Cell and Tissue Journal

Abstract Introduction: Titanium dioxide nanoparticles (TiO₂ NPs) are widely used in industry, medicine, food, and cosmetics. TiO₂ NPs are harmful to the environment and human health. Changes in the environment may especially affect the growing neurological system. Therefore, it is impossible to overlook their impact on the development of the embryo and reproductive success. A sensitive and widely used model for evaluating the teratogenic potential and developmental toxicity of different nanoparticles is the chicken embryo. At the outset of embryogenesis, the application of TiO₂ NPs enables the penetration into various tissues, such as brain precursor cells and structures. Furthermore, the organs are unable to remove the nanoparticles from the egg due to their isolation and enclosure from the mother and the environment. Consequently, the embryos are perpetually exposed to TiO₂ NPs during the 20-day embryogenesis period. In recent years, a growing number of studies have been conducted to examine the possible harmful effects of TiO₂ NPs due to worries about inadvertent exposure of NPs on humans.
Aim: In this study, the embryonic toxicity of various dosages of TiO₂ NPs was investigated in the chicken embryo's brain cortex.
Materials and Methods: In this study, 90 fertilized eggs were divided into five groups: control group (untreated group), four treatment groups that received 0 (sham), 12.5, 25, and 50 μg/mL of TiO₂ nanoparticles. The embryos' morphology, weight, and Crown-rump length (CRL) were assessed after 7, 9, and 13 days.   Tissue samples were collected from the cerebrum and cerebellum of the chick embryos. The specimens were immediately fixed in a 10% neutral buffered formalin solution for 48 hours. Subsequently, they were dehydrated through a series of ascending ethanol concentrations (70%, 80%, 90%, and 100%), with each step lasting two hours. The samples were then cleared in xylene for one hour, embedded in paraffin wax, and sectioned at a thickness of 5μm using a microtome. The sections were then mounted on glass slides.  Finally, all tissue sections were stained with hematoxylin and eosin, and the effects of TiO₂ NPs were examined on the histology, pathology, and histomorphometry of the chick embryo cerebrum and cerebellum tissues.
Results: The findings showed that TiO₂ NPs cause embryo death in all days at 25 and 50 μg/mL. In morphological studies, the weight and length of 13-day-old embryos treated with 50 μg/mL of TiO₂ NPs decreased. Counting of cells (neurons and glial cells) in the cerebral cortex of a 13-day-old chick embryo displayed a significant decrease in the experimental group of 50 μg/ml compared to the control and sham groups. The evaluations showed a decrease in the number of Purkinje cells of the cerebellum cortex in 13-day-old embryos treated with 50 μg/ml of TiO₂ NPs.   The group of 13-day-old embryos treated with 50 µg/ml had a considerable capillary hyperemia in the cerebellar cortex.
Conclusion: It was concluded that the in-ovo-administered TiO₂ NPs given immediately before incubation have adverse effects on the developing cerebellum and cerebrum. So that the increase of damage happened in older embryos, and the highest damage occurred on day 13 of incubation.

Abstract Introduction: Peppermint (Mentha piperita L.) is an economically and medicinally significant plant valued for its essential oil. Silver nanoparticles (AgNPs) act as potent elicitors, triggering defense responses that include the production of reactive oxygen species (ROS) and the accumulation of secondary metabolites. However, the impact of AgNPs on peppermint’s physico-biochemical traits and essential oil performance remains underexplored.
Aims:  This study aimed to evaluate the effects of AgNPs on various physico-biochemical characteristics and essential oil performance in peppermint. The analyzed parameters, including H₂O₂, soluble proteins, phenols, flavonoids, antioxidant enzymes (SOD, CAT, APX), and photosynthetic pigments (chlorophyll and carotenoids), were examined to determine the optimal concentrations for enhanced metabolic activity.
Materials and methods: Rhizomes were planted in pots and grown under controlled greenhouse conditions, including a daytime temperature of 25°C, a photoperiod of 16 h of light and 8 h of darkness, and a relative humidity of 60. At the ten-leaf stage, plants underwent foliar spraying with 0 (control), 1, and 2 mM AgNPs. The AgNPs solution was prepared, characterized via UV-Vis spectroscopy, and SEM (20 nm quasi-spherical particles). A completely randomized design with three replicates was used to assess parameters including H₂O₂ content, soluble proteins, phenols, flavonoids, antioxidant enzymes (SOD, CAT, APX), photosynthetic pigments (chlorophyll, carotenoids), and essential oil performance. Statistical analysis was performed using ANOVA and LSD tests (p<0.05).
