Interaction of Asn297-Linked Glycan Ligands with the Fc Fragment of the Immunoglobulin Class G1: A Molecular Dynamics Simulation Study.

The allosteric modulation of the homodimeric H10-03-6 protein to glycan ligands L1 and L2, and the STAB19 protein to glycan ligands L3 and L4, respectively, has been studied by molecular dynamics simulations and free energy calculations. The results revealed that the STAB19 protein has a significantly higher affinity for L3 (-11.38 ± 2.32 kcal/mol) than that for L4 (-5.51 ± 1.92 kcal/mol). However, the combination of the H10-03-6 protein with glycan L2 (1.23 ± 6.19 kcal/mol) is energetically unfavorable compared with that of L1 (-13.96 ± 0.35 kcal/mol). Further, the binding of glycan ligands L3 and L4 to STAB19 would result in the significant closure of the two CH2 domains of the STAB19 conformation with the decrease of the centroid distances between the two CH2 domains compared with the H10-03-6/L1/L2 complex. The CH2 domain closure of STAB19 relates directly to the formation of new hydrogen bonds and hydrophobic interactions between the residues Ser239, Val240, Asp265, Glu293, Asn297, Thr299, Ser337, Asp376, Thr393, Pro395, and Pro396 in STAB19 and glycan ligands L3 and L4, which suggests that these key residues would contribute to the specific regulation of STAB19 to L3 and L4. In addition, the distance analysis revealed that the EF loop in the H10-03-6/L1/L2 model presents a high flexibility and partial disorder compared with the stabilized STAB19/L3/L4 complex. These results will be helpful in understanding the specific regulation through the asymmetric structural characteristics in the CH2 and CH3 domains of the H10-03-6 and STAB19 proteins.

Mitochondria-targeting two-photon fluorescent probe for sequential recognition of Cu2+ and ATP in neurons and zebrafish.

Highly active mitochondria play a significant role in neuron function. Cu2+ and ATP levels in mitochondria regulate neuronal mitochondrial activity. However, mitochondrial activity was often evaluated by mitochondrial membrane potential. Less is known about the dynamics of Cu2+ and ATP in mitochondria. Herein, we developed a two-photon fluorescence probe (MP), which provided a determination of mitochondrial ATP and Cu2+. The fluorescence of MP showed remarkable quenching in the presence of Cu2+ and then gradually recovered in the presence of ATP, which can be used for sequential recognition. MP has high sensitivity to Cu2+ and ATP, with limits of detection (LOD) close to 0.31 nM and 13.6 nM, respectively. Using this useful probe, we monitor the fluctuation of concentrations of Cu2+ and ATP by fluorescence imaging at single neuron and zebrafish.

Phototheranostics for NIR fluorescence image guided PDT/PTT with extended conjugation and enhanced TICT.

Introducing donor and acceptor into conjugated system can facilitate the intersystem crossing (ISC) rate to increase the generation of ROS. Twisted intramolecular charge transfer (TICT) state could favor enhance the nonradiative transition and photothermal conversion efficiency (PCE). Herein, diketopyrrolopyrrole (DPP) core functionalized benzene (PDDP), thiophene (TDPP), triphenylamine-conjugated benzene (TPA-PDDP) and thiophene (TPA-TDPP) derivatives were designed and synthesized. Electrochemistry experiments revealed the heavy atom effect and the introduction of triphenylamine reduced the energy level of TPA-TDPP and improved the ability to generate 1O2 (1O2 QY = 50%). In addition, in the aggregated state, introduction of thiophene, triphenylamine, and long alkyl chains promoted the twisting effect, preventing the intermolecular π-π interaction and enhancing the PCE of TPA-TDPP (38.7%). In vivo fluorescence imaging showed that TPA-TDPP NPs can target the tumor site with the enhanced permeability and retention (EPR) effect and presented excellent synergistic photodynamic/photothermal therapy.

Research Progress on Small Molecular Inhibitors of the Type 3 Secretion System.

