Inducing Long Lasting B Cell and T Cell Immunity Against Multiple Variants of SARS-CoV-2 Through Mutant Bacteriophage Qß-Receptor Binding Domain Conjugate.

More than 3 years into the global pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant threat to public health. Immunities acquired from infection or current vaccines fail to provide long term protection against subsequent infections, mainly due to their fast-waning nature and the emergence of variants of concerns (VOCs) such as Omicron. To overcome these limitations, SARS-CoV-2 Spike protein receptor binding domain (RBD)-based epitopes are investigated as conjugates with a powerful carrier, the mutant bacteriophage Qß (mQß). The epitope design is critical to eliciting potent antibody responses with the full length RBD being superior to peptide and glycopeptide antigens. The full length RBD conjugated with mQß activates both humoral and cellular immune systems in vivo, inducing broad spectrum, persistent, and comprehensive immune responses effective against multiple VOCs including Delta and Omicron variants, rendering it a promising vaccine candidate.

Melatonin Protects Mouse Type A Spermatogonial Stem Cells against Oxidative Stress via The Mitochondrial Thioredoxin System.

OBJECTIVE: Mitochondrial oxidative stress is an important factor in infertility. The mitochondrial thioredoxin system plays an important role in this condition. N-acetyl-5-methoxy tryptamine (melatonin) plays a role in reducing oxidative stress and apoptosis in spermatogonial stem cells (SSCs). In this study, we explore the probable protective effects of melatonin on the mitochondrial thioredoxin system [thioredoxin 2 (Trx2)/Txnip] in SSCs under oxidative stress. MATERIALS AND METHODS: In this experimental study, SSCs were co-cultured two-dimensionally (2D) with Sertoli cells in DMEM culture medium that contained 10% fetal bovine serum (FBS), 1% antibiotics, and 10 ng/ml glial cell-derived neurotrophic factor (GDNF) for 30 days. The cultured cells were subsequently divided into four groups: control; melatonin (250 µM, 24 hours); melatonin (250 µM, 24 hours)+hydrogen peroxide (H2O2, 50 µM, 24 hours); and H2O2 (50 µM, 24 hours). Intracellular reactive oxygen species (ROS) production was determined by flow cytometry. Malondialdehyde (MDA) levels were measured by Fluorometry. The expressions of apoptotic and antioxidant genes and nuclear factor erythroid 2-related factor 2 (Nrf2), Trx2, and nicotinamide nucleotide transhydrogenase (NNT) proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Adenosine triphosphate (ATP) levels were measured by fluorometry. RESULTS: Melatonin reduced H2O2-induced ROS levels and apoptosis in the SSCs. Melatonin also increased mRNA expression of Nrf2, Trx2, NNT, Sirtuin 3 (Sirt3), and decreased mRNA expression of Txnip, and increased protein expressions of Nrf2, Trx2, NNT thereby increasing activity of the mitochondrial thioredoxin system. In addition, melatonin increased ATP levels. CONCLUSION: Melatonin increased Trx2 expression through the Nrf2 pathway. This study suggests that melatonin may protect SSCs from oxidative stress in diseases related to infertility.

Synergistic Effects of Capsaicin and Quercetin Improved Induced Premature Ovarian Failure in Rat.

OBJECTIVE: Premature ovarian failure (POF) is a heterogeneous disorder. POF is defined as hypergonadotropic hypoestrogenism in women under 40 years. There is no effective treatment to cure POF patients. Antioxidants prevent ovarian damage by reducing the lipid peroxidation cascades affecting folliculogenesis, meiosis and ovulation. Hence; the aim of present study was to investigate the effects of Capsaicin (CAP) and Quercetin (QUR) on cyclophosphamide (CYC)-induced POF in rat model. MATERIALS AND METHODS: In this experimental study, POF was induced by intraperitoneal injection of 200 mg/kg CYC on first day and then 8 mg/kg/day for the following 3 days. After 4 days of CYC administration, rats were randomly divided into five groups (n=6/group) as follows: POF, dimethyl sulfoxide (DMSO), CAP (0.5 mg/kg/day), QUR (100 mg/kg/day) and CAP+QUR. Biochemical, hormonal, gene expression, and histological evaluations were performed on blood serum and tissue samples after 14 days of treatment with the CAP and QUR. RESULTS: CAP, QUR and CAP+QUR groups showed signs of restored ovarian function in the form of a significant increase in serum total antioxidant capacity (TAC), estrogen, progesterone and anti-mullerian hormone (AMH) levels versus POF and DMSO groups and a significant improvement in histological parameters and follicle numbers in treatment groups compared to POF and DMSO groups. Polymerase chain reaction (PCR) analysis demonstrated that CAP and QUR upregulate the expression of BAX gene and decreased the expression of apoptosis inducing genes (BCL-2 and P53). CONCLUSION: CAP and QUR treatment of CYC-induced POF rats showed a positive effect on reducing ovarian damage by improving TAC levels, expression of apoptotic genes, levels of ovarian reserve markers, and histological parameters. Our results suggest that treatment with CAP or QUR may be a conservative treatment approach for CYC -induced POF.

