Insoluble Aggregates Research Articles

Palladium nanoballs coupled with smartphone-thermal reader for photothermal lateral flow immunoassay of Aβ 1-40.

Amyloid beta 1-40 (Aβ 1-40) is one of the most abundant substances in the body with the capacity to form insoluble aggregates and is a universal biomarker for the prediction of Alzheimer's disease. Here, a palladium nanoball (PNB)-strip was developed and coupled with a smartphone-thermal reader as an ultrasensitive and cost-effective platform for Aβ 1-40 detection. In this study, PNB was synthesized and introduced into lateral flow strips as an alternative signal source to gold nanoparticles to improve sensitivity because the PNB has a better heat generation ability. Quantitative analysis was performed using a self-developed smartphone-thermal reader, which is portable and cost-effective. The detection limit of the system was determined to be 20 pg mL-1, which fulfils the need for clinical diagnosis at the point-of-care. This work highlights a PNB-strip coupled smartphone-thermal reader for ultrasensitive and cost-effective Aβ 1-40 detection.

Dual-environment-sensitive probe to detect protein aggregation in stressed laryngeal carcinoma cells and tissues.

The interplay between protein folding and biological activity is crucial, with the integrity of the proteome being paramount to ensuring effective biological function execution. In this study, we report a dual-environment-sensitive probe A1, capable of selectively binding to protein aggregates and dynamically monitoring their formation and degradation. Through in vitro, cellular, and tissue assays, A1 demonstrated specificity in distinguishing aggregated from folded protein states, selectively partitioning into aggregated proteins. Thermal shift assays revealed A1 could monitor the process of protein aggregation upon binding to misfolded proteins and preceding to insoluble aggregate formation. In cellular models, A1 detected stress-induced proteome aggregation in TU212 cells (laryngeal carcinoma cells), revealing a less polar microenvironment within the aggregated proteome. Similarly, tissue samples showed more severe proteome aggregation in cancerous tissues compared to paracancerous tissues. Overall, A1 represents a versatile tool for probing protein aggregation with significant implications for both fundamental research and clinical diagnostics.

Cell invasive amyloid assemblies from SARS-CoV-2 peptides can form multiple polymorphs with varying neurotoxicity.

The neurological symptoms of COVID-19, often referred to as neuro-COVID include neurological pain, memory loss, cognitive and sensory disruption. These neurological symptoms can persist for months and are known as Post-Acute Sequalae of COVID-19 (PASC). The molecular origins of neuro-COVID, and how it contributes to PASC are unknown, however a growing body of research highlights that the self-assembly of protein fragments from SARS-CoV-2 into amyloid nanofibrils may play a causative role. Previously, we identified two fragments from the SARS-CoV-2 proteins, Open Reading Frame (ORF) 6 and ORF10, that self-assemble into neurotoxic amyloid assemblies. Here we further our understanding of the self-assembly mechanisms and nano-architectures formed by these fragments and their biological responses. By solubilising the peptides in a fluorinated solvent, we eliminate insoluble aggregates in the starting materials (seeds) that change the polymorphic landscape of the assemblies. The resultant assemblies are dominated by structures with higher free energies (e.g. ribbons and amorphous aggregates) that are less toxic to cultured neurons but do affect their mitochondrial respiration. We also show the first direct evidence of cellular uptake of viral amyloids. This work highlights the importance of understanding the polymorphic behaviour of amyloids and the correlation to neurotoxicity, particularly in the context of neuro-COVID and PASC.

Mass Spectrometry-Based Characterization of Protein Aggregates in Tissues and Biofluids.

Protein aggregation is a common mechanism in multiple neurodegenerative and heart diseasesand the accumulation of proteins in aggregates is toxic to cells, causing injury and death. The degree of protein aggregation directly correlates with the severity of the disease. Misfolded proteins present thermodynamic barriers that culminate in the loss of structure and function and the exposure of hydrophobic residues. The exposure of hydrophobic residues is the driving force behind protein aggregation, as it reduces surface free energy and increases the propensity for the formation of large insoluble aggregates. Exploring the protein content of aggregates is fundamental to understanding their formation mechanism and pathophysiological effects. We demonstrate here a method for isolating aggregated protein content in human plasma and mouse brain samples. The samples were characterized by mass spectrometry analysis, transmission electron microscopy, and western blotting. We report the identification of proteins associated with neurodegenerative diseases in the isolated pellets. The western blotting analyses of the isolated pellet showed the positivity for CD89 and CD63, consolidated markers of exosomes, confirming the presence of exosomes within the pellet but not in the supernatant in human plasma. Notably, the concomitant isolation of exosomes together with the protein aggregates was feasible starting from 200 μL of human plasma. Moreover, the presented methodology separated albumin from the aggregated pellet, allowing identification of larger diversity of proteins through mass spectrometry analysis.

