Chemoenzymatic Synthesis and Antibody Detection of DNA Glycoconjugates

A chemoenzymatic approach for the efficient synthesis of DNA−carbohydrate conjugates was developed and applied to an antibody-based strategy for the detection of DNA glycoconjugates. A phosphoramidite derivative of N-acetylglucosamine (GlcNAc) was synthesized and utilized to attach GlcNAc sugars to the 5‘-terminus of DNA oligonucleotides by solid-phase DNA synthesis. The resulting GlcNAc−DNA conjugates were used as substrates for glycosyl transferase enzymes to synthesize DNA glycoconjugates. Treatment of GlcNAc−DNA with β-1,4-galactosyl transferase (GalT) and UDP−Gal produced N-acetyllactosamine-modified DNA (LacNAc−DNA), which could be converted quantitatively to the trisaccharide Lewis X (LeX)−DNA conjugate by α-1,3-fucosyltransferase VI (FucT) and GDP−Fuc. The facile enzymatic synthesis of LeX−DNA from GlcNAc−DNA also was accomplished in a one-pot reaction by the combined action of GalT and FucT. The resulting glycoconjugates were characterized by gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and glycosidase digestion experiments. Covalent modification of the 5‘-terminus of DNA with carbohydrates did not interfere with the ability of DNA glycoconjugates to hybridize with complementary DNA, as indicated by UV thermal denaturation analysis. The trisaccharide DNA glycoconjugate, LeX−DNA, was detected by a dual DNA hybridization/monoclonal antibody (mAb) detection protocol (“Southwestern”):  membrane-immobilized LeX−DNA was visualized by Southern detection with a radiolabeled complementary DNA probe and by Western chemiluminescence detection with a mAb specific for the LeX antigen. The efficient chemoenzymatic synthesis of DNA glycoconjugates and the Southwestern detection protocol may facilitate the application of glycosylated DNA to cellular targeting and DNA glycoconjugate detection strategies

Public service operational efficiency and blockchain – A case study of Companies House, UK

 Despite the increasing interest and exploration of the use of blockchain technology in public service organisations (PSOs), academic understanding of its transformative impact on the operational excellence of PSOs remains limited. This study adopts an action design science research methodology to develop a proof of concept (POC) blockchain based application for Companies House, a government agency that is registering companies across UK. The application addresses the operational challenges of Companies House as well as issues citizens face when accessing its services. We draw from the public value framework proposed by Twizeyimana and Andersson (2019) and demonstrate the significance of the emerging blockchain technology in relation to their democratic practices based on six dimensions. We further discuss the related challenges and barriers for its implementation and evaluate the POC with the stakeholders of Companies House. We also present an illustrative case study, where we explored the appropriateness of the POC in relation to the draft legislation, “Registration of Overseas Entities and Beneficial Owners” (ROEBO) bill which proposes the introduction of a register of the beneficial owners of overseas legal entities that own real estate in the UK. Our research is one of the few studies that will provide in-depth empirical insights about the relationship between blockchain and operational excellence of PSOs. </p

Immunogenicity and Vaccine Efficacy Boosted by Engineering Human Heavy Chain Ferritin and Chimeric Hepatitis B Virus Core Nanoparticles

Human heavy-chain ferritin (HFn) and hepatitis B virus core (HBc) are both nanoparticle proteins presenting a well-oriented architecture with constant size and shape, which can be engineered to carry epitopes on the surface of the nanoparticle protein cage, enabling vaccine design. This study aims to investigate the immunogenicity differences between engineered HFn and chimeric HBc bearing the same epitope. As a proof of concept, the model epitope Epstein–Barr nuclear antigen 1 (EBNA1) is inserted at the N-terminus of the HFn and HBc subunit to produce two vaccine candidates named EBNA1–HFn (E1F1) and EBNA1–HBc (E1H1), respectively. From in vivo immunogenicity studies, E1H1 demonstrates the capability to prompt significant humoral and cell-mediated immune responses in adjuvant-free formulation. When formulated with the aluminum hydroxide adjuvant, E1H1 produces approximately 5× higher titer and 2× stronger proliferation index (PI) than E1F1. These results confirm that the HBc carrier induces a stronger humoral immune response than HFn. On the other hand, from lymphocyte activation experiments, E1F1 induces a stronger cell-mediated immune response indicated by 5× more CD8+T cells and 2× more effector memory T cells in the E1F1 group versus the E1H1 group. Through this study, HFn and HBc are shown to be potentially effective vaccine carrier nanoparticles having subtly different immunological responses

