Global and cell type-specific immunological hallmarks of severe dengue progression identified via a systems immunology approach.

Severe dengue (SD) is a major cause of morbidity and mortality. To define dengue virus (DENV) target cells and immunological hallmarks of SD progression in children's blood, we integrated two single-cell approaches capturing cellular and viral elements: virus-inclusive single-cell RNA sequencing (viscRNA-Seq 2) and targeted proteomics with secretome analysis and functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion as well as cell-cell communications point towards increased immune cell migration and inflammation in SD progressors. Concurrently, antigen-presenting cells from SD progressors demonstrate intact uptake yet impaired interferon response and antigen processing and presentation signatures, which are partly modulated by DENV. Increased activation, regulation and exhaustion of effector responses and expansion of HLA-DR-expressing adaptive-like NK cells also characterize SD progressors. These findings reveal DENV target cells in human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.

Hyperoxia prevents the dynamic neonatal increases in lung mesenchymal cell diversity.

Rapid expansion of the pulmonary microvasculature through angiogenesis drives alveolarization, the final stage of lung development that occurs postnatally and dramatically increases lung gas-exchange surface area. Disruption of pulmonary angiogenesis induces long-term structural and physiologic lung abnormalities, including bronchopulmonary dysplasia, a disease characterized by compromised alveolarization. Although endothelial cells are primary determinants of pulmonary angiogenesis, mesenchymal cells (MC) play a critical and dual role in angiogenesis and alveolarization. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung to profile mesenchymal cells during alveolarization and in the context of lung injury. Specific mesenchymal cell subtypes were present at birth with increasing diversity during alveolarization even while expressing a distinct transcriptomic profile from more mature correlates. Hyperoxia arrested the transcriptomic progression of the MC, revealed differential cell subtype vulnerability with pericytes and myofibroblasts most affected, altered cell to cell communication, and led to the emergence of Acta1 expressing cells. These insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world.

Astaxanthin accumulation in Microcystis aeruginosa under different light quality.

The aim of this work was to uncover the astaxanthin biosynthesis mechanism in Microcystis aeruginosa under optimum light quality, and promote astaxanthin production using this alga. Among purple, blue and red light, only purple light promoted M. aeruginosa cell growth compared with white light, due to up-regulating expression of the genes related with DNA replication. An increase was detected in the photosynthetic rate under purple light, which should be caused by the raised carotenoid content and up-regulation of the genes associated with light reaction and carbon fixation. Compared with white light, purple light increased the levels of ß-carotene, zeaxanthin and astaxanthin by up-regulating expression of the genes related with methylerythritol-4-phosphate pathway (MEP) and astaxanthin biosynthesis. For red and blue light, they did not impact or declined the content of astaxanthin and its precursors. Therefore, purple light promoted M. aeruginosa cell growth and astaxanthin production by up-regulating related gene expression.

Transcriptome analysis of well-differentiated laryngeal squamous cell carcinoma cells in below-background environment.

Background: Preliminary research has shown an inhibited growth rate of well-differentiated laryngeal squamous cell carcinoma cells (FD-LSC-1) in below-background radiation (BBR), but how the cells respond to this environmental stress and the potential mechanisms are yet unknown. The current study aimed to reveal the molecular differences in cells grown under BBR conditions and normal radiation at the transcriptional level. Methods: The expression profiles between FD-LSC-1 cells grown in a deep underground laboratory and above ground laboratory collected on day 4 were investigated by whole-transcriptome analysis, including messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs). Functional analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were then implemented for differentially expressed (DE) mRNAs and target genes of lncRNAs and circRNAs. Co-expression levels and the Bayesian network of DE genes were subsequently constructed, and the reliability of expression patterns were validated by quantitative real-time polymerase chain reaction (PCR). Results: The study identified a total of 671 mRNAs, 286 lncRNAs, 489 circRNAs, and 6 miRNAs as significantly expressed in response to the environmental stress. The GO annotations regarding the biological processes category were mainly biological regulation, metabolic process, response to stimulus, cell cycle, and modification process. The KEGG enrichment analysis indicated that TGF-ß and Hippo signaling played a crucial role in the transcriptional regulation of FD-LSC-1 cell growth under background radiation. Further network construction suggested that the enriched KEGG pathways affected this process by regulating cell proliferation-related genes including SMAD, SMAD7, CDH1, EGR1, and BMP2. Conclusions: Below-background radiation can lead to transcriptional changes in FD-LSC-1 cells cultured in the deep underground. The inhibitory growth effect is associated with multiple biological processes as well as canonical pathways of proliferation.