Overview of biological effects of melatonin on testis: A review.

Infertility is a major global health issue and male factors account for half of all infertility cases. One of the causes of male infertility is the loss of spermatogonial stem cells, which may occur because of chemotherapy, radiotherapy or genetic defects. In numerous animal species, the evidence suggests the pineal gland and melatonin secretion in their reproductive activities are involved. Recently, considerable attention has pointed to the usage of melatonin in the treatment of diseases. Melatonin is associated with the regulation of circadian and seasonal rhythmic functions, immune system functions, retinal physiology, spermatogenesis and inhibition of tumour growth in different species. Several studies demonstrated that melatonin acts as an anti-apoptotic, anti-inflammatory, anticancer and antioxidant agent. Melatonin can also protect testicles and spermatogonia against oxidative damage, chemotherapy drugs, environmental radiation, toxic substances, hyperthermia, ischemia/reperfusion, diabetes-induced testicular damage, metal-induced testicular toxicity, improve sperm quality and it affects the testosterone secretion pathway by affecting Leydig cells. Therefore, the objective of this study is to investigate the biological effects of melatonin as a natural antioxidant on testicles and their disorders.

Sperm cryopreservation and DNA methylation: possible implications for ART success and the health of offspring.

Despite the beneficial effects of sperm cryopreservation, increased reactive oxygen species (ROS) production during this process can affect spermatozoon structure and function. Moreover, ROS production is associated with elevated DNA damage and alterations in DNA methylation. There is little information about the effects of cryopreservation on epigenetic modulation in sperm and the health of children born with frozen spermatozoa. Considering the potential consequences of cryopreservation in ART-conceived children, it is necessary to assure that cryopreservation does not modify sperm DNA methylation status. This review summarizes reports on epigenetic modifications of spermatozoa during cryopreservation and the probable effects of this process on offspring health. Contradictory results have reported the influence of sperm cryopreservation on DNA methylation in imprinted genes. Multiclinical studies with larger sample sizes under the same conditions of cryopreservation and DNA methylation analysis are needed to make any definitive conclusion about the effect of the cryopreservation process on sperm DNA methylation.

The Protective Effect of Sulforaphane against Oxidative Stress through Activation of NRF2/ARE Pathway in Human Granulosa Cells.

OBJECTIVE: Sulforaphane (SFN) is a natural free radical scavenger that can reduce oxidative stress (OS) through mediating nuclear factor (erythroid-derived 2)-like 2 (NF-E2-related factor 2 or NRF2)/antioxidant response element (ARE) signaling pathway and the downstream antioxidant enzymes. Here, we intended to study the role of SFN in OSinduced human granulosa cells (GCs) by investigating the intracellular levels of reactive oxygen species (ROS), cell death, and NRF2-ARE pathway. MATERIALS AND METHODS: This experimental study was conducted on GCs of 12 healthy women who had normal menstrual cycles with no history of polycystic ovary syndrome (PCOS), endometriosis, menstrual disorders, hyperprolactinemia, or hormonal therapy. After isolation of GCs, the MTT assay was performed to explore GCs viability after treatment with SFN in the presence or absence of H2O2. Flow cytometry was utilized to determine the intracellular ROS production and the apoptosis rate. Evaluation of the mRNA and protein expression levels of NRF2 and phase II enzymes including superoxide dismutase (SOD) and catalase (CAT) was performed by quantitative real-time polymerase chain reaction (PCR) and western blotting. Finally, the data were analyzed by SPSS software using One-way ANOVA and the suitable post-hoc test. Significance level was considered as P<0.05. RESULTS: Pretreatment of GCs with SFN attenuated intracellular ROS production and apoptosis rate in the H2O2-exposed cells. Moreover, SFN treatment increased the mRNA expression level of NRF2, SOD, and CAT. Higher expression of NRF2 and SOD was also observed at the protein level. CONCLUSION: Our study demonstrated that SFN protects human GCs against H2O2 induced-OS by reducing the intracellular ROS production and the following apoptosis through a mechanism by which NRF2 increases the antioxidant enzymes such as SOD and CAT. This result may have a potential application in assisted reproduction cycles by improving the quality of GCs and the embedded oocyte, especially in PCOS patients.