Results: AgNPs significantly elevated all measured parameters compared to the control in a generally concentration-dependent manner: Treatment with 1 and 2 mM AgNPs increased H₂O₂ content by 2- and 3-fold, respectively. Treatment with 2 mM AgNPs resulted in a 2.4-fold increase in protein content. 1 and 2 mM AgNPs resulted in a 2.8- and 2.6-fold increase in leaf phenolic content, respectively. Concentrations of 1 and 2 mM AgNPs resulted in a 1.8- and 2.6-fold increase in leaf flavonoid content, respectively. Treatment with 2 mM AgNPs resulted in a 3.3-fold increase in SOD activity. Treatment with 2 mM AgNPs had a significant positive effect (about a 2.2-fold) on CAT enzyme activity. However, treatment with 1 mM AgNPs failed to affect CAT enzyme activity. AgNPs 1 and 2 mM increased APX enzyme activity by 1.7 and 2.5 times, respectively. Treatment with 1 and 2 mM AgNPs increased total chlorophyll concentration by 2.4- and 3-fold, respectively. 1 and 2 mM AgNPs increased carotenoid concentration by 1.8- and 3.5-fold, respectively. Treatment with 1 and 2 mM increased essential oil yield by 2.2- and 2.3-fold, respectively.
Discussion: AgNPs significantly increased the level of H₂O₂, soluble proteins, phenols, flavonoids, antioxidant enzymes (SOD, CAT, APX), chlorophyll, carotenoids, and essential oil yield. Although the mechanisms of plant response to AgNP elicitors are not well understood, we proposed that the improvement in peppermint’s physico-biochemical traits in our study was due to the following series of mechanisms: AgNPs induce ROS (e.g., H₂O₂) generation through NADPH oxidase activation and electron leakage from organelles. This oxidative stress activates MAPK cascades and calcium signaling, which upregulate transcription factors (MYB, bHLH). These transcription factors subsequently enhance the expression of phenylpropanoid pathway enzymes (e.g., PAL) and terpenoid biosynthesis genes, leading to increased essential oil biosynthesis and accumulation. AgNP elicitors also improve antioxidant enzyme activity (SOD, CAT, APX) to mitigate ROS damage. In addition to the enzymatic antioxidant system, AgNPs enhance components of the non-enzymatic antioxidant system, including phenols and phenoloids. Furthermore, these nanoparticles upregulate key chlorophyll biosynthesis genes (HEMA1, CHLH), while suppress degradation-related genes (NYC1, PAO), resulting in elevated photosynthetic pigment concentrations.
Conclusion: Foliar application of 2 mM AgNPs significantly enhances peppermint’s antioxidant capacity, photosynthetic efficiency, and essential oil biosynthesis by modulating ROS-mediated signaling. Therefore, this concentration is recommended for maximizing peppermint’s medicinal and economic potential. Future studies should explore broader concentration ranges and early gene expression responses (6–12 h post-treatment).

Abstract Introduction: Mesenchymal stem cells (MSCs) are  important cellular components in the microenvironment of various types of cancers, including multiple myeloma (MM); their dynamic interactions with malignant cells can control the tumor microenvironment (TME) to favor the progression, drug resistance, and survival of cancer cells. It is shown that not only are MM cells influenced by the environmental cells, but also malignant cells affect the behavior of other cells in the TME. Secreted signaling factors are important players in this scenario. Accordingly, MSCs derived from TME, affected by cancer cells, are different from normal MSCs; therefore, in cases of disease modeling and drug screening studies, optimization of MSCs culture condition and their priming with cancer cell-derived secretome are crucial considerations to become more similar to the patient-derived MSCs. In this regard, conditioned medium, which is derived from the supernatant culture medium of cultured cells, contains cell-secreted factors and can be used as a source of cell secretome.
Aims: This study attempted to evaluate the effect of conditioned medium derived from the myeloma cell line U266 (U266-CM) on the proliferation, cell cycle, and differentiation of bone marrow-derived MSCs (BMMSCs).
