Inherited causes of exocrine pancreatic insufficiency in pediatric patients: clinical presentation and laboratory testing.

Pediatric patients with exocrine pancreatic insufficiency (EPI) have symptoms that include abdominal pain, weight loss or poor weight gain, malnutrition, and steatorrhea. This condition can be present at birth or develop during childhood for certain genetic disorders. Cystic fibrosis (CF) is the most prevalent disorder in which patients are screened for EPI; other disorders also are associated with pancreatic dysfunction, such as hereditary pancreatitis, Pearson syndrome, and Shwachman-Diamond syndrome. Understanding the clinical presentation and proposed pathophysiology of the pancreatic dysfunction of these disorders aids in diagnosis and treatment. Testing pancreatic function is challenging. Directly testing aspirates produced from the pancreas after stimulation is considered the gold standard, but the procedures are not standardized or widely available. Instead, indirect tests are often used in diagnosis and monitoring. Although indirect tests are more widely available and easier to perform, they have inherent limitations due to a lack of sensitivity and/or specificity for EPI.

Structure of the toxic core of α-synuclein from invisible crystals.

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face ß-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.

High School Student Driving Perceptions Following Participation in a Distracted Driving Curriculum.

Objectives. Distracted driving is a major public health issue in the United States. In response to requests from high school students participating in a university-based initiative, the authors describe the collaborative development and implementation of a curriculum designed to address distracted driving behaviors among students in four high-needs school districts in the northeastern United States. Method. The curriculum integrates current statistics on distracted and drowsy driving and three interactive learning stations: driving while distracted, walking while distracted, and driving while drowsy. Pre- and postsurveys were conducted to collect student driving data, assess student satisfaction with the program, and assess their likelihood of speaking up as a passenger in a high-risk situation. Results. The majority of students reported that they learned new information and would recommend the program to others. A Wilcoxon signed-rank test showed that students were more likely to speak up as a passenger with a distracted or drowsy driver (p < .001) after the program. Conclusion. This experience demonstrates a voluntary, multidisciplinary, university-based collaboration in the development of a novel public health education initiative. Based on the success of this phase, school districts elected to participate in Train the Trainer sessions to continue the program within their local high-needs school district.

Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS.

ALS is a terminal disease of motor neurons that is characterized by accumulation of proteinaceous deposits in affected cells. Pathological deposition of mutated Cu/Zn superoxide dismutase (SOD1) accounts for ∼20% of the familial ALS (fALS) cases. However, understanding the molecular link between mutation and disease has been difficult, given that more than 140 different SOD1 mutants have been observed in fALS patients. In addition, the molecular origin of sporadic ALS (sALS) is unclear. By dissecting the amino acid sequence of SOD1, we identified four short segments with a high propensity for amyloid fibril formation. We find that fALS mutations in these segments do not reduce their propensity to form fibrils. The atomic structures of two fibril-forming segments from the C terminus, (101)DSVISLS(107) and (147)GVIGIAQ(153), reveal tightly packed ß-sheets with steric zipper interfaces characteristic of the amyloid state. Based on these structures, we conclude that both C-terminal segments are likely to form aggregates if available for interaction. Proline substitutions in (101)DSVISLS(107) and (147)GVIGIAQ(153) impaired nucleation and fibril growth of full-length protein, confirming that these segments participate in aggregate formation. Our hypothesis is that improper protein maturation and incompletely folded states that render these aggregation-prone segments available for interaction offer a common molecular pathway for sALS and fALS.

Uncovering the Mechanism of Aggregation of Human Transthyretin.

The tetrameric thyroxine transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify ß-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy.

ß-Arrestin-Biased Agonists of the GLP-1 Receptor from ß-Amino Acid Residue Incorporation into GLP-1 Analogues.

Activation of a G protein-coupled receptor (GPCR) causes recruitment of multiple intracellular proteins, each of which can activate distinct signaling pathways. This complexity has engendered interest in agonists that preferentially stimulate subsets among the natural signaling pathways ("biased agonists"). We have examined analogues of glucagon-like peptide-1 (GLP-1) containing ß-amino acid residues in place of native α residues at selected sites and found that some analogues differ from GLP-1 in terms of their relative abilities to promote G protein activation (as monitored via cAMP production) versus ß-arrestin recruitment (as monitored via BRET assays). The α → ß replacements generally cause modest declines in stimulation of cAMP production and ß-arrestin recruitment, but for some replacement sets cAMP production is more strongly affected than is ß-arrestin recruitment. The central portion of GLP-1 appears to be critical for achieving bias toward ß-arrestin recruitment. These results suggest that backbone modification via α → ß residue replacement may be a versatile source of agonists with biased GLP-1R activation profiles.

A potent α/ß-peptide analogue of GLP-1 with prolonged action in vivo.

Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic ß cells. GLP-1R agoinsts are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. We describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained ß-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogues of other peptide hormones.

A genetic condition that spans both extremes of the nutritional spectrum.

Prader-Willi syndrome (PWS) is a complex genetic disorder caused by lack of expression of genes on the paternally inherited chromosome 15q11.2-q13 region, known as the Prader Willi critical region. Nutritional clinical manifestations change with age and are described in four different phases. The phases span both extremes of the nutritional spectrum, beginning with an infant with poor sucking reflexes and failure to thrive then progressing to an adolescent who may have hyperphagia and be at risk for obesity. The phenotype is likely due to hypothalamic dysfunction due to genetic changes in the Prader Willi critical region. Researchers are examining the pathological mechanisms that determine the disease course.

Preventing Potential Patient Harm Through Clinical Content Interventions During Oncology Clinical Trial Implementation.

OBJECTIVES: Clinical trial documents are complex and may have inconsistencies, leading to potential site implementation errors and may compromise participant safety. This study characterizes the frequency and type of administrative and potential patient safety interventions (PPSIs) made during the review of oncology trial documents for clinical trial implementation by centralized clinical content specialists. METHODS: A dedicated group of centralized clinical content specialists reviewed trial documents, including the protocol, laboratory manual, and pharmacy/cellular therapy manual, and collected intervention data over a 1-year period. Each trial was categorized by study phase and sponsor type, and multiple interventions could be identified per trial. Interventions were deemed administrative or PPSIs, with PPSIs further subcategorized as medication, laboratory, procedure related, or other. RESULTS: Of 585 clinical trials reviewed, 269 (46%) required intervention(s). Among 1001 interventions, 171 (17.1%) were PPSIs. Most PPSIs were medication related (45.6%), with drug dosing interventions most frequently identified (53.8%). Phase 1 trials had the highest proportion of PPSIs (0.35:1) and administrative interventions (2:1) per trial compared with all other phases. Investigator-initiated trials saw the highest proportion of PPSIs per trial (0.44:1) of all sponsor types. CONCLUSIONS: This study demonstrates a gap in patient safety when assessing trial documents for clinical trial implementation. One solution to address this gap is the utilization of a centralized team of clinical specialists to preemptively review trial documents, thereby enhancing patient safety during clinical trial conduct.

Enhancement of α-helix mimicry by an α/ß-peptide foldamer via incorporation of a dense ionic side-chain array.

We report a new method for preorganization of α/ß-peptide helices, based on the use of a dense array of acidic and basic side chains. Previously we have used cyclically constrained ß residues to promote α/ß-peptide helicity; here we show that an engineered ion pair array can be comparably effective, as indicated by mimicry of the CHR domain of HIV protein gp41. The new design is effective in biochemical and cell-based infectivity assays; however, the resulting α/ß-peptide is susceptible to proteolysis. This susceptibility was addressed via introduction of a few cyclic ß residues near the cleavage site, to produce the most stable, effective α/ß-peptide gp41 CHR analogue identified. Crystal structures of an α- and α/ß-peptide (each involved in a gp41-mimetic helix bundle) that contain the dense acid/base residue array manifest disorder in the ionic side chains, but there is little side-chain disorder in analogous α- and α/ß-peptide structures with a sparser ionic side-chain array. These observations suggest that dense arrays of complementary acidic and basic residues can provide conformational stabilization via Coulombic attractions that do not require entropically costly ordering of side chains.

Structural and biological mimicry of protein surface recognition by alpha/beta-peptide foldamers.

Unnatural oligomers that can mimic protein surfaces offer a potentially useful strategy for blocking biomedically important protein-protein interactions. Here we evaluate an approach based on combining alpha- and beta-amino acid residues in the context of a polypeptide sequence from the HIV protein gp41, which represents an excellent testbed because of the wealth of available structural and biological information. We show that alpha/beta-peptides can mimic structural and functional properties of a critical gp41 subunit. Physical studies in solution, crystallographic data, and results from cell-fusion and virus-infectivity assays collectively indicate that the gp41-mimetic alpha/beta-peptides effectively block HIV-cell fusion via a mechanism comparable to that of gp41-derived alpha-peptides. An optimized alpha/beta-peptide is far less susceptible to proteolytic degradation than is an analogous alpha-peptide. Our findings show how a two-stage design approach, in which sequence-based alpha-->beta replacements are followed by site-specific backbone rigidification, can lead to physical and biological mimicry of a natural biorecognition process.

Comparison of fecal calprotectin and pancreatic elastase assays based on proficiency testing results.