Astaxanthin Protects Human Granulosa Cells against Oxidative Stress through Activation of NRF2/ARE Pathway and Its Downstream Phase II Enzymes.

OBJECTIVE: Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes. MATERIALS AND METHODS: In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (H2O2) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity. RESULTS: This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05). CONCLUSION: Our results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving in vitro fertilization (IVF) outcomes and treatment of infertility.

The Effect of Astaxanthin and Metformin on Oxidative Stress in Granulosa Cells of BALB C Mouse Model of Polycystic Ovary Syndrome.

Reactive oxygen species (ROS), involved in the pathogenesis of the polycystic ovary syndrome (PCOS), play a key role in the onset of apoptosis in follicles and granulosa cells (GCs). We aimed to investigate the antioxidant effects of AST and metformin separately and in combination on GCs using a PCOS mouse model. Forty-eight prepubertal female BALB C mice aged 25-30 days and weighing 12-14 g were studied. The PCOS model was created by subcutaneous injection of the dehydroepiandrosterone (DHEA) hormone in 8 mice of BALB C for 20 consecutive days. Apoptosis and the amount of ROS were evaluated in GCs of the ovaries via flow cytometry. The activity of AKT protein was measured by western blot, and the viability of GCs was investigated using spectrophotometry. Ovarian tissue sections were prepared, stained with H&E, and the morphology of the sections was examined. Statistical analysis was performed by SPSS v22.0 software using one-way ANOVA. We found that AST administration leads to a significant reduction in oxidative stress (P<0.01) and consequently a significant decrease in the rate of apoptosis (P<0.01). While the expression of AKT in the AST group revealed a significant increase (P<0.05), it decreased in the metformin group. However, it was still significantly higher than the control and PCOS groups. Ovulation was confirmed in both metformin and AST groups. Further studies are warranted to prove the efficacy of AST and to introduce it as a complementary therapeutic agent in PCOS.

Ceratonia siliqua (Carob) extract improved in vitro development of vitrified-warmed mouse germinal vesicle oocytes: assessment of possible mechanism.

Oocyte banking is a vital step for safekeeping and spreading genetic resources of animals. It is also used for fertility preservation of human. Oocyte vitrification is closely related to the lower developmental competence which includes the cryo-injury arisen during vitrification. The aim of the present study was to evaluate the maturation, embryonic development and production of reactive oxygen species (ROS) of mice oocytes following the supplementation vitrification media with different concentrations of Ceratonia siliqua (carob) extracts. In this experimental study, germinal vesicle oocytes collected from 8 to 10 week-old female NMRI mice (30-40 gr) were randomly divided into six groups of vitrification media supplemented with 0 (control), 5, 10, 20, 30 and 50 µg/ml C. siliqua. After thawing, oocytes were put in an in vitro maturation medium (IVM) (α-MEM: Alpha Minimum Essential Medium). 3-4 and 24 h (hr) later, the oocyte nuclear maturity was checked. Standard in vitro fertilization was performed on the matured oocytes (MII), and embryonic development was followed. Extra- and intra-cellular ROS was measured in IVM medium after 24 h of oocyte incubation. The addition of 20 and 30 µg/ml C. siliqua extract to vitrification media improved normal morphology of warmed germinal vesicle (GV) oocytes, rate of germinal vesicle break down (GVBD), and metaphase 2 (MII) oocyte formation significantly (p < 0.05). Fertilization rate, (embryonic development to 2 cells stage, 4-8 cells stage, and > 8 cells stage increased in the 30 µg/ml C. siliqua group significantly (p < 0.05). Furthermore, supplementation of 30 µg/ml C. siliqua in vitrification media significantly decreased extra- and intra-cellular of ROS as well as embryonic fragmentation (p < 0.05). In conclusion, supplementation of GV oocyte vitrification media with carob extract improved maturation, fertilization, and embryonic development rate and decreased extra- and intra-cellular ROS levels.

Overview of biological effects of Quercetin on ovary.

