Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides.

Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to RNA transcripts and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization driven, whereas differential expression events are more common and driven by other mechanisms (including spurious experimental variation). We further evaluated the performance of in silico screens for off-target splicing events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate (FDR) of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR of 90%. Despite these large improvements in performance, this approach does not detect the majority of events at an FDR <99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs, and experimental screening by RNA-seq should be the preferred approach.

ATP7B variant c.1934T > G p.Met645Arg causes Wilson disease by promoting exon 6 skipping.

Wilson disease is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3:c.1934T > G (Met645Arg) has been reported as compound heterozygous, and is highly prevalent among Wilson disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading one ClinVar submitter to question its pathogenicity. Here, we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T > G causes ~70% skipping of exon 6. Exon 6 skipping results in frameshift and stop-gain, leading to loss of ATP7B function. The elucidation of the mechanistic effect for this variant resolves any doubt about its pathogenicity and enables the development of genetic medicines for restoring correct splicing.

Allosteric Modulation of Binding Specificity by Alternative Packing of Protein Cores.

Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core composition, illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural analysis and molecular dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core volumes, which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding surface and induced significant conformational changes. These results show that alternative core packing and consequent allosteric modulation of binding interfaces could be used to engineer proteins with novel functions.

Combined immunodeficiency and atopy caused by a dominant negative mutation in caspase activation and recruitment domain family member 11 (CARD11).

BACKGROUND: Combined immunodeficiency (CID) is a T-cell defect frequently presenting with recurrent infections, as well as associated immune dysregulation manifesting as autoimmunity or allergic inflammation. OBJECTIVE: We sought to identify the genetic aberration in 4 related patients with CID, early-onset asthma, eczema, and food allergies, as well as autoimmunity. METHODS: We performed whole-exome sequencing, followed by Sanger confirmation, assessment of the genetic variant effect on cell signaling, and evaluation of the resultant immune function. RESULTS: A heterozygous novel c.C88T 1-bp substitution resulting in amino acid change R30W in caspase activation and recruitment domain family member 11 (CARD11) was identified by using whole-exome sequencing and segregated perfectly to family members with severe atopy only but was not found in healthy subjects. We demonstrate that the R30W mutation results in loss of function while also exerting a dominant negative effect on wild-type CARD11. The CARD11 defect altered the classical nuclear factor κB pathway, resulting in poor in vitro T-cell responses to mitogens and antigens caused by reduced secretion of IFN-γ and IL-2. CONCLUSION: Unlike patients with biallelic mutations in CARD11 causing severe CID, the R30W defect results in a less profound yet prominent susceptibility to infections, as well as multiorgan atopy and autoimmunity.

Data driven flexible backbone protein design.

Protein design remains an important problem in computational structural biology. Current computational protein design methods largely use physics-based methods, which make use of information from a single protein structure. This is despite the fact that multiple structures of many protein folds are now readily available in the PDB. While ensemble protein design methods can use multiple protein structures, they treat each structure independently. Here, we introduce a flexible backbone strategy, FlexiBaL-GP, which learns global protein backbone movements directly from multiple protein structures. FlexiBaL-GP uses the machine learning method of Gaussian Process Latent Variable Models to learn a lower dimensional representation of the protein coordinates that best represent backbone movements. These learned backbone movements are used to explore alternative protein backbones, while engineering a protein within a parallel tempered MCMC framework. Using the human ubiquitin-USP21 complex as a model we demonstrate that our design strategy outperforms current strategies for the interface design task of identifying tight binding ubiquitin variants for USP21.

Delayed diagnosis of celiac stenosis causing hepatic transplant ischaemic necrosis: diagnosis by spectral Doppler findings.

Following presentation with abnormal liver function enzymes, confusion and fatigue, a 65-year-old male with alcoholic cirrhosis underwent spectral Doppler sonography that showed tardus parvus-like morphology in the main and left hepatic arteries, although peak systolic velocities and resistive indices remained normal. The patient's continuing clinical symptoms prompted CT angiography, which demonstrated an unexpected, haemodynamically significant stenosis of the celiac artery. Although the stenosis was successfully stented and the hepatic arterial waveforms normalized, the transplanted liver had already undergone ischaemic necrosis, with resulting failure and the need for retransplantation. Recognition of abnormal waveforms, despite normal peak systolic velocities and resistive indices, with prompt definitive imaging evaluation of the arterial tree beyond just the main hepatic artery, may lead to the diagnosis of unexpected flow-limiting lesions in time to allow revascularization and thus prevent ischaemic transplant failure.

