Earticle

In vivo molecular imaging can measure the average kinetics and movement routes of injected cells through the body. However, owing to non-specific accumulation of the contrast agent and its efflux from the cells, most of these imaging methods inaccurately estimate the distribution of the cells. Here, we show that single human breast cancer cells loaded with mesoporous silica nanoparticles concentrating the 68Ga radioisotope and injected into immunodeficient mice can be tracked in real time from the pattern of annihilation photons detected using positron emission tomography, with respect to anatomical landmarks derived from X-ray computed tomography. The cells travelled at an average velocity of 50 mm s−1 and arrested in the lungs 2–3 s after tail-vein injection into the mice, which is consistent with the blood-flow rate. Single-cell tracking could be used to determine the kinetics of cell trafficking and arrest during the earliest phase of the metastatic cascade, the trafficking of immune cells during cancer immunotherapy and the distribution of cells after transplantation.

α-Synuclein has been proposed to play a key pathogen in Parkinson's disease (PD). Especially, the abnormal accumulation and aggregation of the misfolded α-Synuclein protein is the neuropathological hallmark of all α-Synucleinopathies, including Parkinson's disease. Netrins (netrin-1, netrin-3, and netrin-4) are secreted proteins, which are laminin-related and involved in axon guidance function and cell survival molecular pathway. Remarkably, only netrin-1 is highly expressed in healthy adult substantia nigra brain region and inversely correlates with the expression of α-Synuclein, which led us to investigate the impact of the molecular interaction between α-Synuclein and netrin-1 in dopaminergic neuron fate. Here we show that netrin-1 and α-Synuclein directly interact in pre-formed fibrils (PFFs) generation test and real time binding assay. Strikingly, we observed that the level of netrin-1 was significantly decreased in SNCA Tg mouse brain with dopaminergic cell death, which matched the analysis result from human PD patients database. Our finding suggest that netrin-1 deprivation triggers dopaminergic neuronal cell death with α-Synuclein aggregation during aging process and netrin-1 can be the promising therapeutic or diagnostic molecule in α-Synucleinopathies.

Maltodextrin in specific length is of great interest because of its wide industrial applications. Previously, we have proven that Pyrococcus furiosus thermostable amylase (PFTA) produces high purity maltoheptaose (G7) from β-cyclodextrin (CD) at early reaction time. However, the production of pure G7 is a cost and time-consuming process. Therefore, this work has designed an optimal continuous process for pure G7 production from β-CD using PFTA with immobilization in a packed-bed reactor (PBR). The optimal conditions determined using the response surface method for the maximized G7-purity of 96.3% were predicted at 4.7 ml/min of flow rate, 1.1% substrate, and 48.7 °C. As a result, the immobilized PFTA in PBR retained 85% of the initial activity after six cycles of 24 h intervals, and the product purity was unchanged. Also, maltohexaose (G6) and maltooctaose (G8) at high purity (92-97%) were produced by continuously supplying CDs to the PFTA-PBR under the same optimized condition of G7. In addition, a new method based on preparative reversed-phase liquid chromatography was developed to purify maltodextrins in specific lengths (DP 3-26) with high purity from the reaction mixture of G6-G8 substrates with Thermotoga maritima maltosyltransferase. On the other hand, novel G7-based sugar esters were also synthesized and investigated for emulsifying properties. As a result, G7-fatty acids (C10–C16) esters were produced with a high conversion yield (90-95%) under the optimal conditions (10% of DMSO, 0.2 of G7/ fatty acids molar ratio, 33.5 U of immobilized Candida antarctica lipase B) for 24 h of incubation at 60 °C. Notable, G7-palmitate (PA) exhibited emulsifying properties better than commercial sucrose-PA ester, especially in acidic conditions. It is feasible that the number of the hydroxyl groups and the size of G7 moiety provide that distinctive property of G7-PA. Therefore, applications of G7-PA in foods, cosmetics, and drug delivery under extreme pH conditions are promising.

Glycans, also known as carbohydrates, saccharides, or simply as sugars, play central roles in many biological processes and have useful properties in a variety of applications from biomarker discovery to therapeutics. However, glycans have received little attention from the research community due to the lack of tools to probe their often complex structures and properties. Recently, advances in glycomic and glycoproteomic methods along with the development of technology in mass spectrometry allow us to better understand the role of the glycome in biological processes in areas as broad as biotherapeutics, infection, brain, and cancer. The challenges are no longer scientific but technical. In this lecture, we will cover the glycomic methods that enables in-depth structural characterization of sugar chains and the glycoproteomic methods for obtaining comprehensive site-specific glycosylation information using mass spectrometry-based platforms. In addition, glyco-informatics and database that automate interpretation of glycome data, which are key to the broader implementation of glycoscience, will be discussed.

