Biofunctional lipid nanoparticles for precision treatment and prophylaxis of bacterial infections.

The lack of bacterial-targeting function in antibiotics and their prophylactic usage have caused overuse of antibiotics, which lead to antibiotic resistance and inevitable long-term toxicity. To overcome these issues, we develop neutrophil-bacterial hybrid cell membrane vesicle (HMV)-coated biofunctional lipid nanoparticles (LNP@HMVs), which are designed to transport antibiotics specifically to bacterial cells at the infection site for the effective treatment and prophylaxis of bacterial infection. The dual targeting ability of HMVs to inflammatory vascular endothelial cells and homologous Gram-negative bacterial cells results in targeted accumulation of LNP@HMVs in the site of infections. LNP@HMVs loaded with the antibiotic norfloxacin not only exhibit enhanced activity against planktonic bacteria and bacterial biofilms in vitro but also achieve potent therapeutic efficacy in treating both systemic infection and lung infection. Furthermore, LNP@HMVs trigger the activation of specific humoral and cellular immunity to prevent bacterial infection. Together, LNP@HMVs provide a promising strategy to effectively treat and prevent bacterial infection.

Cell-active, irreversible covalent inhibitors that selectively target the catalytic lysine of EGFR by using fluorosulfate-based SuFEx chemistry.

EGFR signaling is involved in multiple cellular processes including cell proliferation, differentiation and development, making this protein kinase one of the most valuable drug targets for the treatment of non-small cell lung carcinomas (NSCLC). Herein, we describe the design and synthesis of a series of potential covalent inhibitors targeting the catalytically conserved lysine (K745) of EGFR on the basis of Erlotinib, an FDA-approved first-generation EGFR drug. Different amine-reactive electrophiles were introduced at positions on the Erlotinib scaffold proximal to K745 in EGFR. The optimized compound 26 (as well as its close analog 30), possessing a novel arylfluorosulfate group (ArOSO2F), showed excellent in vitro potency (as low as 0.19 nM in independent IC50 determination) and selectivity against EGFR and many of its drug-resistant mutants. Both intact protein mass spectrometry (MS) and site-mapping analysis revealed that compound 26 covalently bound to EGFR at K745 through the formation of a sulfamate. In addition, compound 26 displayed good anti-proliferative potency against EGFR-overexpressing HCC827 cells by inhibiting endogenous EGFR autophosphorylation. The pharmacokinetic studies of compound 26 demonstrated the druggable potential of other ArOSO2F-containing compounds. Finally, competitive activity-based protein profiling (ABPP), cellular thermal shift assay (CETSA), as well as cellular wash-out experiments, all showed compound 26 to be the first cell-active, fluorosulfate-based targeted covalent inhibitor (TCI) of protein kinases capable of covalently engaging the catalytically conserved lysine of its target in live mammalian cells.

Discovery and characterization of hydroxylysine O-glycosylation in an engineered IL-2 fusion protein.

In the present study, an engineered interleukin-2 (IL-2) fusion protein consisting of an anti-human serum albumin nanobody linked by ASTKG and a (G4S)2 linker to IL-2 was constructed. Liquid chromatography-mass spectrometry (LC-MS) characterization was performed on the intact molecule and at the peptide level. The LC-MS molecular mass analysis for the engineered fusion protein showed the appearance of unreported +340 Da peaks, apart from the expected O-glycosylation-related peaks in the IL-2 domain. Through a combination analysis of a K120R mutated molecule (The lysine at the position of 120 was mutated to arginine while the rest amino acid sequence remain unchanged), the possibility of a non-cleaved valine-histidine-serine signal peptide was ruled out and the presence of hydroxylysine (HyK) O-glycosylation in the ASTKG linker was confirmed. HyK O-glycosylation have been reported in other proteins such as collagen, which occurs in the conserved Gly-Xaa-HyK motif and is catalyzed by lysyl hydroxylase-3 complex. The present study showed high similar conserved motif of HyK-O-glycosylation in collagen, implying the HyK O-glycosylation in the engineered IL-2 possibly was catalyzed by the Chinese hamster ovary homolog of enzymes promoting HyK O-glycosylation in collagen. Bioactivity testing results revealed that HyK-O-glycosylation had no obvious effect on the in vitro activity of engineered IL-2. Our study is the first to report HyK-O-glycosylation modifications in therapeutic proteins through LC-MS characterization and in vitro activity analysis, which expands the scope of post-translational modification knowledge of therapeutic proteins.

