The Spectrum of α-Thalassemia Mutations in Kermanshah Province, West Iran.

Thalassemia is a hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin (Hb) polypeptide chains. Although thalassemia mostly affects developing countries, there is limited knowledge of its accurate frequency and distribution in these regions. Knowing the prevalence of thalassemia and the frequency of responsible mutations is therefore an important step in the prevention and control program as well as treatment strategies. α-Thalassemia (α-thal) is prevalent in Middle East Asian populations, including Iran. In this study, 678 unrelated α-thal carriers, attending the Kermanshah Medical Genetics Laboratory, Kermanshah, Iran, were investigated for α-globin gene mutations by multiplex polymerase chain reaction (PCR) and direct sequencing. The most common mutation among our patients was -α(3.7) (rightward) (60.9%) deletion, which is also known to occur in high frequencies in other parts of Iran, in Southeast Asia and Mediterranean countries. Other prevalent α-thal mutations were α(-5 nt) (10.6%), α(polyA4) (9.9%), α(polyA6) (3.7%), - -(MED) (3.2%), -α(4.2) (leftward) (3.1%) deletion and codon 59 (Hb Adana; HBA1: c.179 G > A) (2.5%). These comprehensive new data are useful for establishing a screening strategy for the effective control of α-thal in Kermanshah Province.

"Cyclophilin A" Enzymatic Effect on the Aggregation Behavior of 1N4R Tau Protein: An Overlooked Crucial Determinant that should be Re-considered in Alzheimer's Disease Pathogenesis.

BACKGROUND: Neurodegenerative disorders like Alzheimer's disease (AD) involve the abnormal aggregation of tau protein, which forms toxic oligomers and amyloid deposits. The structure of tau protein is influenced by the conformational states of distinct proline residues, which are regulated by peptidyl-prolyl isomerases (PPIases). However, there has been no research on the impact of human cyclophilin A (CypA) as a PPIase on (non-phosphorylated) tau protein aggregation. METHODS: On the basis of these explanations, we used various spectroscopic techniques to explore the effects of CypA on tau protein aggregation behavior. RESULTS: We demonstrated the role of the isomerization activity of CypA in promoting the formation of tau protein amyloid fibrils with well-defined and highly ordered cross-ß structures. According to the "cistauosis hypothesis," CypA's ability to enhance tau protein fibril formation in AD is attributed to the isomerization of specific proline residues from the trans to cis configuration. To corroborate this theory, we conducted refolding experiments using lysozyme as a model protein. The presence of CypA increased lysozyme aggregation and impeded its refolding process. It is known that proper refolding of lysozyme relies on the correct (trans) isomerization of two critical proline residues. CONCLUSION: Thus, our findings confirmed that CypA induces the trans-to-cis isomerization of specific proline residues, ultimately leading to increased aggregation. Overall, this study highlights the emerging role of isomerization in tau protein pathogenesis in AD.

Synthesis of 4-sulfamoyl phenyl diazocarboxylic acid derivatives as novel non-classical inhibitors of human carbonic anhydrase II activity: an in vitro study.

The first class of carbonic anhydrase inhibitors (CAIs) discovered was sulfonamides, but their clinical use is limited due to side effects caused by their inhibition of multiple CA isoforms. To overcome this, researchers have focused on developing isoform-selective CAIs. This study involved the synthesis and characterization of novel carboxylic acid/sulfonamide derivatives. We investigated the interaction between these compounds and the human carbonic anhydrase II (hCA II) isoform using spectroscopic and computational methods. The synthesized compounds were evaluated based on their IC50, Kd and Ki values, and it was found that the inhibitory potency and binding affinity of the compounds increased with the number of carboxylic acids zinc binding groups. Specifically, the compound C4, with three carboxylic acid groups, showed the strongest inhibitory potency. Fluorescence measurements revealed that all compounds quenched the intrinsic fluorescence of hCA II through a dynamic quenching process, and each compound had one binding site in the hCA II structure. Thermodynamic analysis indicated hydrogen bonds and van der Waals interactions played key roles in the binding of these compounds to hCA II. Docking studies showed that the carboxylic acid groups directly attached to the zinc ion in the active site, displacing water/hydroxide ions and causing steric hindrance. Overall, the strengthening of inhibitory activity and the binding power of these carboxylic acid derivatives for the hCA II makes these compounds interesting for designing novel hCA II inhibitors.Communicated by Ramaswamy H. Sarma.

