Structural insights into the pathogenicity of point mutations in human acyl-CoA dehydrogenase homotetramers.

Acyl-CoA dehydrogenase deficiency (ACAD) is an inherited and potentially fatal disorder with variable clinical symptoms. The relationship between pathogenicity and deleterious point mutations is investigated here in ACAD structures of short (SCAD) and medium-chain (MCAD) types. Structures and dynamic features of native and mutant forms of enzymes models were compared. A total of 2.88 µs molecular dynamics simulations were performed at four different temperatures. Total energy, RMSD, protein ligand interactions and affinity, RMSF measures, secondary structure changes, and important interactions were studied. Mutations in the three main domains of ACADs are pathogenic, while those located at linker turns are not. Mutations affect mostly tetramer formations, secondary structures, and many contacts and interactions. In R206H (MCAD mutant) which is experimentally known to cause a huge turnover decrease, the lack of a single H-bond between substrate and FAD was observed. Secondary structures showed temperature-dependent changes, and SCAD activity was found to be highly correlated to the enzyme helix 3-10 content. Finally, RMSF patterns pointed to one important loop that maintains the substrate close to the active site and is a cause of substrate wobbling upon mutation. Despite similar structure, function, and cellular location, SCAD and MCAD may have different optimum temperatures that are related to the structure taken at that specific temperature. In conclusion, new insight has been provided on the effect of various SCAD and MCAD pathogenic mutations on the structure and dynamical features of the enzymes.

Local Insulin-Derived Amyloidosis Model Confronted with Silymarin: Histological Insights and Gene Expression of MMP, TNF-α, and IL-6.

Amyloidosis is a heterogeneous group of protein deposition diseases associated with the presence of amyloid fibrils in tissues. Analogs of insulin that are used for treating diabetic patients (including regular insulin) can form amyloid fibrils, both in vitro and in vivo as reported in patients. The main purpose of this study was the induction of localized insulin-generated amyloidosis and the observation of silymarin effects on this process. In order to obtain amyloid structures, regular insulin was incubated at 37 °C for 24 h. Congo red absorbance and transmission electron microscopy images validated the formation of amyloid fibrils. Those fibrils were then injected subcutaneously into rats once per day for 6, 12 or 18 consecutive days in the presence or absence of silymarin, and caused development of firm waxy masses. These masses were excised and stained with Hematoxylin and Eosin, Congo red and Thioflavin S. Histological examination showed adipose cells and connective tissue in which amyloid deposition was visible. Amyloids decreased in the presence of silymarin, and the same effect was observed when silymarin was added to normal insulin and injected subsequently. Furthermore, plasma concentrations of MMP2, TNF-α, and IL-6 inflammatory factors were measured, and their gene expression was locally assessed in the masses by immunohistochemistry. All three factors increased in the amyloidosis state, while silymarin had an attenuating effect on their plasma levels and gene expression. In conclusion, we believe that silymarin could be effective in counteracting insulin-generated local amyloidosis.

5-[Aryloxypyridyl (or nitrophenyl)]-4H-1,2,4-triazoles as novel flexible benzodiazepine analogues: Synthesis, receptor binding affinity and lipophilicity-dependent anti-seizure onset of action.

A new series of 5-(2-aryloxy-4-nitrophenyl)-4H-1,2,4-triazoles and 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-triazoles, possessing C-3 thio or alkylthio substituents, was synthesized and evaluated for their benzodiazepine receptor affinity and anti-seizure activity. These analogues revealed similar to significantly superior affinity to GABAA/benzodiazepine receptor complex (IC50 values of 0.04-4.1 nM), relative to diazepam as the reference drug (IC50 value of 2.4 nM). To determine the onset of anti-seizure activity, the time-dependent effectiveness of i.p. administration of compounds on pentylenetetrazole induced seizure threshold was studied and a very good relationship was observed between the lipophilicity (cLogP) and onset of action of studied analogues (r2 = 0.964). The minimum effective dose of the compounds, determined at the time the analogues showed their highest activity, was demonstrated to be 0.025-0.1 mg/kg, relative to diazepam (0.025 mg/kg).

