3D-Printed Microcubes for Catalase Drug Delivery.

Oxidative stress, i.e., excessive production of reactive oxygen species (ROS), plays an important role in the pathogenesis of inflammatory diseases such as cardiovascular diseases, cancer, and neurodegenerative diseases. Catalase, an antioxidant enzyme, has great therapeutic potential; however, its efficacy is limited by its delivery to target cells or tissues. In order to achieve efficient delivery, consistent drug distribution, and drug activity, small and uniformly sized drug delivery vehicles are needed. Here, three-dimensional (3D) microcubes were printed by Nanoscribe Photonic Professional GT2, a high-resolution 3D printer, and the characteristics of 3D-printed microcubes as drug delivery vehicles for the delivery of catalase were investigated. The size of the 3D-printed microcubes was 800 nm in length of a square and 600 nm in height, which is suitable for targeting macrophages passively. Microcubes were also tunable in shape and size, and high-resolution 3D printing could provide microparticles with little variation in shape and size. Catalase was loaded on 3D-printed microcubes by nonspecific adsorption, and catalase on 3D-printed microcubes (CAT-MC) retained 83.1 ± 1.3% activity of intact catalase. CAT-MC also saved macrophages, RAW 264.7, from the cytotoxicity of H2O2 by 86.4 ± 4.1%. As drug delivery vehicles, 3D-printed microparticles are very promising due to their small and uniform size, which provides consistent drug distribution and drug activity. Therefore, we anticipate numerous applications of 3D-printed microparticles for delivering therapeutic proteins.

Alginate-Based Oral Delivery Systems to Enhance Protection, Release, and Absorption of Catalase.

Oxidative stress, overproduction of reactive oxygen species (ROS), plays an important role in the development of inflammatory bowel diseases. Catalase has great therapeutic potential by scavenging hydrogen peroxide, one of the ROSs produced in cellular metabolisms. However, in vivo application to scavenge ROS is currently limited especially in oral administrations. Here, we introduced an alginate-based oral drug delivery system that effectively protected catalase from the simulated harsh conditions of the gastrointestinal (GI) tract, released it in the small intestine mimicked condition, and enhanced its absorption via M cells, highly specialized epithelium cells in the small intestine. First of all, catalase was encapsulated in alginate-based microparticles with different amounts of polygalacturonic acid or pectin, which achieved an encapsulation efficiency of more than 90%. It was further shown that catalase was released from alginate-based microparticles in a pH-dependent manner. Results indicated that alginate-polygalacturonic acid microparticles (60 wt % Alg:40 wt % Gal) released 79.5 ± 2.4% of encapsulated catalase at pH 9.1 in 3 h, while they only released 9.2 ± 1.5% of encapsulated catalase at pH 2.0. Even when catalase was encapsulated in microparticles (60 wt % Alg:40 wt % Gal) and exposed to pH 2.0 followed by pH 9.1, it still retained 81.0 ± 11.3% enzyme activity compared to that in microparticles prior to the pH treatment. We then investigated the efficiency of RGD conjugation to catalase on the catalase uptake by M-like cells, the coculturing of human epithelial colorectal adenocarcinoma; Caco-2 cells and B lymphocyte; Raji cells. RGD-catalase protected M-cells more efficiently from the cytotoxicity of H2O2, a typical ROS. RGD conjugation to catalase enhanced the uptake by M-cells with 87.6 ± 0.8% RGD-catalase, whereas 11.5 ± 9.2% of RGD-free catalase passed across M-cells. From the results of protection, release, and absorption of model therapeutic proteins from the harsh pH conditions, alginate-based oral drug delivery systems will have numerous applications for the controlled release of drugs that are easily degradable in the GI tract.

Structural polymorphism and cytotoxicity of brain-derived ß-amyloid extracts.

