In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein.

COVID-19 is caused by SARS-CoV-2 infection and remains one of the biggest pandemics around the world since 2019. Vaccination has proved to be an effective way of preventing SARS-CoV-2 infection and alleviating the hospitalization burden. Among different forms of COVID-19 vaccine design, the spike protein of SARS-CoV-2 virus is widely used as a candidate vaccine antigen. As a surface protein on the virus envelop, the spike was reported to be heavily N-glycosylated and glycosylation had a great impact on its immunogenicity and efficacy. Besides, N-glycosylation might vary greatly on different expression systems and sequence variant designs. Therefore, comprehensive analysis of spike N-glycosylation is of great significance for better vaccine understanding and quality control. In this study, full characterization of N-glycosylation was performed for a Chinese Hamster Ovary (CHO) cell expressed variant-designed spike protein. The spike protein featured the latest six-proline substitution design together with the incorporation of a combination of mutation sites. Trypsin and Glu-C digestion coupled with PNGase F strategies were adopted, and effective LC-MS/MS methods were applied to analyze samples. As a result, a total of 19 N-glycosites were identified in the recombinant pike protein at intact N-glycopeptide level. Quantitative analysis of released glycan by LC-MS/MS was also performed, and 31 high-abundance N-glycans were identified. Sequencing analysis of glycan was further provided to assist glycan structure confirmation. Moreover, all of the analyses were performed on three consecutive manufactured batches and the glycosylation results on both glycosite and glycans showed good batch-to-batch consistency. Thus, the reported analytical strategy and N-glycosylation information may well facilitate studies on SARS-CoV-2 spike protein analysis and quality studies.

Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here, we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.

Critical Role of Order-Disorder Behavior in Perovskite Ferroelectric KNbO3.

The evolution of local atomic configuration and macroscopic electrical properties of KNbO3 is investigated in detail using ab initio molecular dynamics simulations within the framework of density functional theory. By analyzing the local off-center displacements of K and Nb atoms, the presence of the crossover of displacive and order-disorder mechanisms in the NbO6 octahedron due to orbital hybridization is found, whereas the K is a completely displacive type due to the nonoriented ionic K-O bond. Besides, the order-disorder behavior of Nb atoms is enhanced with the increase in temperature, especially undergoing the phase transition. The predicted high dielectric constant of KNbO3 confirms the key role of the Nb's order-disorder behavior. We discover that the anomalous increase in dielectric constant in the vicinity of the phase boundary is not only from the transformation of the polarized direction but also from the enhancement of order-disorder contribution in the nonpolarized direction. The high dielectric constant with large spontaneous polarization boosts the piezoelectricity at the orthorhombic-tetragonal phase boundary of 500 K, giving rise to the nearly 100% increase than that of 300 K. A rebonding model is developed to illustrate the origin of high piezoelectricity around the tetragonal ferroelectric phase boundary, and the significance of introducing a tetragonal structure is emphasized for developing high piezoelectricity in the inorganic perovskite ferroelectrics.

Dissipation and residue determination of penicillin G and its two metabolites in citrus under field conditions by DSPE/UPLC-MS/MS.

A rapid determination method of residual penicillin G and its two metabolites in citrus was developed and validated by dispersive solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (DSPE/UPLC-MS/MS). The samples were extracted with 80% acetonitrile and purified with octadecylsilane. High linearity was obtained with correlation coefficients (r2 ) >0.9981. The limits of quantification were 0.005-0.01 mg/kg. The recoveries of penicillin G and its metabolites spiked in blank citrus were within 76.7-107%, with relative standard deviations of 1.3-9.6%. The dissipation dynamics and distribution of penicillin G in citrus followed first-order kinetics, with half-life of 1.7-2.7 days. Penicillin G degraded easily in citrus and the metabolite was mainly penilloic acid, which can exist stably for long time. The terminal residues of penicillin G in pulp, whole citrus and peels were 0.015-0.701, 0.047-7.653 and 0.162-13.376 mg/kg, respectively. The hazard indexes for risk assessment of citrus were significantly <1, suggesting that the health risks to humans after consumption of citrus were insignificant and negligible. These results could provide necessary data for evaluating the safe and proper use of penicillin G in citrus.

