Insoluble Deposits Research Articles

Free-standing nitrogen doped graphene/Co(OH)2 composite films with superior catalytic activity for aprotic lithium-oxygen batteries

The recent boom in large-scale energy storage system promotes the development of lithium-oxygen batteries because of their high theoretical energy density. However, their applications are still limited by the sluggish kinetic, insoluble discharge product deposition and the undesired parasitic reaction. Herein, the free-standing nitrogen doped reduced graphene oxide/Co(OH)2 (NRGO/Co(OH)2) composite films were prepared by a facile hydrothermal method. The NRGO/Co(OH)2 composite films display interconnected three-dimensional conductive network, which can not only promote the diffusion of O2 and the transport of electrolyte ions, but also provide abundant storage space for discharge products. Moreover, the introduction of nitrogen-containing functional groups results in improved conductivity and electron adsorption ability, which can facilitate electron transport and enhance the surface catalytic activity. Combining with excellent catalytic performance, the lithium-oxygen batteries with NRGO/Co(OH)2 composite film cathodes deliver low charge overpotential and excellent cycling performance.

Evaluation of Internal and Superficial Self-Healing of Cracks in Concrete with Crystalline Admixtures.

Reinforced concrete structures are prone to cracking. The development of cementitious matrices with the capacity for self-healing soon after these cracks appear represents savings with inspections and repairs of the structures. Self-healing can be stimulated with the use of crystalline admixtures. Such materials easily react with water and increase the density of C-S-H (hydrated-calcium-silicate), forming insoluble deposits blocking existing pores and cracks. In this research, self-healing in concrete cracks was evaluated using three different crystalline admixtures, submitted to two and six wetting–drying cycles. The efficiency of self-healing was evaluated by optical microscopy and using the chloride diffusion test, which allowed calculating the predicted useful life of the concretes. The results highlight two important findings: (i) in optical microscopy, crystalline admixtures were not efficient in promoting self-healing on the surface of cracks in any of the studied concretes; (ii) the passage of chlorides by diffusion was lower for concretes with crystalline admixtures compared to the reference, showing better internal healing of these materials and, consequently, greater prediction of the concrete’s useful life.

Bleeding events in patients with cardiac amyloidosis

Abstract Background Amyloid cardiomyopathy (CA) is a life-threatening disease that arises from the accumulation of insoluble fibrous deposits of misfolded proteins in the myocardium. Infiltration of the conduction system results in a high prevalence of atrial fibrillation (AF). Due to a notable thromboembolic risk, long-term anticoagulation is preferred once AF is detected in amyloidosis. However, anticoagulation may exacerbate the hemorrhagic tendency, previously described in amyloid light-chain (AL) amyloidosis. Therefore, the potential benefits of anticoagulation must be carefully weighed against hemorrhagic complications. Methods We recorded bleeding events in a prospective cohort of CA patients. Results Out of 140 patients (median age of 73 years (IQR 62–77) and 77.1% of male gender) 65 (46.4%) were diagnosed with AL amyloidosis and 69 (49.3%) had transthyretin amyloid deposits (ATTR). Median NYHA functional class was 3 (IQR 2–3) and NT-proBNP 2574 pg/mL (1070–7173). 42.1% of patients (n=59) were treated with OAC with 13 (22.0%) receiving vitamin-K-antagonists (VKA) and 46 (78.0%) novel oral anticoagulants (NOACS). During a median follow up time of 23 months (IQR 9–35) 22 bleeding events occurred. In total, we recorded 1 cerebral bleeding, 7 gastrointestinal bleedings, 8 urogenital bleedings, 4 hematomas and 2 nasal bleedings. Of those, fifteen (68.2%) were observed in orally anticoagulated patients (p=0.198). 4 (30.8%) bleeding events occurred in the VKA treated group and 11 (23.9%) under NOAC treatment (p=0.335). Bleeding events were more prevalent in patients with AL compared to TTR amyloidosis (14, 63.6% vs 8, 36.4%, p=0.011). No difference was observed in terms of CHA2DS2-VASc score: 4 (IQR 2–5) in the bleeding group versus 3 (IQR 1–4) in the control group (p=0.579). On the contrary, HASBLED score was higher in patients with bleeding events: 4 (IQR 4–5) versus 2 (IQR 1–3) (p<0.001). Patients with bleeding events had a higher prevalence of arterial hypertension (p=0.010) and chronic kidney disease (p=0.002). Overall survival was comparable between the groupss (Figure 1). Conclusion Although CA is associated with potentially life-threatening hemorrhage, our cohort did not reveal a higher prevalence of fatal bleeding in patients treated with oral anticoagulation. Patients who experienced bleeding events did not show worse outcomes. Figure 1. Kaplan Meier survival estimates Funding Acknowledgement Type of funding source: None

