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A miniaturized fiber-optic sensor system for real-time biosensing with RGB LED as the light source and photodiode as the receiver.

Protein-carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins. SFY-incorporated Siglec-7 crosslinked with its sialoglycan ligand specifically in vitro and on the surface of cancer cells. Through irreversible cloaking of sialoglycan at the cancer cell surface, SFY-incorporated Siglec-7 enhanced the killing of cancer cells by natural killer cells. Genetically encoding the chemical crosslinking of proteins to carbohydrates (GECX-sugar) offers a solution to address the low affinity and weak strength of protein-sugar interactions.

Breast cancer resistance protein (BCRP/Abcg2 in human, Bcrp/Abcg2 in rat), a member of the ATP-binding cassette (ABC) transporter family, acts as an efflux pump for xenobiotics, with ability to transport various drugs out of cells. Capsaicin may have the potential to modulate the function of Bcrp transport. This study was to evaluate the effects of capsaicin on the pharmacokinetics of sulfasalazine, a Bcrp substrate, in rats and investigate the mechanism of this food-drug interaction.The rats were pre-treated with 5% carboxymethylcellulose sodium (vehicle), capsaicin (3, 8, 25 mg/kg) and cyclosporine A (10 mg/kg) by gastric gavage for 7 days. On day 7, blood, liver and intestine samples were collected after sulfasalazine administered. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to study the effects of capsaicin on the pharmacokinetics of sulfasalazine in rats. RT-PCR and western blotting were used to study the mechanism in biomolecules in rats, respectively.Compared with vehicle group, AUC

Shared autonomous vehicles (SAVs) are revolutionizing the future of urban mobility. This study aims to investigate the effects of artificial intelligence (i.e., autonomy level and anthropomorphic characteristics), human-related, environmental, and societal factors on public trust and acceptance. Structural equation modelling is used to analyze a valid survey sample of 451 participants. Results show that autonomy level can both directly and indirectly (via trust) increase public acceptance; Whereas, anthropomorphic characteristics cannot directly affect public acceptance, but can indirectly increase their acceptance via trust. The other human-related, environmental, and societal factors also positively contribute to public acceptance. Additionally, moderators, including age, gender, income, housing size, COVID-19 history, shared mobility experience, vehicle ownership, and driving experience are also examined. In theory, this study contextualizes the trust-in-automation three-factor model, UTAUT model, and trust theory and includes two domain-specific constructs (i.e., SAV anthropomorphism and SAV autonomy) to study public trust and acceptance towards SAVs. In practice, this study suggests the incorporation of some anthropomorphic features and relatively high autonomy level in SAVs to build public trust and acceptance.

Protein-RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein-RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.

Genetically introducing novel chemical bonds into proteins provides innovative avenues for biochemical research, protein engineering, and biotherapeutic applications. Recently, latent bioreactive unnatural amino acids (Uaas) have been incorporated into proteins to covalently target natural residues through proximity-enabled reactivity. Aryl fluorosulfate is particularly attractive due to its exceptional biocompatibility and multitargeting capability via sulfur(VI) fluoride exchange (SuFEx) reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the only aryl fluorosulfate-containing Uaa that has been genetically encoded. FSY has a relatively rigid and short side chain, which restricts the diversity of proteins targetable and the scope of applications. Here we designed and genetically encoded a new latent bioreactive Uaa, fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coli and mammalian cells. Due to its long and flexible aryl fluorosulfate-containing side chain, FSK was particularly useful in covalently linking protein sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent nanobodies that irreversibly bound to epidermal growth factor receptors (EGFR) on cells, with FSK and FSY targeting distinct positions on EGFR to counter potential mutational resistance. Moreover, we established the use of FSK and FSY for genetically encoded chemical cross-linking to capture elusive enzyme-substrate interactions in live cells, allowing us to target residues aside from Cys and to cross-link at the binding periphery. FSK complements FSY to expand target diversity and versatility. Together, they provide a powerful, genetically encoded, latent bioreactive SuFEx system for creating covalent bonds in diverse proteins in vitro and in vivo, which will be widely useful for biological research and applications.

