Analysis of plasma proteins using 2D gels and novel fluorescent probes: in search of blood based biomarkers for Alzheimer's disease.

BACKGROUND: The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer's disease. METHODS: The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. RESULTS: We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. CONCLUSIONS: This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.

Intraoperative bispectral index monitoring and time to extubation after cardiac surgery: secondary analysis of a randomized controlled trial.

BACKGROUND: Fast track recovery is a care process goal after cardiac surgery. Intraoperative anesthetic depth may impact recovery, but the impact of brain monitoring on time to extubation and intensive care unit (ICU) length of stay after cardiac surgery has not been extensively studied. Our goal was to determine if BIS-guided anesthesia improves time to extubation compared to MAC-guided anesthesia in a cardiac surgery population. METHODS: In this secondary outcome analysis of a randomized controlled study, we analyzed 294 patients undergoing elective coronary bypass grafting, valve replacements, and bypass plus valve replacements at a single tertiary referral center between February 1, 2009 and April 30, 2010. We analyzed cardiac surgery patients that had been randomized to BIS-guided anesthesia alerts (n = 131) or MAC-guided anesthesia alerts (n = 163). The primary outcome measure was time to extubation in the BIS-guided and anesthetic concentration-guided groups. Secondary outcomes were length of stay in the ICU and total postoperative hospital length of stay. RESULTS: Valid extubation time data were available for 247 of 294 patients. The median [IQR] time to extubation was 307 [215 to 771] minutes in the BIS group and 323 [196 to 730] minutes in the anesthetic concentration group (p = 0.61). The median [IQR] ICU length of stay was 54 [29 to 97] hours versus 70 [44 to 99] hours (p = 0.11). In terms of postoperative hospital length of stay, there was no difference between the groups with median [IQR] times of 6 [5-8] days (p = 0.69) in each group. CONCLUSIONS: The use of intraoperative BIS monitoring during cardiac surgery did not change time to extubation, ICU length of stay or hospital length of stay. Data regarding BIS monitoring and recovery in an exclusively cardiac surgery population are consistent with recent effectiveness studies in the general surgical population. TRIAL REGISTRATION: ClinicalTrials.gov number NCT00689091.

Intraoperative blood collection without fluid replacement for cardiac surgery - A retrospective analysis.

Background: Transfusion rates in cardiac surgery are high. Aim: To determine if intraoperative autologous blood removal without volume replacement is associated with fewer homologous blood transfusions without increasing acute kidney injury. Setting and Design: Retrospective, comparative study. Materials and Methods: Adult patients undergoing cardiac surgery, excluding those who underwent ventricular assist device surgery, heart transplants, or cardiac surgery without cardiopulmonary bypass were excluded, who had 1-3 units of intraoperative autologous blood removal were compared to patients without blood removal for determination of volume replacement, vasopressor support, acute kidney injury, and transfusions. Results: Autologous blood removal was associated with fewer patients receiving homologous transfusions: intraoperative red cell transfusions fell from 75% (Control) to 48% (1 unit removed), 40% (2 units), and 30% (3 units), P < 0.001. Total intraoperative and postoperative homologous RBC units transfused were lower in the blood removal groups: median (interquartile range) 3 (1, 6) in Control patients and 0 (0, 2), 0 (0, 2) and 0 (0, 1) in the 1, 2, and 3 units removed groups, P < 0.001. Similarly, plasma, platelet, and cryoprecipitate transfusions decreased. After adjustment for confounders, increased amounts of autologous blood removal were associated with increased intravenous fluids, only when 2 units were removed, and trivially increased vasopressor use. However, it was not associated with acidosis or acute kidney injury. Conclusions: Intraoperative autologous blood removal without volume replacement of 1-3 units for later autologous transfusion is associated with decreased homologous transfusions without acidosis or acute kidney injury.

Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

Lithography of Polymer Nanostructures on Glass for Teaching Polymer Chemistry and Physics.

As nanolithography becomes increasingly important in technology and daily life, a variety of inexpensive and creative methods toward communicating the concepts underpinning these processes in the classroom are necessary. An experiment is described that uses simple CD-Rs, C-clamps, an oven, and a freezer to provide concrete examples and insights into the chemistry and principles of nanolithography. The experiment also has flexibility, making it suitable for a range of classroom levels from high school to more advanced labs in college. Because CD-Rs are composed of grooves of polycarbonate, the experiment provides a basis for discussions and exploration into the chemistry and physics of polymers on the nanoscale.