Aspergillus Section Flavi from Four Agricultural Products and Association of Mycotoxin and Sclerotia Production with Isolation Source.

Many agricultural products are susceptible to contamination by aflatoxin-producing species from Aspergillus section Flavi. The objectives of this study were to determine the occurrence of Aspergillus section Flavi in four agricultural products, such as pistachio, walnut, hazelnut, and dried fruits, collected from market and retail shops in various areas of Kerman County and obtain information on the relationships between isolation source and ability to produce sclerotia and potential for aflatoxin production. Aspergillus species were identified based on morphological characteristics as well as subsequent sequencing of the parts of the ß-tubulin and calmodulin genes. From 207 isolated strains, the following species were identified: A. flavus, A. tamarii A. nomius, A. parasiticus, A. arachidicola, A. caelatus, A. pseudotamarii, and A. leporis. To the best of our knowledge, this is the first report of A. pseudotamarii and A. arachidicola with the potential to produce aflatoxins from dried apricots and hazelnuts, respectively. Sclerotial type was significantly different between isolates from different isolation sources. From 192 tested isolates, 38% were aflatoxin producer from which 5% were scored as strong aflatoxin producers and 33% as average aflatoxin producers. A significant difference in the population of aflatoxin-producing strains across the isolation sources was observed which may reflect host adaptation and thereby different vulnerabilities to aflatoxin-producing species among the examined products.

Investigating the role of endogenous opioid system in chloroquine-induced phospholipidosis in rat liver by morphological, biochemical and molecular modelling studies.

Drug-induced phospholipidosis (DIPL) is characterized by phospholipid storage in the lysosomes of affected tissues. Many severe effects and toxicities have been linked to DIPL. The aim of this study was to determine whether the endogenous opioid system is involved in chloroquine-induced phospholipidosis. The effect of naltrexone as an antagonist of opioid receptors in chloroquine-induced phospholipidosis in rat liver was investigated by morphological, biochemical, and molecular modelling studies. Transmission electron microscopy (TEM) showed that morphological characteristic changes of rat liver, including the number of lamellar bodies, grade of vacuolization and cell steatosis, were markedly attenuated in rats treated with naltrexone alone or in combination with chloroquine, in comparison with chloroquine-treated rats. The results of liquid chromatography mass spectrometry (LC/MS) showed that the concentrations of phenylacetylglycine (PAG) and hippuric acid (HA) were significantly decreased and increased, respectively, in target groups. Besides, the concentration ratio of PAG/HA was significantly decreased. Spectrophotometry resulted in a notable decrease in alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities in target groups. The results from the molecular docking and molecular dynamic simulation studies demonstrated clear chloroquine interaction with the active site cavity of the µ opioid receptor. These data suggest that administration of naltrexone alone, or in combination with chloroquine, notably attenuates the side effects of chloroquine-induced phospholipidosis, as well as demonstrating an increased probability of the endogenous opioid system involvement in chloroquine-induced phospholipidosis in rat liver.

Monocyclic phenolic compounds stabilize human insulin and suppress its amorphous aggregation: In vitro and in vivo study.

Insulin is a small protein with 51 residues that mediates glucose uptake, and an interesting model for studying protein misfolding and aggregation. The aggregated forms of insulin undergo loss of activity and can provoke unwanted immune responses. Use of small molecules is considered to be an affordable method to counteract this aggregation process and stabilize insulin. In this study, aggregated forms of human recombinant insulin have been produced following exposure to high temperature. Aggregation process was followed over time by checking absorbance with spectrophotometry in presence and absence of various concentrations of small phenolic compounds including eugenol and epinephrine. Effects of these compounds on the structure and function of incubated insulin were evaluated by spectrofluorimetry, melting temperature (Tm) measurement and insulin tolerance test on Wistar rats. Formation of heat-induced insulin aggregation can be effectively inhibited by 1 mM eugenol and epinephrine and both compounds were found to preserve insulin activity to a considerable extent. In conclusion, simple aromatic compounds could be tailored to act as potent anti-aggregation compounds for insulin.

