One-pot Synthesis of High-performance Green-emitting Carbon Dots for Cd2+ Sensing and Anti-counterfeiting Applications.

This study presents a facile one-pot solvothermal synthesis of high-performance green fluorescent carbon dots (G-CDs) using o-phenylenediamine and ethylenediamine as precursors. The G-CDs show excellent optical, temporal, and chemical stability. Notably, they exhibit the highest quantum yield of 24.2% in ethanol and a strong green emission peaking at 546 nm under 440-490 nm excitation. In addition, G-CDs have outstanding salt resistance and multi-solvent compatibility. Due to its bright photoluminescence, G-CDs can be used as a secure ink for anti-counterfeiting. More remarkably, Cd2+ ions can efficiently quench the fluorescence of G-CDs with a detection limit of 0.152 µmol/L, enabling accurate quantification of Cd2+ in water systems. The simple synthesis of high-performance G-CDs expands their applicability in sensing and bioimaging.

Strict Twice Iterative Optimization Strategy to Synthesize Ultrabright Fluorescent Carbon Dots for UV and pH Dual-Encryption Fluorescent Ink.

In this work, ultrabright fluorescent carbon dots (U-CDs) were synthesized by using a strict twice iterative optimization strategy. Their relative photoluminescence (PL) quantum yield is close to 100%, exceeding most of the reported fluorescent CDs and greatly boosting the practical applications of fluorescent CDs in many fields. Then serving as fluorescent anti-counterfeiting ink was taken as an example to briefly introduce the application of the U-CDs. The PL emission of the U-CDs is quenched at the range of pH < 4 or pH > 11 and restored at the range of pH = 5-10. This pH-sensitive PL feature allows the U-CDs to be used as fluorescent ink for pH and UV dual information encryption. The written or printed information is invisible under daylight but visible under UV light. After acid treatment or alkali treatment, the information is invisible even under a UV lamp but visible after neutralization treatment. This work provides a standardized scheme for optimizing the synthesis conditions of fluorescent CDs and paves the way for large-scale production of high-performance fluorescent CDs.

Ultrabright carbon dots as a fluorescent nano sensor for Pb2+ detection.

In this work, we synthesized ultrabright carbon dots (U-CDs) with photoluminescence quantum yield (PLQY) up to ∼100% using CA and EDA as precursors. When studying their interaction with the Pb2+ ion, we found that the PL quenching degree is independent of the U-CDs concentration. This feature provides great convenience for practical detection, which allows the standard curve determination and practical detection to be conducted under different U-CDs concentrations with detection error less than 20%. Based on the experimental observations, a possible mechanism is proposed to explain this phenomenon. To our best knowledge, this work has never been reported before and provides a new idea for the design of novel fluorescent sensors.

Molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolates with focus on antimicrobial resistance.

BACKGROUND: The enhancing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP)-mediated infections in Mengchao Hepatobiliary Hospital of Fujian Medical University in 2017 is the motivation behind this investigation to study gene phenotypes and resistance-associated genes of emergence regarding the CRKP strains. In current study, seven inpatients are enrolled in the hospital with complete treatments. The carbapenem-resistant K. pneumoniae whole genome is sequenced using MiSeq short-read and Oxford Nanopore long-read sequencing technology. Prophages are identified to assess genetic diversity within CRKP genomes. RESULTS: The investigation encompassed eight CRKP strains that collected from the patients enrolled as well as the environment, which illustrate that blaKPC-2 is responsible for phenotypic resistance in six CRKP strains that K. pneumoniae sequence type (ST11) is informed. The plasmid with IncR, ColRNAI and pMLST type with IncF[F33:A-:B-] co-exist in all ST11 with KPC-2-producing CRKP strains. Along with carbapenemases, all K. pneumoniae strains harbor two or three extended spectrum ß-lactamase (ESBL)-producing genes. fosA gene is detected amongst all the CRKP strains. The single nucleotide polymorphisms (SNP) markers are indicated and validated among all CRKP strains, providing valuable clues for distinguishing carbapenem-resistant strains from conventional K. pneumoniae. CONCLUSIONS: ST11 is the main CRKP type, and blaKPC-2 is the dominant carbapenemase gene harbored by clinical CRKP isolates from current investigations. The SNP markers detected would be helpful for characterizing CRKP strain from general K. pneumoniae. The data provides insights into effective strategy developments for controlling CRKP and nosocomial infection reductions.