Flavonoid compounds and their synergistic effects: Promising approaches for the prevention and treatment of psoriasis with emphasis on keratinocytes - A systematic and mechanistic review.

Psoriasis, a chronic autoimmune skin disorder, causes rapid and excessive skin cell growth due to immune system dysfunction. Numerous studies have shown that flavonoids have anti-psoriatic effects by modulating various molecular mechanisms involved in inflammation, cytokine production, keratinocyte proliferation, and more. This study reviewed experimental data reported in scientific literature and used network analysis to identify the potential biological roles of flavonoids' targets in treating psoriasis. 947 records from Web of Sciences, ScienceDirect database, Scopus, PubMed, and Cochrane library were reviewed without limitations until June 26, 2023. 66 articles were included in the systematic review. The ten genes with the highest scores, including interleukin (IL)-10, IL-12A, IL-1ß, IL-6, Tumor necrosis factor-α (TNF-α), Janus kinase 2 (JAK 2), Jun N-terminal kinase (JUN), Proto-oncogene tyrosine-protein kinase Src (SRC), Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), and Signal transducer and activator of transcription 3 (STAT3), were identified as the hub genes. KEGG pathway analysis identified connections related to inflammation and autoimmune responses, which are key characteristics of psoriasis. IL-6, STAT3, and JUN's presence in both hub and enrichment genes suggests their important role in flavonoid's effect on psoriasis. This comprehensive study highlights how flavonoids can target biological processes in psoriasis, especially when combined for enhanced effectiveness.

Therapeutic potential of exosomes derived from mesenchymal stem cells for treatment of systemic lupus erythematosus.

Autoimmune diseases are caused by an imbalance in the immune system, producing autoantibodies that cause inflammation leading to tissue damage and organ dysfunction. Systemic Lupus Erythematosus (SLE) is one of the most common autoimmune diseases and a major contributor to patient morbidity and mortality. Although many drugs manage the disease, curative therapy remains elusive, and current treatment regimens have substantial side effects. Recently, the therapeutic potential of exosomes has been extensively studied, and novel evidence has been demonstrated. A direct relationship between exosome contents and their ability to regulate the immune system, inflammation, and angiogenesis. The unique properties of extracellular vesicles, such as biomolecule transportation, biodegradability, and stability, make exosomes a promising treatment candidate for autoimmune diseases, particularly SLE. This review summarizes the structural features of exosomes, the isolation/purification/quantification method, their origin, effect, immune regulation, a critical consideration for selecting an appropriate source, and their therapeutic mechanisms in SLE.

The spectrum of ß-thalassemia mutations in Kermanshah Province in West Iran and its association with hematological parameters.

ß-Thalassemia (ß-thal) is a hereditary autosomal disorder with decreased or absent ß-globin chain synthesis. Two hundred and one unrelated ß-thal carriers, attending the Kermanshah Medical Genetics Laboratory, Kermanshah, Iran, were investigated for ß-globin gene mutations by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and direct sequencing. Eighteen different mutations were identified in these subjects. Four of the mutations accounted for about 75.0% of the studied cases. IVS-II-1 (G>A) was the most frequent (45.8%) followed by codons 8/9 (+G) (15.9%), IVS-I-110 (G>A) (8.0%), IVS-I-6 (T>C) (5.5%), IVS-I-1 (G>A) (3.5%) and codon 44 (-C) (3.5%); the remaining 12 mutations were present with a frequency less than 3.0%. The mean corpuscular volume (MCV) values for males and females were 63.7 ± 3.7 and 63.2 ± 3.2 fL, respectively, while these values were 19.3 ± 1.6 and 19.3 ± 1.4 pg for mean corpuscular hemoglobin (Hb) (MCH). The mean Hb A2 values for males and females were 4.4 ± 0.5 and 4.1 ± 0.6%, respectively. This study provides a distribution guide for ß-thal mutations in Kermanshah Province, West Iran.

Synthesis of 4-hydroxy-L-proline derivatives as new non-classical inhibitors of human carbonic anhydrase II activity: an in vitro study.