Influence of dendrimer surface chemistry and pH on the binding and release pattern of chalcone studied by molecular dynamics simulations.

Poly(amidoamine) (PAMAM) dendrimers are promising nanocarriers that can enhance the solubility of hydrophobic drugs. The surface chemistry of dendrimers is of great relevance as end groups of these nanocarriers can be easily modified to improve the bioavailability and sustained release of the cargo. Therefore, a molecular-level understanding of the host-guest interactions that can give both qualitative and quantitative information is particularly desirable. In this work, fully atomistic molecular dynamics simulations were used to study the association of a bioactive natural product, ie, chalcone, with amine-, acetyl-, and carboxyl-terminated PAMAM dendrimers at physiological and acidic pH environments. Amine- and carboxyl-terminated PAMAM dendrimers have an open microstructure at low pH that is not able to hold the ligand tightly, resulting in an unfavorable encapsulation of the chalcone molecule. In the case of acetyl-terminated dendrimer, chalcone molecule diffuses out of the dendritic cavities a few times during the simulation time and prefers to locate close to the surface of dendrimer. Average center of mass distance values at neutral pH showed that the chalcone molecule bounds firmly in the internal pockets of amine-, acetyl-, and carboxyl-terminated dendrimers and forms stable complexes with these nanovectors. The potential of mean force calculations showed that the release of the ligand from the dendrimers occurs at a controlled rate in the body.

Characterization of surface binding sites in glycoside hydrolases: A computational study.

Structural properties of carbohydrate surface binding sites (SBSs) were investigated with computational methods. Eighty-five SBSs of 44 enzymes in 119 Protein Data Bank (PDB) files were collected as a dataset. On the basis of SBSs shape, they were divided into 3 categories: flat surfaces, clefts, and cavities (types A, B, and C, respectively). Ligand varieties showed the correlation between shape of SBSs and ligands size. To reduce cut-off differences in each SBSs with different ligand size, molecular docking were performed. Molecular docking results were used to refine SBSs classification and binding sites cut-off. Docking results predicted putative ligands positions and displayed dependence of the ligands binding mode to the structural type of SBSs. Physicochemical properties of SBSs were calculated for all docking results with YASARA Structure. The results showed that all SBSs are hydrophilic, while their charges could vary and depended to ligand size and defined cut-off. Surface binding sites type B had highest average values of solvent accessible surface area. Analysis of interactions showed that hydrophobic interactions occur more than hydrogen bonds, which is related to the presence of aromatic residues and carbohydrates interactions.

Atomistic computer simulations on multi-loaded PAMAM dendrimers: a comparison of amine- and hydroxyl-terminated dendrimers.

Poly(amidoamine) (PAMAM) dendrimers have been extensively studied as delivery vectors in biomedical applications. A limited number of molecular dynamics (MD) simulation studies have investigated the effect of surface chemistry on therapeutic molecules loading, with the aim of providing insights for biocompatibility improvement and increase in drug loading capacity of PAMAM dendrimers. In this work, fully atomistic MD simulations were employed to study the association of 5-Fluorouracil (5-FU) with amine (NH2)- and hydroxyl (OH)-terminated PAMAM dendrimers of generations 3 and 4 (G3 and G4). MD results show a 1:12, 1:1, 1:27, and 1:4 stoichiometry, respectively, for G3NH2-FU, G3OH-FU, G4NH2-FU, and G4OH-FU complexes, which is in good agreement with the isothermal titration calorimetry results. The results obtained showed that NH2-terminated dendrimers assume segmented open structures with large cavities and more drug molecules can encapsulate inside the dendritic cavities of amine terminated dendrimers. However, OH-terminated have a densely packed structure and therefore, 5-FU drug molecules are more stable to locate close to the surface of the dendrimers. Intermolecular hydrogen bonding analysis showed that 5-FU drug molecules have more tendency to form hydrogen bonds with terminal monomers of OH-terminated dendrimers, while in NH2-terminated these occur both in the inner region and the surface. Furthermore, MM-PBSA analysis revealed that van der Waals and electrostatic energies are both important to stabilize the complexes. We found that drug molecules are distributed uniformly inside the amine and hydroxyl terminated dendrimers and therefore, both dendrimers are promising candidates as drug delivery systems for 5-FU drug molecules.

