MATERIALS AND METHODS: An overnight fast of 10-hour plasma levels of glutamate, glycine, alanine, and tryptophan were measured in 83 bipolar patients, and were compared to a group of 82 healthy controls.
RESULTS: The mean (SD) age of bipolar patients was 40.9 (12.1), while the mean (SD) age for control groups was 35.6 (7.7) years. The median (25th, 75th percentile) of glutamate and alanine levels in bipolar patients was 111.0 (65.0,176.0) and 530.0 (446.0,629.0), respectively, while the mean (SD) of glycine level in bipolar patients was 304.0 (98.1). Significant higher glutamate, glycine, and alanine levels were found in bipolar disorder patients in the manic episode as compared to the healthy controls.
CONCLUSION: Although the exact relationship between peripheral NMDA receptor co-agonist levels in the pathogenesis of BD is not well understood, these findings should be explored and may enlighten some new paths for BD therapy which could reward the patients also clinicians.
Methods: In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA.
Results: In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls.
Discussion: In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.
METHODS: Endophytes were isolated from plants collected from Kuala Pilah, Negeri Sembilan and the National Park, Pahang and the extracts were tested for BACE1 inhibition. For investigation of biological activity, the pure endophytic cultures were cultivated for 14 days on PDA plates at 28°C and underwent semipolar extraction with ethyl acetate.
RESULTS: Of 212 endophytic extracts (1000 μg/ml), 29 exhibited more than 90% inhibition of BACE1 in the preliminary screening. Four extracts from isolates HAB16R13, HAB16R14, HAB16R18 and HAB8R24 identified as Cytospora rhizophorae were the most active with IC(50(BACE1)) values of less than 3.0 μg/ml. The most active extract HAB16R13 was shown to non-competitively inhibit BACE1 with K(i) value of 10.0 μg/ml. HAB16R13 was considered non-potent against PC-12 and WRL68 (IC(50(CT))) of 60.0 and 40.0 μg/ml, respectively).
CONCLUSIONS: This first report on endophytic fungal extract with good BACE1 inhibitory activity demonstrates that more extensive study is required to uncover the potential of endophytes.
RESULTS: The most abundant monosaccharide in the carbohydrate composition of durian seed gum were galactose (48.6-59.9%), glucose (37.1-45.1%), arabinose (0.58-3.41%), and xylose (0.3-3.21%). The predominant fatty acid of the lipid fraction from the durian seed gum were palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:2). The most abundant amino acids of durian seed gum were: leucine (30.9-37.3%), lysine (6.04-8.36%), aspartic acid (6.10-7.19%), glycine (6.07-7.42%), alanine (5.24-6.14%), glutamic acid (5.57-7.09%), valine (4.5-5.50%), proline (3.87-4.81%), serine (4.39-5.18%), threonine (3.44-6.50%), isoleucine (3.30-4.07%), and phenylalanine (3.11-9.04%).
CONCLUSION: The presence of essential amino acids in the chemical structure of durian seed gum reinforces its nutritional value.
Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.
Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.
Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.