A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo

Fast Large-Tip-Angle Multidimensional and Parallel RF Pulse Design in MRI

Large-tip-angle multidimensional radio-frequency (RF) pulse design is a difficult problem, due to the nonlinear response of magnetization to applied RF at large tip-angles. In parallel excitation, multidimensional RF pulse design is further complicated by the possibility for transmit field patterns to change between subjects, requiring pulses to be designed rapidly while a subject lies in the scanner. To accelerate pulse design, we introduce a fast version of the optimal control method for large-tip-angle parallel excitation. The new method is based on a novel approach to analytically linearizing the Bloch equation about a large-tip-angle RF pulse, which results in an approximate linear model for the perturbations created by adding a small-tip-angle pulse to a large-tip-angle pulse. The linear model can be evaluated rapidly using nonuniform fast Fourier transforms, and we apply it iteratively to produce a sequence of pulse updates that improve excitation accuracy. We achieve drastic reductions in design time and memory requirements compared to conventional optimal control, while producing pulses of similar accuracy. The new method can also compensate for nonidealities such as main field inhomogeneties.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86004/1/Fessler12.pd

Ozone-derived Oxysterols Affect Liver X Receptor (LXR) Signaling: A POTENTIAL ROLE FOR LIPID-PROTEIN ADDUCTS

When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -β and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3. Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects

Treatment of pancreatic ductal adenocarcinoma with tumor antigen specific-targeted delivery of paclitaxel loaded PLGA nanoparticles

Abstract Background Pancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target. Methods Poly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles. Results In vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs. Conclusions The study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment

Treatment of pancreatic ductal adenocarcinoma with tumor antigen specific-targeted delivery of paclitaxel loaded PLGA nanoparticles

Abstract Background Pancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target. Methods Poly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles. Results In vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs. Conclusions The study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment

Home Versus Rehabilitation: Factors that Influence Disposition After Minimally Invasive Surgery in Adult Spinal Deformity Surgery

BACKGROUND: Minimally invasive surgery (MIS) correction for adult spinal deformity (ASD) may reduce the need the need for postoperative skilled nursing facility (SNF) or inpatient rehabilitation (IR) placement following surgery. The likelihood of requiring placement in a facility rather than home disposition may be influenced by various factors. In addition, the associations between discharge location and outcomes and complication rates have not been elucidated in these patients. In this study, we aimed to define factors predicting disposition to an SNF/IR and to elucidate the rates of complications occurring in patients sent to home versus to a facility. METHODS: A retrospective review of a multicenter ASD database, which included patients who underwent surgery between 2009 and 2014. Inclusion criteria were age \u3e18 years, MIS as part of index surgery, location of discharge, and at least 1 of the following: pelvic tilt \u3e20°, sagittal vertical axis \u3e5 cm, pelvic incidence-lumbar lordosis mismatch \u3e10, or lumbar scoliosis \u3e20°. Patients with a 2-year follow-up were included. Preoperative demographic and radiographic data, postoperative (\u3c30 \u3eday) complications, and health-related quality of life were analyzed. RESULTS: A total of 182 patients met our inclusion criteria, including 113 who were discharged to home and 69 who were discharged to an SNF/IR. Older patients (\u3e50 years) were more likely to be discharged to an SNF/IR (P = 0.043). Those aged \u3e70 years were 6-fold more likely to go to an SNF/IR. No association was identified between discharge to an SNF/IR and any radiographic parameters except preoperative pelvic tilt (odds ratio [OR], 1.11; P = 0.009). Staged cases were more likely to be discharged to an SNF/IR (OR, 3.24; 95% confidence interval, 1.11-9.46; P = 0.032); otherwise, there was no difference in levels treated, operating time, estimated blood loss, osteotomy, or length of hospital stay. Patients requiring discharge to an SNF/IR had a higher rate of complications (58% vs. 39.8%; P = 0.017), including major complications (19.5% vs. 42%; P = 0.001), perioperative complications (14.2% vs. 31.9%; P = 0.004) and infections (3.5% vs. 13%; P = 0.016). Patients discharged to an SNF/IR had a higher rate of revision (19.5% vs. 33%; P = 0.035). Health-related quality of life measures were similar regardless of disposition. CONCLUSIONS: Older patients and those undergoing staged MIS deformity correction have a higher likelihood of postoperative disposition to an SNF/IR. Complications occurred more commonly in those patients requiring transfer to an SNF/IR after hospitalization

Race/ethnicity-stratified fine-mapping of the MHC locus reveals genetic variants associated with late-onset asthma.

