Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Portland Press Ltd.  

<jats:title>Abstract</jats:title>
<jats:p>Despite the critical role of bacterial cell walls in maintaining cell shapes, certain environmental stressors can induce the transition of many bacterial species into a wall-deficient state called L-form. Long-term induced Escherichia coli L-forms lose their rod shape and usually hold significant mutations that affect cell division and growth. Besides this, the genetic background of L-form bacteria is still poorly understood. In the present study, the genomes of two stable L-form strains of E. coli (NC-7 and LWF+) were sequenced and their gene mutation status was determined and compared with their parental strains. Comparative genomic analysis between two L-forms reveals both unique adaptions and common mutated genes, many of which belong to essential gene categories not involved in cell wall biosynthesis, indicating that L-form genetic adaptation impacts crucial metabolic pathways. Missense variants from L-forms and Lenski’s long-term evolution experiment (LTEE) were analyzed in parallel using an optimized DeepSequence pipeline to investigate predicted mutation effects (α) on protein functions. We report that the two L-form strains analyzed display a frequency of 6–10% (0% for LTEE) in mutated essential genes where the missense variants have substantial impact on protein functions (α&amp;lt;0.5). This indicates the emergence of different survival strategies in L-forms through changes in essential genes during adaptions to cell wall deficiency. Collectively, our results shed light on the detailed genetic background of two E. coli L-forms and pave the way for further investigations of the gene functions in L-form bacterial models.</jats:p>

DOI： 10.1042/bsr20231227

Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/vaccines11091506

Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

DOI： 10.3389/fphys.2023.1244594

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms24098241

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Insect Biochemistry and Molecular Biology  

O-glycosylation of secreted and membrane-bound proteins is an important post-translational modification that affects recognition of cell surface receptors, protein folding, and stability. However, despite the importance of O-linked glycans, their biological functions have not yet been fully elucidated and the synthetic pathway of O-glycosylation has not been investigated in detail, especially in the silkworm. In this study, we aimed to investigate O-glycosylation in silkworms by analyzing the overall structural profiles of mucin-type O-glycans using LC–MS. We found GalNAc or GlcNAc monosaccharide and core 1 disaccharide (Galβ1-3-GalNAcα1-Ser/Thr) were major components of the O-glycan attached to secreted proteins produced in silkworms. Furthermore, we characterized the 1 b1,3-galactosyltransferase (T-synthase) required for synthesis of the core 1 structure, common to many animals. Five transcriptional variants and four protein isoforms were identified in silkworms, and the biological functions of these isoforms were investigated. We found that BmT-synthase isoforms 1 and 2 were localized in the Golgi apparatus in cultured BmN4 cells and functioned both in cultured cells and silkworms. Additionally, a specific functional domain of T-synthase, called the stem domain, was found to be essential for activity and is presumed to be needed for dimer formation and galactosyltransferase activity. Altogether, our results elucidated the O-glycan profile and function of T-synthase in the silkworm. Our findings allow the practical comprehension of O-glycosylation required for employing silkworms as a productive expression system.

