Rational design and characterization of bioplastics from Hermetia illucens prepupae proteins

In this study proteins extracted from prepupae of Hermetia illucens, also known as black soldier fly, are investigated as promising base for a new type of bioplastics for agricultural purposes. Design of experiments techniques are employed to perform a rational study on the effects of different combination of glycerol as plasticizer, citric acid as cross-linking agent and distilled water as solvent on the capability of proteins to form a free-standing film through casting technique, keeping as fixed the quantity of proteins. Glycerol shows interesting properties as plasticizer contributing to the formation of homogenous and free-standing film. Moreover, mechanical and thermal characterizations are performed to estimate the effect of increasing amounts of proteins on the final properties and thickness of the specimens. Proteins derived from H. illucens can be successfully employed as base for bioplastics to be employed for agricultural purposes.     

Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.     

Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants

There is no conclusive evidence that Helicoverpa spp. (Lepidoptera: Noctuidae) in Australia have evolved significant levels of resistance to Bollgard II^® cotton (which expresses two Bt toxin genes, cry1Ac and cry2Ab). However, there is evidence of surviving larvae on Bollgard II cotton in the field. The distribution and survival of early‐instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae were examined on whole Bollgard II and non‐Bt cotton plants in greenhouse bioassays. The expression of Cry toxins in various parts of Bollgard II plants was compared to the survival of larvae in those locations. Only 1% of larvae survived after 6 days on greenhouse‐grown Bollgard II plants compared to 31% on non‐Bt cotton plants. Overall, and across all time intervals, more larvae survived on reproductive parts (squares, flowers, and bolls) than on vegetative parts (leaves, stems, and petioles) on Bollgard II plants. The concentration of Cry1Ac toxin did not differ between plant structures, whereas Cry2Ab toxin differed significantly, but there was no relationship between the level of expression and the location of larvae. This study provides no evidence that lower expression of Cry toxins in the reproductive parts of plants explains the survival of H. armigera larvae on Bollgard II cotton.     

Cryoprotectant toxicity in Caenorhabditis elegans

We have looked at the effects of the cryoprotectant M22 upon viability in the model organism C. elegans. M22 is a well-known vitrification solution which has been successfully used in the laboratory to preserve organs destined for transplantation. M22 reduces survival of C. elegans in a concentration-dependent manner. M22 at concentrations of 10% (v/v) or higher inhibits progeny production and development. A few mutants in the ILS (insulin-like signaling) pathway of C. elegans are more resistant to the toxic effect of M22 compared to wild-type worms. Afatinib, an anti-cancer drug, protects against M22 toxicity. Afatinib by itself does not increase longevity.     

Synthesis and antitumor activity of three novel ginsenoside M1 derivatives with 3′-ester modifications

Ginsenoside M1 (M1) was considered to be the main antitumor component of ginsenoside metabolites in the body. In order to enhance its potency on antitumor effect, three novel M1 3′–ester derivatives (1c, 2c, 3c) were synthesized and evaluated. The yield of these derivatives was between 41% and 69%. Compared with M1, 2c and 3c can improve the efficacy of the inhibition on breast cancer MCF–7 and MDA–MB–231 cells, especially for MCF–7 (fold: 0.7–4.2, p < 0.0001). Further study suggested that 2c and 3c may cause cell autophagy and promote apoptosis in MCF–7 cells. The results indicated the 3′–ester modified M1 derivatives 2c and 3c possess higher abilities of inhibition growth towards triple–positive breast cancer and provided a new source for synthesis of potential anti-breast cancer drugs.     

Coupling dynamics and evolutionary information with structure to identify protein regulatory and functional binding sites

Binding sites in proteins can be either specifically functional binding sites (active sites) that bind specific substrates with high affinity or regulatory binding sites (allosteric sites), that modulate the activity of functional binding sites through effector molecules. Owing to their significance in determining protein function, the identification of protein functional and regulatory binding sites is widely acknowledged as an important biological problem. In this work, we present a novel binding site prediction method, Active and Regulatory site Prediction (AR-Pred), which supplements protein geometry, evolutionary, and physicochemical features with information about protein dynamics to predict putative active and allosteric site residues. As the intrinsic dynamics of globular proteins plays an essential role in controlling binding events, we find it to be an important feature for the identification of protein binding sites. We train and validate our predictive models on multiple balanced training and validation sets with random forest machine learning and obtain an ensemble of discrete models for each prediction type. Our models for active site prediction yield a median area under the curve (AUC) of 91% and Matthews correlation coefficient (MCC) of 0.68, whereas the less well-defined allosteric sites are predicted at a lower level with a median AUC of 80% and MCC of 0.48. When tested on an independent set of proteins, our models for active site prediction show comparable performance to two existing methods and gains compared to two others, while the allosteric site models show gains when tested against three existing prediction methods. AR-Pred is available as a free downloadable package at https://github.com/sambitmishra0628/AR-PRED_source .     

