Toward an Ultimate Solution for Peptide Retention Time Prediction: The Effect of Column Temperature on Separation Selectivity.

We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of ∼100,000 and ∼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of ∼20% of tryptic peptides that were amenable to MS/MS-based identification.

Full practice authority and burnout among primary care nurse practitioners.

BACKGROUND: Full practice authority (FPA) improves clinical autonomy for nurse practitioners (NPs). Autonomy may reduce burnout. PURPOSE: Estimate the effect of changing from reduced or restricted practice authority to FPA on NP burnout. METHODS: In this quasi-experimental study, we compared NP burnout before (2016) and after (2018) a Veterans Health Administration (VHA) regulation authorized NP FPA. Burnout proportions were estimated for VHA facilities by aggregating responses to the VHA's All Employee Survey from 1,352 primary care NPs. DISCUSSION: Seventy-seven percent of facilities changed to FPA postregulation. Burnout was six points lower among NPs in facilities that changed to FPA compared to facilities that had FPA prior to the regulation; however, this association was not statistically significant. CONCLUSION: NPs are increasingly working under independent practice. While changing to FPA did not reduce NP burnout, this association may vary by health care setting or when burnout is measured for individuals or teams.

Retention Time Prediction for TMT-Labeled Peptides in Proteomic LC-MS Experiments.

We present the first detailed study of chromatographic behavior of peptides labeled with tandem mass tags (TMT and TMTpro) in 2D LC for proteomic applications. Carefully designed experimental procedures have permitted generating data sets of over 100,000 nonlabeled and TMT-labeled peptide pairs for the low pH RP in the second separation dimension and data sets of over 10,000 peptide pairs for high-pH RP, HILIC (amide and silica), and SCX separations in the first separation dimension. The average increase in peptide RPLC (0.1% formic acid) retention upon TMT labeling was found to be 3.3% acetonitrile (linear water/acetonitrile gradients), spanning a range of -4 to 10.3%. In addition to the bulk peptide properties such as length, hydrophobicity, and the number of labeled residues, we found several sequence-dependent features mostly associated with differences in N-terminal chemistry. The behavior of TMTpro-labeled peptides was found to be very similar except for a slightly higher hydrophobicity: an average retention shift of 3.7% acetonitrile. The respective versions of the sequence-specific retention calculator (SSRCalc) model have been developed to accommodate both TMT chemistries, showing identical prediction accuracy (R2 ∼ 0.98) for labeled and nonlabeled peptides. Higher retention for TMT-labeled peptides was observed for high-pH RP and HILIC separations, while SCX selectivity remained virtually unchanged.

Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets.

Immunoprecipitation (IP) is commonly used upstream of mass spectrometry (MS) as an enrichment tool for low-abundant protein targets. However, several aspects of the classical IP procedure such as nonspecific protein binding to the isolation matrix, detergents or high salt concentrations in wash and elution buffers, and antibody chain contamination in elution fractions render it incompatible with downstream mass spectrometry analysis. Here, we discuss an improved IP-MS workflow that is designed to minimize sample prep time and these contaminants. The method employs biotinylated antibodies to the targets of interest and streptavidin magnetic beads that exhibit low background binding. In addition, alterations in the elution protocol and subsequent MS sample prep were made to reduce time and antibody leaching in the eluent, minimizing potential ion suppression effects and thereby maximizing detection of multiple target antigens and interacting proteins.

A preliminary evaluation of full practice authority of advance practice registered nurses in the Veterans Health Administration.

BACKGROUND: Responding to National Academy of Medicine and National Council of State Boards of Nursing recommendations, the Department of Veterans Health Affairs (VHA) implemented full practice authority (FPA) for Advanced Practice Registered Nurses in VHA medical centers (VAMCs) in 2017. PURPOSE: To evaluate FPA policy implementation's impact on quality indicators including access to care as measured by new patient appointments in primary, specialty and mental health services. METHODS: Linear growth models compared early (n = 85) vs. late (n = 55) FPA implementing VAMCs on the trajectories of each of the three quality indicators. FINDINGS: Early FPA implementing VAMCs showed greater rates of improvement over time in new patient appointments completed within 30 days of preferred date for primary care (p = .003), specialty care (p = 0.05), and mental health (p = 0.001). DISCUSSION: VAMCs that started implementation of FPA policy early showed greater improvement in access to care for Veterans over time than VAMCs that did not.

Point-of-care production of therapeutic proteins of good-manufacturing-practice quality.

Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins. We used the device to reproducibly manufacture His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technology may enable the rapid manufacturing of biologics at the point of care.

Anesthesia Care Practice Models in the Veterans Health Administration.

Although the VHA primarily relies on teams for anesthesia care, unsupervised certified registered nurse anesthetists also are used to meet veterans' surgical care needs.

GLUT3 gene expression is critical for embryonic growth, brain development and survival.

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly.

Robust microarray production of freshly expressed proteins in a human milieu.