Long-read nanopore sequencing-based draft genome of a carbapenem-resistant Pseudomonas aeruginosa isolate.

OBJECTIVES: Pseudomonas aeruginosa is a common Gram-negative bacterium causing various serious infections, such as lower respiratory tract infection and urinary tract infection in catheterised patients. Here we report the draft genome sequence of a carbapenem-resistant P. aeruginosa (CRPA) isolate. METHODS: The genome of the CRPA isolate was sequenced using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads. RESULTS: The resulting assembly was highly contiguous, containing a total of 6624003bp with a GC content of 66.21%. Annotation identified 6389 protein-coding genes. Mutations in the oprD and mexR genes conferred resistance to carbapenems in the CRPA isolate. CONCLUSION: The draft genome sequence of this CRPA isolate could provide a solid basis for further research on the resistance mechanisms and the development of drug therapy for drug resistance genes.

T7 RNA Polymerase Discriminates Correct and Incorrect Nucleoside Triphosphates by Free Energy.

RNA polymerase (RNAP) is the primary machine responsible for transcription. Its ability to distinguish between correct (cognate) and incorrect (noncognate) nucleoside triphosphates (NTPs) is important for fidelity control in transcription. In this work, we investigated the substrate selection mechanism of T7 RNAP from the perspective of energetics. The dissociation free energies were determined for matched and unmatched base pairs in the preinsertion complex using the umbrella sampling method. A clear hydrogen-bond-rupture peak is observed in the potential of mean force curve for a matched base pair, whereas no such peaks are present in the position of mean force profiles for unmatched ones. The free-energy barrier could prevent correct substrates from being separated from the active site. Therefore, when NTPs diffuse into the active site, correct ones will stay for chemistry once they establish effective base pairing contacts with the template nucleotide, whereas incorrect ones will be withdrawn from the active site and rejected back to solution. This result provides an important energy evidence for the substrate selection mechanism of RNAP. Then we elucidated energetics and molecular details for correct NTP binding to the active site of the insertion complex. Our observations reveal that strong interactions act on the triphosphate of NTP to constrain its movement, whereas relatively weak interactions serve to position the base in the correct conformation. Triple interactions, hydrophobic contacts from residues M635 and Y639, base stacking from the 3' RNA terminal nucleotide, and base pairing from the template nucleotide act together to position the NTP base in a catalytically competent conformation. At last, we observed that incorrect NTPs cannot be as well-stabilized as the correct one in the active site when they are misincorporated in the insertion site. It is expected that our work can be helpful for comprehensively understanding details of this basic step in genetic transcription.

Novel overlapping subgraph clustering for the detection of antigen epitopes.

Motivation: Antigens that contain overlapping epitopes have been occasionally reported. As current algorithms mainly take a one-antigen-one-epitope approach to the prediction of epitopes, they are not capable of detecting these multiple and overlapping epitopes accurately, or even those multiple and separated epitopes existing in some other antigens. Results: We introduce a novel subgraph clustering algorithm for more accurate detection of epitopes. This algorithm takes graph partitions as seeds, and expands the seeds to merge overlapping subgraphs based on the term frequency-inverse document frequency (TF-IDF) featured similarity. Then, the merged subgraphs are each classified as an epitope or non-epitope. Tests of our algorithm were conducted on three newly collected datasets of antigens. In the first dataset, each antigen contains only a single epitope; in the second, each antigen contains only multiple and separated epitopes; and in the third, each antigen contains overlapping epitopes. The prediction performance of our algorithm is significantly better than the state-of-art methods. The lifts of the averaged f-scores on top of the best existing methods are 60, 75 and 22% for the single epitope detection, the multiple and separated epitopes detection, and the overlapping epitopes detection, respectively. Availability and implementation: The source code is available at github.com/lzhlab/glep/. Supplementary information: Supplementary data are available at Bioinformatics online.