Urine metabolomics analysis of sleep quality in deep-underground miners: A pilot study.

Background: In previous questionnaire surveys of miners, sleep disorders were found among underground workers. The influence of the special deep-underground environment and its potential mechanism are still unclear. Therefore, this study intends to utilize LC-MS metabolomics to study the potential differences between different environments and different sleep qualities. Methods: Twenty-seven miners working at 645-1,500 m deep wells were investigated in this study, and 12 local ground volunteers were recruited as the control group. The Pittsburgh Sleep Quality Index (PSQI) was used to examine and evaluate the sleep status of the subjects in the past month, and valuable basic information about the participants was collected. PSQI scores were obtained according to specific calculation rules, and the corresponding sleep grouping and subsequent analysis were carried out. Through liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics analysis, differences in metabolism were found by bioinformatics analysis in different environments. Results: Between the deep-underground and ground (DUvsG) group, 316 differential metabolites were identified and 125 differential metabolites were identified in the good sleep quality vs. poor sleep quality (GSQvsPSQ) group. The metabolic pathways of Phenylalanine, tyrosine and tryptophan biosynthesis (p = 0.0102) and D-Glutamine and D-glutamate metabolism (p = 0.0241) were significantly enriched in DUvsG. For GSQvsPSQ group, Butanoate metabolism was statistically significant (p = 0.0276). L-Phenylalanine, L-Tyrosine and L-Glutamine were highly expressed in the deep-underground group. Acetoacetic acid was poorly expressed, and 2-hydroxyglutaric acid was highly expressed in good sleep quality. Conclusions: The influence of the underground environment on the human body is more likely to induce specific amino acid metabolism processes, and regulate the sleep-wake state by promoting the production of excitatory neurotransmitters. The difference in sleep quality may be related to the enhancement of glycolytic metabolism, the increase in excitatory neurotransmitters and the activation of proinflammation. L-phenylalanine, L-tyrosine and L-glutamine, Acetoacetic acid and 2-hydroxyglutaric acid may be potential biomarkers correspondingly.

Whole Transcriptome Analysis Revealed a Stress Response to Deep Underground Environment Conditions in Chinese Hamster V79 Lung Fibroblast Cells.

Background: Prior studies have shown that the proliferation of V79 lung fibroblast cells could be inhibited by low background radiation (LBR) in deep underground laboratory (DUGL). In the current study, we revealed further molecular changes by performing whole transcriptome analysis on the expression profiles of long non-coding RNA (lncRNA), messenger RNA (mRNA), circular RNA (circRNA) and microRNA (miRNA) in V79 cells cultured for two days in a DUGL. Methods: Whole transcriptome analysis including lncRNA, mRNAs, circ RNA and miRNA was performed in V79 cells cultured for two days in DUGL and above ground laboratory (AGL), respectively. The differentially expressed (DE) lncRNA, mRNA, circRNA, and miRNA in V79 cells were identified by the comparison between DUGL and AGL groups. Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to verify the selected RNA sequencings. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was analyzed for the DE mRNAs which enabled to predict target genes of lncRNA and host genes of circRNA. Results: With |log2(Fold-change)| ≥ 1.0 and p < 0.05, a total of 1257 mRNAs (353 mRNAs up-regulated, 904 mRNAs down-regulated), 866 lncRNAs (145 lncRNAs up-regulated, 721 lncRNAs down-regulated), and 474 circRNAs (247 circRNAs up-regulated, 227 circRNAs down-regulated) were significantly altered between the two groups. There was no significant difference in miRNA between the two groups. The altered RNA profiles were mainly discovered in lncRNAs, mRNAs and circRNAs. DE RNAs were involved in many pathways including ECM-RI, PI3K-Akt signaling, RNA transport and the cell cycle under the LBR stress of the deep underground environment. Conclusion: Taken together, these results suggest that the LBR in the DUGL could induce transcriptional repression, thus reducing metabolic process and reprogramming the overall gene expression profile in V79 cells.