The hexosamine biosynthesis pathway (HBP) is an important branch of glycolysis to induced protein O-GlcNAcylation and acts as a glucose sensing in many types of cells. Glucosamine (alternatively named 2-amino-2-deoxy-d-glucose), an intermediate metabolite of the HBP, bypasses glycolysis for directly entering HBP. Since glucosamine stimulates glycosaminoglycan production, it has been extensively studied in the context of osteoarthritis and joint pain. Although several clinical studies conducted on oral supplementation of glucosamine demonstrate its beneficial effects in the progression of osteoarthritis and cartilage renewal, the efficacy of glucosamine for osteoarthritis remains controversial. Furthermore, exogenous glucosamine application exhibits therapeutic effects by increasing protein O-GlcNAcylation. Glucosamine regulates glucose metabolism, insulin resistance, diabetic complications and lipid metabolism. Additionally, glucosamine exerts several beneficial effects on oxidative stress, inflammation, cell proliferation, apoptosis, and growth factor signaling. More recent studies demonstrate that exogenous glucosamine can improve the neurodegeneration and cognitive function induced by sleep deprivation, circadian cycle disruption and hypoxia, emerging glucosamine as a therapeutic benefit for neuronal dysfunction. However, whether increased O-GlcNAcylation by exogenous glucosamine is protective to disease or promotes progression of the disease or is still a matter of debate. Due to the abundance and ubiquitous nature of the HBP and protein O-GlcNAcylation in mammalian cells and potential effects to other signaling pathways, exogenous glucosamine may induce various effects depends on doses, metabolic status, cell and tissue types and species.

High-throughput single-cell RNA sequencing (scRNA-seq) technology provide opportunities to identify the entire cellular and molecular difference based on cell-to-cell heterogeneity in gene expression. This study demonstrates the feasibility and utility of scRNA-seq in transgenic Nicotiana tabacum (N. Tabacum) leaves expressing anti-rabies virus mAb(P) SO57 and provides generation gene expression map of the plant leaf at single-cell resolution. Protoplasts isolated from the N. Tabacum leaves at non-transgenic and transgenic plants, and the transcript levels of > 20,000 genes were analyzed in 24,000 single cells from non-transgenic and transgenic plants. Total of 9 putative cell clusters and the cluster-specific marker genes were identified. We performed cell clustering analysis to determine which cell clusters were mainly synthesis exogenous genes. In conclusion, the high-resolution single cell atlas of gene expression shows the developmental landscape of N. tabacum leaf and exogenous gene expression patterns in each clusters.

Protein S-nitrosylation and O-GlcNAcylation are important posttranslational modifications. Biological relevance between S-nitrosylation and O-GlcNAcylation remains unclear. Our goal is to identify the crosstalk between S-nitrosylation and O-GlcNAcylation during heat-shock. Ex vivo heat-shock on mouse tissues together with in vitro heat-shock on culture cells was performed and global levels of S-nitrosylation(SNO) and O-GlcNAcylation were analyzed with Biotin-switch assay and RL2 immunoblots. We found that heat-shock induces hypo-SNO in parallel with hyper-O-GlcNAcylation. Inverted induction of hypo-SNO and hyper-O-GlcNAcylation is globally progressed in a time-dependent manner. Furthermore, heat-shock ubiquitously facilitates S-denitrosylation (SdeNO) of endogenous SNO-proteins including SNO-OGT, SNO-Hsp70, SNO-Hsp90, SNO-Akt, and SNO-actin. Particularly, SdeNO of SNO-OGT leads to enhanced OGT activity. These results provide mechanistic evidence that heat shock induces SdeNO in SNO-OGT, leading to upregulation of OGT activity, resulting in hyper-O-GlcNAcylation.

