Steven W. Cotten

Modulation of nucleobindin-1 and nucleobindin-2 by caspases

Nucleobindin‐1 (NUCB1) and nucleobindin‐2 (NUCB2) are multifunctional proteins that interact with Ca2+, nucleic acids and various regulatory proteins in different signaling pathways. So far, our understanding of the regulation of the biological functions of nucleobindins remains limited. In our proteome‐wide selection for downstream caspase substrates, both NUCB1 and NUCB2 are found to be the downstream substrates of caspases. We report here the detailed analyses of the cleavage of nucleobindins by caspases. Significantly, the caspase cleavage sites are located exactly at one of the Ca2+‐binding EF‐hand motifs. Our results suggest that the functions of nucleobindins could be modulated by caspase‐mediated cleavage in apoptosis.

Drug Testing in the Neonate

Drug testing in newborns comes with analytical, therapeutic, and legal issues, and interpretation of results may be left to physicians, nurses, or social services workers. The unique analytical and legal caveats pose a variety of challenges and therapeutic issues. Positive drug screening results can allow for proper medical management of withdrawal symptoms for certain drug classes. Legal implications and involvement of social services for assessment of child safety surround positive urine or meconium drug samples. Because laboratory results can potentially remove newborns from their biological parents, the caveats and limitations of drug testing in this population are of utmost importance.

mRNA-display-based selections for proteins with desired functions: a protease-substrate case study.

mRNA‐display is an amplification‐based, iterative rounds of in vitro protein selection technique that circumvents a number of difficulties associated with yeast two‐hybrid and phage display. Because of the covalent linkage between the genotype and the phenotype, mRNA‐display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected from a library with a diversity in the range of 1012–1013. In this paper, we briefly review the recent progress in using mRNA‐display to identify affinity reagents, binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. To facilitate the use of mRNA‐display in research laboratories without previous experience, we provide a detailed analysis of the critical steps of an mRNA‐display‐based selection in a case study for the identification of the natural substrates of caspases, including the generation of an mRNA‐displayed proteome library, removal of abundant sequences, and selection of proteins with desired functions. The advantages and technical limitations of mRNA‐display as a general peptide or protein selection tool are also addressed.

Selection of proteins with desired properties from natural proteome libraries using mRNA display

mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca2+-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display–based selection can be accomplished in ∼7 d.

The new collaborative path in medical device development: the medical device innovation consortium.

The United States medical device market is the worlds largest with over

Kids in America: Newborn Screening for Cystic Fibrosis

100 billion in sales in 2011. Despite robust industry growth, the efficiency of the FDA approval process for moderate-risk (Class II) and high-risk devices (Class III) requiring 510(k) submission or pre-market approval (PMA) has been criticized. Recently, the FDAs Center for Devices and Radiological Health (CDRH) announced the creation of a Medical Device Innovation Consortium (MDIC), a public-private partnership (PPP) to share knowledge in regulatory science. Overarching goals include creating a forum for the exchange of ideas among the FDA, industry, and non-profit entities; providing monetary investments for project proposals prioritized by key working groups; and developing tools that support cost effective innovation, data-driven methodology, and implementation strategies. Clinical chemists and clinical laboratory scientists have several unique opportunities to contribute to the MDIC. These laboratory professionals have invaluable experience with the real-life performance of a variety of medical devices and their expertise can recognize unmet needs and contribute towards the acceleration of device development.

Method performance and clinical workflow outcomes associated with meconium and umbilical cord toxicology testing

Within the last year, all 50 states in the United States have adopted newborn screening (NBS) protocols for cystic fibrosis (CF), the most common fatal autosomal recessive disease among Caucasian populations. In this overview, we discuss the rationale for implementing NBS for CF and discuss the different testing algorithms states have adopted. Based on studies in the United States, Australia, and the United Kingdom, these measures will likely lead to less severe disease, prolonged life, and more cost-effective management of CF in the long run.

Evaluation of digital images for identification and characterization of monoclonal immunoglobulins by immunofixation.

