Programming the morphology of DNA origami crystals by magnesium ion strength.

Harnessing the programmable nature of DNA origami for controlling structural features in crystalline materials affords opportunities to bring crystal engineering to a remarkable level. However, the challenge of crystallizing a single type of DNA origami unit into varied structural outcomes remains, given the requirement for specific DNA designs for each targeted structure. Here, we show that crystals with distinct equilibrium phases and shapes can be realized using a single DNA origami morphology with an allosteric factor to modulate the binding coordination. As a result, origami crystals undergo phase transitions from a simple cubic lattice to a simple hexagonal (SH) lattice and eventually to a face-centered cubic (FCC) lattice. After selectively removing internal nanoparticles from DNA origami building blocks, the body-centered tetragonal and chalcopyrite lattice are derived from the SH and FCC lattices, respectively, revealing another phase transition involving crystal system conversions. The rich phase space was realized through the de novo synthesis of crystals under varying solution environments, followed by the individual characterizations of the resulting products. Such phase transitions can lead to associated transitions in the shape of the resulting products. Hexagonal prism crystals, crystals characterized by triangular facets, and twinned crystals are observed to form from SH and FCC systems, which have not previously been experimentally realized by DNA origami crystallization. These findings open a promising pathway toward accessing a rich phase space with a single type of building block and wielding other instructions as tools to develop crystalline materials with tunable properties.

Layer-Controllable "2.5D" DNA Origami Crystals Synthesized by a Hierarchical Assembly Strategy.

The finite periodic arrangement of functional nanomaterials on the two-dimensional scale enables the integration and enhancement of individual properties, making them an important research topic in the field of tuneable nanodevices. Although layer-controllable lattices such as graphene have been successfully synthesized, achieving similar control over colloidal nanoparticles remains a challenge. DNA origami technology has achieved remarkable breakthroughs in programmed nanoparticle assembly. Based on this technology, we proposed a hierarchical assembly strategy to construct a universal DNA origami platform with customized layer properties, which we called 2.5-dimensional (2.5D) DNA origami crystals. Methodologically, this strategy divides the assembly procedure into two steps: 1) array synthesis, and 2) lattice synthesis, which means that the layer properties, including layer number, interlayer distance, and surface morphology, can be flexibly customized based on the independent designs in each step. In practice, these synthesized 2.5D crystals not only pioneer the expansion of the DNA origami crystal library to a wider range of dimensions, but also highlight the technological potential for templating 2.5D colloidal nanomaterial lattices.

Dynamically Reconfigurable DNA Origami Crystals Driven by a Designated Path Diagram.

Constructing adaptable and switchable crystal structures renders it possible to dynamically control the properties and functions of adaptive materials, thereby expanding the potential application of these structures in fields such as optics, biology, and catalysis. Recently, researchers have developed various dynamic crystals possessing phase transition abilities. However, manufacturing switchable crystals with multiple-phase-transition ability by integrating various responsive behaviors into different dimensions of a single lattice remains considerably challenging. Herein, we built a set of dynamically reconfigurable DNA origami crystals by orthogonally integrating multiple dynamic effectors into the prescribed dimensions of the octahedral DNA origami frames. Further, we independently manipulated and logically combined the dynamic behaviors of the effectors in different dimensions. The initial mother phase and three derived daughter phases were interconnected into a path diagram by six elementary paths. Furthermore, these paths could be superimposed under multiple stimulus instructions by design to obtain the desired intricate transition routes. Moreover, finer manipulations were also applied to these paths to obtain extra new phase stations for the path diagram. To conveniently detect these phase transitions, a color-based visualization strategy was developed that converted the microscopic symmetry transformation of the lattices into macroscopic color changes that could be observed via a fluorescence microscope. Hence, this strategy lays the foundation for artificially constructing biomimetic functional crystals.

Confinement in Dual-Chain-Locked DNA Origami Nanocages Programs Marker-Responsive Delivery of CRISPR/Cas9 Ribonucleoproteins.

Delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) offers a powerful tool for therapeutic genome editing. However, precise manipulation of CRISPR/Cas9 RNPs to switch the machinery on and off according to diverse disease microenvironments remains challenging. Here, we present dual-chain-locked DNA origami nanocages (DL-DONCs) that can confine Cas9 RNPs in the inner cavity for efficient cargo delivery and dual-marker-responsive genome editing in the specified pathological states. By engineering of ATP or miRNA-21-responsive dsDNAs as chain locks on the DONCs, the permeability of nanocages and accessibility of encapsulated Cas9 RNPs can be finely regulated. The resulting DL-DONCs enabled steric protection of bioactive Cas9 RNPs from premature release and deactivation during transportation while dismounting the dual chain locks in response to molecular triggers after internalization into tumor cells, facilitating the escape of Cas9 RNPs from the confinement for gene editing. Due to the dual-marker-dominated uncaging mechanism, the gene editing efficiency could be exclusively determined by the combined level of ATP and miRNA-21 in the target cellular environment. By targeting the tumor-associated PLK-1 gene, the DL-DONCs-enveloped Cas9 RNPs have demonstrated superior inhibitory effects on the proliferation of tumor cells in vitro and in vivo. The developed DL-DONCs provide a custom-made platform for the precise manipulation of Cas9 RNPs, which can be potentially applied to on-demand gene editing for classified therapy in response to arbitrary disease-associated biomolecules.

Light Chain Q166C Mutation Permits One-step Site Specific Conjugation on Monoclonal Antibodies.

Engineered cysteines are frequently used for site-specific conjugation in antibody-drug conjugate (ADC) development. When cysteine-engineered mAbs are produced in the cell culture process, the sulfhydryl groups on the engineered cysteines are mostly in an oxidized form. The oxidized cysteines require multiple steps (such as reduction, reoxidation, and buffer exchanges) to reactivate for bioconjugation, which complicates the ADC production process and reduces yields. In this study, we identified a Q166C mutation in the light chain that allows the presence of free sulfhydryl groups during cell culture and purification process. This mutation is in the constant region and away from sites involved in antigen binding or Fc-mediated functions. The free sulfhydryl reacts readily with maleimide in a mild solution at a high conjugation rate. This is only the second such site reported (the first one is Q124C in the light chain). Using the Q166C mutation, we conjugated an anti-angiopoietin-2 (Ang-2) peptide on bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, to construct a peptide antibody conjugate, Ava-Plus, which could block two pro-angiogenic factors simultaneously. Ava-Plus showed high affinity for both VEGF and Ang-2 and demonstrated higher activity than bevacizumab in in vitro cell migration and in vivo mouse xenograft models.

Two-Stage Assembly of Nanoparticle Superlattices with Multiscale Organization.

Self-assembly processes, while promising for enabling the fabrication of complexly organized nanomaterials from nanoparticles, are often limited in creating structures with multiscale order. These limitations are due to difficulties in practically realizing the assembly processes required to achieve such complex organizations. For a long time, a hierarchical assembly attracted interest as a potentially powerful approach. However, due to the experimental limitations, intermediate-level structures are often heterogeneous in composition and structure, which significantly impacts the formation of large-scale organizations. Here, we introduce a two-stage assembly strategy: DNA origami frames scaffold a coordination of nanoparticles into designed 3D nanoclusters, and then these clusters are assembled into ordered lattices whose types are determined by the clusters' valence. Through modulating the nanocluster architectures and intercluster bindings, we demonstrate the successful formation of complexly organized nanoparticle crystals. The presented two-stage assembly method provides a powerful fabrication strategy for creating nanoparticle superlattices with prescribed unit cells.