Over the last few decades, using natural products has been increased to treat different diseases. Today, great attention has been pointed toward the usage of natural products such as flavonoids, especially Quercetin (QUR), in the treatment of diseases. QUR as a natural antioxidant has been traditionally used to prevent or treat a variety of diseases such as cancer, cardiovascular disease, polycystic ovary syndrome (PCOS), obesity, chronic inflammation, and reproductive system dysfunction. Several studies demonstrated that QUR acts as an anti-inflammatory, anti-apoptotic, antioxidant, and anticancer agent. With this in view, in this study, we intended to describe an overview of the biological effects of QUR on the ovary. QUR improves the quality of oocytes and embryos. It affects the proliferation and apoptosis and decreases the oxidative stress in granulosa cells (GCs). Furthermore, QUR can be used as a complementary and alternative therapy in ovarian cancer and it has beneficial effects in the treatment of PCOS patients. It seems that QUR as a supplementary factor has different activities for the treatment of different disorders and it also has bidirectional activities. However, further investigations are needed for understanding the efficacy of QUR in the treatment and improvement of gynecological patients.

Inhibitory effect of resveratrol on the growth and angiogenesis of human endometrial tissue in an In Vitro three-dimensional model of endometriosis.

Endometriosis is a chronic estrogen-dependent disorder and one of the most common causes of infertility in women. Resveratrol (RES) is a polyphenolic and phytoestrogenic compound with anti-oxidative, anti-inflammatory, anti-angiogenic, and anti-estrogenic properties. The aim of the present study was to investigate the effect of different concentrations of RES on human endometrial growth and angiogenesis in an in vitro three-dimensional (3D) model of endometriosis.Human endometrial tissues of endometriosis (endometriotic) and normal (endometrial) subjects (n = 9/groups) were biopsied in sterile conditions and cut into 1 × 2 mm pieces. Tissue fragments of each biopsy were given concentrations of 0 (control), 10, 50, 100 and 200 µM RES for 21 days in 3D culture condition using fibrin as an extracellular matrix. Scoring methods were used for tissue changes, including; cellular invasion, monolayer formation and angiogenesis. Nitric oxide (NO) was measured using Griess's reaction, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the apoptotic gene expression.The mean of growth scores of endometriotic and endometrial tissue showed a significant dose dependent inhibition (P < 0.05). The levels of NO also significantly decreased in different groups. Apoptotic genes (P53, Bax, Bcl2 and caspase 3) and Sirt1 showed a significant increase in various concentrations of RES in both tissues (P < 0.05).RES exert dose- and time-dependent inhibitory effects on human endometrial tissue, and its higher doses suggested it as a natural supplement to inhibit the growth and treatment of endometriosis.

The genetic factors associated with Wnt signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is a common cancer with poor prognosis and high mortality. There is growing information about the factors involved in the pathogenesis of CRC. However, the knowledge of the predisposing factors is limited. The development of CRC is strongly associated with the Wingless/Integrated (Wnt) signaling pathway. This pathway comprises several major target proteins, including LRP5/6, GSK3ß, adenomatous polyposis coli (APC), axis inhibition protein (Axin), and ß-catenin. Genetic variations in these components of the Wnt signaling pathway may lead to the activation of ß-catenin, potentially increasing the proliferation of colorectal cells. Because of the potentially important role of the Wnt signaling pathway in CRC, we aimed to review the involvement of different mutations in the main downstream proteins of this pathway, including LRP5/6, APC, GSK3ß, Axin, and ß-catenin. Determination of the genetic risk factors involved in the progression of CRC may lead to novel approaches for the early diagnosis of CRC and the identification of potential therapeutic targets in the treatment of CRC.

Quercetin protects human granulosa cells against oxidative stress via thioredoxin system.

