Background: The cytokine MIF (Macrophage Migration Inhibitory Factor) has diverse physiological roles and is present at elevated concentrations in numerous disease states. However, its molecular heterogeneity has not been previously investigated in biological samples. Mass Spectrometric Immunoassay (MSIA) may help elucidate MIF post-translational modifications existing in vivo and provide additional clarity regarding its relationship to diverse pathologies.
Results: In this work, we have developed and validated a fully quantitative MSIA assay for MIF, and used it in the discovery and quantification of different proteoforms of MIF in serum samples, including cysteinylated and glycated MIF. The MSIA assay had a linear range of 1.56-50 ng/mL, and exhibited good precision, linearity, and recovery characteristics. The new assay was applied to a small cohort of human serum samples, and benchmarked against an MIF ELISA assay.
Conclusions: The quantitative MIF MSIA assay provides a sensitive, precise and high throughput method to delineate and quantify MIF proteoforms in biological samples.
Background: Height is an important health assessment measure with many applications. In the medical practice and in research settings, height is typically measured with a stadiometer. Although lasers are commonly used by health professionals for measurement including facial imaging, corneal thickness, and limb length, it has not been utilized for measuring height. The purpose of this feasibility study was to examine the ease and accuracy of a laser device for measuring height in children and adults.
Findings: In immediate succession, participant height was measured in triplicate using a stadiometer followed by the laser device. Measurement error for the laser device was significantly higher than that for the stadiometer (0.35 and 0.20 cm respectively). However, the measurement techniques were highly correlated (r2 = 0.998 and 0.990 for the younger [<12 y, n = 25] and older [≥12 y, n = 100] participants respectively), and the estimated reliability between measurement techniques was 0.999 (ICC; 95 % CI: 0.998,1.000) and 0.995 (ICC; 95 % CI: 0.993,0.997) for the younger and older groups respectively. The average differences between the two styles of measurement (e.g., stadiometer minus laser) were significantly different from zero: +0.93 and +0.45 cm for the younger and older groups respectively.
Conclusions: These data demonstrate that laser technology can be adapted to measure height in children and adults. Although refinement is needed, the laser device for measuring height merits further development.
Background: Cystatin C (CysC) is an endogenous cysteine protease inhibitor that can be used to assess the progression of kidney function. Recent studies demonstrate that CysC is a more specific indicator of glomerular filtration rate (GFR) than creatinine. CysC in plasma exists in multiple proteoforms. The goal of this study was to clarify the association of native CysC, CysC missing N-terminal Serine (CysC des-S), and CysC without three N-terminal residues (CysC des-SSP) with diabetic chronic kidney disease (CKD).
Results: Using mass spectrometric immunoassay, the plasma concentrations of native CysC and the two CysC truncation proteoforms were examined in 111 individuals from three groups: 33 non-diabetic controls, 34 participants with type 2 diabetes (DM) and without CKD and 44 participants with diabetic CKD. Native CysC concentrations were 1.4 fold greater in CKD compared to DM group (p = 0.02) and 1.5 fold greater in CKD compared to the control group (p = 0.001). CysC des-S concentrations were 1.55 fold greater in CKD compared to the DM group (p = 0.002) and 1.9 fold greater in CKD compared to the control group (p = 0.0002). CysC des-SSP concentrations were 1.8 fold greater in CKD compared to the DM group (p = 0.008) and 1.52 fold greater in CKD compared to the control group (p = 0.002). In addition, the concentrations of CysC proteoforms were greater in the setting of albuminuria. The truncated CysC proteoform concentrations were associated with estimated GFR independent of native CysC concentrations.
Conclusion: Our findings demonstrate a greater amount of CysC proteoforms in diabetic CKD. We therefore suggest assessing the role of cystatin C proteoforms in the progression of CKD.
Objective: The purpose of this randomized controlled trial was to determine whether adherence to a weight loss app providing intake limits and more food group detail (the Food Lists app) facilitated more weight loss and better diet quality than adherence to a weight loss app based on the MyPlate platform. An additional objective was to examine whether higher app adherence would lead to greater weight loss.