Data_Sheet_1_Genetic Diversity and Population Structure of Schima superba From Southern China.docx

The tree Schima superba is important for afforestation and fire prevention in southern China. The wood of this tree can also be used for furniture and buildings. However, the lack of genetic background and genomic information for this species has lowered wood yield speed and quality improvement. Here, we aimed to discover genome-wide single nucleotide polymorphisms (SNPs) in 302 S. superba germplasms collected from southern China and to use these SNPs to investigate the population structure. Using genotyping by sequencing, a total of 785 high-quality SNP markers (minor allele frequency [MAF] ≥ 0.05) were identified from 302 accessions collected from seven geographical locations. Population structure analyses and principal coordinate analyses (PCoAs) indicated that these germplasm resources can be clearly separated into different populations. The S. superba accessions originating from Yunnan (YN) and Guangxi (GX) fell into the same population, separate from the accessions originating from Guangdong (GD), which indicated that these two regions should be regarded as major provenances of this species. In addition, two independent core germplasm sets with abundant genetic polymorphisms were constructed to support the breeding work. The identification of SNP markers, analyses of population genetics, and construction of core germplasm sets will greatly promote the molecular breeding work of S. superba.</p

Table5_Full-length transcriptome sequencing reveals the molecular mechanism of monoterpene and sesquiterpene biosynthesis in Cinnamomum burmannii.DOCX

Essential oil of Cinnamomum burmannii is rich in monoterpenes and sesquiterpenes and is widely used in cosmetics and medicines. Knowledge about the enzymes that catalyze the formation of monoterpenes and sesquiterpenes in C. burmannii is insufficient. Therefore, anatomy observation of C. burmannii at the four developmental stages (7 days, CBS1; 14 days, CBS2; 21 days, CBS3, and 28 days, CBS4) were conducted to elucidate the origins of essential oil production. Twelve full-length transcriptomes of C. burmannii leaves at the four stages were generated using Oxford Nanopore Technologies. GC-MS analysis revealed 15 monoterpene and sesquiterpenes dramatically increased from CBS1 to CBS4. A weighted correlation network analysis (WGCNA) in association and differentially expressed genes across four developmental stages were performed. A total of 44 differentially expressed genes (DEGs) were involved in terpenoid syntheses during leaf development. Among them, the DEGs of the mevalonate acid (MVA) pathway were predominantly expressed at CBS1, while those of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway showed increased expression from CBS2 to CBS4. Besides, fourteen genes were associated with monoterpene synthesis and nine with sesquiterpene synthesis. Functions of these DEGs were further predicted with regard to gene expression profile and phylogenetic relationship with those characterized in previous studies. In addition, 922 long noncoding RNAs (lncRNAs) were detected, of which twelve were predicted to regulate monoterpene and sesquiterpene biosynthesis. The present study provided new insights the molecular mechanisms of monoterpenoid and sesquiterpenoid syntheses of C. burmannii.</p

Table6_Full-length transcriptome sequencing reveals the molecular mechanism of monoterpene and sesquiterpene biosynthesis in Cinnamomum burmannii.DOCX

Essential oil of Cinnamomum burmannii is rich in monoterpenes and sesquiterpenes and is widely used in cosmetics and medicines. Knowledge about the enzymes that catalyze the formation of monoterpenes and sesquiterpenes in C. burmannii is insufficient. Therefore, anatomy observation of C. burmannii at the four developmental stages (7 days, CBS1; 14 days, CBS2; 21 days, CBS3, and 28 days, CBS4) were conducted to elucidate the origins of essential oil production. Twelve full-length transcriptomes of C. burmannii leaves at the four stages were generated using Oxford Nanopore Technologies. GC-MS analysis revealed 15 monoterpene and sesquiterpenes dramatically increased from CBS1 to CBS4. A weighted correlation network analysis (WGCNA) in association and differentially expressed genes across four developmental stages were performed. A total of 44 differentially expressed genes (DEGs) were involved in terpenoid syntheses during leaf development. Among them, the DEGs of the mevalonate acid (MVA) pathway were predominantly expressed at CBS1, while those of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway showed increased expression from CBS2 to CBS4. Besides, fourteen genes were associated with monoterpene synthesis and nine with sesquiterpene synthesis. Functions of these DEGs were further predicted with regard to gene expression profile and phylogenetic relationship with those characterized in previous studies. In addition, 922 long noncoding RNAs (lncRNAs) were detected, of which twelve were predicted to regulate monoterpene and sesquiterpene biosynthesis. The present study provided new insights the molecular mechanisms of monoterpenoid and sesquiterpenoid syntheses of C. burmannii.</p

