelin, and PYY were significantly decreased and FFA was elevated postoperatively. Insulin sensitivity increased after surgery. The postprandial response increased for C-peptide, GLP-1, GLP-2, PYY, CCK, and glucagon (in response to the mixed meal) and decreased for total and active ghrelin, leptin, and gastrin, but were unchanged for GIP, amylin, PP, and somatostatin after surgery. Dumping symptoms did not differ before and after the operation or between the tests. Conclusions Within 2 weeks after RYGB, we found an increase in insulin secretion and insulin sensitivity. Responses of appetite-regulating intestinal hormones changed dramatically, all in the direction of reducing hunger.

Elevated filling pressure of the left ventricle (LV) defines diastolic dysfunction. The gold standard for diagnosis is represented by the measurement of LV end-diastolic pressure (LVEDP) during cardiac catheterization, but it has the disadvantage of being an invasive procedure. This study aimed to investigate the correlation between LVEDP and cardiac serum biomarkers such as natriuretic peptides (mid-regional pro-atrial natriuretic peptide [MR-proANP], B-type natriuretic peptide [BNP], and N-terminal prohormone BNP [NT-proBNP]), soluble ST2 (sST2), galectin-3 and mid-regional pro-adrenomedullin (MR-proAMD). Consecutive patients hospitalized in a tertiary center and undergoing left cardiac catheterization were included in the study. Diastolic dysfunction was considered present if the end-expiratory LVEDP was ≥ 15 mmHg. Cardiac biomarkers were determined from pre-procedural peripheral venous blood samples. A total of 110 patients were included, of whom 76 (69.0%) were males, with a median age of 65 (55–71) years. Median LVEDP was 13.5 (8–19) mmHg and diastolic dysfunction was present in 50 (45.4%) of the patients. LVEDP correlated with BNP (p < 0.0001, r = 0.39 [0.20–0.53]), NT-proBNP (p < 0.0001, r = 0.40 [0.22–0.55]), MR-proANP (p = 0.001, r = 0.30 [0.11–0.46]), sST2 (p < 0.0001, r = 0.47 [0.30–0.60]), but not with MR-proAMD (p = 0.77) or galectin-3 (p = 0.76). In the final stepwise multivariable binary logistic regression model, diastolic dysfunction was predicted by NT-proBNP, mitral average E/e’, sST2, atrial fibrillation, and left atrium reservoir strain. BNP, NT-proBNP, MR-proANP, and sST2 had predictive value for diastolic dysfunction. In contrast, galectin-3 and MR-proAMD were not associated with increased filling pressures. Furthermore, NT-proBNP and sST2 significantly improved diastolic dysfunction prediction in the final multivariable model.

Heart failure (HF) with preserved ejection fraction (HFpEF) is increasingly recognized as an important clinical entity. Preclinical studies have shown differences in the pathophysiology between HFpEF and HF with reduced ejection fraction (HFrEF). Therefore, we hypothesized that a systematic metabolomic analysis would reveal a novel metabolomic fingerprint of HFpEF that will help understand its pathophysiology and assist in establishing new biomarkers for its diagnosis. Ambulatory patients with clinical diagnosis of HFpEF (n = 24), HFrEF (n = 20), and age-matched non-HF controls (n = 38) were selected for metabolomic analysis as part of the Alberta HEART (Heart Failure Etiology and Analysis Research Team) project. 181 serum metabolites were quantified by LC-MS/MS and 1H-NMR spectroscopy. Compared to non-HF control, HFpEF patients demonstrated higher serum concentrations of acylcarnitines, carnitine, creatinine, betaine, and amino acids; and lower levels of phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins. Medium and long-chain acylcarnitines and ketone bodies were higher in HFpEF than HFrEF patients. Using logistic regression, two panels of metabolites were identified that can separate HFpEF patients from both non-HF controls and HFrEF patients with area under the receiver operating characteristic (ROC) curves of 0.942 and 0.981, respectively. The metabolomics approach employed in this study identified a unique metabolomic fingerprint of HFpEF that is distinct from that of HFrEF. This metabolomic fingerprint has been utilized to identify two novel panels of metabolites that can separate HFpEF patients from both non-HF controls and HFrEF patients. ClinicalTrials.gov NCT02052804.

