A strategy to enhance the water solubility of luminescent ß-diketonate-Europium(III) complexes for time-gated luminescence bioassays.

Luminescent ß-diketonate-europium(III) complexes have been found a wide range of applications in time-gated luminescence (TGL) bioassays, but their poor water solubility is a main problem that limits their effective uses. In this work we propose a simple and general strategy to enhance the water solubility of luminescent ß-diketonate-europium(III) complexes that permits facile synthesis and purification. By introducing the fluorinated carboxylic acid group into the structures of ß-diketone ligands, two highly water-soluble and luminescent Eu3+ complexes, PBBHD-Eu3+ and CPBBHD-Eu3+, were designed and synthesized. An excellent solubility exceeding 20 mg/mL for PBBHD-Eu3+ was found in a pure aqueous buffer, while it also displayed strong and long-lived luminescence (quantum yield φ = 26%, lifetime τ = 0.49 ms). After the carboxyl groups of PBBHD-Eu3+ were activated, the PBBHD-Eu3+-labeled streptavidin-bovine serum albumin (SA-BSA) conjugate was prepared, and successfully used for the immunoassay of human α-fetoprotein (AFP) and the imaging of an environmental pathogen Giardia lamblia under TGL mode, which demonstrated the practicability of PBBHD-Eu3+ for highly sensitive TGL bioassays. The carboxyl groups of PBBHD can also be easily derivatized with other reactive chemical groups, which enables PBBHD-Eu3+ to meet diverse requirements of biolabeling technique, to provide new opportunities for developing functional europium(III) complex biolabels serving for TGL bioassays.

A novel iridium(III) complex-based ratiometric luminescence probe for monitoring hydrogen sulfide in living cells and zebrafish.

Hydrogen sulfide exhibits crucial functions in many biological and physiological processes. The abnormal levels of H2S have been revealed to be associated with numerous human diseases. The majority of existing fluorescent probes toward H2S may still need to be improved in terms of single output signal, water solubility, biotoxicity and photostability. The construction of a ratiometric fluorescent probe based on metal complex is one effective strategy for avoiding the mentioned problems for precisely detecting H2S. Herein, we report an iridium(III) complex-based ratiometric luminescence probe (Ir-PNBD), which is designed by coupling the 7-nitro-2,1,3-benzoxadiazoles (NBD) to one of the bipyridine ligands of Ir (III) complex luminophore through a piperazition moiety. Ir-PNBD owns high selectivity and sensitivity toward H2S, and an excellent ability to target mitochondria. Moreover, Ir-PNBD was further successfully utilized to visualize exogenous and endogenous H2S in HeLa cells and zebrafish. Our work offers new opportunities to gain deeper insights into the construction of transition metal complex-based ratiometric luminescent probes and expands their applications in biomedical imaging and disease diagnosis.

Time-gated luminescent probes for lysosomal singlet oxygen: Synthesis, characterizations and bioimaging applications.

BACKGROUD: Single oxygen (1O2), the molecular oxygen at its excited state, plays a crucial role in the photodynamic therapy (PDT) of some diseases owing to its strong oxidizing property to destroy malignant cells. Although the fluorescent probe technique has proven its powerful application abilities for detection of 1O2 in biological systems, most of the reported fluorescent probes suffered from the interference of background autofluorescence of biological samples. It is clear that the real-time and in situ, background-free fluorescent detection of 1O2 generated in live cells, especially in some organelles, is of great significance for understanding the action mechanism of PDT drugs. RESULTS: By introducing a lysosome-anchoring motif, a morpholine moiety, into a 1O2-specifically-reactive terpyridine polyacid ligand, [4'-(9-anthryl)-2,2':6',2″-terpyridine-6,6″-diyl] bis(methylenenitrilo) tetrakis (acetic acid) (ATTA), and chelating with lanthanide ions (Eu3+ or Tb3+), two lanthanide complex-based "turn-on" luminescent probes that can be used for the background-free time-gated luminescent (TGL) detection of lysosomal 1O2, Lyso-ATTA-Eu3+ and Lyso-ATTA-Tb3+, have been developed. The probes exhibit fast luminescence responses (within 2.5 min) towards 1O2 with high selectivity and sensitivity (<0.75 µM) in a wide pH range (4-11). And the excellent lysosome-localization performance of the probes allowed them to be used for the monitoring of endogenous 1O2 in lysosomes, which enabled the variability of lysosomal-1O2 concentrations induced by different photosensitizers to be successfully discriminated. Furthermore, by doping Lyso-ATTA-Eu3+ into the polyethylene glycol (PEG) hydrogel, the smart luminescent sensor film, PEG-Lyso-ATTA-Eu3+, was prepared, and successfully used for the detection of the on-site 1O2 production during the PDT process of psoriatic disease in model mice. SIGNIFICANT: Two lysosome-targetable background-free TGL probes for 1O2 were firstly reported. The developed smart luminescent sensor film could be a powerful tool for the clinical monitoring of PDT on skin diseases without using sophisticated and expensive instruments.

