Changes in the urinary content of uromodulin oligomeric forms in rats during development of urolithiasis

Введение. Изучение роли олигомерных форм уромодулина в развитии уролитиаза является важной фундаментальной и прикладной задачей. Несмотря на разнообразие моделей уролитиаза на лабораторных животных в настоящее время отсутствует информация относительно динамики концентрации и фракционного состава олигомерных форм уромодулина в моче животных на различных этапах развития патологического процесса. Цель - исследование динамики содержания олигомерных форм уромодулина в моче животных на фоне развития гипероксалатного уролитиаза индуцированного экзогенным введением 1% раствора этиленгликоля в качестве безальтернативного источника питья. Методика. Проводили общеклинический и биохимический анализ образцов крови и мочи на различных этапах развития патологического процесса. До начала моделирования патологии и на фоне экзогенного введения этиленгликоля был исследован осадок мочи. Оценка содержания олигомерных форм уромодулина в моче животных проводилась методом анализа треков наночастиц и динамического рассеяния света. Результаты. Показано, что на фоне развития патологии наблюдается уменьшение концентрации олигомерных форм уромодулина в моче, на начальных этапах развития патологии за счёт увеличения фракции крупных частиц (более 200 нм, олигомерная форма 28 МДа). При дальнейшем развитии патологического процесса на завершающем этапе наблюдается радикальное уменьшение концентрации частиц в моче (более чем в 2 раза). Заключение. Полученные данные показали относительно низкую корреляцию между длительностью моделирования патологии и тяжестью проявления уролитиаза (r-Пирсона = 0,49, p-value = 0,0003). Концентрация олигомеров уромодулина в моче животных уменьшается на фоне увеличения количества кристаллов в осадке мочи, что вероятно связанно с включением уромодулина в структуру кристаллов осадка. Studying uromodulin oligomeric forms in urolithiasis development is important fundamental and applied problem. Despite the variety of in vivo models of urolithiasis, there is currently no information about concentration dynamics and fractional composition of uromodulin oligomeric forms in urine for animals at various stages of pathological process developmen. The purpose We investigate dynamics uromodulin oligomeric forms in urine of animals against the background of development hyperoxalate urolithiasis induced by exogenous administration of 1% ethylene glycol solution as a non-alternative source of drinking. Methods. For urine samples at various stages of pathogenesis, general clinical and biochemical analysis were carried out, for urine samples before the start of pathology modeling and against the background of exogenous administration of ethylene glycol, urine sediment was examined. The study of urine sediment and content uromodulin oligomeric forms was carried out on 0th, 7th, 14th, 21st and 28th days of pathology modeling. Evaluation of the content of uromodulin oligomeric forms in urine of animals was carried out by nanoparticles track analysing and dynamic light scattering. Results. It is shown that against the background of the pathology development there is a decrease in the concentration of oligomeric forms of uromodulin in the urine, at the initial stages of pathology development due to an increase in the fraction of large particles (over 200 nm, oligomeric form 28 МDa). With further development of the pathological process at the final stage, there is a radical decrease in the concentration of particles in the urine (more than 2-fold). Conclusion. The obtained data showed a relatively low correlation between the duration of pathology modeling and the severity of urolithiasis manifestation (Pearson's r = 0.49, p-value = 0.0003). Concentration of uromodulin oligomers in animals urine decreases with an increase in the amount of crystals in urine sediment, which is probably associated with inclusion of uromodulin in structure of sediment crystals.

Research on phenol complex infrared dried grape pomace

Studies have shown that grape pomace is a promising raw material for the functional drinks production with an increased phenols mass concentration. At the same time, the highest mass concentration of phenols was determined in red frape varities pomace. The highest mass concentrations of flovanols were found in white pomace, flavonols in muscat, phenolic acids in red pomace. The mass concentration of oligomeric forms in phenols does not differ significantly in all samples. The highest mass concentration of polymers in phenols is determined in the red pomace. The anthocyanin profile corresponded to the Western European ecological-geographical group grape varieties. Highest mass concentration was determined monoglycoside malvidin.

Immunogenicity Risk Assessment for Multi-specific Therapeutics

The objective of this manuscript is to provide the reader with a hypothetical case study to present an immunogenicity risk assessment for a multi-specific therapeutic as part of Investigational New Drug (IND) application. In order to provide context for the bioanalytical strategies used to support the multi-specific therapeutic presented herein, the introduction focuses on known immunogenicity risk factors. The subsequent hypothetical case study applies these principles to a specific example HC-12, based loosely on anti-TNFα and anti-IL-17A bispecific molecules previously in development, structured as an example immunogenicity risk assessment for submission to health authorities. The risk of higher incidence and safety impact of anti-drug antibodies (ADA) due to large protein complexes is explored in the context of multi-specificity and multi-valency of the therapeutic in combination with the oligomeric forms of the targets.