Post-translational modifications, including O-GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. O-GlcNAcylation is PTMs on proteins that rapidly respond to extracellular stimuli. Although several molecular targets of O-GlcNAcylation associated with pathogen-induced innate immune responses have been reported, it is poorly understood how a host modulates intracellular O-GlcNAcylation to optimize pathogen-induced innate immune response and necroptotic cell death. Here we show that RNA virus infection or gram-negative bacterial infection induces a decrease in O-GlcNAcylation levels in several types of cells. Especially, we show that epithelial cells reduce OGT expression during the late phases of Sendai virus infection via RIG-I-like receptor activation. Downregulation of OGT expression led to a decrease in O-GlcNAcylation of MAVS during RNA virus infection. Moreover, we identified a heavily O-GlcNAcylated serine-rich region between amino acids 249-257 of the mitochondrial antiviral signaling protein (MAVS) and that it inhibits the formation of MAVS aggregates which are crucial for activating MAVS. O-GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-β. On the other hand, host cells suffer from sepsis, a serious inflammatory disease in response to pathogenic infection with Escherichia coli or staphylococcus. In this study, we demonstrate that O-GlcNAcylation suppresses sepsis-induced blood cell dysfunction. Moreover, we observe that the host accelerates its own immune response by reducing cellular O-GlcNAcylation in order to fight pathogen infections. Taken together, we suggest that it is essential to maintain host O-GlcNAc homeostasis in order to avoid excessive or abnormal immune responses.

Several advances in anticancer therapy have demonstrated significant improvements in clinical outcomes, and adoptive cell therapy has emerged as a type of immunotherapy that can modulate immune responses by transferring engineered immune cells. However, it remains a challenge because only a small percentage of respondents have responded. Three-dimensional (3D) in vitro models of the tumor microenvironment (TME) have the potential to provide a platform for assessing and predicting responses to therapy. Here, we propose an in vitro 3D tumor model with clusters of colorectal cancer (CRC) cells around perfusable vascular networks to evaluate immune cell-mediated cytotoxicity against cancer cells. A 3D injection-molded co-culture model consists of 28 microwells where identical vascularized cancer models can be formed in separate wells. It allows robust hydrogel patterning, resulting in high-throughput experiments. Compared to the polydimethylsiloxane (PDMS)-based microfluidic devices, our devices allowed a greater number of experiments to be conducted. A permeability test was also conducted to confirm the characteristics of the tumor vasculature. Primary natural killer (NK) cells were introduced into a tumor vascularized network and monitored using live-cell imaging. The extravasation, migration, and cytotoxic activity of six types of CRC cell lines were examined. Based on the consensus molecular subtypes (CMS) of CRC with distinct immune responses, CMS1 cancer cells were most susceptible to NK cell cytotoxicity. This study indicates the potential of our vascularized tumor model in assessing the responses to adoptive cell therapy by understanding the various steps involved in the immune response.

A diverse antibody repertoire in B cells is generated through the various processes such as V(D)J rearrangement, class switching recombination, and somatic hypermutation at the immunoglobulin(Ig) gene loci. During early B cell development, these Ig genes are initially assembled into functional transcription units by V(D)J rearrangement, a fundamental mechanism for antibody diversity. Several lymphoid-specific factors are recognized to be involved in these recombination processes which can facilitate long-distance interaction of Ig gene segments in germ-line DNA. Proteins within the nucleocytoplasmic compartment are dynamically modified by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc), derived from the end-product uridine 5’-phosphate (UDP)-GlcNAc of the hexosamine biosynthetic pathway (HBP), to serine and threonine hydroxyl groups. Modifications by O-GlcNAcylation can modulate a protein function by altering stability, subcellular localization, protein–protein interaction, phosphorylation status and/or DNA binding ability. Despite the essential role of O-GlcNAcylation in regulating protein function, the linked mechanisms of O-GlcNAcylation and V(D)J recombination remain largely unknown. Here, we show that the long-range interaction in Ig gene loci spanning >3MB is defective during V(D)J recombination in B cells from bone marrow of mice administered with O-GlcNAc inhibitors. Restricted V(D)J recombination is similarly confirmed in a mouse model with nutritional deficiency induced by limiting calorie consumption to 50%. However, defected rearrangement is restored by applying the O-GlcNAc-inducible drugs even during caloric restriction. Significantly, several factors involved in V(D)J recombination such as YY1, SMC1, and SMC3, which can regulate long-range interactions by binding to various positions in the Ig gene loci, are identified as proteins directly modified by O-GlcNAcylation. In addition, DNA binding ability of these proteins is shown to be disrupted once O-GlcNAcylation is inhibited. These results unravel a previously unidentified link between O-GlcNAcylation and V(D)J rearrangement and potentially suggests a crucial role for O-GlcNAc signaling in representing nutritional state to affect antibody diversity.

