HGT is widespread in insects and contributes to male courtship in lepidopterans.

Horizontal gene transfer (HGT) is an important evolutionary force shaping prokaryotic and eukaryotic genomes. HGT-acquired genes have been sporadically reported in insects, a lineage containing >50% of animals. We systematically examined HGT in 218 high-quality genomes of diverse insects and found that they acquired 1,410 genes exhibiting diverse functions, including many not previously reported, via 741 distinct transfers from non-metazoan donors. Lepidopterans had the highest average number of HGT-acquired genes. HGT-acquired genes containing introns exhibited substantially higher expression levels than genes lacking introns, suggesting that intron gains were likely involved in HGT adaptation. Lastly, we used the CRISPR-Cas9 system to edit the prevalent unreported gene LOC105383139, which was transferred into the last common ancestor of moths and butterflies. In diamondback moths, males lacking LOC105383139 courted females significantly less. We conclude that HGT has been a major contributor to insect adaptation.

Communication between the stem cell niche and an adjacent differentiation niche through miRNA and EGFR signaling orchestrates exit from the stem cell state in the Drosophila ovary.

The signaling environment, or niche, often governs the initial difference in behavior of an adult stem cell and a derivative that initiates a path towards differentiation. The transition between an instructive stem cell niche and differentiation niche must generally have single-cell resolution, suggesting that multiple mechanisms might be necessary to sharpen the transition. Here, we examined the Drosophila ovary and found that Cap cells, which are key constituents of the germline stem cell (GSC) niche, express a conserved microRNA (miR-124). Surprisingly, loss of miR-124 activity in Cap cells leads to a defect in differentiation of GSC derivatives. We present evidence that the direct functional target of miR-124 in Cap cells is the epidermal growth factor receptor (EGFR) and that failure to limit EGFR expression leads to the ectopic expression of a key anti-differentiation BMP signal in neighboring somatic escort cells (ECs), which constitute a differentiation niche. We further found that Notch signaling connects EFGR activity in Cap cells to BMP expression in ECs. We deduce that the stem cell niche communicates with the differentiation niche through a mechanism that begins with the selective expression of a specific microRNA and culminates in the suppression of the major anti-differentiation signal in neighboring cells, with the functionally important overall role of sharpening the spatial distinction between self-renewal and differentiation environments.

17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Single-cell expression profile of Drosophila ovarian follicle stem cells illuminates spatial differentiation in the germarium.

BACKGROUND: How stem cell populations are organized and regulated within adult tissues is important for understanding cancer origins and for developing cell replacement strategies. Paradigms such as mammalian gut stem cells and Drosophila ovarian follicle stem cells (FSC) are characterized by population asymmetry, in which stem cell division and differentiation are separately regulated processes. These stem cells behave stochastically regarding their contributions to derivative cells and also exhibit dynamic spatial heterogeneity. Drosophila FSCs provide an excellent model for understanding how a community of active stem cells maintained by population asymmetry is regulated. Here, we use single-cell RNA sequencing to profile the gene expression patterns of FSCs and their immediate derivatives to investigate heterogeneity within the stem cell population and changes associated with differentiation. RESULTS: We describe single-cell RNA sequencing studies of a pre-sorted population of cells that include FSCs and the neighboring cell types, escort cells (ECs) and follicle cells (FCs), which they support. Cell-type assignment relies on anterior-posterior (AP) location within the germarium. We clarify the previously determined location of FSCs and use spatially targeted lineage studies as further confirmation. The scRNA profiles among four clusters are consistent with an AP progression from anterior ECs through posterior ECs and then FSCs, to early FCs. The relative proportion of EC and FSC clusters are in good agreement with the prevalence of those cell types in a germarium. Several genes with graded profiles from ECs to FCs are highlighted as candidate effectors of the inverse gradients of the two principal signaling pathways, Wnt and JAK-STAT, that guide FSC differentiation and division. CONCLUSIONS: Our data establishes an important resource of scRNA-seq profiles for FSCs and their immediate derivatives that is based on precise spatial location and functionally established stem cell identity, and facilitates future genetic investigation of regulatory interactions guiding FSC behavior.

Exploration of immunotherapy in advanced pulmonary lymphoepithelioma-like carcinoma.

