A pH dependence study on the unfolding and refolding of apoazurin

Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a β-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be −27.8±2.4 kJ mol⁻¹. From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1±6.9 kJ/mol and 12.9±8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity—in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.     

Copper impact on heat shock protein 70 expression and apoptosis in rainbow trout hepatocytes

The mechanism underlying copper hepatotoxicity was investigated in primary cultures of rainbow trout hepatocytes maintained in Leibovitz-15 media. CuSO4 treatment (0, 25, 50, 100 and 200 microM) resulted in a dose-dependent elevation in heat shock protein 70 (hsp70) expression at 24 and 48 h post-exposure. There was no effect of copper (200 microM CuSO4) on hepatotoxicity at 24 h, whereas longer exposures (48 h) resulted in increased lactate dehydrogenase (LDH) leakage and apoptosis, demonstrated by fluorescence nuclear staining and DNA fragmentation. Vitamin C (1 mM), a free radical scavenger, inhibited this copper-induced apoptosis implying a role for reactive oxygen species in copper toxicity. However, no parallel inhibition of either LDH leakage or hsp70 protein expression was observed with vitamin C suggesting that at least two independent mechanisms are involved in the cellular response to copper. Also, copper exposed (24 h) cells were unable to mount an hsp70 response to a standardized heat shock (+15 degrees C for 1 h), even in the presence of vitamin C. Together, these results suggest that hepatotoxicity of copper includes impairment of hsp70 response to subsequent stressors and/or signals, which is crucial for protecting cells from proteotoxicity.     

Degradation of methylene blue using co-dopants Mg and Se in an hydroxyapatite composite

In this work, a comparative study was made of different magnesium ion content incorporated into hydroxyapatite (HAP) and modified with selenite ions, with the aim to develop the degradation performance of methylene blue. Although the dopant metal (Mg²⁺) was present at a relatively low ratio, it induced a change in the microstructure, morphology, surface area, external surface charge, particle size, and degradation performance. The effect of magnesium and selenium binary doping on microstructure and degradation of methylene blue was evaluated. The external surface charge measured by zeta potential clarified that the highest negativity was −11.8 mV and this was accomplished in 1.0 Mg/Se-HAP. Furthermore, the roughness average increased from 36.8 nm, reaching 59.2 nm upon the addition of Mg(II). Moreover, transmission electron microscope micrographs showed that compositions were formed as rod shapes. The process of degradation was optimized, showing effectiveness in methylene blue degradation of 62.4% after 150 min of exposure to visible light. Electrostatic attraction and H-bonding, and coordination played vital roles in the adsorption process. The recyclability of the as-prepared compositions demonstrated that the effectiveness had been reduced to ~54.2% after five times of re-use.     

Detection of baleen whale species using kernel dynamic mode decomposition-based feature extraction with a hidden Markov model

The negative effects of human activities within the ecological space of whales remains an issue of concern to marine ecologists. The accurate detection and subsequent classification of whale species are vital in mitigating these negative effects. Automatic detection techniques have come in handy for the efficient detection of the various whale species without human error. Hidden Markov model (HMM) remains one the most efficient detectors of whale species. However, its performance efficiency is greatly influenced by the feature vectors adapted with it. In this work, we propose the use of the kernel dynamic mode decomposition (kDMD) algorithm as a tool to extract features of baleen whale species, which are then adapted with HMM for their detection. Dynamic mode decomposition (DMD) is an eigendecomposition-based algorithm that is capable of extracting latent underlying features of non-linear signals such as those vocalised by whales. However, the underlying cost of DMD is the singular value decomposition (SVD), which adds significant complexity to the modes derivation steps. Thus, this work is introducing the kernel method into the DMD, in order to find a more efficient way of computing DMD without explicitly using the SVD algorithm. Furthermore, the feature formation steps in the original DMD was modified (mDMD) in this work, to make it more generic for datasets with sparse whale sound samples. The performance of the detectors was tested on datasets containing sounds of southern right whales (SRWs) and humpback whales. The results obtained show a high true positive rate (TPR), high precision (PREC) and low error rate (ERR) for both species. The performance of the three DMD-based feature-extraction methods were compared. The kDMD-HMM generally performed better than the mDMD-HMM and DMD-HMM detectors. The methods proposed here can be tailored for the automatic detection and classification of other vocalising animal species through their sounds.     