Inclusion Bodies in Ionic Liquids

The pivotal role of proteins in pharmaceuticals is challenged by stability issues, making the study of inclusion bodies—a source of insoluble protein aggregates—increasingly relevant. This review outlines the critical procedures in inclusion body processing, focusing on ’mild solubilization concepts’ and refolding methodologies. Attention is afforded to the emerging role of ionic liquids with unique and tunable physicochemical properties in optimizing protein unfolding and refolding processes. The review critically assesses the existing literature at the intersection of inclusion bodies and ionic liquids, identifying recent advancements, potential applications, and avenues for future research. This comprehensive analysis aims to elucidate the complexities in efficient protein processing from inclusion bodies.

Aggregation of Disrupted in Schizophrenia 1 arises from a central region of the protein

An emerging approach to studying major mental illness is through proteostasis, with the identification of several proteins that form insoluble aggregates in the brains of patients. One of these is Disrupted in Schizophrenia 1 (DISC1), a neurodevelopmentally-important scaffold protein, and product of a classic schizophrenia risk gene. DISC1 aggregates have been detected in post mortem brain tissue from patients with schizophrenia, bipolar disorder and major depressive disorder, as well as various model systems, although the mechanism by which it aggregates is still unclear. Aggregation of two other proteins implicated in mental illness, TRIOBP-1 and NPAS3, was shown to be dependent on very specific structural regions of the protein. We therefore looked at the domain structure of DISC1, and investigated which structural elements are key for its aggregation. While none of the known structured DISC1 regions (named D, I, S and C respectively) formed aggregates individually when expressed in neuroblastoma cells, the combination of the D and I regions, plus the linker region between them, formed visible aggregates. Further refinement revealed that a region of approximately 30 amino acids between these two regions is critical for aggregation, and deletion of this region is sufficient to abolish the aggregation propensity of DISC1. This finding from mammalian cell culture contrasts with the recent determination that the C-region of DISC1 can aggregate in vitro, although some variations of the C-terminal of DISC1 could aggregate in our system. It therefore appears likely that DISC1 aggregation, implicated in mental illness, can occur through at least two distinct mechanisms.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation.

Serotonin receptors - a potential target for the treatment of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of dementia worldwide, having an increasing impact on aging societies. It is known that the serotonin (5-HT) system of the brain, in addition to its critical role in the control of various physiological functions and behaviors, is involved in the regulation of migration, proliferation, differentiation, maturation and programmed death of neurons. At the same time, increasing evidence indicates the involvement of 5-HT neurotransmission in the mechanisms underlying the formation of insoluble aggregates of β-amyloid and tau protein, which are the main histopathological signs of AD. In this review, we focused our attention on the available data on the participation of various 5-HT receptors and the intracellular signaling cascades induced by them in pathological processes leading to the development of AD. First of all, this concerns information about the involvement of 5-HT receptors in the mechanisms of protein aggregation in AD, which indicate that specific changes in the function of certain 5-HT receptors or associated intracellular signal transduction mediators prevent the accumulation of β-amyloid plaques and neurofibrillary tau protein tangles. Based on the accumulated experimental data, it can be assumed that the use of 5-HT receptors as new drug targets may not only be useful for improving cognitive performance in AD, but will also play an important role in treating the causes of AD-related dementia.