FRET Luminescent Probe for the Ratiometric Imaging of Peroxynitrite in Rat Brain Models of Epilepsy-Based on Organic Dye-Conjugated Iridium(III) Complex.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures globally, imposing a substantial burden on patients and their families. The pathological role of peroxynitrite (ONOO-), which can trigger oxidative stress, inflammation, and neuronal hyperexcitability, is critical in epilepsy. However, the development of reliable, in situ, and real-time optical imaging tools to detect ONOO- in the brain encounters some challenges related to the depth of tissue penetration, background interference, optical bleaching, and spectral overlapping. To address these limitations, we present Ir-CBM, a new one-photon and two-photon excitable and long-lived ratiometric luminescent probe designed specifically for precise detection of ONOO- in epilepsy-based on the Förster resonance energy transfer mechanism by combining an iridium(III) complex with an organic fluorophore. Ir-CBM possesses the advantages of rapid response, one-/two-photon excitation, and ratiometric luminescent imaging for monitoring the cellular levels of ONOO- and evaluating the effects of different therapeutic drugs on ONOO- in the brain of an epilepsy model rat. The development and utilization of Ir-CBM offer valuable insights into the design of ratiometric luminescent probes. Furthermore, Ir-CBM serves as a rapid imaging and screening tool for antiepileptic drugs, thereby accelerating the exploration of novel antiepileptic drug screening and improving preventive and therapeutic strategies in epilepsy research.

Multi-omics analysis reveals substantial linkages between the oral-gut microbiomes and inflamm-aging molecules in elderly pigs.

Introduction: The accelerated aging of the global population has emerged as a critical public health concern, with increasing recognition of the influential role played by the microbiome in shaping host well-being. Nonetheless, there remains a dearth of understanding regarding the functional alterations occurring within the microbiota and their intricate interactions with metabolic pathways across various stages of aging. Methods: This study employed a comprehensive metagenomic analysis encompassing saliva and stool samples obtained from 45 pigs representing three distinct age groups, alongside serum metabolomics and lipidomics profiling. Results: Our findings unveiled discernible modifications in the gut and oral microbiomes, serum metabolome, and lipidome at each age stage. Specifically, we identified 87 microbial species in stool samples and 68 in saliva samples that demonstrated significant age-related changes. Notably, 13 species in stool, including Clostridiales bacterium, Lactobacillus johnsonii, and Oscillibacter spp., exhibited age-dependent alterations, while 15 salivary species, such as Corynebacterium xerosis, Staphylococcus sciuri, and Prevotella intermedia, displayed an increase with senescence, accompanied by a notable enrichment of pathogenic organisms. Concomitant with these gut-oral microbiota changes were functional modifications observed in pathways such as cell growth and death (necroptosis), bacterial infection disease, and aging (longevity regulating pathway) throughout the aging process. Moreover, our metabolomics and lipidomics analyses unveiled the accumulation of inflammatory metabolites or the depletion of beneficial metabolites and lipids as aging progressed. Furthermore, we unraveled a complex interplay linking the oral-gut microbiota with serum metabolites and lipids. Discussion: Collectively, our findings illuminate novel insights into the potential contributions of the oral-gut microbiome and systemic circulating metabolites and lipids to host lifespan and healthy aging.

Ratiometric lysosome-targeting luminescent probe based on a coumarin-ruthenium(II) complex for formaldehyde detection and imaging in living cells and mouse brain tissues.