The overuse of antibiotics has led to severe bacterial drug resistance. Blocking pathogen virulence devices is a highly effective approach to combating bacterial resistance worldwide. Type three secretion systems (T3SSs) are significant virulence factors in Gram-negative pathogens. Inhibition of these systems can effectively weaken infection whilst having no significant effect on bacterial growth. Therefore, T3SS inhibitors may be a powerful weapon against resistance in Gram-negative bacteria, and there has been increasing interest in the research and development of T3SS inhibitors. This review outlines several reported small-molecule inhibitors of the T3SS, covering those of synthetic and natural origin, including their sources, structures, and mechanisms of action.

Cell-membrane-targeted near-infrared fluorescent probe for detecting extracellular ATP.

Extracellular adenosine triphosphate (ATP) as a signal molecule plays a key role in tumor progression and metastasis. Therefore, the development of a fluorescent probe to detect extracellular ATP is crucial for tumor treatment. However, small-molecule fluorescent probes have better advantages than biological probes, such as low price, easy modification, and optical tunability, but still remain highly challenging and rarely explored in extracellular ATP detection. Here, a near-infrared small molecule fluorescent probe (NIR-P) with hydrophobic alkyl chains and hydrophilic macrocyclic polyamines was prepared for the detection of extracellular ATP. The NIR-P exhibited enhanced fluorescence upon binding to ATP by electrostatic interaction and π-π interaction between phosphates and macrocyclic polyamines, adenines and benzene rings with a limit of detection (LOD) of 21 nM. In addition, with similarity and intermiscibility to the cell membrane, the NIR-P can specifically target cell membranes and image extracellular ATP. This work provides a cell-membrane-targeted fluorescent probe used for extracellular ATP detection.

Molecular cloning, characterization, and evolution analysis of the luciferase genes from three sympatric sibling fireflies (Lampyridae: Lampyrinae, Diaphanes).

Firefly adult bioluminescence functions as signal communication between sexes. How sympatric sibling species with similar glow pattern recognize their conspecific mates remains largely unknown. To better understand the role of the luciferases of sympatric fireflies in recognizing mates, we cloned the luciferase genes of three sympatric forest dwelling fireflies (Diaphanes nubilus, Diaphanes pectinealis, and Diaphanes sp2) and evaluated their enzyme characteristics. Our data show that the amino acid (AA) sequences of all three luciferases are highly conserved, including the identities (D. nubilus vs D. pectinealis: 99%; D. nubilus vs Diaphanes sp2: 98.5%; D. pectinealis vs Diaphanes sp2: 99.4%) and the protein structures. Three recombinant luciferases produced in vitro all possess significant luminescence activity at pH 7.8, and similar maximum emission spectrum (D. nubilus: 562 nm; D. pectinealis and Diaphanes sp2: 564 nm). They show the highest activity at 10 °C (D. pectinealis, Diaphanes sp2) and 15 °C (D. nubilus), and completely inactivation at 45 °C. Their KM for D-luciferin and ATP were 2.7 µM and 92 µM (D. nubilus), 3.7 µM and 49 µM (D. pectinealis), 3.5 µM and 46 µM (Diaphanes sp2). Phylogenetic analyses support that D. nubilus is sister to D. pectinealis with Diaphanes sp2 at their base, which further cluster with Pyrocoelia. All combined data indicate that sympatric Diaphanes species have similar luciferase characteristics, suggesting that other strategies (e.g., pheromone, active time, etc.) may be adopted to recognize mates. Our data provide new insights into Diaphanes luciferases and their evolution.

The Fate of SWCNTs in Mouse Peritoneal Macrophages: Exocytosis, Biodegradation, and Sustainable Retention.