Dent disease 1-linked novel CLCN5 mutations result in aberrant location and reduced ion currents

Dent disease is a rare renal tubular disease with X-linked recessive inheritance characterized by low molecular weight proteinuria (LMWP), hypercalciuria, and nephrocalcinosis. Mutations disrupting the 2Cl−/1H+ exchange activity of chloride voltage-gated channel 5 (CLCN5) have been causally linked to the most common form, Dent disease 1 (DD1), although the pathophysiological mechanisms remain unclear. Here, we conducted the whole exome capture sequencing and bioinformatics analysis within our DD1 cohort to identify two novel causal mutations in CLCN5 (c.749 G > A, p. G250D, c.829 A > C, p. T277P). Molecular dynamics simulations of ClC-5 homology model suggested that these mutations potentially may induce structural changes, destabilizing ClC-5. Overexpression of variants in vitro revealed aberrant subcellular localization in the endoplasmic reticulum (ER), significant accumulation of insoluble aggregates, and disrupted ion transport function in voltage clamp recordings. Moreover, human kidney-2 (HK-2) cells overexpressing either G250D or T277P displayed higher cell-substrate adhesion, migration capability but reduced endocytic function, as well as substantially altered transcriptomic profiles with G250D resulting in stronger deleterious effects. These cumulative findings supported pathogenic role of these ClC-5 mutations in DD1 and suggested a cellular mechanism for disrupted renal function in Dent disease patients, as well as a potential target for diagnostic biomarker or therapeutic strategy development.

Aggregation and cytotoxicity of food additive dye (Azorubine)-albumin adducts: a multi-spectroscopic, microscopic and computational analysis

Protein and peptide misfolding is a central factor in the formation of pathological aggregates and fibrils linked to disorders like Alzheimer’s and Parkinson’s diseases. Therefore, it’s essential to understand how food additives, particularly Azorubine, affect protein structures and their ability to induce aggregation. In this study, human serum albumin (HSA) was used as a model protein to investigate the binding and conformational changes caused by azorubine, a common food and drink colorant. The research revealed that azorubine destabilized the conformation of HSA at both physiological (pH 7.4) and acidic (pH 3.5) conditions. The loss of tryptophan fluorescence in HSA suggested significant structural alterations, particularly around aromatic residues. Far UV-CD analysis demonstrated disruptions in HSA's secondary structure, with a notable reduction in α-helical structures at pH 7.4. At pH 3.5, Azorubine induced even more extensive perturbations, resulting in a random coil conformation at higher azorubine concentrations. The study also investigated aggregation phenomena through turbidity measurements, RLS analysis, and TEM imaging. At pH 3.5, larger insoluble aggregates formed, while at pH 7.4, only conformational changes occurred without aggregate formation. Cytotoxicity assessments on neuroblastoma (SH-SY5Y) cells highlighted the concentration-dependent toxicity of albumin aggregates. Molecular dynamics simulations reaffirmed the stable interaction between azorubine and HSA. This research provides valuable insights into the mechanisms by which azorubine influences protein conformations. To further advance our understanding and contribute to the broader knowledge in this area, several future directions can be considered such as exploring other proteins, studying dose-response relationship, gaining mechanistic insights, biological relevance, toxicity assessment, identifying alternative food colorants, and mitigation strategies to prevent adverse effects of azorubine on serum proteins.Communicated by Ramaswamy H. Sarma

Characterization of protein aggregates in cream and skimmed human milk after heat and high-pressure pasteurization treatments.

Preservation processes applied to ensure microbial safety of human milk (HM) can modify the native structure of proteins and their bioactivities. Consequently, this study evaluated the effect of pasteurization methods (Holder pasteurization, high-temperature short-time (HTST), and high hydrostatic pressure (HHP)) of whole human milk (HM) on protein aggregates in skim milk and cream fractions. For heat-treated whole milk, insoluble protein aggregates at milk fat globule membrane (MFGM) were formed by disulfide and non-covalent bonds, but insoluble skim milk protein aggregates were only stabilized by non-covalent interactions. Contrary to heat treatment, the insolubilization of main proteins at the MFGM of HHP-treated HM was only through non-covalent interactions rather than disulfide bonds. Moreover, only heat treatment induced the insoluble aggregation of ⍺-lactalbumin. Overall, compared to heat treatment, HHP produced a milder effect on protein aggregation, validating the use of this process to better preserve the native state of HM bioactive proteins.