Materials and methods: U266 cells were cultured in RPMI-1640 complete culture medium, and their conditioned medium was collected after two days of culture and stored. Since the basal culture medium of the U266 cell line (RPMI-1640) was different from the basal culture medium of BMMSCs (DMEM), in the first step, the impact of changing the culture medium from DMEM to RPMI-1640 on the proliferation and viability of BMMSCs was evaluated. In the next step, the effects of U266-CM treatment on the proliferation, cell cycle, and differentiation of BMMSCs into osteoblasts and adipocytes were assessed. Alkaline phosphatase activity assessment and Alizarin red staining were performed to evaluate the osteogenic differentiation, and Oil red O staining was carried out to assess the adipogenic differentiation. BMMSCs cultured in DMEM complete media were considered as control group.
Results: Changing the culture media from DMEM complete media to RPMI-1640 complete media affected the viability and proliferation rate of BMMSCs; however, up to 48 hours, the viability and proliferation rate of cells cultured in both culture media were maintained. Therefore, 48 hours was selected as the optimum incubation time of BMMSCs with U266-CM. Treatment with U266-CM decreased the proliferation rate of BMMSCs, assessed by cell counting and cell cycle analysis. Moreover, BMMSCs cultured in U266-CM showed slightly increased osteogenic differentiation and maintained their adipogenic propensity.
Discussion: Our results showed that CM derived from the MM cell line affected the proliferation rate and the differentiation potential of BMMSCs. These observations highlighted the importance of a more similar recapitulation of BMMSCs culture condition to the TME, for disease modeling and drug screening studies. Further experiments, including providing dynamic interactions of MSCs with MM cells in direct or indirect co-culture systems, are required to better recapitulate TME conditions for MSC priming. Moreover, for mechanistic analysis, evaluation of the secreted factors and assessment of expressed transcripts and proteins are highly recommended for future studies.
Conclusion: In summary, U266-CM influenced the proliferation rate and the differentiation potential of BMMSCs.

Abstract Introduction: Homeodomain transcription factor 1 (PDX-1) is necessary for proper pancreatic development and β-cell function. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, in conjunction with CRISPR-associated protein 9 (Cas9), facilitates modifications, particularly deletions, within the genome. The use of lentiviral vectors for gene transfer to somatic stem cells has increased significantly. They harbor advantageous features, including a relatively high gene loading capacity, a diminished likelihood of gene expression silencing following integration into the genome, and high efficiency in delivering genes to a variety of target cells. Furthermore, they elicit no undesirable immune reactions and offer a broad selection of alternative envelope proteins.
Aim: This study aims to introduce a mutation in the chicken PDX-1 gene. A guide RNA (gRNA) will be designed to complement a specific region of the PDX-1 gene. This approach aims to create targeted mutations with potential therapeutic applications in gene therapy.
 
Materials and Methods: The PDX-1 gene was cloned using the LentiCRISPRv2GFP vector. To delete and modify the PDX-1 gene, a specific single guide RNA (sgRNA) sequence is designed. This sgRNA sequence is approximately 100 base pairs (bp) in length, with about 76 bp derived from the scaffold segment already integrated into the LentiCRISPRv2GFP vector.
The region responsible for binding the sgRNA to the target genomic sequence is 20 nucleotides long and is complementary to 20 nucleotides in exon one of the PDX-1 gene. The CRISPR Design Tool (http://crispr.mit.edu) was used to design a specific sgRNA sequence targeting the PDX1 locus (sgPDX1).
The double-stranded oligonucleotide fragment and the LentiCRISPRv2GFP vector were subjected to enzymatic digestion using the BsmbI restriction enzyme. To create the LentiCRISPRv2GFP-sgPDX1 vector, the cleaved sgPDX1 sequence was joined with the cleaved vector. This was accomplished by mixing the vector and nucleotide fragment and subjecting them to a ligation reaction using the T4 ligase enzyme. The resulting vector contains a transcript of the sgRNA, specifically sgPDX1.
