Site-Specific N-Glycosylation on the AAV8 Capsid Protein.

Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.

Safety and Tolerability of Intravenous Valproic Acid in Healthy Subjects: A Phase I Dose-Escalation Trial.

BACKGROUND: Valproic acid, a histone deacetylase inhibitor, has beneficial effects in the setting of cancer, neurologic diseases, and traumatic injuries. In animal models of traumatic injury, a single dose of valproic acid has been shown to reduce mortality. The purpose of this trial was to determine the maximum tolerated single dose of intravenous valproic acid in healthy humans. METHODS: A double-blinded, placebo-controlled, dose-escalation trial design was used to identify dose-limiting toxicities in healthy subjects who received a single dose of intravenous valproic acid. Patients were monitored for adverse events and data were collected for pharmacokinetic, pharmacodynamic, and safety profiling of valproic acid. RESULTS: Fifty-nine healthy subjects (mean 30 ± 12 years) were enrolled. Forty-four subjects received valproic acid in doses from 15 to 150 mg/kg. The most common adverse events were hypoacusis (n = 19), chills (n = 18), and headache (n = 16). The maximum tolerated dose was 140 mg/kg. Dose-limiting toxicities included headache and nausea lasting longer than 12 h. No drug-related abnormalities were seen in other safety measures including laboratory tests, hemodynamic parameters, cardiac rhythm monitoring, and cognitive testing. A two-compartment model was predictive of valproic acid concentration-time profiles, with a strong correlation (R 2 = 0.56) observed between the number of reported adverse events and the dose level. CONCLUSIONS: The maximum tolerated dose of intravenous valproic acid in healthy subjects is 140 mg/kg. This is significantly higher than the previously established maximum tolerated dose of 60-75 mg/kg. Next, the safety and tolerability of high-dose valproic acid will be tested in trauma patients in hemorrhagic shock. ClinicalTrials.gov Identifier: NCT01951560.

An Insight into Glyco-Microheterogeneity of Plasma von Willebrand Factor by Mass Spectrometry.

Human plasma von Willebrand Factor (VWF) plays essential roles in primary hemostasis in cooperation with other coagulations factors. There is ample indication that glycosylation affects many biological phases during the protein life cycle. However, comprehensive characterization of all probable N-glycosites simultaneous with O-glycosites is still not fully revealed. Thus, the intention of this exploration was to estimate the occupancy of all canonical N-glycosites besides simultaneous characterization of N- and O-glycoforms. An RP-LC-MS/MS system functionalized with CID and HCD tandem mass was utilized to analyze VWF. N-Glycosite occupancy varied along the protein backbone chain. Out of 257 HCD spectra, 181 characterized glycoforms were specified as either N- or O-glycosites. Sequential cleavage of glycosidic bonds along with Human Database mass matching have confirmed the glycoform structures. A total of 173 glycoforms represented most commonly biantennary and infrequently tri- and tetra-antennary N-glycans beside high mannose, hybrid, ABH antigen-terminated, and sulfated N-glycans. Many glycoforms were common across all N-sites. Noteworthy, previously unreported N-glycosites within domain D'(TIL'-E') showed glycosylation. Moreover, sialylated core 1 and core 2 O-glycans were detected on 2298T. Given subtle characterization of site-specific glycoforms, we can attain a profound understanding of the biological roles of VWF as well as facilitate the production of VWF-based therapeutics.

Effect of hyperglycemia on brain and liver 18F-FDG standardized uptake value (FDG SUV) measured by quantitative positron emission tomography (PET) imaging.

PURPOSE: Blood glucose is routinely measured prior to 18F-fluorodeoxyglucose (FDG) administration in positron emission tomography (PET) imaging to identify hyperglycemia that may affect image quality. In this study we explore the effects of blood glucose levels upon semi-quantitative standardized uptake value (SUV) measurements of target organs and tissues of interest and in particular address the relationship of blood glucose to FDG accumulation in the brain and liver. METHODS: 436 FDG PET/CT consecutive studies performed for oncology staging in 229 patients (226 male) at the Ann Arbor Veterans Administration Healthcare System were reviewed. All patients had blood glucose measured (112.4±34.1mg/dL) prior to injection of 466.2±51.8MBq (12.6±1.4mCi) of FDG. SUV measurements of brain, aortic arch blood-pool, liver, and spleen were obtained at 64.5±10.2min' post-injection. RESULTS: We found a negative inverse relationship of brain SUV with increasing plasma glucose, levels for both absolute and normalized (either to blood-pool or liver) values. Higher blood glucose levels had a mild effect upon liver and blood-pool SUV. By contrast, spleen SUV was independent of blood glucose, but demonstrated the greatest variability (deviation on linear regression). In contrast to other tissues, liver and spleen SUV normalized to blood-pool SUV were not dependent upon blood glucose levels. CONCLUSION: The effects of hyperglycemia upon FDG uptake in brain and liver, over a range of blood glucose values generally considered acceptable for clinical PET imaging, may have measurable effects on semi-quantitative image analysis.