Use of plasma P‐tau217 for enrichment in the Phase 2 PROSPECT‐ALZ study of LY3372689 in early symptomatic Alzheimer’s disease: Cohort selection and baseline characteristics

AbstractBackgroundInhibition of O‐linked N‐acetyl glucosaminidase (OGA) is hypothesized to impede the formation of hyperphosphorylated, insoluble tau aggregates and neurofibrillary tangles. LY3372689 is an orally available, potent inhibitor of OGA being tested in the ongoing PROSPECT‐ALZ study. This is the first study assessing the safety and efficacy of an OGA inhibitor in individuals with early symptomatic Alzheimer’s Disease (AD). To improve efficiencies in identifying individuals with desired levels of tau pathology, PROSPECT‐ALZ implemented a gated screening approach using plasma P‐tau217 as an enrichment step prior to tau PET. We present the study design, findings from the screening process, and baseline characteristics of the PROSPECT‐ALZ study cohort.MethodWith 68 sites across Australia, Canada, Japan, Poland, and the US, PROSPECT‐ALZ is an ongoing multicenter, double‐blind, placebo‐controlled Phase 2 study with 1:1:1 randomization of daily LY3372689 low dose, LY3372689 high dose, or placebo. The primary objective is to evaluate the effect of LY3372689 on AD clinical progression, as assessed by the change from baseline to endpoint (76‐124 weeks with common close design) on the integrated AD Ratings Scale (iADRS).Pre‐screening procedures include the Mini‐Mental State Examination (MMSE, scores of 22‐30) and a plasma P‐tau217 test to determine eligibility for further screening. Subsequent key inclusion requirements to meet were age 60‐85 years, a CDR global score of 0.5 or 1 with a memory box score of ≥ 0.5, and a PET (18F‐flortaucipir) scan demonstrating an intermediate or high level of aggregated brain tau by combined visual and quantitative assessment.ResultAfter consent, 2180 participants entered screening. The top reason for screen failure was a P‐tau217 result below the eligibility threshold. Performance of the P‐tau217 test for pre‐screening patient selection as well as characteristics and demographics of the screening and randomized cohorts will be presented.ConclusionAs blood‐based biomarkers become more accessible, their use in selecting participants for clinical research is desirable. The PROSPECT‐ALZ study successfully implemented plasma P‐tau217 in sequence with tau PET to improve efficiency in identifying trial participants. This study of LY3372689, the first orally administered OGA inhibitor to be investigated in AD, has topline trial results anticipated in 2024.

Serotonin Receptors as a Potential Target in the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia worldwide that has an increasing impact on aging societies. Besides its critical role in the control of various physiological functions and behavior, brain serotonin (5-HT) system is involved in the regulation of migration, proliferation, differentiation, maturation, and programmed death of neurons. At the same time, a growing body of evidence indicates the involvement of 5-HT neurotransmission in the formation of insoluble aggregates of β-amyloid and tau protein, the main histopathological signs of AD. The review describes the role of various 5-HT receptors and intracellular signaling cascades induced by them in the pathological processes leading to the development of AD, first of all, in protein aggregation. Changes in the functioning of certain types of 5-HT receptors or associated intracellular signaling mediators prevent accumulation of β-amyloid plaques and tau protein neurofibrillary tangles. Based on the experimental data, it can be suggested that the use of 5-HT receptors as new drug targets will not only improve cognitive performance in AD, but will be also important in treating the causes of AD-related dementia.

The flavonoid luteolin reduces mutant huntingtin aggregation and cytotoxicity in huntingtin-mutated neuroblastoma cells