Shigellosis caused by Shiga toxin (Stx)-producing Shigella dysenteriae serotype 1 or Stx-producing Escherichia coli (STEC) continues to be a major public health threat and is a particular concern because of the potential to develop life-threatening extra-intestinal complications such as acute renal failure (hemolytic uremic syndrome; HUS) and CNS complications such as seizures, paralysis, and death. Once Shiga toxins (Stxs) are internalized following the toxin-specific receptor (globotriaosylceramide, Gb3) binding on host cellular surface, they are trafficked into the Golgi apparatus and to the ER in retrograde manner to enter the host cell cytosol, leading to various host cellular responses including protein synthesis inhibition, apoptosis through ER stress, autophagy and inflammation associated with the toxin-containing exosome secretion. The distinct investigations on host cell signaling responses activated by Stxs as multi-functional proteins were performed to identify novel targets for intervention in pathogenesis by employing various in-vitro/in vivo models including human renal three dimensional spheroid or organoid. Different forms of circulating Stx2a have been described: either alone as Stx2a in microvesicles shedding from blood cells, or as Stx2a associated with human neutrophil. In addition to this, alternative non canonical receptors to Stx have been proposed whereby the toxin can bind to a host cell. Moreover, many studies present compelling and strong evidences about therapeutic applications to target particular diseases such as tumors by engineering the toxins. In this meeting, we will cover diverse aspects of interactions between host cells mainly focusing on renal tissue, Shiga toxins, and the bacteria that produce the toxins.

Host tp53 mutations are frequently found during the early stages of colitis-associated colorectal cancer (CAC), but whether such mutations induce gut microbiota dysbiosis and chronic intestinal inflammation that contributes to the development of CAC, remains unknown. We found that zebrafish tp53 mutant larvae exhibited elevated intestinal inflammation, by monitoring the NFκB activity in the mid-distal intestines of zebrafish larvae using an NFκB:EGFP transgenic reporter line in vivo as well as neutrophil infiltration into the intestine. This inflammation was due to dysbiotic gut microbiota with reduced diversity, revealed using both 16S rRNA amplicon sequencing and a germfree larva model. In this dysbiosis, Aeromonas spp. were aberrantly enriched as major pathobionts and exhibited the capacity for aggressive colonization in tp53 mutants. Importantly, the ex-germfree experiments supported the causality of the host tp53 mutation for inducing the inflammation. Transcriptome and high performance liquid chromatography analyses of the host gastrointestinal tracts identified dysregulated sialic acid (SA) metabolism concomitant with increased host Neu5Gc levels as the key determinant of aberrant inflammation, which was reversed by the sialidase inhibitors oseltamivir and Philippin A. These results demonstrate a crucial role for host tp53 in maintaining symbiosis and immune homeostasis via SA metabolism. Disturbed SA metabolism via a tp53 mutation may be exploited by specific elements of the gut microbiome, eliciting both dysbiosis and inflammation. Manipulating sialometabolism may therefore provide an efficacious therapeutic strategy for tp53 mutation-induced dysbiosis, inflammation, and, ultimately, related cancers.

Understanding glycan-protein interactions provide valuable information on biological processes in living organisms. However, many roles of glycans in biology are still veiled. The primary reason might be the limitation of access to a glycan library with diverse structures, which hampers extensive studies on molecular interactions of glycans. Here, a functional glycan chip combined with on-chip enzymatic glycosylation was developed to prepare complex glycan sources and apply glycan-involved applications simultaneously. GM1 pentasaccharide and Globo H hexasaccharide-related complex glycans were successfully synthesized on a chip through consecutive glycosyltransferase reactions along with small amounts of enzymes and donors, without any additional processes. Biosynthesized complex glycans were demonstrated to provide information on glycan-related interactions. Thus, the proposed on-chip glycan biosynthesis system can provide a new direction toward obtaining complex glycan sources and complex glycan-involved applications such as glycan-protein interaction analysis and glycan biomarker screening.

Improved methods are required for glyco-characterization of biopharmaceuticals and comparison between their corresponding biologics and biosimilar, where they have multiple glycosylation sites such as Etanercept of a fusion protein. In order to enhance sensitivity of discovery and minimize the sample loss, the combination of a TMT labeling method and fractionation using high flow HPLC has contributed to detect low amount of peptide in data dependent mode of LC-MS/MS. Here, we report a new method combined with TMT labeling, fractionation using ZIC-HILIC HPLC, LC-MS/MS using HCD triggered CID/EThcD, and computational software for identification of site-specific N- and O-glycopeptide and comparison of their corresponding biologics and biosimilar. Of three different Etanercept, such as Enbrel (biologics), Etoloce and Eucept biosimilars), a total of 115 N- and O- glycopeptides were identified and quantified using TMT-11plex labeling including triplicates and 22 ZIC-HILIC fractionation in three and 15 of N- and O- glycosylation sites, respectively. Two O-glycopeptides in different O-glycosylation site and seven core 2 type of O-glycopeptides were first reported. In particular, three kinds of O-glycopeptides with NeuGC which could be a foreign agent of immune response. In depth of quantification, our strategy shows site-specific similarity is quite not similar between biologics and biosimilar.

