mehdi dianatpour

Breast cancer is considered one of the leading causes of mortality in the world. Cancer incidence and consequently, drug consumption can strongly influence gene expressions at the transcriptome level. Therefore, the assessment of the candidate biomarkers’ gene expression can accelerate the diagnosis process and increase the chance of treatment and remission. In this regard, the quantitative assessment of Partner and localizer of BRCA2 (PALB2) and BRCA1 Interacting Helicase 1 (BRIP1) genes expression in the breast cancer cell line under the treatment of Tamoxifen (TAM) was executed in this study.

MCF7 cells were cultured as TAM-treated and control groups. RNA extraction and cDNA synthesis were performed based on the instructions of provided kits. qPCR Hi-ROX Master Mix kit was applied to the Quantitative Real-Time Polymerase Chain Reaction (Q-PCR). The outputs of Q-PCR were analyzed by REST statistical software.

Outcomes derived from data analysis of BRIP1 gene expression did not show any significant difference between the gene expression of control and TAM-treated groups. The expression of PALB2 was significantly higher in the TAM-treated group compared to the control group (P<0.05).

Our findings showed a significant alteration between PALB2 gene expression in the TAM-treated breast cancer cell line and the control cell line. The quantitative assessment of mentioned genes as possible markers could be considered a non-invasive method for breast cancer in the processes of prognostic evaluations, screening, and treatment monitoring.

Androgenetic alopecia (AGA) is a prevalent form of hair loss, mainly caused by follicular sensitivity to androgens. Despite developing different anti-androgen treatment options, the success rate of these treatments has been limited. Using animal models, this study evaluated the therapeutic effects of umbilical cord (UC) stem cell conditioned media (CM) combined with oral anti-androgens for hair regeneration.

In this experimental study, Poloxamer 407 (P407) was used as a drug carrier for subcutaneous testosterone injection. AGA models were treated with oral finasteride, oral flutamide, and CM injections. Samples were thoroughly evaluated and compared using histological, stereological, and molecular analyses.

Injecting CM-loaded hydrogel alone or combined with oral intake of anti-androgens improved hair regeneration. These treatments could promote hair growth by inducing hair follicles in the anagen stage and shortening the telogen and catagen phases. Furthermore, the combination treatment led to an upregulation of hair induction gene expression with a downregulation of inflammation genes.

Through a reduction in inflammation, injection of CM-loaded hydrogel alone or combined with oral intake of anti-androgens induces the hair cell cycle with regeneration in damaged follicles. Hence, this could be a promising therapeutic method for AGA patients.

Valproic acid (VPA), which is often used to treat epilepsy, causes a variety of neurobehavioral impairments that closely resemble the phenotype of autism spectrum disorder (ASD) in prenatally exposed individuals. Although the neurobehavioral effects of extremely low concentrations of VPA have received limited research attention, several investigations have shown that the impact of VPA is connected with the concentration and exposure length.

In the current study, the aim was to find the lowest dose of VPA with the fewest side effects to induce behavioral phenotypes related to ASD in zebrafish.

Zebrafish embryos were first exposed to various concentrations of VPA (i.e., 1, 5, 15, 25, 48, and 75 µM) for 120 hours. Then, 42 days after conception, the survival rate, quality of hatching, and presence of deformity were assessed. Afterward, a 1 µM VPA was chosen based on observations, and behavioral experiments were carried out at 7, 21, and 42 days after fertilization (dpf). Additionally, 7dpf gene expression analysis was evaluated.

According to the obtained findings, behavioral abnormalities resembling ASD were induced in 7 and 21 dpf but not in 42 dpf after 120 hours of exposure to 1µM VPA. Real-time analysis in 7 dpf revealed significant changes in a number of genes linked to ASD, including lrp6, gsk3beta, chd8, and ctnnb.