Mechanism of NTP Binding to the Active Site of T7 RNA Polymerase Revealed by Free-Energy Simulation.

In genetic transcription, molecular dynamic details and energetics of NTP binding to the active site of RNA polymerase (RNAP) are poorly understood. In this article, we investigated the NTP binding process in T7 RNAP using all-atom MD simulation combined with the umbrella sampling technique. Based on our simulations, a two-step mechanism was proposed to explain NTP binding: first, substrate NTP in aqueous solution, which carries a magnesium ion, diffuses through a secondary channel of RNAP to attain a pore region, where it undergoes conformational changes to give a correct orientation; next, the NTP establishes initial basepairing contacts with the template nucleoside (TN). Our free-energy calculations suggest that both steps are spontaneous. This mechanism can easily explain the problem of NTP binding with different orientations. Moreover, it is found that the nascent NTP:TN basepair is fragile and easily broken by thermal disturbance. Therefore, we speculate that the fingers domain will be triggered to close, so as to create a steady environment for the next chemical step. The observations from the work provide valuable information for comprehensively understanding the mechanism of the basic step in genetic transcription.

T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking.

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA-DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.

Molecular dynamics simulation study of the "stay or leave" problem for two magnesium ions in gene transcription.

Two magnesium ions play important roles in nucleotide addition cycle (NAC) of gene transcription. However, at the end of each NAC, why does one ion stay in the active site while the other ion leaves with product pyrophosphate (PPi )? This problem still remains obscure. In this work, we studied the problem using all-atom molecular dynamics simulation combined with steered molecular dynamics and umbrella sampling simulation methods. Our simulations reveal that although both ions are located in the active site after chemistry, their detailed positions are not symmetrical, leading to their different forces from surrounding groups. One ion makes weaker contacts with PPi than the whole protein. Hence, PPi release is less likely to take it away. The other one forms tighter contacts with PPi relative to the protein. The formed (Mg2+ -PPi )2- complex is found to break the contacts with surrounding protein residues one by one so as to dissociate from the active site. This effectively avoids the coexistence of two ions in the active site after PPi release and guarantees a reasonable Mg2+ ion number in the active site for the next NAC. The observations from this work can provide valuable information for comprehensively understanding the molecular mechanism of transcription. Proteins 2017; 85:1002-1007. © 2017 Wiley Periodicals, Inc.

Protein denaturation at a single-molecule level: the effect of nonpolar environments and its implications on the unfolding mechanism by proteases.

Most proteins are typically folded into predetermined three-dimensional structures in the aqueous cellular environment. However, proteins can be exposed to a nonpolar environment under certain conditions, such as inside the central cavity of chaperones and unfoldases during protein degradation. It remains unclear how folded proteins behave when moved from an aqueous solvent to a nonpolar one. Here, we employed single-molecule atomic force microscopy and molecular dynamics (MD) simulations to investigate the structural and mechanical variations of a polyprotein, I278, during the change from a polar to a nonpolar environment. We found that the polyprotein was unfolded into an unstructured polypeptide spontaneously when pulled into nonpolar solvents. This finding was corroborated by MD simulations where I27 was dragged from water into a nonpolar solvent, revealing details of the unfolding process at the water/nonpolar solvent interface. These results highlight the importance of water in maintaining folding stability, and provide insights into the response of folded proteins to local hydrophobic environments.

