Effects of Citicoline,Homotaurine, and Vitamin E on Contrast Sensitivity and Visual-Related Quality of Life in Patients with Primary Open-Angle Glaucoma: A Preliminary Study

The aim of the present study was to evaluate the effects of supplementation with a fixed combination of citicoline 500 mg, homotaurine 50 mg, and vitamin E 12 mg (CIT/HOMO/VITE) on contrast sensitivity and visual-related quality of life in patients with primary open-angle glaucoma (POAG) in mild stage. This was a multicenter, observational, cross-over, short-term, pilot study on POAG patients with stable controlled intraocular pressure (IOP). Patients were randomly assigned to Group 1 (current topical therapy for 4 months and then current topical therapy plus CIT/HOMO/VITE for 4 months) or Group 2 (CIT/HOMO/VITE in addition to current topical therapy for 4 months and then topical therapy alone for 4 months). Best-corrected visual acuity, IOP, visual field, and the Spaeth/Richman contrast sensitivity (SPARCS) test score were recorded at baseline and after 4 and 8 months. The Glaucoma Quality of Life-15 (GQL-15) questionnaire was administered at each check time. Forty-four patients were assigned to Group 1 and 65 to Group 2. Over the follow-up period, there were no significant changes in IOP or visual field findings, whereas SPARCS and GQL-15 findings significantly varied from baseline, both being improved in subjects treated with CIT/HOMO/VITE fixed combination. These results demonstrate that a daily intake of a fixed combination of citicoline, homotaurine, and vitamin E in addition to the topical medical treatment significantly increased the total score of the contrast sensitivity test and the quality of life in patients with POAG.     

Positive Association between Aqueous Humor Hydroxylinoleate Levels and Intraocular Pressure

We previously proposed the total assessment of hydroxylinoleates (HODEs) by LC-MS/MS after saponification and reduction of the biologic samples as biomarkers to investigate pathogenesis, disease progression, and prognosis. In this study, HODE levels were estimated in aqueous humor (AH) samples from 63 eyes (41 Japanese subjects; 15 men; mean age, 77.3 ± 6.8 years) with primary open-angle glaucoma (POAG) or cataracts. The correlations between intraocular HODE levels and background parameters, including intraocular pressure (IOP), were analyzed to assess the possible involvement of oxidative stress in glaucoma pathology. Univariate analyses showed that linoleic acid (LA) (p = 0.034) and arachidonic acid (AA) (p = 0.0041) levels were associated negatively with age; 13-(Z,E)-HODE (p = 0.018) and 13-(E,E)-HODE (p = 0.021) were associated positively with IOP; 9-(Z,E)-HODE (p = 0.039), 13-(Z,E)-HODE (p = 0.021), totally assessed-HODE (t-HODE, p = 0.023), LA (p = 0.0080), and AA (p = 0.0051) were higher in eyes with glaucoma than cataract. No gender differences were seen. A mixed-effect regression model showed that higher 13-(Z,E)-HODE (p = 0.0040) and higher t-HODE (p = 0.040) were associated with glaucoma rather than cataracts; and higher levels of 13-(Z,E)-HODE/LA (p = 0.043), 13-(E,E)-HODE/LA (p = 0.042), 13-(Z,E)-HODE (p = 0.0054), and 13-(E,E)-HODE (p = 0.027) were associated with higher IOP. Linoleate-derived oxidation products were quantified successfully in AH samples from patients with glaucoma and cataracts. A free radical oxidation mechanism can be associated with IOP elevation, while enzymatic oxidation may be involved, specifically, in the pathogenesis of POAG.     

Gliadin effect on fluctuation properties of phospholipid giant vesicles

Gliadin is a fraction of wheat gluten, a protein supramolecular complex known for its remarkable and biotechnologically relevant viscoelastic properties. Very high molecular mass characterise these systems, thus hindering high-resolution structural investigations. It is known, however, that these proteins comprise rather extended, extensively interassociated structures, which respond for their peculiar mechanical behaviour. Besides these properties, some of gluten's fractions, such as gliadin, are also known to be involved in a nutritionally relevant pathology of auto-immune character, the celiac disease, supposedly related to some unusual structural features of the protein. Despite its medical relevance, however, the role played by gliadin in the etiology of the celiac disease is not sufficiently understood to date. In this work, we investigated the role of gliadin on mechanical properties of a membrane model of dioleoylphosphatidylcholine (DOPC) giant unilamellar vesicles. The technique of micropipette aspiration, coupled to videomicroscopy, was applied. The microvesicles, produced by electric field pulsing over metal-covered plates, were suctioned into the micropipettes under varying applied pressures. A significant increase in the values of the bilayer curvature constant, k(c), was observed, with a saturation effect being verified at around 0.02-0.03 gliadin/DOPC mass ratio, indicating that the membrane becomes less elastic in the presence of the protein. Possible correlations between the observed membrane fluctuation properties and the celiac disease etiology are suggested and discussed.     