Investigation of thymol effect on learning and memory impairment induced by intrahippocampal injection of amyloid beta peptide in high fat diet- fed rats.

Obesity and consumption of a high fat diet (HFD) are known to increase the risk of Alzheimer's disease (AD). In the present study, we have examined the protective and therapeutic effects of thymol (main monoterpene phenol found in thyme essential oil) on a HFD-fed rat model of AD. Fourty adult male Wistar rats were randomly assigned to 5 groups:(n = 8 rats/group): group 1, control, consumed an ordinary diet, group 2 consumed a HFD for 8 weeks, then received phosphate-buffered saline (PBS) via intrahippocampal (IHP) injection, group 3 consumed HFD for 8 weeks, then received beta-amyloid (Aß)1-42 via IHP injections to induce AD, group 4 consumed HFD for 8 weeks, then received Aß1-42, and was treated by thymol (30 mg/kg in sunflower oil) daily for 4 weeks, and group 5 consumed HFD for 8 week, then received Aß1-42 after what sunflower oil was administered by oral gavage daily for 4 weeks. Biochemical tests showed an impaired lipid profile and higher glucose levels upon consumption of HFD, which was ameliorated by thymol treatment. In behavioral results, spatial memory in group 3 was significantly impaired, but groups treated with thymol showed better spatial memory compared to group 3 (p ≤ 0.01). In histological results, formation of Aß plaque in hippocampus of group 3 increased significantly compared to group 1 and group 2 (p ≤ 0.05), but group 4 showed decreased Aß plaques compared to group 3 (p ≤ 0.01). In conclusion, thymol decreased the effects of Aß on memory and could be considered as neuroprotective.

Turmeric effect on subcutaneous insulin-induced amyloid mass: an in vivo study.

Protein-derived amyloid structures are associated with a wide variety of pathologies, including neurodegenerative diseases and local amyloidoses. Reports exist on the ability of insulin to form local amyloidoses under specific conditions. In vitro-generated fibrils of insulin have been previously shown to produce amyloid-containing masses upon repetitive subcutaneous injection in mouse. The present study aimed at investigating the effect of insulin fibrils injection in rats, as well as the potential of turmeric in attenuating this process. It was found that subcutaneous amyloid-containing masses could form in rats at a faster rate compared with mice. Upon addition of turmeric to the fibrils, previous to injection, formed masses had a significantly reduced size, as well as less ordered cellular structure. In conclusion, the results of this study show the potential of turmeric in attenuation of local amyloidosis. Furthermore, we suggest that this model could be of use in screening antiamyloid compounds.

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact.

Injection of insulin amyloid fibrils in the hippocampus of male Wistar rats: report on memory impairment and formation of amyloid plaques.

Amyloid fibrils result from a particular type of protein aggregation, and have been linked with various disorders, including neurodegenerative ones. In the case of Alzheimer's disease, amyloid beta (abeta) fibrils are detected in patients' brain, in the amyloid plaques. These fibrils can be produced in vitro, and their injection into animals' brains generates an animal model of Alzheimer's disease. Based on the structural similarity of amyloid fibrils that are formed from different proteins, we hypothesized that injecting insulin amyloid fibrils into rats' brains could result in amyloid plaque formation. Fourteen male Wistar rats were divided into control and experimental groups (n = 7). The experimental group was bilaterally injected with insulin amyloid in the hippocampus. Seven days after injection, a shuttle box test was performed and the experimental group's memory was found to be impaired. Histological investigation of these rats' brain showed the formation of amyloid plaques in the hippocampus. A limited test has provided preliminary evidence for the stability of these plaques up to 35 days. Further complementary studies are required to fully validate the proposed procedure, which is simple and relatively low cost, and could be suggested as an alternative to models generated with abeta fibrils.

