Atomic force microscopy study of the conformational change in immobilized calmodulin.

Maintaining the biological functionality of immobilized proteins is the key to the success of numerous protein-based biomedical devices. To that end, we studied the conformational change in calmodulin (CaM) immobilized on chemical patterns. 1-Cysteine-mutated calmodulin was immobilized on a mercapto-terminated surface through cysteine-Hg-mercapto coupling. Utilizing atomic force microscopy (AFM), the average height of immobilized calmodulin was determined to be 1.87 ± 0.19 nm. After incubation in EGTA solution, the average height of the protein changed to 2.26 ± 0.21 nm, indicating the conformational change of CaM to Apo-CaM. Immobilized CaM also demonstrated a conformational change upon the reaction with known calmodulin antagonist chlorpromazine (CPZ). After incubation in CPZ solution, the average height of CPZ-bound CaM increased to 2.32 ± 0.20 nm, demonstrating that immobilized CaM has a similar response to that in bulk solution. These results show that the immobilization of calmodulin on a solid support does not interfere with the ability of the protein to bind calcium and calmodulin antagonists. Our results demonstrate the feasibility of employing AFM to probe and understand protein conformational changes.

Measuring the oral bioavailability of protein hydrolysates derived from food sources: A critical review of current bioassays.

BACKGROUND: Food proteins are a source of hydrolysates with potentially useful biological attributes. Bioactive peptides from food-derived proteins are released from hydrolysates using exogenous industrial processes or endogenous intestinal enzymes. Current in vitro permeability assays have limitations in predicting the oral bioavailability (BA) of bioactive peptides in humans. There are also difficulties in relating the low blood levels of food-derived bioactive peptides detected in preclinical in vivo models to pharmacodynamic read-outs relevant for humans. SCOPE AND APPROACH: In this review, we describe in vitro assays of digestion, permeation, and metabolism as indirect predictors of the potential oral BA of hydrolysates and their constituent bioactive peptides. We discuss the relationship between industrial hydrolysis processes and the oral BA of hydrolysates and their peptide by-products. KEY FINDINGS: Hydrolysates are challenging for analytical detection methods due to capacity for enzymatic generation of peptides with novel sequences and also new modifications of these peptides during digestion. Mass spectrometry and peptidomics can improve the capacity to detect individual peptides released from complex hydrolysates in biological milieu.

Preservatives from food-For food: Pea protein hydrolysate as a novel bio-preservative against Escherichia coli O157:H7 on a lettuce leaf.

Fresh-cut fruits and vegetables are becoming particularly popular as healthy fast-food options; however, they present challenges such as accelerated rates of decay and increased risk for contamination when compared to whole produce. Given that food safety must remain paramount for producers and manufacturers, research into novel, natural food preservation solutions which can help to ensure food safety and protect against spoilage is on the rise. In this work, we investigated the potential of using a novel protein hydrolysate, produced by the enzymatic hydrolysis of Pisum sativum (PSH), as a novel bio-preservative and its abilities to reduce populations of Escherichia coli O157:H7 after inoculation on a lettuce leaf. While unhydrolyzed P. sativum proteins show no antimicrobial activity, once digested, and purified, the enzymatically released peptides induced in vitro bactericidal effects on the foodborne pathogen at 8 mg/ml. When applied on an infected lettuce leaf, the PSH significantly reduced the number of bacteria recovered after 2 hr of treatment. PSH may be preferred over other preservation strategies based on its natural, inexpensive, sustainable source, environmentally friendly process, nontoxic nature, good batch to batch consistency, and ability to significantly reduce counts of E. coli both in vitro and in a lettuce leaf.

Characterising the efficacy and bioavailability of bioactive peptides identified for attenuating muscle atrophy within a Vicia faba-derived functional ingredient.

Characterising key components within functional ingredients as well as assessing efficacy and bioavailability is an important step in validating nutritional interventions. Machine learning can assess large and complex data sets, such as proteomic data from plants sources, and so offers a prime opportunity to predict key bioactive components within a larger matrix. Using machine learning, we identified two potentially bioactive peptides within a Vicia faba derived hydrolysate, NPN_1, an ingredient which was previously identified for preventing muscle loss in a murine disuse model. We investigated the predicted efficacy of these peptides in vitro and observed that HLPSYSPSPQ and TIKIPAGT were capable of increasing protein synthesis and reducing TNF-α secretion, respectively. Following confirmation of efficacy, we assessed bioavailability and stability of these predicted peptides and found that as part of NPN_1, both HLPSYSPSPQ and TIKIPAGT survived upper gut digestion, were transported across the intestinal barrier and exhibited notable stability in human plasma. This work is a first step in utilising machine learning to untangle the complex nature of functional ingredients to predict active components, followed by subsequent assessment of their efficacy, bioavailability and human plasma stability in an effort to assist in the characterisation of nutritional interventions.

Human cardiac troponin I: a Langmuir monolayer study.