Size-controlled excitonic effects on electronic and optical properties of Sb2S3 nanowires.

In this work, the electronic and optical properties of one-dimensional (1D) Sb2S3 nanowires (NWs) with different sizes are investigated using first-principles calculations. The indirect-direct band transition of Sb2S3 NWs can be tuned effectively by the NW size and various uniaxial strains. In the Sb2S3 NWs, the quantum confinement effects result in wider bandgaps while the significantly enhanced electron-hole interaction that is expected to produce excitonic bound states generates a bandgap narrowing. The exciton binding energies for the Sb2S3 NWs are predicted by the effective masses of electrons and holes to lie in the range of 0-1 eV, which are larger than that of bulk Sb2S3, suggesting that excitons in Sb2S3 NWs may bind possible defects to promote luminescence. The size-controlled absorption edge blueshift and redshift of Sb2S3 NWs suggest that Sb2S3 NWs may be promising in the applications of nanoscale light emitting devices.

Structure features of GH10 xylanase from Caldicellulosiruptor bescii: implication for its thermophilic adaption and substrate binding preference.

Caldicellulosiruptor bescii is the most thermophilic cellulolytic species of organisms known to date. In our previous study, GH10 xylanase CbXyn10B from C. bescii displayed outstanding hydrolytic activity toward various xylans at high temperatures. To understand the structural basis for this protein's catalysis and thermostability, we solved the crystal structures of CbXyn10B and its complexes with xylooligosaccharides. These structural models were used to guide comparison with its mesophilic counterpart PbXyn10B. A distinctive structural feature is that thermophilic CbXyn10B presents a relatively stable interaction between the extended loops L7 and L8 in the catalytic cleft by an extensive hydrogen bonding network, which is mediated by Lys306, Arg314 and three well-ordered water molecules. Moreover, a unique aromatic cluster consisting of Try17, Phe20, Phe21, and Phe337 may enhance the interaction between the N- and C- terminus. Targeted mutagenesis demonstrated that these interactions substantially contribute to enzyme stabilization, as indicated by a considerable decrease in the melting temperature (Tm) of CbXyn10B by substituting critical residues with Ala. Therefore, it was shown that not only the aromatic interaction connecting protein termini but also the extensive hydrogen bonding network formed between surface loops could restrict the local structural flexibility and contribute significantly to the overall stability of enzymes. Furthermore, the xylooligosaccharides were found to tightly bind to the glycone subsites of xylanase, indicating higher affinities at these subsites and reflecting its substrate binding preference. Our results suggest that CbXyn10B is stabilized with distinct rigidity at the catalytic cleft as well as the terminal regions, which provides insights into the evolutionary strategy for accommodating the functional needs of GH10 enzymes to high temperature.

Enhanced enzyme kinetic stability by increasing rigidity within the active site.

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

Physical inability rather than depression and cognitive impairment had negative effect on centenarian prognosis: A prospective study with 5-year follow-up.