The heat shock method was employed to transform Escherichia coli Top10 host cells with the cloned LentiCRISPRv2GFP-sgPDX1 vector. To confirm the presence of the sgPDX-1 gene in the vector, a polymerase chain reaction (PCR) was conducted using primers targeted at a portion of the U6 promoter sequence (forward primer) and a segment downstream of the sgRNA gene region (reverse primer). Additionally, each PCR product was analyzed using enzymatic digestion with EcoRV and BsmBI enzymes. To confirm the successful cloning, the PCR product was sequenced.
Results: The PCR results showed an amplified fragment measuring 267 base pairs (bp), which indicated the presence of the sgPDX-1 gene. This was visualized on an agarose gel. Additionally, the reverse digestion with two restriction enzymes produced a 2000 bp fragment isolated from the vector. The sequencing results confirmed that the cloning process was successful.
Discussion: Genome editing technology has been highlighted in bird species due to their significant potential for economic production and creating models for biological research. The application of CRISPR gene editing technology in avian research has yielded promising advancements, particularly in the modification of somatic cells and tissues in birds. By precisely targeting primordial germ cells (PGCs), researchers can implement specific genetic modifications within the chick genome, thereby facilitating the development of genetically edited chicks. Numerous studies have investigated the role of the PDX-1 gene in gene therapy using the CRISPR/Cas9 system across various organisms. However, this study is the first to design and produce a recombinant construct containing the chicken PDX1 gene using a lentiviral vector with the CRISPR/Cas9 construct. This advancement aims to create a practical research platform for future studies by enabling the direct transfer of this recombinant construct into chick embryos and cells.
Conclusion: The chicken PDX-1 gene was cloned into a lentiviral vector containing the CRISPR/Cas9 construct. The recombinant vector LentiCRISPRv2GFP-sgPDX1 was successfully obtained. The gene construct developed in this study has significant potential for investigating gene function and for use in gene therapy. Consequently, utilizing this construct may enable the creation of an animal model in chicken embryos featuring defects, including an empty organ or tissue cavity. Such a model will allow for the investigation and treatment of the defects using specific drugs and cell lines.

Abstract Introduction: Numerous studies on microRNAs (miRNAs) have indicated that abnormal interactions between miRNAs and mRNAs contribute to the development and worsening of endometriosis. miR-223 influences genes associated with various biological functions such as signaling pathways, gene expression, cellular development, proliferation, angiogenesis, and programmed cell death. Alterations in genes have been demonstrated to be significant in the progression of endometriosis. The p53 gene, recognized as a tumor suppressor, plays a vital role in inhibiting cancer by regulating cell growth and division. Recent investigations suggest a potential connection between the p53 gene and endometriosis. In addition, Forkhead box protein O1 (FOXO1) acts as a cell-specific core transcription factor essential for effective endometrial remodeling throughout the menstrual cycle and may play an important role in the onset of endometriosis.
Aims: This study aimed to explore the expression levels of miR-223-3p and its target genes, TP53 and FOXO1, in cases of ovarian endometriosis through bioinformatic analysis and experimental verification.
Materials and methods: Gene expression data were obtained from the GSE105765 dataset in the Gene Expression Omnibus (GEO) database, which encompasses both normal (eutopic) and ectopic tissue samples from endometriosis patients. Differentially, expressed genes were pinpointed using the "limma" package in R software, with the results visualized as a heat map. Among the various expressed miRNAs, miR-223-3p was chosen for further investigation. Subsequently, the target genes of miR-223-3p were explored utilizing the miRDB database, leading to the identification of FOXO1 and TP53 as target genes. In the experimental component of this study, 40 ectopic tissue samples, 40 eutopic tissue samples from individuals with endometriosis, and 40 normal endometrial samples (control group) were analyzed. Following the extraction of total RNA and synthesis of cDNA, the expression levels of miR-223-3p, TP53, and FOXO1 were assessed using Real-Time PCR.