In the human body, each microbial habitat exhibits a different microbial population pattern, and these distinctive microflorae are highly related to the development of diseases. The microbial interactions from host different niches are becoming crucial regulators to shape the microbiota and their physiological or pathological functions. The oral cavity and gut are the most complex and interdependent microbial habitats. Helicobacter pylori is one of the most important pathogens from digestive tract, especially the stomach, due to its direct relationships with many gastric diseases including gastric cancer. H. pylori infections can destroy the normal gastric environment and make the stomach a livable channel to enhance the microbial interactions between oral cavity and gut, thus reshaping the oral and gut microbiomes. H. pylori can be also detected in the oral and gut, while the interaction between the oral-gut axis microbiota and H. pylori plays a major role in H. pylori’s colonization, infection, and pathogenicity. Both the infection and eradication of H. pylori and its interaction with oral-gut axis microbiota can alter the balance of the microecology of the oral-gut axis, which can affect the occurrence and progress of related diseases. The shift of oral-gut axis microbiota and their interactions with H. pylori maybe potential targets for H. pylori infectious diagnosis and treatment.

Recently, there is an increasing trend to study the impact of anthropomorphism on human trust. However, previous research has shown divergent results. In this study, shared autonomous vehicles (SAVs), an emerging mobility solution, are used to explore the threshold effects of anthropomorphism on human trust. Structural equation modelling is deployed to test the research model based on a valid survey sample of 451 respondents. Results show that a superficial level of anthropomorphism can significantly boost human trust via interaction quality and facilitating condition. By contrast, a deep level of anthropomorphism would decrease human trust. Although the negative effect does not reach statistical significance based on the whole sample, results of the multigroup analysis show that a deep level of anthropomorphism has a significant negative effect on human-SAV interaction quality when respondents possess the following characteristics: (1) male, (2) low-income, (3) low-education, or (4) no-vehicle ownership. Regarding theoretical contribution, this study enriches the literature by identifying the threshold effects of anthropomorphism on human trust. Regarding policy and management implications, this study offers some implications on adding anthropomorphic features to SAVs.

In the human body, each microbial habitat exhibits a different microbial population pattern, and these distinctive microflorae are highly related to the development of diseases. The microbial interactions from host different niches are becoming crucial regulators to shape the microbiota and their physiological or pathological functions. The oral cavity and gut are the most complex and interdependent microbial habitats.

Graphical abstract, Highlights • A reliable Glide docking-based virtual screening (VS) pipeline for P2Y14R was developed. • Several potent P2Y14R antagonists with novel scaffolds were identified utilizing the VS strategy. • P2Y14R inhibitory effect was evaluated by testing cAMP levels in HEK293 cells. • Anti-gout activity of screened compound was detected in MSU-treated THP-1 cells. • The mechanism of test compound in treating acute gouty arthritis was elucidated., P2Y14 nucleotide receptor is a Gi protein-coupled receptor, which is widely involved in physiological and pathologic events. Although several P2Y14R antagonists have been developed thus far, few have successfully been developed into a therapeutic drug. In this study, on the basis of two P2Y14R homology models, Glide docking-based virtual screening (VS) strategy was employed for finding potent P2Y14R antagonists with novel chemical architectures. A total of 19 structurally diverse compounds identified by VS and drug-like properties testing were set to experimental testing. 10 of them showed good inhibitory effects against the P2Y14R (IC50

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here we developed covalent nanobodies capable of binding with SARS-CoV-2 spike protein irreversibly via proximity-enabled reactive therapeutic (PERx) mechanism. A novel latent bioreactive amino acid FFY was designed and genetically encoded into nanobodies to accelerate PERx reaction rate. After covalent engineering, nanobodies binding with the Spike in the down state, but not in the up state, were discovered to possess striking enhancement in inhibiting viral infection. In comparison with the noncovalent wildtype nanobody, the FFY-incorporated covalent nanobody neutralized both authentic SARS-CoV-2 and its Alpha and Delta variants with potency drastically increased over tens of folds. This PERx-enabled covalent nanobody strategy and uncovered insights on potency increase can be valuable to developing effective therapeutics for various viral infections.

Peptide drug development has made great progress in the last decade thanks to new production, modification, and analytic technologies. Peptides have been produced and modified using both chemical and biological methods, together with novel design and delivery strategies, which have helped to overcome the inherent drawbacks of peptides and have allowed the continued advancement of this field. A wide variety of natural and modified peptides have been obtained and studied, covering multiple therapeutic areas. This review summarizes the efforts and achievements in peptide drug discovery, production, and modification, and their current applications. We also discuss the value and challenges associated with future developments in therapeutic peptides.