Carbonic anhydrase (CA) is a zinc metalloenzyme that facilitates the rapid conversion of water and carbon dioxide into proton and bicarbonate ion. CA isozymes have been broadly studied in many pathological/physiological processes. In the current research, a series of 4-hydroxy-L-proline derivatives were designed and chemically synthetized, and interaction of these carboxylic acid-based compounds with hCA II were evaluated. Results indicated that different derivatives had different potencies on hCAII inhibitory activity and among them, compounds 3 b and 3c had the lowest IC50 and Kd values than 4-hydroxy-L-proline and other derivatives and therefore had the most affinity to the hCA II enzyme. As a result, compounds 3 b and 3c were chosen for additional testing in this research. The Kinetic data demonstrated that 3 b and 3c inhibit the hCA II esterase activity in a linear competitive way, with Ki values in the low micromolar range. Fluorescence tests showed that the hCA II surface hydrophobicity is diminished in the presence of compounds 3 b and 3c, as confirmed by the decrease in ANS binding to hCA II in their presence. Docking results revealed that 3 b and 3c had more binding energy than 4-hydroxy-L-proline. Furthermore, these compounds could occupy the active site of hCA II, where they would interact with critical amino acid residues via non-covalent forces to inhibit hCA II. Overall, the strengthening of inhibitory activity and the binding power of these carboxylic acid derivatives (3 b and 3c) for the hCA II makes these compounds interesting for designing novel hCA II inhibitors.Communicated by Ramaswamy H. Sarma.

The induction of tau aggregation is restricted by sulfamethoxazole and provides new information regarding the use of the drug.

The aggregation of tau protein in the form of paired helical filament (PHF) leads to the breakdown of microtubule structure and the development of neurodegenerative disorders, such as Alzheimer's disease. Therefore, inhibiting tau protein aggregation is a potential strategy for preventing the progression of these disorders. In this study, sulfamethoxazole (SMZ), an antibiotic that easily crosses the blood-brain barrier and interacts with tau protein, was tested for its ability to inhibit tau aggregation in vitro. Various multi-spectroscopic techniques including XRD, LDH cytotoxicity colorimetric assay, and microscopic imaging were employed. The results showed that SMZ effectively interacts with tau protein through hydrogen and van der Waals interactions. It also effectively inhibited tau protein aggregation in vitro and significantly reduced toxicity in the SH-SY5Y neuroblastoma cell line. Molecular docking and MD simulation results suggested that SMZ may reduce tau protein aggregation by interacting with the PHF6 motif. Overall, these findings indicate that SMZ has therapeutic potential as a tau protein aggregation inhibitor, at least under in vitro conditions. These findings suggest that SMZ has potential as a treatment for neurodegenerative disorders involving tau protein aggregation. However, further research is needed to confirm these results and assess the effectiveness of SMZ in animal models and clinical trials.Communicated by Ramaswamy H. Sarma.

Effective Reduction of Tau Amyloid Aggregates in the Presence of Cyclophilin from Platanus orientalis Pollens; An Alternative Mechanism of Action of the Allergen.

BACKGROUND: A hallmark pathology of Alzheimer's disease (AD) is the construction of neurofibrillary tangles, which are made of hyperphosphorylated Tau. The cis-proline isomer of the pThr/Ser-Pro sequence has been suggested to act as an aggregation precursor according to the 'Cistauosis' hypothesis; however, this aggregation scheme is not yet completely approved. Various peptidyl-prolyl isomerases (PPIases) may specifically isomerize cis/trans-proline bonds and restitute Tau's ability to attach microtubules and may control Tau amyloid aggregation in AD. METHODS: In this study, we provided experimental evidence for indicating the effects of the plant Cyclophilin (P-Cyp) from Platanus orientalis pollens on the Tau aggregation by various spectroscopic techniques. RESULTS: Our findings disclosed that the rate/extent of amyloid formation in the Tau sample which is incubated with P-Cyp decreased and these observations do not seem to be due to the macromolecular crowding effect. Also, as proven that 80% of the prolines in the unfolded protein are in the trans conformation, urea-induced unfolding analyses confirmed this conclusion and showed that the aggregation rate/extent of urea-treated Tau samples decreased compared with those of the native protein. Also, XRD analysis indicated the reduction of scattering intensities and beta structures of amyloid fibrils in the presence of P-Cyp. Therefore, the ability of P-Cyp to suppress Tau aggregation probably depends on cis to trans isomerization of proline peptide bonds (X-Pro) and decreasing cis isomers in vitro. CONCLUSION: The findings of the current study may inspire possible protective/detrimental effects of various types of cyclophilins on AD onset/progression through direct regulation of intracellular Tau molecules and provides evidence that a protein from a plant source is able to enter the cell cytoplasm and may affect the behavior of cytoplasmic proteins.

