Molecular cloning and Biochemical properties of GH-16 β-agarase from Gilvimarinus agarolyticus JEA5

Agar is complex polysaccharide founds in the cell walls of some red algae and up to 70 % of the algal cell wall can be agar polymers. Agar was formed by a mixture of two polysaccharides named agarose and agaropectin. Agarose can be hydrolyzed by α-agarase (E.C. 3.2.1.158) and by β-agarase (E.C. 3.2.1.81); the former cleaves the α-1, 3 linkage of agarose to generate agaro-oligosaccharides, and the latter cleaves the β-1,4 linkage to generate neoagaro-oligosaccharides. Agarases have been isolated from many sources, including seawater, marine sediments, marine algae, marine mollusks, fresh water and soil. Recently, Givimarinus chinensis, G. polysacchalyticus, G. agarilyticus were identified and their agarolytic activity also reported. However, there are no report published that molecular and functional characterization of agarase from Givimarinus genus. In this study, we first report molecular characterization and biochemical properties of agarase from Gilvimarinus genus. Please click Additional Files below to see the full abstract

Biochemical properties of a novel neoagarotriose-producing β-agarase from Gilvimarinus agarolyticus JEA5

An agar degrading bacterium was isolated from seawater, collected from the east coast of Jeju Island, republic of Korea and identified as Gilvimarinus agarolyticus JEA5. The β-agarase gene from Gilvimarinus agarolyticus JEA5 (rGaa16B) was identified from draft genome sequence by BLAST. Gaa16B has 1800 bp of open reading frame encoding 636 amino acids (aa), and include glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding module 6 (CBM6). The Gaa16b was cloned and overexpressed as a MBP-fusion recombinant β-agarase (without signal peptide and two CBM6) in E. coil. rGaa16B showed highest activity at 60°C and pH 7. After incubation at 45OC for 90 min, rGaa16B showed over than 95% of its initial activity. rGaa16B were enhanced in the presence of MnCl2, KCl2, MgCl2, FeSO4. rGaa16B showed 2112.1 unit/mg in the presence of 2.5 mM of MnCl2. rGaa16B produce mainly neoagartetraose (NA4) and neoagarobiose (NA2). Interestingly, we observed neoagartriose (NA3) from hydrolytic products of rGaa16B. LC/Mass analysis was performed to confirm the hydrolytic products containing neoagarotriose. We found three different hydrolytic products which showed 324.28, 468.41, 630.55 Da of molecular weight, respectively. Please click Additional Files below to see the full abstract

Synergistic effect of acetyl xylan esterase on xylanase reaction originated from Ochrovirga pacifica

Acetyl xylan esterase plays an important role in complete enzymatic hydrolysis of lignocellulosic materials into fermentable sugars. It hydrolyzes ester linkages of acetic acid in xylan polysaccharide and supports to enhance the activity of xylanase. This study was conducted to recognize and overexpress the acetyl xylan esterase gene found from Ochrovirga pacifica strain S85 which was isolated from Chuuk state, Micronesia. The genome sequence was analyzed with genome sequencer-FLX and acetyl xylan esterase gene (Axe) was detected. The gene had an open reading frame of 864 bp encoding a polypeptide of 287 amino acids. Theoretical molecular mass and isoelectric point (pI) were 32 kDa and 5.9, respectively. The deduced amino acid sequence of the Axe showed 35.1% similarity with both endo-1,4-β-xylanase B from Robiginitalea biformata HTCC2501. The mature protein displayed the catalytic residues classically found in enzymes belonged to GH16 family. Axe was cloned into pET11a vector and recombinant protein was expressed in E. coli BL21 (DE3), purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. Commercial xylanase activity was tested after treatment of recombinant acetyl xylan esterase (rAXE) to birchwood xylan substrate. The xylanase activity of rAXE treated sample was about 2 times higher than xylanase only treated sample. Please click Additional Files below to see the full abstract

Recombinant protein production in Escherichia coli by combining of signal peptide originated from Bacillus subtilis

We isolated chitosanase secreting B. subtilis CH2 and identified the chitosanase nucleotide sequence. Analyzed the sequence showed that it consisted of 813 bp, including 87 bp signal sequence. The signal sequence leads the target protein to the cell-membrane of the B. subtilis CH2 and then secret the chitosanase out of the cell. The signal peptide showed 6 amino acids deletion compared to other B. subtilis chitosanase signal peptides. The chitosanase sequence including signal peptide was cloned into pET11a vector without fusion and expressed in E. coli BL21(DE3). The expressed chitosanase in E. coli showed two distinct bands which represent the pro-chitosanase in cytoplasm and mature chitosanase in periplasm. Time frame induction and results showed that muture chitosanase was increased. Subsequently, we linked this chitosanase signal sequence in front of B. subtilis CH2 xylanase and human superoxide distimutase 1 (hSOD1) sequences, and expressed it in E. coli BL21(DE3). The recombinant xylanase and hSOD1 moved to periplasmic space with high efficiency. This signal sequence is useful for bio-medical protein production in E. coli. Please click Additional Files below to see the full abstract

Practical photoacoustic tomography: Realistic limitations and technical solutions

This article offers a perspective on photoacoustic tomography (PAT) under realistic scenarios. While PAT has gained much attention in preclinical and clinical research, most early works used image reconstruction techniques based on ideal assumptions, and thus these techniques may not be fully effective in real environments. In this work, we consider such non-ideal conditions as a limited view, limited bandwidth, lossy medium, or heterogeneous medium. More importantly, we use k-Wave simulation to numerically evaluate the effects of these limiting factors on various image reconstruction algorithms. Then, to enable more reliable PAT image reconstruction, we introduce recent techniques for mitigating each of the limiting conditions. We seek to emphasize the importance of working within these realistic limitations, and we encourage researchers to develop compensating solutions that advance PAT's translation to real clinical environments.11Nsciescopu