Pulmonary lymphoepithelioma-like carcinoma (PLELC) is a rare and histologically distinctive subtype of nonsmall cell lung cancer (NSCLC). High expression of programmed death ligand 1 (PD-L1) and scarcity of druggable driver mutations raise the potential of immunotherapy for advanced PELEC. However, evidence on the clinical impact of immune-checkpoint inhibitors (ICIs) remained limited and unconvincing. The present study retrospectively enrolled advanced PLELC patients who received ICIs either as up-front or salvage therapy in SYSUCC between March 15, 2017 and March 15, 2022. The comparative efficacy of chemoimmunotherapy vs chemotherapy in the first-line setting and chemoimmunotherapy vs ICIs monotherapy in the ≥2 line setting was investigated. A total of 96 patients were finally enrolled; 49 PLELC patients received immunotherapy plus platinum-based chemotherapy, while 45 patients received platinum-based chemotherapy as first-line treatment. Patients with chemoimmunotherapy significantly obtain more survival benefits than those receiving chemotherapy (median progression-free survival [PFS]: 15.6 vs 8.6 months, P = .0015). Additionally, patients with chemoimmunotherapy obtained more PFS benefits than those with ICIs monotherapy in the ≥2 line of therapy (median PFS: 21.7 months vs 7.8 months, P = .094). A significant correlation was observed between prognostic nutritional index (PNI) and favorable treatment outcomes in patients receiving first-line chemoimmunotherapy (median PFS: 17.8 months vs 7.6 months, P < .0001). Likewise, patients in the monocyte-to-lymphocyte ratio (MLR)-high group had significantly shorter PFS than the MLR-low group (median PFS: 11.2 months vs not reached, P = .0009). Our study elucidated the superior efficacy of ICIs therapy, especially chemoimmunotherapy in advanced PLELC, which may provide new insight into the role of immunotherapy in advanced PLELC.

Symbiotic bracovirus of a parasite manipulates host lipid metabolism via tachykinin signaling.

Parasites alter host energy homeostasis for their own development, but the mechanisms underlying this phenomenon remain largely unknown. Here, we show that Cotesia vestalis, an endoparasitic wasp of Plutella xylostella larvae, stimulates a reduction of host lipid levels. This process requires excess secretion of P. xylostella tachykinin (PxTK) peptides from enteroendocrine cells (EEs) in the midgut of the parasitized host larvae. We found that parasitization upregulates PxTK signaling to suppress lipogenesis in midgut enterocytes (ECs) in a non-cell-autonomous manner, and the reduced host lipid level benefits the development of wasp offspring and their subsequent parasitic ability. We further found that a C. vestalis bracovirus (CvBV) gene, CvBV 9-2, is responsible for PxTK induction, which in turn reduces the systemic lipid level of the host. Taken together, these findings illustrate a novel mechanism for parasite manipulation of host energy homeostasis by a symbiotic bracovirus gene to promote the development and increase the parasitic efficiency of an agriculturally important wasp species.

Role of antiangiogenic agents in first-line treatment for advanced NSCLC in the era of immunotherapy.

