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Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2–7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP–sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

Spine surgery outcomes assessment currently relies on patient-reported outcome measures, which satisfy established reliability and validity criteria, but are limited by the inherently subjective and discrete nature of data collection. Physical activity measured from smartphones offers a new data source to assess postoperative functional outcomes in a more objective and continuous manner.To present a methodology to characterize preoperative mobility and gauge the impact of surgical intervention using objective activity data garnered from smartphone-based accelerometers.Smartphone mobility data from 14 patients who underwent elective lumbar decompressive surgery were obtained. A time series analysis was conducted on the number of steps per day across a 2-year perioperative period. Five distinct clinical stages were identified using a data-driven approach and were validated with clinical documentation.Preoperative presentation was correctly classified as either a chronic or acute mobility decline in 92% of patients, with a mean onset of acute decline of 11.8 ± 2.9 weeks before surgery. Postoperative recovery duration demonstrated wide variability, ranging from 5.6 to 29.4 weeks (mean: 20.6 ± 4.9 weeks). Seventy-nine percentage of patients ultimately achieved a full recovery, associated with an 80% ± 33% improvement in daily steps compared with each patient's preoperative baseline (P = .002). Two patients subsequently experienced a secondary decline in mobility, which was consistent with clinical history.The perioperative clinical course of patients undergoing spine surgery was systematically classified using smartphone-based mobility data. Our findings highlight the potential utility of such data in a novel quantitative and longitudinal surgical outcome measure.

PDF file - 114K, Supplemental Figure 5 Cellular distribution of HIF-2alpha under normal oxygen conditions. (a) Representative confocal microscopy images (magnification 400x) showing HIF-2α or Nox4 immunofluorescense, DAPI nuclear staining, or merged images. b) Control (NS) or Nox4 shRNA (KD) cells were imaged following exposure to TEMPOL (0.25 mM), DTT (1mM). c) 786-0 NS cells imaged following transduction with Ad-GFP or Ad-MnSOD. d) 786-0 cells expressing pcDNA-Nox4 imaged with or without treatment with TEMPOL (0.25 mM).

PDF file - 376K, Supplemental Figure 2. Characterization of 786-0 cells stably transfected with the human, full-length Nox4 cDNA. (a) Morphology of 786-0 cells following exogenous expression of Nox4 reveals smaller, denser, more-rounded appearance relative to parental 786-0 cells. (b) Cell viability assay. Equal numbers of untransfected cells or 786-0 Nox4 cells were plated in 96-well plates. After attachment, assay reagent was added, incubated at 37{degree sign}C, and the fluorescent signal read at 590 nm.

PDF file - 224K, Supplemental Figure 1. Quantitative RT-PCR (a) and semi-quantitative RT-PCR (b) for detection of Nox1 in 786-0, RCC4 or LNCaP prostate cancer cells using two published primer pairs(14, 17),. Error bar represents +/- SE.

PDF file - 183K, Supplemental Figure 4. Cytotoxicity was measured by CellTiter-Blue (Promega, Madison, WI). 786-0 NS cells were plated at 1 x 104 cells per well in 96-well plates. After 24 hours, dithiothreitol was added as indicated in 5 replicate wells. 20 �l of the CellTiter-Blue reagent was added to each well and viability quantitated by spectrofluorometer (Spectra Max Plus384, Molecular Devices) and expressed as percentage of viability of media-only controls. Error bars indicate +/- S.E. mean.

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. Cancer Res; 74(13); 3501–11. ©2014 AACR.

PDF file - 98K, Supplemental Figure 3. Lucigenin chemiluminescence assay for superoxide detection performed as described in materials and methods on a) isolated membrane fractions from parental 786-0, RCC4 and Caki-1 cells or isolated cell fractions from 786-0 (b) or RCC4 (c) cells. RLU: relative light units.

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PDF file - 446K, Supplemental Figure 6 Immunohistochemistry for Nox4 and HIF-2alpha in 786-0 NS and KD xenograft explants confirms re-expression of Nox4 in KD tumors with corresponding elevated HIF-2alpha expression. Magnification 200X.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knock-downs sensitize P67L to VX-445 and further enhance the correction of this variant. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.

Purine nucleoside phosphorylase (PNP) gene transfer represents a promising approach to treatment of head and neck malignancies. We tested recombinant adenovirus already in phase I/II clinical testing and leading-edge patient-derived xenografts (PDX) as a means to optimize this therapeutic strategy.Our experiments investigated purine base cytotoxicity, PNP enzyme activity following treatment of malignant tissue, tumor mass regression, viral receptor studies, and transduction by tropism-modified adenovirus.Replication deficient vector efficiently transduced PDX cells and mediated significant anticancer effect following treatment with fludarabine phosphate in vivo. Either 6-methylpurine or 2-fluoroadenine (toxic molecules generated by the PNP approach) ablated head and neck cancer cell proliferation. High levels of adenovirus-3 specific receptors were detected in human tumor models, and vector was evaluated that utilizes this pathway.Our studies provide the scientific foundation necessary to improve PNP prodrug cleavage and advance a new treatment for head and neck cancer.