Design: Thirty seven adults from a campus population were recruited, randomized, and instructed to follow either the Food Lists app (N=20) or the MyPlate app (N=17) for eight weeks. Subjects received one 15 minute session of diet and app training at baseline, and their use of the app was tracked daily. Body mass was measured at baseline and post-test.
Participants/setting: Healthy adults from a university campus population in downtown Phoenix, Arizona with BMI 24 to 40, medically stable, and who owned a smartphone.
Main outcome measures: Outcome measures included weight change, days of adherence, and diet quality change. Secondary measures included BMI, fat %, and waist circumference.
Statistical analysis: Descriptive statistics (means and standard errors); Repeated measures ANOVAs analyzing weight, diet quality, and BMI; Pearson and Spearman correlations analyzing adherence and weight loss.
Results: Repeated measures ANOVAs and correlations revealed no significant mean differences in primary outcome variables of weight loss, adherence, or diet quality (P=0.140; P=0.790; P=0.278). However, there was a significant mean reduction of BMI favoring the group using the Food Lists app (P=0.041).
Conclusion: The findings strengthen the idea that intake limits and food group detail may be associated with weight loss. Further investigation is warranted to determine whether longer use of the Food Lists app can produce more significant dieting successes and encourage healthier behavioral outcomes.
Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.
Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications.
Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI2)]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.
Vegetarian diets are associated with factors that may not support bone health, such as low body mass and low intakes of protein; yet, these diets are alkaline, a factor that favors bone mineral density (BMD). This study compared the correlates of BMD in young, non-obese adults consuming meat-based (n = 27), lacto-ovo vegetarian (n = 27), or vegan (n = 28) diets for ≥1 year. A 24 h diet recall, whole body DXA scan, 24 h urine specimen, and fasting blood sample were collected from participants. BMD did not differ significantly between groups. Protein intake was reduced ~30% in individuals consuming lacto-ovo and vegan diets as compared to those consuming meat-based diets (68 ± 24, 69 ± 29, and 97 ± 47 g/day respectively, p = 0.006); yet dietary protein was only associated with BMD for those following vegan diets. Urinary pH was more alkaline in the lacto-ovo and vegan groups versus omnivores (6.5 ± 0.4, 6.7 ± 0.4, and 6.2 ± 0.4 respectively, p = 0.003); yet urinary pH was associated with BMD in omnivores only. These data suggest that plant-based diets are not detrimental to bone in young adults. Moreover, diet prescriptions for bone health may vary among diet groups: increased fruit and vegetable intake for individuals with high meat intakes and increased plant protein intake for individuals who follow a vegetarian diet plan.
In spite of well-documented health benefits of vegetarian diets, less is known regarding the effects of these diets on athletic performance. In this cross-sectional study, we compared elite vegetarian and omnivore adult endurance athletes for maximal oxygen uptake (VO2 max) and strength. Twenty-seven vegetarian (VEG) and 43 omnivore (OMN) athletes were evaluated using VO2 max testing on the treadmill, and strength assessment using a dynamometer to determine peak torque for leg extensions. Dietary data were assessed using detailed seven-day food logs. Although total protein intake was lower among vegetarians in comparison to omnivores, protein intake as a function of body mass did not differ by group (1.2 ± 0.3 and 1.4 ± 0.5 g/kg body mass for VEG and OMN respectively, p = 0.220). VO2 max differed for females by diet group (53.0 ± 6.9 and 47.1 ± 8.6 mL/kg/min for VEG and OMN respectively, p < 0.05) but not for males (62.6 ± 15.4 and 55.7 ± 8.4 mL/kg/min respectively). Peak torque did not differ significantly between diet groups. Results from this study indicate that vegetarian endurance athletes’ cardiorespiratory fitness was greater than that for their omnivorous counterparts, but that peak torque did not differ between diet groups. These data suggest that vegetarian diets do not compromise performance outcomes and may facilitate aerobic capacity in athletes.