To date, more than 37 amyloidogenic proteins have been found to form toxic aggregates that are implicated in the progression of numerous debilitating protein misfolding diseases including Alzheimer's disease (AD). Extensive literature highlights the role of ß-amyloid (Aß) aggregates in causing excessive neuronal cell loss in the brains of AD patients. In fact, major advances in our understanding of Aß aggregation process, including kinetics, toxicity, and structures of fibrillar aggregates have been revealed by examining in vitro preparations of synthetic Aß peptides. However, ongoing research shows that brain-derived Aß aggregates have specific characteristics that distinguish them from in vitro prepared species. Notably, the molecular structures of amyloid fibrils grown in the human brain were found to be markedly different than synthetic Aß fibrils. In addition, recent findings report the existence of heterogeneous Aß proteoforms in AD brain tissue in contrast to synthetically produced full-length aggregates. Despite their high relevance to AD progression, brain-derived Aß species are less well-characterized compared with synthetic aggregates. The aim of this review is to provide an overview of the literature on brain-derived Aß aggregates with particular focus on recent studies that report their structures as well as pathological roles in AD progression. The main motivation of this review is to highlight the importance of utilizing brain-derived amyloids for characterizing the structural and toxic effects of amyloid species. With this knowledge, brain-derived aggregates can be adopted to identify more relevant drug targets and validate potent aggregation inhibitors toward designing highly effective therapeutic strategies against AD.

Binding Behavior between Transforming-Growth-Factor-Beta1 and Its Receptor Reconstituted in Biomimetic Membranes.

Transforming growth factor ß1 (TGF-ß1) is critical to cell differentiation, proliferation, and apoptosis. It is important to understand the binding affinity between TGF-ß1 and its receptors. In this study, their binding force was measured using an atomic force microscope. Significant adhesion was induced by the interaction between the TGF-ß1 immobilized on the tip and its receptor reconstituted in the bilayer. Rupture and adhesive failure occurred at a specific force around 0.4~0.5 nN. The relationship of the force to loading rate was used to estimate the displacement where the rupture occurred. The binding was also monitored in real time with surface plasmon resonance (SPR) and interpreted with kinetics to acquire the rate constant. Using the Langmuir adsorption, the SPR data were analyzed to estimate equilibrium and association constants to be approximately 107 M-1 and 106 M-1 s-1. These results indicated that the natural release of the binding seldom occurred. Furthermore, the degree of binding dissociation, confirmed by the rupture interpretation, supported that the reverse of the binding hardly happened.

Detection of defects in cellular solids using highly nonlinear solitary waves: a numerical study of the proximal femur.

This study investigates the suitability of a relatively new non-destructive evaluation (NDE) technique for the detection of non-visible defects in cellular solids using highly nonlinear solitary waves (HNSWs) in a one-dimensional granular chain. Specifically, the HNSW-based NDE approach is employed to identify the existence of micro-fractures in trabecular bone within the femoral neck (FN) and the intertrochanteric (IT) region of the proximal femur which are fracture-prone sites due to their relatively low bone density, particularly in osteoporosis patients. The availability of a HNSW-based bone quality assessment tool could not only help in early diagnosis of osteoporosis but also affect surgical decisions and improve clinical outcomes in joint replacement surgeries which motivated this study. To obtain a realistic representation of the trabecular microstructure, high-resolution finite-element (FE) models of the FN and the IT region are first constructed using a topology optimization-based bone reconstruction scheme. Then, artificial defects in the form of fractured ligaments are generated in the FN and IT models by selectively disconnecting various struts within the trabecular network. Using the FE models as the inspection medium, hybrid discrete-element/finite-element (DE/FE) simulations are performed to examine the interaction of the HNSWs with the cellular bone samples through two different inspection modes, i.e., inspection via direct contact with the sample and indirect contact through an adequately chosen face sheet inserted between the cellular sample and the granular chain. The delays and amplitudes of the HNSWs are used to estimate the effective elastic moduli of the cellular samples and these estimates were found to be reasonably accurate only in case the face sheet was applied. For the latter case, it was shown that the HNSW-based modulus estimates can be used as indicators for defect detection, allowing to discern between pristine and damaged cellular solids. These results suggest that HNSW-based NDE is a reliable and cost-effective technique for the identification of defects in cellular solids, and is expected to find applications in various fields, such as non-invasive screening of bone diseases and fractures, or damage detection in additively manufactured cellular structures.

2D MXenes for controlled releases of therapeutic proteins.