Rational design of stable and functional hirudin III mutants with lower antigenicity.

Hirudin is an inhibitor of thrombin and used as an effective anticoagulant, but has a potential to develop unacceptable immune responses. In this study, two computational tools were used to predict T-cell epitopes within Hirudin variant III (HVIII) sequence, and design mutations that would lessen its antigenicity. Homology models of native and mutant HVIII proteins (T4K, S9G, V21G, and V21K) were generated, and further used to assess their interactions with thrombin. The docking experiment showed that all mutants had a suitable pattern of interactions, with similar or lower interaction energies compared with the native protein. These complexes were subsequently subjected to molecular dynamics simulation. All mutants complexes had overall stable structures over simulation time, with RMSD, gyration radius, hydrogen bonds numbers, and accessible surface areas patterns that were comparable with the native HVIII over time. Interestingly, in all mutants, a shorter length was observed for the two salt bridges Arg73-Asp55 and Arg77-Glu57, which are suggested to be important in Hirudin-thrombin complex formation. Best selected mutants expressed in Escherichia coli BL21(DE3), subsequently SDS-PAGE and Western blot analysis confirmed the successful same expression of Hirudin and mutants. In conclusion, we believe that this computational approach could identify potentially safer proteins with preserved or even improved functionality.

Structural stability of myoglobin and glycomyoglobin: a comparative molecular dynamics simulation study.

Glycoproteins are formed as the result of enzymatic glycosylation or chemical glycation in the body, and produced in vitro in industrial processes. The covalently attached carbohydrate molecule(s) confer new properties to the protein, including modified stability. In the present study, the structural stability of a glycoprotein form of myoglobin, bearing a glucose unit in the N-terminus, has been compared with its native form by the use of molecular dynamics simulation. Both structures were subjected to temperatures of 300 and 500 K in an aqueous environment for 10 ns. Changes in secondary structures and RMSD were then assessed. An overall higher stability was detected for glycomyoglobin, for which the most stable segments/residues were highlighted and compared with the native form. The simple addition of a covalently bound glucose is suggested to exert its stabilizing effect via increased contacts with surrounding water molecules, as well as a different pattern of interactions with neighbor residues.

Interaction of Aß(25-35) fibrillation products with mitochondria: Effect of small-molecule natural products.

The 25-35 fragment of the amyloid ß (Aß) peptide is a naturally occurring proteolytic by-product that retains the pathophysiology of its larger parent molecule, whose deposition has been shown to involve mitochondrial dysfunction. Hence, disruption of Aß(25-35) aggregates could afford an effective remedial strategy for Alzheimer's disease (AD). In the present study, the effect of a number of selected small-molecule natural products (polyphenols: resveratrol, quercetin, biochanin A, and indoles: indole-3-acetic acid, indole-3-carbinol (I3C)) on Aß(25-35) fibrillogenesis was explored under physiological conditions, and interaction of the resulting structures with rat brain mitochondria was investigated. Several techniques, including fluorescence, circular dichroism, and transmission electron microscopy were utilized to characterize the aggregation products, and possible mitochondrial membrane permeabilization was determined following release of marker enzymes. Results demonstrate the capacity of Aß(25-35) fibrils to damage mitochondria and suggest how small molecules may afford protection. While I3C appeared more effective in inhibiting the fibrillation process, all natural products behaved similarly in destabilizing preformed aggregates. It is concluded that elucidation of such protection may provide important insights into the development of preventive and therapeutic agents for AD.

Silymarin effect on amyloid-ß plaque accumulation and gene expression of APP in an Alzheimer's disease rat model.

