Biomolecules, Vol. 9, Pages 255: Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

Biomolecules, Vol. 9, Pages 254: A Novel Kunitzin-Like Trypsin Inhibitor Isolated from Defensive Skin Secretion of Odorrana versabilis Protease inhibitors that were identified from amphibian skin secretions with low molecular weights and potent inhibitory activity were thought to be potential candidates for novel peptide drugs. Here, a novel peptide with trypsin inhibitory activity was found in the skin secretion of the Chinese bamboo leaf odorous frog, Odorrana versabilis. Based on the sequence alignments of sequencing results, the novel peptide (ALKYPFRCKAAFC) was named as Kunitzin-OV. The synthetic replicate of Kunitzin-OV was subjected to a series of functional assays, and it exhibited a trypsin inhibitory activity with a Ki value of 3.042 µM, whereas, when Lys-9 at P1 position was substituted by Phe, trypsin inhibitory activity was undetected and the chymotrypsin inhibitory activity was optimized with a Ki value of 2.874 µM. However, its protease-binding loop was catabolized by trypsin during the trypsin cleavage test. In conclusion, Kunizin-OV is a novel peptide with trypsin inhibitory activity as a member of kunitzins, which is a non-typical Kunitz-like trypsin inhibitor with a highly conserved reactive site (K-A) and quite a short sequence.

Biomolecules, Vol. 9, Pages 253: Isoform-Specific Role of Akt in Oral Squamous Cell Carcinoma Protein kinase B (Akt) plays a very significant role in various cancers including oral cancer. However, it has three isoforms (Akt1, Akt2, and Akt3) and they perform distinct functions and even play contrasting roles in different cancers. Therefore, it becomes essential to evaluate the isoform-specific role of Akt in oral cancer. In the present study, an attempt has been made to elucidate the isoform-specific role of Akt in oral cancer. The immunohistochemical analysis of oral cancer tissues showed an overexpression of Akt1 and 2 isoforms but not Akt3. Moreover, the dataset of “The Cancer Genome Atlas” for head and neck cancer has suggested the genetic alterations of Akt1 and 2 tend to be associated with the utmost poor clinical outcome in oral cancer. Further, treatment of oral cancer cells with tobacco and its components such as benzo(a)pyrene and nicotine caused increased mRNA levels of Akt1 and 2 isoforms and also enhanced the aggressiveness of oral cancer cells in terms of proliferation, and clonogenic and migration potential. Finally, silencing of Akt1 and 2 isoforms caused decreased cell survival and induced cell cycle arrest at the G2/M phase. Akt1/2 silencing also reduced tobacco-induced aggressiveness by decreasing the clonogenic and migration potential of oral cancer cells. Moreover, silencing of Akt1 and 2 isoforms was found to decrease the expression of proteins regulating cancer cell survival and proliferation such as cyclooxygenase-2, B-cell lymphoma 2 (Bcl-2), cyclin D1, and survivin. Thus, the important role of Akt1 and 2 isoforms have been elucidated in oral cancer with in-depth mechanistic analysis.

Biomolecules, Vol. 9, Pages 252: A Novel Approach for Investigating Upper Airway Hyperresponsiveness Using Micro-CT in Eosinophilic Upper Airway Inflammation such as Allergic Rhinitis Model Airway hyperresponsiveness (AHR) has been proposed as a feature of pathogenesis of eosinophilic upper airway inflammation such as allergic rhinitis (AR). The measurement system for upper AHR (UAHR) in rodents is poorly developed, although measurements of nasal resistance have been reported. Here we assessed UAHR by direct measurement of swelling of the nasal mucosa induced by intranasal methacholine (MCh) using micro-computed tomography (micro-CT). Micro-CT analysis was performed in both naïve and ovalbumin-induced AR mice following intranasal administration of MCh. The nasal cavity was segmented into two-dimensional horizontal and axial planes, and the data for nasal mucosa were acquired for the region of interest threshold. Then, a ratio between the nasal mucosa area and nasal cavity area was calculated as nasal mucosa index. Using our novel method, nasal cavity structure was clearly identified on micro-CT, and dose-dependent increased swelling of the nasal mucosa was observed upon MCh treatment. Moreover, the nasal mucosa index was significantly increased in AR mice compared to controls following MCh treatment, while ovalbumin administration did not affect swelling of the nasal mucosa in either group. This UAHR following MCh treatment was completely reversed by pretreatment with glucocorticoids. This novel approach using micro-CT for investigating UAHR reflects a precise assessment system for swelling of the nasal mucosa following MCh treatment; it not only sheds light on the mechanism of AR but also contributes to the development of new therapeutic drugs in AR patients.