Background Pharmacy intravenous admixture service (PIVAS) center has emerged as an important department of hospital as it can improve occupational protection and ensure the safety and effectiveness of intravenous infusions. However, medication errors were considered to be a significant challenge in PIVAS, so information-intelligence technologies were introduced to optimize the management of PIVAS. Our article summarized the application of information-intelligence technologies in PIVAS of a large third-class A hospital in China, and provided an example for PIVAS in other hospitals at home and abroad. Methods Prescription-reviewing rules containing intravenous medications and infusion solution guideline were recorded in the database of prescription-cheking system. Drugs information were recorded in the PIVAS management system with special identification and warning labels to reduce intravenous infusion errors. Automatic labeling device was used to label the infusion bags, and the quality control program database of intelligent compounding robot for cytotoxic drugs was established ingeniously. Automatic sorting devices were applied for the third batch of finished infusion admixtures, and intelligent logistics robots were used to transport the infusion to the ward. Results After establishing and implementing of prescription-reviewing rules in the prescription-cheking system database, the number of prescriptions checked by pharmacists increased from 18 to 43 per minute. The success rate of intervention with irrational medical orders increased from 85.89% to 99.06% (P P P Conclusions The application of information-intelligence technologies in PIVAS can improve work efficiency and reduce error risk. However, some intelligent devices have failed to achieve the expected effect in practical use, and further improvements are needed to meet the demands of PIVAS in the future.

Waterway transportation is considered as one of the most critical means of transportation. Risk management is one of the most important ways to ensure the safe operation of waterway transportation systems (WTS). Recently, the concept of resilience has been widely studied as it can be used to measure not only the system's reliability, but also its recoverability. The objectives of this study are to (1) analyze WTS resilience by introducing disruptions at different stages of the resilience recovery cycle through simulation and (2) introduce a comprehensive resilience indicator which comprises ship load, ship delay and recovery cost. A discrete-event based simulation model is developed to quantify WTS resilience. The Yangtze Estuary Deepwater Channel is used as the case study, and the automatic identification system (AIS) data of this channel in 2018 are collected and used as the input parameters of the simulation model. Different scenarios and experiments have been designed to analyze the resilience performance of the WTS when it encounters different accidents. Based on a real accident case, two rescue plans are designed for comparisons, and the results demonstrate the effectiveness of the proposed resilience indicator. Based on the analysis results several practical suggestions are recommended. Submitted/Accepted version

BackgroundPrevious studies have suggested associations between testosterone, genetic factors, and a series of complex diseases, but the associations with the lifespan phenotype, such as health span, remain unclear.MethodsIn this prospective cohort study, we analyzed 145,481 men and 147,733 women aged 38–73 years old from UK Biobank (UKB) to investigate the sex-specific associations of total testosterone (TT), free testosterone (FT), or polygenic risk score (PRS) with health span termination (HST) risk. At baseline, serum testosterone levels were measured. HST was defined by eight events strongly associated with longevity. PRS, an efficient tool combining the effect of common genetic variants to discriminate genetic risk of complex phenotypes, was constructed by 12 single-nucleotide polymorphisms related to health span from UKB (P ≤ 5.0 × 10−8). We used multivariable Cox regression models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs).ResultsWith a median follow-up time of 7.70 years, 26,748 (18.39%) men and 18,963 (12.84%) women had HST. TT was negatively associated with HST in men [HR per standard deviation (SD) increment of log-TT: 0.92, 95% CI: 0.88–0.97]. Inversely, both TT (HR per SD increment of log-TT: 1.05, 95% CI: 1.02–1.08) and FT (HR per SD increment of log-FT: 1.08, 95% CI: 1.05–1.11) presented an increased risk of HST in women. PRS was positively associated with HST risk (quintile 5 versus quintile 1, men, HR: 1.19, 95% CI: 1.15–1.24; women, HR: 1.21, 95% CI: 1.16–1.27). Moreover, men with high TT and low genetic risk showed the lowest HST risk (HR: 0.80, 95% CI: 0.73–0.88), whereas HST risk for women with both high TT and genetic risk increased obviously (HR: 1.32, 95% CI: 1.19–1.46). Similar joint effects were observed for FT in both genders.ConclusionsWe observed sex-specific associations that testosterone was negatively associated with HST risk in men and positively associated with HST risk in women. Genetic factors increased the HST risk, suggesting that participants with both high genetic risk and abnormal testosterone levels (high level in women or low level in men) should be the target for early intervention. Although our findings highlight the associations between testosterone and health span, further mechanistic studies and prospective trials are warranted to explore the causation behind.