A critical residue selectively recruits nucleotides for t7 RNA polymerase transcription fidelity control.

Nucleotide selection is essential for fidelity control in gene replication and transcription. Recent work on T7 RNA polymerase suggested that a small posttranslocation free energy bias stabilizes Tyr(639) in the active site to aid nucleotide selection. However, it was not clear exactly how Tyr(639) assists the selection. Here we report a molecular-dynamics simulation study revealing atomistic detail of this critical selectivity. The study shows first that Tyr(639) blocks the active site at posttranslocation by marginally stacking to the end basepair of the DNA-RNA hybrid. The study then demonstrates that at the nucleotide preinsertion state, a cognate RNA nucleotide does not affect the local Tyr(639) stabilization, whereas a noncognate nucleotide substantially stabilizes Tyr(639) so that Tyr(639) keeps blocking the active site. As a result, further nucleotide insertion into the active site, which requires moving Tyr(639) out of the site, would be hindered for the noncognate nucleotide, but not for the cognate nucleotide. In particular, we note that water molecules assist the ribose recognition in the RNA nucleotide preinsertion, and help Tyr(639) stacking to the end basepair in the case of a DNA nucleotide. It was also seen that a base-mismatched nucleotide at preinsertion directly grabs Tyr(639) for the active site stabilization. We also find that in a mutant polymerase Y639F the strong stabilization of residue 639 in the active site cannot establish upon the DNA nucleotide preinsertion. The finding explains the reduced differentiation between ribo- and deoxyribonucleotides that has been recorded experimentally for the mutant polymerase.

Simulation study of protein-mediated vesicle fusion.

A dissipative particle dynamics simulation method is used to probe the mechanism of protein-mediated membrane fusion. The coarse-grained models for proteins are designed based on the function of fusion proteins. Attractive forces have been introduced to produce a protein complex. The formation of protein complexes provides mechanical forces to bring membranes in proximity and trigger their merging. The whole fusion process is in good agreement with the scaffold hypothesis. Additionally, if self-defined interactions are also imposed on transmembrane segments (TMSs), their association will yield an unstable fusion pore, which can incorporate lipid and water to accomplish membrane fusion. It indicates the formation of a protein-lined pore will promote the stalk-pore transition and accelerate the fusion process.

Behaviors of enzyme immobilization onto functional microspheres.

Micron-grade monodisperse PMMA microspheres, whose surfaces were modified with functional groups by co-polymerisation using functional monomer, were prepared via dispersion polymerisation. Characterized by their large specific surface area, high adsorption ability, favourable biocompatibility, these monodisperse micron-sized PMMA microspheres were employed as the supporting material in the enzyme immobilization in present work. The influential factors on the activity of immobilized enzyme including pH, temperature, time etc were preliminarily investigated. The results concluded from the experiments indicated that the immobilization procedure could promote the resistance of enzyme against temperature, pH shift and some other tough reaction conditions meanwhile prolong the enzymatic lifetime for storage.

Application of scintillation proximity assay in drug discovery.

Scintillation proximity assay (SPA), characterized by its speed, sensitivity, reliability, and the fact that no separation step is required, has become an important technique in high-throughput screening (HTS) for new drugs, and for investigating their biological interactions. The SPA technique now plays a key role in HTS, in that it can be used in many assay formats including radioimmunoassays (RIAs), ligand-receptor binding assays, and enzyme assays. The SPA-based enzyme assay is usually designed in three formats corresponding to different enzymes: signal removal format for hydrolytic enzymes, signal addition format for polymerase and transferase enzymes, and product capture format for antibodies, DNA probes, receptors or other specific binding proteins. The use of SPA in RIAs has been facilitated by new carriers, such as membranes that can be configured in various shapes and sizes, allowing the assay to be performed on samples from many sources including tissue, serum, plasma or cells. This review presents the principles of SPA, discusses supporting materials and quenching effects, as well as detailed examples of the latest advances.