MXenes belong to a new class of two dimensional (2D) functional nanomaterials, mainly encompassing transition-metal carbides, nitrides and carbonitrides, with unique physical, chemical, electronic and mechanical properties for various emerging applications across different fields. To date, the potentials of MXenes for biomedical application such as drug delivery have not been thoroughly explored due to the lack of information on their biocompatibility, cytotoxicity and biomolecule-surface interaction. In this study, we developed novel drug delivery system from MXene for the controlled release of a model therapeutic protein. First, the structural, chemical and morphological properties of as synthesized MXenes were probed with electron microscopy and X-ray diffraction. Second, the potential cytotoxicity of MXene toward the proliferation and cell morphology of murine macrophages (RAW 264.7) were evaluated with MTT assays and electron microscopy, respectively. Moreover, the drug loading capacities and sustained release capabilities of MXene were assessed in conjunction with machine learning approaches. Our results demonstrated that MXene did not significantly induce cellular toxicity at any concentration below 1 mg/ml which is within the range for effective dose of drug delivery vehicle. Most importantly, MXene was efficiently loaded with FITC-catalase for subsequently achieving controlled release under different pHs. The release profiles of catalase from MXene showed higher initial rate under basic buffer (pH 9) compared to that in physiological (pH 7.4) and acidic buffers (pH 2). Taken together, the results of this study lead to a fundamental advancement toward the use of MXene as a nanocarrier for therapeutic proteins in drug delivery applications.

Phosphorylation Induced Conformational Transitions in DNA Polymerase ß.

DNA polymerase ß (pol ß) is a member of the X- family of DNA polymerases that catalyze the distributive addition of nucleoside triphosphates during base excision DNA repair. Previous studies showed that the enzyme was phosphorylated in vitro with PKC at two serines (44 and 55), causing loss of DNA polymerase activity but not DNA binding. In this work, we have investigated the phosphorylation-induced conformational changes in DNA polymerase ß in the presence of Mg ions. We report a comprehensive atomic resolution study of wild type and phosphorylated DNA polymerase using molecular dynamics (MD) simulations. The results are examined via novel methods of internal dynamics and energetics analysis to reveal the underlying mechanism of conformational transitions observed in DNA pol ß. The results show drastic conformational changes in the structure of DNA polymerase ß due to S44 phosphorylation. Phosphorylation-induced conformational changes transform the enzyme from a closed to an open structure. The dynamic cross-correlation shows that phosphorylation enhances the correlated motions between the different domains. Centrality network analysis reveals that the S44 phosphorylation causes structural rearrangements and modulates the information pathway between the Lyase domain and base pair binding domain. Further analysis of our simulations reveals that a critical hydrogen bond (between S44 and E335) disruption and the formation of three additional salt bridges are potential drivers of these conformational changes. In addition, we found that two of these additional salt bridges form in the presence of Mg ions on the active sites of the enzyme. These results agree with our previous study of DNA pol ß S44 phosphorylation without Mg ions which predicted the deactivation of DNA pol ß. However, the phase space of structural transitions induced by S44 phosphorylation is much richer in the presence of Mg ions.

Linking Alzheimer's Disease and Type 2 Diabetes: Characterization and Inhibition of Cytotoxic Aß and IAPP Hetero-Aggregates.

The cytotoxic self-aggregation of ß-amyloid (Aß) peptide and islet amyloid polypeptide (IAPP) is implicated in the pathogenesis of Alzheimer's disease (AD) and Type 2 diabetes (T2D), respectively. Increasing evidence, particularly the co-deposition of Aß and IAPP in both brain and pancreatic tissues, suggests that Aß and IAPP cross-interaction may be responsible for a pathological link between AD and T2D. Here, we examined the nature of IAPP-Aß40 co-aggregation and its inhibition by small molecules. In specific, we characterized the kinetic profiles, morphologies, secondary structures and toxicities of IAPP-Aß40 hetero-assemblies and compared them to those formed by their homo-assemblies. We demonstrated that monomeric IAPP and Aß40 form stable hetero-dimers and hetero-assemblies that further aggregate into ß-sheet-rich hetero-aggregates that are toxic (cell viability <50%) to both PC-12 cells, a neuronal cell model, and RIN-m5F cells, a pancreatic cell model for ß-cells. We then selected polyphenolic candidates to inhibit IAPP or Aß40 self-aggregation and examined the inhibitory effect of the most potent candidate on IAPP-Aß40 co-aggregation. We demonstrated that epigallocatechin gallate (EGCG) form inter-molecular hydrogen bonds with each of IAPP and Aß40. We also showed that EGCG reduced hetero-aggregate formation and resulted in lower ß-sheets content and higher unordered structures in IAPP-Aß40-EGCG samples. Importantly, we showed that EGCG is highly effective in reducing the toxicity of IAPP-Aß40 hetero-aggregates on both cell models, specifically at concentrations that are equivalent to or are 2.5-fold higher than the mixed peptide concentrations. To the best of our knowledge, this is the first study to report the inhibition of IAPP-Aß40 co-aggregation by small molecules. We conclude that EGCG is a promising candidate to prevent co-aggregation and cytotoxicity of IAPP-Aß40, which in turn, contribute to the pathological link between AD and T2D.

