Utilization of Fad Distribution in South Buton Waters As a Fishing App by Purse Sein Fishermen in Kadatua District, Selatan Buton Regency

Fish Aggregating Devices (FAD's) have long been used by fishermen in South Buton Regency. This study aims to determine the utilization of FAD distribution as a fishing aid by purse sein vessels in the waters of Kadatua District, South Buton Regency. This study aims to determine the utilization of FAD distribution as a fishing tool by purse sein vessels in Kadatua District, South Buton Regency. Data collection methods include FAD point data taken from GPS (Global Position System) fishermen and dominant catch data from purse sein fishermen. The results of the study obtained data on the distribution of FADs in Kadatua District spread between a distance of 0-12 miles, during the study the coordinates of FADs were obtained as many as 79 coordinate points owned by fishermen in Kadatua District. FADs used by purse sein fishermen to carry out fishing operations have 24 FAD points, which are spread over a distance of 0 – 12 miles. In April the distribution of FADs used as fishing aids by purse sein vessels was at a distance of ± 0 – 12 miles, while in May and June FADs used as fishing aids were at a distance of ± 2 – 12 miles. The dominant fish catch data for purse sein fishermen is 690 kg in April, 525 kg in May and 735 kg in June. The dominant catch during the study was dominated by scad fish (Decapterus spp). The level of effectiveness of the dominant catch of purse sein fishermen is highest at a distance of 4-12 miles with a total catch of 1,070 kg of the total catch of 1,950 kg.

Molecular Cloning, Sequence Characterization and Expression Studies of Human Intereukin-24

Cancer is one of the major causes accounting for death and illness both in developed and underdeveloped countries. The second largest reason of death in Pakistan is cancer. For the treatment of cancer, a range of biomedical and non-biomedical therapeutic options are being used. However the overall recovery and survival is low. Thus signifying the development of new therapeutic agents and strategies to cure cancer and associated malignancies. Interleukin 24 (IL-24) with its unique proapoptotic and cancer cell selective properties is a potential biopharmaceutical candidate among the recently developed family of protein therapeutics. Since its discovery IL-24 has been prime focus for its recombinant production in various forms to improve biological activity and explore the mechanism of apoptosis induction. The present study reports the recombinant production of IL-24 in biologically active form, using bacterial expression system, its structural and functional characterization for subsequent use in therapeutics as well as in the studies to decipher the mechanisms of unique cancer-specific growth suppressing properties of IL-24. The present study also reports to achieve partial soluble expression of IL-24 as SUMO-IL-24 in E. coli. This study also reports recombinant production of IL-24 with native N-terminus, and recombinant production of fusion protein RGD-IL-24. The cDNA encoding IL-24 was synthesized from the total RNA extract of peripheral blood mononuclear cells using gene specific primers by reverse transcription polymerase chain reaction (RT-PCR). The IL-24 cDNA was cloned in pET-22b(+) expression vector between NdeI and BamHI restriction sites to generate pET-IL-24 recombinant plasmid. To express IL-24 as fusion protein SUMO-IL-24, the IL-24 cDNA was cloned in pE-SUMOstar expression vector between BsaI and XbaI restriction sites to generate pSUMO-IL-24 recombinant plasmid. To express IL-24 as RGD-IL-24 fusion protein a v modified IL-24 cDNA with coding sequence of RGD at 5ʹ end was cloned in pET-22b(+) expression vector between NdeI and XhoI restriction sites to generate pET-RGD-IL-24 recombinant plasmid. The recombinant plasmids pET-IL-24, pET-RGD-IL-24 and pSUMO-IL-24 were used to transform competent E. coli DH5α and positive transformants were selected by ampicillin/kanamycin resistance and further screened by colony PCR. Presence of IL-24 inserts in recombinant plasmids pET-IL-24, pET-RGD-IL-24 and pSUMO-IL-24 was confirmed by restriction analysis. Recombinant plasmids were analyzed for correct orientation and in frame integration of IL-24 insert by DNA sequencing with T7 promoter and T7 terminator primers. Subsequently the recombinant plasmids pET-IL-24, pET-RGD-IL-24 and pSUMO-IL-24 were used to transform competent E. coli BL21 CodonPlus (DE3) RIPL. The expression of recombinant proteins SUMO-IL-24, M-IL-24 and RGD-IL-24 was obtained by induction with 0.5 mM IPTG at an OD600 of 0.5 for three hours in LB broth supplemented with ampicillin/kanamycin. The expression of recombinant proteins was >30% of the total proteins in E. coli cellular extract. The growth of E. coli transformed with recombinant plasmid and expression of recombinant proteins were optimized with respect to media, duration of cultivation, inducer and temperature. M-IL-24 and RGD-IL-24 obtained in the form of insoluble inclusion bodies were solubilized in non ionic detergent NLS. Solubilized M-IL-24 and RGD-IL-24 were refolded and further purified by using anion exchange chromatography on FPLC up to >95 % purity. The fusion protein SUMO-IL-24 was purified using IMAC. SUMOstar protease was used to cleave the fusion protein SUMO-IL-24 into SUMO and IL-24 fragments. Cleaved IL-24 has native N-terminus starting with glutamine (Q) as in human hence it is referred as N-IL-24. N-IL-24 was purified using IMAC to >95 %purity. Refolded and purified recombinant proteins were characterized for secondary structure contents using circular dichroism spectroscopy. Molecular weights of the recombinant vi proteins were characterized by MALDI-TOF MS. Structural modeling of recombinant proteins was performed using in silico computational analysis. Initially biological activity of M-IL-24 was assessed on human cervical cancer cell line HeLa and human colon colorectal carcinoma cell line HCT116. Cytotoxicity of M-IL-24 was further measured on HeLa using neutral red assay and fluorescence activated cell sorting. Cytotoxic effects of N-IL-24 and RGD-IL-24 were initially observed on human cerebrum glioma cell line SF767 and human cervical cancer cell line HeLa. Biological activities of recombinant N-IL-24 and RGD-IL-24 were assessed in detail on HeLa using neutral red assay, FDAPI staining and crystal violet cell adhesion assay. Polyclonal antibodies against IL-24 were raised in Swiss mice and were characterized in post-immune sera using alkaline phosphatase conjugated rabbit anti-mouse IgG antibodies by immunodot blot, ELISA and western blot analysis. Native N-terminus is very important feature of recombinant therapeutic proteins in cancer treatment and the results of present study reveal that N-IL-24 was successfully expressed and purified in biologically active 3-D conformation with native N-terminus. Secondly N-IL-24 recombinant protein showed maximum apoptotic properties among three recombinant forms of IL-24. Decrease in cell survival rate of HeLa, cerebrum glioma cells (SF767) and HCT116 proves N-IL-24 a successful pharmaceutical candidate in future for therapy of various cancers in human. In addition the model mice injected with M-IL-24 subcutaneously retained good health during immunization period. This also proves that recombinant proteins N-IL-24 and M-IL-24 were successfully sufficient purified and safe for bio pharmaceutical applications.