The process for the synthesis of storage polysaccharides, glycogen and starch, respectively, in bacteria and plants, takes place with ADP-glucose(ADP-Glu) as the glucosyl donor for the elongation of the α-1,4-glucosidic chain. In these organisms, ADP-Glc and inorganic pyrophosphate (PPi) are synthesized from ATP and Glc-1-P in a reaction catalyzed by ADP-glucose pyrophosphorylase(AGP). To identify AGP isoforms essential for this biosynthetic process in sink and source tissues of rice plants, we analyzed the rice AGP gene family which consists of two genes, OsAGPS1 and OsAGPS2, encoding small subunits and four genes, OsAGPL1, OsAGPL2, OsAGPL3 and OsAGPL4, encoding large subunits of this enzyme heterotetrameric complex. Subcellular localization, expression profile and functional studies of AGP subunits revealed that OsAGPS2b, an alternative spliced form of OsAGPS2 and OsAGPL2 are endosperm-specifically expressed, localized in the cytosol. Analysis of osagps2 and osagpl2 mutants revealed that a lesion of one of the two cytosolic isoforms, OsAGPL2 and OsAGPS2b, causes a shrunken endosperm due to a remarkable reduction in starch synthesis. Interestingly, they exhibited reduced not only the level of starch but also that of the major seed storage proteins and protein modifying proteins such as protein disulfide isomerase and the dnaK-type endoplasmic reticulum chaperone BiP. They also had much lower levels of malate dehydrogenase and alanine aminotransferase, which likely function in maintaining an adequate carbohydrate supply into storage proteins of seed endosperms under low oxygen condition. Additionally, PPi dependent isoforms, PPi-dependent fructose-6-phosphate 1-phosphotransferase and PPi-dependent pyruvate-orthophosphate dikinase (PPDK) are preferentially expressed in seed endosperm. Surprisingly, the knockout mutants of all these genes show opaque endosperms. These results strongly support the idea that the PPi produced by AGP in the cytosol of seed endosperm, not in the plastid, is necessary not only for the starch synthesis but for the supply of PPi, an alternative energy currency of ATP to produce storage proteins.

Fast and high-throughput validation of protein products is of interest in biopharmaceutical industry. Top-down mass spectrometry (TDMS) allows for directly measuring of the intact form of proteins (i.e., proteoform) which have genetic mutations, alternative RNA splicing events and post-translational modifications. We’ve developed a new interface, called SampleStream, enabling fast and high-throughput measurement of proteoforms coupling to TDMS. The SampleStream interface consists of a fluidic channel that incorporates a molecular weight cutoff (MWCO) membrane to trap and concentrate proteins while allowing rapid buffer-exchange. The resulted proteins can be eluted from the channel then directly analyzed by high-resolution mass spectrometry. The platform offers the reduced sample processing time and quite similar sensitivity to conventional liquid chromatography (LC) technologies. We also combined immunoprecipitation with SampleStream-mass spectrometry (i.e., IP-SampleStream-MS) as a high-throughput workflow for the quantitative analysis of target proteins. We applied IP-SampleStream-MS to human serum samples to quantify proteoforms of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III), which are associated with cardiometabolic charateristics such as high-density lipoprotein cholesterol (HDL-C) and obesity. We found proteoform-to-phenotype associations for ApoC-III, glycoproteoforms of which were characterized in this study.

In eukaryotic cells, organellar proteome biogenesis is pivotal for cellular function. Chloroplasts contain a complex proteome, the biogenesis of which includes post-translational import of nuclear-encoded proteins. Protein-targeting machineries must provide chaperones to protect their membrane-protein substrates from aggregation and to keep them in a translocation-competent state. However, the mechanisms determining when and how nascent chloroplast-targeted membrane proteins are sorted and guided in the cytosol are unknown. Here, we establish the timing and mode of interaction between ankyrin repeat-containing protein 2 (AKR2A), the cytosolic targeting factor of chloroplast outer membrane (COM) proteins, and its interacting partners during translation at the single-molecule level. The targeting signal of a nascent AKR2A client protein residing in the ribosomal exit tunnel induces AKR2A binding to ribosomal RPL23A. Subsequently, RPL23A-bound AKR2A binds to the targeting signal when it becomes exposed from ribosomes. AKR2A-mediated targeting of COM proteins is coupled to their translation, which in turn is crucial for biogenesis of the entire chloroplast proteome. On the other hand, structural analysis, molecular modeling, and mutational analysis of the Ankyrin repeat domain (ARD) of AKR2A identified two adjacent sites for coincidental and synergistic binding of monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG), and this and this binding is very important for the membrane insertion or import into the chloroplast of their cargo proteins. Finally, based on the understanding of the targeting mechanism of these membrane proteins, we investigate the possibility of utilizing organelles as oral delivery vehicle for protein therapuetics such as vaccines and protein drugs through engineering of the chloroplast outer membrane and membrane proteins.