Protein engineering by highly parallel screening of computationally designed variants.

Current combinatorial selection strategies for protein engineering have been successful at generating binders against a range of targets; however, the combinatorial nature of the libraries and their vast undersampling of sequence space inherently limit these methods due to the difficulty in finely controlling protein properties of the engineered region. Meanwhile, great advances in computational protein design that can address these issues have largely been underutilized. We describe an integrated approach that computationally designs thousands of individual protein binders for high-throughput synthesis and selection to engineer high-affinity binders. We show that a computationally designed library enriches for tight-binding variants by many orders of magnitude as compared to conventional randomization strategies. We thus demonstrate the feasibility of our approach in a proof-of-concept study and successfully obtain low-nanomolar binders using in vitro and in vivo selection systems.

Histogram analysis of hepatobiliary phase MR imaging as a quantitative value for liver cirrhosis: preliminary observations.

PURPOSE: To investigate whether histogram analysis of the hepatobiliary phase on gadoxetate enhanced-MRI could be used as a quantitative index for determination of liver cirrhosis. MATERIALS AND METHODS: A total of 63 patients [26 in a normal liver function (NLF) group and 37 in a cirrhotic group] underwent gadoxetate-enhanced MRI, and hepatobiliary phase images were obtained at 20 minutes after contrast injection. The signal intensity of the hepatic parenchyma was measured at four different regions of interest (ROI) of the liver, avoiding vessels and bile ducts. Standard deviation (SD), coefficient of variation (CV), and corrected CV were calculated on the histograms at the ROIs. The distributions of CVs calculated from the ROI histogram were examined and statistical analysis was carried out. RESULTS: The CV value was 0.041±0.009 (mean CV±SD) in the NLF group, while that of cirrhotic group was 0.071±0.020. There were statistically significant differences in the CVs and corrected CV values between the NLF and cirrhotic groups (p<0.001). The most accurate cut-off value among CVs for distinguishing normal from cirrhotic group was 0.052 (sensitivity 83.8% and specificity 88.5%). There was no statistically significant differences in SD between NLF and cirrhotic groups (p=0.307). CONCLUSION: The CV of histograms of the hepatobiliary phase on gadoxetate-enhanced MRI may be useful as a quantitative value for determining the presence of liver cirrhosis.

Indeterminate observations (liver imaging reporting and data system category 3) on MRI in the cirrhotic liver: fate and clinical implications.

OBJECTIVE: The purpose of this article is to retrospectively evaluate the imaging characteristics and outcomes of indeterminate observations (Liver Imaging Reporting and Data System category 3) detected on MRI in patients with cirrhosis. MATERIALS AND METHODS: Sixty-nine indeterminate observations in 52 patients with cirrhosis were imaged with hepatobiliary contrast agent-enhanced MRI. Observations were evaluated retrospectively in terms of the location, size, enhancement pattern, and follow-up results. Each observation was categorized as stable or progressed observations according to serial follow-up MRI. RESULTS: The mean follow-up interval was 11.2 months. Forty-six (67%) of the total observations showed arterial enhancement, and 23 (33%) observations showed isointense signal or low signal intensity on arterial phase. The indeterminate observations were classified as arterial enhancement with fade-out appearance (41 observations [59%]), arterial enhancement with washout appearance (five observations [7%]), and nonhyperenhancing observations (23 observations [33%]). Two of 69 observations (3%) were hyperintense on T2-weighted images, and four of 55 observations (7%) were hyperintense on hepatocellular phase. On the final follow-up MRI examinations, four (6%) observations proved to be probable or definite hepatocellular carcinoma, 55 (80%) remained stable, and 10 (14%) decreased in size or were no longer visible. CONCLUSION: The most common cause of indeterminate observations on MRI is hypervascular pseudolesions that were clinically stable on follow-up imaging.

Readmission after colorectal surgery is related to preoperative clinical conditions and major complications.