Spatially Resolved Molecular Compositions of Insoluble Multilayer Deposits Responsible for Increased Pollution from Internal Combustion Engines.

Internal combustion engines are used heavily in diverse applications worldwide. Achieving the most efficient operation is key to improving air quality as society moves to a decarbonized energy system. Insoluble deposits that form within internal combustion engine components including fuel injectors and filters negatively impact CO2 and pollutant emissions. Understanding the composition, origins, and formation mechanisms of these complex materials will be key to their mitigation however, previous attempts only afforded nondiagnostic chemical assignments and limited knowledge toward this. Here, we uncover the identity and spatial distribution of molecular species from a gasoline direct injector, diesel injector, and filter deposit in situ using a new hyphenation of secondary ion mass spectrometry and the state-of-the-art Orbitrap mass analyzer (3D OrbiSIMS) and elemental analysis. Through a high mass resolving power and tandem MS we unambiguously uncovered the identity, distribution, and origin of species including alkylbenzyl sulfonates and provide evidence of deposit formation mechanisms including formation of longer chain sulfonates at the gasoline deposit's surface as well as aromatization to form polycyclic aromatic hydrocarbons up to C66H20, which were prevalent in the lower depth of this deposit. Inorganic salts contributed significantly to the diesel injector deposit throughout its depth, suggesting contamination over multiple fueling cycles. Findings will enable several strategies to mitigate these insoluble materials such as implementing stricter worldwide fuel specifications, modifying additives with adverse reactivity, and synthesizing new fuel additives to solubilize deposits in the engine, thereby leading to less polluting vehicles.

Correction of microtubule defects within Aβ plaque-associated dystrophic axons results in lowered Aβ release and plaque deposition.

The hallmark pathologies of the Alzheimer's disease (AD) brain are amyloid beta (Aβ)-containing senile plaques and neurofibrillary tangles formed from the microtubule (MT)-binding tau protein. Tau becomes hyperphosphorylated and disengages from MTs in AD, with evidence of resulting MT structure/function defects. Brain-penetrant MT-stabilizing compounds can normalize MTs and axonal transport in mouse models with tau pathology, thereby reducing neuron loss and decreasing tau pathology. MT dysfunction is also observed in dystrophic axons adjacent to Aβ plaques, resulting in accumulation of amyloid precursor protein (APP) and BACE1 with the potential for enhanced localized Aβ generation. We have examined whether the brain-penetrant MT-stabilizing compound CNDR-51657 might decrease plaque-associated axonal dystrophy and Aβ release in 5XFAD mice that develop an abundance of Aβ plaques. Administration of CNDR-51657 to 1.5-month-old male and female 5XFAD mice for 4 or 7 weeks led to decreased soluble brain Aβ that coincided with reduced APP and BACE1 levels, resulting in decreased formation of insoluble Aβ deposits. These data suggest a vicious cycle whereby initial Aβ plaque formation causes MT disruption in nearby axons, resulting in the local accumulation of APP and BACE1 that facilitates additional Aβ generation and plaque deposition. The ability of a MT-stabilizing compound to attenuate this cycle, and also reduce deficits resulting from reduced tau binding to MTs, suggests that molecules of this type hold promise as potential AD therapeutics.