We present the largest whole-genome sequencing (WGS) study of non-small cell lung cancer (NSCLC) to date among 6,004 individuals of Chinese ancestry, coupled with 23,049 individuals genotyped by SNP array. We construct a high-quality haplotype reference panel for imputation and identify 20 common and low-frequency loci (minor allele frequency [MAF] ≥ 0.5%), including five loci that have never been reported before. For rare loss-of-function (LoF) variants (MAF 0.5%), we identify BRCA2 and 18 other cancer predisposition genes that affect 5.29% of individuals with NSCLC, and 98.91% (181 of 183) of LoF variants have not been linked previously to NSCLC risk. Promoter variants of BRCA2 also have a substantial effect on NSCLC risk, and their prevalence is comparable with BRCA2 LoF variants. The associations are validated in an independent case-control study including 4,410 individuals and a prospective cohort study including 23,826 individuals. Our findings not only provide a high-quality reference panel for future array-based association studies but depict the whole picture of rare pathogenic variants for NSCLC.

Lactylation is a new modification discovered on histones. However, whether it can be installed on non-histone proteins remains unclear. Here we report the formation of a signature cyclic immonium ion of lactyllysine, together with the characteristically changed chromatographic behavior, enabling confident protein lactylation assignment by mass spectrometry. This identification strategy was confirmed by affinity-enriched lactylation proteome and revealed lactylation on nuclear non-histone proteins such as nucleolin. Subsequent exploitation of the approach to mining unenriched, deep proteome resources unveiled an understudied lactylation landscape. From the draft map of the Human Proteome, we identified widespread lactylation on DHRS7 among human tissues, and demonstrated site-directed mutagenesis of the lactylated site affects previously unannotated proteinaceous association. Additionally, the Meltome Atlas showed lactylation frequently occurs on glycolytic enzymes and concomitantly induces thermal stability changes on carrier enzymes. Collectively, the identified signatures of protein lactylation enable confident assignment and allow for the discovery of lactylation proteome expanding beyond histones, representing a step to further understand how lactylation governs cells.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

Genetically introducing covalent bonds into proteins in vivo with residue specificity is affording innovative ways for protein research and engineering, yet latent bioreactive unnatural amino acids (Uaas) genetically encoded to date react with one to few natural residues only, limiting the variety of proteins and the scope of applications amenable to this technology. Here we report the genetic encoding of (2 R)-2-amino-3-fluoro-3-(4-((2-nitrobenzyl)oxy) phenyl) propanoic acid (FnbY) in Escherichia coli and mammalian cells. Upon photoactivation, FnbY generated a reactive quinone methide (QM), which selectively reacted with nine natural amino acid residues placed in proximity in proteins directly in live cells. In addition to Cys, Lys, His, and Tyr, photoactivated FnbY also reacted with Trp, Met, Arg, Asn, and Gln, which are inaccessible with existing latent bioreactive Uaas. FnbY thus dramatically expanded the number of residues for covalent targeting in vivo. QM has longer half-life than the intermediates of conventional photo-cross-linking Uaas, and FnbY exhibited cross-linking efficiency higher than p-azido-phenylalanine. The photoactivatable and multitargeting reactivity of FnbY with selectivity toward nucleophilic residues will be valuable for addressing diverse proteins and broadening the scope of applications through exploiting covalent bonding in vivo for chemical biology, biotherapeutics, and protein engineering.

Using personal trajectory information to grasp the spatiotemporal laws of dangerous activities to curb the occurrence of criminal acts is a new opportunity and method for security prevention and control. This paper proposes a novel method to discover abnormal behaviors and judge abnormal behavior patterns using mobility trajectory data. Abnormal behavior trajectory refers to the behavior trajectory whose temporal and spatial characteristics are different from normal behavior, and it is an important clue to discover dangerous behavior. Abnormal patterns are the behavior patterns summarized based on the regular characteristics of criminals’ activities, including wandering, scouting, random walk, and trailing. This paper examines the abnormal behavior patterns based on mobility trajectories. A Long Short-Term Memory Network (LSTM)-based method is used to extract personal trajectory features, and the K-means clustering method is applied to extract abnormal trajectories from the trajectory dataset. Based on the characteristics of different abnormal behaviors, the spatio-temporal feature matching method is used to identify the abnormal patterns based on the filtered abnormal trajectories. Experimental results showed that the trajectory-based abnormal behavior discovery method can realize a rapid discovery of abnormal trajectories and effective judgment of abnormal behavior patterns.

Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage. Integrating GECX with OpenUaa, the direct interactome of thioredoxin is identified in Escherichia coli cells, yielding 289 crosslinked peptides and corresponding to 205 direct binding protein of thioredoxin. These identified direct binders provide evidence for thioredoxin's regulation of redox state and mitochondria energy metabolism. When identifying endogenous conjugation of small ubiquitin-like modifier (SUMO), OpenUaa also markedly improves coverage of SUMOylated peptides by ≈92%, revealing new SUMO targets. GECX-OpenUaa will enable efficient identification of direct interactomes of various proteins in live cells.

In the crime scene, criminals often consciously conceal their facial identity through face-masked disguise, which poses a huge challenge to identity recognition. Existing disguised face recognition techniques aiming for light even slight occlusions are completely invalid for face-masked identification. To this end, this paper proposes a masked face recognition method based on person re-identification association, which converts the masked face recognition problem into an association uncovering problem between the masked face and the appearing faces of the same person. Based on the characteristics that person re-identification technique does not rely solely on facial information, it first takes advantages of re-identification to establish the association between face-masked pedestrians and face-unveiled pedestrians. It further provides an effective face image quality assessment to select the most identifiable faces for subsequent recognition from a variety of appearing candidate faces. Finally, the selected high-quality recognizable faces are used to replace masked faces for identification. The comparison experiments with the existing disguise face recognition methods show its superiority in terms of accuracy.

Site-specific modification of proteins with functional molecules provides powerful tools for researching and engineering proteins. Here we report a new chemical conjugation method which photocages highly reactive but chemically selective moieties, enabling the use of protein-inert amines for selective protein modification. New amino acids FnbY and FmnbY, bearing photocaged quinone methides (QMs), were genetically incorporated into proteins. Upon light activation, they generated highly reactive QM, which rapidly reacted with amine derivatives. This method features a rare combination of desired properties including fast kinetics, small and stable linkage, compatibility with low temperature, photo-controllability, and widely available reagents. Moreover, labeling via FnbY occurs on the β-carbon, affording the shortest linkage to protein backbone which is essential for advanced studies involving orientation and distance. We installed various functionalities onto proteins, and attached a spin label as close as possible to the protein backbone, achieving high resolution in double electron-electron paramagnetic resonance distance measurements.

Irinotecan (CPT-11) is a broad spectrum agent for the treatment of solid tumor malignancies, despite severe diarrhea is limiting its widespread usage. The local effects of SN-38 in the small intestine were considered to be responsible for the irinotecan-induced delayed diarrhea. It was proposed that cyclosporin A (CsA) inhibiting biliary excretion could attenuate this side effect, but in fact, it could not improve the therapeutic index of irinotecan. At present, most studies focused on the inhibition of bile excretion by cyclosporin A through the transporters MRP2 and MDR1 and its effect the irinotecan treatment

Acidic organelles and vesicles, such as endosomes, lysosomes, autophagosomes, trans-Golgi network, and synaptic vesicles, are known to play important roles in a broad range of cellular events. To facilitate studying these multifunctional systems, we describe here an acid-brightening fluorescent protein (abFP), which fluoresces strongly at acidic pH, but is almost nonfluorescent at or above physiological pH, making it well suited for imaging molecules residing in acidic microenvironment in live cells. Specifically, a quinoline-containing unnatural amino acid Qui is incorporated into the chromophore of EGFP via genetic code expansion to generate the abFP. When being exposed to acidic environment, protonation of Qui results in a cationic chromophore and fluorescence increase. Protocols are presented to express abFP in E. coli and mammalian cells, and to fluorescently image the endocytosis of δ opioid receptor-abFP fusion protein in mammalian cells. This strategy may be similarly applicable to other fluorescent proteins to enable acidic imaging.

As natural proteins generally do not bind targets in a covalent mode, the therapeutic potential of covalent protein drugs remains largely unexplored. Recently, latent bioreactive amino acids have been incorporated into proteins through genetic code expansion, which selectively react with nearby natural residues via proximity-enabled reactivity, generating diverse covalent linkages for proteins in vitro and in cells. These new covalent linkages provide novel avenues for protein research and engineering. In addition, a general platform technology, proximity-enabled reactive therapeutics (PERx), has been established for the development of covalent protein drugs. The first covalent protein drug demonstrates advantageous features in cancer immunotherapy in mice. Selective introduction of covalent bonds into proteins will advance biological studies, synthetic biology, and biotherapeutics with the power of biocompatible covalent chemistries.