Biomolecules, Vol. 9, Pages 251: Correlating Lipid Membrane Permeabilities of Imidazolium Ionic Liquids with Their Cytotoxicities on Yeast, Bacterial, and Mammalian Cells Alkyl-imidazolium chloride ionic liquids (ILs) have been broadly studied for biochemical and biomedical technologies. They can permeabilize lipid bilayer membranes and have cytotoxic effects, which makes them targets for drug delivery biomaterials. We assessed the lipid-membrane permeabilities of ILs with increasing alkyl chain lengths from ethyl to octyl groups on large unilamellar vesicles using a trapped-fluorophore fluorescence lifetime-based leakage experiment. Only the most hydrophobic IL, with the octyl chain, permeabilizes vesicles, and the concentration required for permeabilization corresponds to its critical micelle concentration. To correlate the model vesicle studies with biological cells, we quantified the IL permeabilities and cytotoxicities on different cell lines including bacterial, yeast, and ovine blood cells. The IL permeabilities on vesicles strongly correlate with permeabilities and minimum inhibitory concentrations on biological cells. Despite exhibiting a broad range of lipid compositions, the ILs appear to have similar effects on the vesicles and cell membranes.

Biomolecules, Vol. 9, Pages 250: Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One In practice and despite Anfinsen’s dogma, the refolding of recombinant multiple SS-bonded proteins is famously difficult because misfolded species with non-native SS-bonds appear upon the oxidization of their cysteine residues. On the other hand, single SS-bond proteins are thought to be simple to refold because their cysteines have only one SS-bond partner. Here, we report that dengue 4 envelope protein domain 3 (DEN4 ED3), a single SS-bonded protein can be irreversibly trapped into a misfolded species through the formation of its sole intramolecular SS-bond. The misfolded species had a much lower solubility than the native one at pHs higher than about 7, and circular dichroism measurements clearly indicated that its secondary structure content was different from the native species. Furthermore, the peaks in the Heteronuclear Single Quantum Correlation spectroscopy (HSQC) spectrum of DEN4 ED3 from the supernatant fraction were sharp and well dispersed, reflecting the beta-sheeted native structure, whereas the spectrum of the precipitated fraction showed broad signals clustered near its center suggesting no or little structure and a strong tendency to aggregate. The two species had distinct biophysical properties and could interconvert into each other only by cleaving and reforming the SS-bond, strongly suggesting that they are topologically different. This phenomenon can potentially happen with any single SS-bonded protein, and our observation emphasizes the need for assessing the conformation and biophysical properties of bacterially produced therapeutic proteins in addition to their chemical purities.

Biomolecules, Vol. 9, Pages 249: Bioevaluation of Ranatuerin-2Pb from the Frog Skin Secretion of Rana pipiens and Its Truncated Analogues Antimicrobial peptides (AMPs) are considered as a promising agent to overcome the drug-resistance of bacteria. Large numbers of AMPs have been identified from the skin secretion of Rana pipiens, including brevinins, ranatuerins, temporins and esculentins. In this study, the cDNA precursor of a broad-spectrum antimicrobial peptide, ranatuerin-2Pb, was cloned and identified. Additionally, two truncated analogues, RPa and RPb, were synthesised to investigate the structure-activity relationship of ranatuerin-2Pb. RPa lost antimicrobial activity against Candida albicans, MRSA, Enterococcus faecalis and Pseudomonas aeruginosa, while RPb retained its broad-spectrum antimicrobial activity. Additionally, ranatuerin-2Pb, RPa and RPb demonstrated inhibition and eradication effects against Staphylococcus aureus biofilm. RPb showed a rapid bacterial killing manner via membrane permeabilization without damaging the cell membrane of erythrocytes. Moreover, RPb decreased the mortality of S. aureus infected Galleria mellonella larvae. Collectively, our results suggested that RPb may pave a novel way for natural antimicrobial drug design.