Background Boswellic acid (BA), a phytoconstituent obtained from Boswellia serrata, suffers from several limitations after oral administration such as poor systemic absorption, high first-pass metabolism and high frequency of dose requirement, which creates a need to develop an alternative route for drug administration via novel drug delivery formulation. The present research work aims at developing ultradeformable vesicular carriers (transferosomes) for transdermal delivery of boswellic acid to effectively deliver the drug into deeper layers of the skin reaching the target site and thus improving its systemic bioavailability. Ultradeformable vesicles were prepared by thin-film hydration technique, and the formulation was optimized using 32 full factorial design where the amount of lecithin (mg) and concentration of surfactant (%) were considered as independent variables. The formulated boswellic acid-loaded vesicles were incorporated into transdermal film via solvent evaporation technique using the blend of polymers such as starch and HPMC K4M. Results The BA-loaded transferosomes were optimized based on vesicle size (nm) and drug entrapment efficiency (%EE), and the results were found to be 205.4 ± 1.215 nm and 86.39 ± 0.019%, respectively. Transmission electron microscopy (TEM) of optimized batch showed spherical shape of vesicles with identified lamellarity, surface charge of vesicles with high negative value − 15.2 mV that suggests electrostatic repulsion between vesicles, while the formulation showed good deformability index of 11.31 ± 0.032% due to use of Tween 80 as surfactant. In vitro permeation study demonstrated sustained release pattern of 96.53 ± 0.023% up to 24 h. Also, the in vitro drug diffusion study was carried out for transfersomal transdermal film which exhibited enhanced permeation and sustained retention of drug up to 94.71 ± 0.019% for 24 h. Conclusion Accordingly, the research work suggested that the transferosomes provided an efficient nanosized carriers for enhanced permeation of boswellic acid into deeper layers of skin and could successfully exhibit its therapeutic effect.

The aim is to develop and evaluate rizatriptan benzoate (RB) loaded nanostructured lipid carriers (NLCs) that might improve oral bioavailability. NLC was prepared using High-Speed Homogenization (HSH) coupled with the ultrasonication method. Process parameters were selected on basis of the trial-and-error method which is time for homogenization, speed of homogenization, and time for sonication. Using a Design expert, 32 Full Factorial design was prepared using solid lipid to liquid lipid ratio and concentration of surfactant as independent variables. RB-NLC was evaluated on basis of particle size, %Entrapment Efficiency (EE), Zeta potential, Transmission electron microscopy (TEM), In-Vitro permeation study, and Ex-vivo permeation study. Confocal Laser Scanning Microscopy was performed to study the uptake of RB-NLC through M-Cells, and Peyer's Patches. RB-NLC dispersion was converted into free-flowing lyophilized powder using cryoprotectant which is trehalose and in vitro permeation study was performed. Among all the batches of RB-NLC, the Optimized batch was evaluated. The optimized formulation showed a particle size of 412.8 ± 1.15nm with 0.261 PDI and − 36.6mV zeta potential. It showed 84.26 ± 1.03% EE. TEM image showed a spherical shape and smooth surface area. In-Vitro and Ex-vivo studies showed 97.21 ± 0.36%CDR up to 22 hours & 61.02 ± 1.25%CDR up to 6 hours. Lyophilized powder showed 96.37 ± 0.36%CDR up to 22 hours. Based on the data we can conclude that RB-NLC increases the permeation and it may increase its Bioavailability (BA). Lyophilized powder of RB-NLC showed a sustained release pattern and it may reduce the dosing frequency of the drug.

Although prescription opioid abuse is a serious concern, patients are commonly prescribed opioids for postoperative pain management. This study sought to evaluate the efficacy of low-dose steroids to reduce postoperative pain and opioid use after lumbar laminectomy and/or discectomy. In the 10-day period postdischarge, patients prescribed a low-dose steroid reported decreased pain scores compared with those who were not prescribed a low-dose steroid; however, there was no associated significant difference in opioid consumption. Outcomes data suggest that postoperative oral steroid may reduce postoperative pain but does not necessarily decrease short-term opioid use after lumbar laminectomy and/or discectomy.