In conclusion, 120 hours of exposure of zebrafish embryos to 1 µM of VPA might produce suitable VPA induces autism-like behavior models in zebrafish larvae to research early and long-term neurobehavioral and gene expression alterations. Studies on drug development might adopt this approach

Autophagy is a conservative mechanism for cell survival as the main response of cells to stress conditions. The present study aimed to assess the effect of docetaxel on the survival, fertilization, and expression of autophagy-related genes in vitrified oocytes.  The study was conducted in 2018 at the Stem Cells Technology Research Center, Shiraz University of Medical Sciences (Shiraz, Iran). Denuded oocytes were randomly selected and assigned to five groups, namely control (n=133), docetaxel (n=136), docetaxel+cryoprotectants (n=146), docetaxel+vitrification (n=138), and vitrification (n=145). The effect of vitrification on the expression of autophagy-related gene 5 (ATG5) and Beclin-1 was determined using a real-time polymerase chain reaction. Data were analyzed using SPSS software (version 26.0) and GraphPad Prism 9. Survival and fertilization rates in each experimental group were significantly reduced compared to the control group (P=0.001). After in vitro fertilization of oocytes, the 2-cell formation rate was significantly reduced in the docetaxel+vitrification and vitrification groups compared to the control and docetaxel groups (P=0.001 and P=0.001, respectively). Pre-incubation of oocytes with docetaxel reduced gene expression levels of Beclin-1 and ATG5 in the docetaxel+cryoprotectants and docetaxel+vitrification groups (P=0.001 and P=0.019, respectively). The expression level of these genes was also reduced in the docetaxel group compared to the control group (P=0.001).  Incubation of mouse metaphase II oocytes with docetaxel prior to vitrification reduced the expression of autophagy-related genes and increased survival and fertilization rates compared to untreated oocytes.

 The LIPA gene on chromosome 10q23.31 contains 10 exons and encodes lipase A, the lysosomal acid lipase (LAL) containing 399 amino acids. Pathogenic variants in the LIPA result in autosomal recessive Wolman disease and cholesteryl ester storage disease (CESD). Here, we report a novel missense variant (NM_001127605.3:c.928T>A, p.Trp310Arg) of LIPA in an Iranian family with fatty liver disease identified by whole-exome sequencing and confirmed by Sanger sequencing.

 A 28-year-old woman referred with lean NASH cirrhosis and extremely high cholesterol levels. Fatty liver disease was found in six of her family members using vibration-controlled transient elastography (VCTE). Baseline routine laboratory tests were performed and whole-exome sequencing and confirmation by Sanger sequencing were done.

 The index case had severe dyslipidemia and cirrhosis despite a body mass index of 21.09 kg/m2 . Six other family members had dyslipidemia and fatty liver or cirrhosis. A homozygous missense variant (NM_001127605.3:c.928T>A, p.Trp310Arg) of LIPA which caused LAL-D was found to be associated with fatty liver disease and/or cirrhosis.

 A homozygous missense variant (NM_001127605.3:c.928T>A, p.Trp310Arg) of the LIPA gene which caused LAL-D was found to be associated with dyslipidemia, fatty liver disease and/or cirrhosis in six members of an Iranian family. These results should be confirmed by functional studies and extending the study to at least three families.

 Cardiovascular diseases, with an estimated 18.6 million deaths per year, are the leading cause of death worldwide. One of the major risk factors is elevated blood low-density lipoprotein cholesterol (LDL-C) secondary to multiple environmental and genetic factors. Genes involved in LDL-C metabolism are the targets of the most common treatment options. Advanced molecular techniques could pave the way for identifying novel targets in dyslipidemia therapies. The LIM domain and actin-binding 1 (LIMA1) gene binds to the NPC1L1 protein and facilitates its more efficient recycling to the plasma membrane. Inhibition of LIMA1 could disrupt cellular cholesterol hemostasis with a probable decrease in blood LDL-C levels. 

 The present study was designed to knock out exon 2 of the LIMA1 gene using lentiviruses as an in vitro model for reducing cholesterol absorption. 

 A CRISPR/Cas9 system with dual guide RNAs (gRNAs) was designed to completely excise exon 2 of LIMA1. Two gRNAs (gRNA1 and gRNA2) were cloned in the LentiCRISPR v2 vector. LentiCRISPR viruses were produced in the HEK293T cell line to encode the CRISPR/Cas9 complex structure. HepG2 cell lines were transduced with two different LentiCRISPR viruses simultaneously. 

 Exon 2 deletion was detected by PCR, gel electrophoresis, and subsequent Sanger sequencing of the PCR product. Exon 2 deletion caused a frameshift mutation, and the subsequent production of nonfunctional transcripts led to gene knockout. The dual gRNA CRISPR/Cas9 system could be used in gene editing setups. 