Comparison of various photoacoustic imaging reconstruction algorithms under realistic scenarios: a simulation study

Undoubtedly, photoacoustic tomography (PAT) is a promising technique unveiling physiological information in the biomedical imaging field. However, targeting optically/acoustically non-uniform biological tissues and receiving signals with finite aperture/band-limited ultrasonic transducers have been practical limitations that hinder the clinical application of PAT. This study closely analyzes the effects of four main limiting factors named limited view (LV), limited bandwidth (LB), lossy medium (LM), and heterogeneous medium (HM) and compared the adverse effects given from the four factors. First, radiofrequency (RF) data was generated assuming a realistic situation using k-Wave, a MATLAB-based wave propagation-simulating tool. A single 4 mm-sized circular target was used, and physical parameters of the detector array were designed referring to a commercial linear probe (GE9LD, General Electric). Supposing double-aperture, full-band signal reception from the lossless, homogeneous medium as a baseline, RF data were acquired under a successive accumulation of the limiting factors in order of LV, LB, LM, and HM. Second, the obtained RF datasets were reconstructed with 7 representative beamforming algorithms; delay and sum (DAS), delay multiply and sum, pth root DAS, minimum variance, filtered back-projection, frequency-domain reconstruction, and time reversal. Among obtained images, common graphical features for each limiting factor were specified. Lastly, we quantitatively assessed the similarity between the image and the actual target with peak signal-to-noise ratio and structural similarity as performance indices. As a result, the characteristic features from each limiting factor were commonly identified on the reconstructed image, and the performance indices sequentially degraded following the sequence of practical conditions. ? COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.1

Functional photoacoustic imaging: from nano- and micro- to macro-scale

Abstract Functional photoacoustic imaging is a promising biological imaging technique that offers such unique benefits as scalable resolution and imaging depth, as well as the ability to provide functional information. At nanoscale, photoacoustic imaging has provided super-resolution images of the surface light absorption characteristics of materials and of single organelles in cells. At the microscopic and macroscopic scales. photoacoustic imaging techniques have precisely measured and quantified various physiological parameters, such as oxygen saturation, vessel morphology, blood flow, and the metabolic rate of oxygen, in both human and animal subjects. This comprehensive review provides an overview of functional photoacoustic imaging across multiple scales, from nano to macro, and highlights recent advances in technology developments and applications. Finally, the review surveys the future prospects of functional photoacoustic imaging in the biomedical field

New contrast agents for photoacoustic imaging and theranostics: Recent 5-year overview on phthalocyanine/naphthalocyanine-based nanoparticles

The phthalocyanine (Pc) and naphthalocyanine (Nc) nanoagents have drawn much attention as contrast agents for photoacoustic (PA) imaging due to their large extinction coefficients and long absorption wavelengths in the near-infrared region. Many investigations have been conducted to enhance Pc/Ncs' photophysical properties and address their poor solubility in an aqueous solution. Many diverse strategies have been adopted, including centric metal chelation, structure modification, and peripheral substitution. This review highlights recent advances on Pc/Nc-based PA agents and their extended use for multiplexed biomedical imaging, multimodal diagnostic imaging, and image-guided phototherapy. (C) 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).11Nsciescopu

Switchable preamplifier for dual modal photoacoustic and ultrasound imaging

Photoacoustic (PA) imaging is a high-fidelity biomedical imaging technique based on the principle of molecular-specific optical absorption of biological tissue constitute. Because PA imaging shares the same basic principle as that of ultrasound (US) imaging, the use of PA/US dual-modal imaging can be achieved using a single system. However, because PA imaging is limited to a shallower depth than US imaging due to the optical extinction in biological tissue, the PA signal yields a lower signal-to-noise ratio (SNR) than US images. To selectively amplify the PA signal, we propose a switchable preamplifier for acoustic-resolution PA microscopy implemented on an application-specific integrated circuit. Using the preamplifier, we measured the increments in the SNR with both carbon lead and wire phantoms. Furthermore, in vivo whole-body PA/US imaging of a mouse with a preamplifier showed enhancement of SNR in deep tissues, unveiling deeply located organs and vascular networks. By selectively amplifying the PA signal range to a level similar to that of the US signal without contrast agent administration, our switchable amplifier strengthens the mutual complement between PA/US imaging. PA/US imaging is impending toward clinical translation, and we anticipate that this study will help mitigate the imbalance of image depth between the two imaging modalities. © 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.11Nsciescopu

Optimising the DPPH Assay for Cell-Free Marine Microorganism Supernatants

Antioxidants prevent ageing and are usually quantified and screened using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. However, this assay cannot be used for salt-containing samples, such as the cell-free supernatants of marine microorganisms that are aggregated under these conditions. Herein, the DPPH solvent (methanol or ethanol) and its water content were optimized to enable the analysis of salt-containing samples, aggregation was observed for alcohol contents of >70%. The water content of methanol influenced the activities of standard antioxidants but did not significantly affect that of the samples. Based on solution stability considerations, 70% aqueous methanol was chosen as the optimal DPPH solvent. The developed method was successfully applied to the cell-free supernatants of marine bacteria (Pseudoalteromonas rubra and Pseudoalteromonas xiamenensis), revealing their high antioxidant activities. Furthermore, it was concluded that this method would be useful for the screening of marine microorganism–derived antioxidants, which also has numerous potential applications, such as salt-fermented foods