Effect of Fontan operation on liver stiffness in children with single ventricle physiology.

OBJECTIVES: Assess liver stiffness using ultrasound point shear wave elastography (US P-SWE) in children before and after the Fontan operation. METHODS: Eighteen children undergoing the Fontan operation were prospectively enrolled. Eight US P-SWE measurements were obtained from the right hepatic lobe before surgery, and at multiple postoperative time points. Temporally related inferior vena cava pressure (IVC) data was collected from medical records, when available. Changes in mean liver shear wave speed (SWS) were assessed using a mixed-effect model with post hoc Tukey correction. Changes in IVC pressure were evaluated using the Wilcoxon signed-rank test. A p value less than 0.05 was considered significant. RESULTS: Mean age at enrolment was 33.5 ± 10.5 months. Baseline mean liver SWS was normal at 1.18 ± 0.29 m/s, increased to 2.28 ± 0.31 m/s at 2.5 ± 1.2 days (p < 0.0001) and to 2.22 ± 0.38 m/s at 7.5 ± 1.4 days (p < 0.0001). Five subjects returned at a mean of 185 ± 28 days, and mean liver SWS remained elevated at 2.08 ± 0.24 m/s (p < 0.0001). Mean IVC pressure increased from 7.2 ± 2.6 mmHg at baseline to 16.44 ± 3.3 mmHg at 2.2 ± 0.8 days post-surgery (p = 0.004). CONCLUSION: The Fontan operation immediately and chronically increases liver stiffness and IVC pressure. Our study provides further evidence that congestion is a key driver of Fontan-associated liver disease. KEY POINTS: • The Fontan operation triggers immediate hepatic congestion and marked liver stiffening. • Congestion, not fibrosis, drives early increased liver stiffness in Fontan patients. • Hepatic congestion persists chronically for months after the Fontan operation. • Congestion confounds shear wave elastography as a post-Fontan liver fibrosis biomarker.

Value of pelvis CT during follow-up of patients with pancreatic adenocarcinoma.

PURPOSE: The purpose of this study was to determine the frequency in which the pelvis component of an abdominopelvic CT provides information that would influence clinical management in two separate groups of patients: those with previously resected pancreatic ductal adenocarcinoma (PDA) and those with locally advanced unresectable PDA. METHODS: This institutional review-board approved HIPAA compliant retrospective study with waived informed consent included 247 subjects with histologically proven PDA, including 153 subjects post-pancreaticoduodenectomy and 94 subjects with locally advanced unresectable disease. Imaging reports interpreted between January 2005 and December 2013 were obtained from our institution's Radiology Information System by searching a Cancer Registry database of PDA patients separately for the words "whipple" and "unresectable." CT findings were separated by location in the abdomen or pelvis, and subsequently reviewed and graded for their likelihood of representing metastatic disease. The probability of pelvic CT influencing clinical management-i.e., of finding isolated pelvic metastatic disease-was determined using 95% binomial proportion confidence intervals for both the post-pancreaticoduodenectomy and locally advanced unresectable groups. RESULTS: No subjects who had undergone pancreaticoduodenectomy had an isolated pelvic metastasis on follow-up imaging (0%; 95% CI 0-2.38, p = 0.0004); 33 had metastatic disease in the abdomen, and 120 had no or equivocal evidence of abdominopelvic metastatic disease. One subject with locally advanced unresectable PDA had a possible isolated pelvic metastasis on follow-up imaging (1.1%; 95% CI 0.03-5.79, p = 0.048); 20 had metastatic disease in the abdomen, and 73 had no or equivocal evidence of abdominopelvic metastatic disease. CONCLUSION: Isolated pelvic metastatic disease rarely occurs in patients with PDA who have had prior pancreaticoduodenectomy or have a locally advanced unresectable primary tumor, suggesting routine pelvic CT in follow-up imaging of these patients may not be necessary.

An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

O-linked ß-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac34dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/ß 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac34dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

Greater Trochanteric Pain Syndrome: Percutaneous Tendon Fenestration Versus Platelet-Rich Plasma Injection for Treatment of Gluteal Tendinosis.

