Protein footprinting

Single cell cloning is a critical step for cell line development (CLD) for therapeutic protein production, with proof of monoclonality being compulsorily sought in regulatory filings. Among the different single cell deposition technologies, we found that fluorescence activated cell sorting (FACS) offers high probability of monoclonality and can allow selective enrichment of the producer cells. However, FACS instruments are expensive and resource-intensive, have a large footprint, require highly skilled operators and take hours for setup, thereby complicating the cell line generation process...

Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these biohybrid catalysts on support materials to enhance stability and enable their retention, recovery, and reuse. Here, we describe the immobilization of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, e.g., due to poisoning or decomposition, on immobilized metal affinity chromatography resins...

Haberlea rhodopensis, a resurrection species, is the only plant known to be able to survive multiple extreme environments, including desiccation, freezing temperatures, and long-term darkness. However, the molecular mechanisms underlying tolerance to these stresses are poorly studied. Here, we present a high-quality genome of Haberlea and found that ~ 23.55% of the 44,306 genes are orphan. Comparative genomics analysis identified 89 significantly expanded gene families, of which 25 were specific to Haberlea...

OBJECTIVES: To characterize nutritionally adequate, climate-friendly diets that are culturally acceptable across socio-demographic groups. To identify potential equity issues linked to more climate-friendly and nutritionally adequate dietary changes. DESIGN: An optimization model minimizes distance from observed diets subject to nutritional, greenhouse gas emissions (GHGE) and food-habit constraints. It is calibrated to socio-demographic groups differentiated by sex, education and income levels using dietary intake data...

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax...

AIMS: Spinal cord injury (SCI) is a devastating neurological disease that often results in tremendous loss of motor function. Increasing evidence demonstrates that diabetes worsens outcomes for patients with SCI due to the higher levels of neuronal oxidative stress. Mammalian sterile 20-like kinase (MST1) is a key mediator of oxidative stress in the central nervous system; however, the mechanism of its action in SCI is still not clear. Here, we investigated the role of MST1 activation in induced neuronal oxidative stress in patients with both SCI and diabetes...

Effective dewatering of sewage sludge could potentially address the issues of high energy consumption and large carbon footprint inherent in the sludge treatment process, advancing toward carbon neutrality in environmental remediation. Yet, the surface hydrophilic characteristics and water-holding interfacial affinity in sludge led to dwindled sludge-water separation performance. Here, the integration of in-situ generation of iron from zero-valent scrap iron (ZVSI) and sodium percarbonate (SPC) was attempted to attenuate the water-retaining interfacial affinity within sludge, thus achieving superior sludge dewatering performance...

In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein...

Protein dynamics form a critical bridge between protein structure and function, yet the impact of evolution on ultrafast processes inside proteins remains enigmatic. This study delves deep into nanosecond-scale protein dynamics of a structurally and functionally conserved protein across species separated by almost a billion years, investigating ten homologs in complex with their ligand. By inducing a photo-triggered destabilization of the ligand inside the binding pocket, we resolved distinct kinetic footprints for each homolog via transient infrared spectroscopy...

The nanopore sensing method holds the promise of delivering a single molecule technology for identification of biological proteins, direct detection of post-translational modifications, and perhaps de novo determination of a protein's amino acid sequence. The key quantity measured in such nanopore sensing experiments is the magnitude of the ionic current passing through a nanopore blocked by a polypeptide chain. Establishing a relationship between the amino acid sequence of a peptide fragment confined within a nanopore and the blockade current flowing through the nanopore remains a major challenge for realizing the nanopore protein sequencing...

Parkinson's Disease (PD) is a debilitating neurodegenerative disease, causing loss of motor function and, in some instances, cognitive decline and dementia in those affected. The quality of life can be improved, and disease progression delayed through early interventions. However, current methods of confirming a PD diagnosis are extremely invasive. This prevents their use as a screening tool for the early onset stages of PD. We propose a surface imprinted polymer (SIP) electroimpedance spectroscopy (EIS) biosensor for detecting α-Synuclein (αSyn) and its aggregates, a biomarker that appears in saliva and blood during the early stages of PD as the blood-brain barrier degrades...

