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One-dimensional benzo[1,2-b:4,5-b′]dithiophene-based graphdiyne (1D-BDT-GDY) shows enhanced electrochemical adsorption ability for chloramphenicol (CAP).

The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24 °C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24 °C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

In order to timely detect road damage under complex constraints, the road damage mechanism was used to reclassify the types of road damage, the VGG-19 model was applied to identify and detect less road damage images intelligently through convolutional neural network and transfer learning and at the same time, the road damage situation was detected using the Softmax classifier and the feasibility and accuracy of the method were verified on the basis of the detection set. The results show that the detection and recognition accuracy of the model proposed in this paper reaches 86.2 %, an increase of 6.2-49.8 percentage points than the detection results of other convolutional neural network models. Therefore, it can be concluded that the transfer learning- and convolutional neural network-based road damage intelligent detection methods proposed in this paper are feasible, and this research is helpful to realize high-precision real-time intelligent detection of road damage.

The carbon-based metal-free catalyst is one of the ideal alternatives to Pt as electrocatalysts for oxygen reduction reaction, which can reduce the cost of fuel cells and zinc-air batteries. Here, graphdiyne(GDY), a carbon material with uneven charge distribution, was used as substrate. By doping nitrogen and phosphorus, a N-P-GDY catalyst was prepared, which further regulated the electron structure of GDY. The sheet-like morphology of GDY was preserved in N-P-GDY. The N and P were distributed uniformly in the catalyst, whereas defects and active sites were created by doping N and P, as demonstrated by the element mapping images and Raman spectra. X-Ray photoelectron spectroscopy results indicated N and P existed in many forms in N-P-GDY. The N-P-GDY exhibited higher activity for ORR than only N or P doped GDY, due to the synergistic effect of N and P in N-P-GDY. Moreover, the activity of N-P-GDY changed little after a long time cyclic voltammetry test or injecting methanol in the electrolyte. Besides, the four electrons transfer reaction to produce water was the main process for ORR on N-P-GDY catalysts.

Antibiotic resistance has become a global crisis in recent years, while wastewater treatment plants (WWTPs) have been identified as a significant source of both antibiotic resistant bacteria (ARB) ...

A new type of highly active and cost-effective nanocomposite electrocatalyst Ni–Fe–P–FeMnO3 on nickel foam (NF) supports is designed and prepared by electroless composite deposition and rapid dealloying. The nanocomposite electrocatalyst Ni–Fe–P–FeMnO3/NF presents a more remarkable electrocatalytic performance than Pt/C noble metal catalysts towards hydrogen evolution reaction (HER), which demands a quite low overpotential of 16.62 mV at −10 mA cm−2 current density in 1 M KOH. The prepared Ni–Fe–P–FeMnO3/NF electrocatalyst exhibits remarkable oxygen evolution reaction (OER) activity as well, which requires only 297 mV and 219 mV of overpotential at 50 mA cm−2 and 10 mA cm−2 current density, respectively. Moreover, the overall water splitting activity of Ni–Fe–P–FeMnO3/NF as both anode and cathode achieves a low potential of 1.63 V (vs. RHE) at a current density of 10 mA cm−2.

Genome engineering tools in bacteria are limited by their targeting abilities, cargo size capacities, and integration efficiencies. Programmable Cas-directed transposons have been shown to bypass these constraints; however, genome integrations with these Cas-directed transposons require a cargo plasmid carrying the desired DNA payload flanked by directed repeat transposon arms. This cloning pre-requisite significantly hinders the modularity and streamlining capabilities of Cas-directed transposon systems, diminishing their utility for genome engineering. Here, we present a system that can robustly integrate a linear DNA payload into the genome ofE. coliby employing a Type I-F CRISPR-associated transposon fromVibrio cholerae. This system bypasses the traditional limiting factors of Cas-directed transposons by leveraging oligonucleotide design and nested polymerase chain reactions to reconstitute the transposon arms onto a designated cargo. Our findings demonstrate that this programmable linear integration method has high efficiencies in integrating large DNA payloads across multiple genomic loci.