Study of the human plasma proteome of rheumatoid arthritis

In this study, we report a combined proteomic and peptidomic analysis of human plasma from patients with rheumatoid arthritis (RA) and controls. We used molecular weight cut-off filters (MWCO: 10 kDa) to enrich low-molecular-weight (LMW) peptides from human plasma. The peptide fraction was analyzed without trypsin digestion by capillary reversed-phase high-performance liquid chromatography (HPLC) coupled to a linear ion trap–FT-MS system, which identified 771 unique peptides in the peptidome study (from 145 protein progenitors). An anti-albumin/anti-IgG column was used to remove albumin and immunoglobulin G (IgG) from the human plasma. After that, the albumin/IgG-depleted sample was fractionated into a bound fraction and an unbound fraction on a multi-lectin affinity column (M-LAC). LC–MS analysis of the corresponding tryptic digests identified 308 proteins using the M-LAC approach. Relative differences in the following protein classifications were observed in the RA human plasma samples compared with controls: structural proteins, immuno-related proteins, protease inhibitors, coagulation proteins, transport proteins and apolipoproteins. While some of these proteins/peptides have been previously reported to be associated with RA disease such as calgranulin A, B, C and C-reactive protein, several others were newly identified (such as thymosin β4, actin, tubulin, and vimentin), which may further the understanding of the disease pathogenesis. Moreover, we have found that the peptidomic and glycoproteomic approaches were complementary and allow a more complete picture of the human plasma proteome which can be valuable in studies of disease etiology.     

Pan-ROCK and ROCK2 Inhibitors Affect Dexamethasone-Treated 2D- and 3D-Cultured Human Trabecular Meshwork (HTM) Cells in Opposite Manners

Effects of a pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025 on dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells as a model of steroid-induced glaucoma were investigated. In the presence of Rip or KD025, DEX-treated HTM cells were subjected to permeability analysis of 2D monolayer by transendothelial electrical resistance (TEER) and FITC–dextran permeability, physical properties, size and stiffness analysis (3D), and qPCR of extracellular matrix (ECM), and their modulators. DEX resulted in a significant increase in the permeability, as well as a large and stiff 3D spheroid, and those effects were inhibited by Rip. In contrast, KD025 exerted opposite effects on the physical properties (down-sizing and softening). Furthermore, DEX induced several changes of gene expressions of ECM and their modulators were also modulated differently by Rip and KD025. The present findings indicate that Rip and KD025 induced opposite effects toward 2D and 3D cell cultures of DEX-treated HTM cells.     

Parsing disease‐relevant protein modifications from epiphenomena: perspective on the structural basis of SOD1‐mediated ALS

Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well‐understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease‐relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease‐associated mutations and post‐translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn‐superoxide dismutase (SOD1). Previous genetic studies discovered >180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using hydrogen–deuterium exchange with mass spectrometry, we determined that diverse disease‐associated SOD1 mutations cause a common structural defect – perturbation of the SOD1 electrostatic loop. X‐ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta‐barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ~15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ~80% of ALS is not associated with a gene defect) could be caused by post‐translational protein modification of wild‐type SOD1. We developed immunocapture antibodies and high sensitivity top‐down MS methods and characterized PTMs of wild‐type SOD1 using human tissue samples. Using global hydrogen–deuterium exchange, X‐ray crystallography and neurotoxicology, we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof‐of‐concept that post‐translational modification can cause disease. We show that numerous mutations (N➔D; Q➔E), which result in the same chemical structure as the PTM deamidation, cause multiple diseases. Copyright © 2017 John Wiley & Sons, Ltd.     

Investigating and characterizing the binding activity of the immobilized calmodulin to calmodulin-dependent protein kinase I binding domain with atomic force microscopy

Protein–protein interactions are responsible for many biological processes, and the study of how proteins undergo a conformational change induced by other proteins in the immobilized state can help us to understand a protein’s function and behavior, empower the current knowledge on molecular etiology of disease, as well as the discovery of putative protein targets of therapeutic interest. In this study, a bottom-up approach was utilized to fabricate micro/nanometer-scale protein patterns. One cysteine mutated calmodulin (CaM), as a model protein, was immobilized on thiol-terminated pattern surfaces. Atomic Force Microscopy (AFM) was then employed as a tool to investigate the interactions between CaM and CaM kinase I binding domain, and show that the immobilized CaM retains its activity to interact with its target protein. Our work demonstrate the potential of employing AFM to the research and assay works evolving surface-based protein–protein interactions biosensors, bioelectronics or drug screening.     