Ratiometric luminescence probes have attracted widespread attention because of their self-calibration capability. However, some defects, such as small emission shift, severe spectral overlap and poor water solubility, limit their application in the field of biological imaging. In this study, a unique luminescence probe, Ru-COU, has been developed by combining tris(bipyridine)ruthenium(II) complex with coumarin derivative through a formaldehyde-responsive linker. The probe exhibited a large emission shift (Δλ > 100 nm) and good water solubility, achieving ratiometric emission responses at 505 nm and 610 nm toward formaldehyde under acidic conditions. Besides, ratiometric luminescence imaging of formaldehyde in living cells and Alzheimer disease mouse's brain slices demonstrates the potential value of Ru-COU for the diagnosis and treatment of formaldehyde related diseases.

A tumor-targetable probe based on europium(III)/gadolinium(III) complex-conjugated transferrin for dual-modal time-gated luminescence and magnetic resonance imaging of cancerous cells in vitro and in vivo.

The synergy of magnetic resonance imaging (MRI) and time-gated luminescence imaging (TGLI) provides a robust platform with extensive spatial resolution (from submicrometer to hundred-micron) and unlimited penetration depth for visual detection of lesion tissues and target biomolecules. In this work, highly stable lanthanide (Eu3+ and Gd3+) complexes with a terpyridine polyacid ligand, CNSTTA-Ln3+, were chosen as signal reporters for TGLI (Ln3+ = Eu3+) and MRI (Ln3+ = Gd3+), respectively. After conjugating CNSTTA-Ln3+ with a tumor-targetable glycoprotein, transferrin (Tf), the obtained bioconjugate, showed low cytotoxicity and high stability and exhibited strong long-lived luminescence (Tf-CNSTTA-Eu3+, ϕ = 10.8%, τ = 1.27 ms), high magnetic resonance relaxivity (Tf-CNSTTA-Gd3+, r1 = 8.70 mM-1 s-1, r2 = 10.90 mM-1 s-1), and high binding affinity toward Tf receptor-overexpressed cancerous cells. On the basis of these features, a tumor-targetable probe was constructed by simply mixing Tf-CNSTTA-Eu3+ and Tf-CNSTTA-Gd3+, and successfully used for the bimodal TGLI and MRI of tumor cells in tumor-bearing mice. The bimodal imaging simultaneously provided the anatomical and molecular information of the tumor, which enabled the accuracy for tumor diagnosis to be mutually verified, and revealed the potential of Tf-CNSTTA-Gd3+/Eu3+ for the monitoring of cancer cells in vivo.

An activatable nanoprobe based on nanocomposites of visible-light-excitable europium(III) complex-anchored MnO2 nanosheets for bimodal time-gated luminescence and magnetic resonance imaging of tumor cells.

Bimodal imaging probes that combine magnetic resonance imaging (MRI) and photoluminescence imaging are quite appealing since they can supply both anatomical and molecular information to effectively ameliorate the accuracy of detection. In this study, an activatable nanoprobe, [Eu(BTD)3(DPBT)]@MnO2, for bimodal time-gated luminescence imaging (TGLI) and MRI has been constructed by anchoring visible-light-excitable Eu3+ complexes on lamellar MnO2 nanosheets. Due to the luminescence quenching effect and non-magnetic resonance (MR) activity of MnO2 nanosheets, the developed nanoprobe presents quite weak TGL and MR signals. After exposure to H2O2 or GSH, accompanied by the transformation from MnO2 to Mn2+, the nanoprobe exhibits rapid, sensitive, and selective "turn-on" responses towards GSH and H2O2 in TGL and MR detection modes. Furthermore, the nanoprobe displays high stability, low cytotoxicity, good biocompatibility and water dispersion. Given the high contents of GSH and H2O2 in cancer cells, the nanoprobe was used for the identification of cancer cells by TGLI of intracellular GSH and H2O2, as well as for the tracing of tumor cells in tumor-bearing mice by tumor-targeting in vivo MRI and TGLI of tumor tissues. The research outcomes proved the potential of [Eu(BTD)3(DPBT)]@MnO2 as a useful nanoprobe for the tracing and accurate detection of cancer cells in vitro and in vivo via bimodal TGLI and MRI.

Mitochondria-Targetable Ratiometric Time-Gated Luminescence Probe Activated by Selenocysteine for the Visual Monitoring of Liver Injuries.

