In vitro antioksidacijska, citotoksična i antidijabetička aktivnost hidrolizata proteina iz Reevesove barske kornjače (Chinemys reevesii)

Research background. Cardiovascular diseases and diabetes are the biggest causes of death globally. Bioactive peptides derived from many food proteins using enzymatic proteolysis and food processing have a positive impact on the prevention of these diseases. The bioactivity of Chinese pond turtle muscle proteins and their enzymatic hydrolysates has not received much attention, thus this study aims to investigate their antioxidant, antidiabetic and cytotoxic activities. Experimental approach. Chinese pond turtle muscles were hydrolysed using four proteolytic enzymes (Alcalase, Flavourzyme, trypsin and bromelain) and the degrees of hydrolysis were measured. High-performance liquid chromatography (HPLC) was conducted to explore the amino acid profiles and molecular mass distribution of the hydrolysates. The antioxidant activities were evaluated using various in vitro tests, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical scavenging activity, reducing capacity, chelating Fe2+ and lipid peroxide inhibition activity. Antidiabetic activity was evaluated using α-amylase inhibition and α-glucosidase inhibition assays. Besides, cytotoxic effect of hydrolysates on human colon cancer (HT-29) cells was assessed. Results and conclusions. The amino acid composition of the hydrolysates revealed higher mass fractions of glutamic, aspartic, lysine, hydroxyproline and hydrophobic amino acids. Significantly highest inhibition of lipid peroxidation was achieved when hydrolysate obtained with Alcalase was used. Protein hydrolysate produced with Flavourzyme had the highest radical scavenging activity measured by DPPH (68.32%), ABTS (74.12%) and FRAP (A700 nm=0.300) assays, α-glucosidase (61.80%) inhibition and cytotoxic effect (82.26%) on HT-29 cell line at 550 µg/mL. Hydrolysates obtained with trypsin and bromelain had significantly highest (p<0.05) hydroxyl radical scavenging (92.70%) and Fe2+ metal chelating (63.29%) activities, respectively. The highest α-amylase (76.89%) inhibition was recorded when using hydrolysates obtained with bromelain and Flavourzyme. Novelty and scientific contribution. Enzymatic hydrolysates of Chinese pond turtle muscle protein had high antioxidant, cytotoxic and antidiabetic activities. The findings of this study indicated that the bioactive hydrolysates or peptides from Chinese pond turtle muscle protein can be potential ingredients in pharmaceuticals and functional food formulations.Pozadina istraživanja. Kardiovaskularne bolesti i dijabetes najčešći su uzroci smrti na svijetu. Bioaktivni peptidi dobiveni proteolizom i preradom hrane imaju pozitivan učinak na prevenciju tih bolesti. Biološka aktivnost proteina iz mišića Reevesove barske kornjače i njihovih hidrolizata nije dovoljno istražena, stoga je svrha ovoga rada bila ispitati njihovu antioksidacijsku, antidijabetičku i citotoksičnu aktivnost. Eksperimentalni pristup. Mišići Reevesove barske kornjače hidrolizirani su pomoću proteolitičkih enzima (Alcalase, Flavourzyme, tripsin i bromelain), te su mjereni stupnjevi hidrolize proteina. Aminokiselinski sastav i distribucija molekularne mase hidrolizata ispitani su pomoću visokodjelotvorne tekućinske kromatografije. Antioksidacijska aktivnost određena je različitim testovima in vitro, uključujući sposobnost uklanjanja 1,1-difenil-2-pikrilhidrazila (DPPH), 2,2’-azino-bis(3-etilbenzotiazolin-6-sumporne kiseline) (ABTS) i hidroksil radikala, keliranja Fe2+ i inhibicije lipidne peroksidacije. Antidijabetička aktivnost ispitana je testovima inhibicije α-amilaze i α-glukozidaze. Osim toga, analiziran je citotoksični učinak hidrolizata na stanice tumora debelog crijeva (HT-29). Rezultati i zaključci. Analizom aminokiselinskog sastava hidrolizata pronađeni su veći maseni udjeli glutaminske i asparaginske kiseline, lizina, hidroksiprolina te hidrofobnih aminokiselina od onih u nehidroliziranim proteinima. Hidrolizat proteina dobiven pomoću proteolitičkog enzima Alcalase bitno je inhibirao peroksidaciju lipida. Pri koncentraciji od 550 µg/mL, hidrolizat proteina dobiven pomoću enzima Flavourzyme imao je najveću sposobnost uklanjanja slobodnih radikala mjerenu pomoću DPPH (68,32 %), ABTS (74,12 %) i FRAP (A700 nm=0,300) metoda, inhibicije α-glukozidaze (61,80 %) te najveći citotoksični učinak na stanične linije HT-29 (82.26 %). Hidrolizat proteina dobiven pomoću tripsina imao je znatnu (p<0,05) aktivnost uklanjanja hidroksilnih radikala (92,70 %), a onaj dobiven pomoću bromelaina najveću aktivnost keliranja Fe2+ (63,29 %). Najveća inhibicija α-amilaze postignuta je pomoću hidrolizata proteina dobivenih djelovanjem bromelaina i enzima Flavourzyme. Novina i znanstveni doprinos. Hidrolizati proteina mišića Reevesove barske kornjače dobiveni enzimskom hidrolizom imali su veliku antioksidacijsku, citotoksičnu i antidijabetičku aktivnost. Rezultati istraživanja pokazuju da se ti hidrolizati ili peptidi zbog svojih bioaktivnih svojstava mogu upotrijebiti kao sastojak u farmaceutskim i funkcionalnim prehrambenim proizvodima