Dual-functional AIE fluorescent probes for imaging β-amyloid plaques and lipid droplets

Alzheimer’s disease (AD) is a chronic neurodegenerative disease. Better imaging and early diagnosis of biomarkers of AD is extremely important for therapeutic interventions. The amyloid cascade hypothesis and its revised version identify insoluble β-amyloid deposition as a good diagnostic biomarker for AD. Moreover, lipid droplets may also act as an auxiliary biomarker related to AD pathology based on recent studies. Herein, two quinoline-based AIE probes were designed and synthesized for the imaging of Aβ plaques and lipid droplets. The probes exhibited remarkable turn-on fluorescence enhancements with the Aβ aggregates. The lipid droplets-targeting probe FB exhibited high selectivity and binding affinity towards the Aβ aggregates with a detection limit as low as 26.9 nM. Furthermore, FB was capable of readily imaging Aβ plaques and lipid droplets at the cellular level and in brain sections of transgenic AD mice. The probe FB can serve as a promising tool for developing early diagnosis and innovative therapeutics of AD.

Proteasomal degradation of the intrinsically disordered protein tau at single-residue resolution.

Intrinsically disordered proteins (IDPs) can be degraded in a ubiquitin-independent process by the 20S proteasome. Decline in 20S activity characterizes neurodegenerative diseases. Here, we examine 20S degradation of IDP tau, a protein that aggregates into insoluble deposits in Alzheimer's disease. We show that cleavage of tau by the 20S proteasome is most efficient within the aggregation-prone repeat region of tau and generates both short, aggregation-deficient peptides and two long fragments containing residues 1 to 251 and 1 to 218. Phosphorylation of tau by the non-proline-directed Ca2+/calmodulin-dependent protein kinase II inhibits degradation by the 20S proteasome. Phosphorylation of tau by GSK3β, a major proline-directed tau kinase, modulates tau degradation kinetics in a residue-specific manner. The study provides detailed insights into the degradation products of tau generated by the 20S proteasome, the residue specificity of degradation, single-residue degradation kinetics, and their regulation by posttranslational modification.

Amyloid Aggregation of Insulin: An Interaction Study of Green Tea Constituents

Exogenous insulin, used as a therapeutic agent for diabetes, forms insoluble deposits containing amyloid fibrillar structures near the administration site. We have analyzed the in vitro anti-amyloid activity of four green tea constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF), and their equimolar mixtures. Regarding individually tested compounds, only EGCG inhibited the fibrillization process. The individual EC, GA, and CF molecules were ineffective. The presence of EGCG in equimolar combinations with GA, EC, or CF was required for the inhibitory activity of most mixtures. Molecular docking revealed that EGCG interacts with an essential amyloidogenic region of insulin chain B. Individually inactive GA had a potentiating effect on the activity of EGCG. In contrast, EC and CF had a negative impact on the activity of the mixtures. We have observed diverse morphology and the amount of insulin amyloid aggregates formed in the presence of studied compounds. The distinct types of amyloid aggregates created in vitro in the presence of EGCG and other green tea constituents were characterized. Results indicate that the biological activity of individual molecules is not directly applicable to the pooled samples effects prediction.

Coiled-coil inspired functional inclusion bodies

BackgroundRecombinant protein expression in bacteria often leads to the formation of intracellular insoluble protein deposits, a major bottleneck for the production of soluble and active products. However, in recent years, these bacterial protein aggregates, commonly known as inclusion bodies (IBs), have been shown to be a source of stable and active protein for biotechnological and biomedical applications. The formation of these functional IBs is usually facilitated by the fusion of aggregation-prone peptides or proteins to the protein of interest, leading to the formation of amyloid-like nanostructures, where the functional protein is embedded.ResultsIn order to offer an alternative to the classical amyloid-like IBs, here we develop functional IBs exploiting the coiled-coil fold. An in silico analysis of coiled-coil and aggregation propensities, net charge, and hydropathicity of different potential tags identified the natural homo-dimeric and anti-parallel coiled-coil ZapB bacterial protein as an optimal candidate to form assemblies in which the native state of the fused protein is preserved. The protein itself forms supramolecular fibrillar networks exhibiting only α-helix secondary structure. This non-amyloid self-assembly propensity allows generating innocuous IBs in which the recombinant protein of interest remains folded and functional, as demonstrated using two different fluorescent proteins.ConclusionsHere, we present a proof of concept for the use of a natural coiled-coil domain as a versatile tool for the production of functional IBs in bacteria. This α-helix-based strategy excludes any potential toxicity drawback that might arise from the amyloid nature of β-sheet-based IBs and renders highly active and homogeneous submicrometric particles.

Cationic Carbosilane Dendritic Systems as Promising Anti-Amyloid Agents in Type 2 Diabetes.