The development of electrocatalysts with high activity and low Tafel slope for overall water splitting has become a crucial challenge to exploit the sustainable energy. Herein, we construct a Fe–Co–P–Gr catalyst on nickel foam (NF) support through electroless composite plating to realize the co-deposition of Fe–Co–P alloys and graphene quantum dots. Interestingly, graphene quantum dots exhibit obvious effects on electron mobility and active sites of Fe–Co–P–Gr/NF catalyst. In oxygen evolution reaction, the Fe–Co–P–Gr/NF catalyst exhibits a small overpotential of 230 mV at 10 mA cm−2 and fast kinetics with Tafel slope of 37.8 mV dec−1. Meanwhile, the Fe–Co–P–Gr/NF also has a superior hydrogen evolution reaction performance in 1.0 M KOH. Compared with the Fe–Co–P alloys, the Fe–Co–P–Gr/NF both as the anode and cathode require only 1.58 V to reach a current density of 10 mA cm−2. The successful preparation of Fe–Co–P–Gr/NF electrode through electroless composite deposition provides a new path to manufacture electrocatalysts for overall water splitting.

The development of highly efficient and superior durability electrocatalysts is vital to expedite hydrogen evolution reaction (HER). Herein, a mixed amorphous and nano-crystalline Ni–Co–Fe–P alloy on Ni foam after 75 s dealloying in 3 M HCl (Ni–Co–Fe–P/NF-3-75) is synthesized by the preparation strategy of two-step method consisting of electroless deposition and dealloying process. Ni–Co–Fe–P/NF-3-75 shows an excellent HER performance and high durability in both alkaline and neutral conditions by optimizing the composition of the catalysts, acid concentration, and the time of dealloying. Benefitting from the high conductivity of Ni foam carrier, coordination between polymetallic phases, and the large exposure of defects, the as-prepared Ni–Co–Fe–P/NF-3-75 requires only a low overpotential of 56 mV and 104 mV to reach the current density of 10 mA cm−2 in 1.0 M KOH and 1.0 M phosphate buffer (PBS), respectively. Remarkably, the Ni–Co–Fe–P/NF-3-75 electrode exhibits superior cycling stability and long-term robust durability without obvious overpotential decline. The successful preparation of the Ni–Co–Fe–P/NF-3-75 catalyst indicates that this method provides an efficient way to synthesize polymetallic phosphides for hydrogen evolution reaction.

Significance During pathogenic invasion, neutralizing antibodies (nAbs) are involved in regulating immune clearance and evoking the host-protective response. We previously reported a highly potent nAb 8C11 against HEV, an RNA virus with an icosahedral capsid and associated with abundant acute hepatitis. Structural analysis demonstrates that the binding of 8C11 to HEV VLPs would result in tremendous spatial clashing with the capsid. Cryo-EM analysis showed that 8C11 binding leads to complete disorder of the outer rim of the VLP at earlier stages (∼15 min) and causes the dissociation of HEV VLPs into homodimer species within 2 h. Similar 8C11-mediated dissociation was observed for the native HEV virion. Our results categorize a viral neutralization mechanism and suggest a strategy to generate 8C11-like antibodies., In adaptive immunity, organisms produce neutralizing antibodies (nAbs) to eliminate invading pathogens. Here, we explored whether viral neutralization could be attained through the physical disruption of a virus upon nAb binding. We report the neutralization mechanism of a potent nAb 8C11 against the hepatitis E virus (HEV), a nonenveloped positive-sense single-stranded RNA virus associated with abundant acute hepatitis. The 8C11 binding flanks the protrusion spike of the HEV viruslike particles (VLPs) and leads to tremendous physical collision between the antibody and the capsid, dissociating the VLPs into homodimer species within 2 h. Cryo-electron microscopy reconstruction of the dissociation intermediates at an earlier (15-min) stage revealed smeared protrusion spikes and a loss of icosahedral symmetry with the capsid core remaining unchanged. This structural disruption leads to the presence of only a few native HEV virions in the ultracentrifugation pellet and exposes the viral genome. Conceptually, we propose a strategy to raise collision-inducing nAbs against single spike moieties that feature in the context of the entire pathogen at positions where the neighboring space cannot afford to accommodate an antibody. This rationale may facilitate unique vaccine development and antimicrobial antibody design.

Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium butyricum, induces DNA interference between homologous sequences and generates double-stranded breaks (DSBs) in target DNAs. This mechanism enables the host to defend against invading DNAs such as plasmids and viruses. However, whether such a CbAgo-mediated DNA cleavage is mutagenic remains unexplored. Here we demonstrate that CbAgo, directed by plasmid-encoded guide sequences, can cleave genome target sites and induce chromosome recombination between downstream homologous sequences in Escherichia coli. The recombination rate correlates well with pAgo DNA cleavage activity and the mechanistic study suggests the recombination involves DSBs and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. We anticipate this novel DNA-guided interference to be useful in broader genetic manipulation. Our study also provides an in vivo assay to characterize or engineer pAgo DNA cleavage activity.

BackgroundThe various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli.ResultsOf 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD­—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. ConclusionReengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Background Bacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design. Various outer membrane proteins, such as outer membrane protein A (OmpA), OmpC and outer membrane pore protein E precursor (PhoE), have been used as carriers for surface display, fused to the proteins or peptides of interest in Gram-negative bacteria. Here, we investigated the utility of constitutively expressed OmpF for the display of foreign immune epitopes on the Escherichia coli cell surface and then compared it with plasmid-induced expression of OmpF and OmpC. Results Enhanced expression of OmpF was linked to a mutation in the OmpF promoter sequence. This mutation rendered OmpF an ideal carrier protein for the enriched display of a target of interest on the bacterial surface. To this end, we grafted two peptides, harboring important epitopes of the hepatitis B virus (HBV) S antigen and human papilloma virus (HPV) L2 protein, onto OmpF of E. coli by genome editing. The resultant fused OmpF proteins were constitutively expressed in the edited E. coli and purified by membrane component extraction. The epitope that displayed on the bacterial surface was verified by SDS-PAGE, western blotting, flow cytometry, and immunoelectron microscopy of the intact bacteria. We further compared this constitutive expression with plasmid-induced expression of OmpF and OmpC in bacterial cells using the same methods for verification. We found that plasmid-induced expression is much less efficient than constitutive expression of OmpF from the bacterial genome. Conclusions Enhanced expression of OmpF in a plasmid-independent manner provides an amenable way to display epitopes on the bacterial surface and sheds light on ways to engineer bacteria for biotechnological applications. Electronic supplementary material The online version of this article (10.1186/s12934-019-1120-2) contains supplementary material, which is available to authorized users.

In this paper, we report a method that the transition element nickel is doped into the interstitial position of CdZnS (CZS) nanoparticles for their photocatalytic hydrogen evolution activity boosting. It is demonstrated that CdZnNiS nanocrystalline materials (with 0.5 wt% of Ni) could achieve the highest photocatalytic H2-evolution activity of 25.4 mmol·g−1·h−1 in sulfide and sulfite solution under visible light which value is 1.16 times higher than that of Pt modified CdZnS particles (with 3 wt% of Pt). This work evidences the possibility of substitution of noble metal cocatalyst by adding a suitable inexpensive Ni to achieve high-efficient visible light driven water splitting hydrogen production.

The magnesium oxide modified TiO2 nanotube arrays (TiO2 NTs) was prepared to investigate the photoelectrochemical (PEC) cathodic protection performance to 304 stainless steel (SS 304). With the annealing treatment at different temperature, the surface of the modified materials changed remarkably. The film-like Mg(OH)2 obtained at 400 °C transformed to MgO particles after treatment at 500 °C and 600 °C. Further thermal processing at 700 °C enables MgTixOy@TiO2 shell-core structure. Using photoelectrochemical approaches to study the series samples, the cathodic protection performance of the modified TiO2 NTs has significantly improved. The magnesium oxides modified TiO2 NTs treated at 600 °C (Ti-Mg-O 600) shows the best PEC cathodic protection to the underneath steel. Further investigation shows that the highly enhanced PEC cathodic protection performance of Ti-Mg-O 600 was attributed to the effective separation of photogenerated carriers, the increase of carrier concentration and the negative shift of the conduction band potential.

