A fluorescent probe for STED microscopy to study NIP-specific B cells.

We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the functional principles of B cell antigen receptors (BCRs). We successfully used them in flow-cytometry, confocal and super-resolution microscopy techniques.

Phospholipase-Cγ1 Signaling Protein Down-Regulation by Oligoclonal-VHHs based Immuno-Liposome: A Potent Metastasis Deterrent in HER2 Positive Breast Cancer Cells.

OBJECTIVE: The purpose of this study was to develop multivalent antibody constructs via grafting anti-HER2 antibodies, including Herceptin and oligoclonal-variable domain of heavy chain antibodies (VHHs), onto liposome membranes to enhance antibody activity and compare their effect on phospholipase C (PLC) signaling pathway with control. MATERIALS AND METHODS: In this experimental study, SKBR3 and BT-474 cell lines as HER2 positive and MCF10A cell line as normal cell were screened with anti-HER2 antibodies, including constructs of multivalent liposomal antibody developed with Herceptin and anti-HER2 oligoclonal-VHHs. To confirm the accuracy of the study, immunofluorescent assay, migration assay and immuno-liposome binding ability to HER2 were evaluated. Finally, the antibodies effect on PLCγ1 protein level was measured by an immunoassay method (ELISA). RESULTS: In the present study, by using multivalent form of antibodies, we were able to significantly inhibit the PLCγ1 protein level. Interestingly, the results of migration assay, used for study the motility of different types of cell, shows correspondingly decreased number of immigrated cells in SKBR3 and BT-474 cell lines. Since MCF10A cells show no overexpression of HER2, as expected, the result did not show any change in PLCγ1 level. Moreover, immunofluorescent assay has confirmed high expression of HER2 in SKBR3 and BT-474 cell lines and low HER2 expression on MCF10A cell line. High binding of immuno-liposome to SKBR3 and BT-474 cells and low binding to MCF10A confirmed that in this study anti-HER2 antibodies have conserved binding ability to HER2 even after conjugation with liposome. CONCLUSION: PLCγ1 protein levels did indeed decrease after treatment with immuno-liposome form of compounds in both two tested cell lines, verifying the inhibition ability of them. Moreover, an elevated antibody activity is associated with liposomes conjugation suggesting that immuno-liposome may be a potential target for enhancing the antibody activity.

The Impact of Genetic Variation and Gene Expression Level of The Follicle-Stimulating Hormone Receptor on Ovarian Reserve.

OBJECTIVES: Ovarian reserve is defined as the capacity of the ovary to provide fertile oocytes. Diminished ovarian reserve (DOR) is a disorder in which ovaries are prone to go through early menopause. Where this loss of function occurs before the age of 40, it results in the premature ovarian failure (POF) disease. Throughout folliculogenesis, the follicle-stimulating hormone receptor (FSHR) starts a signaling cascade in the granulosa cells where its inactivation leads to the arrest of follicle maturation and therefore adversely affects ovarian reserve. The aim of this study was to investigate the association of genetic variation (polymorphisms and inactivating mutations) of FSHR with POF and DOR. MATERIALS AND METHODS: This case-control study comprised 84 POF, 52 DOR and 80 fertile Iranian women. To determine the presence of the 566C>T mutation and the -29G>A polymorphism in FSHR, PCR-RFLP method was used. SSCP-sequencing was used to identify any allelic variants in exon 10. The expression of human FSHR at the transcript level was also compared between DOR and fertile controls by real time-polymerase chain reaction (PCR). RESULTS: The 566C>T polymorphism was normal in all the cases. All genotypes of -29G>A and 919G>A (exon 10) polymorphisms were observed. Statistically significant differences were seen in the genotypic distribution of both polymorphisms when comparing the control group with the DOR patient group. A decrease was observed in FSHR expression of DOR patients compared with the control group but was not significant. CONCLUSIONS: We conclude that the -29G>A and 919G>A polymorphisms in FSHR may be associated with DOR. Although these polymorphisms had significant differences at the genic level, no significant variation was found at the transcript level.

High Expression of Sphingosine Kinase 1 in Estrogen and Progesterone Receptors-Negative Breast Cancer.

BACKGROUND & OBJECTIVE: Breast cancer is the leading cause of cancer related death in females. Sphingosine kinase 1 (SPHK1) and its product sphingosine-1-phosphate (S1P) are the essential key regulator molecules in breast cancer through their ability to promote cell proliferation, angiogenesis, cell proliferation, and lymphagiogenesis. SPHK1 is overexpressed in multiple types of cancer including breast cancer and is associated with resistance to treatment. The current study aimed at investigating the expression of SPHK1 in estrogen and progesterone receptors (ER, PR) negative in comparison to ER, and PR positive breast cancer and their normal controls, and also finding the relationship between SPHK1 expression and high body index (BMI) in the selected groups with breast cancer. METHODS: A total of 120 human breast cancer tissue specimens were analyzed for SPHK1 expression using Quantitative Real-Time Polymerase Chain Reaction (q RT-PCR) assay. Detection of hormonal status of breast cancer tissue samples was conducted by immunohistochemical assay. RESULT: The current study findings showed that the level of SPHK1expression in the breast cancer tissue was significantly higher in patients with estrogen and progesterone negative receptors, compared to the ones without them (P-value< 0.05). The obtained data confirmed that the obesity in patients with ER negative was higher than the ones with positive receptors (BMI> 25). CONCLUSION: The current study showed that expression of SPHK1gene was higher in the patients with ER and PR negative breast cancer and high BMI, compared with other groups.

Association of MOV10L1 gene polymorphisms and male infertility in azoospermic men with complete maturation arrest.

PURPOSE: The present research was undertaken to study probable genetic variations of MOV10L1 in 30 infertile men that had complete maturation arrest in their spermatocyte levels and 70 fertile men as the control group. METHODS: We performed polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) on extracted DNAs and sequencing was used to confirm the results. Identified polymorphisms in the MOV10L1 were further subjected to a haplotype analysis. RESULTS: We identified eight single nucleotide polymorphisms (SNPs): one missense (rs2272837) and four nonsense polymorphisms (rs2272836, rs11704548, rs2272838, rs138271) in the exonic sequences and three polymorphisms (rs12170772, rs2272840, rs17248147) in the intronic regions. With the exception of rs2272838, there was a statistically significant association in all polymorphisms between study population (P < 0.05). The result of haplotyping analysis showed ten possible haplotypes, from which five were significantly increased in infertile patients compared with the control group. CONCLUSIONS: Our results suggest that MOV10L1 gene polymorphisms in the studied infertile males with complete maturation arrest are linked to infertility.