Proteomics provides insights into the inhibition of Chinese hamster V79 cell proliferation in the deep underground environment.

As resources in the shallow depths of the earth exhausted, people will spend extended periods of time in the deep underground space. However, little is known about the deep underground environment affecting the health of organisms. Hence, we established both deep underground laboratory (DUGL) and above ground laboratory (AGL) to investigate the effect of environmental factors on organisms. Six environmental parameters were monitored in the DUGL and AGL. Growth curves were recorded and tandem mass tag (TMT) proteomics analysis were performed to explore the proliferative ability and differentially abundant proteins (DAPs) in V79 cells (a cell line widely used in biological study in DUGLs) cultured in the DUGL and AGL. Parallel Reaction Monitoring was conducted to verify the TMT results. γ ray dose rate showed the most detectable difference between the two laboratories, whereby γ ray dose rate was significantly lower in the DUGL compared to the AGL. V79 cell proliferation was slower in the DUGL. Quantitative proteomics detected 980 DAPs (absolute fold change ≥ 1.2, p < 0.05) between V79 cells cultured in the DUGL and AGL. Of these, 576 proteins were up-regulated and 404 proteins were down-regulated in V79 cells cultured in the DUGL. KEGG pathway analysis revealed that seven pathways (e.g. ribosome, RNA transport and oxidative phosphorylation) were significantly enriched. These data suggest that proliferation of V79 cells was inhibited in the DUGL, likely because cells were exposed to reduced background radiation. The apparent changes in the proteome profile may have induced cellular changes that delayed proliferation but enhanced survival, rendering V79 cells adaptable to the changing environment.

Proteomic Characterization of Proliferation Inhibition of Well-Differentiated Laryngeal Squamous Cell Carcinoma Cells Under Below-Background Radiation in a Deep Underground Environment.

Background: There has been a considerable concern about cancer induction in response to radiation exposure. However, only a limited number of studies have focused on the biological effects of below-background radiation (BBR) in deep underground environments. To improve our understanding of the effects of BBR on cancer, we studied its biological impact on well-differentiated laryngeal squamous cell carcinoma cells (FD-LSC-1) in a deep underground laboratory (DUGL). Methods: The growth curve, morphological, and quantitative proteomic experiments were performed on FD-LSC-1 cells cultured in the DUGL and above-ground laboratory (AGL). Results: The proliferation of FD-LSC-1 cells from the DUGL group was delayed compared to that of cells from the AGL group. Transmission electron microscopy scans of the cells from the DUGL group indicated the presence of hypertrophic endoplasmic reticulum (ER) and a higher number of ER. At a cutoff of absolute fold change ≥ 1.2 and p < 0.05, 807 differentially abundant proteins (DAPs; 536 upregulated proteins and 271 downregulated proteins in the cells cultured in the DUGL) were detected. KEGG pathway analysis of these DAPs revealed that seven pathways were enriched. These included ribosome (p < 0.0001), spliceosome (p = 0.0001), oxidative phosphorylation (p = 0.0001), protein export (p = 0.0001), thermogenesis (p = 0.0003), protein processing in the endoplasmic reticulum (p = 0.0108), and non-alcoholic fatty liver disease (p = 0.0421). Conclusion: The BBR environment inhibited the proliferation of FD-LSC-1 cells. Additionally, it induced changes in protein expression associated with the ribosome, gene spliceosome, RNA transport, and energy metabolism among others. The changes in protein expression might form the molecular basis for proliferation inhibition and enhanced survivability of cells adapting to BBR exposure in a deep underground environment. RPL26, RPS27, ZMAT2, PRPF40A, SNRPD2, SLU7, SRSF5, SRSF3, SNRPF, WFS1, STT3B, CANX, ERP29, HSPA5, COX6B1, UQCRH, and ATP6V1G1 were the core proteins associated with the BBR stress response in cells.

