Ali Akbar Shabani

Compatible osmolytes like trimethylamine N-oxide (TMAO), known for preferentially stabilizing proteins in their folded states, offer a potential solution; however, the precise molecular mechanisms underlying their stabilizing effects are not fully understood. On the other hand, the poor thermal stability of pharmaceutical enzymes such as urate oxidase (UOX) in aqueous solutions remains a significant challenge. This study explored how TMAO modulates the enzyme's environment to enhance UOX's catalytic efficiency and stability. By combining experimental and computational approaches, we evaluated the kinetic, thermodynamic, and structural changes in urate oxidase with and without the osmolyte. Kinetic analysis revealed enhanced enzymatic activity in the presence of TMAO. Thermodynamic studies indicated negative values for enthalpy change (ΔH°) and entropy change (ΔS°), suggesting the involvement of van der Waals forces and hydrogen bonding in UOX-TMAO interactions. The negative value of free energy change (ΔG°) further confirms spontaneous binding. Fluorescence spectroscopy indicated structural modifications in the enzyme due to osmolyte binding, supported by a complex quenching mechanism. Molecular dynamics (MD) simulations demonstrated that TMAO increased UOX's structural compactness and enzyme stability, along with a rise in secondary structure content, which helped preserve the integrity of the active site. Moreover, molecular docking revealed a favorable binding of TMAO to UOX, primarily mediated by non-covalent interactions, thereby corroborating our experimental findings. These findings provide atomic insights into how TMAO can improve the stability and catalytic function of UOX.

Introduction Methamphetamine (METH) is an addictive psychostimulant with deleterious effects on the central nervous system. Chronic use of METH in high doses impairs cognition, attention and executive functions, but the underlying mechanisms are still unclear. Sirtuin 1 (SIRT1) is a post-translational regulator that is downregulated following METH neurotoxicity. Melatonin is a neuroprotective hormone that enhances mitochondrial metabolism. Here, we evaluated the effect of melatonin on METH-induced attention deficits disorder and the involvement of the miR-181/SIRT1 axis in melatonin neuroprotection. Methods and results METH at a dose of 5 mg/kg was injected for 21 consecutive days. The animals were assigned to receive either melatonin or the vehicle after METH injections. Attention levels were evaluated with abject-based attention test. In the prefrontal cortex, the expression levels of miR-181a-5p, SIRT1, p53 and CCAR2, as well as the mtDNA copy numbers were evaluated using qRT-PCR and western blotting. The outcomes revealed that melatonin treatment following METH injections improved METH-induced attention deficits. METH toxicity can be associated with changes in the miR-181/SIRT1 axis, elevated levels of p53 and COXII, and decreased levels of mtDNA in the prefrontal cortex of adult rats. Interestingly, administration of melatonin can improve the expression of these molecules and reduces the toxic effects of METH. Conclusion Melatonin ameliorated the neurotoxicity of METH in the prefrontal cortex and the miR-181/SIRT1 axis is involve in the protective effects of melatonin. However, melatonin can be potentially administrated to improve attention impairment in METH use disorders.

Diabetes mellitus type 1 is a chronic condition characterized by the loss or dysfunction of β-cells in the pancreas, resulting in insufficient insulin production. This mini-review examines current treatment approaches and explores the potential of gene therapy as interventions for type 1 diabetes mellitus. The discussed strategies include β-cell sensitization, β-cell regeneration from various cell sources, stem cell therapies, and the promotion of β-cell replication. The article emphasizes the importance of understanding the pathways involved in β-cell proliferation and the factors influencing their replication. Stem cell therapies, particularly using embryonic stem cells and induced pluripotent stem cells, hold promise for generating β-cells and replacing damaged or lost cells. Additionally, gene therapy offers a novel approach by manipulating genes involved in insulin production and glucose metabolism. However, ethical considerations, tumorigenic risks, and the translation of these therapies into clinical trials pose challenges. Nonetheless, the ongoing research and advancements in these areas provide hope for improved management and treatment of type 1 diabetes mellitus.