At present, it is difficult for electrocatalytic electrode materials with high-Performance to be prepared at low cost and large area under mild conditions. Therefore, we adopt a facile electroless plating method to deposit the Fe Co P alloys on the nickel foam (NF) with different areas of 1 cm2, 4 cm2, 8 cm2 and 16 cm2. The Fe Co P/NF catalysts exhibit extraordinary catalytic activity for the oxygen evolution reaction (OER) in alkaline media and are comparable to the state-of-the-art IrO2 in 1.0 M KOH, capable of yielding a current density of 10 mA cm−2 at an overpotential of only 250 mV. Furthermore, the Fe Co P/NF catalysts show efficient activity towards the hydrogen evolution reaction (HER) with an overpotential of 163 mV at j = 10 mA cm−2 as well. Remarkably, when used as both the anode and cathode, a low potential of 1.68 V (vs. RHE) is required to reach the current density of j = 10 mA cm−2, making the Fe Co P/NF alloys as an active bifunctional electrocatalyst for overall water splitting. The Fe Co P/NF alloy catalysts with high catalytic activity, facile preparation and low cost would provide a new pathway for the design and large-scale application of high-performance bifunctional catalysts for electrochemical water splitting.

Amorphous materials have been extensively studied in the field of catalysis due to their unique properties. Herein, we report a nanoporous and amorphous Ni–Fe–P array on Ni foam, which acts as a highly efficient overall water splitting catalyst synthesized by electroless deposition and fast dealloying.

We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7–10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR–Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes. This protocol describes a recombination-based approach for accurate replacement of genomic DNA with large synthetic DNA fragments in E. coli (REXER). Iterative repetition of REXER (GENESIS) combined with genome assembly methods enables the creation of custom synthetic E. coli genomes.

BackgroundTheEscherichia coliER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of theE. coliER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure.ResultsThe reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3′ non-coding region) positioned 19-bp away from thelacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing.ConclusionThe ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of theE. coliER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications.

Additional file 12: Figure S2. The workflow of CDSs reannotation.

Hepatitis E virus (HEV) is associated with acute hepatitis disease. Numerous truncated HEV capsid proteins have been successfully expressed using different expression systems. Among these, p495, a protein truncated at its N- and C-termini by 111 and 54 amino acids (aa), respectively (HEV ORF2 aa 112–606) can self-assemble into T = 1 virus-like particles (VLPs) when expressed by insect cells. A shorter p239 (aa 368–606) protein is a particulate antigen that we have previously used in our commercialized HEV vaccine, Hecolin. Here, we sought to express p495 in its soluble form (named Ep495) in E. coli and in baculovirus-infected Tn5 insect cells (named BTp495) as a back-to-back control. Characterization of p495 particles derived from these two expression systems showed similarities in particle size, morphology, and sedimentation coefficient. Antigenicity assays using a panel of anti-HEV monoclonal antibodies also showed similar strong reactivities for Ep495 and BTp495, as well as similar binding profiles that were congruent with p239. Mouse immunization results showed that Ep495 particles had comparable immunogenicity with that of BTp495 VLPs, as well as p239. Overall, our findings suggest that p495 particles produced in E. coli are ideal for the development of next-generation prophylactic vaccines against hepatitis E.

Exploring high-performance noble-metal-free bifunctional electrocatalysts is vitally crucial for hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) in water electrolysis. Herein, the defect-enriched multistage morphology Ni-Fe-P-Ni3S2 heterogeneous catalyst growth on the Ni Foam (Ni-Fe-P-Ni3S2/NF) is synthesized for the first time through electroless composite plating. Attributed to the heterogeneous structure between Ni-Fe-P alloys and Ni3S2, abundant lattice defects, exposed highly open multistage skeleton morphology, the optimal Ni-Fe-P-Ni3S2/NF catalyst only requires 65 and 219 mV to reach the current density of 10 mA cm−2 towards HER and OER in alkaline solution, respectively. With regard to the overall water splitting, the alkaline electrolyzer assembled by Ni-Fe-P-Ni3S2/NF electrodes exhibits a low cell voltage of 1.5 V at 10 mA cm−2. This research provides a new perspective for the preparation of heterogeneous catalysts between alloys and sulfides for overall water splitting.