Liver injury can result from various risk factors including diabetes, virus, alcohol, drugs, and other toxins, which is mainly responsible for global mortality and morbidity. Selenocysteine (Sec), as the main undertaker of selenium function in the life system, features prominently in a series of hepatic injuries and has close association with the pathological progression of liver injuries. Here, we report a mitochondria-targetable lanthanide complex-based probe, Mito-NPTTA-Tb3+/Eu3+, that can be used for accurately determining Sec in live cells and laboratory animals via the ratiometric time-gated luminescence (TGL) technique. This probe is composed of 2,2':6',2″-terpyridine-Tb3+/Eu3+ mixed complexes as the luminophore, 2,4-dinitrophenyl (DNP) as the responsive moiety and a lipophilic triphenylphosphonium cation (PPh3+) as the mitochondria-targeting moiety. Upon reaction with Sec, accompanied by the cleavage of DNP from the probe molecule, the I540/I690 ratio of the probe increased by 55 times, which enabled Sec to be detected with the ratiometric TGL method. After being incubated with living cells, the probe molecules were selectively accumulated in mitochondria to allow the mitochondrial Sec to be successfully imaged under the ratiometric TGL mode. Importantly, using this probe coupled with the ratiometric TGL imaging technique, the fluctuations of liver Sec in various liver injuries of model mice induced by diabetes, drug, toxin, and alcohol were precisely monitored, revealing that Sec plays an important antioxidant role during the oxidative stress process in liver injury, and the Sec levels have a close interrelationship with the degree of liver injury. All the results suggest that the new probe Mito-NPTTA-Tb3+/Eu3+ could be a potential tool for the accurate diagnosis of liver injury.

Detection of rare prostate cancer cells in human urine offers prospect of non-invasive diagnosis.

Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4-10 cells/mL. Blinded analysis of each patient urine sample, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3-20 PCa cells per mL by TGiA, 4-13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine samples of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa.

The Effect of Dexmedetomidine on Postoperative Nausea and Vomiting in Patients Undergoing Thoracic Surgery-A Meta-Analysis of a Randomized Controlled Trial.

Background: Dexmedetomidine reduces the occurrence of postoperative nausea and vomiting (PONV); however, the effect of dexmedetomidine on PONV in patients undergoing thoracic surgery remains inconclusive. In addition, the effect of different dexmedetomidine application methods, anesthetics, and surgical procedures on the effects of dexmedetomidine on PONV remains unclear. Therefore, the purpose of this meta-analysis was to study the effect of dexmedetomidine on PONV in patients undergoing thoracic surgery. Methods: Electronic databases were searched to identify randomized controlled trials studying the effects of dexmedetomidine on nausea and vomiting after thoracic surgery. In total, 12 articles that met the inclusion criteria were obtained. The primary outcome of this comprehensive analysis was the incidence of PONV; secondary outcomes included the incidence of postoperative nausea, the incidence of postoperative vomiting, postoperative visual analog score (VAS), the amount of intraoperative sufentanil, and the number of times postoperative salvage analgesia was administered. Results: Twelve trials involving 905 participants undergoing thoracic surgery were included. Compared with placebo, dexmedetomidine reduced the incidence of nausea and vomiting after thoracic surgery [12 trials; 905 participants; risk ratio (RR) = 0.32; 95% CI (0.23, 0.44); P < 0.00001, I2 = 0%]. The subgroup analysis revealed that dexmedetomidine reduces the occurrence of PONV in both thoracotomy and thoracoscopic surgery. In addition, both intravenous and local infusion of dexmedetomidine can reduce the occurrence of PONV, and intravenous or inhaled anesthetics do not affect the effect of dexmedetomidine on reducing PONV. Dexmedetomidine can reduce the postoperative resting VAS of patients, and no statistically significant differences in the amount of intraoperative sufentanil and the number of salvage analgesia procedures after surgery were noted. Conclusion: Compared with placebo, dexmedetomidine can reduce the occurrence of PONV in patients undergoing thoracic surgery, and this effect is not affected by the method of dexmedetomidine administration, use of minimally invasive surgery, and use of a combination of intravenous or inhalation anesthetics. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/#myprospero, PROSPERO, identifier: CRD42021269358.

Comparative transcriptome analysis of experimental cryptorchidism: of mice and cynomolgus monkeys.