 The in vitro knockout model of LIMA1 could be considered as preliminary work to study the role and mechanism of action of the LIMA1 protein, along with its potential as a target for hypercholesterolemia therapy.

Freezing is a long-term egg storage method that plays an important role in assisted reproductive methods. The aim of the present study is to investigate the effect of freezing solutions and docetaxel on the expression changes of autophagy genes such as Atg5 and Beclin-1 in mouse MII oocytes after glass freezing by cryotop method. To achieve this goal, mouse MII oocytes were collected and frozen in two different concentrations of 15% ethylene glycol, 15% dimethyl sulfoxide and 0.5 M sucrose in group A (VS1) and 7.5% ethylene glycol, glycerol. 7.5% and 0.5 M sucrose were frozen in group B (VS2) and some groups were affected by docetaxel before freezing. After thawing, the eggs were fertilized.The percentage of survival and fertilization of frozen and thawed oocytes was evaluated and the expression changes of genes (Atg5 and Beclin-1) were investigated by RT-PCR method. The results showed that there are significant differences between the percentage of survival and the percentage of fertilization in the freezing groups compared to the control group (P<0.05). The percentage of survival and fertilization in the VS1 group decreased compared to the VS2 group. Also, the percentage of survival and conception of the groups pre-incubated with Docetaxel was higher than the non-incubated groups. This study showed that vitrification with cryotop changes the transcript levels of autophagy genes in frozen-thawed MII oocytes, and pre-incubation of oocytes with docetaxel before vitrification can decrease the transcript levels of Atg5 and Beclin-1 in the experimental groups and above Increase the percentage of survival and the percentage of formation of two-celled embryos.

Human tears can be used as a noninvasive source of genetic materials and biomarkers in the prognosis and diagnosis of ocular and non-ocular diseases. The present protocol is a novel direct RNA extraction method from tears. This study aims to provide a suitable method for direct extraction of RNA from tears with high quality and quantity. In this study, we develop a TRIzol base protocol for direct RNA extraction from human tears. quality and quantity of extracted RNA measured by calculation of 260/280 UV absorption ratio using Nanodrop and real-time PCR. RNA was extracted with this modified method and a purified (260/280 UV absorption ratio between 1.8 to 2 and a high yield of total RNA, on average 95 μg, from tears was extracted. In conclusion, we developed an easy and suitable method for direct extraction of total RNA from tears with high quality and quantity.

Stem cell therapy is considered as a promising strategy to treat neurological disorders. Amongst different cell types that are recruited under these devastating conditions, epidermal neural crest stem cells (EPI-NCSCs) are known as potential candidates. Acetylsalicylic acid (ASA or aspirin) is one of the commonly prescribed drugs that might affect the therapeutic potential of the transplanted stem cells. Hence, the present study aimed to evaluate the effects of ASA on the expression of fundamental growth factors involved in restorative pathways expressed by EPI-NCSCs in vitro for possible combination therapy’s purpose.

EPI-NCSCs were obtained from the rat’s hair follicle. The appropriate ASA concentration to treat the cells was defined based on the MTT assay and then the obtained cells were treated with 80 or 800µM ASA for 1, 3 or 7 days. The relative expressions of Bdnf, Gdnf, Ngf, Neurotrophin-3, Vegf, Gfap, and doublecortin were finally assessed by qRT-PCR.

The obtained data revealed that the growth factors expressions are influenced by concentration and duration of the treatment applied. One-day ASA treatment was found to be able to increase the expression of all the evaluated genes, except Gdnf and doublecortin, which elevated three days later. Herein, seven-day treatment of stem cells with 800µM ASA resulted in higher levels of Bdnf, Vegf, and doublecortin.

Therefore, combination of aspirin and EPI-NCSCs might increase the therapeutic potential of these stem cells to treat neurological disorders.