The understanding of toxicological and pharmacological profiles of nanomaterials is an important step for the development and clinical application of nanomedicines. Carbon nanotubes (CNTs) have been extensively explored as a nanomedicine agent in pharmaceutical/biomedical applications, such as drug delivery, bioimaging, and tissue engineering. The biological durability of CNTs could affect the function of CNTs-based nanomedicines as well as their toxicity in cells and tissues. Therefore, it is crucial to assess the fate of nanomedicine in phagocytes. Herein, we investigated the candidate fate of acid-oxidized single-walled carbon nanotubes (SWNCTs) in non-activated primary mouse peritoneal macrophages (PMQ). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the intracellular SWCNTs continued growing from 4 to 36 h in PMQ. After replacing the exposure medium, we found the exosome induced by SWCNTs on the surface of macrophages according to scanning electron microscope (SEM) observation. The near-infrared (NIR) absorption increase of the supernatant samples after post-exposure indicates that SWCNTs exocytosis occurred in PMQ. The decreasing intracellular SWCNTs amount suggested the incomplete biodegradation in PMQ, which was confirmed by Raman spectroscopy and transmission electron microscopy (TEM). The combined data reveal that SWCNTs could be retained for more than 60 h in macrophages. Then sustainable retention of SWCNTs in primary macrophages was coexist with exocytosis and biodegradation. The findings of this work will shed light on the bioimaging, diagnosis and other biomedical applications of CNTs-based nanomedicines.

Biomimetic Modification and In Vivo Safety Assessment of Superparamagnetic Iron Oxide Nanoparticles.

The efficacy of superparamagnetic iron oxide nanoparticles (SPIONs) for biomedical applications depends on the magnetic properties, long time stability in biological fluids, and specific targeting capacity. The properties of SPIONs were generally improved by surface modification, but common modification technologies were usually conducted with multi-steps under rigid conditions. In this work, a facile and simple approach to synthesize functionalized SPIONs contrast agents was set up. First of all, SPIONs were prepared by an improved ultrasonic co-precipitation method. Then the surfaces of these SPIONs were modified biomimeticly by dopamine (DA) with strong adhesion. At last, the c(RGDyK), a biomolecule with the capacity of specific targeting capacity towards liver tumor cells, were coupled with DA on SPIONs via Mannich reaction. Thus the novel magnetic composite nanoparticles (abbreviated as c(RGDyK)-PDA-SPIONs) were successfully prepared. The as-synthesized nanoparticles were characterized by scanning electron microscope (SEM), dynamic light scattering, magnetic hysteresis loop measuring instrument. As a result, that the c(RGDyK)-PDA-SPIONs had an average size of about 50 nm and uniform distribution, and had superparamagnetic properties, good water dispersion stability. The acute toxicity test of the assynthesized c(RGDyK)-PDA-SPIONs to mice was also investigated. It was observed that LD50 of c(RGDyK)-PDA-SPIONs was 4.38 g/kg, with a 95% confidence interval ranging from 3.49 g/kg to 5.87 g/kg. These results indicated the novel c(RGDyK)-PDA-SPIONs had excellent biocompatibility, which was endowed with a potential capacity to serve as MRI contrast agents in diagnosis and treatment of the liver tumor.

Gene expression profiling analysis of locus coeruleus in idiopathic Parkinson's disease by bioinformatics.

This study aimed to explore the underlying molecular mechanisms of idiopathic Parkinson's disease (IPD) by bioinformatics analysis. Gene expression profile GSE34516 was downloaded from the Gene Expression Omnibus. Eight locus coeruleus post-mortem tissue samples derived from four IPD patients and four neurological healthy controls were used to identify the differentially expressed genes (DEGs) by paired t test. Based on the DEGs, principal components were analyzed. The Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway analysis of the genome microarray data were then performed. Finally, protein-protein interaction (PPI) network of the DEGs was constructed. Total 261 DEGs including 195 up-regulated and 66 down-regulated DEGs were identified. Intracellular protein transport and RNA splicing via transesterification reactions were selected as the most two significantly enriched functions. Mismatch repair, N-glycan biosynthesis, spliceosome and nucleotide excision repair were the significantly enriched pathways. In the PPI network, CTSS, CD53, IGSF6, PTPRC and LAPTM5 were the hub nodes. Intracellular protein transport and RNA splicing via transesterification reactions were closely associated with IPD. The DEGs, such as CX3CR1, SLC5A7, CD53 and PTPRC may be the potential targets for IPD diagnosis and treatment.

Single-walled carbon nanotubes and graphene oxides induce autophagosome accumulation and lysosome impairment in primarily cultured murine peritoneal macrophages.

