Development strategy of non-GMO organism for increased hemoproteins in Corynebacterium glutamicum: a growth-acceleration-targeted evolution.

Heme, found in hemoproteins, is a valuable source of iron, an essential mineral. The need for an alternative hemoprotein source has emerged due to the inherent risks of large-scale livestock farming and animal proteins. Corynebacterium glutamicum, regarded for Qualified Presumption of Safety or Generally Recognized as Safe, can biosynthesize hemoproteins. C. glutamicum single-cell protein (SCP) can be a valuable alternative hemoprotein for supplying heme iron without adversely affecting blood fat levels. We constructed the chemostat culture system to increase hemoprotein content in C. glutamicum SCP. Through adaptive evolution, hemoprotein levels could be naturally increased to address oxidative stress resulting from enhanced growth rate. In addition, we used several specific plasmids containing growth-accelerating genes and the hemA promoter to expedite the evolutionary process. Following chemostat culture for 15 days, the plasmid in selected descendants was cured. The evolved strains showed improved specific growth rates from 0.59 h-1 to 0.62 h-1, 20% enhanced resistance to oxidative stress, and increased heme concentration from 12.95 µg/g-DCW to 14.22-15.24 µg/g-DCW. Notably, the putative peptidyl-tRNA hydrolase-based evolved strain manifested the most significant increase (30%) of hemoproteins. This is the first report presenting the potential of a growth-acceleration-targeted evolution (GATE) strategy for developing non-GMO industrial strains with increased bio-product productivity.

Development of a Normal Porcine Cell Line Growing in a Heme-Supplemented, Serum-Free Condition for Cultured Meat.

A key element for the cost-effective development of cultured meat is a cell line culturable in serum-free conditions to reduce production costs. Heme supplementation in cultured meat mimics the original meat flavor and color. This study introduced a bacterial extract generated from Corynebacterium that was selected for high-heme expression by directed evolution. A normal porcine cell line, PK15, was used to apply the bacterial heme extract as a supplement. Consistent with prior research, we observed the cytotoxicity of PK15 to the heme extract at 10 mM or higher. However, after long-term exposure, PK15 adapted to tolerate up to 40 mM of heme. An RNA-seq analysis of these heme-adapted PK15 cells (PK15H) revealed a set of altered genes, mainly involved in cell proliferation, metabolism, and inflammation. We found that cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), lactoperoxidase (LPO), and glutathione peroxidase 5 (GPX5) were upregulated in the PK15H heme dose dependently. When we reduced serum serially from 2% to serum free, we derived the PK15H subpopulation that was transiently maintained with 5-10 mM heme extract. Altogether, our study reports a porcine cell culturable in high-heme media that can be maintained in serum-free conditions and proposes a marker gene that plays a critical role in this adaptation process.

Methylococcus mesophilus sp. nov., the first non-thermotolerant methanotroph of the genus Methylococcus, from a rice field.

Strain 16-5T, a mesophilic methanotroph of the genus Methylococcus, was isolated from rice field soil sampled in Chungcheong Province, Republic of Korea. Strain 16-5T had both particulate and soluble methane monooxygenases and could only grow on methane and methanol as electron donors. Strain 16-5 T cells are Gram-negative, white to light tan in color, non-motile, non-flagellated, diplococcoid to cocci, and have the typical type I intracytoplasmic membrane system. Strain 16-5T grew at 18-38 °C (optimum, 27 °C) and at pH 5.0-8.0 (optimum, pH 6.5-7.0). C16 : 1 ω7c (38.8%), C16 : 1 ω5c (18.8%), C16 : 1 ω6c (16.8%) and C16 : 0 (16.9%) were the major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phospholipid were the major polar lipids. The main respiratory quinone was methylene-ubiquinone-8. Strain 16-5T displayed the highest 16S rRNA gene sequence similarities to other taxonomically recognized members of the genus Methylococcus, i.e. Methylococcus capsulatus TexasT (98.62%) and Methylococcus geothermalis IM1T (98.49 %), which were its closest relatives. It did, however, differ from all other taxonomically described Methylococcus species due to some phenotypic differences, most notably its inability to grow at temperatures above 38 °C, where other Methylococcus species thrive. Its 4.34 Mbp-sized genome has a DNA G+C content of 62.47 mol%, and multiple genome-based properties such as average nucleotide identity and digital DNA-DNA hybridization value distanced it from its closest relatives. Based on the data presented above, this strain represents the first non-thermotolerant species of the genus Methylococcus. The name Methylococcus mesophilus sp. nov. is proposed, and 16-5T (=JCM 35359T=KCTC 82050T) is the type strain.