OBJECTIVE Neonatal abstinence syndrome (NAS) is a rising concern with unknown long-term effects. It is apparent that higher cost of care, impact on the community and reduced quality of life are associated with similar etiologies (e.g., fetal alcohol syndrome). Detection of drug exposure in utero allows for earlier intervention to potentially reduce undesired outcomes. Umbilical cord tissue (UCT) has been documented as a readily accessible specimen for detection of drug exposure and has emerged as an alternative specimen to meconium. METHODS The analytical and clinical impact of umbilical cord tissue relative to meconium was evaluated for assessment of in utero drug exposure. Quality metrics relating to turnaround-time and diagnosis of NAS were investigated after switching from meconium to UCT. RESULTS Umbilical cord tissue showed higher clinical sensitivity but lower specificity for prediction of NAS diagnosis. Birth to result time decreased with adoption of UCT. CONCLUSIONS Birth to result time decreased by the switching to UTC as well as the number of missed collections. The clinical sensitivity and negative predictive value for NAS increased with UCT; however both meconium and UTC samples were negative for opiates for a significant percentage of newborns with a diagnosis of NAS.

Multiple Positive Sweat Chloride Tests in an Infant Asymptomatic for Cystic Fibrosis

OBJECTIVES High resolution digital imaging systems were recently introduced to capture and visualize serum protein electrophoresis results. In this study, we compared the performance of five, experienced interpreters using digital images and physical gels to identify and characterize monoclonal gammopathies by immunofixation. DESIGN AND METHODS Immunofixation gels were generated using Sebias HYDRASYS and digital images were captured with Sebias Gelscan system. Interpreters blindly reviewed 200 consecutively obtained immunofixation results using physical gels, low resolution (LR) images, and high resolution (HR) images. RESULTS Interpretations of the physical gels were significantly more sensitive (p≤0.01) than LR and HR images, and significantly more specific (p<0.001) than the LR images. Interpreters had a sensitivity of 82.0% (45.8-95.7) using the LR images and a specificity of 71.0% (47.8-91.3); using the HR images interpreters had a sensitivity of 80.4% (68.1-86.8) and specificity of 91.8% (80.3-97.8). There was 73.6% agreement between the HR digital images and the physical gel for immunoglobulin isotype characterization. Interpreters using digital images collectively missed 19 patients with monoclonal immunoglobulins that were identified using physical gels. CONCLUSIONS Interpreters using digital images had significantly different performance than when using physical agarose gels. Differences were most pronounced for low concentration monoclonal gammopathies (<0.3 g/dL) and for complex patterns. Between-interpreter agreement was also lower using digital images. While digital images may serve as a useful resource for retrospective analysis and review of previous results, they are not equivalent to physical gels. Additional studies are warranted to explore the clinical impact of these observed differences.

An Introduction to Drug Testing: The Expanding Role of Mass Spectrometry

Patient: 1-month-old male. Chief Complaint: Multiple positive sweat chloride tests in the absence of signs or symptoms suggestive of cystic fibrosis (CF). History of Present Illness: The patient was referred for sweat chloride testing after a positive newborn screen for immunoreactive trypsinogen (IRT). Immunoreactive trypsinogen is suggestive of CF when the value is within the top 5% of a period-specific floating cutoff value1 (Figure 1). The patient’s sweat chloride levels were elevated (>60 mmol/L). Preliminary screening for CF transmembrane conductance regulator (CFTR) mutations using a 40 mutation screen identified 1 mutation in intron 11 (1585 -1G>A, legacy name 1717 -1G>A). Subsequent full gene CFTR sequencing revealed a second CFTR mutation in intron 22. Past Medical History: The infant was full term, weighing 8 lbs 6 oz at birth, and experienced an uncomplicated delivery. The infant was discharged following a standard 2-day stay in the newborn nursery. Physical Exam: A temperature of 36.4°C (axillary), blood pressure of 120/80 mm Hg, heart rate of 148, and respirations of 40 per minute were measured for the patient. The patient’s O2 saturation was 100% on room air. The patient’s length was 61.2 cm (90th percentile), weight 6.27 kg (95th percentile), and head circumference was 39.7 cm (50th percentile). His weight-to-length ratio was at the 50th percentile. The patient appeared healthy with no labored respiration and no apparent digestive issues. Drug History: None Family History: No family history of chronic diseases. Principal Laboratory Findings: The patient underwent sweat chloride testing on sweat collected by pilocarpine iontophoresis from both arms (Table 1). These tests were repeated several weeks later yielding similar results (not shown). 1. What are this patient’s most striking laboratory and clinical findings (Table 1)? 2. How do you explain the patient’s most striking clinical and laboratory findings? 3. What conditions are typically associated …