High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

The Fc glycosylation of therapeutic antibodies is crucial for their effector functions and their behavior in pharmacokinetics and pharmacodynamics. To monitor the Fc glycosylation in bioprocess development and characterization, high-throughput techniques for glycosylation analysis are needed. Here, we describe the development of a largely automated high-throughput glycosylation profiling method with multiplexing capillary-gel-electrophoresis (CGE) with laser induced fluorescence (LIF) detection using a DNA analyzer. After PNGaseF digestion, the released glycans were labeled with 9-aminopyrene-1,3,6-trisulfonic acid (APTS) in 96-well plates, which was followed by the simultaneous analysis of up to 48 samples. The peak assignment was conducted by HILIC-UPLC-MS/MS of the APTS-labeled glycans combined with peak fractionation and subsequent CGE-LIF analysis of the MS-characterized fractions. Quantitative data evaluation of the various IgG glycans was performed automatically using an in-house developed software solution. The excellent method accuracy and repeatability of the test system was verified by comparison with two UPLC-based methods for glycan analysis. Finally, the practical value of the developed method was demonstrated by analyzing the antibody glycosylation profiles from fermentation broths after small scale protein A purification.     

NMR structure of the Vibrio vulnificus ribosomal protein S1 domains D3 and D4 provides insights into molecular recognition of single-stranded RNAs

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.     

Stresses affect inbreeding depression in complex ways: disentangling stress-specific genetic effects from effects of initial size in plants

The magnitude of inbreeding depression (ID) varies unpredictably among environments. ID often increases in stressful environments suggesting that these expose more deleterious alleles to selection or increase their effects. More simply, ID could increase under conditions that amplify phenotypic variation (CV²), e.g., by accentuating size hierarchies among plants. These mechanisms are difficult to distinguish when stress increases both ID and phenotypic variation. We grew in- and outbred progeny of Mimulus guttatus under six abiotic stress treatments (control, waterlogging, drought, nutrient deficiency, copper addition, and clipping) with and without competition by the grass Poa palustris. ID differed greatly among stress treatments with δ varying from 7% (control) to 61% (waterlogging) but did not consistently increase with stress intensity. Poa competition increased ID under nutrient deficiency but not other stresses. Analyzing effects of initial size on performance of outbred plants suggests that under some conditions (low N, clipping) competition increased ID by amplifying initial size differences. In other cases (e.g., high ID under waterlogging), particular environments amplified the deleterious genetic effects of inbreeding suggesting differential gene expression. Interestingly, conditions that increased the phenotypic variability of inbred progeny regularly increased ID whereas variability among outbred progeny showed no relationship to ID. Our study reconciles the stress- and phenotypic variability hypotheses by demonstrating how specific conditions (rather than stress per se) act to increase ID. Analyzing CV² separately in inbred and outbred progeny while including effects of initial plant size improve our ability to predict how ID and gene expression vary across environments.Subject terms: Genetic variation, Evolutionary ecology, Plant ecology     

Determinants of elevated carbohydrate antigen 125 in patients with severe symptomatic aortic valve stenosis referred for transcatheter aortic valve implantation

Purpose: Elevated carbohydrate antigen 125 (CA125) predicts adverse outcome after transcatheter aortic valve implantation (TAVI). While known underlying pathophysiological mechanisms of elevated CA125 include serosal effusions and inflammatory stimuli, clinical determinants associated with elevated CA125 in patients referred for TAVI remain unknown. Therefore, we investigated clinical, laboratory and echocardiographic determinants of elevated CA125 in patients with severe aortic valve stenosis referred for TAVI.

Methods: This study includes 650 patients with severe aortic stenosis referred for TAVI. Baseline CA125 was determined by an immunoassay and dichotomized (elevated versus normal) based on the manufacturer cutoff value (>35?U/mL).

Results: CA125 elevation was present in 28% (181/650). Patients with elevated CA125 had an overall worse clinical profile and were more symptomatic with a higher rate of NYHA class III/IV (80% versus 58%; p?Conclusion: Elevated CA125 levels in patients referred for TAVI summarize a subset of patients with an overall worse clinical profile who are more symptomatic.     

Absence of PKC-Alpha Attenuates Lithium-Induced Nephrogenic Diabetes Insipidus

Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI) in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1) and water channel (AQP2) expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO) and strain-matched wild type (WT) controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy.     

LPS-Stimulated Human Skin-Derived Stem Cells Enhance Neo-Vascularization during Dermal Regeneration

High numbers of adult stem cells are still required to improve the formation of new vessels in scaffolds to accelerate dermal regeneration. Recent data indicate a benefit for vascularization capacity by stimulating stem cells with lipopolysaccharide (LPS). In this study, stem cells derived from human skin (SDSC) were activated with LPS and seeded in a commercially available dermal substitute to examine vascularization in vivo. Besides, in vitro assays were performed to evaluate angiogenic factor release and tube formation ability. Results showed that LPS-activated SDSC significantly enhanced vascularization of the scaffolds, compared to unstimulated stem cells in vivo. Further, in vitro assays confirmed higher secretion rates of proangiogenic as well as proinflammatoric factors in the presence of LPS-activated SDSC. Our results suggest that combining activated stem cells and a dermal substitute is a promising option to enhance vascularization in scaffold-mediated dermal regeneration.