Genomic and biological characteristics of a novel lytic phage, vB_MscM-PMS3, infecting the opportunistic zoonotic pathogen Mammaliicoccus sciuri.

Mammaliicoccus sciuri is an opportunistic zoonotic pathogen in humans and animals. We isolated the Mammaliicoccus phage vB_MscM-PMS3, which was also able to infect and lyse M. sciuri and M. lentus. The phage genome is a linear dsDNA that is 147,811 bp in length and contains 206 ORFs and three tRNA genes. It showed low genome coverage (< 17%) and sequence identity (< 91.3%) to other phage genomes. Phylogenetic analysis based on the whole genome and major capsid protein revealed that this phage clustered with members of the subfamily Twortvirinae of the family Herelleviridae, but it was distinctly separated from the other members, indicating its uniqueness.

Spatiotemporally controlled drug delivery via photothermally driven conformational change of self-integrated plasmonic hybrid nanogels.

BACKGROUND: Spatiotemporal regulation is one of the major considerations for developing a controlled and targeted drug delivery system to treat diseases efficiently. Light-responsive plasmonic nanostructures take advantage due to their tunable optical and photothermal properties by changing size, shape, and spatial arrangement. RESULTS: In this study, self-integrated plasmonic hybrid nanogels (PHNs) are developed for spatiotemporally controllable drug delivery through light-driven conformational change and photothermally-boosted endosomal escape. PHNs are easily synthesized through the simultaneous integration of gold nanoparticles (GNPs), thermo-responsive poly (N-isopropyl acrylamide), and linker molecules during polymerization. Wave-optic simulations reveal that the size of the PHNs and the density of the integrated GNPs are crucial factors in modulating photothermal conversion. Several linkers with varying molecular weights are inserted for the optimal PHNs, and the alginate-linked PHN (A-PHN) achieves more than twofold enhanced heat conversion compared with others. Since light-mediated conformational changes occur transiently, drug delivery is achieved in a spatiotemporally controlled manner. Furthermore, light-induced heat generation from cellular internalized A-PHNs enables pinpoint cytosolic delivery through the endosomal rupture. Finally, the deeper penetration for the enhanced delivery efficiency by A-PHNs is validated using multicellular spheroid. CONCLUSION: This study offers a strategy for synthesizing light-responsive nanocarriers and an in-depth understanding of light-modulated site-specific drug delivery.

Optical Detection of Copper Ions via Structural Dissociation of Plasmonic Sugar Nanoprobes.

Heavy metal ions are known to cause environmental pollution and several human diseases because of their inherent toxicity. Among them, Cu2+ is an essential element for the human body, but its continuous exposure and accumulation may cause adverse effects. Thus, copper ion levels in aquatic environments are strictly regulated by international standards. Herein, we demonstrate a simple optical method for detecting Cu2+ using plasmonic sugar nanoprobes (PSNs) composed of gold nanoparticles and polysaccharides. Gold precursors were reduced to nanoparticles and spontaneously embedded in the sugar-based polymeric network with the sulfated residues of carrageenan during the polymerization procedure. Owing to the abundant functional residues of PSNs and their affinity toward Cu2+, we observed the Cu2+-mediated preferential dissociation of the PSNs, resulting in absorbance spectral shifts and scattering shifts of the PSNs. Based on these plasmon band shifts, Cu2+ below the EPA regulation level of 20 µM can be easily detected by the optimized experimental condition. Additionally, the reaction mechanism between the PSNs and Cu2+ was elucidated by indepth spectroscopic analyses, which revealed that the increased binding of Cu2+ to the sulfate groups in the PSNs induces the eventual decomposition of the PSNs.

Colloidal Multiscale Assembly via Photothermally Driven Convective Flow for Sensitive In-Solution Plasmonic Detections.