In utero xenotransplantation of stem cells in the abnormal fetuses is effectively used to treat several genetic illnesses. The current research was aimed to evaluate structural and morphological alterations in the liver of rabbit fetuses following xenotransplantation of human Wharton’s jelly-derived mesenchymal stromal/stem cells (hWJ-MSCs), using a stereological technique. hWJ-MSCs were isolated from human umbilical cord and their authenticity was established by flow-cytometry and differentiation. At gestational day 14, the rabbits were anesthetized and hWJ-MSCs were injected into uteri of 24 fetuses. 22 fetuses were born successfully. Ten rabbit liver specimens were prepared from injected fetuses including eight rabbits on day 3 following birth and two rabbits on the 21st post-natal day. The non-injected fetuses were considered as positive controls. The livers of the control and hWJ-MSCs-treated rabbits were fixed, processed, stained, and examined through stereological approaches. In the hWJ-MSCs-treated group, the mean of liver weight and volume enhanced ~42% and ~78% comparing with the control ones. The total volume of the hepatocytes increased ~63% and that of sinusoids almost triplicated in the treated rabbits. The total volume of the central veins increased ~70%. The total number corresponding to hepatocytes in the experimental group enhanced ~112% in comparing with control rabbits. The total volume of the hepatocyte nuclei in the experimental group enhanced ~117% in comparing with control rabbits. In conclusion, after xenotransplantation of human MSCs, host tissue microenvironments (here the rabbit liver) altered quantitative factors corresponding to the liver tissue and hepatocyte morphometric indices.

CRISPR-Cas9 is the most important tool in genome engineering in recent years. The efficiency of this instrument on active and non-active genes is variable. Programmed cell death protein 1(PD-1) is a surface acceptor on T cells, B cells, and dendritic cells. This protein has an important role in the production of inducing tolerance in lymphocytes. Nowadays, this characteristic is used in cell therapy and immunotherapy of cancer. In the present study, the peripheral blood mononuclear cells and HEK293 cells were selected as expression and non-expression cells of the PD-1 gene. Six pairs of sgRNA were designed for the PD-1 gene. The transfected cells were sorted by the FACS machine. A common pair of primers were used for amplification of cute regions. Px458 was used as an expressional vector for the transfection of PBMCs and HEK293. Transfection was done using lipofectamine and electroporation methods. In PBMCs, 2 guides, sgRNA (3+1) and sgRNA (3+5) were able to disrupt the PD-1 gene. In contrast, in HEK293, none of the 6 guides were able to disrupt it. According to the results obtained, the PD-1 gene cutting in HEK293 cells was failed. However, it was successful in PBMCs. Therefore, it can be told that the heterochromatin region or other genome remodeling mechanisms such as epigenetic remodeling inhibit the PD-1 gene cutting by CRISPR-Cas9 in HEK293 cells.

The aim of the present study was to investigate the effect of docetaxel on the survival rate and in vitro fertilization of oocytes after vitrification by two cryopreservation solution.

For this NMRI mice (8-10 weeks old) were superovulated by injecting PMSG and HCG. Oocytes are surrounded by cumulus and corona cells and must be denuded by 0.1% hyaluronidase enzyme. The oocytes were then divided into 8 experimental groups including control, docetaxel, docetaxel + vitrification 1 solution; docetaxel + vitrification1; vitrification1; docetaxel + vitrification 2 solution; docetaxel + vitrification2; vitrification2. Mature oocytes were vitrified in ethylene glycol and dimethyl sulfoxide solutions at 15% concentration and 0.5 M sucrose in cryopreservation solution1 and ethylene glycol and glycerol at 7.5 concentration and 0.5 M sucrose in cryopreservation solution2. After thawing, their survival and fertilization rates were assessed up to the two-cell stage. Staining of the microtubules in the oocytes was performed with alpha-tubulin antibody.

The results showed a significant difference in survive and fertilization rates compared to the control group (P<0.05). The rate of survival and formation of 2-cell embryos in the first cryopreservation group decreased compared to the second cryopreservation group but the two groups were not statistically significant. The results showed that survival and fertilization rates in pre-incubated groups with docetaxel were higher than non-incubated groups.

Docetaxel could improve reproductive techniques by reducing the damage to the oocyte cytoskeleton.

Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the DMD gene, resulting in the absence of dystrophin expression leading to membrane fragility and myofibril necrosis in the muscle cells. Because of progressive weakness in the skeletal and cardiac muscles, premature death is inevitable.  There is no curative treatment available for DMD. In recent years, advances in genetic engineering tools have made it possible to manipulate gene sequences and accurately modify disease-causing mutations. CRISPR/Cas9 technology is a promising tool for gene editing because of its ability to induce double-strand breaks in the DNA.