3D bioprinting is the technique to precisely control 3D tissue constructs, combining with cells, growth factors, and/or other biomolecules. It provides tissue-like 3D complexity, which meets critical needs in drug discovery, cosmetics testing, medical research, and artificial organ replacement by mimicking anatomical shapes and environments. Nevertheless, there have been only a few examples of protein-based bioinks due to its poor printability. Here the silk protein was blended with carrageenan to resolve this restrictions. Carrageenan, a sulphated linear polysaccharide from marine, has shown the temperature - derived reversible sol/gel transition. Since its viscosity varies readily by temperature, its rheological properties are manipulatable to satisfy the properties of a printable bioink. The interaction between silk protein and carageenan is physically induced, and the interaction within silk is generated by photo-crosslinking reaction. The bioink presented suitable viscosity and fast sol/gel transition to stack tens of layers, as well as durable modulus which is similar to skin. The sulfate chains of carrageenan was also advantageous to recruit cell binding, and exhibited improved cell viability.

Ammonia-oxidizing archaea (AOA) from the phylum Thaumarchaeota are ubiquitous in marine ecosystems and play an important role in the carbon and nitrogen cycling. Although viruses are known to have a key impact on the functioning and mortality of their hosts, thereby regulating the global biogeochemical cycles, not a single virus infecting thaumarchaea has been isolated thus far. Here we report on the isolation and characterization of the Nitrosopumilus spindle-shaped viruses (NSV) which infect a marine AOA and are distinct from other known marine viruses. Their morphology, genome architecture and life cycle indicate that they are distantly related to spindle-shaped viruses infecting hyperthermophilic and hyperhalophilic archaea. However, NSVs do not share appreciable sequence similarity to other archaeal viruses, except for the protein-primed family B DNA polymerase, and are likely to represent a new virus family. NSVs have high adsorption rate to host cells and are not lytic. These properties might be important for the predation on chemolithoautotrophic hosts in resource-poor environments. We show that NSV infection results in cessation of ammonia oxidation, although host cells are not lysed. Widespread distribution of NSV in marine sediments indicates that viral predation regulates the diversity and dynamics in the AOA community.

Numerous biological or chemical substances have posed a serious threat to human beings and the demand for decentralized, point-of-care (POC) diagnostic systems has accordingly increased. Among many POC devices that are commercially available on the market, a personal glucose meter (PGM) with high portability, low cost, simple operation, and reliable quantitative ability has been regarded as one of the most successful. Recently, we developed a label-free and washing-free method for biomolecular detection using a personal glucose meter (PGM). ATP was selected as a model target, and cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were adopted to link the amount of ATP to glucose that is detectable by a hand-held PGM. In principle, the presence of target ATP enables hexokinase to catalyze the conversion of glucose to glucose 6-phosphate, and thus the amount of glucose is decreased in proportion to the amount of ATP. The regenerated ATP from ADP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to signal amplification. As a result, the change of glucose amount that is dependent to ATP amount is simply measured by a hand-held PGM. By employing this strategy, we successfully detected various targets containing nucleic acids, proteins, enzymes activity, small molecules with high sensitivity and selectivity even in real samples. Importantly, the developed system does not require expensive modification and washing steps but is conveniently operated with a commercially available PGM, which would pave the way for the development of a simple and cost-effective sensing platform.