Genome-wide identification and comprehensive analysis of Excretory/Secretory proteins in nematodes provide potential drug targets for parasite control

Nematodes are responsible for causing severe diseases in plants, humans and other animals. Infection is associated with the release of Excretory/Secretory (ES) proteins into host cytoplasm and interference with the host immune system which make them attractive targets for therapeutic use. The identification of ES proteins through bioinformatics approaches is cost- and time-effective and could be used for screening of potential targets for parasitic diseases for further experimental studies. Here, we identified and functionally annotated 93,949 ES proteins, in the genome of 73 nematodes using integration of various bioinformatics tools. 30.6% of ES proteins were found to be supported at RNA level. The predicted ES proteins, annotated by Gene Ontology terms, domains, metabolic pathways, proteases and enzyme class analysis were enriched in molecular functions of proteases, protease inhibitors, c-type lectin and hydrolases which are strongly associated with typical functions of ES proteins. We identified a total of 452 ES proteins from human and plant parasitic nematodes, homologues to DrugBank-approved targets and C. elegans RNA interference phenotype genes which could represent potential targets for parasite control and provide valuable resource for further experimental studies to understand host-pathogen interactions.     

pLoc_bal-mGpos: Predict subcellular localization of Gram-positive bacterial proteins by quasi-balancing training dataset and PseAAC

Knowledge of protein subcellular localization is vitally important for both basic research and drug development. With the avalanche of protein sequences emerging in the post-genomic age, it is highly desired to develop computational tools for timely and effectively identifying their subcellular localization purely based on the sequence information alone. Recently, a predictor called “pLoc-mGpos” was developed for identifying the subcellular localization of Gram-positive bacterial proteins. Its performance is overwhelmingly better than that of the other predictors for the same purpose, particularly in dealing with multi-label systems in which some proteins, called “multiplex proteins”, may simultaneously occur in two or more subcellular locations. Although it is indeed a very powerful predictor, more efforts are definitely needed to further improve it. This is because pLoc-mGpos was trained by an extremely skewed dataset in which some subset (subcellular location) was over 11 times the size of the other subsets. Accordingly, it cannot avoid the bias consequence caused by such an uneven training dataset. To alleviate such bias consequence, we have developed a new and bias-reducing predictor called pLoc_bal-mGpos by quasi-balancing the training dataset. Rigorous target jackknife tests on exactly the same experiment-confirmed dataset have indicated that the proposed new predictor is remarkably superior to pLoc-mGpos, the existing state-of-the-art predictor in identifying the subcellular localization of Gram-positive bacterial proteins. To maximize the convenience for most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc_bal-mGpos/, by which users can easily get their desired results without the need to go through the detailed mathematics.     

The role of mitochondria in sepsis-induced cardiomyopathy

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Myocardial dysfunction, often termed sepsis-induced cardiomyopathy, is a frequent complication and is associated with worse outcomes. Numerous mechanisms contribute to sepsis-induced cardiomyopathy and a growing body of evidence suggests that bioenergetic and metabolic derangements play a central role in its development; however, there are significant discrepancies in the literature, perhaps reflecting variability in the experimental models employed or in the host response to sepsis. The condition is characterised by lack of significant cell death, normal tissue oxygen levels and, in survivors, reversibility of organ dysfunction. The functional changes observed in cardiac tissue may represent an adaptive response to prolonged stress that limits cell death, improving the potential for recovery. In this review, we describe our current understanding of the pathophysiology underlying myocardial dysfunction in sepsis, with a focus on disrupted mitochondrial processes.     

Novel MicroRNA-455-3p and its protective effects against abnormal APP processing and amyloid beta toxicity in Alzheimer's disease

The purpose of our study is to understand the protective role of miR-455-3p against abnormal amyloid precursor protein (APP) processing, amyloid beta (Aβ) formation, defective mitochondrial biogenesis/dynamics and synaptic damage in AD progression. In-silico analysis of miR-455-3p has identified the APP gene as a putative target. Using mutant APP cells, miR-455-3p construct, biochemical and molecular assays, immunofluorescence and transmission electron microscopy (TEM) analyses, we studied the protective effects of miR-455-3p on – 1) APP regulation, amyloid beta (Aβ)(1–40) & (1–42) levels, mitochondrial biogenesis & dynamics; 3) synaptic activities and 4) cell viability & apoptosis. Our luciferase reporter assay confirmed the binding of miR-455-3p at the 3’UTR of APP gene. Immunoblot, sandwich ELISA and immunostaining analyses revealed that the reduced levels of the mutant APP, Aβ(1–40) & Aβ(1–42), and C99 by miR-455-3p. We also found the reduced levels of mRNA and proteins of mitochondrial biogenesis (PGC1α, NRF1, NRF2, and TFAM) and synaptic genes (synaptophysin and PSD95) in mutant APP cells; on the other hand, mutant APP cells that express miR-455-3p showed increased mRNA and protein levels of biogenesis and synaptic genes. Additionally, expression of mitochondrial fission proteins (DRP1 and FIS1) were decreased while the fusion proteins (OPA1, Mfn1 and Mfn2) were increased by miR-455-3p. Our TEM analysis showed a decrease in mitochondria number and an increase in the size of mitochondrial length in mutant APP cells transfected with miR-455-3p. Based on these observations, we cautiously conclude that miR-455-3p regulate APP processing and protective against mutant APP-induced mitochondrial and synaptic abnormalities in AD.