In most mammalian species, the testis descends from the abdomen into the scrotum during fetal or neonatal life. The failure of testicular descent, a pathological condition known as cryptorchidism, has long been the subject of scientific interest in a wide range of fields, including medicine, developmental biology, and evolutionary biology. In this study, we analyzed global gene expression changes associated with experimental cryptorchidism in mice by using RNA-seq. A total of 453 differentially expressed genes were identified, of which 236 genes were upregulated, and 217 genes were downregulated. Gene ontology, pathway, and gene network analysis highlighted the activation of inflammatory response in experimental cryptorchidism. By examining the promoter regions of differentially expressed genes, we identified 12 causal transcription factors. In addition, we also induced experimental cryptorchidism in two cynomolgus monkeys and performed RNA-seq. A cross-species comparison was performed at the gene expression level. Our study provides a valuable resource for further understanding the molecular mechanisms of cryptorchidism in mammals.

Development of a fluorescein modified ruthenium(II) complex probe for lysosome-targeted ratiometric luminescence detection and imaging of peroxynitrite in living cells.

Ratiometric luminescence (fluorescence/phosphorescence) probes have attracted widespread attention of researchers in the field of biological detection and noninvasive imaging of bioactive molecules in living systems. However, most of them suffer from some defects such as small emission shift, different excitation wavelength and spectral overlap, which eventually affect the luminescence ratio, thus leading to limitations in ratiometric bioimaging applications. In this paper, we present a novel "ruthenium(II) complex-fluorescein" scaffold probe (Ru-FL-ONOO) for ratiometric luminescence detection of peroxynitrite (ONOO-), in which a Ru(II) complex was conjugated to fluorescein serving as the dual-emissive moiety and the spirocyclic structure of fluorescein-phenylhydrazine was used as the specifically-reactive moiety for recognizing ONOO-. The probe possesses not only favourable specificity but also high sensitivity for responding to ONOO-, exhibiting a large emission shift (Δλem > 120 nm) at a single excitation wavelength. After being transferred into living cells, the probe localized within lysosomes, allowing ONOO- therein to be imaged at ratiometric mode. The imaging results reveal that the ratiometric probe bearing the Ru(II) complex-fluorescein scaffold could be a useful approach for overcoming the drawback of spectral overlap of dual-emissive moiety under single-wavelength excitation so as to improve the signal-to-noise ratio, thus benefiting the development of ratiometric bioimaging.

The TLR4/NF-κB/MAGI-2 signaling pathway mediates postoperative delirium.

PURPOSE: To evaluate the TLR4/NF-κB/MAGI-2 signaling pathway in postoperative delirium. METHODS: Elderly patients aged 65-80 years who received unilateral hip arthroplasty under subarachnoid anesthesia were included. Pre-anesthesia cerebrospinal fluid and perioperative blood samples were collected. After follow-up, patients were divided into two groups according to the occurrence of postoperative delirium (POD) after surgery. The potential differentially expressed proteins in the two groups were determined by proteomics assay and subsequent western blot validation. A POD model of aged mice was established, and the TLR4/NF-κB/MAGI-2 signaling pathway was determined. MAIN FINDINGS: The IL-1ß and TNF-α levels in pre-anesthesia cerebrospinal fluid and postoperative blood were higher in patients who developed POD than in those patients who did not. Compared with non-POD patients, MAGI-2 was highly expressed in POD patients, as validated by proteomics assays and western blotting. Higher p-NF-κB-p65, TLR4 and MAGI-2 in POD patients were detected by western blot. The POD model in aged mice was successfully established and verified by three behavioral tests. Postoperative inflammatory cytokines and the TLR4/NF-κB/MAGI-2 signaling pathway were increased in mice with POD. Inhibiting TLR4/NF-κB/MAGI-2 signaling pathway could reduce postoperative delirium. CONCLUSIONS: The TLR4/NF-κB/MAGI-2 signaling pathway mediates POD.

The Effects of Sevoflurane vs. Propofol for Inflammatory Responses in Patients Undergoing Lung Resection: A Meta-Analysis of Randomized Controlled Trials.