PURPOSE: In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT (HCIVT)) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu. EXPERIMENTAL DESIGN: Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by HCIVT compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine. RESULTS: HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than ten times higher than RRL, in both Western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array. CONCLUSION AND CLINICAL RELEVANCE: HCIVT is a robust in vitro transcription and translation system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer.

Optimizing treatment outcomes with phosphodiesterase type 5 inhibitors for erectile dysfunction: opening windows to enhanced sexual function and overall health.

PURPOSE: Phosphodiesterase type 5 (PDE5) inhibitors have proved to be efficacious, safe, and well tolerated, in clinical trials and practice, for men with erectile dysfunction (ED). However, many patients are not satisfied with treatment and discontinue it prematurely. This review discusses evidence-based strategies that nurse practitioners (NPs) can use to improve diagnosis of ED, optimize patient outcomes, and identify opportunities to detect other potentially serious comorbid conditions. DATA SOURCES: This article was based on a previously published review, which involved a PubMed-MEDLINE search of the clinical literature from January 1, 1998 (year of sildenafil's approval in many markets), through August 30, 2008 (date of search). CONCLUSIONS: Strategies to optimize responses to PDE5 therapy are summarized by the mnemonic "EPOCH": Evaluating and educating to ensure realistic expectations of therapy; Prescribing a treatment individualized to the couple's needs and preferences; Optimizing drug dose/regimen and revisiting key educational messages at follow-up visits; Controlling comorbidities via lifestyle counseling, medications, and/or referrals; and Helping patients and their partners to seek other forms of therapy if they have decided not to use a PDE5 inhibitor. IMPLICATIONS FOR PRACTICE: The "EPOCH" mnemonic may remind NPs of steps to optimize treatment outcomes with PDE5 inhibitors.

Akt2 modulates glucose availability and downstream apoptotic pathways during development.

Glucose is the primary energy substrate for eukaryotic cells and the predominant substrate for the brain. Studies suggest that glucose serves an additional role in the regulation of cellular functions, including viability. Zebrafish is a tractable system for defining the cellular and molecular mechanisms perturbed by impaired glucose transport and metabolism. Previously, we demonstrated a critical role for the facilitative glucose transporter, Glut1, in the regulation of embryonic brain development. In this study, we aim to identify mediators in this Glut1-sensitive process by investigating the role of the antiapoptotic kinase, Akt2. Results show that abrogating expression of akt2 causes a phenotype strikingly similar to that observed when glut1 expression is inhibited. akt2-deficient embryos exhibit increased neuronal apoptosis, impaired glucose uptake, and death by 72 h postfertilization. Similar to what was observed in the glut1 morphants, inhibiting the expression of the proapoptotic protein, bad, in the context of impaired akt2 expression results in the inhibition of apoptosis and rescue of the morphant embryos. Intriguingly, overexpression of glut1 in the akt2 morphants was also able to rescue these embryos. Quantitative reverse transcription-PCR analysis revealed decreased glut1 transcript expression in akt2 morphant embryos. Taken together, these data suggest that Akt2 modulates glucose availability by regulating Glut1 expression at the transcript level. These data support a role for akt2 in an integrative pathway directly linking glucose, Glut1 expression, and activation of apoptosis and demonstrate the dependence of akt2 on glucose availability for the maintenance of cellular viability, particularly in the central nervous system.

GLUT1 deficiency links nutrient availability and apoptosis during embryonic development.

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.

Alpha-synuclein protects naive but not dbcAMP-treated dopaminergic cell types from 1-methyl-4-phenylpyridinium toxicity.

The pre-synaptic protein, alpha-synuclein, has been associated with the pathogenesis of Parkinson's disease. The present study indicates that alpha-synuclein, but not its mutants (A53T, A30P), can protect CNS dopaminergic cells from the parkinsonism-inducing drug 1-methyl-4-phenylpyridinium (MPP+), whereas it cannot protect from the dopaminergic toxin, 6-hydroxydopamine, hydrogen-peroxide, or the beta-amyloid peptide, A-beta. Protection from MPP+ was directly correlated with the preservation of mitochondrial function. Specifically, alpha-synuclein rescued cells from MPP+ mediated decreases in mitochondrial dehydrogenase activity and loss of ATP levels by utilizing ketosis. It also prevented toxin-induced activation of the creatine kinase/creatine phosphate system. Similarly, alpha-synuclein protected cells from the complex I inhibitor rotenone and 3-nitroproprionic acid, a complex II inhibitor. Wild-type alpha-synuclein-mediated neuroprotection and subsequent alterations in energy were not found in dbcAMP-differentiated cells. These results suggest that the normal physiological role for alpha-synuclein may change during development.

Health care clinicians in sexual health medicine: focus on erectile dysfunction.

A consensus guidelines model was developed for managing erectile dysfunction (ED) by urology nurses and other health care clinicians. The model emphasizes identification of ED in all males older than 40, education and counseling as keys to intervention, and inclusion of partners whenever possible. These guidelines are intended to provide a comprehensive care model for ED patients and their partners, which would be optimally cost effective and clinically relevant. If successful, this new model will allow quality sexual health care to be provided to increasing numbers of patients and their partners.