Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation,Membrane Association,and Internalization

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.     

Characterization of AhMITE1 transposition and its association with the mutational and evolutionary origin of botanical types in peanut (Arachis spp.)

AhMITE1 is an active miniature inverted repeat transposable element (MITE) in peanut (Arachis hypogaea L). Its transpositional activity from a particular (FST1-linked) site within the peanut genome was checked using AhMITE1-specifc PCR, which used a forward primer annealing to the 5??-flanking sequence and a reverse primer binding to AhMITE1. It was found that transposition activation was induced by stresses such as ethyl methane sulfonate (EMS), gamma irradiation, environmental conditions, and tissue culture. Excision and insertion of AhMITE1 at this particular site among the mutants led to gross morphological changes resembling alternate subspecies or botanical types. Analysis of South American landraces revealed the presence of AhMITE1 at the site among most of the spp. fastigiata types, whereas the element was predominantly missing from spp. hypogaea types, indicating its strong association. Four accessions of the primitive allotetraploid, A. monticola were devoid of AhMITE1 at the site, indicating only recent activation of the element, possibly because of the ??genomic shock?? resulting from hybridization followed by allopolyploidization.     

Understanding the mechanism of copurification of “difficult to remove” host cell proteins in rituximab biosimilar products

Host cell proteins (HCPs) are considered a critical quality attribute and are linked to safety and efficacy of biotherapeutic products. Researchers have identified 10 HCPs in Chinese hamster ovary (CHO) that exhibit common characteristics of product association, coelution, and age-dependent expression and therefore are “difficult to remove” during downstream purification. These include cathepsin D, clusterin, galectin-3-binding protein, G-protein coupled receptor 56, lipoprotein lipase, metalloproteinase inhibitor, nidogen-1 secreted protein acidic and rich in cysteine (SPARC), sulfated glycoprotein, and insulin-like growth factor-2 RNA-binding protein. While the levels of HCPs in the investigated biosimilars were within the acceptable range of <100 ppm, certain “difficult to remove” HCPs were found in the biosimilar samples. This article aims to elucidate the underlying interactions between these “difficult to remove” HCPs and the mAb product. Surface study of rituximab exhibited unstable discontinuous patches of residues on the protein surface that have high propensity to get buried and lower the solvent exposed area. The higher order structure and the receptor binding were not affected, except for one of the biosimilars, owing to extremely low-HCP levels in its final drug product. Finally, based on the combined experimental and computational data from this study, a probable mechanism of retention for the 10 HCPs is proposed. The results presented here can guide downstream process design or avenues for protein engineering during product discovery to achieve more effective removal of the impurities.     

Integration of evidence across human and model organism studies: A meeting report

The National Institute on Drug Abuse and Joint Institute for Biological Sciences at the Oak Ridge National Laboratory hosted a meeting attended by a diverse group of scientists with expertise in substance use disorders (SUDs), computational biology, and FAIR (Findability, Accessibility, Interoperability, and Reusability) data sharing. The meeting's objective was to discuss and evaluate better strategies to integrate genetic, epigenetic, and 'omics data across human and model organisms to achieve deeper mechanistic insight into SUDs. Specific topics were to (a) evaluate the current state of substance use genetics and genomics research and fundamental gaps, (b) identify opportunities and challenges of integration and sharing across species and data types, (c) identify current tools and resources for integration of genetic, epigenetic, and phenotypic data, (d) discuss steps and impediment related to data integration, and (e) outline future steps to support more effective collaboration—particularly between animal model research communities and human genetics and clinical research teams. This review summarizes key facets of this catalytic discussion with a focus on new opportunities and gaps in resources and knowledge on SUDs.     

Observations on the Varanasi wall flora

Summary This study deals with the ecology of a man-made habitat, i.e., old walls and buildings of Varanasi, India. The wall flora of Varanasi includes 135 species of angiosperms, 1 species of pteridophyte, 4 species of bryophytes and one lichen. Abundant wall plants likeArthraxon lancifolius, Lindenbergia polyantha, Aristida funiculata and lichens are never seen off the walls. The vegetation of the three types of walls, viz., brick mortar, brick mud and mud walls, classified according to the materials of construction, show sharp differences, despite many common features. Analysis of the wall flora, according to Raunkiaer's life-form system, revealed a marked contrast from the adjoining ground flora in having a very high percentage of therophytes.It is concluded, that the distinctiveness of the wall flora is due to the unique topography, microclimate and xeric nature of the wall habitat. Differences in the physico-chemical nature of the three types of walls are responsible for the variation in their vegetation.     

Membrane-Mediated Regulation of the Intrinsically Disordered CD3? Cytoplasmic Tail of the TCR

The regulation of T-cell-mediated immune responses depends on the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on T-cell receptors. Although many details of the signaling cascades are well understood, the initial mechanism and regulation of ITAM phosphorylation remains unknown. We used molecular dynamics simulations to study the influence of different compositions of lipid bilayers on the membrane association of the CD3ϵ cytoplasmic tails of the T-cell receptors. Our results show that binding of CD3ϵ to membranes is modulated by both the presence of negatively charged lipids and the lipid order of the membrane. Free-energy calculations reveal that the protein-membrane interaction is favored by the presence of nearby basic residues and the ITAM tyrosines. Phosphorylation minimizes membrane association, rendering the ITAM motif more accessible to binding partners. In systems mimicking biological membranes, the CD3ϵ chain localization is modulated by different facilitator lipids (e.g., gangliosides or phosphoinositols), revealing a plausible regulatory effect on activation through the regulation of lipid composition in cell membranes.     

Advances in bioreactor control for production of biotherapeutic products

Advanced control strategies are well established in chemical, pharmaceutical, and food processing industries. Over the past decade, the application of these strategies is being explored for control of bioreactors for manufacturing of biotherapeutics. Most of the industrial bioreactor control strategies apply classical control techniques, with the control system designed for the facility at hand. However, with the recent progress in sensors, machinery, and industrial internet of things, and advancements in deeper understanding of the biological processes, coupled with the requirement of flexible production, the need to develop a robust and advanced process control system that can ease process intensification has emerged. This has further fuelled the development of advanced monitoring approaches, modeling techniques, process analytical technologies, and soft sensors. It is seen that proper application of these concepts can significantly improve bioreactor process performance, productivity, and reproducibility. This review is on the recent advancements in bioreactor control and its related aspects along with the associated challenges. This study also offers an insight into the future prospects for development of control strategies that can be designed for industrial-scale production of biotherapeutic products.     

Experimental model of staphylococcal osteomyelitis in dogs

Osteomyelitis was induced in 45 male dogs by inoculating hemolytic strain of Staphylococcus aureus alone into the tibial marrow cavity. Clinical, radiological and bacteriological studies were conducted to evaluate the progress of disease up to 15 weeks. Clinical signs consisted of localized soft tissue swelling, pain, pyrexia and lameness which later developed an open wound with purulent exudation. Predominant radiographic features were extensive periosteal reaction, cortical lysis, new bone formation, frequent development of sequestrum and formation of localized abscess pockets in advanced cases. Staphylococci were recovered from the tibial marrow cavity for as long as 15 weeks after onset of the infection.