Visual Cue Integration for Small Target Motion Detection in Natural Cluttered Backgrounds

The robust detection of small targets against cluttered background is important for future artificial visual systems in searching and tracking applications. The insects’ visual systems have demonstrated excellent ability to avoid predators, find prey or identify conspecifics – which always appear as small dim speckles in the visual field. Build a computational model of the insects’ visual pathways could provide effective solutions to detect small moving targets. Although a few visual system models have been proposed, they only make use of small-field visual features for motion detection and their detection results often contain a number of false positives. To address this issue, we develop a new visual system model for small target motion detection against cluttered moving backgrounds. Compared to the existing models, the small-field and wide-field visual features are separately extracted by two motion-sensitive neurons to detect small target motion and background motion. These two types of motion information are further integrated to filter out false positives. Extensive experiments showed that the proposed model can outperform the existing models in terms of detection rates

High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Fibroblast growth factor 21 (FGF21) is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21) in <it>Escherichia coli (E. coli) </it>is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the <it>FGF21 </it>with <it>SUMO </it>(Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and expressed the fused gene in <it>E. coli </it>BL21(DE3).</p> <p>Results</p> <p>By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC) was shown to be higher than 96% with low endotoxin level (<1.0 EU/ml). The results of <it>in vivo </it>animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ) injection.</p> <p>Conclusions</p> <p>This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in <it>E. coli</it>, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.</p

A bioinspired angular velocity decoding neural network model for visually guided flights

Efficient and robust motion perception systems are important pre-requisites for achieving visually guided flights in future micro air vehicles. As a source of inspiration, the visual neural networks of flying insects such as honeybee and Drosophila provide ideal examples on which to base artificial motion perception models. In this paper, we have used this approach to develop a novel method that solves the fundamental problem of estimating angular velocity for visually guided flights. Compared with previous models, our elementary motion detector (EMD) based model uses a separate texture estimation pathway to effectively decode angular velocity, and demonstrates considerable independence from the spatial frequency and contrast of the gratings. Using the Unity development platform the model is further tested for tunnel centering and terrain following paradigms in order to reproduce the visually guided flight behaviors of honeybees. In a series of controlled trials, the virtual bee utilizes the proposed angular velocity control schemes to accurately navigate through a patterned tunnel, maintaining a suitable distance from the undulating textured terrain. The results are consistent with both neuron spike recordings and behavioral path recordings of real honeybees, thereby demonstrating the model’s potential for implementation in micro air vehicles which have only visual sensors

A novel Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) grafted Polyethyleneimine(PCFC-g-PEI), Part 1, synthesis, cytotoxicity, and in vitro transfection study

<p>Abstract</p> <p>Background</p> <p>Polyethyleneimine (PEI), a cationic polymer, is one of the successful and widely used vectors for non-viral gene transfection <it>in vitro</it>. However, its <it>in vivo </it>application was greatly limited due to its high cytotoxicity and short duration of gene expression. To improve its biocompatibility and transfection efficiency, PEI has been modified with PEG, folic acid, and chloroquine in order to improve biocompatibility and enhance targeting.</p> <p>Results</p> <p>Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) (PCFC) was synthesized by ring-opening polymerization, and PCFC-<it>g</it>-PEI was obtained by Michael addition reaction with GMA-PCFC-GMA and polyethyleneimine (PEI, 25 kD). The prepared PCFC-<it>g</it>-PEI was characterized by ¹H-NMR, SEC-MALLS. Meanwhile, DNA condensation, DNase I protection, the particle size and zeta potential of PCFC-<it>g</it>-PEI/DNA complexes were also determined. According to the results of flow cytometry and MTT assay, the synthesized PCFC-<it>g</it>-PEI, with considerable transfection efficiency, had obviously lower cytotoxicity against 293 T and A549 cell lines compared with that of PEI 25 kD.</p> <p>Conclusion</p> <p>The cytotoxicity and <it>in vitro </it>transfection study indicated that PCFC-<it>g</it>-PEI copolymer prepared in this paper was a novel gene delivery system with lower cytotoxicity and considerable transfection efficiency compared with commercial PEI (25 kD).</p

Towards Computational Models and Applications of Insect Visual Systems for Motion Perception: A Review

