Enrichment of Bioactive Lipids in Urinary Extracellular Vesicles and Evidence of Apoptosis in Kidneys of Hypertensive Diabetic Cathepsin B Knockout Mice after Streptozotocin Treatment.

Cathepsin B (CtsB) is a ubiquitously expressed cysteine protease that plays important roles in health and disease. Urinary extracellular vesicles (uEVs) are released from cells associated with urinary organs. The antibiotic streptozotocin (STZ) is known to induce pancreatic islet beta cell destruction, diabetic nephropathy, and hypertension. We hypothesized that streptozotocin-induced diabetic kidney disease and hypertension result in the release of bioactive lipids from kidney cells that induce oxidative stress and renal cell death. Lipidomics was performed on uEVs isolated from CtsB knockout mice treated with or without STZ, and their kidneys were used to investigate changes in proteins associated with cell death. Lysophosphatidylethanolamine (LPE) (18:1), lysophosphatidylserine (LPS) (22:6), and lysophosphatidylglycerol (LPG) (22:5) were among the bioactive lipids enriched in uEVs from CtsB knockout mice treated with STZ compared to untreated CtsB mice (n = 3 uEV preparations per group). Anti-oxidant programming was activated in the kidneys of the CtsB knockout mice treated with STZ, as indicated by increased expression of glutathione peroxidase 4 (GPX4) and the cystine/glutamate antiporter SLC7A11 (XCT) (n = 4 mice per group), which was supported by a higher reactivity to 4-hydroxy-2-nonenal (4-HNE), a marker for oxidative stress (n = 3 mice per group). Apoptosis but not ferroptosis was the ongoing form of cell death in these kidneys as cleaved caspase-3 levels were significantly elevated in the STZ-treated CtsB knockout mice (n = 4 mice per group). There were no appreciable differences in the pro-ferroptosis enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) or the inflammatory marker CD93 in the kidneys (n = 3 mice per group), which further supports apoptosis as the prevalent mechanism of pathology. These data suggest that STZ treatment leads to oxidative stress, inducing apoptotic injury in the kidneys during the development of diabetic kidney disease and hypertension.

Effective Liquid Chromatography-Trapped Ion Mobility Spectrometry-Mass Spectrometry Separation of Isomeric Lipid Species.

Lipids are a major class of molecules that play key roles in different biological processes. Understanding their biological roles and mechanisms remains analytically challenging due to their high isomeric content (e.g., varying acyl chain positions and/or double bond locations/geometries) in eukaryotic cells. In the present work, a combination of liquid chromatography (LC) followed by high resolution trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to investigate common isomeric glycerophosphocholine (PC) and diacylglycerol (DG) lipid species from human plasma. The LC dimension was effective for the separation of isomeric lipid species presenting distinct double bond locations or geometries but was not able to differentiate lipid isomers with distinct acyl chain positions. High resolution TIMS-MS resulted in the identification of lipid isomers that differ in the double bond locations/geometries as well as in the position of the acyl chain with resolving power ( R) up to ∼410 ( R ∼ 320 needed on average). Extremely small structural differences exhibiting collision cross sections (CCS) of less than 1% (down to 0.2%) are sufficient for the discrimination of the isomeric lipid species using TIMS-MS. The same level of performance was maintained in the complex biological mixture for the biologically relevant PC 16:0/18:1 lipid isomers. These results suggest several advantages of using complementary LC-TIMS-MS separations for regular lipidomic analysis, with the main emphasis in the elucidation of isomer-specific lipid biological activities.

Circular dichroism spectroscopy: Enhancing a traditional undergraduate biochemistry laboratory experience.

The undergraduate biochemistry laboratory curriculum is designed to provide students with experience in protein isolation and purification protocols as well as various data analysis techniques, which enhance the biochemistry lecture course and give students a broad range of tools upon which to build in graduate level laboratories or once they begin their careers. One of the most common biochemistry protein purification experiments is the isolation and characterization of cytochrome c. Students across the country purify cytochrome c, lysozyme, or some other well-known protein to learn these common purification techniques. What this series of experiments lacks is the use of sophisticated instrumentation that is rarely available to undergraduate students. To give students a broader background in biochemical spectroscopy techniques, a new circular dichroism (CD) laboratory experiment was introduced into the biochemistry laboratory curriculum. This CD experiment provides students with a means of conceptualizing the secondary structure of their purified protein, and assessments indicate that students' understanding of the technique increased significantly. Students conducted this experiment with ease and in a short time frame, so this laboratory is conducive to merging with other data analysis techniques within a single laboratory period. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):515-520, 2017.

A novel risk scoring system reliably predicts readmission after pancreatectomy.

BACKGROUND: Postoperative readmissions have been proposed by Medicare as a quality metric and can impact provider reimbursement. Because readmission after pancreatectomy is common, we sought to identify factors associated with readmission to establish a predictive risk scoring system. STUDY DESIGN: A retrospective analysis of 2,360 pancreatectomies performed at 9 high-volume pancreatic centers between 2005 and 2011 was performed. Forty-five factors strongly associated with readmission were identified. To derive and validate a risk scoring system, the population was randomly divided into 2 cohorts in a 4:1 fashion. A multivariable logistic regression model was constructed and scores were assigned based on the relative odds ratio (OR) of each independent predictor. A composite Readmission after Pancreatectomy (RAP) score was generated and then stratified to create risk groups. RESULTS: Overall, 464 (19.7%) patients were readmitted within 90 days. Eight pre- and postoperative factors, including earlier MI (OR = 2.03), American Society of Anesthesiologists class ≥ 3 (OR = 1.34), dementia (OR = 6.22), hemorrhage (OR = 1.81), delayed gastric emptying (OR = 1.78), surgical site infection (OR = 3.31), sepsis (OR = 3.10), and short length of stay (OR = 1.51) were independently predictive of readmission. The 32-point RAP score generated from the derivation cohort was highly predictive of readmission in the validation cohort (area under the receiver operating curve = 0.72). The low-risk (0 to 3), intermediate-risk (4 to 7), and high-risk (>7) groups correlated with 11.7%, 17.5%, and 45.4% observed readmission rates, respectively (p < 0.001). CONCLUSIONS: The RAP score is a novel and clinically useful risk scoring system for readmission after pancreatectomy. Identification of patients with increased risk of readmission using the RAP score will allow efficient resource allocation aimed to attenuate readmission rates. It also has potential to serve as a new metric for comparative research and quality assessment.