BackgroundHuntington's disease is an inherited progressive neurodegenerative disorder caused by an expansion of the polyglutamine tract leading to malformation and aggregation of the mutant huntingtin protein in the cell cytoplasm and nucleus of affected brain regions. The development of neuroprotective agents from plants has received considerable research attention. ObjectiveOur study aims to investigate the neuroprotective effects of luteolin and the mechanisms that underline its potential mediated protection in the mutant htt neuroblastoma cells. MethodsThe mutant htt neuroblastoma cells were transfected with 160Q, and the control wild-type neuroblastoma cells were transfected with 20Q htt for 24 h and later treated with luteolin. Cell viability was determined by MTT and PI staining in both groups, while western blotting was used to evaluate caspase 3 protein expression. Aggregation formation was assessed via immunofluorescence microscopy. Also, western blotting was utilized to measure the protein expression of mutant htt aggregated and soluble protein, Nrf2 and HO-1. The impact of Nrf2 on luteolin-treated neuroblastoma cells was assessed using small interfering RNAs. ResultsOur study reports that luteolin can protect cultured cells from mutant huntingtin cytotoxicity, evidenced by increased viability and decreased apoptosis. Also, luteolin reduced the accumulation of soluble and insoluble mutant huntingtin aggregates in mutant htt neuroblastoma cells transfected with 160Q compared to the control wild-type. The mutant htt aggregate reduction mediated by luteolin appeared to be independent of the Nrf2 –HO-1 antioxidant pathway. ConclusionLuteolin presents a new potential therapeutic and protective agent for the treatment and decreasing the cytotoxicity in neurodegenerative diseases such as Huntington's disease.

Alkyl Derivatives of Perylene Photosensitizing Antivirals: Towards Understanding the Influence of Lipophilicity.

Amphipathic perylene derivatives are broad-spectrum antivirals against enveloped viruses that act as fusion inhibitors in a light-dependent manner. The compounds target the lipid bilayer of the viral envelope using the lipophilic perylene moiety and photogenerating singlet oxygen, thereby causing damage to unsaturated lipids. Previous studies show that variation of the polar part of the molecule is important for antiviral activity. Here, we report modification of the lipophilic part of the molecule, perylene, by the introduction of 4-, 8-, and 12-carbon alkyls into position 9(10) of the perylene residue. Using Friedel-Crafts acylation and Wolff-Kishner reduction, three 3-acetyl-9(10)-alkylperylenes were synthesized from perylene and used to prepare 9 nucleoside and 12 non-nucleoside amphipathic derivatives. These compounds were characterized as fluorophores and singlet oxygen generators, as well as tested as antivirals against herpes virus-1 (HSV-1) and vesicular stomatitis virus (VSV), both known for causing superficial skin/mucosa lesions and thus serving as suitable candidates for photodynamic therapy. The results suggest that derivatives with a short alkyl chain (butyl) have strong antiviral activity, whereas the introduction of longer alkyl substituents (n = 8 and 12) to the perylenyethynyl scaffold results in a dramatic reduction of antiviral activity. This phenomenon is likely attributable to the increased lipophilicity of the compounds and their ability to form insoluble aggregates. Moreover, molecular dynamic studies revealed that alkylated perylene derivatives are predominately located closer to the middle of the bilayer compared to non-alkylated derivatives. The predicted probability of superficial positioning correlated with antiviral activity, suggesting that singlet oxygen generation is achieved in the subsurface layer of the membrane, where the perylene group is more accessible to dissolved oxygen.

Approaches to produce and characterize recombinant protein VP1-2A of HAV for serological rapid test application

Studies reporting the expression of hepatitis A virus (HAV) structural proteins, specifically recombinant VP1-2A containing an immunogenic activity, use the Escherichia coli system. Recombinant HAV proteins may represent a source of less expensive antigens for application in different diagnostic platforms. However, the formation of insoluble aggregates is an obstacle to obtaining large amounts of HAV proteins in their native form. To overcome this obstacle, some approaches were applied in this study to improve purification, solubility, and protein expression levels. Critical properties were evaluated. The introduction of another insertion codon to increase the protein concentration and vector activity was observed and verified by SDS-PAGE. The expression was established with 0.4 mM IPTG for 4 h at 37 °C. The VP1 protein was partially soluble at an isoeletric point (pI) of 6.45. The majority of HAV VP1-2A proteins measured 45.19 kDa in size and had a homogeneity of 53.58%. Multi-antigen print immunoassay (MAPIA) showed antigenicity at different HAV VP1-2A concentrations, and microsphere-based immunoassays showed a specificity of 100% and a sensitivity of 84%. HAV VP1-2A was characterized using different sensitivity methods to prove its biological activity, indicating its use as a tool for the diagnosis of Hepatitis A virus infection.