OBJECTIVES: The purpose of this study was to compare ultrasound-guided percutaneous tendon fenestration to platelet-rich plasma (PRP) injection for treatment of greater trochanteric pain syndrome. METHODS: After Institutional Review Board approval was obtained, patients with symptoms of greater trochanteric pain syndrome and ultrasound findings of gluteal tendinosis or a partial tear (<50% depth) were blinded and treated with ultrasound-guided fenestration or autologous PRP injection of the abnormal tendon. Pain scores were recorded at baseline, week 1, and week 2 after treatment. Retrospective clinic record review assessed patient symptoms. RESULTS: The study group consisted of 30 patients (24 female), of whom 50% were treated with fenestration and 50% were treated with PRP. The gluteus medius was treated in 73% and 67% in the fenestration and PRP groups, respectively. Tendinosis was present in all patients. In the fenestration group, mean pain scores were 32.4 at baseline, 16.8 at time point 1, and 15.2 at time point 2. In the PRP group, mean pain scores were 31.4 at baseline, 25.5 at time point 1, and 19.4 at time point 2. Retrospective follow-up showed significant pain score improvement from baseline to time points 1 and 2 (P< .0001) but no difference between treatment groups (P= .1623). There was 71% and 79% improvement at 92 days (mean) in the fenestration and PRP groups, respectively, with no significant difference between the treatments (P >.99). CONCLUSIONS: Our study shows that both ultrasound-guided tendon fenestration and PRP injection are effective for treatment of gluteal tendinosis, showing symptom improvement in both treatment groups.

Ultrasound of the Gruberi Bursa With Cadaveric and MRI Correlation.

OBJECTIVE: The purpose of this study was to describe the appearance and ultrasound characteristics of the Gruberi bursa using a cadaveric model and retrospective ultrasound imaging review. MATERIALS AND METHODS: For the cadaveric study, ultrasound of the dorsolateral ankle of a foot-ankle specimen was performed and was followed by injection of latex between the extensor digitorum longus (EDL) tendons and the talus and dissection. For the ultrasound imaging review, the radiology database was searched for ultrasound studies performed from September 15, 2000, through April 1, 2015, to identify subjects with a dorsolateral foot or ankle fluid collection detected on ultrasound. Images were retrospectively reviewed to characterize the location and size of the fluid collection, assess for the number of locules, and evaluate the compressibility of the fluid collection. It was determined whether the ultrasound findings were significantly different from chance: CI and p values were obtained from performing a test for one proportion. RESULTS: Dissection of a cadaveric specimen revealed latex within a well-defined region between the EDL tendons and the dorsolateral talus; this location is consistent with a Gruberi bursa. For the image review, the imaging examinations of 162 subjects (age range, 16-88 years; 31 male subjects and 131 female subjects) were reviewed. On the ultrasound images, a fluid collection with its epicenter between the dorsolateral talus and EDL was found in 93% of ankles. Of the fluid collections identified on ultrasound, 98% were unilocular and 94% were anechoic. Of these fluid collections, 133 were assessed for compressibility, and 89% were compressible. The positive findings for a Gruberi bursa that were different from chance (p < 0.0001) were a fluid collection being located between the EDL tendons and the dorsolateral talus and being unilocular, anechoic, and compressible. CONCLUSION: The Gruberi bursa characteristically is located between the EDL and the talus; on ultrasound, the Gruberi bursa is most commonly unilocular, anechoic, and compressible.

Ultrasound-Guided Fenestration of Tendons About the Hip and Pelvis: Clinical Outcomes.

OBJECTIVES: Percutaneous ultrasound-guided needle fenestration has been used to treat tendinopathy of the elbow, knee, and ankle with promising results. The purpose of this study was to evaluate the clinical outcome of ultrasound-guided fenestration of tendons about the hip and pelvis. METHODS: After Institutional Review Board approval, a retrospective search of imaging reports from January 1, 2005, to June 30, 2011, was completed to identify patients treated with ultrasound-guided tendon fenestration about the hip or pelvis. Subsequent clinic notes were retrospectively reviewed to determine whether the patient showed marked improvement, some improvement, no change, or worsening symptoms. RESULTS: The study group consisted of 22 tendons in 21 patients with an average age of 55.8 years (range, 26.7-77.0 years). The treated tendons included 11 gluteus medius (9 tendinosis and 2 partial tears), 2 gluteus minimus (both tendinosis), 8 hamstring (6 tendinosis and 2 partial tears), and 1 tensor fascia latae (tendinosis). The average interval to clinical follow-up was 70 days (range, 7-813 days). There was marked improvement in 45.5% (10 of 22), some improvement in 36.4% (8 of 22), no change in symptoms in 9.1% (2 of 22), and worsening symptoms in 9.1% (2 of 22). There were no patient variables (age, chronicity of symptoms, sex, tendon, tendinosis versus tear, prior physical therapy, and prior corticosteroid injection) that were significantly different between patients who improved and those who did not. There were no cases of a subsequent tendon tear or infection. CONCLUSIONS: Clinical follow-up after ultrasound-guided fenestration of the gluteus medius, gluteus minimus, proximal hamstring, and tensor fascia latae tendons showed that 82% of patients had improvement in their symptoms.