Generation and Characterization of Monoclonal Antibodies against Swine Acute Diarrhea Syndrome Coronavirus Spike Protein.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a member of the family Coronaviridae and the genus Alphacoronavirus, primarily affects piglets under 7 days old, causing symptoms such as diarrhea, vomiting, and dehydration. It has the potential to infect human primary and passaged cells in vitro, indicating a potential risk of zoonotic transmission. In this study, we successfully generated and purified six monoclonal antibodies (mAbs) specifically targeting the spike protein of SADS-CoV, whose epitope were demonstrated specificity to the S1A or S1B region by immunofluorescence assay and enzyme-linked immunosorbent assay. Three of these mAbs were capable of neutralizing SADS-CoV infection on HeLa-R19 and A549. Furthermore, we observed that SADS-CoV induced the agglutination of erythrocytes from both humans and rats, and the hemagglutination inhibition capacity and antigen-antibody binding capacity of the antibodies were assessed. Our study reveals that mAbs specifically targeting the S1A domain demonstrated notable efficacy in suppressing the hemagglutination phenomenon induced by SADS-CoV. This finding represents the first instance of narrowing down the protein region responsible for SADS-CoV-mediated hemagglutination to the S1A domain, and reveals that the cell attachment domains S1A and S1B are the main targets of neutralizing antibodies.

Dopamine depletion and subcortical dysfunction disrupt cortical synchronization and metastability affecting cognitive function in Parkinson's disease.

Parkinson's disease (PD) is primarily characterized by the loss of dopaminergic cells and atrophy in subcortical regions. However, the impact of these pathological changes on large-scale dynamic integration and segregation of the cortex are not well understood. In this study, we investigated the effect of subcortical dysfunction on cortical dynamics and cognition in PD. Spatiotemporal dynamics of the phase interactions of resting-state blood-oxygen-level-dependent signals in 159 PD patients and 152 normal control (NC) individuals were estimated. The relationships between subcortical atrophy, subcortical-cortical fiber connectivity impairment, cortical synchronization/metastability, and cognitive performance were then assessed. We found that cortical synchronization and metastability in PD patients were significantly decreased. To examine whether this is an effect of dopamine depletion, we investigated 45 PD patients both ON and OFF dopamine replacement therapy, and found that cortical synchronization and metastability are significantly increased in the ON state. The extent of cortical synchronization and metastability in the OFF state reflected cognitive performance and mediates the difference in cognitive performance between the PD and NC groups. Furthermore, both the thalamic volume and thalamocortical fiber connectivity had positive relationships with cortical synchronization and metastability in the dopaminergic OFF state, and mediate the difference in cortical synchronization between the PD and NC groups. In addition, thalamic volume also reflected cognitive performance, and cortical synchronization/metastability mediated the relationship between thalamic volume and cognitive performance in PD patients. Together, these results highlight that subcortical dysfunction and reduced dopamine levels are responsible for decreased cortical synchronization and metastability, further affecting cognitive performance in PD. This might lead to biomarkers being identified that can predict if a patient is at risk of developing dementia.

Environment-Resistant DNA Origami Crystals Bridged by Rigid DNA Rods with Adjustable Unit Cells.

The crystallization methodology of DNA origami frames has found salient utility in large-scalely integrating multifarious functional components following organized arrangements, thus opening up the possibilities for optical, biological, and other interdisciplinary applications. However, the single strand-dominated spacing region between adjacent DNA origami units has extremely restricted the adjustment of DNA origami separations, leading the soft crystals susceptible to environmental influences. Herein, we developed a cocrystallization pathway by incorporating rigid DNA rods into a DNA origami assembly system to achieve mutually ordered bridging on a three-dimensional scale. The intervention of DNA rods significantly improved the rigidity and crushing resistance of entire cocrystals and rendered DNA origami units exhibiting different spacing distances within the obtained crystal phase when varying DNA rod structures artificially. Such a tuning strategy that uses DNA rods as allosteric factors would provide a rational method for accessing diverse crystalline states and even modulating the tailorable properties of materials on demand.

Inhibition of N-Acetyltransferase 10 Suppresses the Progression of Prostate Cancer through Regulation of DNA Replication.

