sj-docx-1-trr-10.1177_03611981231201113 – Supplemental material for Analysis of the Key Factors Influencing Automation Transformation in Container Terminals Based on the Dempster–Shafer Evidence Interval Method

Supplemental material, sj-docx-1-trr-10.1177_03611981231201113 for Analysis of the Key Factors Influencing Automation Transformation in Container Terminals Based on the Dempster–Shafer Evidence Interval Method by Nanxi Wang, Kum Fai Yuen, Daofang Chang and Yinping Gao in Transportation Research Record</p

sj-docx-2-trr-10.1177_03611981231201113 – Supplemental material for Analysis of the Key Factors Influencing Automation Transformation in Container Terminals Based on the Dempster–Shafer Evidence Interval Method

Supplemental material, sj-docx-2-trr-10.1177_03611981231201113 for Analysis of the Key Factors Influencing Automation Transformation in Container Terminals Based on the Dempster–Shafer Evidence Interval Method by Nanxi Wang, Kum Fai Yuen, Daofang Chang and Yinping Gao in Transportation Research Record</p

Fluorescent Protein-Based Turn-On Probe through a General Protection–Deprotection Design Strategy

We demonstrated a general protection–deprotection strategy for the design of fluorescent protein biosensors through the construction of a turn-on Hg²⁺ sensor. A combination of fluorescent protein engineering and unnatural amino acid mutagenesis was used. Unlike previously reported fluorescent protein-based Hg²⁺ sensors that relied on the binding of Hg²⁺ to the sulfhydryl group of cysteine residues, a well-established chemical reaction, oxymercuration, was transformed into biological format and incorporated into our sensor design. This novel Hg²⁺ sensor displayed good sensitivity and selectivity both in vitro and in live bacterial cells. Over 60-fold change in fluorescence signal output was observed in the presence of 10 μM Hg²⁺, while such a change was undetectable when nine other metal ions were tested. This new design strategy could expand the repertoire of fluorescent protein-based biosensors for the detection of small-molecule analytes

Controlling Multicycle Replication of Live-Attenuated HIV‑1 Using an Unnatural Genetic Switch

A safe and effective human immunodeficiency virus type 1 (HIV-1) vaccine is urgently needed, but remains elusive. While HIV-1 live-attenuated vaccine can provide potent protection as demonstrated in rhesus macaque-simian immunodeficiency virus model, the potential pathogenic consequences associated with the uncontrolled virus replication preclude such vaccine from clinical applications. We investigated a novel approach to address this problem by controlling live-attenuated HIV-1 replication through an unnatural genetic switch that was based on the amber suppression strategy. Here we report the construction of all-in-one live-attenuated HIV-1 mutants that contain genomic copy of the amber suppression system. This genetic modification resulted in viruses that were capable of multicycle replication <i>in vitro</i> and could be switched on and off using an unnatural amino acid as the cue. This stand-alone, replication-controllable attenuated HIV-1 virus represents an important step toward the generation of a safe and efficacious live-attenuated HIV-1 vaccine. The strategy reported in this work can be adopted for the development of other live-attenuated vaccines

Controlling the Replication of a Genomically Recoded HIV‑1 with a Functional Quadruplet Codon in Mammalian Cells

Large efforts have been devoted to genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells. Intriguingly, the trend of quadruplet codon decoding efficiency among the tested tRNA variants in mammalian cells was largely the same as that in <i>E. coli</i>. We subsequently demonstrate the utility of quadruplet codon decoding by the construction of the first HIV-1 mutant that lacks any in-frame amber nonsense codons and can be precisely activated by the decoding of a genomically embedded UAGA codon with an unnatural amino acid. Such conditionally activatable HIV-1 mutant can likely facilitate both fundamental investigations of HIV-1 as well as vaccine developments. The use of quadruplet codon, instead of an amber nonsense codon, to control HIV-1 replication has the advantage in that the correction of a frameshift caused by a quadruplet codon is much less likely than the reversion of an amber codon back into a sense codon in HIV-1

DNA methylation marker identification and poly-methylation risk score in prediction of healthspan termination

Supplementary figures 1-3Supplementary tables 1-9Supplementary materials and methods</p

Oxidation-Induced Protein Cross-Linking in Mammalian Cells

A proximity-enabled protein cross-linking strategy with additional spatiotemporal control is highly desirable. Here, we report an oxidation-induced protein cross-linking strategy involving the incorporation of a vinyl thioether group into proteins in both Escherichia coli and mammalian cells via genetic code expansion. We demonstrated that vinyl thioether can be selectively induced by exogenously added oxidant or by reactive oxygen species from the cellular environment, as well as by photocatalysts, and converted into a Michael acceptor, enabling fluorescence labeling and protein cross-linking

In Situ Generation of Fluorescent Amino Acids and Peptides via Double C–H Activation/Annulation

Unnatural fluorescent amino acids have been synthesized to obtain better emission wavelengths, fluorescence lifetime, and quantum yields. Despite major advances, most of them face inherent restrictions as fluorophores and are limited to the methods from coupling between amino acids and fluorophores. Herein, we develop a Rh­(III)-catalyzed double C–H activation/annulation reaction of diverse benzamides with alkynes for the synthesis of tricyclic-fused aromatic hydrocarbon carbocations. The robustness of this strategy is demonstrated by the diversification of Lys-based amino acids and peptides, in situ generating tricyclic fluorophores. This method features broad substrate scope and high atom and step economy as well as high chemo- and site selectivity. Unsymmetrical double C–H activation/annulation employing two different alkynes is well tolerated to produce the unnatural fluorescent amino acids in high yields. These tricyclic fluorophores display tunable fluorescence emission, low cytotoxicity, and the potential for specifically targeting lysosomes

Genetically Encoding Fluorosulfate‑l‑tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins <i>in Vivo</i>

Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering <i>in vivo</i>. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity <i>in vivo</i>, a chemical functionality that is biocompatible and able to react with multiple natural residues under physiological conditions is highly desirable. Here we report the genetic encoding of fluorosulfate-l-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins <i>in vivo</i>. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein cross-link of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins <i>in vivo</i>. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chemistry, SuFEx, which will afford broad utilities in chemical biology, drug discovery, and biotherapeutics

Genetically Encoding Fluorosulfate‑l‑tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins <i>in Vivo</i>

Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering <i>in vivo</i>. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity <i>in vivo</i>, a chemical functionality that is biocompatible and able to react with multiple natural residues under physiological conditions is highly desirable. Here we report the genetic encoding of fluorosulfate-l-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins <i>in vivo</i>. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein cross-link of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins <i>in vivo</i>. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chemistry, SuFEx, which will afford broad utilities in chemical biology, drug discovery, and biotherapeutics