In this study for the exon-skipping approach, we designed a new pair of guide RNAs (gRNA) to induce large deletion of exons 48 to 53 in the DMD gene in the human skeletal muscle cell line (HSkMC), in order to correct the frame of the gene.

Data showed successful editing of DMD gene by deletion of exons 48 to 53 and correction of the reading frame in edited cells. Despite a large deletion in the edited DMD gene, the data of real-time PCR, immune florescent staining demonstrated successful expression of truncated dystrophin in edited cells.

This study demonstrated that the removal of exons 48-53 by the CRISPR / Cas9 system did not alter the expression of the DMD gene due to the preservation of the reading frame of the gene.

Out of frame mutations in DMD gene cause Duchenne Muscular Dystrophy (DMD) which is a neuromuscular progressive genetic disorder. In DMD patients, lack of dystrophin causes progressive muscle degeneration, which results in heart and respiratory failure leading to premature death. At present, there is no certain treatment for DMD. DMD gene is the largest gene in human genome by 2.2 mega base pairs and contains 79 exons. In the past few years, gene therapy has been considered a promising DMD treatment, and among various gene-editing technologies, CRISPR/Cas9 system is shown to be more precise and reliable. The aim of this study was to assess the possibility of knocking out exon 48 by using a pair of sgRNAs.

A pair of guide RNAs (gRNAs) was designed to cleave DMD gene and induce deletion of exon 48. gRNAs were transfected to the HEK-293 cell line and then the deletion in genomic DNA was analyzed by PCR and subsequent Sanger sequencing.

Exon 48 was successfully deleted and therefore exon 47 was joined to exon 49.

This result indicated that CRISPR/Cas9 system could be used to edit DMD gene precisely.

As a stabilizing agent, docetaxel can potentially reduce the damage to the oocyte cytoskeleton during vitrification. The aim of the present study was to investigate the effect of docetaxel on the survival rate and in vitro fertilization of oocytes after vitrification. NMRI mice (8-10 weeks old) were superovulated by injecting PMSG and HCG. Oocytes are surrounded by cumulus and corona cells and must be denuded by 0.1% hyaluronidase enzyme. The oocytes were then divided into 5 experimental groups including control, docetaxel, docetaxel+vitrification solution; docetaxel+ vitrification and vitrification. Mature oocytes were vitrified in ethylene glycol and dimethyl sulfoxide solutions at 15% concentration and 0.5 M sucrose. After thawing, their survival and fertilization rates were assessed up to the two-cell stage. Staining of the microtubules in the oocytes was performed with alpha-tubulin antibody. The fertilization rate of each group showed a significant decrease compared to the control group (P=0.001). The rate of formation of 2-cell embryos in both vitrified groups (docetaxel+ vitrified and vitrified vitrified) was significantly lower than non-vitrified (control (P=0.001) and docetaxel ((P=0.004)). The results showed that survival and fertilization rates in pre-incubated groups with docetaxel were higher than non-incubated groups, so docetaxel could improve reproductive techniques by reducing the damage to the oocyte cytoskeleton.

Checkpoint blocking is considered a revolutionary method in cancer treatment. This method eliminates cancer cells by maintaining the sensitivity of immune cells. Today, cell therapy through checkpoint blocking is known as the most efficient method of cancer treatment. The programmed cell death protein-1(PD-1), as an immune check protein, has a vital role in weakening the immune responses by reducing the number of stimulated T cells. In normal situations, a decline in the immune responses can cause induced tolerance and prevent autoimmune diseases. In this study, to reduce the induction of tolerance due to PDL-1 binding to PD-1, the PD-1 gene was destroyed in PBMCs by the means of CRISPR-Cas9 and dual-transfection of two plasmids containing the Cas 9 gene and two different sgRNAs specific to two region of PD-1 gene in order to produce a deletion mutation.  Six different sgRNA were designed and cloned in PX-458 plasmid vector, and PBMCs were transfected using lipofectamine 2000 and electroporation. Indels were evaluated by gel electrophoresis and Sanger sequencing.   We showed the PD-1 gene in PBMCs was knocked out successfully by CRISPR-Cas9 and dual-transfection of two sgRNAs. The minimum interval between the two sgRNAs was 448 nucleotides. The results of this research demonstrated that the use of dual-transfection of CRISPR-Cas9 sgRNA is a suitable method to knock out the PD-1 gene and prevention of inducing tolerance in PBMCs.

