Method for Label-Free Quantitative Proteomics for <Emphasis Type="Italic">Sorghum bicolor</Emphasis> L. Moench

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.     

Aphid population abundance and pestiferous effect on various bean plant species

The population abundance, infestation, and harmful effects of the aphid Aphis craccivora Koch (Hemiptera: Aphididae) were studied on four bean plant species, namely the country bean (Lablab purpureus var. BARI Seem 1), the yard‐long bean (Vigna sesquipedalis var. BARI Borboti 1), the hyacinth bean (Dolichos lablab var. BARI Seem 6), and the bush bean (Phaseolus vulgaris var. BARI Jar Seem 3). Aphid abundance and infestation on the leaves, inflorescences, flowers, and pods differed significantly among the bean plant species, with P. vulgaris and V. sesquipedalis having the lowest and highest results, respectively. Aphid severity grade and the number of trichomes of the bean plant species were negatively correlated. The duration of the growth stages among the bean plant species were significantly different, with V. sesquipedalis having the shortest durations. Aphid abundance and infestation significantly affected the physical and phytochemical characteristics of the bean plant species. The highest reduction of number of leaves, flower inflorescences, and pod inflorescences per plant, and moisture and chlorophyll content in the leaves was found in L. purpureus. The results for V. sesquipedalis revealed the highest reduction in plant height, seed weight, and pH, while those of D. lablab showed the highest reduction in leaf area.     

Separation of Antioxidant-Rich Alternanthera Sessilis Red Extracts by Sephadex LH-20 and Identification of Polyphenols Using HPLC-QToF-MS/MS

This study aimed to fractionate Alternanthera sessilis Red (ASR) crude extracts and determine their antioxidant activities as well as the related active components in the whole plant. ASR was extracted with water and ethanol, and further separated using a Sephadex LH-20 column. Following the assessments of the polyphenolic contents and antioxidant activities of crude extracts (H₂O_ASR and EtOH_ASR) and fractions, a HPLC-QToF analysis was performed on the crude extracts and selected fractions (H₂O_ASR FII and EtOH_ASR FII). Three water fractions (H₂O_ASR FI, FII and FIII) and four ethanolic fractions (EtOH_ASR FI, FII, FIII and FIV) were derived from their crude extracts, respectively. EtOH_ASR FII exhibited the greatest total phenolic content (120.41 mg GAE/g fraction), total flavonoid content (223.07 mg RE/g fraction), and antioxidant activities (DPPH IC₅₀=159.43 μg/mL; FRAP=1.93 mmol Fe²⁺/g fraction; TEAC=0.90 mmol TE/g fraction). Correlation analysis showed significant (p<0.01) positive correlations between both TPC (r=0.748–0.970) and TFC (r=0.686–0.949) with antioxidant activities in the crude extracts and fractions. Flavonoids were the major compounds in the four selected samples tentatively identified using HPLC-QToF-MS/MS, with the highest number of 30 polyphenol compounds detected in the most active fraction, EtOH_ASR FII.     

Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme

The Drosophila γ-glutamyl carboxylase (dγC) has substrate recognition properties similar to that of the vertebrate γ-carboxylase (γC), and its carboxylated product yield, in vitro, was shown to be more than that obtained with the human enzyme. However, whether the Drosophila enzyme is able to γ-carboxylate the human vitamin K-dependent (VKD) proteins, such as the human coagulation factor IX (hFIX), as synthesized in cultured Drosophila cells was not known. To examine this possibility, the Drosophila Schnider (S2) cell line was transfected with a metallothionein promoter-regulated hFIX-expressing plasmid. After induction with copper ion, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assey (ELISA) and coagulation test on the culture supernatant of the transfected S2 cells during 72 h of postinduction. In comparison with Chinese hamster ovary cell line, S2 cells showed higher (≈ 12-fold) expression level of the hFIX. The γ-carboxylation of the Drosophila-derived hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. The biological activity of the S2 cell-derived hFIX indicated the capability of S2 cells to fulfill the required γ-carboxylation of the expressed hFIX. Coexpression of the human γ-glutamyl carboxylases (hγC) was also shown to improve both expression and γ-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the dγC to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active γ-carboxylated VKD proteins.     

Effect of Feeding Palm Oil By-Products Based Diets on Muscle Fatty Acid Composition in Goats

The present study aims to evaluate the effects of feeding palm oil by-products based diets on different muscle fatty acid profiles in goats. Thirty-two Cacang × Boer goats were randomly assigned to four dietary treatments: (1) control diet (CD), (2) 80% decanter cake diet (DCD), (3) 80% palm kernel cake diet (PKCD) and (4) CD plus 5% palm oil (PO) supplemented diet (CPOD). After 100 days of feeding, four goats from each group were slaughtered and longissimus dorsi (LD), infraspinatus (IS) and biceps femoris (BF) were sampled for analysis of fatty acids. Goats fed the PKCD had higher (P<0.05) concentration of lauric acid (C12:0) than those fed the other diets in all the muscles tested. Compared to the other diets, the concentrations of palmitic acid (C16:0) and stearic acid (C18:0) were lower (P<0.05) and that of linoleic acid (C18:2 n-6) was higher (P<0.05) in the muscles from goats fed the CD. It was concluded that palm kernel cake and decanter cake can be included in the diet of goats up to 80% with more beneficial than detrimental effects on the fatty acid profile of their meat.     

Nanovaccine: A novel approach in immunization

Despite great advances in the field of vaccination, there are still needs for novel and effective vaccines because still no effective vaccines have been produced for some diseases such as malaria, acquired immune deficiency syndrome (AIDS), and tuberculosis. Furthermore, many of the existing vaccines have disadvantages such as failure to stimulate completely the immune system, in vivo instability, high toxicity, the need for cold chain, and multiple administrations. Nanotechnology has been raised as a powerful tool for solving these problems in this regard. Generally, nanovaccines are a new generation of vaccines using nanoparticles (NPs) as carriers and/or adjuvants. Due to the similar scale (size) between the NPs and pathogens, the immune system can be stimulated well, resulting in triggered cellular and humoral immunity responses. Other benefits of the nanovaccines include their better stability in blood flow to increase the shelf life in blood, enhanced immune system stimulation, no need for booster doses, no need to maintain the cold chain, and ability to create active targeting. In addition, nanovaccines have raised the hope to treat diseases such as rheumatoid arthritis, AIDS, malaria, and chronic autoimmune, and so forth.     

Cardioprotective role of extracellular vesicles: A highlight on exosome beneficial effects in cardiovascular diseases

Extracellular vesicles (EVs) are nano-sized vesicles, released from many cell types including cardiac cells, have recently emerged as intercellular communication tools in cell dynamics. EVs are an important mediator of signaling within cells that influencing the functional behavior of the target cells. In heart complex, cardiac cells can easily use EVs to transport bioactive molecules such as proteins, lipids, and RNAs to the regulation of neighboring cell function. Cross-talk between intracardiac cells plays pivotal roles in the heart homeostasis and in adaptive responses of the heart to stress. EVs were released by cardiomyocytes under baseline conditions, but stress condition such as hypoxia intensifies secretome capacity. EVs secreted by cardiac progenitor cells and cardiosphere-derived cells could be pinpointed as important mediators of cardioprotection and cardiogenesis. Furthermore, EVs from many different types of stem cells could potentially exert a therapeutic effect on the damaged heart. Recent evidence shows that cardiac-derived EVs are rich in microRNAs, suggesting a key role in the controlling of cellular processes. EVs harboring exosomes may be clinically useful in cell-free therapy approaches and potentially act as prognosis and diagnosis biomarkers of cardiovascular diseases.