The most common denominator of many of the neurodegenerative diseases is badly folded protein accumulation, which results in the formation of insoluble protein deposits located in different parts of the organism, causing cell death and tissue degeneration. Dendritic systems have turned out to be a promising new therapeutic approach for the treatment of these diseases due to their ability to modulate the folding of these proteins. With this perspective, and focused on type 2 diabetes (T2D), characterized by the presence of deposits containing the amyloidogenic islet amyloid polypeptide (IAPP), we demonstrate how different topologies of cationic carbosilane dendrimers inhibit the formation of insoluble protein deposits in pancreatic islets isolated from transgenic Tg-hIAPP mice. Also, the results obtained by the modification of dendritic carbosilane wedges with the chemical chaperone 4-phenylbutyric acid (4-PBA) at the focal point confirmed their potential as anti-amyloid agents with a concentration efficiency in their therapeutic action five orders of magnitude lower than that observed for free 4-PBA. Computational studies, which determined the main interaction between IAPP and dendrimers at the atomic level, support the experimental work.

Mechanistic insights into the thermal deposition of highly thermal-stable jet fuel

Highly thermal-stable jet fuel plays an important role in the advanced supersonic aircrafts, but its oxidation to the insoluble deposits is a serious problem when serving as a coolant. However, there are few reports as to the deep insights of insoluble deposits formation. Here we apply the PetroOxy device to investigate the thermal oxidation and deposition behaviors of highly thermal-stable jet fuel, using decalin as the model jet fuel, in order to explore the formation mechanism of insoluble deposits. All oxidation products (gas, liquid and solid) of fuel at different oxidation stages were separated and carefully characterized. The initial oxidation of fuel produces hydroperoxides, which then decompose rapidly to produce gas products (H2, CO or CO2) as well as large amounts of polar species (soluble oxidized products, e.g., alcohols, ketones, and molecular growth products). Meanwhile, these polar monomer species condense to produce soluble macromolecular oxidatively reactive species (SMORS) and insoluble deposits. The SMORS amount increases during the overall oxidation process, but the amount of insoluble deposits increases only in the presence of oxygen. The characterizations of liquid chromatography/electrospray ionization mass spectrometry and solution-state 2D heteronuclear single quantum coherence NMR confirm that the concentration of highly polar macromolecular species in insoluble deposits is much higher than that in SMORS, which causes the precipitation of the insoluble deposits from the bulk fuel. We further propose the detailed formation process and chemical structures of the insoluble deposits during the autoxidation of highly thermal-stable jet fuel, which would be helpful for a better understanding of the deposition mechanism of jet fuel and the improvement of its thermal stability.

Lipids Lay Thyselves Down: Redox-Induced Lipid Vesicle Fusion Onto Ferrocene-Terminated Self-Assembled Monolayers

Due to the attractive electrochemical properties of ferrocene (fast electron transfer, low oxidation potential, and two stable redox states), ferrocenylalkanethiolates on gold are the most studied and employed electroactive self-assembled monolayer (SAM) system. Oxidation of the SAM-confined ferrocenes to ferroceniums is accompanied by the formation of ion-pairs with counter-ions in solution [1,2]. Ferrocene-terminated SAMs exhibit a reversible electrochemistry in the presence of anionic surfactants such as n-alkyl sulfates and sulfonates, while the less chaotropic n-alkyl phosphates and carboxylates lead to the irreversible oxidation of the SAM-confined ferrocene (Fig. A) [3]. Cyclic voltammetry indicates that these latter surfactants form strong ion pairs with the oxidized ferroceniums; an oxidation peak is observed for the first anodic potential sweep but no reduction peak is detected during the reverse sweep. No redox peaks are observed in subsequent sweeps. The surface plasmon resonance signal does not return back to its original value during reduction, indicating an irreversible adsorption of material to the surface, which in turn suggests the formation of a stable and insoluble ferrocenium-surfactant anion complex or deposit that blocks the redox process. In this work, atomic force microscopy is used to investigate the association of sodium n-dodecanoate to the SAM surface. This electrochemical approach appears to be an effective strategy for triggering the formation of solid-supported lipid membranes by the fusion of phospholipid vesicles onto the ferrocenylalkanethiolate SAM. In fact, phospholipids are double-tailed amphiphiles and these can associate with the oxidized ferrocene (Fig. B). A potential-step hold at 0,64 V for only five minutes induces the immediate adsorption of phosphatidylserine (DMPS, POPS and DOPS) at the SAM-gold/electrolyte interface with a full surface coverage. This technique is a versatile tool that offers many benefits such as, a faster supported lipid bilayers formation mimicking the biological membranes, a larger variety of lipid composition, structure and chemical nature, and a possibility of using metal surfaces considering their potential applications in electrochemical techniques, spectroscopic techniques and surface plasmon resonance. In this work, AFM-based nano-shaving, force spectroscopy, and surface plasmon resonance are combined to determine the thickness of the obtained lipid membrane.