Animals can play an important role in preparing tissues for human through the development of xenotransplantation protocols. The most common problem with liver transplantation like any other organ transplantation is organ supply shortage.

To evaluate the in utero xenotransplantation of mouse bone marrow-derived stromal/stem cells (BMSCs) to the liver of rat fetus to produce mouse liver tissue.

BMSCs were isolated and confirmed from enhanced green fluorescent protein (eGFP)-genetic labeled mice. Using a microinjection protocol, mice BMSCs were injected into the liver of rat fetuses in utero on day 14 of pregnancy. After birth, livers were collected and the presence of mice eGFP-positive cells in rat livers was evaluated through polymerase chain reaction.

The eGFP mRNA was detected in the liver of injected infant rats. BMSCs of adult mice were capable to remain functional probably as hepatocyte-like cells in liver of infant rats after in utero xenotransplantation.

BMSCs have the potential for intrauterine xenotransplantation for the treatment of liver dysfunction before birth. This method can also be used for xenoproduction of liver tissue for transplantation.

CAD is a major cause of death in worldwide. Both vitamin D (vit D) and Vitamin-D receptor (VDR) gene polymorphisms have been reported to be associated with Coronary artery disease (CAD). Because of high prevalence of vit D deficiency and mortality caused by cardiovascular diseases in our country, Iran, in this study we aimed to determine the frequency of two known VDR gene polymorphisms (BsmI and ApaI) in patients undergoing Percutaneous Coronary Intervention (PCI) in Iranian populations. Blood samples were collected from 150 patients performing elective PCI (102 males and 48 females). VDR genotypes were determined by RFLP method. Serum vit D levels were measured using HPLC method and patients were divided into three groups as follows: subjects with a total vit D concentration 30 ng/ml> were described as normal, 20-30 ng/ml as insufficient and < 20 ng/ml as deficient. Among 150 samples analyzed for ApaI and BsmI polymorphisms the following genotypic frequency was observed: AA 44.67%, AC 44.67%, and CC 10.66% for ApaI and GG 47.33%, GA 37.33%, AA 15.34% for BsmI Levels of active vitamin D could be influenced by both environmental and genetic factors. Our results also revealed that VDR gene polymorphisms (ApaI and BsmI) may vary across different ethnic groups in CAD patients.

The placenta provides nutrients and oxygen to embryo and removes waste products from embryo’s blood. As far as we know, the effects of exposure to Wi-Fi (2.4 GHz) signals on placenta have not been evaluated. Hence, we examined the effect of prenatal exposure to Wi-Fi signals on anti-oxidant capacity, expressions of CDKNA1, and GADD45a as well as apoptosis in placenta and pregnancy outcome.

Pregnant mice were exposed to Wi-Fi signal (2.4 GHz) for 2 and 4 hr. Placenta tissues were examined to measure the MDA and SOD levels. To measure SOD, CDKNA1, GADD45a, Bax, and Bcl-2 expressions were compared by real-time PCR analysis. TUNEL assay was used to assess apoptosis in placenta tissues. The results were analyzed by one-way analysis of variance (ANOVA) using Prism version 6.0 software.

MDA and SOD levels had significantly increased in exposed Wi-Fi signal groups (P-value< 0.05). Also, quantitative PCR experiment showed that SOD mRNA expression significantly increased in Wi-Fi signal groups. The data showed that CDKN1A and GADD45a genes were increased in Wi-Fi groups (P-value<0.05). The quantitative PCR and the TUNEL assay showed that apoptosis increased in Wi-Fi groups (P-value<0.05).

Our results provide evidence that Wi-Fi signals increase lipid peroxidation, SOD activity (oxidative stres), apoptosis and CDKN1A and GADD45a overexpression in mice placenta tissue. However, further experimental studies are warranted to investigate other genes and aspects of pregnancy to determine the role of Wi-Fi radiation on fertility and pregnancy.