Peer reviewed: TrueAcknowledgements: We would like to thank PEGS participants for their contributions. We would like to thank Hannah Collins Cakar for assistance with manuscript preparation.Introduction: Asthma is a chronic disease of the airways that impairs normal breathing. The etiology of asthma is complex and involves multiple factors, including the environment and genetics, especially the distinct genetic architecture associated with ancestry. Compared to early-onset asthma, little is known about genetic predisposition to late-onset asthma. We investigated the race/ethnicity-specific relationship among genetic variants within the major histocompatibility complex (MHC) region and late-onset asthma in a North Carolina-based multiracial cohort of adults. Methods: We stratified all analyses by self-reported race (i.e., White and Black) and adjusted all regression models for age, sex, and ancestry. We conducted association tests within the MHC region and performed fine-mapping analyses conditioned on the race/ethnicity-specific lead variant using whole-genome sequencing (WGS) data. We applied computational methods to infer human leukocyte antigen (HLA) alleles and residues at amino acid positions. We replicated findings in the UK Biobank. Results: The lead signals, rs9265901 on the 5' end of HLA-B, rs55888430 on HLA-DOB, and rs117953947 on HCG17, were significantly associated with late-onset asthma in all, White, and Black participants, respectively (OR = 1.73, 95%CI: 1.31 to 2.14, p = 3.62 × 10-5; OR = 3.05, 95%CI: 1.86 to 4.98, p = 8.85 × 10-6; OR = 19.5, 95%CI: 4.37 to 87.2, p = 9.97 × 10-5, respectively). For the HLA analysis, HLA-B*40:02 and HLA-DRB1*04:05, HLA-B*40:02, HLA-C*04:01, and HLA-DRB1*04:05, and HLA-DRB1*03:01 and HLA-DQB1 were significantly associated with late-onset asthma in all, White, and Black participants. Conclusion: Multiple genetic variants within the MHC region were significantly associated with late-onset asthma, and the associations were significantly different by race/ethnicity group

Impact of case type, length of stay, institution type, and comorbidities on Medicare diagnosis-related group reimbursement for adult spinal deformity surgery

OBJECTIVE The aim of this study was to educate medical professionals about potential financial impacts of improper diagnosis-related group (DRG) coding in adult spinal deformity (ASD) surgery. METHODS Medicare's Inpatient Prospective Payment System PC Pricer database was used to collect 2015 reimbursement data for ASD procedures from 12 hospitals. Case type, hospital type/location, number of operative levels, proper coding, length of stay, and complications/comorbidities (CCs) were analyzed for effects on reimbursement. DRGs were used to categorize cases into 3 types: 1) anterior or posterior only fusion, 2) anterior fusion with posterior percutaneous fixation with no dorsal fusion, and 3) combined anterior and posterior fixation and fusion. RESULTS Pooling institutions, cases were reimbursed the same for single-level and multilevel ASD surgery. Longer stay, from 3 to 8 days, resulted in an additional $1400 per stay. Posterior fusion was an additional$6588, while CCs increased reimbursement by approximately $13,000. Academic institutions received higher reimbursement than private institutions, i.e., approximately$14,000 (Case Types 1 and 2) and approximately $16,000 (Case Type 3). Urban institutions received higher reimbursement than suburban institutions, i.e., approximately$3000 (Case Types 1 and 2) and approximately $3500 (Case Type 3). Longer stay, from 3 to 8 days, increased reimbursement between$208 and $494 for private institutions and between$1397 and $1879 for academic institutions per stay. CONCLUSIONS Reimbursement is based on many factors not controlled by surgeons or hospitals, but proper DRG coding can significantly impact the financial health of hospitals and availability of quality patient care