Rheum khorasanicum. Hydroalcoholic root extract induces cell death in human colorectal adenocarcinoma: An in vitro and in silico study.

Colorectal cancer (CRC) is the second greatest cause of cancer-related death in the world and chemotherapy, as an important part of CRC treatment, has some drawbacks, including systemic toxicity. Therefore, it is crucial to discover new and more effective CRC treatment plans. Rheum khorasanicum (R. khorasanicum) is a medicinal plant with high flavonoids, stilbenes, and anthraquinone contents, so it can be a potential source of antioxidants and can be used for therapeutic purposes and trigger apoptosis in cancer cells. In this study, we investigated the effects of hydroalcoholic root extract of R. khorasanicum treatment on inducing mitochondrial apoptosis of HT-29 and Caco-2 human colorectal adenocarcinoma cells. Firstly, the total phenolic and flavonoid content was determined. Then, the cytotoxic effects of R. khorasanicum on cells of three different types, including HT-29 and Caco-2 colon cancer cells as well as normal 3T3 cells were assessed using the MTT assay. To investigate the characteristics of cellular death, flow cytometry, and western blotting were performed. The results of this study indicated considerable phenolic (356.4±9.4 GAE/gDW) and flavonoid (934.55±17.1 QE/gDW) contents in R. khorasanicum. MTT assay's finding indicated that 100, 60, and 30µg/mL concentrations of R. khorasanicum reduce cell viability in HT-29 and Caco-2 cell lines significantly (P<0.05). It has been also revealed that R. khorasanicum extract induces apoptosis rather than necrosis in these cell lines. Moreover, Bcl-2 expression was significantly reduced in both HT-29 and Caco-2 cell lines, while Bax and cleaved caspase-3 expression soared considerably in the groups under R. khorasanicum treatment (P<0.05). In conclusion, our findings have suggested that high phenol and flavonoid contents of R. khorasanicum root extract possibly play an important role in cell cytotoxicity and apoptosis induction in HT-29 and Caco-2 colon cancer cells.

Impact of a 50bp insertion/deletion polymorphism of the superoxide dismutase-1 on oxidative stress status and risk of keratoconus.

Keratoconus (KC) is characterized by the predominant primary ectatic disease, affecting the cornea, necessitating corneal transplants in some cases. While some loci associated with KC risk have been identified, the understanding of the disease remains limited. Superoxide dismutase (SOD) enzymes play a crucial role in countering the reactive oxygen species and providing protection against oxidative stress (OS). Accordingly, the objective of this study was to investigate a potential association of a 50 nucleotide base pairs (bp) insertion/deletion (I/D) within the SOD1 promoter, and the located 1684 bp upstream of the SOD1 ATG, with KC in the Iranian population. Additionally, an assessment was conducted on SOD activity and the total antioxidant capacity (TAC), as determined by the ferric reducing-antioxidant power assay, along with malondialdehyde (MDA) levels. In this case-control study, genomic DNA was extracted from the blood cells of KC (n = 402) and healthy (n = 331) individuals. The genotype of this gene was determined using the PCR technique. Furthermore, the amount of SOD enzyme activity and the MDA and TAC levels were measured in the serum of the study groups. The (I/I) genotype was present in 84.23%, the (I/D) genotype in 15.06%, and the (D/D) genotype in 0.69% of both groups. A statistically significant relationship was seen between different genotypes and TAC, MDA, and SOD1 activity indices (P < 0.05). Individuals with the D/D genotype exhibited a decrease in total antioxidant capacity, an increase in the amount of MDA, and a decrease in SOD1 enzyme activity (P < 0.05). Moreover, the logistic regression analysis of KC development indicated that elevated levels of MDA increased the risk of KC incidence in the patient group compared to the healthy group, while a higher activity of SOD1 and greater values of TAC decreased the KC risk. The removal of the 50 bp fragment reduced SOD1 activity and elevated OS levels, thereby impacting the oxidant-antioxidant balance. This could potentially play a significant role in individuals afflicted by KC.

Lithium and zinc levels along with oxidative status in myocardial infarction: A case-control study.