Oat protein-shellac nanoparticles as a delivery vehicle for resveratrol to improve bioavailability in vitro and in vivo.

Aim: Oat protein-shellac nanoparticles (NPs) were developed as a delivery system for resveratrol to improve bioavailability. Materials & methods: The NPs were prepared from w/w emulsion followed by cold-gelation. In vitro release and cell uptake mechanism of NPs were estimated by HPLC and confocal laser scanning microscopy. In vivo bioavailability and hepatoprotective activity of encapsulated resveratrol were studied using rat models. Results & conclusion: NPs (90-300 nm) protected resveratrol in gastric fluid, while allowing controlled release into small intestine in vitro. The optimized NPs showed improvement in resveratrol cell uptake and transport when compared with free resveratrol. NP-100S increased resveratrol bioavailability up to 72.4%, and the absorbed resveratrol effectively prevented CCl4-induced hepatotoxicity by attenuating oxidative stress.

Epithelia transmembrane transport of orally administered ultrafine drug particles evidenced by environment sensitive fluorophores in cellular and animal studies.

Little is known about the in vivo fate of drug particles taken orally, in particular, the drug release kinetics and interaction with the gastrointestinal (GI) membrane. Lacking is analytical means that can reliably identify the integrity of drug particles under the complexity of biological environment. Herein, we explored fluorescent probes whose signals become quenched upon being released from drug carriers. Taking advantage of so-called the aggregation caused quenching (ACQ), particles may be identified by the integrated fluorophores, which are "turned off" when the particles become destructed and dyes are released. In the current study, ultrafine amorphous particles (UAPs) of cyclosporin A (CsA) were prepared with synthesized ACQ dyes physically entrapped. The fluorescence intensity of suspension of these UAPs was found correlated well with the dissolution of the particles. When given to rats orally, it was found that some of the administered UAPs could survive the animal's GI tracts for as long as 18h. Whole-body fluorescence imaging detected fluorescent signals in the liver and lungs. Particularly noticed in sections of jejunum and ileum, the detection suggested the possibility of direct absorption of UAPs through epithelial membranes. Moreover, 250nm particles were absorbed faster via transepithelia than larger ones (550nm), while the latter were preferably taken up by M cells in the follicle-associated epithelium (FAE) region of Peyer's patches. In vitro permeation studies with Caco-2 cells confirmed the transmembrane transport of the dye-integrated UAPs. Our study supports the idea of using ACQ fluorophores for imaging and characterizing the fate of intact particles in a biological environment.

Preparation and Optimization of Amorphous Ursodeoxycholic Acid Nano-suspensions by Nanoprecipitation based on Acid-base Neutralization for Enhanced Dissolution.

BACKGROUND: Ursodeoxycholic acid, usually used to dissolve cholesterol gallstones in clinic, is a typical hydrophobic drug with poor oral bioavailability due to dissolution rate-limited performance. The objective of this study was to increase the dissolution of ursodeoxycholic acid by amorphous nanosuspensions. METHODS: Nanoprecipitation based on acid-base neutralization was used to prepare the nanosuspensions with central composite design to optimize the formula. The nanosuspensions were characterized by particle size, morphology, crystallology and dissolution. RESULTS: The ursodeoxycholic acid nanosuspensions showed mean particle size around 380 nm with polydispersion index value about 0.25. Scanning electron microscope observed high coverage of HPMC-E50 onto the surface of the nanosuspensions. Differential scanning calorimetry and powder X-ray diffractometry revealed amorphous structure of the ursodeoxycholic acid nanosuspensions. A significant increase of dissolution in acidic media was achieved by the amorphous nanosuspensions compared with the physical mixture. CONCLUSION: It can be predicted that the amorphous nanosuspensions show great potential in improving the oral bioavailability of ursodeoxycholic acid.