Both vitamin D and inflammation were investigated as important players in the pathogenesis of postmenopausal osteoporosis. This study compared vitamin D, inflammatory the biomarkers serum levels and their association with bone mineral density (BMD) in case and control groups to evaluate the possible immune-regulatory effect of vitamin D in this population.

Participants in post-menopausal age, were categorized to 44 osteoporotic vs. 44 healthy aged-matched women according to WHO criteria. Total BMD, T- scores, Z-scores as well as fracture risk were measured in both groups, using Hologic system Dual-energy X-ray absorptiometry (DEXA). Serum 25-OH vitamin D, high sensitive CRP (hs-CRP) and serum amyloid A (SAA) were compared between groups. The association between serum biomarkers level and BMD were also investigated. The same evaluations were performed for vitamin D deficient (<20 ng/mL) and non-deficient (≥20 ng/mL) subgroups.

Vitamin D deficiency was higher in the osteoporotic group (32.6%) in comparison with the control group (25.6%), but the differences were not significant (P=0.47). There were no significant differences in serum levels of hs-CRP and SAA (P=0.83 and P=0.39) as well. No significant association between serum inflammatory biomarkers, vitamin D, and BMD were detected (P≥0.05). The results were the same for vitamin D deficient and non-deficient subgroups (P≥0.05).

In the current study, the beneficial effects of vitamin D as a result of its immune-regulatory mechanisms was not reached. Larger scale studies might pave the way to define vitamin D benefits in postmenopausal osteoporosis.

Gamete cryopreservation is an inseparable part of assisted reproductive technology, and vitrification is an effective approach to the cryopreservation of oocytes. The aim of this study was to investigate vitrification effects on the expression levels of mitochondrial transcription factor A (Tfam) and mitochondrial-encoded cytochrome c oxidase subunit 1 (Cox1) in mouse metaphase II oocytes. Oocytes were selected by simple random sampling and distributed amongst five experimental groups (control [n=126], docetaxel [n=132], docetaxel+cryoprotectant agent [CPA] [n=134], docetaxel+vitrification [n=132], and vitrification [n=123]). After the warming process, the oocytes were fertilized and cultured into a 2-cell stage. Then, the effects of vitrification on the expression of the Tfam and Cox1 genes were determined via real-time reverse transcriptase polymerase chain reaction. Each group was compared with the control group. The data were analyzed with ANOVA using GraphPad and SPSS, version 21. A significant decrease was observed in the fertilization rate of each group in comparison with the control group (P=0.001). The rate of 2-cell formation after in vitro fertilization was significantly lower in both vitrification groups (docetaxel+vitrification and vitrification) than in the non-vitrification groups (fresh control and docetaxel) and control group (P=0.001 and P=0.004). The expression level of Cox1 was significantly higher in the vitrification group than in the control group (P=0.01), while it was lower in the docetaxel group than that in the control group (P=0.04). The expression level of the Tfam gene was significantly high in the vitrification group (vitrification+docetaxel) and the non-vitrified group (docetaxel+CPA) in comparison with the control group (P=0.01). This study indicated that the vitrification of mouse MII oocytes increased the expression of the Tfam and Cox1 genes.

CRISPR/Cas9 system is a powerful gene editing tool in vivo and in vitro. Currently, CRISPR/Cas9 delivery cells or tissue with different vehicles are available, and Adeno- associated virus (AAV) in one of them. Due to AAV packaging size limitation, AAV base vectors that carry CRISPR/Cas9 system do not have florescent tag like GFP for simple detection and navigation of cells, containing AAV. The aim of this study was to modify and synthesis AAV base vector for CRISPR/cas9 system containing sgRNA and GFP.Px602 plasmid was double digested with NcoI and HindIII restriction enzyme. Gfp gene was amplified from px458 plasmid. Linear digested px602 and amplified Gfp gene were ligated together. After transformation and colony PCR on white colonies, plasmid was extracted and transfected to HEK-293 cell line. Gfp expression was monitored by florescent microscopy. After transfection of modified plasmid, florescent microscopy of HEK-293 cells showed shining green florescent cells, which indicate that Gfp gene, was replaced in the correct place according to our design.We modified an AAV base vector carrying CRISPR/Cas9 system, and synthesized a new vector carrying Gfp gene and sgRNA that can be packaged as reporter AAV for navigation and detection of cells, containing AAV.