Cancer suppression through the inhibition of N-acetyltransferase 10 (NAT10) by its specific inhibitor Remodelin has been demonstrated in a variety of human cancers. Here, we report the inhibitory effects of Remodelin on prostate cancer (PCa) cells and the possible associated mechanisms. The prostate cancer cell lines VCaP, LNCaP, PC3, and DU145 were used. The in vitro proliferation, migration, and invasion of cells were measured by a cell proliferation assay, colony formation, wound healing, and Transwell assays, respectively. In vivo tumor growth was analyzed by transplantation into nude mice. The inhibition of NAT10 by Remodelin not only suppressed growth, migration, and invasion in vitro, but also the in vivo cancer growth of prostate cancer cells. The involvement of NAT10 in DNA replication was assessed by EdU labeling, DNA spreading, iPOND, and ChIP-PCR assays. The inhibition of NAT10 by Remodelin slowed DNA replication. NAT10 was detected in the prereplication complex, and it could also bind to DNA replication origins. Furthermore, the interaction between NAT10 and CDC6 was analyzed by Co-IP. The altered expression of NAT10 was measured by immunofluorescence staining and Western blotting. Remodelin markedly reduced the levels of CDC6 and AR. The expression of NAT10 could be altered under either castration or noncastration conditions, and Remodelin still suppressed the growth of in vitro-induced castration-resistant prostate cancers. The analysis of a TCGA database revealed that the overexpression of NAT10, CDC6, and MCM7 in prostate cancers were correlated with the Gleason score and node metastasis. Our data demonstrated that Remodelin, an inhibitor of NAT10, effectively inhibits the growth of prostate cancer cells under either no castration or castration conditions, likely by impairing DNA replication.

CSAD Ameliorates Lipid Accumulation in High-Fat Diet-Fed Mice.

Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic disease manifested in hepatic steatosis, inflammation, fibrosis, etc., which affects over one-quarter of the population around the world. Since no effective therapeutic drugs are available to cope with this widespread epidemic, the functional research of genes with altered expression during NAFLD helps understand the pathogenesis of this disease and the development of new potential therapeutic targets for drugs. In the current work, we discovered via the analysis of the Gene Expression Omnibus (GEO) dataset that cysteine sulfinic acid decarboxylase (CSAD) decreased significantly in NAFLD patients, which was also confirmed in multiple NAFLD mouse models (HFD-fed C57BL/6J, db/db and HFHFrHC-fed C57BL/6J mice). Next, CSAD's function in the progression of NAFLD was explored using AAV-mediated liver-directed gene overexpression in an HFD-fed mouse model, where the overexpression of CSAD in the liver could alleviate NAFLD-associated pathologies, including body weight, liver/body weight ratio, hepatic triglyceride and total cholesterol, and the degree of steatosis. Mechanically, we found that the overexpression of CSAD could increase the expression of some genes related to fatty acid ß-oxidation (Acad1, Ppara, and Acox1). Furthermore, we also detected that CSAD could improve mitochondrial injury in vitro and in vivo. Finally, we proposed that the effect of CSAD on lipid accumulation might be independent of the taurine pathway. In conclusion, we demonstrated that CSAD is involved in the development of NAFLD as a protective factor, which suggested that CSAD has the potential to become a new target for drug discovery in NAFLD.

pH-Induced Symmetry Conversion of DNA Origami Lattices.

DNA nanotechnology has provided credible approaches for assembly of three-dimensional (3D) lattices with complex patterns. However, the symmetries are strictly dependent on their initial configurations and difficult to alter via non-thermal treatments. While switchable nucleic acid structures have been employed to construct deformable DNA motifs, it remains challenging to arrange them anisotropically in 3D lattices to trigger directed collective shape transition and dynamic symmetry conversion. In this work, we used octahedral DNA origami frames to synthesize four DNA origami lattices by placing the pH-reactive i-motif sequences in the desired dimensions. Thereinto, lattices with an anisotropic design can switch between simple cubic (SC) and simple tetragonal (ST) upon pH change. Small angle X-ray scattering (SAXS) results reveal the feasibility of obtaining 3D lattices with sensitive responses to external stimuli, expanding the way to obtain low-symmetry lattices.

miR-552-3p modulates transcriptional activities of FXR and LXR to ameliorate hepatic glycolipid metabolism disorder.