The Value of Songs for Teaching and Learning Cardiopulmonary Resuscitation (CPR) Competencies: A Systematic Review.

We sought to summarize, in a systematic review, the effectiveness of songs to support learning, performance, and recall of quality characteristics of cardiopulmonary resuscitation (CPR) compression rate, and depth. We systematically reviewed the literature from eight academic indexes from the fields of medicine, nursing, allied health, and education, from 2014 to 2020 to identify studies that evaluated an intervention of song use during CPR training against control and reported outcomes of compression rate and depth. There were 185 studies initially identified for review, eight met criteria for inclusion and analysis. For the critical outcome of compression depth, a pooled song group (n=446) when compared to a non-song group (n=443) demonstrated higher odds of being in the recommended range (OR 3.47). All studies, however, performed an average compression depth shallower than recommended guidelines in each arm. The available literature, we found, utilized heterogenous methodology and was at high risk of bias. When pooled, there were trends towards improved CPR metric performance in groups who were exposed to songs during treatment, though this only reached significance when groups were tested at >30 days from initial exposure. Findings of lower compression rates in the song groups suggest that song selection should favor beats per minute closer to the midpoint of the 100-120 ideal range to allow for variation when used as mental metronomes.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

Cyclic 1,2-diketones as core building blocks: a strategy for the total synthesis of (-)-terpestacin.

We report a full account of our work towards the total synthesis of (-)-terpestacin (1), a sesterterpene originally isolated from fungal strain Arthrinium sp. FA1744. Its promising anti-HIV and anti-cancer activity, as well as its novel structure, make terpestacin an attractive synthetic target. A strategy based on the unique reactivity of cyclic 1,2-diketones (diosphenols) was developed and total synthesis of 1 was achieved in 20 steps, in the longest linear sequence, from commercially available 2-hydroxy-3-methyl-2-cyclopenten-1-one. The key feature of our synthesis is the double usage of a "Pd AAA-Claisen" protocol (AAA=asymmetric allylic alkylation), first in the early stages to generate the C1 quaternary center and then in the late stages to install the side chain. In addition, a rather unusual ene-1,2-dione moiety was synthesized and utilized as an excellent Michael acceptor to attach the C15 substituent. Several possible routes towards the total synthesis have been examined and carefully evaluated. During our exploration many interesting chemoselectivity issues have been addressed, such as a highly selective ring-closing metathesis and a challenging oxidation of a disubstituted olefin in the presence of three trisubstituted ones.

Quantitation of free and total N-acetylcysteine amide and its metabolite N-acetylcysteine in human plasma using derivatization and electrospray LC-MS/MS.

Studies of N-acetylcysteine amide (NACA) in nonclinical models have demonstrated various antioxidant, anti-apoptotic, anti-inflammatory and neuroprotective effects, and it is currently being developed as a treatment for retinitis pigmentosa. Sensitive LC-MS/MS methods were developed and validated to quantitate reduced and total NACA and its major metabolite, N-acetylcysteine (NAC), in human plasma to support clinical studies involving NACA. To trap and stabilize reduced NACA and NAC at the time of collection, whole blood was immediately treated with 2-chloro-1-methylpyridinium iodide (CMPI) to convert free thiols to 1-methylpyridinyl thioether derivatives. Plasma was harvested and frozen until samples were assayed using protein precipitation and an LC-MS/MS separation based on hydrophilic-interaction chromatography (HILIC). To process NACA and NAC present as disulfides, an intermediate portion of the extract was further subjected to reduction with tris(2-carboxyethyl) phosphine; the released thiols were then reacted with CMPI, extracted, and analyzed as before, to measure total thiols. The method for NACA and NAC, whether free/reduced or total, covered a range from 50 ng/mL to 50 µg/mL in human plasma and required a single 25 µL plasma sample. Up to 180 samples could be assayed in a single session. The inter-run mean bias and precision (%CV) were within ±5% for the free thiol method and within ±8.5% for the total thiol method. Benchtop, freeze/thaw, and long-term stability were evaluated and acceptable. The NAC/NACA method applied to a clinical study demonstrated incurred sample reproducibility of 95.5% for NAC and 99.1% for NACA.

Modules for the Technical Skills Section of the OSCE Component of the American Board of Anesthesiology APPLIED Examination.

