Search

Cross-linking mass spectrometry methods have not been successfully applied to protein-protein interaction discovery at a proteome-wide level mainly due to the computation complexity (O (n2)) issue. In a previous report, we proposed a decision tree searching strategy (DTSS), which can reduce complexity by orders of magnitude. In this study, we further found that the monolinked peptides carry out the information on the retention time of the corresponding cross-linked pairs; therefore, the retention time of cross-linked peptide pairs can be predicted accurately. By utilizing the retention time as an extra filter, the false positive rate can be reduced by around 86% with a sensitivity loss of 10%. The method combined with DTSS (T-DTSS) not only benefits improving identification confidence but also leads to lower cutoff scores and facilitates substantially increasing inter-cross-link identification. T-DTSS was successfully applied to the identification of inter-cross-links obtained from Escherichia coli cell lysate cross-linked by a newly synthesized enrichable cross-linker, pDSBE. The approach can be applicable to both cleavable and noncleavable methods.

Edwardsiella piscicida (E. piscicida) is a common pathogen in freshwater and marine environments and an important intestinal pathogen of fish. Fucose large presence in the intestines could possibly regulate relevant genes to promote bacterial infectivity. In this study, the wild-type E. piscicida and the fucP gene-deleted strain ΔfucP were used to identify its virulence-regulating pathway and colonization. The results showed that the gene transcription levels of key FusKR and type III secretion system (T3SS) in E. piscicida were upregulated by different concentrations of fucose. Both the wild-type and the ΔfucP strain grew faster with 30 mM fucose added than that without fucose added. In addition, the penetration of the wild-type E. piscicida with fucose added was significantly stronger than that of both ΔfucP with fucose and the wild-type E. piscicida without fucose. Meanwhile, the gene transcription levels of T3SS-related genes eseB, eseC, eseD, eseE, and eseG in E. piscicida were significantly downregulated after the fucP gene mutant. Finally, tilapia infected with the wild-type strain showed a significant increase in the E. piscicida colonization compared to those infected with the mutant strain. All results indicate that FucP of E. piscicida mediated fucose to promote the growth, motility, and intestinal colonization in tilapia.

Edwardsiella piscicida (E. piscicida) is an important zoonotic pathogen that mostly infects fish by colonizing the intestine. Fucose within the intestine could regulate the pathogenicity of some intestinal pathogens. Here we identified the function of fucose permease in E. piscicida and its physiological influence. The results showed that E. piscicida grew faster in the presence of l -fucose. The fucose permease gene fucP mutant strain ΔfucP significantly decreased penetration and motility in mucin compared with the wild-type strain. The gene expression levels of esaB, esaD, esaL and esaK in ΔfucP had significantly downregulated when compared with the wild-type strain. Most importantly, the survival rate of zebrafish after being infected with ΔfucP was significantly higher than those of the wild type. In a word, fucose promoted the growth and motility of E. piscicida and the fucP gene mutant decreased the pathogenicity of E. piscicida in zebrafish.

Poroelastic road surface is a type of pavement surface which makes full use of waste rubber and at the same time effectively reduces traffic noise. However, the comprehensive performance of polyurethane rubber particle mixture (PRPM) prepared with different kinds of rubber particles and polyurethane is rarely studied. This study aims to evaluate the comprehensive performance of PRPMs, such as the high-temperature performance, anti-aging performance, skid resistance performance, etc. Rubber particles and four different kinds of polyurethane were adopted to prepare polyurethane rubber particle mixtures. A dog bone shape mold for forming specimens was self-designed, and a slab specimen forming device was created to form slab specimens. Different test methods were conducted to evaluate the comprehensive performance of the mixtures. The results showed that four types of polyurethane rubber particle mixtures exhibited high rutting resistance and high temperature performance. Mixture F5 had the best anti-aging performance and noise reduction performance. The skid-resistance of the polyurethane rubber particle mixture was slightly lower than that of conventional asphalt mixture, but it still met the road specification requirements. The polyurethane rubber particle mixture had more interconnected pores, which was found to have greater drainage function and was expected to reduce noise. Polyurethane rubber particle mixture F5 had superior comprehensive performance, except for slightly lower skid resistance, and it was determined to be the most suitable polyurethane rubber particle mixture type for future application.