Genome-Wide Identification of the Highly Conserved INDETERMINATE DOMAIN (IDD) Zinc Finger Gene Family in Moso Bamboo (Phyllostachys edulis).

INDETERMINATE DOMAIN (IDD) proteins, a family of transcription factors unique to plants, function in multiple developmental processes. Although the IDD gene family has been identified in many plants, little is known about it in moso bamboo. In this present study, we identified 32 PheIDD family genes in moso bamboo and randomly sequenced the full-length open reading frames (ORFs) of ten PheIDDs. All PheIDDs shared a highly conserved IDD domain that contained two canonical C2H2-ZFs, two C2HC-ZFs, and a nuclear localization signal. Collinearity analysis showed that segmental duplication events played an important role in expansion of the PheIDD gene family. Synteny analysis indicated that 30 PheIDD genes were orthologous to those of rice (Oryza sativa). Thirty PheIDDs were expressed at low levels, and most PheIDDs exhibited characteristic organ-specific expression patterns. Despite their diverse expression patterns in response to exogenous plant hormones, 8 and 22 PheIDDs responded rapidly to IAA and 6-BA treatments, respectively. The expression levels of 23 PheIDDs were closely related to the outgrowth of aboveground branches and 20 PheIDDs were closely related to the awakening of underground dormant buds. In addition, we found that the PheIDD21 gene generated two products by alternative splicing. Both isoforms interacted with PheDELLA and PheSCL3. Furthermore, both isoforms could bind to the cis-elements of three genes (PH02Gene17121, PH02Gene35441, PH02Gene11386). Taken together, our work provides valuable information for studying the molecular breeding mechanism of lateral organ development in moso bamboo.

A self-delivery chimeric peptide for high efficient cell membrane-targeting low-temperature photothermal/photodynamic combinational therapy and metastasis suppression of tumor.

Cellular barriers such as the cell membranes, lysosomes or nuclear pores of tumor cells hinder the drugs delivery and weaken the efficiency of traditional tumor therapies. Targeted destructing tumor cell membranes can quickly destroy cell homeostasis and kill cells without facing intracellular delivery barriers. Herein, we designed a self-delivery phototherapeutic chimeric peptide (CCP) for high efficient cell membrane-targeting combinational low-temperature photothermal therapy (LTPTT) and photodynamic therapy (PDT). The self-assembled CCP nanoparticles display remarkable tumor accumulation after systemic administration without additional carriers, avoiding the carriers related side toxicities. The CCPs are able to generate reactive oxygen species (ROS) and mild heat (<45 °C) locally at cell membrane and quickly induce immunogenic cell death to achieve efficient combinational LTPTT/PDT. The damage-associated molecular patterns released after cell membrane rupture effectively elicit antitumor immunity to eradicate residual tumor cells. With a single dosage and short-term near-infrared (NIR) light irradiation, CCPs significantly inhibit growth and metastasis of tumor, and prolong survival time of tumor-bearing mice. This work presents a unique cell membrane-targeting phototherapy strategy to kill tumor and suppress metastasis in an effective, safe and minimally invasive manner.

Autophagy-inhibiting biomimetic nanodrugs enhance photothermal therapy and boost antitumor immunity.

The instinctive protective stress responses of tumor cells hamper low-temperature photothermal therapy (LTPTT), resulting in tumor recurrence and metastasis. The rapid blood clearance and low-efficiency tumor enrichment of nanomedicines also decrease the efficacy of LTPTT. In this study, we fabricated coassembled photothermal agents (indocyanine green, ICG) and autophagy inhibitors (chloroquine, CQ) and red blood cell and cancer cell hybrid membrane (RCm)-camouflaged ICGCQ@RCm nanoparticles (ICGCQ@RCm NPs) to enhance tumor LTPTT. The ICGCQ@RCm NPs exhibited prolonged blood drug circulation and markedly enhanced drug accumulation in tumor tissues. The ICGCQ@RCm NPs reduced the thermal tolerance of tumor cells to sensitize ICG-mediated LTPTT by inhibiting protective autophagy. The ICGCQ@RCm NPs exerted strong immunogenic cell death (ICD) after efficient LTPTT to activate antitumor immunity. In addition, ICGCQ@RCms optimized the therapeutic efficacy by imaging-guided LTPTT, taking advantage of the near-infrared (NIR) fluorescence of ICG. Consequently, the ICGCQ@RCm NPs effectively inhibited tumors under mild LTPTT, significantly suppressed tumor metastasis and prolonged the survival time of tumor-bearing mice. Furthermore, the ICGCQ@RCm NPs showed high biosafety in vitro and in vivo. The ICGCQ@RCm NPs demonstrated tumor-targeting and imaging-guided autophagy inhibition-sensitized LTPTT using two Food and Drug Administration (FDA)-approved drugs, which have great potential for clinical application.