The effects of some natural products compared to synthetic products on the metabolic activity,proliferation, viability,migration, and wound healing in sheep tenocytes

BackgroundTendinopathy or tendon injuries can affect many people, causing a huge impact on their movements and maintaining standing posture. Treatment options include physiotherapy, anti-inflammatory drugs, and alternative medicine. The use of physiotherapy or anti-inflammatory drugs may cause some side effects like pain and liver failure, respectively, therefore, alternative medicine will be a better choice.MethodTenocytes were isolated from sheep Achilles tendon and used in Alamar blue assay to assess the metabolic activity, proliferation, and viability of tenocytes over 24 hrs. and 48 hrs., using natural and synthetic products [i.e., olive oil, oleic acid, corn oil, Inula viscosa oil, Inula viscosa extract, Nigella sativa oil, naproxen sodium, and paracetamol and LED photobiomodulation]. Furthermore, tenocytes viability was assessed by FDA/PI stain. For migration and healing of a wound, the scratch assay was used.ResultsAlamar blue assay over 24 hrs. showed that Nigella sativa oil increased the metabolic activity, proliferation, and viability of tenocytes significantly, while Alamar blue over 48 hrs. showed that oleic acid, LED, and their combination increased these parameters for tenocytes significantly. Olive oil increased the viability of tenocytes significantly using FDA/PI stains. Scratch assay revealed that Inula viscosa oil, Inula viscosa extract, and paracetamol increased tenocyte migration and healing significantly.ConclusionNigella sativa oil, olive oil, oleic acid, Inula viscosa oil, and Inula viscosa extract may be used as an alternative therapy for tendinopathy with less side effects.     

Assembly of the cytochrome bo3 complex

An understanding of the mechanisms that govern the assembly of macromolecular protein complexes is fundamental for studying their function and regulation. With this in mind, we have determined the assembly pathway for the membrane-embedded cytochrome bo(3) of Escherichia coli. We show that there is a preferred order of assembly, where subunits III and IV assemble first, followed by subunit I and finally subunit II. We also show that cofactor insertion catalyses assembly. These findings provide novel insights into the biogenesis of this model membrane protein complex.     

Free Marine Natural Products Databases for Biotechnology and Bioengineering

Marine organisms and micro‐organisms are a source of natural compounds with unique chemical features. These chemical properties are useful for the discovery of new functions and applications of marine natural products (MNPs). To extensively exploit the potential implementations of MNPs, they are gathered in chemical databases that allow their study and screening for applications of biotechnological interest. However, the classification of MNPs is currently poor in generic chemical databases. The present availability of free‐access‐focused MNP databases is scarce and the molecular diversity of these databases is still very low when compared to the paid‐access ones. In this review paper, the current scenario of free‐access MNP databases is presented as well as the hindrances involved in their development, mainly compound dereplication. Examples and opportunities for using freely accessible MNP databases in several important areas of biotechnology are also assessed. The scope of this paper is, as well, to notify the latent potential of these information sources for the discovery and development of new MNPs in biotechnology, and push future efforts to develop a public domain MNP database freely available for the scientific community.     

Mitochondrial diseases: Drosophila melanogaster as a model to evaluate potential therapeutics

While often presented as a single entity, mitochondrial diseases comprise a wide range of clinical, biochemical and genetic heterogeneous disorders. Among them, defects in the process of oxidative phosphorylation are the most prevalent. Despite intense research efforts, patients are still without effective treatment. An important part of the development of new therapeutics relies on predictive models of the pathology in order to assess their therapeutic potential. Since mitochondrial diseases are a heterogeneous group of progressive multisystemic disorders that can affect any organ at any time, the development of various in vivo models for the different diseases-associated genes defects will accelerate the search for effective therapeutics. Here, we review existing Drosophila melanogaster models for mitochondrial diseases, with a focus on alterations in oxidative phosphorylation, and discuss the potential of this powerful model organism in the process of drug target discovery.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Microhabitat use by mountain pygmy‐possum (Burramys parvus): Implications for the conservation of small mammals in alpine environments

The Australian alpine region harbours a wide range of species, many of which are endemic and of high conservation value. Among these species, the endangered mountain pygmy‐possum, Burramys parvus, is of particular interest because this specialized marsupial is highly sensitive to extreme temperatures. The selection of microhabitats by B. parvus is a critical but poorly understood element of its biological characteristics. To understand the microhabitat preferences of B. parvus, we performed detailed investigations of the thermal properties of alpine boulder fields. The selection of a preferred microclimate was demonstrated by comparing temperatures and environmental conditions in preferred and non‐preferred boulder fields. The variability of the daily temperature depended on the depth at which measurements were made within the boulder fields. Temperatures were more stable as depth increased. The results suggest that B. parvus prefers to occupy deep boulder fields at high elevations with good rock structure (small rock and cavity size with multiple layers) and long snow duration because these boulder fields can provide a favourable microclimate. At 1 m depth, the maximum temperatures in the hottest part of the year were 1.27°C cooler in preferred compared to non‐preferred boulder fields. In the coldest part of the year, immediately following the melting of persistent snow cover, the minimum temperatures at a depth of 1 m were 1.67°C warmer in preferred compared to non‐preferred boulder fields. On average, the snow duration was 27 days greater in the boulder fields preferred by B. parvus than in non‐preferred boulder fields. Our results emphasize the value of boulder field microhabitats as thermal refuges for small mammals in rocky habitats within alpine environments in the light of continuing habitat loss and climate change.