PURPOSE During magnetic resonance-guided focused ultrasound (MRgFUS) thalamotomy for refractory tremor, high temperatures must be achieved and sustained for tissue necrosis. We assessed the impact of both patient-specific as well as procedure-related factors on the efficiency of acoustic energy transfer, or heating efficiency (HE). METHODS Retrospective analysis of 92 consecutive patients (857 sonications) with essential tremor or tremor-dominant Parkinson's disease treated at a single institution. Temperature elevations at the target were measured for each sonication with MR thermometry. HE of each sonication was defined as the ratio of peak temperature elevation and the delivered energy. HE was analyzed with respect to patient skull features (area, thickness, skull density ratio [SDR]), computed from CT scans, as well as demographic and clinical variables (age, sex, diagnosis, and duration of symptoms). RESULTS Across the full range of sonication energies that can be delivered with current devices (up to 36 kJ), average sonication HE was diminished in patients with lower SDR. In individual subjects, there was a progressive loss in HE as sonication energy was titrated up throughout the course of treatment, with a more rapid decline in patients with higher SDR. This energy-dependent loss in HE was not related to procedural factors, namely, the number of previous sonications, or the cumulative energy deposited during previous sonications. In contrast to SDR, neither skull area nor thickness was an independent predictor of average HE or the rate of its decline with increasing energies. In 11% of patients, all of whom with SDR

There is a gap in understanding how to ensure opioid stewardship while managing postoperative neurosurgical pain.To describe self-reported opioid consumption and pain intensity after common neurosurgery procedures gathered using an automated text messaging system.A prospective, observational study was performed at a large, urban academic health system in Pennsylvania. Adult patients (≥ 18 years), who underwent surgeries between October 2019 and May 2020, were consented. Data on postoperative pain intensity and patient-reported opioid consumption were collected prospectively for 3 months. We analyzed the association between the quantity of opioids prescribed and consumed.A total of 517 patients were enrolled. The median pain intensity at discharge was 5 out of a maximum of pain score of 10 and was highest after thoracolumbar fusion (median: 6, interquartile range [IQR]: 4-7). During the follow-up period, patients were prescribed a median of 40 tablets of 5-mg oxycodone equivalent pills (IQR: 28-40) and reported taking a median of 28 tablet equivalents (IQR: 17-40). Responders who were opioid-naive vs opioid-tolerant took a similar median number of opioid pills postoperatively (28 [IQR: 17-40] vs 27.5 [17.5-40], respectively). There was a statistically significant positive correlation between the quantity of opioids prescribed and used during the 3-month follow-up (Pearson R = 0.85, 95% CI [0.80-0.89], P.001). The correlation was stronger among patients who were discharged to a higher level of care.Using real-time, patient-centered pain assessment and opioid consumption data will allow for the development of evidence-based opioid prescribing guidelines after spinal and nerve surgery.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding “correctors” that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)—the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in cis exhibits positive epistatic effects on some CF disease–causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain–domain interactions that are otherwise perturbed by each individual mutation.

The presence of PAHs in soil and the close proximity of soils to humans may lead to human exposure through various pathways. Therefore, levels of priority sixteen PAHs in soils were measured for human and environmental health risk assessments. No recent study on human and environmental health effects due to PAHs in soils is available for Delhi. The levels of total PAHs in soils ranged between 213–851 μg kg−1 with the mean of 550 ± 55 μg kg−1, which was comparatively lower in rural area. Seven carcinogenic PAHs (7CPAHs) accounted for 28.6% of ∑16PAHs. However, benzo(a)pyrene toxicity equivalency (BaPTE) of 7CPAHs accounted for 98.2% to ∑BaPTE. Lifetime average daily dose (LADD) of PAHs through soil for human adults and children was 9.43 × 10−8 mg kg−1 d−1 and 3.53 × 10−7 mg kg−1 d−1, respectively. The estimated incremental lifetime cancer risk (ILCR) for children (4.80 × 10−5) was comparatively higher than adults (1.29 × 10−5). The index of additive cancer risk (IACR), potential threat to potable groundwater water quality from leaching of carcinogenic PAHs from soil were also estimated and presented. The observed levels of PAHs in soils and their human health risk and environmental health hazard at different locations were assessed using recommended guidelines.

Objective Enhanced recovery after surgery (ERAS) pathways have previously been shown to be feasible and safe in elective spinal procedures. As publications on ERAS pathways have recently emerged in elective neurosurgery, long-term outcomes are limited. We report on our 18-month experience with an ERAS pathway in elective spinal surgery. Methods A historical cohort of 149 consecutive patients was identified as the control group, and 1,141 patients were prospectively enrolled in an ERAS protocol. The primary outcome was the need for opioid use one month postoperation. Secondary outcomes were opioid and nonopioid consumption on postoperative day (POD) 1, opioid use at three and six months postoperation, inpatient pain scores, patient satisfaction scores, postoperative Foley catheter use, mobilization/ambulation on POD0–1, length of stay, complications, and intensive care unit admissions. Results There was significant reduction in use of opioids at one, three, and six months postoperation (38.6% vs 70.5%, P Conclusions ERAS protocols for all elective spine and peripheral nerve procedures are both possible and effective. This standardized approach to patient care decreases opioid usage, eliminates the use of PCAs, mobilizes patients faster, and reduces length of stay.