Solid-phase extraction based on chloromethylated polystyrene magnetic nanospheres followed by gas chromatography with mass spectrometry to determine phthalate esters in beverages.

An ultrasound-assisted magnetic solid-phase extraction procedure with chloromethylated polystyrene-coated Fe3 O4 nanospheres as magnetic adsorbents has been developed to determine eight phthalate esters (bis(4-methyl-2-pentyl) phthalate, dipentyl phthalate, dihexyl phthalate, benzyl butyl phthalate, bis(2-butoxyethyl) phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, and dinonyl phthalate) simultaneously in beverage samples, in combination with gas chromatography coupled to tandem mass spectrometry for the first time. Several factors related to magnetic solid-phase extraction efficiencies, such as amount of adsorbent, extracting time, ionic strength, and desorption conditions were investigated. The enrichment factors of the method for the eight analytes were over 2482. A good linearity was observed in the range of 10-500 ng/L for bis(2-butoxyethyl) phthalate and 2-500 ng/L for the other phthalate esters with correlation coefficients ranging from 0.9980 to 0.9998. The limits of detection and quantification for the eight phthalate esters were in the range of 0.20-2.90 and 0.67-9.67 ng/L, respectively. The mean recoveries at three spiked levels were 75.8-117.7%, the coefficients of variations were <11.6%. The proposed method was demonstrated to be a simple and efficient technique for the trace analysis of the phthalate esters in beverage samples.

Identifying the proton transfer reaction mechanism via a proton-bound dimeric intermediate for esomeprazoles by a kinetic method combined with density functional theory calculations.

RATIONALE: Esomeprazole analogs are a class of important proton pump inhibitors for the treatment of gastro-esophageal reflux diseases. Understanding the fragmentation reaction mechanism of the protonated esomeprazole analogs will facilitate the characterization of their complex metabolic fate in humans. In this paper, the kinetic method and theoretical calculations were applied to evaluate the fragmentation of protonated esomeprazole analogs. METHODS: All collision-induced dissociation (CID) mass spectrometry experiments were carried out using electrospray ionization (ESI) ion trap mass spectrometry in positive ion mode. Also the accurate masses of fragments were measured on by ESI quadrupole time-of-flight (QTOF) MS in positive ion mode. Theoretical calculations were carried out by the density functional theory (DFT) method with the 6-31G(d) basis set in the Gaussian 03 program. RESULTS: In the fragmentation of the protonated esomeprazole analogs, C-S bond breakage is observed, which gives rise to protonated 2-(sulfinylmethylene)pyridines and protonated benzimidazoles. DFT calculations demonstrate that the nitrogen atom of the pyridine part is the thermodynamically most favorable protonation site, and the C-S bond cleavage is triggered by the transfer of this ionizing proton from the nitrogen atom of the pyridine part to the carbon atom of the benzimidazole part to which the sulfinyl is attached. Moreover, with the kinetic plot, the intensity ratios of two protonated product ions yield a linear relationship with the differences in proton affinities of the corresponding neutral molecules, which provides strong experimental evidence that the reaction proceeds via proton-bound 2-(sulfinylmethylene)pyridine/benzimidazole complex intermediates. CONCLUSIONS: The kinetic method combined with theoretical calculations was successfully applied to probe the proton transfer reaction by proton-bound 2-(sulfinylmethylene)pyridine/benzimidazole complexes in the fragmentation of protonated esomeprazole analogs by ESI CID MS, which is a strong evidence that the kinetic method can be applied in identifying a proton-bound dimeric intermediate in the fragmentation of protonated ions.

Ultrasound-assisted magnetic solid-phase extraction based ionic liquid-coated Fe3O4@graphene for the determination of nitrobenzene compounds in environmental water samples.