AIM: Scarce study has involved the effects of physical inability, depression and cognitive impairment on the prognosis of older individuals, especially in Chinese centenarians. This prospective study was designed to investigate the effects with 5-year follow-up in Chinese centenarians. METHODS: According to the list of centenarians provided by Department of Civil Affairs, an household survey was conducted on all centenarians residing in 18 cities and counties of Hainan province. A total of 423 centenarians were followed up, including 84 survival centenarians and 261 dead centenarians, with 78 cases lost to follow-up. RESULTS: Dead centenarians had less females and more physical inability than survival centenarians (P < 0.05 for all). Univariable Cox regression analyses indicated that physical inability [EXP(B): 2.038, 95 % confidence interval (CI): 1.413-2.939], urea nitrogen [EXP(B): 1.116, 95 % CI: 1.039-1.199], and creatinine [EXP(B): 1.006, 95 % CI: 1.001-1.012] had negative effects on the prognosis of centenarians (all P < 0.05). Gender [EXP(B): 0.606, 95 % CI: 0.391-1.940] and albumin [EXP(B): 0.939, 95 % CI: 0.896-0.985] had positive effects on the prognosis of centenarians (all P < 0.05). Multivariable Cox regression analysis indicated that physical inability [EXP(B): 2.148, 95 % CI: 1.454-3.173] and urea nitrogen [EXP(B): 1.114, 95 % CI: 1.020-1.216] had negative effects on the prognosis of centenarians (all P < 0.05). CONCLUSIONS: For Chinese centenarians, this prospective study demonstrated that physical inability rather than depression and cognitive impairment had negative effect on the long-term mortality rate and survival time. This result suggested that in order to improve the prognosis of older adults, it could be mainly achieved by improving physical ability.

Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides.

The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (k(cat)/K(m)) for the OP pesticide ethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 10(5)-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

A potent, broadly protective vaccine against SARS-CoV-2 variants of concern.

Since the first outbreak in December 2019, SARS-CoV-2 has been constantly evolving and five variants have been classified as Variant of Concern (VOC) by the World Health Organization (WHO). These VOCs were found to enhance transmission and/or decrease neutralization capabilities of monoclonal antibodies and vaccine-induced antibodies. Here, we successfully designed and produced a recombinant COVID-19 vaccine in CHO cells at a high yield. The vaccine antigen contains four hot spot substitutions, K417N, E484K, N501Y and D614G, based on a prefusion-stabilized spike trimer of SARS-CoV-2 (S-6P) and formulated with an Alum/CpG 7909 dual adjuvant system. Results of immunogenicity studies showed that the variant vaccine elicited robust cross-neutralizing antibody responses against SARS-CoV-2 prototype (Wuhan) strain and all 5 VOCs. It further, stimulated a TH1 (T Helper type 1) cytokine profile and substantial CD4+ T cell responses in BALB/c mice and rhesus macaques were recorded. Protective efficacy of the vaccine candidate was evaluated in hamster and rhesus macaque models of SARS-CoV-2. In Golden Syrian hamsters challenged with Beta or Delta strains, the vaccine candidate reduced the viral loads in nasal turbinates and lung tissues, accompanied by significant weight gain and relieved inflammation in the lungs. In rhesus macaque challenged with prototype SARS-CoV-2, the vaccine candidate decreased viral shedding in throat, anal, blood swabs over time, reduced viral loads of bronchus and lung tissue, and effectively relieved the lung pathological inflammatory response. Together, our data demonstrated the broadly neutralizing activity and efficacy of the variant vaccine against both prototype and current VOCs of SARS-CoV-2, justifying further clinical development.

Emergence and Tuning of Multiple Flat Bands in Twisted Bilayer γ-Graphyne.

Using twisted bilayer γ-graphyne (TBGY) as an example, we show that it is possible to create multiple flat bands in carbon allotropes without the requirement of a specified magic angle. The origin of the flat bands can be understood by a simple two-level coupling model. The narrow bandwidth and strong localization of the flat band states might lead to strong correlation effects, which make TBGY a good platform for studying correlation physics. On the basis of the two-level coupling model, we further propose that the width and extent of localization of flat bands can be tuned by an energy mismatch ΔE between the two layers of TBGY, which can be realized by either applying a perpendicular electric field or introducing a heterostrain. This allows continuous modulation of TBGY from the strong-correlation regime to the medium- or weak-correlation regime, which could be utilized to study the quantum phase transition.

Development of recombinant COVID-19 vaccine based on CHO-produced, prefusion spike trimer and alum/CpG adjuvants.