Hormonal Regulation of Novel Glycogen Metabolic Proteins Hari Priya Vemana, Ehab M. Abo‐Ali, John L. Croft, Shraddha Bhutkar, Michael Woolford, Vikas V. DukhandeDepartment of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA

Lafora disease (LD) is a rare autosomal recessive disorder manifesting as a progressive myoclonic epilepsy. LD is characterized by the formation of Lafora bodies, insoluble deposits containing abnormal glycogen‐like material in many tissues. LD is caused by mutations in the genes encoding dual‐specificity phosphatase laforin (EPM2A) or the E3 ubiquitin ligase malin (EPM2B / NHLRC1). Several research studies on LD have shown that laforin functions as a phosphatase for glycogen. Malin forms a functional complex with laforin and either malin alone or the laforin‐malin complex is known to ubiquitinate multiple proteins involved in glycogen metabolism. It is well established that hormones such as insulin regulates glycogen metabolism but hormonal regulation of laforin and malin is not completely elucidated. In type‐2 diabetes, defects in insulin responsiveness leads to a state called insulin resistance. Our objective is to study the effect of hormones and nutrients on novel glycogen metabolic proteins laforin and malin in physiology and diabetes pathophysiology. We used liver cells in culture and a rodent model of diabetes using high‐fat diet and low dose streptozotocin (HF‐STZ). We measured gene expression, protein expression, protein posttranslational modifications, and functional activities of laforin and malin. Our preliminary data indicates that both diabetic conditions and insulin modulate the expression of LD proteins. Our ongoing studies are directed towards delineating the molecular mechanisms of these alterations. This study can serve to find novel potential targets for the pharmacotherapy of type‐2 diabetes.Support or Funding InformationThis study is supported by an award from National Institute of General Medical Sciences of the National Institute of Health, Award number: SC2GM125550.

Dipeptide repeat derived from C9orf72 hexanucleotide expansions forms amyloids or natively unfolded structures in vitro

The abnormal repetition of the hexanucleotide GGGGCC within the C9orf72 gene is the most common genetic cause of both Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Different hypothesis have been proposed to explain the pathogenicity of this mutation. Among them, the production of aberrant proteins called Dipeptide Repeat Proteins (DPR) from the repeated sequence. Those proteins are of interest, as they are toxic and form insoluble deposits in patient brains. In this study, we characterized the structural features of three different DPR encoded by the hexanucleotide repeat GGGGCC, namely poly-GA, poly-GP and poly-PA. We showed that DPR are natively unstructured proteins and that only poly-GA forms in vitro fibrillary aggregates. Poly-GA fibrils are of amyloid nature as revealed by their high content in beta sheets. They neither bind Thioflavin T nor Primuline, the commonly used amyloid fluorescent dyes. Remarkably, not all of the poly-GA primary structure was part of fibrils amyloid core.

Strategically designed macromolecules as additives for high energy-density hydrocarbon fuels

To improve oxidative stability and heat sink of hydrocarbon fuels, a series of versatile macromolecular additives, BHPEI and CBHPEI, were synthesized by modification of hyperbranched poly(ethyleneimine) (HPEI). These macromolecules were used as circumstance-dependent additives in JP-10, a high energy–density hydrocarbon fuel. Each strategically designed macromolecule serves not only as a radical scavenger to improve the thermal-oxidative stability of hydrocarbon fuels at relatively low temperature, but also as a cracking initiator for heat sink enhancement as the temperature rises. According to the ASTM E1858, with the addition of BHPEI-10K at 500 ppm, the oxidation induction time of JP-10 under 175 °C could increase from 9.3 min to 14.8 min. The insoluble deposits could be reduced by 73% with the addition of 500 ppm BHPEI-10K according to accelerated oxidation tests. The superior antioxidation capability of macromolecular antioxidants were further elucidated by density functional theory (DFT) calculations. Furthermore, the BHPEI-10K performed decently as a macromolecular initiator for supercritical cracking of JP-10, with a dosage of 0.1 wt%, the conversion of JP-10 at 675 °C can be elevated from 16.4% to 33.4% and the corresponding heat sink can be improved from 1.98 MJ/kg to 2.15 MJ/kg.

