Identifying substrate proteins for GAN1 and Keap1

Abstract only availableGAN1 and Keap1 are proteins characterized by a N-terminal BTB domain and a C-terminal Kelch repeat domain. Both of these domains are protein-protein interaction domains, suggesting that these BTB-Kelch proteins form signaling complexes in cells. Previous work has suggested that BTB-Kelch proteins function as substrate adaptor proteins for Cul3-dependent E3 ubiquitin ligase complexes. The goal of this project was to identify substrate proteins of GAN1 and Keap1. This information will be particularly useful when for understanding Giant Axonal Neuropathy, a sensorimotor disease characterized by excessive accumulation of neurofilaments in neurons that contain mutated GAN1 genes. We used an affinity purification approach to identify candidate substrate proteins for GAN1 and Keap1. Recombinant GAN1 and Keap1 genes containing a C-terminal chitin binding domain (CBD) were inserted into pBabe puro vectors. These vectors were used to generate virus stocks, which were used to infect a microglial cell line, BV-2. Stable cell lines were generated using puromycin selection. A mock-infected cell line was generated in parallel. When the cell lines were confluent, the cells were lysed using a 0.1% SDS RIPA solution and chitin beads were used to precipitate the CBD-tagged proteins. Western blot analyses were performed to determine if the purification of the CBD-tagged proteins was successful. No CBD-tagged proteins were identified in our first pull-down experiment. We are currently reexamining the precipitation protocol and preparing to lysate the same set of cells.Life Sciences Undergraduate Research Opportunity Progra

Fluoroscopy-based tracking of femoral kinematics with statistical shape models

Precise knee kinematics assessment helps to diagnose knee pathologies and to improve the design of customized prosthetic components. The first step in identifying knee kinematics is to assess the femoral motion in the anatomical frame. However, no work has been done on pathological femurs, whose shape can be highly different from healthy ones

Resequencing microarray probe design for typing genetically diverse viruses: human rhinoviruses and enteroviruses

<p>Abstract</p> <p>Background</p> <p>Febrile respiratory illness (FRI) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. A particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (HRV) and enteroviruses (HEV) which are frequent causes of FRI. Resequencing Pathogen Microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking.</p> <p>Results</p> <p>Using HRV and HEV as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. A minimal number of probe sequences (26 for HRV and 13 for HEV), which were potentially capable of detecting all serotypes of HRV and HEV, were determined and implemented on the Resequencing Pathogen Microarray RPM-Flu v.30/31 (<it>Tessarae RPM-Flu</it>). The specificities of designed probes were validated using 34 HRV and 28 HEV strains. All strains were successfully detected and identified at least to species level. 33 HRV strains and 16 HEV strains could be further differentiated to serotype level.</p> <p>Conclusion</p> <p>This study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse RNA viruses with a minimal number of prototype sequences. The results demonstrated that the newly designed RPM-Flu v.30/31 can provide comprehensive and specific analysis of HRV and HEV samples which implicates that this design strategy will be applicable for other genetically diverse viruses.</p

Bone Marrow Stem Cell Treatment for Ischemic Heart Disease in Patients with No Option of Revascularization: A Systematic Review and Meta-Analysis

PMCID: PMC3686792This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; correlates with aging, it may not be causative

Sex Differences in Primary and Secondary Prevention of Cardiovascular Disease in China