Hydrogen phosphate selectively induces MDA MB 231 triple negative breast cancer cell death in vitro.

Phosphate ions are the most abundant anions inside the cells, and they are increasingly gaining attention as key modulators of cellular function and gene expression. However, little is known about the effect of inorganic phosphate ions on cancer cells, particularly breast cancer cells. Here, we investigated the toxicity of different phosphate compounds to triple-negative human breast cancer cells, particularly, MDA-MB-231, and compared it to that of human monocytes, THP-1. We found that, unlike dihydrogen phosphate (H2PO4-), hydrogen phosphate (HPO42-) at 20 mM or lower concentrations induced breast cancer cell death more than immune cell death, mainly via apoptosis. We correlate this effect to the fact that phosphate in the form of HPO42- raises pH levels to alkaline levels which are not optimum for transport of phosphate into cancer cells. The results in this study highlight the importance of further exploring hydrogen phosphate (HPO42-) as a potential therapeutic for the treatment of breast cancer.

Nanocomposite Conductive Bioinks Based on Low-Concentration GelMA and MXene Nanosheets/Gold Nanoparticles Providing Enhanced Printability of Functional Skeletal Muscle Tissues.

There is a growing need to develop novel well-characterized biological inks (bioinks) that are customizable for three-dimensional (3D) bioprinting of specific tissue types. Gelatin methacryloyl (GelMA) is one such candidate bioink due to its biocompatibility and tunable mechanical properties. Currently, only low-concentration GelMA hydrogels (≤5% w/v) are suitable as cell-laden bioinks, allowing high cell viability, elongation, and migration. Yet, they offer poor printability. Herein, we optimize GelMA bioinks in terms of concentration and cross-linking time for improved skeletal muscle C2C12 cell spreading in 3D, and we augment these by adding gold nanoparticles (AuNPs) or a two-dimensional (2D) transition metal carbide (MXene nanosheets) for enhanced printability and biological properties. AuNP and MXene addition endowed GelMA with increased conductivity (up to 0.8 ± 0.07 and 0.9 ± 0.12 S/m, respectively, compared to 0.3 ± 0.06 S/m for pure GelMA). Furthermore, it resulted in an improvement of rheological properties and printability, specifically at 10 °C. Improvements in electrical and rheological properties led to enhanced differentiation of encapsulated myoblasts and allowed for printing highly viable (97%) stable constructs. Taken together, these results constitute a significant step toward fabrication of 3D conductive tissue constructs with physiological relevance.

Effect of Exogenous pH on Cell Growth of Breast Cancer Cells.