In-silico studies of glutathione peroxidase4 activators as candidate for multiple sclerosis management

Multiple sclerosis (MS) is an autoimmune and inflammatory demyelinating disease of the central nervous system (CNS) that affects approximately 2.8 million people worldwide. Although numerous studies have been conducted to investigate novel therapeutic targets and lead compounds, few drug choices are available to treat MS patients. The etiology of this disease is still poorly understood. However, oxidative stress is proposed as one of the underlining pathology. The neuronal antioxidant enzyme glutathione peroxidase 4 (GPx4) is responsible for scaffolding toxic peroxide phospholipids and reducing neuronal death within the CNS. Therefore, screening for lead compounds able to activate this essential enzyme might protect neuronal cells from damage and slow the disease progression. This study aimed to identify potential activators of GPx4, an essential inhibitor to ferroptosis, as a novel neuroprotective strategy in MS treatment. For understanding the binding of the four selected compounds to GPX4 protein showing the mechanism of the interaction, molecular docking analysis and molecular dynamic (MD) simulation were used. The study was carried out through various computational methods using Autodock Vina for docking of the protein and ligand and Desmond for MD simulation. The four tested compounds used to activate GPx4 are as follows: ferrostatin, lapatinib, liproxstatin-1, and PKUMDL-LDL-102. Results showed that the lapatinib had greater log P value (6.17) which indicates higher permeability through blood brain barrio (BBB) to exirt the proposed neurological effect. In the molecular docking analysis, the best docking scores was displayed by Lapatinib (?7.6 kcal/mol). Ferrostatin, Lapatinib, and Liproxstatin-1 almost bind in the similar sites of the target protein, while PKUMDL-LC-102 binds at a different site. Furthermore, MD simulation study showed a stable system for lapatinib and liproxstatin-1 as confirmed by RMSD and RMSF values during 100 ns trajectories. Additionally, the most negative ΔG Bind score (the lowest) which considered the best was exhibited by lapatinib (?47.52 Kcal/mol). The test compounds were further inspected for their intersction with GPx4 in terms of hydrophobic, hydrogen and other bonding types beside the stability of these bonds by observing the protein–ligand contact within 100 ns trajectories. Interestingly, the receptor–ligand complex showed deep continuous bands for Lapatinib with Lys127 and Gly128. In conclusion, among the four studied compounds Lapatinib could be a promising scaffold for developing effective leads capable of activating GPx4 and assist in the treatment of MS.     

Photosensitizing Activity of Endogenous Eye Lens Chromophores: An Attempt to Unravel Their Contributions to Photo‐Aging and Cataract Disease

UVA‐visible light has been proposed as a risk factor in the photo‐aging of the human eye lens, as well as in the etiology of cataract disease. There is accumulating evidence indicating that photosensitizing reactions mediated by endogenous chromophores, which are generated during human eye lens aging, can play an important role in the generation of these processes. These reactions can lead to protein impairment by inducing non‐enzymatic post‐translational modifications such as protein oxidation and crosslinking. Although numerous chromophores have been characterized as both bound to human eye lens proteins and as unbound low‐molecular‐mass compounds, their contribution to eye lens photoaging and cataract disease is not completely understood. In this article we discuss the photochemical contribution of UV‐filters derived from tryptophan catabolism and advanced glycation end products (AGEs) to human eye lens aging and cataract disease. We also discuss the recently described photosensitizing capacity of chromophores derived from newly discovered glucose and ascorbate degradation as a parallel pathway to their role in AGEs generation.     

Biophysical,Biochemical, and Molecular Docking Investigations of Anti-Glycating,Antioxidant, and Protein Structural Stability Potential of Garlic

Garlic has been reported to inhibit protein glycation, a process that underlies several disease processes, including chronic complications of diabetes mellitus. Biophysical, biochemical, and molecular docking investigations were conducted to assess anti-glycating, antioxidant, and protein structural protection activities of garlic. Results from spectral (UV and fluorescence) and circular dichroism (CD) analysis helped ascertain protein conformation and secondary structure protection against glycation to a significant extent. Further, garlic showed heat-induced protein denaturation inhibition activity (52.17%). It also inhibited glycation, advanced glycation end products (AGEs) formation as well as lent human serum albumin (HSA) protein structural stability, as revealed by reduction in browning intensity (65.23%), decrease in protein aggregation index (67.77%), and overall reduction in cross amyloid structure formation (33.26%) compared with positive controls (100%). The significant antioxidant nature of garlic was revealed by FRAP assay (58.23%) and DPPH assay (66.18%). Using molecular docking analysis, some of the important garlic metabolites were investigated for their interactions with the HSA molecule. Molecular docking analysis showed quercetin, a phenolic compound present in garlic, appears to be the most promising inhibitor of glucose interaction with the HSA molecule. Our findings show that garlic can prevent oxidative stress and glycation-induced biomolecular damage and that it can potentially be used in the treatment of several health conditions, including diabetes and other inflammatory diseases.     

Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).     

Advances in Molecular Pathology of Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) is a common syndrome that features a complex etiology and set of mechanisms. Here we summarized the molecular pathogenesis of OSA, especially the prospective mechanism of upper? airway dilator fatigue and the current breakthroughs. Additionally, we also introduced the molecular mechanism of OSA in terms of related studies on the main signaling pathways and epigenetics alterations, such as microRNA, long non-coding RNA, and DNA methylation. We also reviewed small molecular compounds, which are potential targets for gene regulations in the future, that are involved in the regulation of OSA. This review will be beneficial to point the way for OSA research within the next decade.     

Methods for generating protein molecular ions in ToF-SIMS

One of the greatest challenges in mass spectrometry lies in the generation and detection of molecular ions that can be used to directly identify the protein from the molecular weight of the molecular ion. Typically, proteins are large (MW > 1000), nonvolatile, and/or thermally labile, but the vaporization process produced by many mass spectrometry techniques including time-of-flight secondary ion mass spectrometry (ToF-SIMS) is inherently limited to generating ions from smaller compounds or fragments of the parent molecule, making the identification of proteins complex. The application of specific molecules to aid in the generation of high molecular weight ions in ToF-SIMS has been recognized for some time. In this study we have developed a matrix-SAM substrate preparation technique based on the self-assembly of a matrix-like molecule, mercaptonicotinic acid (MNA), on gold. We then compare this substrate with two existing ToF-SIMS sample preparation techniques, cationized alkane thiol and matrix-enhanced SIMS (MESIMS). The results of this study illustrate that while there is a range of methods that can be used to improve the molecular ion yield of proteins in ToF-SIMS, their efficacy and reproducibility vary considerably and crucially are linked to the sample preparation and/or protein application methods used. Critically, the MNA modified substrate was able to simultaneously induce molecular ions for each protein present in a multicomponent solution, suggesting that this sample preparation technique may have future application in proteomics and DNA analysis.     

Inorganic nanoparticles as carriers of nucleic acids into cells

The transfer of nucleic acids (DNA or RNA) into living cells, that is, transfection, is a major technique in current biochemistry and molecular biology. This process permits the selective introduction of genetic material for protein synthesis as well as the selective inhibition of protein synthesis (antisense or gene silencing). As nucleic acids alone are not able to penetrate the cell wall, efficient carriers are needed. Besides viral, polymeric, and liposomal agents, inorganic nanoparticles are especially suitable for this purpose because they can be prepared and surface-functionalized in many different ways. Herein, the current state of the art is discussed from a chemical viewpoint. Advantages and disadvantages of the available methods are compared.     

Multi-scale top-down approach for modelling epileptic protein-protein interaction network analysis to identify driver nodes and pathways

Protein - Protein Interaction Network (PPIN) analysis unveils molecular level mechanisms involved in disease condition. To explore the complex regulatory mechanisms behind epilepsy and to address the clinical and biological issues of epilepsy, in silico techniques are feasible in a cost- effective manner. In this work, a hierarchical procedure to identify influential genes and regulatory pathways in epilepsy prognosis is proposed. To obtain key genes and pathways causing epilepsy, integration of two benchmarked datasets which are exclusively devoted for complex disorders is done as an initial step. Using STRING database, PPIN is constructed for modelling protein-protein interactions. Further, key interactions are obtained from the established PPIN using network centrality measures followed by network propagation algorithm -Random Walk with Restart (RWR). The outcome of the method reveals some influential genes behind epilepsy prognosis, along with their associated pathways like PI3 kinase, VEGF signaling, Ras, Wnt signaling etc. In comparison with similar works, our results have shown improvement in identifying unique molecular functions, biological processes, gene co-occurrences etc. Also, CORUM provides an annotation for approximately 60% of similarity in human protein complexes with the obtained result. We believe that the formulated strategy can put-up the vast consideration of indigenous drugs towards meticulous identification of genes encoded by protein against several combinatorial disorders.     