Granulosa Cells (GCs) are sensitive to excessive production of reactive oxygen species (ROS). Quercetin (QUR) is a free radical scavenger which can alleviate oxidative stress through nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant response element (ARE) pathway and thioredoxin (Trx) system. We aimed to explore the probable protective role of QUR on cultured human GCs treated with hydrogen peroxide (H2O2) as an inducer of oxidative stress. MTT assay was applied for evaluating the cell cytotoxicity of QUR and H2O2. The rate of apoptotic cells and intracellular ROS generation were determined by Annexin V-FITC/PI staining and 2'-7'-dichlorodihydrofluorescein diacetate fluorescent probes (DCFH-DA), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blot analysis were used to evaluate the gene and protein expression of Nrf2 and kelch-like ech-associated protein 1 (Keap1)1. The Nrf2 and Trx activities were measured by Enzyme-linked Immunosorbent Assay (ELISA). The results indicated that QUR pretreatment can decrease ROS production and apoptosis induced by H2O2. In addition, QUR increased Nrf2 gene and protein expression, as well as its nuclear translocation. Moreover, in QUR-treated group, a lower level of Keap1 protein was observed, which was not reported as significant. The results also indicated a significant correlation between the expression of Nrf2 and Keap1 in QUR-treated group. Further, QUR protected GCs from oxidative stress by increasing Trx gene expression and activity. This study suggests that QUR as a supplementary factor may protect GCs from oxidative stress in diseases related to this condition.

Synergistic Anticancer Effect of Paclitaxel and Noscapine on Human Prostate Cancer Cell Lines.

Paclitaxel is one of the most common chemotherapeutic drugs used for the treatment of prostate cancer. However, its current clinical utility has been limited due to numerous serious side effects and drug resistance. Noscapine is an antitussive opium alkaloid that showed antitumor activity against a variety of cancer while it has not exhibited severe side effects. This study investigates the anticancer activity of noscapine in combination with paclitaxel against two LNCaP and PC-3 human prostate cancer cell lines. LNCaP and PC-3 cells were treated with noscapine or paclitaxel or combination. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test. Apoptosis was assessed by acridine orange/ ethidium bromide (AO/EB) staining. The mRNA expression of Bax, Bcl-2, AR, and PSA in the cellular response to treatments was investigated. MTT assay indicated the combination treatment of paclitaxel and noscapine significantly decreased viability compared to single-agent treatment and control groups. The results obtained with AO/EB double staining showed increased percentages of apoptotic cells in the presence of the combination of paclitaxel and noscapine. Furthermore, combinational treatment of paclitaxel and noscapine showed significant decrease in the mRNA expression of B-cell CLL/Lymphoma (Bcl-2) and increase in the mRNA expression of Bcl-2-associated X protein (Bax(, and Bax/Bcl-2 ratio in LNCaP and PC-3 cells (P<0.05.( The mRNA expression of androgen receptor (AR) and prostate specific antigen (PSA) decreased in paclitaxel and noscapine combination-treated of LNCaP cells (P<0.05). This study provides a novel concept of combination treatment of paclitaxel and noscapine to improve efficiency in human prostate cancer treatment.

Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine.

OBJECTIVES: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. MATERIALS AND METHODS: In this in vitro study, endometrial biopsies from endometriosis patients (n=9) were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml). Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0), 10, 25, 50 and 100 micromole/liter (µM) concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO) concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO)-Ethidium Bromide (EB) double staining and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. Data were analyzed by one-way ANOVA. RESULTS: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P<0.05) and apoptotic index increased in 25, 50 and 100 µM noscapine concentrations in 48 hr significantly (P<0.05). Concentrations of NO didn't show a significant decrease. CONCLUSION: Noscapine increased endometriotic epithelial and stromal cell death and can be suggested as a treatment for endometriosis.

Thymoquinone Could Increase The Efficacy of Tamoxifen Induced Apoptosis in Human Breast Cancer Cells: An In Vitro Study.

OBJECTIVE: Thymoquinone (TQ), as the main component of Nigella Sativa plant, shows anticancer properties. This study was aimed to evaluate the combined effect of TQ and Tamoxifen (TAM) on viability and apoptosis of human breast cancer cell lines. MATERIALS AND METHODS: In this experimental study, estrogen positive MCF-7 and estrogen negative MDA-MB-231 human breast cancer cell lines were induced by TAM (2 µM) or different doses of TQ (50, 75, 100, 150 µM), individually or in combination. Cell viability and apoptosis were investigated by MTT assay and TdT-mediated deoxy-uracil nick end labeling (TUNEL) assay; Acridine orange (AO)/Ethidium bromide (EB) staining respectively. Data were analyzed by one way ANOVA and P<0.05 was considered significant. RESULTS: In 24 hours treatment, TAM and all doses of TQ, solely or in combination, significantly reduced cell viability of both cell lines, except in MCF-7 cells treated with 50 µM TQ, and MDA-MB-231 cells treated with 50 or 75 µM TQ (P<0.01). After 48 hours treatment, cell viability of both cell lines was reduced in all treated groups (P<0.05). Remarkable apoptotic index was observed in combination treatment of MCF-7 or MDA-MB-231 cell lines with TAM and TQ (P<0.001). CONCLUSION: The synergistic effect of TQ and TAM on human breast cancer cell lines showed cell viability reduction as well as apoptosis induction, independent to estrogen.