Breast cancer is the most common type of cancer in women and the most life-threatening cancer in females worldwide. One key feature of cancer cells, including breast cancer cells, is a reversed pH gradient which causes the extracellular pH of cancer cells to be more acidic than that of normal cells. Growing literature suggests that alkaline therapy could reverse the pH gradient back to normal and treat the cancer; however, evidence remains inconclusive. In this study, we investigated how different exogenous pH levels affected the growth, survival, intracellular reactive oxygen species (ROS) levels and cell cycle of triple-negative breast cancer cells from MDA-MB-231 cancer cell lines. Our results demonstrated that extreme acidic conditions (pH 6.0) and moderate to extreme basic conditions (pH 8.4 and pH 9.2) retarded cellular growth, induced cell death via necrosis and apoptosis, increased ROS levels, and shifted the cell cycle away from the G0/G1 phase. However, slightly acidic conditions (pH 6.7) increased cellular growth, decreased ROS levels, did not cause significant cell death and shifted the cell cycle from the G0/G1 phase to the G2/M phase, thereby explaining why cancer cells favored acidic conditions over neutral ones. Interestingly, our results also showed that cellular pH history did not significantly affect the subsequent growth of cells when the pH of the medium was changed. Based on these results, we suggest that controlling or maintaining an unfavorable pH (such as a slightly alkaline pH) for cancer cells in vivo could retard the growth of cancer cells or potentially treat the cancer.

Predictions of the elastic modulus of trabecular bone in the femoral head and the intertrochanter: a solitary wave-based approach.

This paper deals with the numerical prediction of the elastic modulus of trabecular bone in the femoral head (FH) and the intertrochanteric (IT) region via site-specific bone quality assessment using solitary waves in a one-dimensional granular chain. For accurate evaluation of bone quality, high-resolution finite element models of bone microstructures in both FH and IT are generated using a topology optimization-based bone microstructure reconstruction scheme. A hybrid discrete element/finite element (DE/FE) model is then developed to study the interaction of highly nonlinear solitary waves in a granular chain with the generated bone microstructures. For more robust and reliable prediction of the bone's mechanical properties, a face sheet is placed at the interface between the last chain particle and the bone microstructure, allowing more bone volume to be engaged in the dynamic deformation during interaction with the solitary wave. The hybrid DE/FE model was used to predict the elastic modulus of the IT and FH by analysing the characteristic features of the two primary reflected solitary waves. It was found that the solitary wave interaction is highly sensitive to the elastic modulus of the bone microstructure and can be used to identify differences in bone density. Moreover, it was found that the use of a relatively stiff face sheet significantly reduces the sensitivity of the wave interaction to local stiffness variations across the test surface of the bone, thereby enhancing the robustness and reliability of the proposed method. We also studied the effect of the face sheet thickness on the characteristics of the reflected solitary waves and found that the optimal thickness that minimizes the error in the modulus predictions is 4 mm for the FH and 2 mm for the IT, if the primary reflected solitary wave is considered in the evaluation process. We envisage that the proposed diagnostic scheme, in conjunction with 3D-printed high-resolution bone models of an actual patient, could provide a viable solution to current limitations in site-specific bone quality assessment.

Multi-Compartment Lymph-Node-on-a-Chip Enables Measurement of Immune Cell Motility in Response to Drugs.

Organs On-a-Chip represent novel platforms for modelling human physiology and disease. The lymph node (LN) is a relevant immune organ in which B and T lymphocytes are spatially organized in a complex architecture, and it is the place where the immune response initiates. The present study addresses the utility of a recently designed LN-on-a-chip to dissect and understand the effect of drugs delivered to cells in a fluidic multicellular 3D setting that mimics the human LN. To do so, we analyzed the motility and viability of human B and T cells exposed to hydroxychloroquine (HCQ). We show that the innovative LN platform, which operates at a microscale level, allows real-time monitoring of co-cultured B and T cells by imaging, and supports cellular random movement. HCQ delivered to cells through a constant and continuous flow induces a reduction in T cell velocity while promotes persistent rotational motion. We also find that HCQ increases the production of reactive oxygen species in T cells. Taken together, these results highlight the potential of the LN-on-a-chip to be applied in drug screening and development, and in cellular dynamics studies.

Inhibition of SARS-CoV-2 Entry into Host Cells Using Small Molecules.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a virus belonging to the Coronavirus family, is now known to cause Coronavirus Disease (Covid-19) which was first recognized in December 2019. Covid-19 leads to respiratory illnesses ranging from mild infections to pneumonia and lung failure. Strikingly, within a few months of its first report, Covid-19 has spread worldwide at an exceptionally high speed and it has caused enormous human casualties. As yet, there is no specific treatment for Covid-19. Designing inhibitory drugs that can interfere with the viral entry process constitutes one of the main preventative therapies that could combat SARS-CoV-2 infection at an early stage. In this review, we provide a brief introduction of the main features of coronaviruses, discuss the entering mechanism of SARS-CoV-2 into human host cells and review small molecules that inhibit SARS-CoV-2 entry into host cells. Specifically, we focus on small molecules, identified by experimental validation and/or computational prediction, that target the SARS-CoV-2 spike protein, human angiotensin converting enzyme 2 (ACE2) receptor and the different host cell proteases that activate viral fusion. Given the persistent rise in Covid-19 cases to date, efforts should be directed towards validating the therapeutic effectiveness of these identified small molecule inhibitors.