Motion perception is a critical capability determining a variety of aspects of insects' life, including avoiding predators, foraging and so forth. A good number of motion detectors have been identified in the insects' visual pathways. Computational modelling of these motion detectors has not only been providing effective solutions to artificial intelligence, but also benefiting the understanding of complicated biological visual systems. These biological mechanisms through millions of years of evolutionary development will have formed solid modules for constructing dynamic vision systems for future intelligent machines. This article reviews the computational motion perception models originating from biological research of insects' visual systems in the literature. These motion perception models or neural networks comprise the looming sensitive neuronal models of lobula giant movement detectors (LGMDs) in locusts, the translation sensitive neural systems of direction selective neurons (DSNs) in fruit flies, bees and locusts, as well as the small target motion detectors (STMDs) in dragonflies and hover flies. We also review the applications of these models to robots and vehicles. Through these modelling studies, we summarise the methodologies that generate different direction and size selectivity in motion perception. At last, we discuss about multiple systems integration and hardware realisation of these bio-inspired motion perception models

Angular Velocity Estimation of Image Motion Mimicking the Honeybee Tunnel Centring Behaviour

Insects use visual information to estimate angular velocity of retinal image motion, which determines a variety of flight behaviours including speed regulation, tunnel centring and visual navigation. For angular velocity estimation, honeybees show large spatial-independence against visual stimuli, whereas the previous models have not fulfilled such an ability. To address this issue, we propose a bio-plausible model for estimating the image motion velocity based on behavioural experiments of the honeybee flying through patterned tunnels. The proposed model contains mainly three parts, the texture estimation layer for spatial information extraction, the delay-and-correlate layer for temporal information extraction and the decoding layer for angular velocity estimation. This model produces responses that are largely independent of the spatial frequency in grating experiments. And the model has been implemented in a virtual bee for tunnel centring simulations. The results coincide with both electro-physiological neuron spike and behavioural path recordings, which indicates our proposed method provides a better explanation of the honeybee’s image motion detection mechanism guiding the tunnel centring behaviour

Open source board based acoustofluidic transwells for reversible disruption of the blood–brain barrier for therapeutic delivery

Background: Blood–brain barrier (BBB) is a crucial but dynamic structure that functions as a gatekeeper for the central nervous system (CNS). Managing sufficient substances across the BBB is a major challenge, especially in the development of therapeutics for CNS disorders. Methods: To achieve an efficient, fast and safe strategy for BBB opening, an acoustofluidic transwell (AFT) was developed for reversible disruption of the BBB. The proposed AFT was consisted of a transwell insert where the BBB model was established, and a surface acoustic wave (SAW) transducer realized using open-source electronics based on printed circuit board techniques. Results: In the AFT device, the SAW produced acousto-mechanical stimulations to the BBB model resulting in decreased transendothelial electrical resistance in a dose dependent manner, indicating the disruption of the BBB. Moreover, SAW stimulation enhanced transendothelial permeability to sodium fluorescein and FITC-dextran with various molecular weight in the AFT device. Further study indicated BBB opening was mainly attributed to the apparent stretching of intercellular spaces. An in vivo study using a zebrafish model demonstrated SAW exposure promoted penetration of sodium fluorescein to the CNS. Conclusions: In summary, AFT effectively disrupts the BBB under the SAW stimulation, which is promising as a new drug delivery methodology for neurodegenerative diseases. Graphical Abstract

Effectiveness of 2009 pandemic influenza A(H1N1) vaccines: a systematic review and meta-analysis

Background: The clinical effectiveness of monovalent influenza A(H1N1)pdm09 vaccines has not been comprehensively summarised. We undertook a systematic review and meta-analysis to assess vaccine effectiveness (VE) for adjuvanted and unadjuvanted vaccines. Methods: We searched healthcare databases and grey literature from 11 June 2009 to 12 November 2014. Two researchers independently assessed titles and abstracts to identify studies for full review. Random effects meta-analyses estimated the pooled effect size of vaccination compared to placebo or no vaccination for crude and adjusted odds ratios (OR) to prevent laboratory confirmed influenza illness (LCI) and related hospitalization. VE was calculated as (1-pooled OR) ∗ 100. Narrative synthesis was undertaken where meta-analysis was not possible. Results: We identified 9229 studies of which 38 at moderate risk of bias met protocol eligibility criteria; 23 were suitable for meta-analysis. Pooled adjusted VE against LCI with adjuvanted and unadjuvanted vaccines both reached statistical significance (adjuvanted: VE = 80%; 95% confidence interval [CI] 59–90%; unadjuvanted: VE = 66%; 95% CI 47–78%); in planned secondary analyses, VE in adults often failed to reach statistical significance and pooled point estimates were lower than observed in children. Overall pooled adjusted VE against hospitalization was 61% (95% CI 14–82%); in planned secondary analyses, adjusted VE attained statistical significance in adults aged 18–64 years and children for adjuvanted vaccines. Adjuvanted vaccines were significantly more effective in children compared to adults for both outcomes. Conclusions: Adjuvanted and unadjuvanted monovalent influenza A(H1N1)pdm09 vaccines were both effective in preventing LCI. Overall, the vaccines were also effective against influenza-related hospitalization. For both outcomes adjuvanted vaccines were more effective in children than in adults