Biomolecules, Vol. 9, Pages 248: Melanins of Inonotus Obliquus: Bifidogenic and Antioxidant Properties Extracts and melanins from Inonotus obliquus are widely used in medicine due to their high antioxidant properties. This study is dedicated to define the influence of the physicochemical and antioxidant properties of Inonotus obliquus melanins and their bifidogenic effects on Bifidobacterium bifidum 1 and Bifidobacterium animalis subsp. lactis. For this purpose, melanins precipitated from Inonotus obliquus aqueous extracts, obtained by a few methods, and separated melanin fractions by organic solvents were used. For the melanin physicochemical properties analysis spectrophotometry, electron paramagnetic resonance (EPR) spectroscopy and dynamic light scattering methods were applied. Melanins and their fractions difference in particle size and charge, antioxidant properties, and redox potential were revealed. It was shown that the redox potential, the size of melanin particles and the z-potential had maximum influence on bifidobacteria growth. The greatest activating effect on bifidobacteria was established by using melanin isolated from aqueous microwave extracts in concentrations of 10−13, 10−10, 10−5 g/cm3. The use of this melanin with antioxidant activity 0.67 ± 0.06 mg/g (expressed as ascorbic acid equivalent), and with redox potential −5.51 ± 2.22 mV as a prebiotic allowed the growth of Bifidobacterium bifidum 1 s to increase by 1.4 times in comparison with ascorbic acid by 24 h of cultivation.

Biomolecules, Vol. 9, Pages 247: Cross-Talk between Cadmium and Selenium at Elevated Cadmium Stress Determines the Fate of Selenium Uptake in Rice Cadmium (Cd) is a well-known metal imposing threats to human health, and it can be accumulated in polished rice over the permitted range of 0.2 mg kg−1 (GB 2762-2017). It has been reported that selenium (Se) application decreases Cd uptake. Se-rich diets have gained attention recently, but the potential of Se-rich rice in mitigating Cd stress needs further investigation. In this study, a pot experiment in the field was conducted to assess the influence of environmental factors and exogenous split application of Se on the nutritional status of rice under Cd stress. The results indicated that the increased fertilizer treatment in soil bulk linearly increased the metal content in rice grains. Approximately 50–70% of metal was recovered in rice tissues, while 5–20% of the metal that was applied leached down into the soil. A Se concentration of 0.4 mg kg−1 could significantly improve the total Se content in grain and mitigate Cd toxicity (1 mg kg−1) below the permitted range. Panicles and roots were more active for total Se accumulation in Se-rich and non-Se-rich rice, respectively. Polishing and milling operations can significantly reduce the Cd content, as rice bran in rice tissues accumulated most of the metal’s residues. The late matured rice cultivars consumed more heat units, and more metal contents were found in them. Collectively, it was found that Se can mitigate Cd toxicity, but the rice cultivation at T2 (high Cd; 2 mg kg−1 and Se; 1 mg kg−1) increased the metal uptake capability and health-risk index in polished rice, with its Se content heightened over permitted range of 0.04 to 0.30 mg kg−1 (GB/T 22499-2008). However, further molecular studies are required, in order to completely access the inverted Se accumulation behavior in rice tissues at high Cd soil stress.

Biomolecules, Vol. 9, Pages 246: Secretome Profiling Reveals Virulence-Associated Proteins of Fusarium proliferatum during Interaction with Banana Fruit Secreted proteins are vital for the pathogenicity of many fungi through manipulating their hosts for efficient colonization. Fusarium proliferatum is a phytopathogenic fungus infecting many crops, vegetables, and fruit, including banana fruit. To access the proteins involved in pathogen–host interaction, we used label-free quantitative proteomics technology to comparatively analyze the secretomes of F. proliferatum cultured with and without banana peel in Czapek’s broth medium. By analyzing the secretomes of F. proliferatum, we have identified 105 proteins with 40 exclusively secreted and 65 increased in abundance in response to a banana peel. These proteins were involved in the promotion of invasion of banana fruit, and they were mainly categorized into virulence factors, cell wall degradation, metabolic process, response to stress, regulation, and another unknown biological process. The expressions of corresponding genes confirmed the existence of these secreted proteins in the banana peel. Furthermore, expression pattern suggested variable roles for these genes at different infection stages. This study expanded the current database of F. proliferatum secreted proteins which might be involved in the infection strategy of this fungus. Additionally, this study warranted the further attention of some secreted proteins that might initiate infection of F. proliferatum on banana fruit.