The work presented in this report demonstrates that amphiphilic polysaccharide-clasped self-assembly (Amp-SA) with nanometer size, encapsulating hydrophobic nanoparticles (NPs) can be generated via electrohydrodynamic spraying. It is observed that the formation of hydrophobic NP-encapsulated Amp-SA is dependent on the surface chemistry of NPs. The citrate-coated magnetic NPs (MNPs-Cit) were also prepared and compared. The hydrophobic magnetic NP-encapsulated Amp-SA (Amp-SA-M) exhibited around 2.7−2.8-fold higher values in r2 relaxivity than that of MNPs-Cit. In addition, the resulting Amp-SA-M achieved ∼17.2-fold higher values in r2/r1 ratios than MNPs-Cit. The enhanced performances in magnetic transverse (r2) relaxivity and r2/r1 ratio as well as the in vivo behavior of Amp-SA-M suggest the potential of Amp-SA-M as a promising MRI nanoprobe. This approach based on the nature-originated amphiphilic biopolymers may provide a novel insight into electrohydrodynamic techniques that have the ability to create various nanostructures, encapsulating high-quality hydrophobic nanomaterials for applications in diverse biotechnology.

To cope with multiple stresses caused by various biotic and abiotic stimuli and consequently increased accumulation of misfolded proteins, eukaryotic cells activate a series of adaptive mechanisms that are known as the unfolded protein response (UPR). In response to cellular stresses, phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) attenuates general protein synthesis. However, it also favors the selective translation of mRNAs with upstream open reading frames (uORFs) such as ATF4, a key regulator of the integrated stress response (ISR), in mammalian cells. The ISR is an evolutionarily conserved translational and transcriptional program initiated upon phosphorylation of eIF2α. However, no studies to date have been conducted on the ISR mediated by phosphorylation of eIF2α, and key regulators responsible for the ISR have not been identified in plants. In this study, we performed in silico analysis to investigate functional orthologs of the ATF4, and confirmed that some genes belong to bZIP family containing uORFs in the 5′UTR are potential candidates. However, it is not well understood whether they are involved in plant ISR mediated by phosphorylation of eIF2α. During prolonged ER stress, transcription of these genes increased by 2-3 fold. Arabidopsis plants overexpressing showed a more sensitive phenotype than Col-0 and knockout mutant plants under tunicamycin-induced ER stress condition, while plants overexpressing with uORF did not show any difference. In addition, the expression level of bZIP GFP fusion protein without uORF is increased under ER stress conditions compare to normal conditions. These results suggest that uORF-containing bZIP genes may have a role in plant ISR. * Corresponding

Rheumatoid arthritis, which is known as an autoimmune phenomenon as the main mechanism, is a chronic inflammatory disease that causes inflammation in several joints, such as the hands, wrists, feet, and ankles. Humira, a monoclonal antibody, recognizes tumor necrosis factor-alpha (TNF-α) and is used to treat rheumatoid arthritis. Humira is mostly produced in animal cell-based production systems that have high investment costs, high production costs, and risk of contamination. In this study, Humira was produced using plants to overcome the problems of the existing production system. In order to improve the efficacy of the antibody and reduce side effects, glycoengineered plants without plant-specific residues developed in previous studies were used. In addition, in order to solve the low expression level of the recombinant protein, a vector containing the antibody gene was constructed using a virus-based expression system in which expression is regulated by an inducible promoter. The expression vector was introduced into Agrobacterium to transform wild-type plants and glycoengineered plants, and the transformed plants were selected using an antibiotic-containing medium and a reporter gene. The expression of the antibody expressed in the plant was confirmed using an antibody specific to the protein extracted from the leaf. It was identified at a size of approximately 50 kDa and 27 kDa, respectively, representing the heavy and light chains of the antibody. Single copy plants were selected through the segregation test, and homozygous lines were established through the selection process. The expression level of the recombinant protein by the inducible promoter was compared according to the concentration and time of the inducer. These results show the potential for lower production costs and safer production of rheumatoid arthritis drugs using plants.

Gaucher's disease is a metabolic disorder in which a functional deficiency of an enzyme occurs due to a mutation in the gene encoding glucocerebrosidase (GC), resulting in multiple organ malfunctions. Gaucher's disease is being treated with enzyme replacement therapy, which compensates for the enzyme deficiency by administering activated GCs produced in mammalian cells. However, there is a potential of contamination by pathogenic viruses or prions in the mammalian cell-based manufacturing method, as well as a high production cost. In this study, GCs were expressed in plants that produce customized N-glycans to overcome the limitations of the mammalian cell-based production system and to produce a safe treatment drug for Gaucher's disease. A human cDNA library was used to clone a region expressing a GC gene, and sequence analysis was performed. In addition, a binary vector was created and introduced into plants using Agrobacterium to insert the sequence-confirmed GC gene. Plants into which the GC gene was introduced were selected using an antibiotic-containing media, and the presence or absence of the GC gene introduction into the transformed plants was confirmed using polymerase chain reaction (PCR). Furthermore, proteins were collected from the plant leaves into which the gene was inserted, and GC expression levels in the transformed plants were measured using a GC antibody. Finally, a single cloned plant was selected through the antibiotic-resistant isolate ratio. These findings suggest that employing plants, it is possible to overcome the disadvantages of the present mammalian cell-based manufacturing method, decreasing the production cost of pricey Gaucher's disease therapy, and effectively generating safer enzyme treatment.