Acidic vesicles and organelles play fundamental roles in a broad range of cellular events such as endocytosis, lysosomal degradation, synaptic transmission, pathogen fate, and drug delivery. Fluorescent reporters will be invaluable for studying these complex and multifunctional systems with spatiotemporal resolution, yet common fluorescent proteins are generally nonfluorescent at acidic conditions due to the decrease of anionic chromophores upon protonation, but are fluorescent at physiological pH, creating interfering fluorescence from nonvesicle regions. Here we developed a novel acid-brightening fluorescent protein (abFP) that fluoresces strongly at acidic pH but is nonfluorescent at or above neutral pH, boasting a pH profile opposite to that of common fluorescent proteins. Through expansion of the genetic code, we incorporated a quinoline-containing amino acid Qui into the chromophore of EGFP to reverse the chromophore charge. Protonation of Qui rendered a cationic chromophore, which resulted in unique fluorescence increase only at acidic pH in vitro, in E. coli cells, and on the mammalian cell surface. We further demonstrated that abFP-tagged δ opioid receptors were fluorescently imaged in lysosome showing distinct features and without background fluorescence from other cellular regions, whereas EGFP-tagged receptors were invisible in lysosome. This Qui-rendered cationic chromophore strategy may be generally applied to other fluorescent proteins to generate a palette of colors for acidic imaging with minimal background, and these abFPs should facilitate the study of molecules in association with various acidic vesicles and organelles in different cells and model organisms.

Capsaicin (CAP), the main ingredient of chili peppers, displays potent anti-tumor activity in several human cancers. Irinotecan, an anticancer agent with narrow therapeutic index, is subjected to disposition by several enzymes or transporters modulated by CAP. This study systematically investigated the CAP–drug interactions in vitro, in situ and in animals. Results indicated that after 7 days of 3 mg/kg CAP treatment, the AUC0-∞ of SN-38 was significantly increased to 1.48-fold compared to the vehicle treated rats. Meanwhile, it caused a significant plasma protein binding displacement of SN-38, lowered the liver to plasma concentration ratio and slight reduction of biliary excretion after CAP treatment. These results demonstrated that ingestion of CAP would increase the systemic exposure and toxicity of SN-38 to a significant extent in rats by affecting multiple enzymes and transporters at various stages.

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.

While the combined antiretroviral therapy has resulted in a significant decrease in HIV‐1 related morbidity and mortality, the HIV‐1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV‐1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV‐1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live‐attenuated vaccines (LAVs) provided excellent protection in a non‐human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV‐1 LAVs through controlling HIV‐1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid‐mediated suppression of amber nonsense codon to generate conditionally replicating HIV‐1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies. © 2016 Society of Chemical Industry

With improved machine learning models, studies on bankruptcy prediction show improved accuracy. This paper proposes three relatively newly-developed methods for predicting bankruptcy based on real-life data. The result shows among the methods (support vector machine, neural network with dropout, autoencoder), neural network with added layers with dropout has the highest accuracy. And a comparison with the former methods (logistic regression, genetic algorithm, inductive learning) shows higher accuracy.

Small molecule cross-linkers are invaluable for probing biomolecular interactions and for cross-linking mass spectrometry (CXMS) in addressing large protein complexes and intrinsically disordered proteins. Existing chemical cross-linkers target only a small selection of amino acid residues, limiting the number and type of cross-links, while conventional photocross-linkers target virtually all residues non-selectively, complicating data analysis. Here we report photocaged quinone methide (PQM)-based cross-linkers that are able to multitarget nine nucleophilic residues through specific Michael addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM cross-linkers notably cross-linked Gln, Arg, and Asn hitherto untargetable by existing chemical cross-linkers, markedly increasing the number of residues targetable with a single cross-linker. Such multiplicity of cross-links will increase the abundance of cross-linked peptides for CXMS identification and afford ample constraints to facilitate structural deciphering. PQM cross-linkers were used in vitro, in E. coli, and in mammalian cells to cross-link dimeric proteins and endogenous membrane receptors. The cross-linker NHQM could directly cross-link proteins to DNA, for which few cross-linkers exist. The photoactivatable and multitargeting reactivity of these PQM cross-linkers will substantially enhance chemical cross-linking based technologies for studies of protein-protein and protein-DNA networks and for structural biology.