Small supernumerary marker chromosomes (sSMCs), or markers, are abnormal chromosomal fragments that can be hereditary or de novo. Despite the importance of sSMCs diagnosis, de novo sSMCs are rarely detected during the prenatal diagnosis process. Usually, prenatally diagnosed de novo sSMCs cannot be correlated with a particular phenotype without knowing their chromosomal origin and content; therefore, molecular cytogenetic techniques are applied to achieve this goal. The present study aimed to characterize an sSMC in a case of Klinefelter syndrome using an in-house microsatellite analysis method and fluorescent in situ hybridization (FISH) technique. Amniotic fluid was collected from a pregnant woman who was considered to have risk factors for trisomy higher than the screening cut-off. Karyotype analysis was followed by the amplification of different microsatellite loci and FISH technique. Karyotype analysis identified a fetus with an extra X chromosome and also an sSMC with unknown identity. Further investigation of the parents showed that the sSMC is de novo. Microsatellite amplification by quantitative fluorescent PCR (QF-PCR) and FISH analysis showed that the sSMC is a derivative of chromosome 18. Eventually, the patient decided to terminate the pregnancy. Here, the first case of the coincidence of sSMC 18 in a Klinefelter fetus is reported.

Pterygium is one of the most common eye conditions without any clear etiology. Some studies have suggested an association between sun exposure and pterygium, but others have proposed the role of genetic variations in its pathogenesis. To date, no study has investigated the association of inflammatory transcription factor, NFκB genes with pterygium in the Middle East. We examined the changes in expression of 3 inflammatory related NFκB1, NFκB2, and RELA genes in patients with pterygium. Thirty patients with pterygium and 30 age and sex-matched controls were enrolled in this case-control study. None of the participants showed any clinical signs of inflammation in their conjunctiva. Demographic information was obtained and the expression levels of three genes including NFκB1, NFκB2, and RELA were measured in their conjunctiva by real-time RT-PCR using gene-specific primers. Mean expression level of NFκB1, NFκB2 and RELA genes in patients were 2.4±0.3, 1.9± 0.5, and 1.8±0.4 times higher than normal subjects, respectively. Higher levels of gene expression were observed in individuals with more outdoor activity and sun exposure. Moreover, a significant correlation was observed between the expression levels of NFκB2 and RELA genes, suggesting a possible NFκB2- RELA heterodimer formation in patients with pterygium. This study has indicated a significant association between expressions of inflammatory-related NFκB1, NFκB2 and RELA genes, and pterygium. Further studies to verify the role of inflammation in the pathogenesis of pterygium, may provide new targets for managing pterygia.

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) with more potent immunomodulatory effects, greater proliferative potential and secretion of growth factors and cytokines in comparison with bone marrow derived MSCs are more appropriate for cell therapy. The aims of the present study were to evaluate the histomorphometric effect of AT-MSCs allotransplantation on regeneration of germinal layer cells of seminiferous tubules in busulfan-induced azoospermic hamsters.
In the present experimental case-control study, AT-MSCs were isolated from adipose tissue of two female and six male donor albino hamsters, and testes of the males were simultaneously used as negative control group. Six mature male recipient hamsters received two doses of busulfan with three weeks interval to stop endogenous spermatogenesis. Right testis of hamsters was intratubular injected with AT-MSCs via efferent duct 35 days after induction of azoospermia and was used as cell therapy group. The left testis without cell therapy was served as azoospermia group.
After 35 days, testes and epididymis in all groups were removed for histological evaluation. Histomorphometric analyses of AT-MSCs-treated testes and epididymis showed that the epithelial tissue of seminiferous tubules was normally repaired in most cell-treated seminiferous tubules, and spermatozoa were present in epididymis tubes in comparison with intact testes. The untreated seminiferous tubules and epididymis tubes of azoospermia group were empty.
Allotransplanted AT-MSCs could successfully induce spermatogenesis in azoospermic seminiferous tubules of hamster. Therefore, AT-MSCs can be suggested as an attractive candidate in cell transplantation of azoospermia.