Novel bone morphogenetic protein 15 (BMP15) gene variants implicated in premature ovarian insufficiency.

BACKGROUND: Bone morphogenetic protein 15 (BMP15) is expressed in oocytes and plays a crucial role in the reproduction of mono-ovulating species. In humans, BMP15 gene mutations lead to imperfect protein function and premature ovarian insufficiency. Here we investigated the BMP15 gene variants in a population of Iranian women with premature ovarian insufficiency. We conducted predictive bioinformatics analysis to further study the outcomes of BMP15 gene alterations. METHODS: Twenty-four well-diagnosed premature ovarian insufficiency cases with normal karyotype participated in this study. The entire coding sequence and exon-intron junctions of the BMP15 gene were analyzed by direct sequencing. In-silico analysis was applied using various pipelines integrated into the Ensembl Variant Effect Predictor online tool. The clinical interpretation was performed based on the approved guidelines. RESULTS: By gene screening of BMP15, we discovered p.N103K, p.A180T, and p.M184T heterozygous variants in 3 unrelated patients. The p.N103K and p.M184T were not annotated on gnomAD, 1000 Genome and/or dbSNP. These mutations were not identified in 800 Iranians whole-exome sequencing that is recorded on Iranom database. We identified the p.N103K variant in a patient with secondary amenorrhea at the age of 17, elevated FSH and atrophic ovaries. The p.M184T was detected in a sporadic case with atrophic ovaries and very high FSH who developed secondary amenorrhea at the age of 31. CONCLUSIONS: Here we newly identified p.N103K and p.M184T mutation in the BMP15 gene associated with idiopathic premature ovarian insufficiency. Both mutations have occurred in the prodomain region of protein. Despite prodomain cleavage through dimerization, it is actively involved in the mature protein function. Further studies elucidating the roles of prodomain would lead to a better understanding of the disease pathogenesis.

Microencapsulated mammalian cells for simultaneous production of VEGF165b and IFNα.

Targeted and simultaneous delivery VEGF165b and IFN alpha in anti-angiogenic and other applications could offer several advantages. For this a system was design using artificial cell alginate-poly-L-lysine- alginate (APA) microcapsules. Result confirms the ability of this system for simultaneous production of these proteins for 28-days. The IFN alpha on a 3 days period increased from 8 ± 0.36 µg/ml at day 10 to 27 ± 2.4 µg/ml at day 16 and then dropped to 6.5 ± 0.5 µg/ml. The VEGF165b on a 3 days period increased from 2.7 ± 0.7 µg/ml at day 10 to 6.9 ± 1 µg/ml at day 16.

Encapsulation of magnetotactic bacteria for targeted and controlled delivery of anticancer agents for tumor therapy.

We showed that magnetotactic bacteria (MTB) have great potentials to be used as microcarriers for targeted delivery of therapeutic agents. Indeed, magnetotaxis inherent in MTB can be exploited to direct them towards a tumor while being propelled by their own flagellated molecular motors. Nonetheless, although the thrust propelling force above 4 pN of the MC-1 MTB showed to be superior compared to other technologies for displacement in the microvasculature, MTB becomes much less efficient when travelling in larger blood vessels due to higher blood flow. In the latter case, a new technique developed by our group and referred to as Magnetic Resonance Navigation (MRN), has been successfully applied in larger vessels using synthetic microcarriers nut proved to be less efficient in the microvasculature due mainly to technological constraints. These findings called for the need to integrate both approaches by encapsulating MTB in special MRN-compatible microcarriers to be release in the vicinity of microvascular networks where they becomes more effective for targeting purposes in tumoral lesions. In this study Magnetococcus strain MC-1 were encapsulated in giant vesicles. The survival of the encapsulated bacteria was monitored. The release of bacteria from giant vesicles was also studied in different time intervals and conditions.

Investigation of antiangiogenic tumor therapy potential of microencapsulated HEK293 VEGF165b producing cells.

To investigate the antiangiogenic potential of encapsulated VEGF165b producing HEK293 cells, Human Embryonic Kidney 293 (HEK293) cells were stably transfected to produce VEGF165b. Then they were encapsulated in alginate-polylysine-alginate (APA) microcapsules. VEGF165b productivity and viability of encapsulated cells were analyzed and compared with the non-encapsulated cells. Results showed that encapsulated cells proliferated and remained viable within the microcapsules throughout the 28-day period of the experiment. The quantity of VEGF165b increased from 6.5 ± 1.2 µg/ml at day 13 to 13 ± 0.96 µg/ml at day 16. Then it gradually dropped to 5 ± 1.2 µg/ml for the last 3 days period as measured at day 28. Production of VEGF165b from encapsulated and non-encapsulated cells was similar. The effect of VEGF165b harvested from encapsulated cells on Human Umbilical Vein Endothelial cells (HUVECs) proliferation were also examined.The same inhibitory effects on HUVECs proliferation was seen when the cells were incubated with a mixture of VEGF165b and a 2-fold VEGF165b or with VEGF165b and 2-fold excess VEGF165b released from encapsulated cells. Subcutaneous injection of microencapsulated VEGF165b producing cells in tumor site of nude mice resulted in the reduction of the number of vessels around the tumors.

Microencapsulated engineered Lactococcus lactis cells for heterologous protein delivery: preparation and in vitro analysis.

This article demonstrates the potential of encapsulated, engineered Lactococcus lactis as a vehicle for the oral delivery of therapeutic proteins. Using alginate-poly-l-lysine-alginate membrane-encapsulated L. lactis engineered to secrete the reporter protein Staphylococcal aureus nuclease, we show comparable viability and protein secretion between free and immobilized cells. After 12 h, microcapsules with a cell density of 4.8 x 10(5) colony forming unit (CFU) ml(-1) grew to 2.2 x 10(8) CFU ml(-1) and released 0.24 arbitrary unit (AU) ml(-1) of nuclease, producing similar results as free cells, which grew from 3.4 x 10(5) to 1.9 x 10(8) CFU ml(-1) and secreted 0.21 AU ml(-1) of nuclease. Moreover, encapsulated cells at a density of 4.4 x 10(7) CFU ml(-1) grew to 2.2 x 10(10) CFU ml(-1) in 12 h and secreted 15.3 AU ml(-1) of nuclease although 3.1 x 10(7) CFU ml(-1)of free cells reached only 2.3 x 10(9) CFU ml(-1) and released 5.6 AU ml(-1) of nuclease. We also show the sustained stability of the microcapsules during storage at 4 degrees C over 8 weeks.

Impact of orally administered microcapsules on gastrointestinal microbial flora: in-vitro investigation using computer controlled dynamic human gastrointestinal model.

Oral administration of artificial cell microcapsules has been proposed for various therapy procedures using biologically active materials. Recently we have designed novel APPPA microcapsules using alginate, poly-L-lysine, pectin, poly-L-lysine and alginate that have shown superior oral delivery features. This article investigates, in-vitro using a computer controlled dynamic gastrointestinal (GI) model, effects of APPPA microcapsules on health of gastrointestinal (GI) microbial flora. The impact of APPPA microcapsules on GI bacterial population, total anaerobes, total aerobes, Escherichia coli, Lactobacillus sp. and Staphylococcus sp. has been analyzed. In addition, the effects of microcapsules on GI microbial extracellular enzymatic activities have been investigated. Result shows the altered activities of microbial flora and enzymes due to the use of APPPA microcapsule. The most disparity is observed in the colon ascendans microbial activities. This study would have significant impact on future microcapsule design. However, further in-vivo studies are required.