Background: Coronary artery disease (CAD) and myocardial infarction (MI) are the most prevalent diseases globally. While several risk factors for MI are well assessed, the influence of trace elements on MI has not been thoroughly studied. This study aimed to evaluate lithium (Li) and zinc (Zn) levels in MI patients and healthy control and assess their relationship with oxidative stress (OS) parameters, such as nitric oxide (NO) and total antioxidant capacity (TAC). Methods: This case-control study was performed on 182 patients with MI and 83 healthy subjects at Shafa Hospital in Kerman, Iran. MI patients were divided into two groups based on the angiography results: those with coronary artery block above 50 % (CAB >50 %, n = 92) and those with coronary artery block below 50 % (CAB <50 %, n = 90). A flame atomic absorption spectrometer was used to detect Li and Zn levels, and biochemical indices were measured by an autoanalyzer. Also, ferric reducing antioxidant power assay and the Griess method were used to measure the amounts of NO and TAC. Results: The levels of TAC and Li were significantly higher in the control group than in the patient groups (in both CAB >50 % and CAB <50 % groups). Furthermore, in the CAB <50 % group, TAC and Li levels were significantly higher than in the CAB >50 % group. In the Zn levels evaluation, higher concentration was seen in the CAB >50 % group compared to the CAB <50 % group (P < 0.05). Moreover, Zn and NO levels were significantly higher in both CAB groups compared to controls. In continue, Li levels had a positive association with TAC and ejection fraction percentage (EF%) as well as a negative association with NO levels and Zn levels had a significant positive association with NO and a negative association with TAC. In logistic regression analysis, Li, TAC, and high-density lipoprotein-cholesterol significantly decreased the odds ratio (OR) of MI, whereas Zn, NO, total cholesterol, triglyceride, low-density lipoprotein-cholesterol, and high-sensitivity C-reactive protein (hs-CRP) significantly increased the OR of MI. Furthermore, the area under the curve (AUC) analysis indicated that Li had the highest AUC for the diagnosis of CAB >50 % (Li < 167 ng/mL), and Zn ≥ 1810 µg/mL increased disease severity. Conclusion: Our investigation revealed that Li had a protective effect against CAD by decreasing OS and increasing EF%. However, Zn at concentrations higher than 1810 µg/mL was found to be cytotoxic and increased the risk of MI through increased OS. Taken togather, it could be concluded that Li supplementation may decrease the risk of CAD.

Genetically engineered bacteria: a new frontier in targeted drug delivery.

Genetically engineered bacteria (GEB) have shown significant promise to revolutionize modern medicine. These engineered bacteria with unique properties such as enhanced targeting, versatility, biofilm disruption, reduced drug resistance, self-amplification capabilities, and biodegradability represent a highly promising approach for targeted drug delivery and cancer theranostics. This innovative approach involves modifying bacterial strains to function as drug carriers, capable of delivering therapeutic agents directly to specific cells or tissues. Unlike synthetic drug delivery systems, GEB are inherently biodegradable and can be naturally eliminated from the body, reducing potential long-term side effects or complications associated with residual foreign constituents. However, several pivotal challenges such as safety and controllability need to be addressed. Researchers have explored novel tactics to improve their capabilities and overcome existing challenges, including synthetic biology tools (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) and bioinformatics-driven design), microbiome engineering, combination therapies, immune system interaction, and biocontainment strategies. Because of the remarkable advantages and tangible progress in this field, GEB may emerge as vital tools in personalized medicine, providing precise and controlled drug delivery for various diseases (especially cancer). In this context, future directions include the integration of nanotechnology with GEB, the focus on microbiota-targeted therapies, the incorporation of programmable behaviors, the enhancement in immunotherapy treatments, and the discovery of non-medical applications. In this way, careful ethical considerations and regulatory frameworks are necessary for developing GEB-based systems for targeted drug delivery. By addressing safety concerns, ensuring informed consent, promoting equitable access, understanding long-term effects, mitigating dual-use risks, and fostering public engagement, these engineered bacteria can be employed as promising delivery vehicles in bio- and nanomedicine. In this review, recent advances related to the application of GEB in targeted drug delivery and cancer therapy are discussed, covering crucial challenging issues and future perspectives.

Nanophotocatalysts in biomedicine: Cancer therapeutic, tissue engineering, biosensing, and drug delivery applications.