Evolution of the folding landscape of effector caspases

Caspases are a family of cysteinyl proteases that control programmed cell death and maintain homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because all caspases are produced as zymogens (procaspases) that must be activated to gain full activity; the protein structures are conserved through hundreds of millions of years of evolution; and some allosteric features arose with the early ancestor while others are more recent evolutionary events. The apoptotic caspases evolved from a common ancestor into two distinct subfamilies: monomers (initiator caspases) or dimers (effector caspases). Differences in activation mechanisms of the two subfamilies, and their oligomeric forms, play a central role in the regulation of apoptosis. Here, we examine changes in the folding landscape by characterizing human effector caspases and their common ancestor. The results show that the effector caspases unfold by a minimum three-state equilibrium model at pH 7.5, where the native dimer is in equilibrium with a partially folded monomeric (procaspase-7, common ancestor) or dimeric (procaspase-6) intermediate. In comparison, the unfolding pathway of procaspase-3 contains both oligomeric forms of the intermediate. Overall, the data show that the folding landscape was first established with the common ancestor and was then retained for >650 million years. Partially folded monomeric or dimeric intermediates in the ancestral ensemble provide mechanisms for evolutionary changes that affect stability of extant caspases. The conserved folding landscape allows for the fine-tuning of enzyme stability in a species-dependent manner while retaining the overall caspase-hemoglobinase fold.

Limited variation between SARS-CoV-2-infected individuals in domain specificity and relative potency of the antibody response against the spike glycoprotein

The spike protein of SARS-CoV-2 is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The variation between infected individuals in domain specificity of the antibodies and in their relative neutralization efficacy is still poorly characterized. To this end, we tested serum and plasma samples from 85 COVID-19 convalescent subjects using 7 immunoassays that employ different domains, subunits and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. We observed strong correlations between the levels of NTD- and RBD-specific antibodies, with a fixed ratio of each type to all anti-spike antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects, and was not associated with the overall amount of anti-spike antibodies produced. Accordingly, the ability of immunoassays that use RBD, NTD and different forms of S1 or S1/S2 as capture antigens to estimate the neutralizing efficacy of convalescent samples was largely similar. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency.

Dendritic/Post-synaptic Tau and Early Pathology of Alzheimer’s Disease

Microtubule-associated protein tau forms insoluble neurofibrillary tangles (NFTs), which is one of the major histopathological hallmarks of Alzheimer’s disease (AD). Many studies have demonstrated that tau causes early functional deficits prior to the formation of neurofibrillary aggregates. The redistribution of tau from axons to the somatodendritic compartment of neurons and dendritic spines causes synaptic impairment, and then leads to the loss of synaptic contacts that correlates better with cognitive deficits than amyloid-β (Aβ) aggregates do in AD patients. In this review, we discuss the underlying mechanisms by which tau is mislocalized to dendritic spines and contributes to synaptic dysfunction in AD. We also discuss the synergistic effects of tau and oligomeric forms of Aβ on promoting synaptic dysfunction in AD.

Conformational distortion in a fibril-forming oligomer arrests alpha-Synuclein fibrillation and minimizes its toxic effects

The fibrillation pathway of alpha-Synuclein, the causative protein of Parkinson’s disease, encompasses transient, heterogeneous oligomeric forms whose structural understanding and link to toxicity are not yet understood. We report that the addition of the physiologically-available small molecule heme at a sub-stoichiometric ratio to either monomeric or aggregated α-Syn, targets a His50 residue critical for fibril-formation and stabilizes the structurally-heterogeneous populations of aggregates into a minimally-toxic oligomeric state. Cryo-EM 3D reconstruction revealed a ‘mace’-shaped structure of this monodisperse population of oligomers, which is comparable to a solid-state NMR Greek key-like motif (where the core residues are arranged in parallel in-register sheets with a Greek key topology at the C terminus) that forms the fundamental unit/kernel of protofilaments. Further structural analyses suggest that heme binding induces a distortion in the Greek key-like architecture of the mace oligomers, which impairs their further appending into protofilaments and fibrils. Additionally, our study reports a novel mechanism of prevention as well as reclamation of amyloid fibril formation by blocking an inter-protofilament His50 residue using a small molecule.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple βα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one β-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Label-Free Protein Detection by Micro-Acoustic Biosensor Coupled with Electrical Field Sorting. Theoretical Study in Urine Models

Diagnostic devices for point-of-care (POC) urine analysis (urinalysis) based on microfluidic technology have been actively developing for several decades as an alternative to laboratory based biochemical assays. Urine proteins (albumin, immunoglobulins, uromodulin, haemoglobin etc.) are important biomarkers of various pathological conditions and should be selectively detected by urinalysis sensors. The challenge is a determination of different oligomeric forms of the same protein, e.g., uromodulin, which have similar bio-chemical affinity but different physical properties. For the selective detection of different types of proteins, we propose to use a shear bulk acoustic resonator sensor with an additional electrode on the upper part of the bioliquid-filled channel for protein electric field manipulation. It causes modulation of the protein concentration over time in the near-surface region of the acoustic sensor, that allows to distinguish proteins based on their differences in diffusion coefficients (or sizes) and zeta-potentials. Moreover, in order to improve the sensitivity to density, we propose to use structured sensor interface. A numerical study of this approach for the detection of proteins was carried out using the example of albumin, immunoglobulin, and oligomeric forms of uromodulin in model urine solutions. In this contribution we prove the proposed concept with numerical studies for the detection of albumin, immunoglobulin, and oligomeric forms of uromodulin in urine models.