Monitoring of Systemic Light-Chain Amyloidosis Patients Using Mass Spectrometry (M-inSight) in Serum

Introduction: Systemic light-chain amyloidosis (AL) is a rare disorder characterized by extracellular deposition of monoclonal light-chain immunoglobulins forming insoluble aggregates in different organs. Kidney, heart and liver are principal organs affected by the disease inducing dysfunctions and failures. In this disease, even a low level of circulating light chain can cause significant damages, making challenging the measure of disease burden via peripheral blood. Although a bone marrow aspiration is required to confirm the diagnosis, the evolution of the disease is usually monitored using free light chains dosage, serum and urine electrophoresis, as well as immunofixation. However, these techniques have demonstrated some limitations in terms of sensitivity. Sebia had recently introduced M-inSight TM, a novel, ultra-sensitive and non-invasive assay for monitoring low level disease on blood using targeted mass spectrometry. In this pilot study, M-inSight TM technology is employed to assess the use of mass spectrometry to monitor AL patients. Methods: 14 AL patients were selected to assess the use of M-InSight to detect and quantify AL light chain. 8/14 patients were selected from the AMYDARA study (NCT02816476), consisting in refractory patients treated with Daratumumab. Serum free light-chain assay (Freelite TM), immunofixation on serum/urine as well as bone marrow collection for DNA (ClonoSeq) and RNA (RACE-RepSeq) immunoglobulin next generation sequencing were used to monitor the disease at baseline and end of treatment (EOT) and compared to M-inSight TM. Additionally, 6/14 multicenter patients with normal free light chain level at diagnosis were selected to evaluate the sensitivity of M-InSight. Total mRNA was extracted from BM samples, retrotranscribed to cDNA and then used for immunoglobulin repertoire high-throughput sequencing assay (RACE-RepSeq). cDNA assembly pipeline using IMGT/HighV-QUEST and Vidjil software were used to construct clonotypes and to identify clonal molecular fingerprints and finally their clonotypic peptides for M-InSight analysis. This technology consists of sequence the AL light chain from serum sample and choose clonotypic peptides from the hypervariable regions which then tracked down in the follow-up sample. Results Clonotypic peptides were identified in all eight patients from the baseline sample, which were then confirmed by mass spectrometry. Out of the 8 AMYDARA patients, 5/8 patients achieved very good partial response (VGPR), 2/8 achieved partial response (PR) and 1/8 resulted in complete response (CR). 6 patients had a decrease of the dFLC ratio measure with Freelite TM of 80% or more. 6 out of these 8 patients had MS data highly concordant with that of the serum free light-chain assay. 2 patients had a strong decrease in dFLC while MS measurements were stable or increased. However, detection of the clonotype using RACE-RepSeqand Clonoseq remained elevated, which was more in line with MS than Freelite TM. All patients with normalization of their dFLC were still detected using M-inSight, even those reaching MRD negativity (1 patient) or low number of cells (~10-5, 2 patients) by ClonoSeq. These results were consistent with the RACE-RepSeq which detected the light chain clonotypes in all patients at EOT. Clonotypic peptides using M-InSight were also detected in all 7 patients with normal free light chain at diagnosis. However, in one bi-clonal patient, peptides from only one of both chains were detected, suggesting that only one clone was secreting light chain. Conclusion The goal of this study was to evaluate the M-InSight technique to monitor the evolution of systemic AL amyloidosis in blood samples. The results showed a high concordance with bone marrow MRD measurement and a higher sensitivity than dFLC to monitor the disease. This study shows that MRD monitoring on blood is feasible, using an ultra-sensitive MS technique, avoiding invasive bone-marrow aspirate. M-inSight can track low level AL disease.

Effect of pH on the formation mechanisms, emulsifying properties and curcumin encapsulation of oat protein isolate–high methoxy pectin complexes