In vitro inhibitory effect of crab shell extract on human umbilical vein endothelial cell.

Angiogenesis plays an important role in the tissue repair, rheumatoid arthritis, retinopathies, and growth and metastasis of cancer. Endothelial cell proliferation has been introduced as a model of angiogenesis. Crab sell was suggested for cancer treatment in traditional medicine. The aim of the present study was to investigate the in vitro effect of crab shell hydroethanolic extract on the human umbilical vein endothelial cell (HUVEC) proliferation. Crab sell extract was prepared and the effect of its various concentrations (0, 100, 200, 400, 800, 1000 µg/ml) on HUVECs were surveyed in three periods of 24, 48 and 72 h. HUVEC viability, Nitric oxide (NO) secretion and apoptosis were assessed by MTT, Griess and Tunnel methods, respectively. Data were compared by one-way ANOVA. In this study we found HUVECs viability was reduced in dose and time depended manner significantly and HUVECs NO production decreased significantly, too. Apoptosis index was increased significantly. These findings reveal that high concentrations of crab shell extract have anti-proliferative effects on HUVECs and can be used for cancer treatment.

Antiproliferatory Effects of Crab Shell Extract on Breast Cancer Cell Line (MCF7).

PURPOSE: Breast cancer is the most common type of cancer in women. Despite various pharmacological developments, the identification of new therapies is still required for treating breast cancer. Crab is often recommended as a traditional medicine for cancer. This study aimed to determine the in vitro effect of a hydroalcoholic crab shell extract on a breast cancer cell line. METHODS: In this experimental study, MCF7 breast cancer cell line was used. Crab shell was powdered and a hydroalcoholic (70° ethanol) extract was prepared. Five concentrations (100, 200, 400, 800, and 1,000 µg/mL) were added to the cells for three periods, 24, 48, and 72 hours. The viability of the cells were evaluated using trypan blue and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Cell apoptosis was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling method. Nitric oxide (NO) level was assessed using the Griess method. Data were analyzed using analysis of variance, and p<0.05 was considered significant. RESULTS: Cell viability decreased depending on dose and time, and was significantly different in the groups that were treated with 400, 800, and 1,000 µg/mL doses compared to that in the control group (p<0.001). Increasing the dose significantly increased apoptosis (p<0.001). NO secretion from MCF7 cells significantly decreased in response to different concentrations of the extract in a dose- and time-dependent manner (p<0.050). CONCLUSION: The crab shell extract inhibited the proliferation of MCF7 cells by increasing apoptosis and decreasing NO production.

Hydrostatic pressure affects in vitro maturation of oocytes and follicles and increases granulosa cell death.

OBJECTIVE: This study examines the effects of hydrostatic pressure on in vitro maturation (IVM) of oocytes derived from in vitro grown follicles. MATERIALS AND METHODS: In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal Essential Medium (α-MEM) under mineral oil for 12 days. Then, follicles were induced for IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We assessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student's t test. RESULTS: The percentage of metaphase II oocytes (MII) increased in hydrostatic pressuretreated follicles compared to controls (p<0.05). Cumulus cell viability reduced in hydrostatic pressure-treated follicles compared to controls (p<0.05). Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05). CONCLUSION: Hydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.

Synthesis of chromeno[3,4-B]quinoline derivatives by heterogeneous [Cu(II)BHPPDAH] catalyst without being immobilized on any support under mild conditions using PEG 300 as green solvent.

The efficient synthesis of chromeno[3,4-[Formula: see text]]quinoline derivatives by condensation of O-propargylated salicylaldehyde, or corresponding compounds and primary aromatic amine derivatives using a catalytic amount of heterogeneous Cu(II)BHPPDAH complex without being immobilized on any supports (5.0 mol%) in PEG 300 as a "green" solvent is described. The remarkable features of this protocol are good to high yields in all cases, short reaction times, a cleaner reaction profile in an environmentally benign solvent (PEG 300), and the method is applicable to large-scale operation without any problem. Furthermore, the catalyst was quantitatively recovered from the reaction mixture by simple filtration and reused at least seven times with almost consistent activity.