Introduction: To assess communication and professionalism, as well as technical skills related to patient care, the American Board of Anesthesiology (ABA) has begun administering an Objective Structured Clinical Examination (OSCE) portion of the APPLIED Examination in addition to the Standard Oral Examination component. Methods: We created video modules and a curriculum for anesthesiology resident OSCE preparation for the Interpretation of Monitors and Interpretation of Echocardiography components. The modules can be used individually by trainees or included as part of an OSCE workshop led by faculty educators with seven individual stations matching the content of the actual ABA examination. These modules are recommended for all levels of anesthesiology trainees so that they can gain exposure to the format and the fast pace of the examination. Results: Sixty-six junior and senior anesthesiology residents, fellows, and junior faculty successfully participated in these modules. Seventy-three percent of the participants agreed that after completing these modules, they now had a good understanding of the Interpretation of Monitors and Interpretation of Echocardiography technical skills stations. More than 90% of participants reported that the modules were useful, and more than 70% reported that they now felt prepared for these stations of the OSCE. Discussion: Developing technical skills stations for deliberate practice and preparation for the ABA OSCE is resource intensive. Finding time and faculty to facilitate OSCE preparation is also challenging. With the video modules and scripts included in this publication, residents can practice independently or as part of larger preparation course.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

To die, to sleep: US physicians' religious and other objections to physician-assisted suicide, terminal sedation, and withdrawal of life support.

This study analyzes data from a national survey to estimate the proportion of physicians who currently object to physician-assisted suicide (PAS), terminal sedation (TS), and withdrawal of artificial life support (WLS), and to examine associations between such objections and physician ethnicity, religious characteristics, and experience caring for dying patients. Overall, 69% of the US physicians object to PAS, 18% to TS, and 5% to WLS. Highly religious physicians are more likely than those with low religiosity to object to both PAS (84% vs 55%, P < .001) and TS (25% vs 12%, P < .001). Objection to PAS or TS is also associated with being of Asian ethnicity, of Hindu religious affiliation, and having more experience caring for dying patients. These findings suggest that, with respect to morally contested interventions at the end of life, the medical care patients receive will vary based on their physicians' religious characteristics, ethnicity, and experience caring for dying patients.

The independent effects of anemia and transfusion on mortality after coronary artery bypass.

BACKGROUND: Both anemia and transfusions (Tx) are associated with mortality after cardiac operations. However, the relative contributions of anemia and Tx and their interaction on late mortality have not been determined. METHODS: 922 patients who underwent isolated coronary artery bypass grafting (CABG) were retrospectively studied. Anemia (A+) was defined as hemoglobin<12 g/dL for men and <11 g/dL for women. Patients who received (Tx+) and did not receive (Tx-) transfusions were compared; patient characteristics were controlled for by the use of Cox analysis and then by matching Tx+ to Tx- patients based on identical hemoglobin levels at admission and by propensity matching. RESULTS: 5.3% of Tx- patients died, compared with 11% of Tx+ patients (p=0.001). The interaction of anemia and Tx was associated with a greater hazard of dying. In particular, A+Tx+ (anemic, received transfusion) patients had a threefold hazard of death (2.918, 95% confidence interval=1.512-5.633, p=0.001) compared with A-Tx- (nonanemic, no transfusion) patients. A+Tx+ patients had twice the hazard of dying as did A+Tx- (anemic, no transfusion) (hazard ratio=2.087, 95% confidence interval=1.004-4.336, p=0.049). In populations matched by preoperative hemoglobin levels or by propensity scores, similar results were seen: a significant interaction between anemia and transfusion of red blood cells. A+Tx+ patients fared significantly worse than did the other three groups. Although there was no difference in mortality between A- patients who did or did not receive transfusions, A+T+ patients had triple the risk as A+T- patients, whereas A+Tx- patients had a similar risk of late mortality as A-Tx- patients. CONCLUSIONS: The anemia-transfusion interaction was associated with an increased hazard of late mortality.

Imidazolylsulfonates: electrophilic partners in cross-coupling reactions.

Aryl imidazolylsulfonates participate as electrophilic coupling partners in palladium-mediated cross-coupling reactions. The aryl imidazolylsulfonates display good stability while maintaining good reactivity in a variety of palladium-catalyzed coupling reactions. Imidazolylsulfonates are a practical and economic alternative to triflates.

Synthesis and evaluation of aminomethyl dihydrocinnamates as a new class of PPAR ligands.

PPAR ligands with varied subtype selectivity have been synthesized using an achiral aminomethyl dihydrocinnamate template. Several compounds in this series have demonstrated potent plasma glucose and triglyceride lowering capability in rodent models of type 2 diabetes.