BACKGROUND & AIMS: The nuclear location of miRNAs has been known for more than a decade, but the exact function of miRNAs in the nucleus has not been fully elucidated. We previously discovered that intranuclear miR-552-3p has an inhibitory role on gene transcription and contains a particular AGGTCA-like sequence, the cis-elements of the NR1 subfamily of nuclear receptors. Here, we aim to explore the potential effect of miR-552-3p and its AGGTCA-like sequence on NR1s and its possible application in improving hepatic glycolipid metabolism. METHODS: RNA-seq, mass spectrometry, and bioinformatics analysis were used to reveal the possible pathways influenced by miR-552-3p. High fat-high fructose diet-fed mice and db/db mice transfected with AAV2/8-miR-552-3p were established to investigate the in vivo effects of miR-552-3p on hepatic glycolipid metabolism. Fluorescence resonance energy transfer, pull-down, electrophoretic mobility shift, and chromatin immunoprecipitation assays were performed to explore the mechanism by which miR-552-3p regulates NR1s. RT-PCR was conducted to analyse miR-552-3p levels in liver biopsies from patients with NAFLD and normal controls. RESULTS: MiR-552-3p could inhibit metabolic gene expression in vitro and displayed beneficial effects on glycolipid metabolism in vivo. Intranuclear miR-552-3p primarily regulated the LXRα and FXR pathways; this was achieved by its binding to the complementary sequence of AGGTCA to modulate the transcriptional activities of LXRα and FXR. Moreover, LXRα and FXR ligands could restore the effects of miR-552-3p on gene expression and glycolipid metabolism. Additionally, the hepatic miR-552-3p level was significantly decreased in liver samples from patients with NAFLD compared to normal controls. CONCLUSIONS: The mechanism by which miR-552-3p modulates LXRα and FXR has revealed a new method of miRNA-mediated gene regulation. In addition, the beneficial effects in vivo and clinical relevance of miR-552-3p suggest that it might be a potential therapeutic target for the treatment of glycolipid metabolic disease. LAY SUMMARY: Glycolipid metabolic diseases, which have become a major public health concern worldwide, are triggered by abnormalities in lipid and glucose metabolism. Herein, we show that miR-552-3p has the ability to ameliorate hepatic glycolipid metabolic diseases by modulating the transcriptional activities of LXRα and FXR in the nucleus. These findings provide evidence that miR-552-3p may serve as a potential therapeutic target.

Potent Bispecific Neutralizing Antibody Targeting Glycoprotein B and the gH/gL/pUL128/130/131 Complex of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause developmental disorders following congenital infection and life-threatening complications among transplant patients. Potent neutralizing monoclonal antibodies (MAbs) are promising drug candidates against HCMV infection. HCMV can infect a broad range of cell types. Therefore, single neutralizing antibodies targeting one HCMV glycoprotein often lack either potency or broad cell-type coverage. We previously characterized two human-derived HCMV neutralizing MAbs. One was the broadly neutralizing MAb 3-25, which targets the antigenic domain 2 of glycoprotein B (gB). The other was the highly potent MAb 2-18, which specifically recognizes the gH/gL/pUL128/130/131 complex (pentamer). To combine the strengths of gB- and pentamer-targeting MAbs, we developed an IgG-single-chain variable fragment (scFv) bispecific antibody by fusing the 2-18 scFv to the heavy-chain C terminus of MAb 3-25. The resulting bispecific antibody showed high-affinity binding to both gB and pentamer. Functionally, the bispecific antibody demonstrated a combined neutralization breadth and potency of the parental MAbs in multiple cell lines and inhibited postinfection viral spreading. Furthermore, the bispecific antibody was easily produced in CHO cells at a yield above 1 g/liter and showed a single-dose pharmacokinetic profile comparable to that of parental MAb 3-25 in rhesus macaques. Importantly, the bispecific antibody retained broadly and potent neutralizing activity after 21 days in circulation. Taken together, our research provides a proof-of-concept study for developing bispecific neutralizing antibody therapies against HCMV infection.