Paocai as a traditional side dish in Asia gains its popularity owing to the favorable tastes, which are bestowed by microbial metabolisms. The microorganisms involved in fermentation are mainly lactic acid bacteria and Saccharomyces, while low-quality Paocai may contain spoilage and potentially pathogenic microorganisms such as Escherichia coli. To monitor and control food safety for direct vat inoculation of Paocai, this study intended to develop propidium monoazide based quantitative PCR to enumerate fermentation starter Lactobacillus plantarum NCU116, as well as Saccharomyces and potentially present pathogenic bacteria. Universal and specific primers targeting different microorganisms were designed to identify and quantify total lactic acid bacteria, Lactobacillus plantarum, Saccharomyces, and Escherichia coli by establishment of standard curves, respectively. Plate counting was performed and showed comparable results to quantitative PCR analysis, indicating the applicability and efficiency of absolute quantitative PCR in fast detection of microbial composition in Paocai system. Nitrite as a hazardous substance in Paocai was also investigated by spectrophotometry and reverse transcription quantitative PCR. The mechanism of nitrite synthesis and degradation was studied, revealing nitrite reductase in lactic acid bacteria as an effective enzyme in reducing nitrite present in the fermented vegetable.

Edwardsiella piscicida (E. piscicida) is an important zoonotic pathogen, which infects animals by colonizing the intestine. Glucose 6-phosphate (Glu6P) was an important carbohydrate in intestine and could be used as a regulate signal. Here we identify a virulence-regulating pathway named Glu6P transport regulatory protein UhpA, which affects the virulent genes of hemolysins, flagellar, T3SS, T6SS and metabolism related genes how to promote E. piscicida infect the host. The results showed that the metabolism related gene expression of cysteine synthase (orf 1134) and sulfate transporter (ychM) in the uhpA mutant strain ΔuhpA was 0.76-fold and 0.68-fold lower than the ones in the wild strains (P < 0.05), the gene expression of ethA and ethB in the ΔuhpA strain was 0.80-fold and 0.72-fold lower than the ones in the wild strains (P < 0.05). However, the gene expression of fliC and flgN in the ΔuhpA was 1.51-fold and 1.21-fold higher than the ones in the wild strains (P < 0.05), the gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the ΔuhpA was 1.27-fold, 1.13-fold 1.28-fold and 1.23-fold higher than the ones in the wild strains (P < 0.05). Besides, the survival rate of fish challenged with E. piscicida EIB202 and ΔuhpA was 50% and 30% respectively. These suggested that although the uhpA gene deletion decreased the metabolic level and the hemolysins related gene expression in E. piscicida, the uhpA gene could down regulate the key virulent gene expression to decrease the pathogenicity of E. piscicida in fish.

A number of vinylsulfonamides were synthesized and screened to identify reagents that can be used to modify octreotide under biological pH and room temperature with improved efficiency. N-Phenyl-N-aceto-vinylsulfonamide exhibits higher reactivity and has emerged as an efficient reagent that has the ability to realize the selective modification of peptides and proteins at the N-terminus via aza-Michael addition. We showed that, after conjugation of peptides and proteins with the reagent containing a bioorthogonal functional group, the derivatives could be further labelled by functionalities, including fluorescent tags, modified drugs and polyethylene glycol (PEG) polymers without the need for prior treatment. Somatostatin, lysozyme, and RNaseA were selectively modified at the N-terminus, which illustrated the application of the method.