An ultrasound-assisted magnetic solid-phase extraction procedure with the [C7MIM][PF6] ionic liquid-coated Fe3O4-grafted graphene nanocomposite as the magnetic adsorbent has been developed for the determination of five nitrobenzene compounds (NBs) in environmental water samples, in combination with high performance liquid chromatography-photodiode array detector (HPLC-PDA). Several significant factors that affect the extraction efficiency, such as the types of magnetic nanoparticle and ionic liquid, the volume of ionic liquid and the amount of magnetic nanoparticles, extraction time, ionic strength, and solution pH, were investigated. With the assistance of ultrasound, adsorbing nitrobenzene compounds by ionic liquid and self-aggregating ionic liquid onto the surface of the Fe3O4-grafted graphene proceeded synchronously, which made the extraction achieved the maximum within 20 min using only 144 µL [C7MIM][PF6] and 3 mg Fe3O4-grafted graphene. Under the optimized conditions, satisfactory linearities were obtained for all NBs with correlation coefficients larger than 0.9990. The mean recoveries at two spiked levels ranged from 80.35 to 102.77%. Attributed to the convenient magnetic separation, the Fe3O4-grafted graphene could be recycled many times. The proposed method was demonstrated to be feasible, simple, solvent-saving and easy to operate for the trace analysis of NBs in environmental water samples.

Ultrasound-assisted magnetic SPE based on Fe3O4-grafted graphene for the determination of polychlorinated biphenyls in water samples.

An ultrasound-assisted magnetic SPE procedure with an Fe3 O4 -grafted graphene nanocomposite as the magnetic adsorbent has been developed to determine seven polychlorinated biphenyls (PCBs; PCB28, PCB52, PCB101, PCB118, PCB138, PCB153, and PCB180) simultaneously in 200 mL environmental water samples, in combination with GC-MS/MS. Several factors related to magnetic SPE efficiencies, such as the superparamagnetic intensity and amount of adsorbent, extraction time, sample pH, and desorption conditions were investigated. With the assistance of ultrasound, the extraction achieved the maximum within only 20 s, attributed to the powerful adsorptive ability of the magnetic adsorbent toward the PCBs. Under the optimized conditions, an excellent linearity was observed in the range of 0.1-100 ng/L for PCB28, 0.2-100 ng/L for PCB52, and 0.5-100 ng/L for the other five PCBs with the correlation coefficients ranging from 0.9988 to 0.9996. The mean recoveries at spiked levels of 5.0 and 10.0 ng/L were 84.9-108.5%, the coefficients of variations were <6.5%. With convenient magnetic separation, the synthesized magnetic adsorbent could be recycled more than ten times. The proposed method was demonstrated to be feasible, simple, rapid, and easy to operate for the trace analysis of the PCBs in environmental water samples.

Single electron redox via an ion-neutral complex in the fragmentation of protonated benzoylferrocenes.

RATIONALE: Ferrocene derivatives have become very popular molecules for biological applications. Although considerable experimental and theoretical calculation studies have demonstrated that ferrocene derivatives are easily oxidized during electrospray ionization (ESI), the details of the single electron redox reaction for protonated benzoylferrocenes in collision-induced dissociation (CID) mass spectrometry (MS) has not been obtained. Characterizing this mechanism is useful for further understanding the properties of ferrocene-containing biomaterials. METHODS: All CID MS experiments were carried out using ESI ion trap mass spectrometry in positive ion mode. In addition, the accurate mass of fragments was measured on a ESI quadrupole time-of-flight (QTOF) mass spectrometer in positive ion mode. Theoretical calculations were carried out by the density functional theory (DFT) method with a hybrid basis set consisting of 6-31G (d) and ECP LanL2DZ in the Gaussian 03 program. RESULTS: In the fragmentation of protonated benzoylferrocenes, the characterized ferrocinium cation was observed, which was proposed from the cleavage of the bond between the ipso-carbon atom and the carbonyl carbon followed by a single electron redox in [substituted benzoyl/ferrocene] complexes. Moreover, when the complex contained an oxidant (substituted benzoyl cation) with higher electron affinity, the single electron redox reaction was more efficient. A correlation was established between the intensities of the two competitive product ions and the electron affinities of substituted benzoyl cations. CONCLUSIONS: The single electron redox reaction by the [substituted benzoyl/ferrocene] complexes was proposed by CID MS and theoretical calculations, which provided potential evidence to further understand the reversible reduction characteristics of ferrocene-containing biomaterials.