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-L-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter- and intra-molecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

Sensitive and comprehensive measurement of systemic metabolites of tryptophan, phenylalanine and glutamate metabolism in biological samples is effective for understanding the pathogenesis of depression and other neurological diseases. Therefore, this study developed an underivatized liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous monitoring the 3 components of glutamate metabolism in rat hippocampus and 11 components of tryptophan and phenylalanine metabolism in rat hippocampus, plasma and urine, and applied it to investigate their changes in rats induced by chronic unpredictable mild stress (CUMS). The investigated analytes are as follows: tryptophan, serotonin, 5-hydroxyindoleacetic acid, kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, phenylalanine, tyrosine, tyramine, glutamate, glutamine and gamma-aminobutyric acid. The method was verified to be sensitive and effective with satisfactory linearity, accuracies in the range of 78.2%-120.4%, and precisions less than 17.8% for all identified analytes. A series of significant changes in CUMS-induced rats can be detected: tryptophan, serotonin and tyrosine levels decreased and quinolinic acid increased in both hippocampus and plasma. In addition, the kynurenine/tryptophan ratios increased in hippocampus and plasma, the kynurenic acid/quinolinic acid ratios of plasma and urine were significantly reduced. These findings demonstrated that the CUMS procedure could lead to the central and peripheral imbalances of tryptophan and phenylalanine metabolism. In conclusion, a LC-MS/MS method for simultaneous measurement of several neurotransmitters in rat hippocampus, plasma and urine was developed and successfully applied to investigation of the central and peripheral changes in CUMS-induced rats. The method would be expected to provide applicability to the study of the mechanisms of depression and other related diseases associated with these neurotransmitters.

The anticancer agent irinotecan (CPT-11) is widely used in several cancer regimens. However, drug–drug interaction effects and considerable interindividual variability in the pharmacokinetics of CPT-11 and its metabolites have been observed, leading to side effects. To solve these problems and promote CPT-11 treatment, we established and validated a simple, rapid, and sensitive LC–MS/MS method for the simultaneous quantitative determination of CPT-11 and its main metabolites (SN-38, SN-38G, and APC). Protein precipitation with methanol was selected for sample preparation. All separations were performed on an Agilent ZORBAX Eclipse XDB-C18 column (2.1 × 50 mm, 3.5 μm) under isocratic elution with a mobile phase consisting of acetonitrile and 0.1% formic acid. The mass spectrometer was operated with multiple reaction monitoring in positive ion mode. The method was validated as linear over the concentration ranges 16.9–6760 ng mL−1 for CPT-11, 2.3–920 ng mL−1 for SN-38, 2.5–1000 ng mL−1 for SN-38G, and 1.25–250 ng mL−1 for APC. The intra- and interbatch precisions and accuracies of the validated method were within acceptable limits (

With the continuous development of international communication and world economy, the number of vessels, together with the vessels’ size have continued to increase, which cause a dramatic increase in channel capacity and pose huge pressure to fairway management. Therefore, maintaining the safety level of the fairway as the continuous growth of the fairway capacity has become a new challenge for managers. In order to solve the above problems, this paper proposes a framework for channel management optimization and the details are as follows: 1.In view of the complexity of the marine transportation system, traffic simulation are used to explore the relationship between influencing factors and channel risk; 2.A novel method of risk assessment based on a classic model is provided, in which a simple and reliable algorithm of calculating the number of ship domain conflicts is introduced; 3.A case study of the Yangtze Estuary Deepwater Channel is developed, in which the historical AIS data in 2018 have been collected and analyzed to ensure reasonable and reliable input parameters; 4.Based on the analysis results, the relationship between key influencing factors and channel risk is quantified, and appropriate recommendations for speed management of channel and risk assessment design of experts are proposed.

Summary Small molecule covalent drugs provide desirable therapeutic properties over noncovalent ones for treating challenging diseases. The potential of covalent protein drugs, however, remains unexplored due to protein’s inability to bind targets covalently. We report a proximity-enabled reactive therapeutics (PERx) approach to generate covalent protein drugs. Through genetic code expansion, a latent bioreactive amino acid fluorosulfate-L-tyrosine (FSY) was incorporated into human programmed cell death protein-1 (PD-1). Only when PD-1 interacts with PD-L1 did the FSY react with a proximal histidine of PD-L1 selectively, enabling irreversible binding of PD-1 to only PD-L1 in vitro and in vivo. When administrated in immune-humanized mice, the covalent PD-1(FSY) exhibited strikingly more potent antitumor effect over the noncovalent wild-type PD-1, attaining therapeutic efficacy equivalent or superior to anti-PD-L1 antibody. PERx should provide a general platform technology for converting various interacting proteins into covalent binders, achieving specific covalent protein targeting for biological studies and therapeutic capability unattainable with conventional noncovalent protein drugs.