Oat protein isolate (OPI) has high nutritional value but poor emulsifying properties. The purpose of modifying the OPI with high methoxyl pectin (HMP) was to improve emulsification. In this work, an oat protein isolate (OPI)–high methoxyl pectin (HMP) complex was constructed by changing the pH, and the interactions, formation mechanism and potential use of the OPI–HMP complex as a food emulsifier were explored. The presence of HMP caused structural changes in the OPI, and the changes were pH dependent. When the OPI to HMP mass ratio was 3:1, the critical pH for the phase behavior of OPI–HMP was 5.0. When the pH was greater than 5.0, the OPI and HMP were codissolved in the composite system mainly through electrostatic repulsion. When the pH was 5.0, a soluble OPI–HMP complex was formed by a combination of electrostatic attractions, hydrophobic interactions and hydrogen bonding. At pH values below 5.0, other aggregates of HMP and OPI were generated through electrostatic attraction and hydrogen bonding and formed insoluble aggregates. When the OPI to HMP mass ratio was 3:1 and the pH was 5.0, the particle sizes of the emulsion were the smallest at 8.75 μm due to the strong electrostatic interactions. The emulsification activity and stability were much higher than those of the OPI, and they formed dense osmotic networks, which protected the curcumin. The rates for curcumin encapsulation and retention reached 83.87 ± 1.50% and 88.70 ± 2.50%, respectively, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging rate was maintained at a high level (approximately 72%). These results confirmed the possibility of using the OPI-HMP complex emulsions as excellent and stable nutraceutical delivery systems for lipid soluble bioactive compounds in food and biomedical applications.

Regulation of the proteostasis network by the neuronal system.

The protein homeostasis (proteostasis) network is a nexus of molecular mechanisms that act in concert to maintain the integrity of the proteome and ensure proper cellular and organismal functionality. Early in life the proteostasis network efficiently preserves the functionality of the proteome, however, as the organism ages, or due to mutations or environmental insults, subsets of inherently unstable proteins misfold and form insoluble aggregates that accrue within the cell. These aberrant protein aggregates jeopardize cellular viability and, in some cases, underlie the development of devastating illnesses. Hence, the accumulation of protein aggregates activates different nodes of the proteostasis network that refold aberrantly folded polypeptides, or direct them for degradation. The proteostasis network apparently functions within the cell, however, a myriad of studies indicate that this nexus of mechanisms is regulated at the organismal level by signaling pathways. It was also discovered that the proteostasis network differentially responds to dissimilar proteotoxic insults by tailoring its response according to the specific challenge that cells encounter. In this mini-review, we delineate the proteostasis-regulating neuronal mechanisms, describe the indications that the proteostasis network differentially responds to distinct proteotoxic challenges, and highlight possible future clinical prospects of these insights.

Pharmacological Inhibition of the AQP4 Water Channel Activity Causes an Aggravation of Alpha-Synuclein Pathology in the Substantia Nigra in a Rat Model of Parkinson’s Disease

The misfolding of the protein α-synuclein, which leads to the formation of neurototoxic oligomers and aggregates, is one of the main causes of loss of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) in Parkinson’s disease (PD). We previously found that pharmacological inhibition of the water channel aquaporin-4 (AQP4), participating in the mechanisms of brain clearance of amyloidogenic proteins, caused the aggravation of neurodegeneration in the nigrostriatal system and the development of motor disturbances in a lactacystin model of PD. It was hypothesized that the progression of neurodegeneration can be a result of the excessive accumulation of pathologic forms of α-synuclein due to the AQP4 inhibition. The aim of this study is to determine whether pharmacological inhibition of AQP4 activity in a rat model of preclinical PD leads to an aggravation in α-synuclein pathology. The experiments were performed on male Wistar rats. AQP4 activity was suppressed using the intracerebroventricular injection of inhibitor TGN-020. To reproduce the model of the preclinical stage of PD, a specific proteasome inhibitor lactacystin (LC) was used. It was injected bilaterally into the SNpc. Immunoblotting methods and confocal microscopy were applied. The LC model of PD was characterized by a pathologic accumulation of total water-soluble and Ser129-phosphorylated forms of α-synuclein, as well as by formation of insoluble α-synuclein aggregates in the DA-neurons of SNpc. TGN-020 caused a significant aggravation of α-synuclein pathology in the LC model of PD. It was manifested by a marked increase in the level of water-soluble and modified forms of α-synuclein and by the 1.9-fold rise in the amount of α-synuclein aggregates in SN. We suppose that the disfunction of AQP4 which is involved in glymphatic system functioning, can be one of the mechanisms leading to the neurodegeneration and accumulation of amyloidogenic proteins in brain parenchyma during PD. The water channel AQP4 might be a target for the development of new therapeutic approaches aimed at attenuation of the cytotoxicity, accumulation and distribution of α-synuclein during the development of PD-like pathology.