Table3_Full-length transcriptome sequencing reveals the molecular mechanism of monoterpene and sesquiterpene biosynthesis in Cinnamomum burmannii.DOCX

Essential oil of Cinnamomum burmannii is rich in monoterpenes and sesquiterpenes and is widely used in cosmetics and medicines. Knowledge about the enzymes that catalyze the formation of monoterpenes and sesquiterpenes in C. burmannii is insufficient. Therefore, anatomy observation of C. burmannii at the four developmental stages (7 days, CBS1; 14 days, CBS2; 21 days, CBS3, and 28 days, CBS4) were conducted to elucidate the origins of essential oil production. Twelve full-length transcriptomes of C. burmannii leaves at the four stages were generated using Oxford Nanopore Technologies. GC-MS analysis revealed 15 monoterpene and sesquiterpenes dramatically increased from CBS1 to CBS4. A weighted correlation network analysis (WGCNA) in association and differentially expressed genes across four developmental stages were performed. A total of 44 differentially expressed genes (DEGs) were involved in terpenoid syntheses during leaf development. Among them, the DEGs of the mevalonate acid (MVA) pathway were predominantly expressed at CBS1, while those of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway showed increased expression from CBS2 to CBS4. Besides, fourteen genes were associated with monoterpene synthesis and nine with sesquiterpene synthesis. Functions of these DEGs were further predicted with regard to gene expression profile and phylogenetic relationship with those characterized in previous studies. In addition, 922 long noncoding RNAs (lncRNAs) were detected, of which twelve were predicted to regulate monoterpene and sesquiterpene biosynthesis. The present study provided new insights the molecular mechanisms of monoterpenoid and sesquiterpenoid syntheses of C. burmannii.</p

Modulation of Naturally Occurring Linear Dipeptide Chirality to Reduce the Affinity for Oligopeptide Transporter 1 and Increase Intestinal Stability for an Enhanced Colon-Targeting Effect in the Treatment of Inflammatory Bowel Disease: An Application of <i>trans</i>-4‑l‑Hydroxyprolyl‑l‑serine

The naturally occurring linear dipeptide JBP923 (trans-4-l-Hyp-l-Ser, HS-tLL) with anti-inflammatory effects showed potential for the treatment of inflammatory bowel disease (IBD). However, colon-specific delivery after oral administration is still a challenge because its absorption is mediated by oligopeptide transporter 1 (PEPT1) in the upper small intestine and because of its instability in the gastrointestinal tract. Therefore, we aimed to enhance the colon-targeting efficiency by modulating HS-tLL chirality to synthesize eight enantiomers. Among these enantiomers, trans-4-d-Hyp-d-Ser, cis-4-l-Hyp-d-Ser, cis-4-d-Hyp-l-Ser, and cis-4-d-Hyp-d-Ser did not work as substrates of PEPT1 and were stable in the gastrointestinal tract, resulting in enhanced colonic accumulation through the paracellular pathway due to the loose tight junctions in IBD. Interestingly, cis-4-d-Hyp-d-Ser exerted the most potent therapeutic effect on IBD. Our findings revealed the impact of chirality on the colonic accumulation of the linear dipeptide, providing strategies for the colon-targeted delivery of the linear dipeptide for the treatment of IBD

Radiographic analyses of mice reveal multiple subcutaneous ossifications.

<p>X-rays of 12 month +/−p and WT mice. A) 12 month +/−p female with no ossifications visualized; B) 12 month +/−p male, inset and arrows demonstrate areas consistent with ossifications; C) 12 month WT without areas of ossifications.</p

Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2

<p>The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C₂H₂-type zinc-finger, and only the C₂H₂-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C₂H₂-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations.</p