Background: Despite improvements in diagnostic and therapeutic interventions to combat cardiovascular disease (CVD) in recent decades, there are significant ongoing access gaps and sex disparities in prevention that have not been adequately quantified in China. Methods: A representative, cross-sectional, community-based survey of adults (aged ≥45 years) was conducted in 7 geographic regions of China between 2014 and 2016. Logistic regression models were used to determine sex differences in primary and secondary CVD prevention, and any interaction by age, education level, and area of residence. Data are presented as adjusted odds ratios (ORs) and 95% CIs. Results: Of 47 841 participants (61.3% women), 5454 (57.2% women) had established CVD and 9532 (70.5% women) had a high estimated 10-year CVD risk (≥10%). Only 48.5% and 48.6% of women and 39.3% and 59.8% of men were on any kind of blood pressure (BP)-lowering medication, lipid-lowering medication, or antiplatelet therapy for primary and secondary prevention, respectively. Women with established CVD were significantly less likely than men to receive BP-lowering medications (OR, 0.79 [95% CI, 0.65-0.95]), lipid-lowering medications (OR, 0.69 [95% CI, 0.56-0.84]), antiplatelets (OR, 0.53 [95% CI, 0.45-0.62]), or any CVD prevention medication (OR, 0.62 [95% CI, 0.52-0.73]). Women with established CVD, however, had better BP control (OR, 1.31 [95% CI, 1.14-1.50]) but less well-controlled low-density lipoprotein cholesterol (OR, 0.66 [95% CI, 0.57-0.76]), and were less likely to smoke (OR, 13.89 [95% CI, 11.24-17.15]) and achieve physical activity targets (OR, 1.92 [95% CI, 1.61-2.29]). Conversely, women with high CVD risk were less likely than men to have their BP, low-density lipoprotein cholesterol, and bodyweight controlled (OR, 0.46 [95% CI, 0.38-0.55]; OR, 0.60 [95% CI, 0.52-0.69]; OR, 0.55 [95% CI, 0.48-0.63], respectively), despite a higher use of BP-lowering medications (OR, 1.21 [95% CI, 1.01-1.45]). Younger patients (<65 years) with established CVD were less likely to be taking CVD preventive medications, but there were no sex differences by area of residence or education level. Conclusions: Large and variable gaps in primary and secondary CVD prevention exist in China, particularly for women. Effective CVD prevention requires an improved overall nationwide strategy and a special emphasis on women with established CVD, who have the greatest disparity and the most to benefit

Hemoglobin and Clinical Outcomes in the Vericiguat Global Study in Patients With Heart Failure and Reduced Ejection Fraction (VICTORIA)

BACKGROUND: In the VICTORIA trial (Vericiguat Global Study in Patients with Heart Failure with Reduced Ejection Fraction), anemia occurred more often in patients treated with vericiguat (7.6%) than with placebo (5.7%). We explored the association between vericiguat, randomization hemoglobin, development of anemia, and whether the benefit of vericiguat related to baseline hemoglobin. METHODS: Anemia was defined as hemoglobin <13.0 g/dL in men and <12.0 g/dL in women (World Health Organization Anemia). Adverse events reported as anemia were also evaluated. We assessed the risk-adjusted relationship between hemoglobin and hematocrit with the primary outcome (composite of cardiovascular death or heart failure hospitalization) and the time-updated hemoglobin relationship to outcomes. RESULTS: At baseline, 1719 (35.7%) patients had World Health Organization anemia; median hemoglobin was 13.4 g/L (25th, 75th percentile: 12.1, 14.7 g/dL). At 16 weeks from randomization, 1643 patients had World Health Organization anemia (284 new for vericiguat and 219 for placebo), which occurred more often with vericiguat than placebo (P<0.001). After 16 weeks, no further decline in hemoglobin occurred over 96 weeks of follow-up and the ratio of hemoglobin/hematocrit remained constant. Overall, adverse event anemia occurred in 342 patients (7.1%). A lower hemoglobin was unrelated to the treatment benefit of vericiguat (versus placebo) on the primary outcome. In addition, analysis of time-updated hemoglobin revealed no association with the treatment effect of vericiguat (versus placebo) on the primary outcome. CONCLUSIONS: Anemia was common at randomization and lower hemoglobin was associated with a greater frequency of clinical events. Although vericiguat modestly lowered hemoglobin by 16 weeks, this effect did not further progress nor was it related to the treatment benefit of vericiguat. Registration: URL: https://www.clinicaltrials.gov: Unique identifier: NCT02861534