Self-discriminating fluorescent hybrid nanocrystals: efficient and accurate tracking of translocation via oral delivery.

The in vivo fate of nanocrystals is a controversial topic, i.e. dissolving versus integral absorption through the intestinal membrane. This is due to the lack of functional strategies to identify integral nanocrystals. In this study, the in vivo fate of quercetin hybrid nanocrystals (QT-HNCs) via the oral route is explored by physically embedding an environment-responsive probe in the crystal lattices of quercetin. The specific property of the probe is the water-initiated aggregation-caused quenching (ACQ) ability, by which integral QT-HNCs can be self-discriminated. Instead of dissolving instantly, QT-HNCs can be retained in the gastrointestinal tract for 12-16 h, and can then be absorbed and distributed into various organs with the liver as the primary terminal. The ileum provides better absorption than the jejunum. Cellular studies prove that both trans-epithelial and M cell-mediated routes are involved in the absorption of integral QT-HNCs, which may be impeded by the mucous layer. Moreover, the particle size affects the in vivo behavior and the ex vivo cellular interaction of QT-HNCs, with moderate size, such as 550 nm, being preferred. The results not only validate the idea of using ACQ fluorophores for bioimaging of integral nanocrystals but also support the intestinal absorption of nanocrystals.

Enhanced antitumor efficacy by d-glucosamine-functionalized and paclitaxel-loaded poly(ethylene glycol)-co-poly(trimethylene carbonate) polymer nanoparticles.

The poor selectivity of chemotherapeutics for cancer treatment may lead to dose-limiting side effects that compromise clinical outcomes. To solve the problem, surface-functionalized polymer nanoparticles are regarded as promising tumor-targeting delivery system. On the basis of glucose transporter (GLUT) overexpression on cancer cells, d-glucosamine-conjugated and paclitaxel-loaded poly(ethylene glycol)-co-poly(trimethylene carbonate) copolymer nanoparticles (DGlu-NP/PTX) were developed as potential tumor-targeting drug delivery system in this study. Because of the high affinity between d-glucosamine and GLUT, DGlu-NP/PTX could target to tumor tissue through GLUT-mediated endocytosis to improve the selectivity of PTX. DGlu-NP/PTX was prepared by emulsion/solvent evaporation technique and characterized in terms of morphology, size, and zeta potential. In vitro evaluation of two-dimensional cells and three-dimensional tumor spheroids revealed that DGlu-NP/PTX was more potent than those of plain nanoparticles (NP/PTX) and Taxol. In vivo multispectral fluorescent imaging indicated that DGlu-NP had higher specificity and efficiency on subcutaneous xenografts tumor of mouse. Furthermore, DGlu-NP/PTX showed the greatest tumor growth inhibitory effect on in vivo subcutaneous xenografts model with no evident toxicity. Therefore, these results demonstrated that DGlu-NP/PTX could be used as potential vehicle for cancer treatment.

Nanoparticles of 2-deoxy-D-glucose functionalized poly(ethylene glycol)-co-poly(trimethylene carbonate) for dual-targeted drug delivery in glioma treatment.