BACKGROUND: Fecal calprotectin and fecal pancreatic elastase assays are not standardized because of a lack of suitable reference material. Laboratories may have difficulty in switching assays because different manufacturers do not compare well with each other despite having similar reference intervals. Data from proficiency testing performed in Germany (Fecal Diagnostics 01 Survey, INSTAND eV) were investigated to understand how results differed across eight calprotectin and five pancreatic elastase manufacturers. METHODS: Data were collected from participating laboratories in external quality assessment schemes from 2015 to 2020 for calprotectin and 2017 to 2020 for pancreatic elastase. The manufacturer group mean values and standard deviations were calculated. Reference points were created for each external quality assessment scheme by calculating the average of all manufacturer group means. Deming regression analyses were used to observe the differences across manufacturers. RESULTS: The slopes of the Deming regression spanned 0.37-1.91 for calprotectin and 0.84-1.33 for pancreatic elastase. The calprotectin assays had a high degree of variability in quantitative results by manufacturer. However, pancreatic elastase assays appear to be harmonized across the different manufacturer when considering the qualitative interpretation. CONCLUSIONS: Both calprotectin and pancreatic elastase assays could be improved by standardization efforts. Given the clinical utility and our data demonstrating high inter-manufacturer variability, calprotectin should be prioritized over pancreatic elastase in standardization efforts.

Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS.

Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing's syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement.We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotope-labeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection.

Comparison of Next-Generation Assays for Fecal Calprotectin vs the PhiCal Assay.

OBJECTIVES: To compare the PhiCal assay (CALPRO), the first US Food and Drug Administration-approved assay for fecal calprotectin, to 4 next-generation assays. METHODS: Stool samples from 50 patients were selected, and relevant clinical information was collected. Comparisons were performed using the PhiCal, fCAL turbo (BÜHLMANN), LIAISON Calprotectin (DiaSorin), QUANTA Lite Calprotectin ELISA (Inova Diagnostics), and Calprotectin Chemiluminescence ELISA (ALPCO) assays. RESULTS: All 4 assays had acceptable agreement with PhiCal when qualitatively categorizing results. Within the PhiCal reportable range of 16 to 1,250 µg/g, the DiaSorin, Inova Diagnostics, and ALPCO assays had Spearman correlation coefficients of 0.98, 0.97, and 0.95 and positive biases of 17%, 20%, and 15%, respectively. The BÜHLMANN assay ran approximately 2-fold higher than the PhiCal assay but had a correlation coefficient of 0.98, with similar result categorization. CONCLUSIONS: Our results demonstrate good comparison between PhiCal and 4 next-generation assays. Laboratories performing fecal calprotectin assays may have compelling reasons to adopt next-generation fecal calprotectin testing, such as greater automation, a decreased number of replicates needed per test, and the use of stool-extraction devices. These benefits could decrease turnaround times and lower costs. Although the results of the assays correlated, they are not standardized. Laboratories adopting the newer assays will need to further investigate their performance through validation studies.

Iatrogenic Cushing syndrome in 24-hour urine free cortisol measurement.

Cushing syndrome (CS) is caused by an excess of glucocorticoids that results in a variety of symptoms such as central obesity, moon facies, hirsutism, and reddish-purple stretch marks. Cortisol is the most potent endogenous glucocorticoid, and measuring the total amount excreted in the urine over a 24-hour period is useful to screen for CS caused by a tumor. However, most cases of CS are believed to be caused by exogenous glucocorticoids, such as prednisone and prednisolone, which are administered for anti-inflammatory and immunosuppressive treatments. This is often referred to as iatrogenic (drug-related or exogenous) CS. We modified an LC-MS/MS method for urine free cortisol to detect the presence of prednisone and prednisolone in patient samples. We wanted to understand the potential prevalence of exogenous CS in our patient population.

Clinical and Analytical Characterization of the DiaSorin and ScheBo Fecal Pancreatic Elastase 1 Assays.

OBJECTIVES: Fecal pancreatic elastase (PE) assays are screening tests for exocrine pancreatic insufficiency (EPI). We analytically evaluated a new PE assay and retrospectively analyzed data from an academic hospital and reference laboratory to understand the clinical utility. METHODS: Forty stool samples with different PE concentrations were tested on the ScheBo enzyme-linked immunosorbent assay (ELISA) versus DiaSorin LIAISON immunoassay; a simple-to-use extraction device was assessed. The cross-reactivity of porcine enzymes was investigated in the immunoassay. Charts of 207 patients with PE results less than 250 µg/g at an academic hospital were reviewed, and data were analyzed for 5136 patients with repeat PE results from a reference laboratory. RESULTS: The LIAISON immunoassay gave comparable results to the ScheBo ELISA, with 87.5% agreement of PE results in classifying as sufficient, mild/moderate insufficiency, or severe insufficiency. The extraction device worked well compared with manual weighing, and no cross reactivity with porcine enzymes was observed. In agreement with prior studies, our clinical data suggested that PE assays were most useful in detecting severe EPI. CONCLUSIONS: The new DiaSorin LIAISON immunoassay preforms similarly to the well-known ScheBo ELISA. Pancreatic elastase assays can help identify patients with severe EPI but are not as useful in classifying mild/moderate EPI.