Photocatalysis can be considered as a green technology owing to its excellent potential for sustainability and fulfilling several principles of green chemistry. This process uses light radiation as the primary energy source, preventing or reducing the requirement for artificial light sources and exogenous catalytic entities. Photocatalysis has promising applications in biomedicine such as drug delivery, biosensing, tissue engineering, cancer therapeutics, etc. In targeted cancer therapeutics, photocatalysis can be employed in photodynamic therapy to form reactive oxygen species that damage cancerous cells' structure. Nanophotocatalysts can be used in targeted drug delivery, showing potential applications in nuclear-targeted drug delivery along with specific delivery of chemotherapeutics to cancer cells or tumor sites. On the other hand, in tissue engineering, nanophotocatalysts can be employed in designing scaffolds that promote cell growth and tissue regeneration. However, some important challenges pertaining to the performance of photocatalysis, large-scale production of nanophotocatalysts, optimization of reaction/synthesis conditions, long-term biosafety issues, stability, clinical translation, etc. still need further explorations. Herein, the most recent advancements pertaining to the biomedical applications of nanophotocatalysts are reflected, focusing on drug delivery, tissue engineering, biosensing, and cancer therapeutic potentials.

Bacterial Cellulose-Based Materials: A Perspective on Cardiovascular Tissue Engineering Applications.

Today, a wide variety of bio- and nanomaterials have been deployed for cardiovascular tissue engineering (TE), including polymers, metal oxides, graphene/its derivatives, organometallic complexes/composites based on inorganic-organic components, among others. Despite several advantages of these materials with unique mechanical, biological, and electrical properties, some challenges still remain pertaining to their biocompatibility, cytocompatibility, and possible risk factors (e.g., teratogenicity or carcinogenicity), restricting their future clinical applications. Natural polysaccharide- and protein-based (nano)structures with the benefits of biocompatibility, sustainability, biodegradability, and versatility have been exploited in the field of cardiovascular TE focusing on targeted drug delivery, vascular grafts, engineered cardiac muscle, etc. The usage of these natural biomaterials and their residues offers several advantages in terms of environmental aspects such as alleviating emission of greenhouse gases as well as the production of energy as a biomass consumption output. In TE, the development of biodegradable and biocompatible scaffolds with potentially three-dimensional structures, high porosity, and suitable cellular attachment/adhesion still needs to be comprehensively studied. In this context, bacterial cellulose (BC) with high purity, porosity, crystallinity, unique mechanical properties, biocompatibility, high water retention, and excellent elasticity can be considered as promising candidate for cardiovascular TE. However, several challenges/limitations regarding the absence of antimicrobial factors and degradability along with the low yield of production and extensive cultivation times (in large-scale production) still need to be resolved using suitable hybridization/modification strategies and optimization of conditions. The biocompatibility and bioactivity of BC-based materials along with their thermal, mechanical, and chemical stability are crucial aspects in designing TE scaffolds. Herein, cardiovascular TE applications of BC-based materials are deliberated, with a focus on the most recent advancements, important challenges, and future perspectives. Other biomaterials with cardiovascular TE applications and important roles of green nanotechnology in this field of science are covered to better compare and comprehensively review the subject. The application of BC-based materials and the collective roles of such biomaterials in the assembly of sustainable and natural-based scaffolds for cardiovascular TE are discussed.

Carbon dots with tissue engineering and regenerative medicine applications.

Carbon dots (CDs) with unique physicochemical features such as exceptional biocompatibility, low cost, eco-friendliness, abundant functional groups (e.g., amino, hydroxyl, and carboxyl), high stability, and electron mobility have been broadly investigated in nano- and biomedicine. In addition, the controlled architecture, tunable fluorescence emission/excitation, light-emitting potential, high photostability, high water solubility, low cytotoxicity, and biodegradability make these carbon-based nanomaterials suitable for tissue engineering and regenerative medicine (TE-RM) purposes. However, there are still limited pre- and clinical assessments, because of some important challenges such as the scaffold inconsistency and non-biodegradability in addition to the lack of non-invasive methods to monitor tissue regeneration after implantation. In addition, the eco-friendly synthesis of CDs exhibited some important advantages such as environmentally friendly properties, low cost, and simplicity compared to the conventional synthesis techniques. Several CD-based nanosystems have been designed with stable photoluminescence, high-resolution imaging of live cells, excellent biocompatibility, fluorescence properties, and low cytotoxicity, which make them promising candidates for TE-RM purposes. Combining attractive fluorescence properties, CDs have shown great potential for cell culture and other biomedical applications. Herein, recent advancements and new discoveries of CDs in TE-RM are considered, focusing on challenges and future perspectives.