Based on the facilitative glucose transporter (GLUT) over-expression on both blood-brain barrier (BBB) and glioma cells, 2-deoxy-d-glucose modified poly(ethylene glycol)-co-poly(trimethylene carbonate) nanoparticles (dGlu-NP) were developed as a potential dual-targeted drug delivery system for enhancing the BBB penetration via GLUT-mediated transcytosis and improving the drug accumulation in the glioma via GLUT-mediated endocytosis. In vitro physicochemical characterization of the dual-targeted nanoparticulate system presented satisfactory size of 71 nm with uniform distribution, high encapsulation efficiency and adequate loading capacity of paclitaxel (PTX). Compared with non-glucosylated nanoparticles (NP), a significantly higher amount of dGlu-NP was internalized by RG-2 glioma cells through caveolae-mediated and clathrin-mediated endocytosis. Both of the transport ratios across the in vitro BBB model and the cytotoxicity of RG-2 cells after crossing the BBB were significantly greater of dGlu-NP/PTX than that of NP/PTX. In vivo fluorescent image indicated that dGlu-NP had high specificity and efficiency in intracranial tumor accumulation. The anti-glioblastoma efficacy of dGlu-NP/PTX was significantly enhanced in comparison with that of Taxol and NP/PTX. Preliminary safety tests showed no acute toxicity to hematological system, liver, kidney, heart, lung and spleen in mice after intravenous administration at a dose of 100 mg/kg blank dGlu-NP per day for a week. Therefore, these results indicated that dGlu-NP developed in this study could be a potential dual-targeted vehicle for brain glioma therapy.

Integrin-facilitated transcytosis for enhanced penetration of advanced gliomas by poly(trimethylene carbonate)-based nanoparticles encapsulating paclitaxel.

The treatment of cerebral tumor, especially advanced gliomas, represents one of the most formidable challenges in oncology. In this study, integrin-mediated poly(trimethylene carbonate)-based nanoparticulate system (c(RGDyK)-NP) was proposed as a delivery vehicle for enhancing drug penetration and chemotherapy of malignant gliomas. Following the recognition by integrin proteins on cell surface, c(RGDyK)-NP could be energy-dependently internalized by human U87MG glioma cells through a multiple endocytic pathway. The tumor penetration, homing specificity and anticancer efficacy of PTX-loaded c(RGDyK)-NP (c(RGDyK)-NP/PTX) were performed on the 3D glioma spheroids, the U87MG glioma cells and the intracranial glioma mice model, respectively. Compared with conventional nanoparticles (NP/PTX) and Taxol, c(RGDyK)-NP/PTX showed the strongest penetration and accumulation into 3D glioma spheroids, an obvious microtubule stabilization effect to U87MG glioma cells, a significant homing specificity to malignant glioma in vivo, and an extended median survival time in the intracranial glioma-bearing mice. Furthermore, preliminary in vivo subacute toxicity was also evaluated by measuring the histopathology, blood cell counts and clinical biochemistry parameters, and the results revealed no obvious subacute toxicity to hematological system, major organs or tissues were observed post successive intravenous injection of c(RGDyK)-NP. Therefore, our results suggested that cyclic RGD-conjugated PEG-PTMC nanoparticle could be a promising vehicle for enhancing the penetration and cxhemotherapy of high-grade malignant gliomas.

Solid tumor penetration by integrin-mediated pegylated poly(trimethylene carbonate) nanoparticles loaded with paclitaxel.

Limited penetration of antineoplastic agents is one of the contributing factors for chemotherapy failure of many solid tumors. In order to enhance drug penetration into solid cancer, especially, into the avascular regions inside tumors, we proposed cyclic RGD peptide functionalized PEGylated poly(trimethylene carbonate) nanoparticles (c(RGDyK)-NP). By integrin-mediated transcytosis and enhanced drug permeation, c(RGDyK)-NP could access the neoplastic cells distant from blood vessels, and consequently, avoiding the capability of cancer regeneration from these tumor cells. In the present study, the solid tumor penetration, homing specificity and anticancer efficacy were evaluated both on the ex vivo 3D tumor spheroids and on the subcutaneous xenograft mice model. In comparison with conventional nanoparticles (NP/PTX) and Taxol, c(RGDyK)-NP/PTX showed the strongest penetration and accumulation into 3D tumor spheroids, a marked tumor-homing specificity in vivo and the greatest tumor growth inhibitory effect in vitro and in vivo. Histochemistry analysis revealed that no obvious histopathological abnormalities or lesions were observed in major organs after intravenous administration with the treatment doses. In conclusion, cyclic RGD peptide-conjugated PEG-PTMC nanoparticle could facilitate drug penetration and accumulation in tumor tissues and may be a promising vehicle for enhancing the chemotherapy of solid cancers.