Numerical predictions of the interaction between highly nonlinear solitary waves and the microstructure of trabecular bone in the femoral head.

The unique properties of highly nonlinear solitary waves in granular chains have prompted extensive research in the area of non-destructive testing and led to the development of new diagnostic schemes with potential applications in the healthcare industry. Here, we study numerically the interaction between highly nonlinear solitary waves in a granular chain and the microstructure of trabecular bone in the femoral head. High-resolution finite element models of bone microstructures with varying bone volume fraction are generated using a topology optimization-based bone microstructure reconstruction scheme. The obtained FE models of the trabecular bone were then used to develop a hybrid discrete/finite element model able to simulate the propagation of highly nonlinear solitary waves in a vertical array of steel particles, and their interaction with the adjacent bone microstructure model was studied. Two test modes were considered, one where the granular chain was placed in direct contact with the bone microstructure model, while in the second test mode, a face sheet was included between the chain and the bone model. For both test modes, we found that the characteristic features of the reflected solitary waves are sensitive to the effective compressive modulus of the bone microstructure models and follow similar trends than those obtained for a homogeneous, non-porous solid. It was also found that the use of the face sheet substantially reduces the sensitivity of the predictions to small changes in the bone topology, making it a robust and reliable method for non-destructive evaluation of the effective elastic modulus of cellular materials with small structural dimensions, as it is required for the site-specific evaluation of the mechanical properties of trabecular bone.

Aggregation and Cellular Toxicity of Pathogenic or Non-pathogenic Proteins.

More than 20 unique diseases such as diabetes, Alzheimer's disease, Parkinson's disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, ß-amyloid (Aß), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins.

Inhibition of lysozyme aggregation and cellular toxicity by organic acids at acidic and physiological pH conditions.

The misfolding of proteins can lead to fibrillar and non-fibrillar deposits that are the hallmark of numerous human diseases. Inhibition of protein aggregation is considered as a promising strategy for the prevention of such diseases. Here we induced the fibrillar and non-fibrillar aggregates of hen egg white lysozyme (HEWL) at acidic (pH 3) and physiological (pH 7.4) environments. HEWL formed non-fibrillar aggregates rapidly at pH 7.4, whereas fibrillar HEWL aggregates were formed slowly at pH 3. Both fibrillar and non-fibrillar aggregates had cytotoxic effects on PC12 cells. Next, four organic acids, succinic acid, maleic acid, tartaric acid and citric acid, were tested for their inhibition potencies against fibrillar and non-fibrillar HEWL species. The four inhibitors were found to prevent the aggregation of HEWL at pH 7.4 with a reduction rate of over 95% as compared with the reduction rate of 42-58% for HEWL aggregation at pH 3. Other biophysical and computational analyses reveal that the candidate inhibitors have higher inhibition efficacy against HEWL monomers incubated at pH 7.4 than at pH 3. These results emphasize the importance of validating the newly identified aggregation drugs against different aggregate species, which would enhance the understanding of small molecules-induced protein aggregation inhibition.

Computational and Experimental Approaches to Design Inhibitors of Amylin Aggregation.