Human cardiac troponin I (cTnI) is the preferred biomarker in the assessment of myocardial infarction. It is known to interact with troponin C and T to form a trimeric complex. Whereas small amounts are found in the cytoplasm, most of cTnI is in the form of a complex with actin located in myofilaments. To understand these interactions of cTnI better, we first investigated the surface chemistry of cTnI as a Langmuir monolayer spread at the air-water interface. We investigated the optimal conditions for obtaining a stable Langmuir monolayer in terms of changing the ionic strength of the subphase using different concentrations of potassium chloride. Monolayer stability was investigated by compressing the cTnI monolayer to a specific surface pressure and keeping the surface pressure constant while measuring the decrease in the molecular area as a function of time. Aggregation and/or domain formation was investigated by using compression-decompression cycles, in situ UV-vis spectroscopy, Brewster angle microscopy (BAM), and epifluorescence microscopy. To ensure that the secondary structure is maintained, we used infrared reflection-absorption spectroscopy (IRRAS) directly at the air-subphase interface. It was found that cTnI forms a very stable monolayer (after more that 5000 s) that does not aggregate at the air-subphase interface. The cTnI molecules maintain their secondary structure and, on the basis of the low reflectivity observed using BAM measurements and the low reflection-absorption intensities measured with IRRAS spectroscopy, lie flat on the subphase with the alpha-helices parallel to the air-subphase interface.

Preclinical Evaluation of a Food-Derived Functional Ingredient to Address Skeletal Muscle Atrophy.

Skeletal muscle is the metabolic powerhouse of the body, however, dysregulation of the mechanisms involved in skeletal muscle mass maintenance can have devastating effects leading to many metabolic and physiological diseases. The lack of effective solutions makes finding a validated nutritional intervention an urgent unmet medical need. In vitro testing in murine skeletal muscle cells and human macrophages was carried out to determine the effect of a hydrolysate derived from vicia faba (PeptiStrong: NPN_1) against phosphorylated S6, atrophy gene expression, and tumour necrosis factor alpha (TNF-α) secretion, respectively. Finally, the efficacy of NPN_1 on attenuating muscle waste in vivo was assessed in an atrophy murine model. Treatment of NPN_1 significantly increased the phosphorylation of S6, downregulated muscle atrophy related genes, and reduced lipopolysaccharide-induced TNF-α release in vitro. In a disuse atrophy murine model, following 18 days of NPN_1 treatment, mice exhibited a significant attenuation of muscle loss in the soleus muscle and increased the integrated expression of Type I and Type IIa fibres. At the RNA level, a significant upregulation of protein synthesis-related genes was observed in the soleus muscle following NPN_1 treatment. In vitro and preclinical results suggest that NPN_1 is an effective bioactive ingredient with great potential to prolong muscle health.

Tissue-protective effects of fullerenol C60(OH)24 and amifostine in irradiated rats.

Polyhydroxylated fullerenes, named fullerenols (C(60)(OH)(n); n=12-26) are excellent antioxidants. Harmful effects of ionizing radiation on living organism are mainly mediated by free radical species and fullerenols attract an attention as a potential radioprotectors. Our preliminary investigations on mice and rats subjected to radiation injury show that fullerenol C(60)(OH)(24) provides high survival rate of irradiated small rodents. Radioprotective effect was comparable to that of the standard radioprotector amifostine. The aim of this study was to compare the efficacy of fullerenol C(60)(OH)(24) (10 and 100mg/kg i.p.) and amifostine (300 mg/kg i.p.) in protection of rats against harmful effects of ionizing radiation. The animals were whole-body irradiated by X-rays (8 MV). Both compounds were given 30 min before irradiation. In order to evaluate the general radioprotective efficacy of fullerenol and amifostine rats were irradiated with an absolutely lethal dose of X-rays (8 Gy) and their survival and body mass gain were monitored during the period of 30 days after irradiation. The aim of the second part of the study is to investigate the tissue-protective effects of tested compounds (100 mg/kg i.p. of fullerenol and 300 mg/kg i.p. of amifostine, 30 min before irradiation). It was carried out on rats irradiated with a sublethal dose of X-rays (7 Gy). Influence of ionizing radiation on hematopoesis as well as the radioprotective efficiency of the compounds given were evaluated by determining blood cell count during 28 days after irradiation. For this purpose the blood was taken from tail vein before irradiation and on the 3rd, 7th, 14th, 21st and 28th day after irradiation. In order to estimate the radioprotective effects of fullerenol and amifostine on other rat tissue, the animals were sacrificed on the 7th and 28th day after irradiation and their main organs (lung, heart, liver, kidney, small intestine and spleen) were taken for histopathological analysis. In the experiment in which the general radioprotective efficacy of fullerenol and amifostine was examined, fullerenol given in a dose of 100mg/kg produced better protection than given in a dose of 10mg/kg. This effect was comparable to that of amifostine. The results of hematological investigations showed that fullerenol better than amifostine prevented radiation-induced reduction in the white cell count (granulocytes and lymphocytes), particularly in the first 7 days after irradiation. Pathohistology examinations revealed better radioprotective effects of fullerenol compared to those of amifostine on the spleen, small intestine and lung, while amifostine had better radioprotective effects than fullerenol in protection of the heart, liver and kidney. These results confirm satisfactory radioprotective efficacy of fullerenol and encourage further investigations as a potential radioprotector.