The assembly of metal nanoparticles and targets to be detected in a small light probe volume is essential for achieving sensitive in-solution surface-enhanced Raman spectroscopy (SERS). Such assemblies generally require either chemical linkers or templates to overcome the random diffusion of the colloids unless the aqueous sample is dried. Here, a facile method is reported to produce 3D multiscale assemblies of various colloids ranging from molecules and nanoparticles to microparticles for sensitive in-solution SERS detection without chemical linkers and templates by exploiting photothermally driven convective flow. The simulations suggest that colloids sub 100 nm in diameter can be assembled by photothermally driven convective flow regardless of density; the assembly of larger colloids up to several micrometers by convective flow is significant only if their density is close to that of water. Consistent with the simulation results, the authors confirm that the photothermally driven convective flow is mainly responsible for the observed coassembly of plasmonic gold nanorods with either smaller molecules or larger microparticles. It is further found that the coassembly with the plasmonic nanoantennae leads to dramatic Raman enhancements of molecules, microplastics, and microbes by up to fivefold of magnitude compared to those measured in solution without the coassembly.

Protein kinase C-δ interacts with and phosphorylates ARD1.

Protein kinase C-δ (PKCδ) is a diacylglycerol-dependent, calcium-independent novel PKC isoform that is engaged in various cell signaling pathways, such as cell proliferation, apoptosis, inflammation, and oxidative stress. In this study, we searched for proteins that bind PKCδ using a yeast two-hybrid assay and identified murine arrest-defective 1 (mARD1) as a binding partner. The interaction between PKCδ and mARD1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated full-length mARD1 protein. The NetPhos online prediction tool suggested PKCδ phosphorylates Ser80 , Ser108 , and Ser114 residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKCδ. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI-TOF MS/MS confirmed that Ser80 and Ser108 were major phosphorylation sites. The alanine mutations of Ser80 and Ser108 abolished the phosphorylation of mARD1 by PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser80 of ARD1 was phosphorylated by PKCßI and PKCζ isotypes with the highest selectivity, while Ser108 and/or Ser114 were phosphorylated by PKCγ with activities comparable to that of the PKCδ isoform. Overall, these results suggest the possibility that PKCδ transduces signals by regulating phosphorylation of ARD1.

Production of juvenile masu salmon (Oncorhynchus masou) from spermatogonia-derived sperm and oogonia-derived eggs via intraperitoneal transplantation of immature germ cells.

No effective cryopreservation technique exists for fish eggs and embryos; thus, the cryopreservation of germ cells (spermatogonia or oogonia) and subsequent generation of eggs and sperm would be an alternative solution for the long-term preservation of piscine genetic resources. Nevertheless, in our previous study using rainbow trout, we showed that recipients transplanted with XY spermatogonia or XX oogonia produced unnatural sex-biased F1 offspring. To overcome these obstacles, we transplanted immature germ cells (XX oogonia or XY spermatogonia; frozen for 33 days) into the body cavities of triploid hatchlings, and the transplanted germ cells possessed a high capacity for differentiating into eggs and sperm in the ovaries and testes of recipients. Approximately 30% of triploid recipients receiving frozen germ cells generated normal salmon that displayed the donor-derived black body color phenotype, although all triploid salmon not receiving transplants were functionally sterile. Furthermore, F1 offspring obtained from insemination of the oogonia-derived eggs and spermatogonia-derived sperm show a normal sex ratio of 1:1 (female:male). Thus, this method presented a critical technique for practical conservation projects for other teleost fish species and masu salmon.

Dual Mode Rapid Plasmonic Detections of Chemical Disinfectants (CMIT/MIT) Using Target-Mediated Selective Aggregation of Gold Nanoparticles.

Chemical disinfectants such as 5-chloro-2-methylisothiazol-3(2H)-one/2-methyl-4-isothiazolin-3-one (CMIT/MIT) have been widely used in commercial products and humidifiers to prevent the growth of microorganisms. However, as continuous inhalation of CMIT/MIT is a fatal health risk, the concentration of its commercially available form is strictly regulated. Nonetheless, there are limited reports on effective methods for the quick and easy detection of CMIT/MIT. In this study, we have demonstrated rapid and convenient plasmonic methods for the dual-mode detection of CMIT/MIT using gold nanoplasmonic particles (GNPs) and understood the underlying molecular mechanism via additional analyses with microscopic and spectroscopic tools. In the presence of CMIT/MIT, the GNPs can rapidly aggregate due to molecular specific interactions with their capping agents and resultant reaction products. This target-mediated aggregation of the GNPs is represented by a visible color change of the solution from red to purple within just 3 min. By adjusting the reaction ratio between the CMIT/MIT and the GNPs, we could observe a marked color change at the regulation level (15 ppm) with naked eyes without any instruments. In addition, the concentration-dependent Raman spectral change in the reaction solution allows us to crosscheck the observed colorimetric responses both quantitatively and qualitatively based on molecular fingerprint spectra. Therefore, our detection protocol provides a powerful way to develop a high-throughput screening method to ensure that the level of the CMIT/MIT ingredients remains within the regulatory concentration.