BACKGROUND: Hospital readmission is increasingly perceived as a marker of quality and is poorly investigated in patients receiving colorectal surgery. OBJECTIVE: The objective of this study was to describe patterns and etiology of readmission, to determine the rate of readmission, and to identify risk factors for readmission after colorectal surgery. DESIGN: This study is a retrospective medical chart review. Significant (p < 0.1) preoperative and perioperative factors associated with readmission on univariate analysis were examined in a multivariable model. SETTING: The investigation was conducted in a tertiary care hospital. PATIENTS: Patients included adults undergoing major colorectal operations by colorectal surgeons at the University of Minnesota in 2008-2009. MAIN OUTCOME MEASURES: The primary outcome measure was hospital readmission at 60 days. RESULTS: The study included 220 patients. Common surgical indications were inflammatory bowel disease (21%), colorectal cancer (39%), and diverticular disease (13%), and 11% were emergencies. Readmissions at 60 days occurred in 25% (n = 54), mostly because of major complications (57%), nonspecific nausea, vomiting and/or pain (18%), dehydration (11%), and wound infections (11%). Predictors of readmission in multivariable analysis were major complications (OR, 13.0), female sex (OR, 5.9), prednisone use (OR, 4.3), BMI ≥30 (OR, 2.6), and preoperative weight loss (OR, 3.4). Age and comorbidity (Charlson score) were not predictors. LIMITATIONS: This was a retrospective study at a single institution, with a small sample size. CONCLUSIONS: Predictors of readmission were major complications and immediate preoperative condition of the patients. Comorbidity profiling does not capture readmission risk. Because most readmissions relate to complications, further efforts to prevent these will improve readmission rates.

c-MET and HGF mRNA expression in hepatocellular carcinoma: correlation with clinicopathological features and survival.

BACKGROUND/AIM: Data on the clinicopathological features and prognostic impact of c-N-Methyl-N'-nitro-N-nitroso-guanidine HOS Transforming gene (c-MET) and hepatocyte growth factor (HGF) in hepatocellular carcinoma (HCC) are inconsistent. We assessed c-MET and HGF expression in 49 patients with early-stage HCC and correlated the results with disease characteristics and survival. MATERIALS AND METHODS: Expression of c-MET and HGF mRNA in tumor (T) and non-tumor (NT) tissues was assessed. Results were correlated with patient characteristics and overall and recurrence-free survival. RESULTS: Median relative tumor c-MET and HGF expressions were 3.23 (T/NT ratio 6.46) and 9.07 (T/NT ratio 0.77), respectively. c-MET and HGF were overexpressed in early-stage disease with favorable characteristics although there was no association with survival. CONCLUSION: Contrary to other studies, in our series increased tumor c-MET and HGF expressions were associated with favorable disease attributes but not with survival. The prognostic and therapeutic applications of this knowledge to HCC are under active investigation.

Distinct types of disorder in the human proteome: functional implications for alternative splicing.

Intrinsically disordered regions have been associated with various cellular processes and are implicated in several human diseases, but their exact roles remain unclear. We previously defined two classes of conserved disordered regions in budding yeast, referred to as "flexible" and "constrained" conserved disorder. In flexible disorder, the property of disorder has been positionally conserved during evolution, whereas in constrained disorder, both the amino acid sequence and the property of disorder have been conserved. Here, we show that flexible and constrained disorder are widespread in the human proteome, and are particularly common in proteins with regulatory functions. Both classes of disordered sequences are highly enriched in regions of proteins that undergo tissue-specific (TS) alternative splicing (AS), but not in regions of proteins that undergo general (i.e., not tissue-regulated) AS. Flexible disorder is more highly enriched in TS alternative exons, whereas constrained disorder is more highly enriched in exons that flank TS alternative exons. These latter regions are also significantly more enriched in potential phosphosites and other short linear motifs associated with cell signaling. We further show that cancer driver mutations are significantly enriched in regions of proteins associated with TS and general AS. Collectively, our results point to distinct roles for TS alternative exons and flanking exons in the dynamic regulation of protein interaction networks in response to signaling activity, and they further suggest that alternatively spliced regions of proteins are often functionally altered by mutations responsible for cancer.

Nonalcoholic fatty liver disease: MR imaging of liver proton density fat fraction to assess hepatic steatosis.