Investigation of the role of prolines 232/233 in RTPPK motif in tau protein aggregation: An in vitro study.

Disease-related tau protein in Alzheimer's disease is hyperphosphorylated and aggregates into neurofibrillary tangles. The cis-proline isomer of the pSer/Thr-Pro sequence has been proposed to act as a precursor of aggregation ('Cistauosis' hypothesis), but this aggregation scheme is not yet entirely accepted. Hence to investigate isomer-specific-aggregation of tau, proline residues at the RTPPK motif were replaced by alanine residues (with permanent trans configuration) employing genetic engineering methods. RTPAK, RTAPK, and RTAAK mutant variants of tau were generated, and their in vitro aggregation propensity was investigated using multi-spectroscopic techniques. Besides, the cell toxicity of oligomers/fibrils was analyzed and compared to those of the wild-type (WT) tau. Analyses of mutant variants have shown to be in agreement (to some degree) to the theory of the 'cistauosis' hypothesis. The results showed that the trans isomer in the 232-rd residue (P232A mutant rather than P233A) had reduced aggregation propensity. However, this study did not illustrate any statistically significant difference between the wild and the mutant protein aggregations concerning cell toxicity.

Synthesis, in vitro antibacterial and carbonic anhydrase II inhibitory activities of N-acylsulfonamides using silica sulfuric acid as an efficient catalyst under both solvent-free and heterogeneous conditions.

Silica sulfuric acid catalyzes efficiently the reaction of sulfonamides with both carboxylic acid anhydrides and chlorides under solvent-free and heterogeneous conditions. All the reactions were done at room temperature and the N-acylsulfonamides were obtained with high yields and purity via an easy work-up procedure. This method is attractive and is in a close agreement with green chemistry. These compounds were also investigated for antibacterial activity, including Gram-positive cocci and Gram-negative bacilli, and carbonic anhydrase II inhibitory activity.

Comparative experimental/theoretical studies on the EGFR dimerization under the effect of EGF/EGF analogues binding: Highlighting the importance of EGF/EGFR interactions at site III interface.

Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play significant roles in cell proliferation and signaling. All the members of the EGFR/ErbB family are primary goals for cancer therapy, particularly for tumors of breast, cervix, ovaries, kidney, esophagus, prostate and non-small-cell lung carcinoma and head and neck tumors. However, the therapeutic ability of accessible anti-ErbB agents is limited. Therefore, recognizing EGF analogues or small organic molecules with high affinity for the extracellular domain of the EGFR is a critical target on cancer research. An effective EGF analogue should have a comparable binding affinity for EGFR in order to create an effective ligand competitive inhibition against circulating wild EGF while fails to transduce appropriate downstream signaling into the cancer cell. In our earlier study we have developed a mutant form of human EGF (mEGF, lacking the four critical amino acid residues; Gln43, Tyr44, Arg45 and Asp46 at the C-terminal of the protein) and its binding properties and mitogenic activity were assessed. The mEGF showed high affinity for EGFR binding domains but caused poor EGFR dimerization and phosphorylation and especially, mEGF induced EGFR internalization. However, underlying mechanism of action of EGF analogues is still unclear and thus considered to be worthwhile for further study. With regard to different effects of the EGF analogue on EGFR activating process, computational analysis of wild EGF/EGFR and mEGF/EGFR complexes (along with EGFt/EGFR complex) were done. Results of the protein dissection identified several interactions within "ligand/EGFR" that are common among EGF and EGFt/mEGF. These results disclose that while several interactions are conserved within EGF/EGFR interfaces, EGF/EGFR interactions on site III interface controls the affinity, EGFR dimerization and subsequent downstream signaling through a heterogeneous set of non-covalent interactions. These findings not only represent the EGFR dynamics complexity but also smooth the path for structure-based design of therapeutics targeting C-terminal region of EGF (and the related domain within the receptor) or EGFR-based imaging probes.

Critical effects on binding of epidermal growth factor produced by amino acid substitutions.