A Low Frequency Vibration Energy Harvester Using ZnO Nanowires on Elastic Interdigitated Electrodes.

This paper presents a low frequency piezoelectric vibration energy harvester using ZnO nanowires on elastic interdigitated electrodes. The interdigitated electrodes are formed using electroplated Ni and have suspended parts at the edges that are elastic and deformable by applying external force. A spherical Ni ball is used as a proof mass, which transforms a low frequency mechanical vibration into the force applied to deform the elastic electrodes. The ZnO nanowires are grown selectively on the electrodes and can generate a piezoelectric potential when the elastic electrodes are deformed by the proof mass activated by the external mechanical vibration. The proposed operation concept is demonstrated using two different types of energy harvesters, which have simple suspended part and cantilever array structures added to the electrodes, respectively. The output voltage of the fabricated harvesters is measured using a vibration exciter at 6 Hz sinusoidal vibration with an acceleration of 0.5 g. Maximum output power of 12.8 pW and 18.8 pW was generated with a load resistance of 1 MΩ for the harvesters using the simple suspended structure and cantilever array, respectively.

Development of an alternative zebrafish model for drug-induced intestinal toxicity.

An evaluation of intestinal toxicity is important because the mucosal lining of the gastrointestinal tract is the first barrier for oral xenobiotics. Until now, a rat model has been recommended as the standard intestinal toxicity model and the Caco-2 cell line, originated from a human colon adenocarcinoma, has been used as an alternative to this model, but there are limitations regarding cost-effectiveness and the need for mimicry of the human system. In this study, we investigated whether zebrafish could be a valid alternative to rats and Caco-2 cells as an intestinal toxicity model. We focused on intestinal gene expression of cytochrome P450 3A65, oxidative stress, apoptosis, inflammation, and intestinal function. Reverse transcription-quantitative polymerase chain reaction analysis was conducted using three models: zebrafish, Sprague-Dawley rats and Caco-2 cells, and the transcript levels and patterns of indicator genes were analyzed in conjunction with histopathological changes. Our results suggested that representative intestinal toxicants, indomethacin, diclofenac and methotrexate, induced significant transcript level changes in marker genes such as CYP3A, inducible nitric oxide synthase, heme oxygenase 1, superoxide dismutase 1, glutathione peroxidase 1, BCL2 associated X, B-cell lymphoma 2, caspase 9, tumor protein p53, nuclear factor-κB, interleukin-1ß, tumor necrosis factor-alphaα and toll-like receptor 2 in the zebrafish model as in the rat and Caco-2 cells models. These results suggest that zebrafish model is sufficiently worth developing as an intestinal toxicity model that can replace or compensate the rat model or Caco-2 cell model.

Fabrication of Localized Surface Plasmon Resonance Sensor Based on Optical Fiber and Micro Fluidic Channel.

This paper proposes Fiber-Optic Localized Surface Plasmon Resonance (FO LSPR) sensor combined with a micro fluidic channel, which enables continuous supply of fluid for bio-reaction. The proposed method prevents degradation of the sensing performance due to changes in measurement conditions. The feasibility of the FO LSPR sensor with a micro fluidic channel was demonstrated by computational fluid dynamics (CFD) simulation. Also, the proposed method was assessed by measuring the output intensity of the FO LSPR sensor at various refractive index solutions. Finally, a prostate-specific antigen (PSA) immunoassay was performed to evaluate the possibility of the fabricated sensor system as a biosensor.

Novel microbial photobioelectrochemical cell using an invasive ultramicroelectrode array and a microfluidic chamber.