PURPOSE: To evaluate the diagnostic performance of magnetic resonance (MR) imaging-estimated proton density fat fraction (PDFF) for assessing hepatic steatosis in nonalcoholic fatty liver disease (NAFLD) by using centrally scored histopathologic validation as the reference standard. MATERIALS AND METHODS: This prospectively designed, cross-sectional, internal review board-approved, HIPAA-compliant study was conducted in 77 patients who had NAFLD and liver biopsy. MR imaging-PDFF was estimated from magnitude-based low flip angle multiecho gradient-recalled echo images after T2* correction and multifrequency fat modeling. Histopathologic scoring was obtained by consensus of the Nonalcoholic Steatohepatitis (NASH) Clinical Research Network Pathology Committee. Spearman correlation, additivity and variance stabilization for regression for exploring the effect of a number of potential confounders, and receiver operating characteristic analyses were performed. RESULTS: Liver MR imaging-PDFF was systematically higher, with higher histologic steatosis grade (P < .001), and was significantly correlated with histologic steatosis grade (ρ = 0.69, P < .001). The correlation was not confounded by age, sex, lobular inflammation, hepatocellular ballooning, NASH diagnosis, fibrosis, or magnetic field strength (P = .65). Area under the receiver operating characteristic curves was 0.989 (95% confidence interval: 0.968, 1.000) for distinguishing patients with steatosis grade 0 (n = 5) from those with grade 1 or higher (n = 72), 0.825 (95% confidence interval: 0.734, 0.915) to distinguish those with grade 1 or lower (n = 31) from those with grade 2 or higher (n = 46), and 0.893 (95% confidence interval: 0.809, 0.977) to distinguish those with grade 2 or lower (n = 58) from those with grade 3 (n = 19). CONCLUSION: MR imaging-PDFF showed promise for assessment of hepatic steatosis grade in patients with NAFLD. For validation, further studies with larger sample sizes are needed.

Computational analysis of interactomes: current and future perspectives for bioinformatics approaches to model the host-pathogen interaction space.

Bacterial and viral pathogens affect their eukaryotic host partly by interacting with proteins of the host cell. Hence, to investigate infection from a systems' perspective we need to construct complete and accurate host-pathogen protein-protein interaction networks. Because of the paucity of available data and the cost associated with experimental approaches, any construction and analysis of such a network in the near future has to rely on computational predictions. Specifically, this challenge consists of a number of sub-problems: First, prediction of possible pathogen interactors (e.g. effector proteins) is necessary for bacteria and protozoa. Second, the prospective host binding partners have to be determined and finally, the impact on the host cell analyzed. This review gives an overview of current bioinformatics approaches to obtain and understand host-pathogen interactions. As an application example of the methods covered, we predict host-pathogen interactions of Salmonella and discuss the value of these predictions as a prospective for further research.

Network evolution: rewiring and signatures of conservation in signaling.

The analysis of network evolution has been hampered by limited availability of protein interaction data for different organisms. In this study, we investigate evolutionary mechanisms in Src Homology 3 (SH3) domain and kinase interaction networks using high-resolution specificity profiles. We constructed and examined networks for 23 fungal species ranging from Saccharomyces cerevisiae to Schizosaccharomyces pombe. We quantify rates of different rewiring mechanisms and show that interaction change through binding site evolution is faster than through gene gain or loss. We found that SH3 interactions evolve swiftly, at rates similar to those found in phosphoregulation evolution. Importantly, we show that interaction changes are sufficiently rapid to exhibit saturation phenomena at the observed timescales. Finally, focusing on the SH3 interaction network, we observe extensive clustering of binding sites on target proteins by SH3 domains and a strong correlation between the number of domains that bind a target protein (target in-degree) and interaction conservation. The relationship between in-degree and interaction conservation is driven by two different effects, namely the number of clusters that correspond to interaction interfaces and the number of domains that bind to each cluster leads to sequence specific conservation, which in turn results in interaction conservation. In summary, we uncover several network evolution mechanisms likely to generalize across peptide recognition modules.

Sensor-driven position-adaptive spinal cord stimulation for chronic pain.