Amylin is a neuroendocrine peptide hormone secreted by pancreatic ß-cells; however, amylin is toxic to ß-cells when it is aggregated in type 2 diabetes mellitus (T2DM). It is important to understand amylin's structures and aggregation mechanism for the discovery and design of effective drugs to inhibit amylin aggregation. In this review, we investigated experimental and computational studies on amylin structures and inhibitors. Our review provides some novel insights into amylin, particularly for the design of its aggregation inhibitors to treat T2DM. We detailed the potential inhibitors that have been studied hitherto and highlighted the neglected need to consider different amylin attributes that depend on the presence/absence of physiologically relevant conditions, such as membranes. These conditions and the experimental methods can greatly influence the results of studies on amylininhibitor complexes. Text-mining over 3,000 amylin-related PubMed abstracts suggests the combined therapeutic potential of amylin with leptin and glucagon-like peptide-1, which are two key hormones in obesity. The results also suggest that targeting amylin aggregation can contribute to therapeutic efforts for Alzheimer's disease (AD). Therefore, we have also reviewed the role of amylin in other conditions including obesity and AD. Finally, we provided insights for designing inhibitors of different types (small molecules, proteins, peptides/mimetics, metal ions) to inhibit amylin aggregation.

Genetic Associations With Diabetic Retinopathy and Coronary Artery Disease in Emirati Patients With Type-2 Diabetes Mellitus.

Aim: Type 2 Diabetes Mellitus (T2DM) is associated with both microvascular complications such as diabetic retinopathy (DR), and macrovascular complications like coronary artery disease (CAD). Genetic risk factors have a role in the development of these complications. In the present case-control study, we investigated genetic variations associated with DR and CAD in T2DM patients from the United Arab Emirates. Methods: A total of 407 Emirati patients with T2DM were recruited. Categorization of the study population was performed based on the presence or absence of DR and CAD. Seventeen Single Nucleotide Polymorphisms (SNPs), were selected for association analyses through search of publicly available databases, namely GWAS catalog, infinome genome interpretation platform and GWAS Central database. A multivariate logistic regression test was performed to evaluate the association between the 17 SNPs and DR, CAD, or both. To account for multiple testing, significance was set at p < 0.00294 using the Bonferroni correction. Results: The SNPs rs9362054 near the CEP162 gene and rs4462262 near the UBE2D1 gene were associated with DR (OR = 1.66, p = 0.001; OR = 1.37, p = 0.031; respectively), and rs12219125 near the PLXDC2 gene was associated (suggestive) with CAD (OR = 2.26, p = 0.034). Furthermore, rs9362054 near the CEP162 gene was significantly associated with both complications (OR = 2.27, p = 0.0021). The susceptibility genes for CAD (PLXDC2) and DR (UBE2D1) have a role in angiogenesis and neovascularization. Moreover, association between the ciliary gene CEP162 and DR was established in terms of retinal neural processing, confirming previous reports. Conclusions: The present study reports associations of different genetic loci with DR and CAD. We report new associations between CAD and PLXDC2, and DR with UBE2D1 using data from T2DM Emirati patients.

Inhibition of Human Amylin Aggregation and Cellular Toxicity by Lipoic Acid and Ascorbic Acid.

More than 30 human degenerative diseases result from protein aggregation such as Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Islet amyloid deposits, a hallmark in T2DM, are found in pancreatic islets of more than 90% of T2DM patients. An association between amylin aggregation and reduction in ß-cell mass was also established by post-mortem studies. A strategy in preventing protein aggregation-related disorders is to inhibit the protein aggregation and associated toxicity. In this study, we demonstrated that two inhibitors, lipoic acid and ascorbic acid, significantly inhibited amylin aggregation. Compared to amylin (15 µM) as 100%, lipoic acid and ascorbic acid reduced amylin fibril formation to 42.1 ± 17.2% and 42.9 ± 12.8%, respectively, which is confirmed by fluorescence and TEM images. In cell viability tests, both inhibitors protected RIN-m5f ß-cells from the toxicity of amylin aggregates. At 10:1 molar ratio of lipoic acid to amylin, lipoic acid with amylin increased the cell viability to 70.3%, whereas only 42.8% RIN-m5f ß-cells survived in amylin aggregates. For ascorbic acid, an equimolar ratio achieved the highest cell viability of 63.3% as compared to 42.8% with amylin aggregates only. Docking results showed that lipoic acid and ascorbic acid physically interact with amylin amyloidogenic region (residues Ser20-Ser29) via hydrophobic interactions; hence reducing aggregation levels. Therefore, lipoic acid and ascorbic acid prevented amylin aggregation via hydrophobic interactions, which resulted in the prevention of cell toxicity in vitro.