Tenebrio molitor (TM) is considered as one of the most promising protein sources for replacing fish meal in aquafeeds, among other things because it is rich in protein, a good source of micronutrients and has a low carbon footprint and land use. However, the main drawback of TM is its fatty acid profile, in particular its low content of n-3 PUFA. This study evaluates the effects of partially replacing plant or marine-derived with full-fat TM meal at two different levels on growth performance and lipid profiles of Senegalese sole ( Solea senegalensis )...

OBJECTIVE: To investigate the effect of M2 microglia (M2-MG) transplantation on spinal cord injury (SCI) repair in mice. METHODS: Primary MG were obtained from the cerebral cortex of 15 C57BL/6 mice born 2-3 days old by pancreatic enzyme digestion and identified by immunofluorescence staining of Iba1. Then the primary MG were co-cultured with interleukin 4 for 48 hours (experimental group) to induce into M2 phenotype and identified by immunofluorescence staining of Arginase 1 (Arg-1) and Iba1...

This study explores microalgae-based bioremediation for treating black gunpowder production effluents, an understudied yet environmentally significant stream. Two native microalgae, Chlorella sp. MC18 (CH) and Scenedesmus sp. MJ23-R (SC), were assessed for growth kinetics and nutrient removal capabilities in culture media containing different proportions of untreated raw wastewater. Results show both species thrived in 100 % raw wastewater, displaying robust growth and substantial biomass production in parallelepiped-shaped photobioreactors...

Despite the large carbon footprint of livestock production, animal protein consumption has grown over the past several decades, necessitating new approaches to sustainable animal protein production. In this techno-economic analysis, single cell protein (SCP) produced via gas fermentation of carbon dioxide, oxygen, and hydrogen is studied as an animal feed source to replace fishmeal or soybean meal. Using wind-powered water electrolysis to produce hydrogen and oxygen with carbon dioxide captured from corn ethanol, the minimum selling price (MSP) of SCP is determined to be $2070 per metric ton...

A comprehensive understanding of the role of dietary fibre in non-ruminant animal production is elusive. Equivocal and conflated definitions of fibre coupled with significant analytical complexity, interact with poorly defined host and microbiome relationships. Dietary fibre is known to influence gut development, feed intake and passage rate, nutrient absorption, microbiome taxonomy and function, gut pH, endogenous nutrient loss, environmental sustainability, animal welfare and more. Whilst significant gaps persist in our understanding of fibre in non-ruminant animal production, there is substantial interest in optimizing the fibre fraction of feed to induce high value phenotypes such as improved welfare, live performance and to reduce the environmental footprint of animal production systems...

Synaptic function is modulated by local translation of mRNAs that are transported to distal portions of axons and dendrites. The Metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) is broadly expressed across cell types, almost exclusively as a nuclear non-coding RNA. We found that in differentiating neurons, a portion of Malat1 RNA redistributes to the cytoplasm. Depletion of Malat1 from neurons stimulated expression of particular pre- and post-synaptic proteins, implicating Malat1 in their regulation...

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF...

OBJECTIVES: We investigated the potential of serum proteins to distinguish clinical and molecular subtypes in patients with giant cell arteritis (GCA). METHODS: Proximity extension assays were used to analyse 1463 proteins in serum samples from patients with new-onset GCA (n = 16) and after achieving remission (n = 13). Unsupervised and supervised cluster analyses were performed. RESULTS: Unsupervised cluster analysis identified three distinct clusters based on the protein signature...

Genetic selection has remarkably helped U.S. dairy farms to decrease their carbon footprint by more than doubling milk production per cow over time. Despite the environmental and economic benefits of improved feed and milk production efficiency, there is a critical need to explore phenotypical variance for feed utilization to advance the long-term sustainability of dairy farms. Feed is a major expense in dairy operations, and their enteric fermentation is a major source of greenhouse gases in agriculture. The challenges to expanding the phenotypic database, especially for feed efficiency predictions, and the lack of understanding of its drivers limit its utilization...