An efficient strategy to recellularization of a rat aorta scaffold: an optimized decellularization, detergent removal, and Apelin-13 immobilization.

BACKGROUND: Tissue engineering of native vessels is an alternative approach for patients with vascular disease who lack sufficient saphenous vein or other suitable conduits for autologous vascular graft. Moreover, the harvest of vessels prolongs the surgical procedure and it may lead to the morbidity of donor site in elder patients: therefore, it seems that the use of tissue-engineered vessels would be an attractive and less invasive substitute for autologous vascular grafts. Apelin-13 plays a pivotal role in cell proliferation, survival, and attachment; therefore, covalent attachment of apelin-13 to the acellular scaffolds might be a favorable approach for improving recellularization efficacy. METHODS: In the present study, the decellularization process was performed using various detergents. Afterward, the efficacy of decellularization procedure was evaluated using multiple approaches including assessment of DNA, hydroxyproline, and GAG content as well as Masson's trichrome and orcein staining used for collagen and elastin determination. Subsequently, the scaffold was bioconjugated with apelin-13 using the EDC-NHS linker and acellular scaffolds were recellularized using fibroblasts, endothelial cells, and smooth muscle cells. SEM images and characterization methods were also used to evaluate the effect of apelin-13 attachment to the acellular scaffold on tissue recellularization. We also developed a novel strategy to eliminate the remnant detergents from the scaffold and increase cell viability by incubating acellular scaffolds with Bio-Beads SM-2 resin. Testometric tensile testing machine was also used for the assessment of mechanical properties and uniaxial tensile strength of decellularized and recellularized vessels compared to that of native tissues. RESULTS: Our results proposed 16-h perfusion of 0.25% sodium dodecyl sulfate (SDS) + 0.5% Triton X-100 combination to the vessel as an optimal decellularization protocol in terms of cell elimination as well as extracellular matrix preservation. Furthermore, the results demonstrated considerable elevation of cell adhesion and proliferation in scaffolds bioconjugated with apelin-13. The immunohistochemical (IHC) staining of CD31, α-SMA, and vimentin markers suggested placement of seeded cells in the suitable sites and considerable elevation of cell attachment within the scaffolds bioconjugated with apelin-13 compared to the non-bioconjugated, and decellularized groups. Moreover, the quantitative analysis of IHC staining of CD31, α-SMA, and vimentin markers suggested considerable elevation in the number of endothelial, smooth muscle, and fibroblast cells in the recellularized scaffolds bioconjugated with apelin-13 group (1.4% ± 0.02, 6.66% ± 0.23, and 9.87% ± 0.13%, respectively) compared to the non-bioconjugated scaffolds (0.03% ± 0.01, 0.28% ± 0.01, and 1.2% ± 0.09%, respectively) and decellularized groups (0.03% ± 0.007, 0.05% ± 0.01, and 0.13% ±0.005%, respectively). Although the maximum strain to the rupture was reduced in tissues decellularized using 0.5% SDS and CHAPS compared to that of native ones (116% ± 6.79, 139.1% ± 3.24, and 164% ± 8.54%, respectively), ultimate stress was decreased in all decellularized and recellularized groups. Besides, our results indicated that cell viability on the 1st, 3rd, and 7th day was 100.79% ± 0.7, 100.34% ± 0.08, and 111.24% ± 1.7% for the decellularized rat aorta conjugated with apelin-13, which was incubated for 48-h with Bio-Beads SM-2, and 73.37% ± 7.99, 47.6% ± 11.69, and 27.3% ± 7.89% for decellularized rat aorta scaffolds conjugated with apelin-13 and washed 48-h by PBS, respectively. These findings reveal that the incubation of the scaffold with Bio-Beads SM-2 is a novel and promising approach for increasing cell viability and growth within the scaffold. CONCLUSIONS: In conclusion, our results provide a platform in which xenograft vessels are decellularized properly in a short time, and the recellularization process is significantly improved after the bioconjugation of the acellular scaffold with apelin-13 in terms of cell adhesion and viability within the scaffold.

A comparative study on the effect of blood collection tubes on stress oxidative markers.