Background: Diversion of prescription opioids pills is a significant contributor to opioid misuse and the opioid epidemic. The goal of this study was to determine the frequency and quantity of excess opioid pills among patients undergoing spine surgery. Further, we wanted to determine the frequency of appropriate opioid disposal. Methods: This was a prospective cohort study of patients undergoing elective spine surgery within a multi-hospital, academic, urban university health system enrolled in a text-messaging program used to track postoperative opioid disposal. Patients who self-reported discontinuation of opioid use but with leftover pills were contacted via telephone and surveyed on opioid disposal. Results: Of the 291 patients who enrolled in the text-messaging program, 192 (66%) patients reported discontinuing opioids within 3 months of surgery. Although 76 (40%) reported excess opioid pills after cessation of use, only 47 (62%) participated in the telephone survey regarding opioid disposal. The median number of leftover pills among these 47 patients was 5 (5, 15) and 64% had not disposed of their prescription. Conclusion: Among the 47 telephone survey participants, a persistent gap remained in postoperative opioid excess and improper disposal. Future efforts must focus on initiatives to improve opioid disposal rates to reduce the quantity of opioids at risk for diversion and to reduce excess prescribing.

Small molecules that address fundamental defects underlying cystic fibrosis (CF), including modulators such as the approved drugs ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have advanced dramatically over the past few years and are transforming care and prognosis among individuals with this disease. The new treatment strategies are predicated on established scientific insight concerning pathogenesis, and applying "personalized" or "precision" interventions for specific abnormalities of the cystic fibrosis transmembrane conductance regulator (CFTR). Even with the advent of highly effective triple drug combinations-which hold great promise for the majority of patients with CF worldwide-barriers to precision therapy remain. These include refractory CFTR variants (premature truncation codons, splice defects, large indels, severe missense mutations, and others) not addressed by available modulators, and access to leading-edge therapeutic compounds for patients with ultrarare forms of CF. In addition to describing the remarkable progress that has occurred regarding CF precision medicine, this review outlines some of the remaining challenges. The CF experience is emblematic of many conditions for which personalized interventions are actively being sought.

Treatment with fludarabine phosphate (9-β-D-arabinofuranosyl-2-F-adenine 5'-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (EcPNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for EcPNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP (TvPNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing TvPNP (D54/TvPNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing EcPNP, although much less enzyme was expressed per cell in the TvPNP transduced condition. As a test of safety and efficacy using TvPNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing TvPNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/TvPNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding TvPNP. In that setting, despite higher F-araA cleavage activity using TvPNP, tumor responses were similar to those obtained with EcPNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that TvPNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.

Elderly patients are a vulnerable patient population in elective spinal surgery. Older patients have more medical comorbidities and are also more sensitive to opiate medications. Despite this, spine and peripheral nerve surgery is still feasible in these patients, and an Enhanced Recovery After Surgery (ERAS) regimen can further enhance the safety profile.This is a before and after cohort study at a single institution on elderly patients who underwent elective spine and peripheral nerve surgery. Patients were prospectively enrolled in a novel ERAS protocol from April 2017 to December 2018. The control group was a historical cohort of patients who underwent surgery from September 2016 to December 2016. The primary outcome was self-reported opioid use at 1- and 3-months postoperatively. The secondary outcome was compliance with the ERAS protocol across several measures including patient-controlled (PCA) use, patient-reported pain scores, mobilization and ambulation status, and Foley catheter use.Among 504 patients aged 65 and older compared to historic controls there was a significant reduction in the use of post-operative opioids at one month (36.2% vs. 71.7%, p 0.001) and 3 months after surgery (33.0% vs. 80.0%, p 0.001). 504 consecutive elderly patients were included in the ERAS protocol compared to a control group of 60. The two groups had similar surgical procedures and baseline demographics, with similar mean ages (ERAS 73.2 years vs. control 73.5 years, p = 0.67). The ERAS group showed improved mobilization and ambulation on POD 0 in compliance with our protocol compared to the control group (mobilization: 60.0% vs. 10.0%, p 0.001; ambulation: 36.1% vs. 10.0%, p 0.001), with no inpatient falls reported for either group.ERAS facilitates reduction in opiate use at 1- and 3-month intervals postoperatively in patients greater than 65 years old undergoing elective spine and peripheral nerve surgery. Early mobilization and ambulation are safe and feasible in this population.

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. Cancer Res; 74(13); 3501–11. ©2014 AACR.