Background Although separate lines of research indicated a moderating role of sex in both sleep-wake disruption and in the interindividual vulnerability to Alzheimer’s disease (AD)-related processes, the quantification of sex differences in the interplay between sleep-wake dysregulation and AD pathology remains critically overlooked. Here, we examined sex-specific associations between circadian rest-activity patterns and AD-related pathophysiological processes across the adult lifespan. Methods Ninety-two cognitively unimpaired adults (mean age = 59.85 ± 13.77 years, range = 30–85, 47 females) underwent 10 days of actigraphic recordings, and blood drawing. Standard non-parametric indices of 24-h rest-activity rhythm fragmentation (intradaily variability, IV) and stability (interdaily stability, IS) were extracted from actigraphy data using the GGIR package. Plasma concentrations of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), amyloid-β 42/40 (Aβ 42/40 ), total tau, and tau phosphorylated at threonine 181 (p-tau 181 ) or threonine 231 (p-tau 231 ) were measured using Single molecule array technology. Multiple linear regression models were adjusted for age, sex, education, body mass index, and actigraphic recording duration. Results Higher IV, indicating worse 24-h rest-activity rhythm fragmentation, was associated with elevated levels of plasma NfL ( t (85) = 4.26, P  < 0.0001), GFAP ( t (85) = 2.49, P  = 0.01), and at trend level with lower Aβ 42/40 ratio values ( t (85) = -1.95, P  = 0.054). Lower IS, reflecting less day-to-day stability in the 24-h rest-activity rhythm, was linked to elevated levels of plasma NfL ( t (85) = -2.24, P  = 0.03), but not with the other plasma biomarkers. Importantly, interaction models demonstrated that male participants were driving the observed relationships between IV and plasma NfL ( t (84) = 4.05, P  < 0.001) or GFAP ( t (84) = 3.60, P  < 0.001), but also revealed a male vulnerability in models testing interactions with p-tau 181 (IV: t (76) = 3.71, P  < 0.001; IS: t (76) = -3.30, P  = 0.001) and p-tau 231 (IV: t (82) = 3.28, P  = 0.002). Sensitivity analyses further showed that accounting for potential confounding factors such as APOE genotype, depression, and self-reported symptoms of possible sleep apnea did not modify the observed relationships. Conclusions These findings suggest that the association between disrupted circadian rest-activity patterns and AD pathophysiological processes may be more evident in cognitively unimpaired males. Our results contribute to the precision medicine approach, and they have clinical implications for improved early detection and selection of at-risk individuals to be enrolled in preventive interventions.

Background The development of blood-based biomarker tests that are accurate and robust for Alzheimer’s disease (AD) pathology have the potential to aid clinical diagnosis and facilitate enrollment in AD drug trials. We developed a high-resolution mass spectrometry (MS)-based test that quantifies plasma Aβ42 and Aβ40 concentrations and identifies the ApoE proteotype. We evaluated robustness, clinical performance, and commercial viability of this MS biomarker assay for distinguishing brain amyloid status. Methods We used the novel MS assay to analyze 414 plasma samples that were collected, processed, and stored using site-specific protocols, from six independent US cohorts. We used receiver operating characteristic curve (ROC) analyses to assess assay performance and accuracy for predicting amyloid status (positive, negative, and standard uptake value ratio; SUVR). After plasma analysis, sites shared brain amyloid status, defined using diverse, site-specific methods and cutoff values; amyloid PET imaging using various tracers or CSF Aβ42/40 ratio. Results Plasma Aβ42/40 ratio was significantly ( p  < 0.001) lower in the amyloid positive vs. negative participants in each cohort. The area under the ROC curve (AUC-ROC) was 0.81 (95% CI = 0.77–0.85) and the percent agreement between plasma Aβ42/40 and amyloid positivity was 75% at the optimal (Youden index) cutoff value. The AUC-ROC (0.86; 95% CI = 0.82–0.90) and accuracy (81%) for the plasma Aβ42/40 ratio improved after controlling for cohort heterogeneity. The AUC-ROC (0.90; 95% CI = 0.87–0.93) and accuracy (86%) improved further when Aβ42/40, ApoE4 copy number and participant age were included in the model. Conclusions This mass spectrometry-based plasma biomarker test: has strong diagnostic performance; can accurately distinguish brain amyloid positive from amyloid negative individuals; may aid in the diagnostic evaluation process for Alzheimer’s disease; and may enhance the efficiency of enrolling participants into Alzheimer’s disease drug trials.

Plasma biomarkers of amyloid, tau, and neurodegeneration (ATN) need to be characterized in cognitively unimpaired (CU) elderly individuals. We therefore tested if plasma measurements of amyloid-β (Aβ)42/40, phospho-tau217 (P-tau217), and neurofilament light (NfL) together predict clinical deterioration in 435 CU individuals followed for an average of 4.8 ± 1.7 years in the BioFINDER study. A combination of all three plasma biomarkers and basic demographics best predicted change in cognition (Pre-Alzheimer’s Clinical Composite; R 2  = 0.14, 95% CI [0.12–0.17]; P < 0.0001) and subsequent AD dementia (AUC = 0.82, 95% CI [0.77–0.91], P < 0.0001). In a simulated clinical trial, a screening algorithm combining all three plasma biomarkers would reduce the required sample size by 70% (95% CI [54–81]; P < 0.001) with cognition as trial endpoint, and by 63% (95% CI [53–70], P < 0.001) with subsequent AD dementia as trial endpoint. Plasma ATN biomarkers show usefulness in cognitively unimpaired populations and could make large clinical trials more feasible and cost-effective. Plasma biomarkers of amyloid, tau and neurodegeneration (ATN) need to be characterized in cognitively unimpaired (CU) elderly individuals. Here, the authors show plasma ATN biomarkers predict clinical deterioration and cognitive decline and show in a simulated clinical trial combining all three biomarkers reduced the required sample size.