Programmable genome engineering The model bacterium Escherichia coli has a single circular chromosome. Wang et al. created a method to fragment the E. coli genome into independent chromosomes that can be modified, rearranged, and recombined. The efficient fission of the unmodified E. coli genome into two defined, stable pairs of synthetic chromosomes provides common intermediates for large-scale genome manipulations such as inversion and translocation. Fusion of synthetic chromosomes from distinct cells generated a single genome in a target cell. Precise, rapid, large-scale genome engineering operations are useful tools for creating diverse synthetic genomes. Science , this issue p. 922

We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7-10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR-Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.

OBJECTIVE To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.

International audience; A sequence of nine almost equidistant quadrupole transitions is observed in Nd137. The sequence represents an extremely regular rotational band that extends to a spin of about 75/2 and an excitation energy of ≈4.5MeV above yrast. Cranked mean-field calculations of the Nilsson-Strutinsky type suggest an oblate shape for the band. They reproduce the observed I(I+1) dependence of the rotational energy whereas predicting a pronounced decrease in the deformation, which is the hallmark of antimagnetic rotation.

Additional file 1: Text S1. Nucleotide sequences of the OmpF locus and the DNA fragments used in gene manipulation.

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon—out of up to 6 synonyms—to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme—with simple corrections at just seven positions—to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA.

Aim: Nanoparticulate design is important for the production of nanotechnological materials and passive immunogens. Using lessons from our hepatitis E vaccine, we herein design protein-based nanoparticles through incorporation of an N-terminal hydrophobic tail (NHT, located on HEV ORF2 aa368–460). Materials & methods: Flu HA1, HIV gp41/gp120/p24, HBsAg and HPV16 L2 were fused with NHT, expressed in Escherichia coli and subjected to self-assembly in vitro. Nanosized particles were characterized by size-exclusion chromatography and negative electron microscopy. Immunogenicity was assessed in mice. Results: All the NHT-fused proteins spontaneously formed nanoparticulates and presented with immunogenicity approximately 2-log over their nonassembling forms. Conclusion: Protein self-assembly provides an attractive means to create nanosized particles that bear specific antigens. Our strategy outlines a novel and shared method for the design of immunogenic nanoparticles.

The Hepatitis E vaccine (Hecolin, licensed in China) harbors a potent particulate immunogen, p239, designed from a 26-aa N-terminal extension of its poorly immunogenic parental protein, E2. Although an effective vaccine, we sought to design a fusion protein in a non-particulate form that could improve the delivery and immunogenicity of E2 epitopes. The non-toxic mutant of diphtheria toxin, CRM197 (Cross-Reacting Material 197) has been successfully used as a carrier protein for conjugated vaccines to enhance the immunogenicity of polysaccharides. Here, we designed a fusion non-particulate protein of E2 and the catalytic domain (fragment A) of CRM197 and evaluated its antigenicity, immunogenicity and disease prevention efficacy in primates. This fusion protein, named CRM197(A)-E2, was bacterially expressed and purified by chromatography. CRM197(A)-E2 presented as a homodimer in solution, similar to its parental E2 protein, and exhibited excellent antigenicity against representative neutralizing monoclonal antibodies, like E2 and p239. However, CRM197(A)-E2 manifested higher immunogenicity in mice compared with that achieved by the particulate p239, as indicated by the 10-times lower ED50 value and 2-log higher HEV-specific antibody level that could persist for at least 28 weeks. In addition, both the 1 μg and 10 μg doses of CRM197(A)-E2 adjuvanted with aluminum could protect vaccinated monkeys against HEV challenge, matching that achieved with only the higher (10 μg) dose of the p239 vaccine. These results suggest that the CRM197 fragment A alone serves as an intra-molecular adjuvant to remarkably enhance the immunogenicity of the target of interest in a non-particulate form. These findings may pave the way for rational vaccine design, especially in cases where particulates are not accessible.