Oxidative stress has a major role in disease pathogenesis. However, limited studies have investigated the effect of various sample collection tubes on oxidative biomarkers. The present study aimed to evaluate the effect of different collection tubes on the variation of malondialdehyde (MDA), nitric oxide (NO), total thiol (t-SH), and ferric reducing ability of plasma (FRAP) levels. A total of 35 individuals participated in this study and each collected sample was separated into three different tubes: glass tubes (GTs), plain plastic tubes (PTs), and gel separator tubes (GSTs). The results of PTs and GSTs were compared to those of GTs as the reference tube. The comparison between the means of biomarkers in various tubes indicated that there was no significant difference in MDA results between tubes. In contrast, t-SH and NO content were significantly decreased in GSTs and PTs compared to GTs. However, the Bland-Altman analysis showed an acceptable concordance for the mentioned analytes and the statistically significant differences were not clinically significant for NO, MDA, and t-SH antioxidant parameters. Moreover, the FRAP level was considerably lower in GSTs compared to GTs. Nevertheless, the Bland-Altman analysis showed a high bias percentage for the FRAP assay when using PTs and GSTs. According to the present results, it can be concluded that switching to plastic blood collection tubes or serum separation tubes could influence the FRAP results. However, there was no interference for the interpretation of other antioxidant assays in different types of collection tubes. Hence, it is suggested to use GTs for total antioxidant capacity evaluations, especially the FRAP assay.

Nanoliposomal formulation of pistachio hull extract: preparation, characterization and anti-cancer evaluation through Bax/Bcl2 modulation.

BACKGROUND: Pistachio is one of the main crops in Iran. Pistachio green hull, as a by-product of this fruit, is obtained in large quantities after the processing of pistachios. This novel work was designed to examine the possible anti-cancer impact of the pistachio hull extract in the liposomal form (PHEL) on HepG2 cells. METHODS AND RESULTS: The thin-film hydration approach was used for preparing liposomes and the physicochemical features of the liposomes were subsequently characterized. Afterward, apoptosis and the expression of genes related to apoptosis were assessed using flow cytometry assay and quantitative real-time polymerase chain reaction (qPCR), respectively. According to the results, the size range of PHEL was between 198 and 201 nm with a negative surface charge of - 39.2 to - 42.9 mV. As revealed by the flow cytometry results, this liposomal extract exhibits good potential for the induction of apoptosis. Moreover, the qPCR results demonstrated the up-regulation of p53 and Bax expressions and the down-regulation of Bcl-2 expression with an associated Bax/Bcl-2 ratio up-regulation. CONCLUSION: The flow cytometry and real-time PCR results indicated the potential of this liposomal extract as an anti-cancer drug candidate for the treatment of liver cancer in the future, and the mitochondrial pathway involving the up-regulation of the Bax/Bcl-2 ratio can mediate its impact.

Iron Chelator or Iron Supplement Consumption in COVID-19? The Role of Iron with Severity Infection.

Iron is a trace element that is used to replicate the virus and has a role in the vital functions of the body and the host's innate immune system. The mechanism of iron in COVID-19 severity is still not well understood. The aim of this study was to evaluate the association of the iron with COVID-19 severity. A case-control study was performed on 147 patients with a positive PCR test result and 39 normal individuals admitted to the Persian Gulf Martyrs Hospital in Bushehr, Iran. The iron profiles and related tests were measured along with hematological analytes. Hemoglobin (Hb), Fe, and saturated transferrin decreased in all the groups compared to the controls, but ferritin increased in the patient groups. After adjusting for age and sex, we found that increased ferritin levels augmented the odds ratio (OR) of the disease in the moderate (OR = 2.95, P = 0.007), severe (OR = 6.1, P < 0.001), and critical groups (OR = 8.34, P < 0.001). The decreased levels of Fe reduced the OR of the disease in the mild (OR = 0.96, P < 0.001), moderate (OR = 0.96, P < 0.001), severe (OR = 0.95, P < 0.001), and critical (OR = 0.98, P = 0.001) groups. Fe (AUC = 85.95, cutoff < 75.5 µg/dL, P < 0.001) and ferritin (AUC = 84.45, cutoff > 157.5 ng/dL, P < 0.001) have higher AUC for disease prognosis, but only ferritin (AUC = 74.89, cutoff > 261.5 ng/dL, P < 0.001) has higher AUC for disease severity assays. It could be concluded that the use of iron chelators to reduce iron intake can be considered a therapeutic goal. In addition, measuring Fe and ferritin is beneficial for the diagnosis of the disease and determining its severity.