Amyloidosis in Alzheimer’s Disease: Pathogeny,Etiology, and Related Therapeutic Directions

The amyloid hypothesis of Alzheimer’s disease has long been the predominant theory, suggesting that Alzheimer’s disease is caused by the accumulation of amyloid beta protein (Aβ) in the brain, leading to neuronal toxicity in the central nervous system (CNS). Because of breakthroughs in molecular medicine, the amyloid pathway is thought to be central to the pathophysiology of Alzheimer’s disease (AD). Currently, it is believed that altered biochemistry of the Aβ cycle remains a central biological feature of AD and is a promising target for treatment. This review provides an overview of the process of amyloid formation, explaining the transition from amyloid precursor protein to amyloid beta protein. Moreover, we also reveal the relationship between autophagy, cerebral blood flow, ACHE, expression of LRP1, and amyloidosis. In addition, we discuss the detailed pathogenesis of amyloidosis, including oxidative damage, tau protein, NFTs, and neuronal damage. Finally, we list some ways to treat AD in terms of decreasing the accumulation of Aβ in the brain.     

Trident Nano-Indexing the Proteomics Table: Next-Version Clustering of Iron Carbide NPs and Protein Corona

Protein corona composition and precise physiological understanding of differentially expressed proteins are key for identifying disease biomarkers. In this report, we presented a distinctive quantitative proteomics table of molecular cell signaling differentially expressed proteins of corona that formed on iron carbide nanoparticles (NPs). High-performance liquid chromatography/electrospray ionization coupled with ion trap mass analyzer (HPLC/ESI-Orbitrap) and MASCOT helped quantify 142 differentially expressed proteins. Among these proteins, 104 proteins showed upregulated behavior and 38 proteins were downregulated with respect to the control, whereas 48, 32 and 24 proteins were upregulated and 8, 9 and 21 were downregulated CW (control with unmodified NPs), CY (control with modified NPs) and WY (modified and unmodified NPs), respectively. These proteins were further categorized on behalf of their regularity, locality, molecular functionality and molecular masses using gene ontology (GO). A STRING analysis was used to target the specific range of proteins involved in metabolic pathways and molecular processing in different kinds of binding functionalities, such as RNA, DNA, ATP, ADP, GTP, GDP and calcium ion bindings. Thus, this study will help develop efficient protocols for the identification of latent biomarkers in early disease detection using protein fingerprints.     

THE REACTION OF SINGLET OXYGEN WITH PROTEINS, WITH SPECIAL REFERENCE TO CRYSTALLINS

Photosensitized oxidation of the eye lens proteins, the crystallins, is thought to lead to protein crosslinks and high molecular weight aggregates. Such protein modifications may be important factors in the formation of lens opacities or cataracts. We focus attention here on type 2 photo-oxidation involving the reaction of singlet oxygen (1O2) with crystallins and some "control" proteins. We find that: (1) trp residues are oxidized to N-formyl kynurenine and related products, but this in itself does not lead to the production of high molecular weight protein aggregates of the protein; (2) tyr residues react with 1O2 but we do not detect dihydroxyphenylalanine or bityrosine nor are protein crosslinks formed as a result; (3) oxidation of his residues appears necessary for high molecular weight protein covalent aggregates to form. Proteins devoid of his, e.g. melittin or bovine pancreatic trypsin inhibitor, do not form high molecular weight products upon reaction with 1O2. Prior reaction and blocking of his inhibits the crosslinking reactions. (4) The oxidized protein is seen to be more acidic than the parent and has an altered tertiary structure. (5) Among the crystallins, reactivity towards 1O2 varies in the order gamma greater than beta greater than alpha and also gamma A/E greater than gamma D greater than gamma B crystallin.     

低分子量紫色酸性磷酸酶多克隆抗体的制备及应用

将最近从地瓜中提出来的低分子量紫色酸性磷酸酶（smPAP)基因克隆到GST融合蛋白表达载体pGEX－2T中，在大肠杆菌BL21codon plus中进行表达，用表达的融合蛋白免疫兔子产生多克隆抗体，用抗血清在昆虫细胞中表达的smPAP和地瓜提取液中分离纯化的PAP进行检测，产生了良好的交叉反应。     

Electrophoresis of mutant proteins in inherited diseases

The electrophoretic approaches for detection of mutant proteins in inherited diseases are briefly reviewed and discussed. Mutation of a protein, known to be associated with a specific inherited disease, is detected by immunoblotting, immunoprecipitation or enzyme staining, combined with various electrophoretic techniques. Some instrumental and technological devices for two-dimensional electrophoresis have been reported for the screening of mutant proteins in diseases of currently unknown etiology.