Aluminum Surface Inclusions of Insoluble Lead Enhanced through Mechanical Attrition of Al Substrates

Preparation of aluminum substrates for surface segregation enhancement of insoluble lead deposition was achieved. Sever plastic deformation 'SPD' of Al sheets was done using surface mechanical attrition treatment 'SMAT' in air. Scanning electron microscope SEM of etched Al substrates cuts showed micro-cavities both on the surface and in-depth. Orientation effects and surface inclusions of Pb inside Al surface found at 40 and 50 Hz - SMAT Al by X-Ray diffraction and energy dispersive of X-Rays EDX. Concluding that SMAT frequency limits used enhanced surface inclusions without annealing that could improve adhesion of industrial protective Pb coatings.

BLINDED BY THE LIGHT: TRANSTHYRETIN CARDIAC AMYLOID MISDIAGNOSED AS AL AMYLOID

Cardiac amyloidosis is caused by insoluble protein deposition into the myocardium. Distinction between the different subtypes has significant management implications, thus emphasizing the importance of establishing an accurate diagnosis. A 75 year old male presented with anemia. Work up revealed

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals - Using Unconjugated Primary Antibodies from the Same Host Species.

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.

Microautophagy regulates proteasome homeostasis.

Proteasomes are highly abundant protein complexes that are responsible for most regulated protein degradation in cells under favorable growth conditions. When yeast cells are under nutritional stress, most proteasomes exit the nucleus and either accumulate in cytoplasmic condensates called proteasome storage granules (PSGs) or are directed to the vacuole by autophagy. Nitrogen starvation does not cause PSG formation but leads to degradation of proteasomes through the classical macroautophagy pathway. By contrast, carbon starvation or extended incubation in stationary phase results in both PSG formation and macroautophagy of proteasomes. Unexpectedly, we found that glucose limitation also causes proteasomes to be taken up directly into vacuoles by a microautophagy mechanism. Macro- and micro-autophagy occur in parallel in glucose-starved cells, and microautophagy appears biased toward aberrant or inactive proteasomes, leaving functional proteasomes to accumulate in PSGs. PSGs dissolve and proteasomes remobilize to the nucleus within minutes after glucose refeeding. We showed that AMP-activated protein kinase (AMPK) and endosomal-sorting-complex-required-for-transport (ESCRT) factors are required for proteasome microautophagy and also impact PSG dissipation and nuclear reimport of proteasomes after glucose refeeding. The insoluble protein deposit (IPOD) compartment provides an alternative means of proteasome homeostasis, including when microautophagy is impaired. Our findings reveal a surprising diversity of mechanisms for proteasome quality and quantity control during starvation. A mechanistic understanding of the AMPK-regulated ESCRT-mediated microautophagy pathway could provide new avenues for manipulating proteasome homeostasis and treating human disease.

Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre.

Systemic amyloidosis is a serious disease which is caused when normal circulating proteins misfold and aggregate extracellularly as insoluble fibrillary deposits throughout the body. This commonly results in cardiac, renal and neurological damage. The tissue target, progression and outcome of the disease depends on the type of protein forming the fibril deposit, and its correct identification is central to determining therapy. Proteomics is now used routinely in our centre to type amyloid; over the past 7 years we have examined over 2000 clinical samples. Proteomics results are linked directly to our patient database using a simple algorithm to automatically highlight the most likely amyloidogenic protein. Whilst the approach has proved very successful, we have encountered a number of challenges, including poor sample recovery, limited enzymatic digestion, the presence of multiple amyloidogenic proteins and the identification of pathogenic variants. Our proteomics procedures and approaches to resolving difficult issues are outlined.