DOI： 10.1016/j.ibmb.2023.103936

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Scopus

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media {SA}  

<jats:p>Recent progress has been made dramatically in decorating virus-like particles (VLPs) on the surface or inside with functional molecules, such as antigens or nucleic acids. However, it is still challenging to display multiple antigens on the surface of VLP to meet the requirement as a practical vaccine candidate. Herein this study, we focus on the expression and engineering of the capsid protein VP2 of canine parvovirus for VLP display in the silkworm-expression system. The chemistry of the SpyTag/SpyCatcher (SpT/SpC) and SnoopTag/SnoopCatcher (SnT/SnC) are efficient protein covalent ligation systems to modify VP2 genetically, where SpyTag/SnoopTag are inserted into the N-terminus or two distinct loop regions (Lx and L2) of VP2. The SpC-EGFP and SnC-mCherry are employed as model proteins to evaluate their binding and display on six SnT/SnC-modified VP2 variants. From a series of protein binding assays between indicated protein partners, we showed that the VP2 variant with SpT inserted at the L2 region significantly enhanced VLP display to 80% compared to 5.4% from N-terminal SpT-fused VP2-derived VLPs. In contrast, the VP2 variant with SpT at the Lx region failed to form VLPs. Moreover, the SpT (Lx)/SnT (L2) double-engineered chimeric VP2 variants showed covalent conjugation capacity to both SpC/SnC protein partners. The orthogonal ligations between those binding partners were confirmed by both mixing purified proteins and co-infecting cultured silkworm cells or larvae with desired recombinant viruses. Our results indicate that a convenient VLP display platform was successfully developed for multiple antigen displays on demand. Further verifications can be performed to assess its capacity for displaying desirable antigens and inducing a robust immune response to targeted pathogens.</jats:p>

DOI： 10.3389/fbioe.2023.1096363

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms24043243

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00253-022-12278-2

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.bbrc.2022.06.035

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Portland Press Ltd.  

<jats:p>Baculovirus expression vector system (BEVS) has been recognized as a potent protein expression system in engineering valuable enzymes and vaccines. Various fusion tags facilitate protein purification, leaving the potential risk to influence the target protein’s biological activity negatively. It is of great interest to consider removing the additional tags using site-specific proteases, such as human rhinoviruses (HRV) 3C protease. The current study validated the cleavage activity of 3C protease in Escherichia coli and silkworm-BEVS systems by mixing the cell or fat body lysates of 3C protein and 3C site containing target protein in vitro. Further verification has been performed in the fat body lysate from co-expression of both constructs, showing remarkable cleavage efficiency in vivo silkworm larvae. We also achieved the glutathione-S-transferase (GST) tag-cleaved product of the VP15 protein from the White spot syndrome virus after purification, suggesting that we successfully established a coinfection-based recognition-and-reaction BEVS platform for the tag-free protein engineering.</jats:p>

DOI： 10.1042/BSR20220739

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

<jats:p>Contaminations frequently occur in bacterial cultures, which significantly affect the reproducibility and reliability of the results from whole-genome sequencing (WGS). Decontaminated WGS data with clean reads is the only desirable source for detecting possible variants correctly. Improvements in bioinformatics are essential to analyze the contaminated WGS dataset. Existing pipelines usually contain contamination detection, decontamination, and variant calling separately. The efficiency and results from existing pipelines fluctuate since distinctive computational models and parameters are applied. It is then promising to develop a bioinformatical tool containing functions to discriminate and remove contaminated reads and improve variant calling from clean reads. In this study, we established a Python-based pipeline named CleanSeq for automatic detection and removal of contaminating reads, analyzing possible genome variants with proper verifications via local re-alignments. The application and reproducibility are proven in either simulated, publicly available datasets or actual genome sequencing reads from our experimental evolution study in Escherichia coli. We successfully obtained decontaminated reads, called out all seven consistent mutations from the contaminated bacterial sample, and derived five colonies. Collectively, the results demonstrated that CleanSeq could effectively process the contaminated samples to achieve decontaminated reads, based on which reliable results (i.e., variant calling) could be obtained.</jats:p>

DOI： 10.3390/app12126209

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

<jats:title>Abstract</jats:title><jats:p>The morphology of primitive cells has been the subject of extensive research. A spherical form was commonly presumed in prebiotic studies but lacked experimental evidence in living cells. Whether and how the shape of living cells changed are unclear. Here we exposed the rod-shaped bacterium <jats:italic>Escherichia coli</jats:italic> to a resource utilization regime mimicking a primordial environment. Oleate was given as an easy-to-use model prebiotic nutrient, as fatty acid vesicles were likely present on the prebiotic Earth and might have been used as an energy resource. Six evolutionary lineages were generated under glucose-free but oleic acid vesicle (OAV)-rich conditions. Intriguingly, fitness increase was commonly associated with the morphological change from rod to sphere and the decreases in both the size and the area-to-volume ratio of the cell. The changed cell shape was conserved in either OAVs or glucose, regardless of the trade-offs in carbon utilization and protein abundance. Highly differentiated mutations present in the genome revealed two distinct strategies of adaption to OAV-rich conditions, i.e., either directly targeting the cell wall or not. The change in cell morphology of <jats:italic>Escherichia coli</jats:italic> for adapting to fatty acid availability supports the assumption of the primitive spherical form.</jats:p>