Aiming at the design requirement of miniature reflective optical system,a new type of off-axis three-mirror anastigmat (TMA) and freeform surface are adopted, the F#1.8,and diagonal field of view7.5° is realized.Light overlap many times within the optical system,the full field average RMS Wavefront abberations is 0.017λ(λ=10μm). Compared with other optical system, the new freeform TMA has small size, high transmittance, large field of view, large relative aperture, no centered obscuration, low stray, radiation, wide spectral band, etc.

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a “plant-and-cast” cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues “planting” the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then “cast” across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme–substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

Aprepitant (APT), an antiemetic drug belonging to the class of substance P antagonists is efficiently used in both acute and delayed chemotherapy-induced nausea and vomiting. Nausea and vomiting induced by imatinib (IMA) as a chemotherapeutic drug could be reduced by APT. This study investigated the effect of APT on the pharmacokinetics of IMA and its major metabolite N-desmethyl imatinib (N-D IMA) in rats and the mechanism of this drug-drug interaction. The results indicated that after 3 days of pretreatment with APT (10 mg/kg), the blood concentration of IMA was decreased in both of oral and intravenous routes of IMA administration compared to vehicle treated rats, whereas the blood concentration of N-D IMA was not significantly changed. The total clearance (CL/F) of oral and intravenous given IMA was increased by 1.41 and 1.32-fold, and the bioavailability was greatly decreased about 30.43% and 24.40% respectively. At this time, the P-gp and the hepatic CYP3A1 were increased at both the mRNA and protein levels. These results demonstrated that ingestion of APT will decrease the bioavailability of IMA to a significant extent in rats and the drug-drug interaction between APT and IMA appears to be due to modulation of P-gp and CYP3A1.

Large efforts have been devoted to the 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 E. coli. 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.

We report a strategy to introduce a reactive electrophile into proteins through the conversion of a chemically inert group into a bioreactive one in response to an inducer molecule. This strategy was demonstrated by an oxidation-induced and proximity-enhanced protein-protein crosslinking in the presence of a large access of free nucleophiles.

Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. 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 in vivo, 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 in vivo. 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 in vivo. 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.

Depression is highly prevalent worldwide and a leading cause of disabilty. However, the medications currently available to treat depression fail to adequately relieve depressive symptoms for a large number of patients. Research into the aberrant overactivation of the kynurenine pathway and the production of various active metabolites has brought new insight into the progression of depression. IDO and TDO are the first and rate-limiting enzymes in the kynurenine pathway and regulate the production of active metabolites. There is substantial evidence that TDO and IDO enzyme are activated during depression, and therefore, IDO and TDO inhibitors have been identified as ideal therapeutic targets for depressive disorder. Hence, this review will focus on the kynurenine branch of tryptophan metabolism and describe the role of IDO and TDO in the pathology of depression. In addition, this review will compare the relative imbalance between KYNA and neurotoxic kynurenine metabolites in different psychiatric disorders. Finally, this review is also directed toward assessing whether IDO and TDO are potential therapeutic target in depression associated with other diseases such as diabetes and/or cancer, as well as the development of potent IDO and TDO inhibitors.

While the combined antiretroviral therapy has resulted in a significant decrease in HIV-1 related morbidity and mortality, the HIV-1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV-1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV-1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live-attenuated vaccines (LAVs) provided excellent protection in a non-human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV-1 LAVs through controlling HIV-1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid-mediated suppression of amber nonsense codon to generate conditionally replicating HIV-1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies.

A linearization double-group linkage zoom lens was presented based on the 640×512 cooled FPA. This configuration’s zoom groups and compensate groups are moved linear zing, omit zoom cam which control the movement of zoom curve make common double-linkage zoom lens easily. This configuration is similarity to optical compensate zoom has a little image displacement, but it is not influence the image quality when the image displacement control in allow range. This lens was placed at cold shield and 100% cold shield efficiency had reached. Moreover, detailed design and image quality were given by CODE optical software. After analysis, MTF approaches diffraction limit. The results show that this optical system has large zoom ratio, and excellent image quality.