BACKGROUND: Variation in the intensity of neurostimulation due to body position is a practical problem for many patients implanted with spinal cord stimulation (SCS) systems because positional changes may result in overstimulation or understimulation that leads to frequent need for compensatory manual programming adjustments. OBJECTIVES: The purpose of this study was to assess the safety and effectiveness of a novel type of SCS therapy designed to automatically adapt stimulation amplitude in response to changes in a patient's position or activity.The primary objective of the study was to demonstrate that automatic position adaptive SCS benefited patients in terms of pain relief and/or convenience compared with neurostimulation adjusted with conventional manual programming. Secondary objectives included assessment of worsened pain relief with automatic adjustment; change in pain score; and the number of manual programming adjustments with position-adaptive neurostimulation compared with manual programming. STUDY DESIGN: Prospective, multicenter, open-label, randomized crossover study. SETTING: Ten interventional pain management centers in the US. METHODS: Patients were enrolled a minimum of one week after a successful SCS screening trial. They were then implanted with the RestoreSensor neurostimulation device (Medtronic, Inc., Minneapolis, MN) that could be programmed to either automatic position-adaptive stimulation (AdaptiveStim) or manual adjustment of stimulation parameters. After implant, all devices were programmed to conventional manual adjustment for a 4-week postoperative period. The patients were then randomized to either conventional manual programming adjustment or position-adaptive stimulation with crossover to the opposite treatment arm occurring at 6 weeks after randomization. The patients were followed for another 6 weeks after crossover. This study was conducted under an FDA-approved Investigational Device Exemption (IDE) and approval of the responsible Institutional Review Boards (IRBs) of the study centers. RESULTS: Seventy-nine patients were enrolled in the study. In an intent-to-treat analysis, 86.5% of patients achieved the primary objective of improved pain relief with no loss of convenience or improved convenience with no loss of pain relief using automatic position-adaptive stimulation compared with using conventional manual programming adjustment alone. This was statistically significantly greater than the predefined minimum success rate of 25%, p < 0.001 (exact one-sided 97.5% lower confidence limit was 76.5%). Only 2.8% of patients reported worsened pain relief during position-adaptive stimulation compared with manual programming. There was a statistically significant reduction in the mean numeric pain rating scale score compared with baseline scores in both treatment arms. Additionally, position-adaptive stimulation demonstrated a statistically significant 41% reduction in the daily average number of programming button presses for amplitude adjustment compared with manual programming (18.2 per day versus 30.7 per day, P = 0.002). Functional improvements reported with position-adaptive stimulation included: improved comfort during position changes (80.3%); improved activity (69%); and improved sleep (47.9%). Adverse events associated with uncomfortable sensations from stimulation did not differ significantly between treatment arms. The incidence of device-related serious adverse events was 3.9%. LIMITATIONS: Patients and physicians were not blinded to whether devices were programmed to automatic position-adaptive stimulation or manual adjustment. Responses to assessment questionnaires were based on patient recall. CONCLUSIONS: The study demonstrated that automatic position-adaptive stimulation is safe and effective in providing benefits in terms of patient-reported improved pain relief and convenience compared with using manual programming adjustment alone. CLINICAL TRIAL: NCT01106404.

Autofluorescence imaging of living pancreatic islets reveals fibroblast growth factor-21 (FGF21)-induced metabolism.

Fibroblast growth factor-21 (FGF21) has therapeutic potential for metabolic syndrome due to positive effects on fatty acid metabolism in liver and white adipose tissue. FGF21 also improves pancreatic islet survival in excess palmitate; however, much less is known about FGF21-induced metabolism in this tissue. We first confirmed FGF21-dependent activity in islets by identifying expression of the cognate coreceptor Klothoß, and by measuring a ligand-stimulated decrease in acetyl-CoA carboxylase expression. To further reveal the effect of FGF21 on metabolism, we employed a unique combination of two-photon and confocal autofluorescence imaging of the NAD(P)H and mitochondrial NADH responses while holding living islets stationary in a microfluidic device. These responses were further correlated to mitochondrial membrane potential and insulin secretion. Glucose-stimulated responses were relatively unchanged by FGF21. In contrast, responses to glucose in the presence of palmitate were significantly reduced compared to controls showing diminished NAD(P)H, mitochondrial NADH, mitochondrial membrane potential, and insulin secretion. Consistent with the glucose-stimulated responses being smaller due to continued fatty acid oxidation, mitochondrial membrane potential was increased in FGF21-treated islets by using the fatty acid transport inhibitor etomoxir. Citrate-stimulated NADPH responses were also significantly larger in FGF21-treated islets suggesting preference for citrate cycling rather than acetyl-CoA carboxylase-dependent fatty acid synthesis. Overall, these data show a reduction in palmitate-induced potentiation of glucose-stimulated metabolism and insulin secretion in FGF21-treated islets, and establish the use of autofluorescence imaging and microfluidic devices to investigate cell metabolism in a limited amount of living tissue.