The wide application of carbon nanomaterials in various fields urges in-depth understanding of the toxic effects and underlying mechanisms of these materials on biological systems. Cell autophagy was recently recognized as an important lysosome-based pathway of cell death, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles, but the underlying mechanisms are still uncertain due to the fact that autophagosome accumulation can result from autophagy induction and/or autophagy blockade. In this study, we first evaluated the toxicity of acid-functionalized single-walled carbon nanotubes and graphene oxides, and found that both carbon nanomaterials induced adverse effects in murine peritoneal macrophages, and GOs were more potent than AF-SWCNTs. Both carbon nanomaterials induced autophagosome accumulation and the conversion of LC3-I to LC3-II. However, degradation of the autophagic substrate p62 protein was also inhibited by both nanomaterials. Further analyses on lysosomes revealed that both carbon nanomaterials accumulated in macrophage lysosomes, leading to lysosome membrane destabilization, which indicates reduced autophagic degradation. The effects of AF-SWCNTs and GOs on cell autophagy revealed by this study may shed light on the potential toxic mechanism and suggest caution on their utilization.

Exposure of single-walled carbon nanotubes impairs the functions of primarily cultured murine peritoneal macrophages.

It is increasingly important to understand the single-walled carbon nanotubes' (SWCNTs) immune response as their increasingly biomedical researches and applications. Macrophages and T cells play important roles in scavenging foreign materials and pathogens and regulating immune response. In this work, primarily cultured murine peritoneal macrophages and purified splenic T cells were utilised to determine the toxic effects of SWCNTs and acid-functionalised SWCNTs (AF-SWCNTs) on the immune system, especially on macrophage functions. Macrophages were exposed to 0-50 µg/ml of CNTs for 24 h and no significant cytotoxicity was found by live/dead and annexin-V-FITC/PI analyses. The TEM images revealed that AF-SWCNTs were engulfed mostly through phagocytosis and located in lysosomes of macrophages. Measurement of mitochondrial membrane potential and proteasome subunit gene expression demonstrated that 10 and 50 µg/ml AF-SWCNTs could damage mitochondrial function and proteasome formation in a concentration-dependent manner. Functional analyses revealed that the percentage of phagocytic cells were affected significantly by 20 µg/ml CNTs, and 5 µg/ml AF-SWCNTs inhibited the phagocytic efficiency of latex beads in macrophages. The accessory cell function was affected by both AF-SWCNTs and SWCNTs at concentrations of 10 and 50 µg/ml, respectively. Furthermore, AF-SWCNT biased naïve T-cell differentiation to Th1 type by inducing the production of IFN-γ and TNF, implying the potential risk of Th1-associated diseases (e.g. autoimmune diseases and inflammation) on AF-SWCNT exposure.

In vitro toxicity of acid-functionalized single-walled carbon nanotubes: effects on murine macrophages and gene expression profiling.

Single-walled carbon nanotubes (SWCNTs) are widely used in industrial and medical sectors, and the increasing exposure of SWCNTs necessitates the studies of their potential environmental and health effects. Considerable efforts have been made to improve the dispersion of SWCNTs by chemical modifications. However, the toxicological effects of such modifications on SWCNTs are mostly unknown. This study was designed to determine the influences of acid functionalization on SWCNT toxicity and to understand the molecular toxic mechanisms. RAW264.7 cells were exposed to 0-50 µg/mL of as-synthesized SWCNTs or acid-functionalized SWCNTs (AF-SWCNTs) for 24 hours and then their toxicities were compared via viability analysis. After that the global gene expression profiles of cells exposed to AF-SWCNTs were obtained and analyzed. The results showed that AF-SWCNTs penetrated cell membrane and aggregated in cell cytoplasm and nuclear areas, resulting in enhanced toxicity. In addition, AF-SWCNTs altered the expression of genes related to ribosome, mitochondria, inflammatory response, cell cycle/apoptosis, and proteasome pathway. The gene expression study excluded the interference of metallic impurities and suggested similar toxic mechanism to that of ultra-fine particulate matters.