Identification of adipocyte plasma membrane-associated protein as a novel modulator of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause disability in newborns and serious clinical diseases in immunocompromised patients. HCMV has a large genome with enormous coding potential; its viral particles are equipped with complicated glycoprotein complexes and can infect a wide range of human cells. Although multiple host cellular receptors interacting with viral glycoproteins have been reported, the mechanism of HCMV infection remains a mystery. Here we report identification of adipocyte plasma membrane-associated protein (APMAP) as a novel modulator active in the early stage of HCMV infection. APMAP is necessary for HCMV infection in both epithelial cells and fibroblasts; knockdown of APMAP expression significantly reduced HCMV infection of these cells. Interestingly, ectopic expression of human APMAP in cells refractory to HCMV infection, such as canine MDCK and murine NIH/3T3 cells, promoted HCMV infection. Furthermore, reduction in viral immediate early (IE) gene transcription at 6 h post infection and delayed nucleus translocation of tegument delivered pp65 at 4 h post infection were detected in APMAP-deficient cells but not in the wildtype cells. These results suggest that APMAP plays a role in the early stage of HCMV infection. Results from biochemical studies of APMAP and HCMV proteins suggest that APMAP could participate in HCMV infection through interaction with gH/gL containing glycoprotein complexes at low pH and mediate nucleus translocation of tegument pp65. Taken together, our results suggest that APMAP functions as a modulator promoting HCMV infection in multiple cell types and is an important player in the complex HCMV infection mechanism.

New PCSK9 inhibitor miR-552-3p reduces LDL-C via enhancing LDLR in high fat diet-fed mice.

PCSK9 has emerged as a promising new therapeutic target for hyperlipidemia. The efficacy of PCSK9 siRNA in clinic trials clues the feasibility of exploring more PCSK9 inhibitors based on genetic inhibition in the treatment of hyperlipidemia. MicroRNAs (miRNAs) as a class of endogenous non-coding small RNAs can regulate genes at transcriptional and/or translational level. Here, we screened miRNAs from the prediction of TargetScan database with possible inhibitory activities in PCSK9 protein level via AlphaLISA and Western blotting, in which miR-552-3p was selected out for its strongest inhibitory effect. MiR-552-3p could bind to the 3' untranslated region (3'-UTR) of PCSK9 to inhibit translation and interact with the promoter of PCSK9 to suppress transcription. Further in vitro and in vivo experiments proved the effects of miR-552-3p on PCSK9 and downstream effectors: it could increase LDLR protein level, promote LDL-C uptake in HepG2 cells and lower serum LDL-C in high fat diet (HFD)-fed mice. In conclusion, our findings firstly identified miR-552-3p as a new PCSK9 inhibitor with the dual-inhibition mechanism, which suggested the possible application of miR-552-3p in the treatment of hyperlipidemia.

GADD45ß stabilized by direct interaction with HSP72 ameliorates insulin resistance and lipid accumulation.

Growth arrest and DNA damage-inducible 45ß (GADD45ß) belongs to the GADD45 family which is small acidic proteins in response to cellular stress. GADD45ß has already been reported to have excellent capabilities against cancer, innate immunity and neurological diseases. However, there is little information regard GADD45ß and non-alcoholic fatty liver disease (NAFLD). In the current work, we found that the expression of GADD45ß was markedly decreased in the livers of NAFLD patients via analyzing Gene Expression Omnibus (GEO) dataset and in mouse model through detecting its mRNA in high-fat-high-fructose diet (HFHFr)-fed mice. Moreover, the results from in vivo experiment demonstrated that overexpression of GADD45ß by AAV8-mediated gene transfer in HFHFr-fed mouse model could reduce the level of serum and hepatic triglyceride (TG), and alleviate insulin resistance. Subsequently, by combining immunoprecipitation (IP) and mass spectrometry, we identified that HSP72 directly interacted with GADD45ß to prevent GADD45ß from being degraded by the proteasome pathway. Finally, the benefits of GADD45ß in regulating key factors of TG synthesis and insulin signaling pathway were abolished after HSP72 knockdown. In conclusion, GADD45ß stabilized by the interaction with HSP72 could alleviate the NAFLD-related pathologies, suggested it might be a potential target for the treatment of NAFLD.