Objective: Inflammatory cytokines are increased during one-lung ventilation in patients undergoing lung resection, and this increase can be fatal. Propofol and sevoflurane are the main anesthetics used for these patients. Unfortunately, there is no consensus on the best choice of an anesthetic agent concerning an inflammatory response in patients undergoing lung resection. This meta-analysis aimed to compare the effects of propofol and sevoflurane on the inflammatory response in patients undergoing lung resection. Methods: We searched electronic databases to identify randomized controlled trials comparing the effects of different anesthetics (sevoflurane vs. propofol) on the inflammatory response. The primary outcome concerned the concentration of systemic inflammatory cytokines. The secondary outcomes concerned the concentrations of inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid from the dependent and independent lung. Random effects analysis of the meta-analyses were performed to synthesize the evidence and to assess the concentrations of inflammatory factors in the sevoflurane and propofol groups. Results: Eight trials involving 488 participants undergoing lung resection with one-lung ventilation were included. There was no significant difference in the concentrations of systemic interleukin (IL)-6, IL-10, or tumor necrosis factor α between the sevoflurane and propofol groups. Compared with the propofol group, BAL levels of IL-6 in the dependent ventilated lung were decreased in the sevoflurane group (three trials, 256 participants; standardized mean difference [SMD], -0.51; 95% confidence interval [CI], -0.90 to -0.11; p = 0.01; I 2 = 46%). The BAL levels of IL-6 in the independent ventilated lung were also decreased by sevoflurane (four trials, 362 participants; SMD, -0.70; 95% [CI], -0.93 to -0.47; p < 0.00001; I 2 = 0%). Conclusions: There was no difference in the systemic inflammatory response between the sevoflurane and propofol groups. However, compared with propofol, sevoflurane can reduce the local alveolar inflammatory response. Additional research is necessary to confirm whether the inflammatory response is direct or indirect.

Natural killer cell exhaustion in lung cancer.

OBJECTIVES: Lung cancer is one of the most frequently diagnosed cancers worldwide. However, the potential causes of lung cancer oncogenesis are still unclear. This study aims to explore the phenomenon and mechanism of NK cell exhaustion in lung cancer and lay the foundation for developing a targeting strategy to ameliorate immune cell exhaustion in cancer. MATERIALS AND METHODS: NK cells were isolated from the blood samples of lung cancer patients and healthy volunteers. After culture in vitro, the colony forming ability, cytotoxicity, apoptosis and receptor expression of NK cells in the peripheral blood from the lung cancer patients and the volunteers were analyzed by flow cytometry and the corresponding methods. The correlation between the NK cell profile and lung cancer occurrence was analyzed as well. RESULTS: The colony formation and cytotoxicity of the NK cells from the lung cancer group were significantly decreased compared to whose of the NK cells from volunteers. The expression of NKG2A was upregulated and CD226 was downregulated significantly in the lung cancer group compared with the control group. Furthermore, through correlation analysis, the colony forming level, cytotoxicity and CD226 expression level were significantly negatively correlated with lung cancer, and the expression level of NKG2A was significantly positively correlated with lung cancer. Moreover, the impaired colony formation of NK cells was significantly correlated with NK cell functional exhaustion in lung cancer. CONCLUSIONS: The downregulated CD226 expression and the upregulated NKG2A expression may serve as potential markers of NK cells exhaustion in lung cancer.

Bioconjugates of versatile ß-diketonate-lanthanide complexes as probes for time-gated luminescence and magnetic resonance imaging of cancer cells in vitro and in vivo.

Magnetic resonance imaging (MRI) and optical imaging (OI) are attractive for constructing bimodal probes due to their complementary imaging characteristics. The combination of these two techniques could be a useful tool to simultaneously obtain both anatomical and molecular information as well as to significantly improve the accuracy of detection. In this study, we found that ß-diketonate-lanthanide complexes, BHHBCB-Ln3+, could covalently bind to proteins to exhibit long-lived and intense luminescence (Ln3+ = Eu3+, τ = 0.52 ms, Φ = 0.40) and remarkably high relaxivity (Ln3+ = Gd3+, r1 = 35.67 mM-1 s-1, r2 = 43.25 mM-1 s-1) with excellent water solubility, stability and biocompatibility. Hence, we conjugated BHHBCB-Ln3+ with a tumor-targetable biomacromolecule, transferrin (Tf), to construct the probes, Tf-BHHBCB-Ln3+, for time-gated luminescence (TGL, Ln3+ = Eu3+) and MR (Ln3+ = Gd3+) imaging of cancerous cells in vitro and in vivo. As expected, the as-prepared probes showed high specificity to bind with the transferrin receptor-overexpressed cancerous cells, to enable the probe molecules to be accumulated in these cells. Using Tf-BHHBCB-Ln3+ as probes, the cultured cancerous cells and the tumors in tumor-bearing mice have been clearly visualized by background-free TGL and in vivo MR imaging. The research outcomes suggested the potential of ß-diketonate-lanthanide complexes for use in constructing bimodal TGL/MR imaging bioprobes.