Objectives Among the most promising antibody formats in terms of inhibiting carcinogenesis are single-stranded variable fragments, whose targeted binding to the Fzd7 receptor has been proven effective at suppressing tumorigenesis. In this study, we investigated the effectiveness of an anti-Fzd7 antibody fragment against both tumor growth and metastasis of breast cancer cells. Methods To develop anti-Fzd7 antibodies, bioinformatics approaches were used and the antibodies were expressed recombinantly in E. coli BL21 (DE3). The expression of anti-Fzd7 fragments was verified by Western blotting. Analysis of the antibody's binding capacity to Fzd7 was conducted by flow cytometry. Cell death and apoptosis were assessed by MTT and Annexin V/PI assays. The transwell migration and invasion assays, as well as the scratch method, were used to evaluate cell motility and invasiveness. Results The anti-Fzd7 antibody was expressed successfully as a single band of 31 kDa. It could bind to 21.5% of MDA-MB-231 cells, as opposed to only 0.54% of SKBR-3 cells as negative control. According to MTT assay, induced apoptosis was 73.7% in MDA-MB-231 cells, compared with 29.5% in SKBR-3 cells. Also, the antibody exerted a significant inhibitory effect of 76% and 58% on migration and invasion of MDA-MB-231 cells, respectively. Conclusion The recombinantly developed anti-Fzd7 scFv of this study could exhibit significant antiproliferative and antimigratory properties, along with a high apoptosis-inducing potential, making it suitable for the immunotherapy of triple negative breast cancer.

Osteoarthritis, which typically arises from aging, traumatic injury, or obesity, is the most common form of arthritis, which usually leads to malfunction of the joints and requires medical interventions due to the poor self-healing capacity of articular cartilage. However, currently used medical treatment modalities have reported, at least in part, disappointing and frustrating results for patients with osteoarthritis. Recent progress in the design and fabrication of tissue-engineered microscale/nanoscale platforms, which arises from the convergence of stem cell research and nanotechnology methods, has shown promising results in the administration of new and efficient options for treating osteochondral lesions. This paper presents an overview of the recent advances in osteochondral tissue engineering resulting from the application of micro- and nanotechnology approaches in the structure of biomaterials, including biological and microscale/nanoscale topographical cues, microspheres, nanoparticles, nanofibers, and nanotubes.

Cancer is a leading cause of death and a vital health care challenge in the world. Hence, this work was conducted to determine the in vitro anticancer property and also the molecular mechanism of aqueous and organic extracts of Ipomoea purpurea leaves in three human cancer cell lines, including A-549 (human lung cancer), HepG-2 (human liver cancer), MDA-MB-231 (human breast cancer), and MCF-10A (breast normal cell line). In vitro cytotoxic potential of organic extracts, such as hexane, chloroform, ethyl-acetate, methanol, and aqueous extract was examined using a standard (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) MTT method in both monolayer two-dimensional (2D) and spheroids multicellular three-dimensional (3D) cultures. The MTT assay data showed that methanol and chloroform extracts of I. purpurea leaves had the antiproliferative effect on lung and breast cancer cells with IC₅₀ of 53.62±0.07 and 124.5±0.01µg/mL, respectively. The results of further examinations, such as dual acridine orange/ethidium bromide, Annexin V-FITC/PI, and caspase-3 colorimetric assay, confirmed that methanol and chloroform extracts of I. purpurea as the most potent cytotoxic extracts might contain a variety of phytochemicals, promoting apoptosis in lung and breast cancer cells. The present research findings suggested that methanolic extract of I. purpurea leaves induced S-phase cell cycle arrest and intrinsic pathway of apoptosis in A-549 lung cancer cells. The study further showed that I. purpurea could be a helpful candidate for cancer treatment.