OBJECTIVE: To fabricate a novel microbial photobioelectrochemical cell using silicon microfabrication techniques. RESULTS: High-density photosynthetic cells were immobilized in a microfluidic chamber, and ultra-microelectrodes in a microtip array were inserted into the cytosolic space of the cells to directly harvest photosynthetic electrons. In this way, the microbial photobioelectrochemical cell operated without the aid of electron mediators. Both short circuit current and open circuit voltage of the microbial photobioelectrochemical cell responded to light stimuli, and recorded as high as 250 pA and 45 mV, respectively. CONCLUSION: A microbial photobioelectrochemical cell was fabricated with potential use in next-generation photosynthesis-based solar cells and sensors.

Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish.

Successful cryopreservation of spermatogonia in critically endangered Manchurian trout (Brachymystax lenok).

Because of the lack of cryopreservation techniques for fish eggs and embryos, cryopreservation of fish spermatogonia and subsequent generation of eggs and sperm would be an exit strategy for the long-term preservation of genetic resources. This study aimed to optimize cryoprotectants, cooling rates, and thawing temperatures for slow freezing of spermatogonia from endangered Manchurian trout (Brachymystax lenok). Whole testes were frozen with a cryomedium containing 1.3 M methanol, 0.2 M trehalose, and 10% egg yolk at a cooling rate of -1 °C/min and then stored in liquid nitrogen for 2 days. After thawing at 30 °C in a water bath, testicular cells from thawed testes were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted spermatogonia migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency did not significantly differ between frozen and fresh testes, demonstrating that Manchurian trout spermatogonia can be successfully cryopreserved in liquid nitrogen.

Long-term (5 years) cryopreserved spermatogonia have high capacity to generate functional gametes via interspecies transplantation in salmonids.

Although sperm cryopreservation is a powerful tool widely applicable in biodiversity conservation and broodstock management, cryopreservation of teleost eggs and embryos remains challenging. In the present study, we demonstrated that spermatogonia of rainbow trout (Oncorhynchus mykiss) cryopreserved for 5 years possessed the ability to differentiate into functional eggs or sperm in the gonads of triploid recipient masu salmon (Oncorhynchus masou). After cryopreservation for 5 years in liquid nitrogen, intraperitoneally transplanted spermatogonia migrated toward, and incorporated into, the gonads of xenogeneic recipients. The transplanted spermatogonia resumed spermatogenesis and oogenesis in male and female recipients, respectively, and differentiated into sperm or eggs within the gonads of male and female recipients at 2 years posttransplantation. The differentiated sperm and eggs generated normal rainbow trout representative of donor phenotypes. Thus, cryopreservation of spermatogonia is a powerful and reliable method for long-term preservation of fish genetic resources.

Generation of functional eggs and sperm from cryopreserved whole testes.

The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozen-thawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.

Ovotestis Isolation and Cryopreservation of Nesiohelix samarangae (Oriental Snail) as a Snail Model for Conserving Other Endangered Snail Species.

This study aimed to develop a cryopreservation system for the reproductive organs of Nesiohelix samarangae (oriental snail) to support the conservation of their species. The reproductive glands of N. samarangae are divided into numerous acini by acinar boundaries. Within each acinus, the presence of spermatogonia, spermatocytes, spermatids, and sperm were observed, indicating various stages of sperm development. The spermatocytes were irregular in shape and possessed large nuclei. Spermatids, on the other hand, were predominantly located within the lumen of the tissue and exhibited densely packed nuclei. Furthermore, sperm with tails attached were observed within the tissue. In order to preserve the oriental snail species, we utilized the vitrification method to freeze the reproductive organs. Comparing the two methods, it was observed that cryopreservation of ovotestis using 2% alginate encapsulation exhibited superior viability following thawing, surpassing the viability achieved with the non-encapsulated approach. In this study, the establishment of a cryopreservation system for the reproductive organs of the oriental snail not only contributes to the genetic conservation of the endangered snail species but also plays a role in maintaining genetic resources and diversity.

Data and videos for the comparison of thermal propagation and cycle performance of multiple lithium-ion batteries in air and insulating oil.

The propagation test of lithium-ion battery pack was conducted in an environment of air and insulating oil. The test results showed the difference in the phenomenon in which fire propagation to surrounding cells, when a cell composing a battery pack is thermal runaway in two environments. The temperature of the cells in the battery pack was measured during propagation test. A cycle test was also conducted to check whether there was an abnormality in cell performance immersed in insulating oil. The residual capacity and internal resistance, insulation resistance data of the cell are presented in the two environments.