Calibration for quantitative Fc-glycosylation analysis of therapeutic IgG1-type monoclonal antibodies by using glycopeptide standards.

Fc-glycosylation has crucial impact on the efficacy and safety of IgG-type therapeutic monoclonal antibodies (mAbs). In order to enhance the performance of MS-based bottom-up quantitation strategy, a library of glycopeptide standards containing 26 common IgG1-type Fc-glycoforms has been constructed via modified two-dimensional hydrophilic interaction liquid chromatography (HILIC) purification. Taking advantage of the acquired glycopeptide standards, calibrated quantitation strategy for Fc-glycosylation analysis of mAbs was established and evaluated on the basis of three LC-MS-based methods, including HILIC-MRM (multiple reaction monitoring), HILIC-SIM (selected ion monitor) and RPLC-SIM. Molar concentrations of eleven individual Fc-glycoforms (0.03 ± 0.001-13.77 ± 0.64 nmol mg-1) as well as degree of fucosylation (75.44-97.04%), galactosylation (3.39-49.47%) and mannosylation (1.12-21.22%) in six IgG1-type mAbs were achieved. In addition, Fc-glycosylation site occupancy was also determined from 98.05% to 99.83%. Compared with traditional MS-based quantitation via peak area normalization, the quantitation accuracy and precision of the calibrated strategy had been remarkably improved, especially when combining with HILIC separation. In addition, the transferability of calibrated quantitation as assessed by using MRM-based method had also been significantly enhanced on different instruments from different laboratories. This calibrated quantitation strategy using glycopeptide standards as calibrators will be useful for Fc-glycosylation analysis of IgG1-type mAbs with multiple glycosylation sites.

A Ruthenium(II) complex-based probe for colorimetric and luminescent detection and imaging of hydrogen sulfide in living cells and organisms.

The development of reliable bioanalytical probes for sensitive and specific detection of hydrogen sulfide (H2S) plays important role for better understanding the roles of this biomolecule in living cells and organisms. Taking advantages of unique photophysical properties of ruthenium(II) (Ru(II)) complex, this work presents the development of a responsive Ru(II) complex probe, Ru-PNBD, for colorimetric and luminescent analysis of H2S in living cells and organisms. In aqueous solution, Ru-PNBD is yellow color and non-luminescent because of the photoinduced electron transfer (PET) process from Ru(II) complex luminophore to NBD moiety. The H2S-triggered specific nucleophilic substitution reaction with Ru-PNBD cleaves the NBD moiety to form pink NBD-SH and highly luminescent Ru-PH. The color of the solution thus changes from yellow to pink for colorimetric analysis and the emission intensity is about 65-fold increased for luminescent analysis. Ru-PNBD has high sensitivity and selectivity for H2S detection, low cytotoxicity and good permeability to cell membrane, which allow the application of this probe for H2S imaging in living cells, Daphnia magna, and larval zebrafish. Collectively, this work provides a useful tool for H2S analysis and expands the scope of transition metal complex probes.

Purification of natural neutral N-glycans by using two-dimensional hydrophilic interaction liquid chromatography × porous graphitized carbon chromatography for glycan-microarray assay.

Glycan microarray for studying carbohydrate-protein interactions requires diverse classes of well-defined glycan standards. In this study, a purification strategy was established based on two-dimensional hydrophilic interaction liquid chromatography and porous graphitized carbon chromatography (HILIC × PGC) for the acquisition of neutral N-glycan standards from natural source. A total of thirty-one N-glycan compounds including seven pairs of isomers with the amounts from 0.7 to 230.0 nmol were isolated from ovalbumin as the model glycoconjugate. The purified N-glycans covered high-mannose, hybrid as well as multi-antenna asymmetric complex types. The purity of majority of these N-glycans was higher than 90%. Detailed structures of the N-glycan compounds were verified via negative ion tandem MS analysis, in which specific diagnostic ions including D- and E-ions were used to identify isomeric and terminal fine structures. The tag-free glycan compounds with well-defined structures, purity and amounts were finally assembled on the glass slide through neoglycolipid technology. Microarray binding assay of purified glycans with WGA lectin indicated the potential of the established strategy in glycan library expansion and functional glycomics.