DOI： 10.1038/s42003-021-02954-w

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

<jats:title>Abstract</jats:title><jats:p>White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15<jats:sub>(1–25)</jats:sub>, VP15<jats:sub>(26–57)</jats:sub>, VP15<jats:sub>(58–80)</jats:sub>, and VP15<jats:sub>(1–25,58–80)</jats:sub>. The purified proteins from the <jats:italic>E. coli</jats:italic> expression system were assayed as potential protective agents in Kuruma shrimp (<jats:italic>Marsupenaeus japonicus</jats:italic>) using the prime-and-boost strategy. Among the four truncated constructs, VP15<jats:sub>(26–57)</jats:sub> provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15<jats:sub>(26–57)</jats:sub> were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host <jats:italic>M. japonicus</jats:italic> were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15<jats:sub>(26–57)</jats:sub>, and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.</jats:p>

DOI： 10.1038/s41598-021-92002-8

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.aspen.2021.04.011

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

DOI： 10.3390/insects12060517

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.34565/seibutsukogaku.99.4_207

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.csbj.2021.04.030

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

(1) Background: giant vesicles (GVs) are widely employed as models for studying physicochemical properties of bio-membranes and artificial cell construction due to their similarities to natural cell membranes. Considering the critical roles of GVs, various methods have been developed to prepare them. Notably, the water-in-oil (w/o) inverted emulsion-transfer method is reported to be the most promising, owning to the relatively higher productivity and better encapsulation efficiency of biomolecules. Previously, we successfully established an improved approach to acquire detailed information of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-derived GVs with imaging flow cytometry (IFC); (2) Methods: we prepared GVs with different lipid compositions, including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and PC/PE mixtures by w/o inverted emulsion methods. We comprehensively compared the yield, purity, size, and encapsulation efficiency of the resulting vesicles; (3) Results: the relatively higher productivities of GVs could be obtained from POPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), DOPC: DLPE (7:3), and POPC: DLPE (6:4) pools. Furthermore, we also demonstrate that these GVs are stable during long term preservation in 4 °C. (4) Conclusions: our results will be useful for the analytical study of GVs and GV-based applications.

DOI： 10.3390/life11030223

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

<jats:title>Abstract</jats:title>
<jats:p>Purification of recombinant proteins is often a challenging matter because high purity and high recovery are desired. If the expressed recombinant protein is also in a complex matrix, such as from the silkworm expression system, purification becomes more challenging. Even if purification from the silkworm expression system is troublesome, it benefits from a high capacity for the production of recombinant proteins. In this study, magnetic nanoparticles (MNPs) were investigated as a suitable tool for the purification of proteins from the complex matrix of the silkworm fat body. The MNPs were modified with nickel so that they have an affinity for His-tagged proteins, as the MNP purification protocol itself does not need special equipment except for a magnet. Among the three different kinds of investigated MNPs, MNPs with sizes of 100 nm to 200 nm and approximately 20 nm-thick nickel shells were the most suitable for our purpose. With them, the total protein amount was reduced by up to at least approximately 77.7%, with a protein recovery of around 50.8% from the silkworm fat body. The minimum binding capacity was estimated to be 83.3 µg protein/mg MNP. Therefore, these MNPs are a promising tool as a purification pretreatment of complex sample matrices.</jats:p>