Considering the worldwide increasing prevalence of resistance to traditional antibiotics, it is necessary to find new antibiotics to deal with this issue. Recently, antimicrobial peptides (AMPs) have been proposed as new antimicrobial agents. Aureins are a family of AMPs that are isolated from Green and Golden Bell Frogs. These peptides have a favorable antibacterial activity against Gram‐positive bacteria. We designed two peptides derived from natural Aurein enjoying alignment‐based design method. After synthesis of the peptides, their secondary structure was checked by circular dichroism. Consequently, the antibacterial effects of these peptides were investigated by determining the minimum inhibitory concentration (MIC) and bactericidal concentration. Eventually, the toxicity of these peptides was determined by MTT (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide) assay on normal human skin cells (Hu02 cell line). Natural Aurein1.2 was used as a natural control to compare the properties in all stages. The results indicated that these new peptides had medium‐upward antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis (MIC of 8–64 μg/mL) and weak bactericidal activity against Staphylococcus aureus (MIC of 128–256 μg/mL). Also, MTT assays results showed that AureinN2 is less toxic than AureinN1 and Aurein1.2. Toxicity of AureinN2 for Hu02 cell lines was between 20 and 40% at the concentration of 8–500 μg/mL. In this study, we were able to improve antimicrobial activity of two synthetic derivatives of the Aurein family against Gram‐negative bacteria by using machine‐learning algorithm and other in silico methods.

Growth factors have a pivotal role in chondrogenic differentiation of stem cells. The differential effects of known growth factors involved in the maintenance and homeostasis of cartilage tissue have been previously studied in vitro. However, there are few reported researches about the interactional effects of growth factors on chondrogenic differentiation of stem cells. The aim of this study is to examine the combined effects of four key growth factors on chondrogenic differentiation of mesenchymal stem cells (MSCs). Isolated and expanded rabbit bone marrow-derived MSCs underwent chondrogenic differentiation in a micromass cell culture system that used a combination of the following growth factors: transforming growth factor beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP2), parathyroid hormone related protein (PTHrP), and fibroblast growth factor 2 (FGF2) according to a defined program. The chondrogenic differentiation program was analyzed by histochemistry methods, quantitative RT-PCR (qRT-PCR), and measurement of matrix deposition of sulfated glycosaminoglycan (sGAG) and collagen content at days 16, 23, and 30. The results showed that the short-term combination of TGF-β1 and BMP-2 increased sGAG and collagen content, Alkaline phosphates (ALP) activity, and type X collagen (COL X) expression. Application of either PTHrP or FGF2 simultaneously decreased TGF-β1/BMP-2 induced hypertrophy and chondrogenic markers (at least for FGF2). However, successive application of PTHrP and FGF2 dramatically maintained the synergistic effects of TGF-β1/BMP-2 on the chondrogenic differentiation potential of MSCs and decreased unwanted hypertrophic markers. This new method can be used effectively in chondrogenic differentiation programs.

Background: BaParathyroid hormone-related protein (PTHrP) is a multi-functional protein with a sequence “aa 1-34” similar to that of parathyroid hormone (PTH). In spite of growth factor activity, there is no standardized analytical method to determine the biological activity of recombinant PTHrP. Methods: The current study isolated the cDNA encoding 141 N-terminal amino acid sequence of PTHrP, cloned it in plasmid vector pET32a, and produced recombinant PTHrP in Escherichia coli. The biological activity analysis of the recombinant protein was evaluated using a precise and practical method that measured PTHrP-mediated cell proliferation of a breast cancer cell line MCF-7. Additionally, to investigate the accuracy and precision of MCF-7 method used, chondrogenic differentiation condition of PTHrP treated mesenchymal stem cells (MSCs) was evaluated. Results: DNA sequencing and protein mass spectrometry analysis confirmed the accuracy of cloned cDNA sequence and expressed the protein. Biological activity analysis showed the increasing effect of recombinant PTHrP on cell proliferation of cancer cell line MCF-7. Alkaline phosphatase and gene expression analysis validated the anti-hypertrophy effects of recombinant PTHrP on chondrogenic differentiation of MSCs. Conclusions: The current study method removed potential challenges and errors in comparability and reproducibility studies focusing on stem cell differentiation and pathogenesis of breast cancer arising from PTHrP biological activity analysis. Keywords: Recombinant PTHrP; Biological Activity; MCF-7; Chondrogenesis; Hypertrophy