Impact of Unconjugated estriol (uE3) assay interference on prenatal screening tests.

BACKGROUND: Unconjugated estriol (uE3) is an important biomarker in second trimester prenatal screening. Previous studies from our laboratory identified rare interference in the Beckman uE3 assay due to anti-ALP antibodies, which could be mitigated with a scavenger or heat-inactivated ALP (hALP). In the current study, 160 de-identified patient samples previously submitted for the Quad screen with low uE3 multiples of the median (MoM ≤0.50) were investigated for potential interference. METHODS: A reagent pack spiking strategy with hALP was employed to understand if the interference could be identified and mitigated in a scalable manner. The 160 samples were measured using uE3 lot #920861 previously known to be subject to interference, lot #920861 spiked with hALP, and the vendor reformulated lot #922579. Samples were suspected to have interference if the percent difference in uE3 measurements was >50%. Pseudo-risks were calculated using a test patient environment to understand the screening impact due to the change in uE3 result. RESULTS: Seventeen of the 160 samples had uE3 results that were >50% different between the hALP spiked and non-spiked reagent pack. Both original lot #920861 with hALP and reformulated lot #922579 identified the same 17 patients as having interference in lot #920861. Analysis of screening risks using a test patient environment showed that assay interference could result in false positives for one trisomy 21 and three trisomy 18 post-test risk calculations. CONCLUSION: Our experiment of reagent pack spiking with hALP produced similar uE3 results to a reformulated reagent designed to address potential interference, demonstrating that this is a feasible strategy to screen for interference in a scalable manner. The vendor-provided reformulation addressed anti-ALP interference and improved the performance of the screen.

Broad distribution of energetically important contacts across an extended protein interface.

Infection of cells by HIV depends upon profound structural rearrangements within the trimeric viral protein gp41. Critical to this process is the formation of a six-helix bundle in which a set of three N-terminal heptad repeat (NHR) helices assemble to form a core displaying long grooves that provide docking sites for three C-terminal heptad repeat (CHR) helices. We report experiments designed to discriminate between two alternative hypotheses regarding the source of affinity between individual CHR helices and the complementary groove: (1) affinity is dominated by interactions of a small cluster of side chains at one end of the CHR helix; or (2) affinity depends upon interactions distributed across the long CHR helix. We have employed two complementary experimental designs, and results from both favor the latter hypothesis.

Hereditary pancreatitis in a young adult: Acute to chronic.

This report investigates an unusual case of recurrent pancreatitis. A 22-year-old female was admitted to the emergency room for severe abdominal pain, nausea, and weight loss. She reported having these symptoms since she was a toddler. The clinician ordered fecal pancreatic elastase-1, fat-soluble vitamins, molecular studies, and imaging of the pancreas by computed tomography. The screening test result for fecal pancreatic elastase-1 revealed severe pancreatic exocrine insufficiency, and the concentrations of fat-soluble vitamins were also low. Imaging showed scattered calcifications in the pancreas. These findings supported a diagnosis of chronic pancreatitis. Due to the rarity of chronic pancreatitis in young adults, molecular studies were performed. The patient was found to be homozygous for a mutation in the SPINK1 gene, which is associated with hereditary pancreatitis. This case report discusses hereditary pancreatitis and highlights data on the utilization of fecal pancreatic elastase-1 to assess pancreatic exocrine insufficiency.

Retrospective comparison of results for simultaneous orders for LDL particle count, apolipoprotein B, and LDL-C.

BACKGROUND: Analysis of lipoprotein size and composition by nuclear magnetic resonance (NMR) has been advocated as a method for identifying individuals at high CVD risk. We compared risk stratification between NMR-based LDL particle number (LDL-PNUM), LDL-cholesterol (LDL-C), and apolipoprotein B (apoB). METHODS: Retrospective data from patients with simultaneous orders for LDL-PNUM, LDL-C, and apoB were analyzed and included data from an NMR assay (Numares). Quantitative and qualitative analyses were performed. Additional lipid parameters were investigated for patients with discordant risk classifications in LDL-related measurements. The percent change of LDL-PNUM was compared to the percent change of LDL-C or apoB for patients with serial measurements. RESULTS: We observed good quantitative and qualitative correlation when comparing LDL-PNUM to either LDL-C or apoB (Spearman's ρ ≥ 0.83, percent agreements ≥ 85%). Among the patients with discordant risk stratification, most had increased LDL-PNUM and normal LDL-C and apoB. For patients with serial measurements, a strong correlation between the LDL-PNUM percent change and the LDL-C or apoB percent change was observed (Spearman's ρ > 0.93). CONCLUSION: For many patients, risk stratification of LDL-PNUM is similar to apoB or LDL-C using cut-offs proposed by guidelines.