COVID-19 pandemic has severely impacted the public health and social economy worldwide. A safe, effective, and affordable vaccine against SARS-CoV-2 infections/diseases is urgently needed. We have been developing a recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminium hydroxide and CpG 7909. The spike protein was expressed in Chinese hamster ovary (CHO) cells, purified, and prepared as a stable formulation with the dual adjuvant. Immunogenicity studies showed that candidate vaccines elicited robust neutralizing antibody responses and substantial CD4+ T cell responses in both mice and non-human primates. And vaccine-induced neutralizing antibodies persisted at high level for at least 6 months. Challenge studies demonstrated that candidate vaccine reduced the viral loads and inflammation in the lungs of SARS-CoV-2 infected golden Syrian hamsters significantly. In addition, the vaccine-induced antibodies showed cross-neutralization activity against B.1.1.7 and B.1.351 variants. These data suggest candidate vaccine is efficacious in preventing SARS-CoV-2 infections and associated pneumonia, thereby justifying ongoing phase I/II clinical studies in China (NCT04982068 and NCT04990544).

Effect of sodium butyrate on ABC transporters in lung cancer A549 and colorectal cancer HCT116 cells.

Histone deacetylase (HDAC) inhibitors and DNA alkylators are effective components of combination chemotherapy. The aim of the present study was to investigate the possible mechanism of their synergism by detecting the effect of HDAC inhibitors on the expression levels of drug transporters that export DNA alkylators. It was demonstrated that the HDAC inhibitor sodium butyrate (NaB) induced the differential expression of multidrug resistant ATP-binding cassette (ABC) transporters in lung cancer and colorectal cancer cells. Specifically, NaB increased the mRNA expression levels of ABC subfamily B member 1 (ABCB1), ABCC10 and ABCC12, and protein expression levels of multidrug resistance-1 (MDR1), multidrug resistance-associated protein 7 (MRP7) and MRP9. Moreover, NaB decreased the expression levels of ABCC1, ABCC2 and ABCC3 mRNAs, as well as those of MRP1, MRP2 and MRP3 proteins. The molecular mechanism underlying this process was subsequently investigated. NaB decreased the expression of HDAC4, but not HDAC1, HDAC2 or HDAC3. In addition, NaB promoted histone H3 acetylation and methylation at lysine 9, as well as MDR1 acetylation, suggesting that acetylation and methylation may be involved in NaB-mediated ABC transporter expression. Thus, the present results indicated that the synergism of the HDAC inhibitors with the DNA alkylating agents may due to the inhibitory effect of MRPs by HDAC inhibitors. The findings also suggested the possibility of antagonistic effects following the combined treatment of HDAC inhibitors with MDR1 ligands.

Modulation of the thermostability and substrate specificity of Candida rugosa lipase1 by altering the acyl-binding residue Gly414 at the α-helix-connecting bend.

Candida rugosa Lipase1 (LIP1) is widely used in industrial applications. Optimizing its catalytic performance is still a challenging goal for protein engineers. Mutagenesis of key residues in the active site of the enzyme may provide an effective strategy for enhancing stability and altering substrate specificity. In this study, multiple sequence alignment and structural analysis revealed that the acyl-binding residue, Gly414, of LIP1, which is located at a bend connecting α-helixes, was the non-conserved residue in five other isoenzymes. Using saturation mutagenesis, four mutants with improved stability (G414A, G414M, G414H and G414W) were obtained. Compared to the wild type, the best mutant (G414W) exhibited a remarkable 6.5-fold enhancement in half-life at 60 °C and a 14 °C higher T50(15). Its optimum temperature was increased by 15 °C. Simultaneously, G414W displayed a shift in substrate preference from medium-chain to short-chain pNP-ester. Modeling analysis showed that the multiple interactions formed by hydrophobic clusters and hydrogen bonds in the acyl-binding tunnel might lead to the observed thermostability improvement. Additionally, the bulky tryptophan substitution formed a strong steric hindrance to the accommodation of long-chain substrates in the tunnel. These results indicate that the key acyl-binding residues at the α-helix-connecting bend could mediate enzyme stability and catalytic substrate spectra.

Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Šoutward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.