Epidermal growth factor (EGF) plays important roles in multiple biological processes, such as the regulation of cell growth, proliferation, and differentiation. EGF exerts their pharmacologic effects via receptor-mediated mechanism associated with high affinity to epidermal growth factor receptor (EGFR) on the cell surface. Overexpression of EGFR has been reported and implicated in the pathogenesis of many human cancers. The current study addresses the effects of mutations on binding properties of EGF to EGFR. Two mutant structures with three point mutations of conserved residues, Ile23, Arg41 and Leu47, which have been found to be important for the receptor binding, were built using homology modeling. The "wild type" (WT) and the mutant structures, after structural validations, were subjected to molecular dynamics simulations (MDSs). The primary aim of MDS was to investigate the possible impact of mutations on the protein structure and function. Analysis of root mean square deviation (RMSD), other time dependent structural properties and their averages provided some insights into the possible structural characteristics of the mutant and the WT forms of the EGF. RMSD analysis showed that WT EGF was more stable than the mutant structures. The docking analysis revealed that the binding energy of mutant EGFs to EGFR is lower than WT. Combination of the used computational approaches provides a way in understanding the impact of deleterious mutations in altering the EGF and EGFR interactions.

Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: A comparative in vitro study using EGFR-positive breast cancer cells.

Epidermal growth factor (EGF) is a local growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. EGF and EGFR are involved in many aspects of the development of carcinomas. Because EGFR has been found to be over-expressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. In this study we designed a mutated form of hEGF (mEGF) with a deletion of four amino acids residues (Gln43, Tyr44, Arg45 and Asp46) in order to show importance of Leu spatial location for EGFR binding/activation. Expression vector pET32a+ and E. Coli, strain Rosetta-gami B (DE3) were used to enhance solubility of the recombinant protein with yielding approximately 10mg/l of cell culture. The purified cleaved hEGF as well as non-cleaved fusion protein were biologically active, which was confirmed by their equal ability to stimulate proliferation of MCF7 cells. The mEGF showed specificity and high affinity for EGFR binding, however binding affinity of mEGF for EGFR was reduced about 11.5 fold compared with that of hEGF. The mEGF effect on the MCF7 cell proliferation had a relatively different outcome; mEGF simulated differential cell growth in a dose dependent manner. On the other hand, in MDA-MB468 cells, hEGF and mEGF induced growth inhibition, which was much more severe for hEGF than that of mEGF. Also, hEGF strongly induced the phosphorylation of EGF receptor in MDA-MB468 cells while mEGF induced poor EGFR phosphorylation. The same observations were also made for migration of cancer cells, especially induction of MDA-MB468 migration by mEGF was significantly lower than that of hEGF, suggesting a connection between tyrosine phosphorylation of EGFR and cell migration. Docking analysis revealed that the binding affinity and the buried surface area of mEGF to EGFR complex are lower than those of hEGF/EGFR. Although theoretical studies confirmed reduction in mEGF-EGFR binding affinity, the data of the present study indicate that mEGF is a potential EGFR blocker but may highlight it as excellent delivery agent of protein/non-protein toxins as well as for α-, ß-, γ-emitting radio-immunotherapy.

Spectroscopic study on the interaction of celecoxib with human carbonic anhydrase II: thermodynamic characterization of the binding process.

The effect of celecoxib, a sulfonamide drug, on the structure and function of human carbonic anhydrase II (hCA II) was investigated by various spectroscopic techniques such as UV-Vis, fluorescence and circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC), in 20 mM Tris, pH 7.75 at 27 degrees C. Kinetic results revealed that celecoxib inhibits the esterase activity of hCA II in a linear competitive manner with K(i) = 61.61 + or -3.05 nM. DSC data indicated that the thermal stability of the enzyme has a minor increment in the presence of celecoxib. Fluorescence measurements showed that the celecoxib acts as a quencher of the enzyme fluorescence and calculation of the protein surface hydrophobicity (PSH), using 1-anilinonaphthalene-8-sulfonate (ANS), revealed the decrement of its PSH upon interaction with the drug. Acrylamide quenching experiments indicated the less accessibility of the tryptophan residues to the acrylamide due to the presence of celecoxib. Stern-Volmer analysis of quenching data at different temperatures elucidated that the quenching of intrinsic fluorescence of hCA II is occurred through a static quenching mechanism. Analysis of the thermodynamic parameters of binding showed that hydrogen bonding and hydrophobic interactions play the major role in stabilization of the enzyme-drug complex. The Job's plot confirmed the existence of one binding site for celecoxib in hCA II. The far- and near-UV CD experiments indicated that celecoxib causes a little increment in alpha-helicity content of hCA II whereas its flexibility is decreased somewhat upon celecoxib binding.