Can Estradiol and Ghrelin Play a Protective Role in Epithelial Ovarian Cancer Incidence in Postmenopausal Women?

OBJECTIVE: The present study aimed to investigate the association between estradiol, n-octanoylated, des-octanoylated, total ghrelin, and ghrelin/des-octanoylated ghrelin ratio levels along with pathological parameters and epithelial ovarian cancer (EOC) odds in postmenopausal women. MATERIALS AND METHODS: A case-control study was carried out on 45 patients with EOC and 33 age-matched postmenopausal women as the control group. Plasma levels of estradiol, n-octanoylated, des-octanoylated, and total ghrelin were measured by ELISA method. RESULTS: Estradiol's plasma levels were significantly higher in patients with EOC than in control women (p <0.001). Although the ratio levels of n-octanoylated, des-octanoylated, total ghrelin, and ghrelin/des-octanoylated ghrelin were not associated with EOC in logistic regression models, estradiol levels were significantly related to the increase in EOC odds (OR: 1.083, 95% CI: 1.037-1.13, p <0.001). However, estradiol levels in the two first quartiles (Q1, Q2) were associated with decreased odds of EOC (OR: 0.011, 95% CI: 0.001-0.118, p <0.001, and OR: 0.030, 95% CI: 0.003-0.284, p = 0.002, respectively). For those patients in the third quartile of plasma des-octanoylated and total ghrelin compared to those in the highest (Q4), the multivariate odds ratios of EOC were respectively 0.192 and 0.25. CONCLUSION: In conclusion, higher concentrations of des-octanoylated and total ghrelin might be associated with the decreased EOC odds. Furthermore, the findings suggest that high levels of estradiol might be a potential odds factor in EOC, however, lower estradiol levels may have a protective effect on EOC development.

Association of Klotho gene polymorphism with hypertension and coronary artery disease in an Iranian population.

BACKGROUND: Klotho, possibly an age-regulating protein, is considered an important factor contributing to the lifespan and pathophysiology of hypertension and coronary artery disease (CAD). The present study was carried out aiming to investigate the association of Klotho-rs564481 (C1818T) gene polymorphism with hypertension and CAD. METHODS: A total of 286 CAD-suspicious subjects were entered into this case-control study. The polymorphism was investigated in hypertensive patients with no CAD (H-Tens, n = 60); hypertensive patients with CAD (CAD + H-Tens, n = 95); CAD patients with no hypertension (CAD, n = 61); and non-hypertensive non-CAD subjects, which were regarded as the control group (Ctrl, n = 70). Genotype and allele frequencies were assessed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: A significant difference was found in allele frequency of Klotho C1818T among the four research groups (P = 0.03). It was also found that wild-type homozygote subjects were negatively associated with hypertension as compared to heterozygote ones (OR = 0.07 [95% CI: 0.008-0.69] P = 0.02). Moreover, in the subgroups older than 57 years old, dominant genetic model demonstrated a negative association with CAD combined with hypertension (OR = 0.31 [95% CI: 0.10-0.95] P = 0.04). CONCLUSIONS: In conclusion, Klotho C1818T variant may be associated with a decreased risk of hypertension. Moreover, aging enhanced positive effects of the Klotho polymorphism on CAD combined with hypertension, indicating the possibility that the KLOTHO gene might play a part in the age-related occurrence of CAD combined with hypertension.

The spa typing of methicillin-resistant Staphylococcus aureus isolates by High Resolution Melting (HRM) analysis.

Molecular typing is an important tool for control and prevention of infection. A suitable molecular typing method for epidemiological investigation must be easy to perform, highly reproducible, inexpensive, rapid and easy to interpret. In this study, two molecular typing methods including the conventional PCR-sequencing method and high resolution melting (HRM) analysis were used for staphylococcal protein A (spa) typing of 30 Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from clinical samples. Based on PCR-sequencing method results, 16 different spa types were identified among the 30 MRSA isolates. Among the 16 different spa types, 14 spa types separated by HRM method. Two spa types including t4718 and t2894 were not separated from each other. According to our results, spa typing based on HRM analysis method is very rapid, easy to perform and cost-effective, but this method must be standardized for different regions, spa types, and real-time machinery.