Results: The average relative expression of miR-223-3p in the ectopic group was measured at 4.13±0.72, while in the eutopic group it was 1.10±0.10, and in the control group it was 1.00±0.09. The relative expression of miR-223-3p was significantly higher in the ectopic group compared to both the eutopic and control groups (p<0.0001), whereas no significant differences were found between the control and eutopic groups (p=0.54). The average expression of FOXO1 in the ectopic group (0.47±0.12) was significantly lower than that in the eutopic group (1.02±0.06) and the control group (1.001±0.11). One-way ANOVA revealed a significant reduction in FOXO1 expression in the ectopic group when compared to the eutopic group and the control group (p<0.0001). However, there was no significant difference in FOXO1 expression between the eutopic and control groups (p=0.53). The relative expression level of the TP53 gene was analyzed in the ectopic group (0.335±0.14), the eutopic group (1.05±0.09), and the control group (1.01±0.12). Statistical evaluations showed that TP53 gene expression was significantly lower in the ectopic group compared to both the eutopic and control groups (p<0.0001). Conversely, no significant difference was observed in TP53 expression between the eutopic and control groups (p=0.28).
Conclusion: The findings indicate that miR-223-3p expression in ectopic samples was significantly elevated compared to both the eutopic and the control samples, while the expression of the FOXO1 and TP53 genes in the ectopic group was notably reduced relative to the eutopic and control groups. These results suggest that the expressions of miR-223-3p, TP53, and FOXO1 genes are changed in the endometrium of individuals with endometriosis, highlighting their potential roles in the disease's pathogenesis and their therapeutic implications.

Abstract Introduction: Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder among women of reproductive age. It is characterized by hormonal imbalances, irregular menstrual cycles, hyperandrogenism, and insulin resistance. The prevalence of this condition is on the rise, largely due to changes in lifestyle, poor dietary habits, and increasing rates of obesity. PCOS not only affects reproductive health but also poses long-term metabolic risks, including type 2 diabetes, cardiovascular disease, and dyslipidemia. Despite its high prevalence, the exact cause of PCOS remains unclear, and its heterogeneous nature makes diagnosis and management challenging. Early identification and a multidisciplinary approach to treatment are essential for improving patient outcomes and preventing long-term complications. Aim: Given the prevalence of polycystic ovary syndrome and its importance as a major problem affecting women's health and fertility, the present study aimed to investigate the relationship between anti-mullerian hormone with insulin resistance and reproductive hormones in women with this syndrome. Materials and Methods: A total of 100 women aged 18 to 45 years, including both healthy individuals and those diagnosed with PCOS, participated in this study. The diagnosis of PCOS was confirmed by a gynecologist based on clinical examination and transvaginal or pelvic ultrasound findings. For all participants, the following parameters were recorded: age, weight, height, waist circumference, hip circumference, fasting blood glucose, insulin levels, and serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogen, progesterone, free testosterone, and anti-Müllerian hormone (AMH). Results: There was no significant difference in mean age and FSH levels between the experimental groups. However, women with PCOS showed a significant decrease in estradiol and progesterone levels, along with a significant increase in body mass index (BMI), LH, free testosterone, AMH, and insulin resistance compared to the control group. Pearson correlation analysis revealed no significant relationship between AMH and the hormones LH, FSH, testosterone, or progesterone in either the control group or the PCOS group. However, a significant positive correlation between AMH and estradiol was observed in the control group, while a significant negative correlation was found in the PCOS group. Discussion: Overweight, obesity, and hormonal imbalances are common characteristics observed in women with PCOS. In the control group, a positive and significant correlation between serum AMH and estradiol levels suggests a normal physiological relationship between follicular activity and estrogen production. In contrast, the significant negative correlation observed in the PCOS group indicates a disruption in the hypothalamic-pituitary-ovarian (HPO) axis and reflects abnormal folliculogenesis. This dysregulation may lead to anovulation and persistent immature follicles, which are hallmarks of PCOS. Moreover, elevated AMH levels in PCOS patients are often linked with increased ovarian follicle count, yet without proper maturation, resulting in hormonal imbalance and infertility. These findings highlight the importance of early detection and hormonal monitoring in women with PCOS to prevent long-term reproductive and metabolic complications. Conclusion: These findings underscore the potential utility of AMH–estradiol correlation patterns as a supplementary diagnostic marker for PCOS. Given the complex and heterogeneous nature of PCOS, the identification of reliable biomarkers is crucial for early diagnosis and tailored treatment strategies. Further studies with larger sample sizes and diverse populations are needed to validate the role of AMH in PCOS diagnosis and its relationship with estradiol levels. Moreover, exploring the underlying mechanisms of AMH dysregulation in PCOS could provide valuable insights into potential therapeutic targets for managing both reproductive and metabolic complications associated with the condition.