Genome-scale functional genomics screening highlights genes impacting protein fucosylation in Chinese hamster ovary cells.

N-linked glycosylation is a common post-translational modification that has various effects on multiple types of proteins. The extent to which an N-linked glycoprotein is modified and the identity of glycans species involved is of great interest to the biopharmaceutical industry, since glycosylation can impact the efficacy and safety of therapeutic monoclonal antibodies (mAbs). mAbs lacking core fucose, for example, display enhanced clinical efficacy through increased antibody-dependent cellular cytotoxicity. We performed a genome-wide CRISPR knockout screen in Chinese hamster ovary (CHO) cells, the workhorse cell culture system for industrial production of mAbs, aimed at identifying novel regulators of protein fucosylation. Using a lectin binding assay, we identified 224 gene perturbations that significantly alter protein fucosylation, including well-known glycosylation genes. This functional genomics framework could readily be extended and applied to study the genetic pathways involved in regulation of other glycoforms. We hope this resource will provide useful guidance toward the development of next generation CHO cell lines and mAb therapeutics.

A functional regression analysis of vessel source level measurements from the Enhancing Cetacean Habitat and Observation (ECHO) database.

Measurements of the source levels of 9880 passes of 3188 different large commercial ships from the Enhancing Cetacean Habitat and Observation (ECHO) program database were used to investigate the dependencies of vessel underwater noise emissions on several vessel design parameters and operating conditions. Trends in the dataset were analyzed using functional regression analysis, which is an extension of standard regression analysis and represents a response variable (decidecade band source level) as a continuous function of a predictor variable (frequency). The statistical model was applied to source level data for six vessel categories: cruise ships, container ships, bulk carriers, tankers, tugs, and vehicle carriers. Depending on the frequency band and category, the functional regression model explained approximately 25%-50% of the variance in the ECHO dataset. The two main operational parameters, speed through water and actual draft, were the predictors most strongly correlated with source levels in all of the vessel categories. Vessel size (represented via length overall) was the design parameter with the strongest correlation to underwater radiated noise for three categories of vessels (bulkers, containers, and tankers). Other design parameters that were investigated (engine revolutions per minute, engine power, design speed, and vessel age) had weaker but nonetheless significant correlations with source levels.

PEAPOD repressors modulate and coordinate developmental responses to light intensity in Arabidopsis.

Higher plants adapt to different light intensities by altering hypocotyl elongation, stomatal density, seed size, and flowering time. Despite the importance of this developmental plasticity, knowledge of the underlying genetic and molecular mechanisms modulating and coordinating responses to light intensity remains incomplete. Here, I report that in Arabidopsis the PEAPOD (PPD) repressors PPD1 and PPD2 prevent exaggerated responses to light intensity. Genetic and transcriptome analyses, of a ppd deletion mutant and a PPD1 overexpression genotype, were used to identify how PPD repressors modulate the light signalling network. A ppd1/ppd2 deletion mutant has elongated hypocotyls, elevated stomatal density, enlarged seed, and delayed flowering, whereas overexpression of PPD1 results in the reverse. Transcription of both PPD1 and PPD2, upregulated in low light and downregulated in higher light, is activated by PHYTOCHROME INTERACTING FACTOR 4. I found PPDs modulate light signalling by negative regulation of SUPPRESSOR OF phyA-105 (SPA1) transcription. Whereas PPDs coordinate many of the responses to light intensity - hypocotyl elongation, flowering time, and stomatal density - by repression/de-repression of SPA1, PPD regulation of seed size occurs independent of SPA1. In conclusion PPD repressors modulate and coordinate developmental responses to light intensity by altering light signal transduction.

Genetic and functional evidence links a missense variant in B4GALT1 to lower LDL and fibrinogen.

Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.9 milligrams per deciliter lower LDL-C (P = 4.1 × 10­19) and 29 milligrams per deciliter lower plasma fibrinogen (P = 1.3 × 10­5). B4GALT1 gene­based analysis in 544,955 subjects showed an association with decreased coronary artery disease (odds ratio = 0.64, P = 0.006). The mutant protein had 50% lower galactosyltransferase activity compared with the wild-type protein. N-linked glycan profiling of human serum found serine 352 allele to be associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. B4galt1 353Ser knock-in mice showed decreases in LDL-C and fibrinogen. Our findings suggest that targeted modulation of protein galactosylation may represent a therapeutic approach to decreasing cardiovascular disease.

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.

Multifaceted assessment of rituximab biosimilarity: The impact of glycan microheterogeneity on Fc function.

