Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates

Immunoconjugates, including antibody-drug conjugates and Fc-conjugates, represent a rapidly growing class of therapeutics undergoing clinical development. Despite their growing popularity, the high intrinsic heterogeneity of immunoconjugates often complicates the development process and limits their widespread application. In particular, immunoconjugate charge variants exhibit markedly different colloidal stabilities, solubilities, pharmacokinetics and tissue distributions. Charge variants arise spontaneously due to degradation and, depending on the type of drug, linker and conjugation site, through drug conjugation. Electrostatic changes in naked antibodies often result in poor performance characteristics, and therefore charge alterations due to degradation are critical to control. Charge properties are expected to be equally important to producing well-behaved ADCs. Charge-based methods of analysis, such as isoelectric focusing and ion exchange chromatography, are capable of probing the underlying complexities within immunoconjugate drug products. Despite the utility of these methods, there are only a few published reports of charge-based assays applied to immunoconjugates. In the present study, we sought to identify the effects of chemical conjugation on the electrostatic properties of Fc-conjugates. In order to minimize the effects of post-translational modifications (e.g. deamidation), a single Fc charge variant was isolated prior to conjugation of a fluorescent probe, Alexa Fluor 350, to the side chains of lysine residues. The resulting Fc-conjugates were assessed by a variety of analytical techniques, including isoelectric focusing and ion exchange chromatography, to determine their charge properties

Rapid transport of muco-inert nanoparticles in cystic fibrosis sputum treated with N -acetyl cysteine

Sputum poses a critical diffusional barrier that strongly limits the efficacy of drug and gene carriers in the airways of individuals with cystic fibrosis (CF). Previous attempts to enhance particle penetration of CF sputum have focused on either reducing its barrier properties via mucolytics, or decreasing particle adhesion to sputum constituents by coating the particle surface with non-mucoadhesive polymers, including polyethylene glycol (PEG). Neither approach has enabled particles to penetrate expectorated sputum at rates previously observed for non-mucoadhesive nanoparticles in human cervicovaginal mucus. Here, we sought to investigate whether a common mucolytic, N-acetyl cysteine (NAC), in combination with dense PEG coatings on particles, can synergistically enhance particle penetration across fresh undiluted CF sputum

Why monitor the neonatal brain-that is the important question

A key goal of neonatal neurocritical care is improved outcomes, and brain monitoring plays an essential role. The recent NEST trial(1) reported no outcome benefits using aEEG monitoring compared to clinical seizure identification among neonates treated for seizures. However, the study failed to prove the effects of monitoring on seizure treatment in the first place.Non peer reviewe

Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research

Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community

Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research

Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community

DNA nanoparticle-mediated thymulin gene therapy prevents airway remodeling in experimental allergic asthma

Thymulin has been shown to present anti-inflammatory and anti-fibrotic properties in experimental lung diseases. We hypothesized that a biologically active thymulin analog gene, methionine serum thymus factor, delivered by highly compacted DNA nanoparticles may prevent lung inflammation and remodeling in a mouse model of allergic asthma. The DNA nanoparticles are composed of a single molecule of plasmid DNA compacted with block copolymers of poly-L-lysine and polyethylene glycol (CK30PEG), which have been found safe in a human phase I/II clinical trial. Thymulin plasmids were detected in the lungs of ovalbumin-challenged asthmatic mice up to 27days after administration of DNA nanoparticles carrying thymulin plasmids. A single dose of DNA nanoparticles carrying thymulin plasmids prevented lung inflammation, collagen deposition and smooth muscle hypertrophy in the lungs of a murine model of ovalbumin-challenged allergic asthma, leading to improved lung mechanics. In the present model of chronic allergic asthma, highly compacted DNA nanoparticles using thymulin analog gene modulated the inflammatory and remodeling processes improving lung mechanics.Instituto de Investigaciones Bioquímicas de La Plat

Biomechanics and anterior cruciate ligament reconstruction

For years, bioengineers and orthopaedic surgeons have applied the principles of mechanics to gain valuable information about the complex function of the anterior cruciate ligament (ACL). The results of these investigations have provided scientific data for surgeons to improve methods of ACL reconstruction and postoperative rehabilitation. This review paper will present specific examples of how the field of biomechanics has impacted the evolution of ACL research. The anatomy and biomechanics of the ACL as well as the discovery of new tools in ACL-related biomechanical study are first introduced. Some important factors affecting the surgical outcome of ACL reconstruction, including graft selection, tunnel placement, initial graft tension, graft fixation, graft tunnel motion and healing, are then discussed. The scientific basis for the new surgical procedure, i.e., anatomic double bundle ACL reconstruction, designed to regain rotatory stability of the knee, is presented. To conclude, the future role of biomechanics in gaining valuable in-vivo data that can further advance the understanding of the ACL and ACL graft function in order to improve the patient outcome following ACL reconstruction is suggested

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men