BACKGROUND: The deposition of amyloid peptides is associated with Alzheimer's disease (AD). These amyloid peptides are derived from the amyloid protein precursor (APP). Silymarin, a standardized extract of milk thistle, which is currently used in liver diseases, may be effective in the inhibition of amyloid formation. However, its effect has not been assessed on APP expression. RESULTS: In this study, first, the effect of silymarin was examined on the passive avoidance learning in a rat model of AD. This model was induced by the intracerebroventricular injection of Aß peptide (Aß1-42) in Wistar rats. Rats were treated with 70 and 140 mg/kgof the extract, once a day, for 4 weeks. Memory function that was evaluated in a shuttle-cage test, showed improvement upon administration of this extract. Brain amyloid plaques had also decreased upon administration of the extract. Furthermore, APP gene expression was compared in treated and untreated groups. The result showed that silymarin was able to suppress APP expression. CONCLUSION: Our results are in accordance with the in vitro tests concerning the positive antiamyloidogenic property of the main component of silymarin, namely silibinin. We suggest that the beneficial effect of sylimarin in the AD model is related to its capacity to disaggregate amyloid plaques and to suppress APP expression. Considering the limited side effects of silymarin, this compound could be of use in AD therapy.

Phenylalanine as an effective stabilizer and aggregation inhibitor of Bacillus amyloliquefaciens alpha-amylase.

Aromatic compounds are known anti-amyloid aggregates. Their effect on amorphous aggregates of proteins is, however, less studied. We chose aromatic amino acids Trp, Tyr, and Phe, as well as another known stabilizer (i.e. Arg), as potential compatible solvents to be tested on Bacillus amyloliquefaciens alpha-amylase (BAA). Among these additives, Phe was the only one to be effective on the thermal inactivation and amorphous aggregation of BAA, while preserving its intrinsic activity. A concentration of 50 mM Phe was used to test its potential in counteracting the deleterious effect of BAA amorphous aggregates in vivo. After 21 days of daily subcutaneous injections of the native enzyme to mice, amorphous aggregates of BAA, as well as aggregates produced in presence of 50 mM Phe, the tissues located at the site of injection were studied histologically. Amorphous aggregates caused an increase in macrophages and lipid droplets. Serum levels of IL6 and TNF-α were also accordingly elevated and indicative of an inflammation state. Aggregates also resulted into increased levels of glucose, triglycerides and cholesterol, as well as liver enzymes SGOT and SGPT. On the other hand, the presence of Phe prevented this exacerbated inflammatory state and the subsequent impairment of biochemical parameters. In conclusion, Phe is an interesting compound for both stabilizing proteins and counteracting the pathological effect of amorphous aggregates.

Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

Comprehensive investigation of insulin-induced amyloidosis lesions in patients with diabetes at clinical and histological levels: A systematic review.

INTRODUCTION: Insulin-derived amyloidosis (AIns), a skin complication in patients with diabetes, causes impaired insulin absorption. This systematic review aims to get a better understanding of this overlooked condition. METHODS: Comprehensive literature searches were performed in Scopus, PubMed, EMBASE, and Web of Science databases until June 17, 2023. From 19,343 publications, duplicate and irrelevant records were eliminated by title, and the full texts of the remaining studies were examined for validity. Clinical, pathological, and therapeutic findings were extracted from 44 papers. RESULTS: Forty-four articles were studied that covered 127 insulin-treated patients with diabetes. From the 62 patients with reported age and sex, males had a mean age of 58 years, and females 68.5 years. While AIns were twice as likely to develop in men (66.13 %) as in women (33.87 %), the administered insulin dose was significantly higher in males (p = 0.017). The most common insulin injection site was the abdominal wall (77.63 %). Histological findings showed the presence of amorphous material with the occasional presence of lymphocytes, plasma cells, macrophages, adipocytes, histocytes, and giant cells. The mean HbA1c level was 8.8 % and the need for receiving insulin was increased in AIns. Changing the site of insulin injections and/or surgically removing the nodules were the most common treatments to obtain better insulin uptake and controlled serum glucose levels. CONCLUSION: This study highlights the importance of AIns, proper rotation of insulin injection site, and post-treatment patient follow-up to recognize and prevent the development of amyloid nodules.