Biosimilars are poised to reduce prices and increase patient access to expensive, but highly effective biologic products. However, questions still remain about the degree of similarity and scarcity of information on biosimilar products from outside of the US/EU in the public domain. Thus, as an independent entity, we performed a comparative analysis between the innovator, Rituxan® (manufactured by Genentech/Roche), and a Russian rituximab biosimilar, Acellbia® (manufactured by Biocad). We evaluated biosimilarity of these two products by a variety of state-of-the-art analytical mass spectrometry techniques, including tandem MS mapping, HX-MS, IM-MS, and intact MS. Both were found to be generally similar regarding primary and higher order structure, though differences were identified in terms of glycoform distribution levels of C-terminal Lys, N-terminal pyroGlu, charge variants and soluble aggregates. Notably, we confirmed that the biosimilar had a higher level of afucosylated glycans, resulting in a stronger FcγIIIa binding affinity and increased ADCC activity. Taken together, our work provides a comprehensive comparison of Rituxan® and Acellbia®.

Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc.

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc2Man(8+n) [n = 0-4]), Man5 (GlcNAc2Man5), GlcNAc1, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc1 > Man5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39-0.52 µM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.

Return-to-Learn Accommodations and Concussion Perceptions Among Indiana High School Principals.

OBJECTIVE: Care after concussion is important for all patients, although especially critical in children and adolescents because of continued rapid brain growth and maturation. Postconcussion symptoms often lead to impaired school performance. Conflicting data regarding best return-to-learn practices make it difficult for school administrators to develop policies to best accommodate concussed students. We sought to assess high school principals' knowledge of concussion, the personnel responsible for implementing accommodations, and the overall willingness to enact recommended academic accommodations. METHODS: In our cross-sectional study, we surveyed 410 Indiana high school principals (157 responded). Assistant principals were excluded. RESULTS: One-third of the respondents received academic accommodations training for concussed students and more than 80% were somewhat or very comfortable with academic management. Greater than 90% were willing to provide accommodations as long as necessary. However, nearly 40% of responding principals were unlikely or unwilling to implement accommodations for standardized testing. National and state data suggest the median Indiana high school should expect 30 and more concussions per year; more than 90% of principals estimated that fewer than 30 concussions occurred each year at their school. CONCLUSIONS: The underestimation of concussion frequency highlights an opportunity for further education of high school principals to ensure all concussed students receive appropriate return-to-learn accommodations.

Synthesis of a Bifunctional Peptide Inhibitor-IgG1 Fc Fusion That Suppresses Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a neurodegenerative disease that is estimated to affect over 2.3 million people worldwide. The exact cause for this disease is unknown but involves immune system attack and destruction of the myelin protein surrounding the neurons in the central nervous system. One promising class of compounds that selectively prevent the activation of immune cells involved in the pathway leading to myelin destruction are bifunctional peptide inhibitors (BPIs). Treatment with BPIs reduces neurodegenerative symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this work, as an effort to further improve the bioactivity of BPIs, BPI peptides were conjugated to the N- and C-termini of the fragment crystallizable (Fc) region of the human IgG1 antibody. Initially, the two peptides were conjugated to IgG1 Fc using recombinant DNA technology. However, expression in yeast resulted in low yields and one of the peptides being heavily proteolyzed. To circumvent this problem, the poorly expressed peptide was instead produced by solid phase peptide synthesis and conjugated enzymatically using a sortase-mediated ligation. The sortase-mediated method showed near-complete conjugation yield as observed by SDS-PAGE and mass spectrometry in small-scale reactions. This method was scaled up to obtain sufficient quantities for testing the BPI-Fc fusion in mice induced with EAE. Compared to the PBS-treated control, mice treated with the BPI-Fc fusion showed significantly reduced disease symptoms, did not experience weight loss, and showed reduced de-myelination. These results demonstrate that the BPI peptides were highly active at suppressing EAE when conjugated to the large Fc scaffold in this manner.

Developing oxygen-enhanced magnetic resonance imaging as a prognostic biomarker of radiation response.