Comparison of N-linked glycosylation on hemagglutinins derived from chicken embryos and MDCK cells: a case of the production of a trivalent seasonal influenza vaccine.

N-linked glycosylation plays critical roles in folding, receptor binding, and immunomodulating of hemagglutinin (HA), the main antigen in influenza vaccines. Chicken embryos are the predominant production host for influenza vaccines, but Madin-Darby canine kidney (MDCK) cells have emerged as an important alternative host. In this study, we compared glycosylation patterns, including the occupancy of potential glycosylation sites and the distribution of different glycans, on the HAs of three strains of influenza viruses for the production a trivalent seasonal flu vaccine for the 2015-2016 Northern Hemisphere season (i.e., A/California/7/2009 (H1N1) X179A, A/Switzerland/9715293/2013 (H3N2) NIB-88, and B/Brisbane/60/2008 NYMC BX-35###). Of the 8, 12, and 11 potential glycosylation sites on the HAs of H1N1, H3N2, and B strains, respectively, most were highly occupied. For the H3N2 and B strains, MDCK-derived HAs contained more sites being partially occupied (<95%) than embryo-derived HAs. A highly sensitive glycan assay was developed where 50 different glycans were identified, which was more than what has been reported previously, and their relative abundance was quantified. In general, MDCK-derived HAs contain more glycans of higher molecular weight. High-mannose species account for the most abundant group of glycans, but at a lower level as compared to those reported in previous studies, presumably due to that lower abundance, complex structure glycans were accounted for in this study. The different glycosylation patterns between MDCK- and chicken embryo-derived HAs may help elucidate the role of glycosylation on the function of influenza vaccines. KEY POINTS: • For the H3N2 and B strains, MDCK-derived HAs contained more partially (<95%) occupied glycosylation sites. • MDCK-derived HAs contained more glycans of higher molecular weight. • A systematic comparison of glycosylation on HAs used for trivalent seasonal flu vaccines was conducted.

Applications of DNA-Functionalized Proteins.

As an important component that constitutes all the cells and tissues of the human body, protein is involved in most of the biological processes. Inspired by natural protein systems, considerable efforts covering many discipline fields were made to design artificial protein assemblies and put them into application in recent decades. The rapid development of structural DNA nanotechnology offers significant means for protein assemblies and promotes their application. Owing to the programmability, addressability and accurate recognition ability of DNA, many protein assemblies with unprecedented structures and improved functions have been successfully fabricated, consequently creating many brand-new researching fields. In this review, we briefly introduced the DNA-based protein assemblies, and highlighted the limitations in application process and corresponding strategies in four aspects, including biological catalysis, protein detection, biomedicine treatment and other applications.

DNA-Grafted 3D Superlattice Self-Assembly.

The exploitation of new methods to control material structure has historically been dominating the material science. The bottom-up self-assembly strategy by taking atom/molecule/ensembles in nanoscale as building blocks and crystallization as a driving force bring hope for material fabrication. DNA-grafted nanoparticle has emerged as a "programmable atom equivalent" and was employed for the assembly of hierarchically ordered three-dimensional superlattice with novel properties and studying the unknown assembly mechanism due to its programmability and versatility in the binding capabilities. In this review, we highlight the assembly strategies and rules of DNA-grafted three-dimensional superlattice, dynamic assembly by different driving factors, and discuss their future applications.