Antibody-drug conjugates (ADCs) have emerging as efficient agents to target deliver cytotoxic drugs and reduce their off-target side effects. Triptolide has attracted attention to be used in ADC development. Herein, three rationally designed triptolide drug-linkers have been synthesized for use in site-specific construction of ADCs. Carbamates that were supposed to be more stable than carbonates were introduced to attach triptolide to the linkers. PEG and discrete PEG chains were incorporated to improve the hydrophilicity of drug-linkers. The ADCs were finally site-specifically prepared by conjugation of the drug-linkers to trastuzumab through disulfide re-bridging approach. The preliminary anti-tumor activities of these ADCs were evaluated and they displayed high potencies against HER2-targeted cancer in vitro and in vivo.

A series of vinylsulfonamides were synthesized and screened for site-selective modification of the ε-amino group of lysine-bearing free α-amine residues. N-Methyl-N-phenylethenesulfonamide has emerged as an applicable reagent and has been developed for efficient and highly selective modification of the lysine residue of native peptides in the presence of a free N-terminus via aza-Michael addition. We demonstrated that functional N-phenylvinylsulfonamide derivatives with a fluorescent moiety or drug could also be conjugated to the lysine residue of octreotide and insulin with high specificity, without modifying the N-terminus. Our method provides a promising strategy for site-selective lysine functionalization in native peptides with a free N-terminus.

A novel water soluble, phosphate-based enrichable, retro-Michael addition-driven MS-cleavable and multi-targeting cross-linker was developed., Chemical cross-linking mass spectrometry (XL-MS) is a powerful technology for obtaining protein structural information and studying protein–protein interactions. We report phospho-bisvinylsulfone (pBVS) as a novel water-soluble, MS-cleavable, phosphate-based enrichable and multi-targeting cross-linker. In this approach, the fragmentation of pBVS cross-linked peptides occurs in situ through retro-Michael addition. The phosphate group is successfully used as a small affinity tag to isolate cross-linked peptides from the highly abundant non-cross-linked peptides. In addition, the linker targets multiple types of amino acid residues, including cysteine, lysine and histidine. This method was applied to cross-link bovine serum albumin (BSA), myoglobin and Lbcpf1 demonstrating the ability to yield accurate and abundant information to facilitate protein structure elucidation.

A number of novel sterol derivatives with dipeptide-like side chains were synthesized using an Ugi four-component condensation method and assayed to test their anti-inflammatory effects in activated microglial cells. Compound 18b ((3S,10R,13S)-N-((R)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthrene-17-carboxamide) was identified as the most potent anti-inflammatory agent in the series of compounds analyzed. Compound 18b markedly inhibited the expression of proinflammatory factors, including inducible nitric oxide synthase, interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and cyclooxygenase-2 in lipopolysaccharide-stimulated microglial cells. Further studies showed that compound 18b significantly suppressed the transcriptional activity of AP-1 and NF-κB in activated microglial cells, which was likely mediated by the inhibition of the p38 MAPK and JNK signal transduction pathways. In addition, compound 18b displayed neuroprotective effects in a microglial-conditioned medium/neuron coculture and an experimental focal ischemic mouse model.

A new class of N-phenyl-divinylsulfonamides which can be easily prepared have been successfully developed and utilized as efficient linkers in the field of disulfide bond modification. Functional divinylsulfonamides provide opportunities for the specific introduction of various functionalities, including affinity probes, fluorescent tags, and drugs, into peptides.

A picosecond (ps) mid-infrared (MIR) optical parametric amplifier (OPA) with LiInSe2 crystal was demonstrated for the first time. The MIR OPA was pumped by a 30 ps 1064 nm Nd:YAG laser and injected by a barium boron oxide (BBO)-based widely tunable near-infrared seed. A maximum idler pulse energy of 433 μJ at 4 μm has been obtained under a pump energy of 17 mJ, and the corresponding pulse duration was estimated to be ~13 ps. To our knowledge, this is the highest single pulse energy generated by LiInSe2 crystal. Furthermore, an idler spectrum tuning from 3.6 to 4.8 μm was investigated at fixed pump energy of 15 mJ.