Our objective was to establish a robust method for the expression and purification of hepatitis E virus(HEV)p495protein using a baculovirus-based insect cell expression system; to determine the properties and cryo-EM structure of the resulting virus-like particles(VLPs);and to compare their immunogenicity with p239 particles in the commercial hepatitis E vaccine (Hecolin). The sequence spanning HEV ORF2 amino acids 112-606 in the genotype I HEV isolate was cloned into baculovirus to express recombinant p495 protein. ELISA, analytical ultracentrifugation, size-exclusion chromatography and negative-staining transmission electron microscopy(TEM)were carried out to characterize the physicochemical properties of p495.Recombinant p495 VLPs were obtained successfully from the insect cell expression system with purity of95%and yield of 15mg/L.The recombinant HEV p495 protein was homogeneous in solutions. The 3Dstructure of p495 VLPs was determined by cryo-EM;it was icosahedral with T=1arrangement,and showed good congruency with the crystal structure in the literature(PDB ID:2ZZQ).In mouse vaccination experiments,p495 conferred comparable immunogenicity with that of p239 antigen in Hecolin. Thus, a robust and scalable approach to obtain homogeneous, immunogenic HEV p495 VLPs has been established. This study may assist investigations of HEV receptors, epitope mapping, vaccine improvement and so on.

Evidence for chiral doublet bands has been observed for the first time in the even-even nucleus 136 Nd . One chiral band was firmly established. Four other candidates for chiral bands were also identified, which can contribute to the realization of the multiple pairs of chiral doublet bands ( M χ D ) phenomenon. The observed bands are investigated by the constrained and tilted axis cranking covariant density functional theory (TAC-CDFT). Possible configurations have been explored. The experimental energy spectra, angular momenta, and B ( M 1 ) / B ( E 2 ) values for the assigned configurations are globally reproduced by TAC-CDFT. Calculated results support the chiral interpretation of the observed bands, which correspond to shapes with maximum triaxiality induced by different multiquasiparticle configurations in 136 Nd . peerReviewed

The protein encoded by ORF2 in hepatitis E virus (HEV) is the only capsid protein for this single-stranded RNA virus. It was previously shown that 148 aa (aa 459-606) was needed for dimer formation, whereas 239 aa (aa 368-606) was necessary to form virus-like particles (VLPs). The self-assembled VLPs of p239 were characterized with a series of methods including high performance size-exclusion chromatography to demonstrate the particulate nature of purified and properly refolded p239. A neutralizing and protective mouse monoclonal antibody (mAb) 8C11 was previously shown to bind three discontinuous peptide segments in the dimer. In addition to the good binding activity to recombinant dimeric form, E2s or E2, and VLP form p239, we demonstrated that 8C11 was able to capture the authentic HEV virions. The capability of virus capturing was demonstrated with a titration curve from 10(5) to 10(7) HEV genome copies, making binding activity to 8C11 a surrogate marker of virion-like epitopes on recombinant VLPs as well as vaccine efficacy in eliciting protective and neutralizing antibodies. Taken together, it was demonstrated that Escherichia coli expressed pORF2 proteins, p239 in particular, maintain the virion-like epitopes on VLP surface. This is consistent with the fact that p239 was demonstrated to be an effective prophylactic vaccine (recently licensed as Hecolin(®) in China) against HEV-induced hepatitis in a large scale clinical trial.

One of the main challenges for electrochemical water splitting is to develop low-cost and efficient oxygen evolution anode materials. In this work, the Co–P alloys as efficient electrocatalysts for oxygen evolution reaction in alkaline media have been successfully synthesized by using electroless plating under mild conditions. SEM, TEM and XRD characterization show that the amorphous Co–P/NF electrocatalysts have rough surface structures, exposing more active sites for oxygen evolution reaction. The electrochemical measurements confirm that Co–P alloys have excellent activity for oxygen evolution reaction, with an overpotential of 306 mV (at j = 10 mA cm −2 ), a lower Tafel slope of 51.1 mV dec −1 and long-term stability (>25 h) under alkaline conditions (1.0 M KOH).

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.