DOI： 10.1186/s12951-020-00715-1

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiotec.2020.08.002

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

White spot syndrome virus (WSSV) is known as one of the most lethal pathogenic viruses in shrimp causing massive damage to shrimp aquaculture industries. To date, no effective treatment or prevention has been found. In this study, five recombinant viral proteins VP15, VP19, VP24, VP26, and VP28 were expressed and purified in E. coli, which were employed as candidates against WSSV in Kuruma shrimp Marsupenaeus japonicus. In vivo antiviral assay in this study newly revealed that VP15 of major nucleocapsid protein, being known as a DNA-binding protein provided the substantial protection against the viral infection when pre-injected into shrimps. Furthermore, we also verified the immunogenic effects of purified VP15 and VP19 proteins produced in a silkworm-bacmid expression system. Taken together, our study identified VP15 as an effective candidate against WSSV infection in the Kuruma shrimp. It is interesting to uncover why and how VP15 is involved in the immune memory in shrimp in the future study.

DOI： 10.1016/j.fsi.2020.03.061

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jchromb.2019.121964

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PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2019.11.056

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Portland Press Ltd.  

A protocell is a synthetic form of cellular life that is constructed from phospholipid vesicles and used to understand the emergence of life from a nonliving chemical network. To be considered ‘living’, a protocell should be capable of self-proliferation, which includes successive growth and division processes. The growth of protocells can be achieved via vesicle fusion approaches. In this review, we provide a brief overview of recent research on the formation of a protocell, fusion and division processes of the protocell, and encapsulation of a defined chemical network such as the genetic material. We also provide some perspectives on the challenges and future developments of synthetic protocell research.

DOI： 10.1042/bst20190576

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

DOI： 10.3390/ijms20174228

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.pep.2019.03.010

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s12033-019-00184-4

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Other Link： https://link.springer.com/article/10.1007/s12033-019-00184-4/fulltext.html

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DOI： 10.1016/j.aspen.2019.02.008

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DOI： 10.3390/nano9040593

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Authorship：Lead author   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.vaccine.2019.09.002

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DOI： 10.1016/j.aspen.2019.01.009

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Authorship：Lead author   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00253-018-9309-6

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DOI： 10.1016/j.aspen.2018.05.002

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12033-018-0127-y

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DOI： 10.1007/s12033-017-0003-1

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DOI： 10.1007/s12033-017-0008-9

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DOI： 10.1038/s41598-017-15884-7

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DOI： 10.1016/j.aspen.2016.07.007

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DOI： 10.1016/j.bbrc.2016.09.151

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DOI： 10.1016/j.aspen.2016.03.014

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DOI： 10.1016/j.ibmb.2015.01.008

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DOI： 10.1007/s11010-015-2529-5

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DOI： 10.1007/s12033-015-9866-1

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DOI： 10.1016/j.ibmb.2015.07.011

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DOI： 10.1016/j.aspen.2015.01.006

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DOI： 10.1016/j.aspen.2015.03.006

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DOI： 10.1038/srep18103

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DOI： 10.1016/j.cbd.2015.07.003

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DOI： 10.1016/j.ibmb.2014.05.008

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DOI： 10.1007/s12010-014-0814-5

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DOI： 10.1007/s12033-013-9694-0

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DOI： 10.1016/j.ibmb.2014.10.001

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DOI： 10.1016/j.aspen.2013.10.009

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Authorship：Lead author   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00253-013-5437-1

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Publishing type：Research paper (scientific journal)  

DOI： 10.1111/imb.12023

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Authorship：Lead author   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00253-013-4785-1

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DOI： 10.1007/s00253-012-4583-1

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DOI： 10.11416/jibs.82.1_019

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DOI： 10.1007/s00253-013-5279-x

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DOI： 10.1016/j.ibmb.2013.04.004

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DOI： 10.1371/journal.pone.0052320

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DOI： 10.1002/arch.21091

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DOI： 10.1371/journal.pone.0034330

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DOI： 10.1007/s13355-012-0109-7

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DOI： 10.1007/s11033-011-1362-5

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