Variability in computed tomography diameter measurements of solid renal masses.

PURPOSE: To determine whether the frequency of intra-observer measurement discrepancies ≥5 mm for solid renal masses varies by renal mass characteristics and CT contrast phase. MATERIALS AND METHODS: This HIPAA-compliant retrospective study was approved by our IRB. We selected single CT images performed during the nephrographic phase (NP) of renal enhancement in 97 patients, each with a single solid renal mass. Mass location, margin, heterogeneity, and growth pattern were assessed. Six readers measured each mass on two occasions >3 weeks apart. Readers also measured the masses on images in 50 patients who had corticomedullary phase (CMP) images obtained during the same study. Results were assessed using Chi-square/Fisher's exact and Wilcoxon Signed Rank tests, and logistic regression analyses. RESULTS: For NP to NP comparisons, intra-reader measurement differences ≥5 mm were seen for 3.7% (17/463) of masses <4 cm, but increased to 16.8% (20/119) for masses >4 cm (p < 0.0001). Masses with poorly defined margins (15.9% [22/138] vs. 3.4% [15/444] for well-defined margins, p < 0.0001) and heterogeneity (15.3% [22/144], vs. 5.0% [14/282] for minimally heterogeneous, vs. 0.6% [1/156] for homogeneous, p < 0.0001), were more frequently associated with measurement differences ≥5 mm. Differences ≥5 mm were more frequent when only CMP images were utilized (14% [42/299]), or when CMP images were compared with NP images (26% [77/299]). CONCLUSIONS: A ≥5 mm intra-reader variation in measured size of solid renal masses <4 cm is uncommon for NP to NP comparisons. Variation increases when masses are ≥4 cm, poorly defined, or heterogeneous; or when CMP images are utilized.

The peroxisome proliferator activated receptor-γ pioglitazone improves vascular function and decreases disease activity in patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is associated with heightened mortality due to atherosclerotic cardiovascular disease (CVD). Inflammatory pathways in RA negatively affect vascular physiology and promote metabolic disturbances that contribute to CVD. We hypothesized that the peroxisome proliferator activated receptor-γ (PPAR-γ) pioglitazone could promote potent vasculoprotective and anti-inflammatory effects in RA. METHODS AND RESULTS: One hundred forty-three non-diabetic adult RA patients (76.2% female, age 55.2 ± 12.1 [mean ± SD]) on stable RA standard of care treatment were enrolled in a randomized, double-blind placebo controlled crossover trial of 45 mg daily pioglitazone versus placebo, with a 3-month duration/arm and a 2-month washout period. Pulse wave velocity of the aorta (PWV), brachial artery flow mediated dilatation (FMD), nitroglycerin mediated dilatation (NMD), microvascular endothelial function (reactive hyperemia index [RHI]), and circulating biomarkers of inflammation, insulin resistance, and atherosclerosis risk all were quantified. RA disease activity was assessed with the 28-Joint Count Disease Activity Score (DAS-28) C-reactive protein (CRP) and the Short Form (36) Health Survey quality of life questionnaire. When added to standard of care RA treatment, pioglitazone significantly decreased pulse wave velocity (ie, aortic stiffness) (P=0.01), while FMD and RHI remained unchanged when compared to treatment with placebo. Further, pioglitazone significantly reduced RA disease activity (P=0.02) and CRP levels (P=0.001), while improving lipid profiles. The drug was well tolerated. CONCLUSIONS: Addition of pioglitazone to RA standard of care significantly improves aortic elasticity and decreases inflammation and disease activity with minimal safety issues. The clinical implications of these findings remain to be established. CLINICAL TRIAL REGISTRATION URL: ClinicalTrials.gov Unique Identifier: NCT00554853.