Oxygen-Enhanced Magnetic Resonance Imaging (OE-MRI) techniques were evaluated as potential non-invasive predictive biomarkers of radiation response. Semi quantitative blood-oxygen level dependent (BOLD) and tissue oxygen level dependent (TOLD) contrast, and quantitative responses of relaxation rates (ΔR1 and ΔR2*) to an oxygen breathing challenge during hypofractionated radiotherapy were applied. OE-MRI was performed on subcutaneous Dunning R3327-AT1 rat prostate tumors (n=25) at 4.7 T prior to each irradiation (2F × 15 Gy) to the gross tumor volume. Response to radiation, while inhaling air or oxygen, was assessed by tumor growth delay measured up to four times the initial irradiated tumor volume (VQT). Radiation-induced hypoxia changes were confirmed using a double hypoxia marker assay. Inhaling oxygen during hypofractionated radiotherapy significantly improved radiation response. A correlation was observed between the difference in the 2nd and 1st ΔR1 (ΔΔR1) and VQT for air breathing rats. The TOLD response before the 2nd fraction showed a moderate correlation with VQT for oxygen breathing rats. The correlations indicate useful prognostic factors to predict tumor response to hypofractionation and could readily be applied for patient stratification and personalized radiotherapy treatment planning.

C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.

The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.

Tumor radio-sensitivity assessment by means of volume data and magnetic resonance indices measured on prostate tumor bearing rats.

PURPOSE: Radiation therapy is one of the most common treatments in the fight against prostate cancer, since it is used to control the tumor (early stages), to slow its progression, and even to control pain (metastasis). Although many factors (e.g., tumor oxygenation) are known to influence treatment efficacy, radiotherapy doses and fractionation schedules are often prescribed according to the principle "one-fits-all," with little personalization. Therefore, the authors aim at predicting the outcome of radiation therapy a priori starting from morphologic and functional information to move a step forward in the treatment customization. METHODS: The authors propose a two-step protocol to predict the effects of radiation therapy on individual basis. First, one macroscopic mathematical model of tumor evolution was trained on tumor volume progression, measured by caliper, of eighteen Dunning R3327-AT1 bearing rats. Nine rats inhaled 100% O2 during irradiation (oxy), while the others were allowed to breathe air. Second, a supervised learning of the weight and biases of two feedforward neural networks was performed to predict the radio-sensitivity (target) from the initial volume and oxygenation-related information (inputs) for each rat group (air and oxygen breathing). To this purpose, four MRI-based indices related to blood and tissue oxygenation were computed, namely, the variation of signal intensity ΔSI in interleaved blood oxygen level dependent and tissue oxygen level dependent (IBT) sequences as well as changes in longitudinal ΔR1 and transverse ΔR2(*) relaxation rates. RESULTS: An inverse correlation of the radio-sensitivity parameter, assessed by the model, was found with respect the ΔR2(*) (-0.65) for the oxy group. A further subdivision according to positive and negative values of ΔR2(*) showed a larger average radio-sensitivity for the oxy rats with ΔR2(*)<0 and a significant difference in the two distributions (p < 0.05). Finally, a leave-one-out procedure yielded a radio-sensitivity error lower than 20% in both neural networks. CONCLUSIONS: While preliminary, these specific results suggest that subjects affected by the same pathology can benefit differently from the same irradiation modalities and support the usefulness of IBT in discriminating between different responses.

Endovascular Interventional Cardiology: 2015 in Review.

Minimally invasive interventional procedures have become an appealing option for patients with endovascular disease historically referred to surgery. This article reviews the major advancements and trials published in 2015.

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis.

Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated high-mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, size exclusion HPLC, and capillary isoelectric focusing confirmed that the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor, FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods using biolayer interferometry, 1 with protein G-immobilized IgG1 Fc and the other with streptavidin-immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The high-mannose IgG1 Fc and Man5-IgG1 Fc glycoforms were highly similar to one another with high affinity for FcγRIIIa, whereas GlcNAc-Fc had weak affinity, and the nonglycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These 4 IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles and then used as a model system to mathematically assess overall biosimilarity, as described in a series of companion articles.

Dynamic oxygen challenge evaluated by NMR T1 and T2*--insights into tumor oxygenation.

There is intense interest in developing non-invasive prognostic biomarkers of tumor response to therapy, particularly with regard to hypoxia. It has been suggested that oxygen sensitive MRI, notably blood oxygen level-dependent (BOLD) and tissue oxygen level-dependent (TOLD) contrast, may provide relevant measurements. This study examined the feasibility of interleaved T2*- and T1-weighted oxygen sensitive MRI, as well as R2* and R1 maps, of rat tumors to assess the relative sensitivity to changes in oxygenation. Investigations used cohorts of Dunning prostate R3327-AT1 and R3327-HI tumors, which are reported to exhibit distinct size-dependent levels of hypoxia and response to hyperoxic gas breathing. Proton MRI R1 and R2* maps were obtained for tumors of anesthetized rats (isoflurane/air) at 4.7 T. Then, interleaved gradient echo T2*- and T1-weighted images were acquired during air breathing and a 10 min challenge with carbogen (95% O2 -5% CO2). Signals were stable during air breathing, and each type of tumor showed a distinct signal response to carbogen. T2* (BOLD) response preceded T1 (TOLD) responses, as expected. Smaller HI tumors (reported to be well oxygenated) showed the largest BOLD and TOLD responses. Larger AT1 tumors (reported to be hypoxic and resist modulation by gas breathing) showed the smallest response. There was a strong correlation between BOLD and TOLD signal responses, but ΔR2* and ΔR1 were only correlated for the HI tumors. The magnitude of BOLD and TOLD signal responses to carbogen breathing reflected expected hypoxic fractions and oxygen dynamics, suggesting potential value of this test as a prognostic biomarker of tumor hypoxia.

Diverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs.

In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a lacZ reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.

Comparison of the usefulness of enoxaparin versus warfarin for prevention of left ventricular mural thrombus after anterior wall acute myocardial infarction.

Left ventricular (LV) thrombus is one of the most common complications in patients with anterior acute myocardial infarction (AMI) and LV dysfunction. Although anticoagulation is frequently prescribed, data regarding the appropriate drug, duration, risks, and effect on echocardiographic indices of thrombus are lacking. Moreover, given the difficulty in obtaining adequate anticoagulation with warfarin, it is possible that short-term treatment with a more predictable agent would be effective. We randomized 60 patients at high risk of developing LV mural thrombus (anterior acute myocardial infarction with Q waves and ejection fraction≤40%) to receive either enoxaparin 1 mg/kg (maximum 100 mg) subcutaneously every 12 hours for 30 days or traditional anticoagulation (intravenous heparin followed by oral warfarin for 3 months). Clinical evaluations and transthoracic echocardiograms were obtained at baseline, in-hospital, and at 3.5 months. There were no differences between the groups regarding baseline demographics, acute echocardiographic findings, and in-hospital outcomes. The length of hospital stay tended to be shorter for the enoxaparin group (4.6 vs 5.6; p=0.066) and the corresponding hospital costs ($25,837 vs $34,666; p=0.18). At 3 months, bleeding and thromboembolic events were rare and similar between enoxaparin and warfarin groups. Although more patients had probable mural thrombus in the enoxaparin group compared with warfarin at 3.5 months (15% vs 4%; p=0.35), this was not significantly different. In conclusion, the use of enoxaparin tends to shorten hospitalization and lower cost of care. However, at 3.5 months, there appears to be numerically higher (but statistically insignificant) rates of LV thrombus in the enoxaparin group.

Mathematical modeling of tumor response to radiation: radio-sensitivity correlation with BOLD, TOLD, ΔR1 and ΔR2* investigated in large Dunning R3327-AT1 rat prostate tumors.

Tumor response to radiation therapy can vary highly across patients. Several factors, both tumor- and environment-specific, can influence the radio-sensitivity, one of the most well-known being hypoxia. In this work, we investigated possible correlations between the radio-sensitivity parameters determined by means of a simple mathematical model of tumor volume evolution, and the MRI-based indicators of oxygenation in Dunning R3327-AT1 rats. Prior to irradiation the rats were subjected to an oxygen-breathing challenge, which was evaluated by MRI. The tumors were administered a single irradiation dose (30 Gy), while breathing air or oxygen. Despite a poor fitting performance, the model was able to identify two different tumor volume regression patterns. Moreover, the radio-sensitivity of the oxygen-breathing group was found to correlate with the variation of the transverse relaxation rate ΔR2* (-0.89). This suggests that MRI-based indices of tumor oxygenation may provide information about radio-sensitivity.

Contact surface motion paths associated with leg length inequality following unilateral total hip replacement.

In the past, there has been little research into leg length inequality (LLI) and its effect on hip arthroplasty bearing longevity. This investigation aimed to determine the effects of post-operative LLI on hip motions during gait and to postulate the subsequent influence on the wear of the artificial hip joint replacement. Motion data from a clinical gait analysis were processed with an in-house computational model to plot graphs showing the movement of loci of 20 points on the femoral head during one gait cycle for two cohorts: 19 LLI patients and 38 normal healthy patients. Loci paths were quantified by calculating the aspect ratio (AR) of the path shape. It was found that on average, LLI patients had a reduction in flexion/extension and abduction/adduction. Furthermore, the AR of LLI patients was found to be 8% smaller than the normal group. The shorter, more multidirectional, motion paths in LLI patients would suggest the potential for greater wear in a polyethylene bearing compared to an asymptomatic, non-LLI patient. The results have potential implications towards preclinical wear testing of joint replacements.