Various environmental stresses lead to accumulation of unfolded proteins in the endoplasmic reticulum (ER) of plants, resulting in ER stress. In eukaryotic cells, an effective mechanism known as the unfolded protein response (UPR) has evolved to deal with this problem. In plants, UPR facilitates in the folding or degradation of unfolded proteins and regulates the expression of various stress-related genes. Although the stress response induced by phosphorylation of the translation attenuation factor (TAF) is well understood, the mechanism of protein translation regulation is unclear. In this study, to find out the mechanism of protein translation regulation by TAF, the characteristics of stress-related gene expression were analyzed. To analyze the expression of stress-related genes, 5’ UTR of a gene including uORF was fused with green fluorescent protein (GFP), and GFP expression was analyzed under ER stress conditions. As a result of analyzing the protein expression of the gene, it was confirmed that in the case of the gene containing uORF, the protein translation was increased compared to the normal growth condition. However, there was no significant difference at the transcription level. Additional experiments were conducted to confirm whether the regulation of protein translation by uORF is regulated by phosphorylation of TAF. In plants, a mutant of GCN2 (gcn2), known as an TAF kinase, was crossed and the gene expression regulation by TAF was analyzed. As a result, it was confirmed that dephosphorylation of TAF reduced the protein translation of the gene including uORF. However, there was no significant difference at the transcription level. These results showed the consistent results under various stress conditions. Taken together, these results show that plants regulate gene expression at the translation level to adapt to various environmental stresses, suggesting that TAF phosphorylation plays a key role in translation regulation. * Corresponding

Chronic hypernutrition and lack of physical activity promote lipid accumulation in the white adipose tissue, and excessive lipid accumulation leads to obesity. An increase in the number and size of adipocytes, a characteristic of obesity, is closely associated with adipose dysfunction. Recent in vitro and in vivo studies have proven that probiotics may prevent this dysfunction by regulating lipid metabolism. However, the mechanisms of action of probiotics in obesity are not fully understood, and hence, their usage for treating obesity remains limited. In this study, Bifidobacterium lactis IDCC 4301 was selected for its anti-obesity potential after evaluating inhibitory activity of pancreatic lipase and cholesterol reducing activity. Next, we investigated the roles of B. lactis IDCC 4301 on lipid metabolism in 3T3-L1 cells and high-fat diet (HFD)-fed mice. Our results show that B. lactis IDCC 4301 inhibits cell differentiation and lipid accumulation by suppressing the expression of adipogenic enzymes in 3T3-L1 cells. Moreover, the administration of B. lactis IDCC 4301 resulted in decreased body and epididymal adipose tissue weight, improvement in serum lipid levels, and downregulated adipogenic mRNA expression in HFD-fed mice. Additionally, metabolomic analysis suggested that 2-ketobutyrate might be a possible target compound against obesity. Collectively, these results support that B. lactis IDCC 4301 could be used as an alternative treatment for obesity.

EpCAM is a tumor-related antigen and is a cell surface glycoprotein expressed in colon cancer. EpCAM has been considered a potential target for tumor vaccines. In this study, Agrobacterium-mediated transformation was applied to produce genetically modified Chinese cabbage plants to express EpCAM-IgM FcK. To avoid the plant-specific N-glycosylation process occurring in Golgi devices, the KDEL ER sequence was attached to the C-terminal of Fc to refair the glycoprotein of ER. The recombianat protein productivity was maximized by developing a safe and efficient plant cell factory system using a genetically modified plant cell line expressing EpCAM-IgM FcK protein. In addition, plant seedlings expressing both EpCAM-IgM FcK and J-chain K were obtained by crossing. PCR confirmed that the EpCAM-IgM FcK gene present in the seedlings. Western Blot screened seedlings with high expression. Chinese cabbage protoplasts were obtained by successfully optimizing the protoplast separation protocol to perform single cell transcription analysis. Single cell transcription analysis was performed to reveal the cell types that highly express EpCAM-IgM FcK protein. Cell lines with high expression will be applied to plant cell factory systems to produce EpCAM-IgM FcK.