BACKGROUND & OBJECTIVE: "Anti-angiogenetic drugs plus chemotherapy" (anti-angio-chemo) and "immune checkpoint inhibitors plus chemotherapy" (ICI-chemo) are superior to traditional chemotherapy in the first-line treatment of patients with advanced non-small-cell lung cancer (NSCLC). However, in the absence of a direct comparison of ICI-chemo with anti-angio-chemo, the superior one between them has not been decided, and the benefit of adding anti-angiogenetic agents to ICI-chemo remains controversial. This study aimed to investigate the role of antiangiogenic agents for advanced NSCLC in the era of immunotherapy. METHODS: Eligible randomized controlled trials (RCTs) comparing chemotherapy versus therapeutic regimens involving ICIs or anti-angiogenetic drugs were included. Outcomes included progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and rate of grade 3-4 toxicity assessment. R-4.3.1 was utilized to perform the analysis. RESULTS: A total of 54 studies with a sample size of 25,046 were finally enrolled. "Atezolizumab + Bevacizumab + Chemotherapy" significantly improved the ORR compared with "Atezolizumab + Chemotherapy" (Odds ratio (OR) = 2.73, 95% confidence interval (CI): 1.27-5.87). The trend also favored "Atezolizumab + Bevacizumab + Chemotherapy" in PFS and OS (hazard ratio (HR) = 0.71, 95% CI: 0.39-1.31; HR = 0.94, 95% CI: 0.77-1.16, respectively). In addition, "Pembrolizumab + Chemotherapy" and "Camrelizumab + Chemotherapy" significantly prolonged the PFS compared to "Bevacizumab + Chemotherapy" (HR = 0.65, 95% CI: 0.46-0.92; HR = 0.63, 95% CI: 0.41-0.97; respectively). Meanwhile, "Pembrolizumab + Chemotherapy" and "Sintilimab + Chemotherapy" yielded more OS benefits than "Bevacizumab + Chemotherapy" (HR = 0.69, 95% CI: 0.56-0.83; HR = 0.64, 95%CI: 0.46-0.91; respectively). Scheme between "Atezolizumab + Bevacizumab + Chemotherapy" and "Atezolizumab + Chemotherapy" made no significant difference (OR = 1.18, 95%CI: 0.56-2.42) concerning the rate of grade 3-4 toxicity. It seemed that ICI-chemo yielded more improvement in quality-adjusted life-year (QALY) than "Bevacizumab + Chemotherapy" in cost-effectiveness analysis. CONCLUSION: Our results suggest that ICI-chemo is associated with potentially longer survival, better cost-effectiveness outcomes, and comparable safety profiles than anti-angio-chemo. Also, adding bevacizumab to ICI-chemo seemed to provide additional therapeutic benefits without adding treatment burden. Our findings would supplement the current standard of care and help the design of future clinical trials for the first-line treatment of patients with advanced NSCLC.

Natural exosome-like nanoparticles derived from ancient medicinal insect Periplaneta americana L. as a novel diabetic wound healing accelerator.

Along with the recognized therapeutic outcomes of regenerative medicine, extracellular vesicles and their exosome subsets have become an alternative option for wound healing. Periplaneta americana L. (PA), an ancient and traditional medicinal insect, has been around for 300 million years, and displays magic formidable vitality and environmental adaptive ability. The linkage between intrinsic amputation regeneration feature and the acknowledged wound healing medicinal benefit of PA has never been revealed. Herein, inspired by the ability of exosomes to participate in the interkingdom communication, we explored whether this effect was ascribed to PA derived exosome-like nanoparticles (PA-ELNs). PA-ELNs were extracted by differential velocity centrifugation approach and characterized by DLS, NTA and TEM. Their cargoes were analyzed by LC-MS/MS proteomics and small RNA-seq analysis. The wound healing activity was verified in vivo and in vitro. PA-ELNs with a concentration of 2.33x109±6.35x107 particles/mL exhibited a lipid bilayer-bound membrane structure with an average size of 104.7 nm. Furthermore, the miRNA cargoes in PA-ELNs participate in some wound healing related signal pathways such as TGF-beta, mTOR, and autophagy. As expected, the in vitro tests indicated that PA-ELNs were apt to be internalized in HUVECs, L929 and RAW 264.7 cells and contributed to cell proliferation and migration. Most importantly, we demonstrated that the topical administration of PA-ELNs could remarkably accelerate wound healing in a diabetic mouse model, and was involved in anti-inflammatory, re-epithelialization and autophagy regulation. This study provides clear evidence for the first time that PA-ELNs, as diabetic wound healing accelerators, are the "bioactive code" of this ancient medicinal insect.

Efficacy and potential resistance mechanisms of afatinib in advanced non-small cell lung cancer patients with EGFR G719X/L861Q/S768I.

BACKGROUND: Afatinib is the only currently approved EGFR-tyrosine kinase inhibitors for advanced non-small cell lung cancer (NSCLC) patients with EGFR G719X/L861Q/S768I. However, there are limited real-world data concerning the benefits and resistance mechanisms of afatinib in patients with these nonclassical mutations. To fill this gap, the present study was conducted. METHODS: All NSCLC patients treated with afatinib were screened, and patients with EGFR G719X/L861Q/S768I were enrolled into the analysis. Either tumor tissue or blood specimens were detected by the commercial next-generation sequencing (NGS) panels or amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) to figure out the mutation genotype. RESULTS: A total of 106 advanced NSCLC patients with EGFR G719X/L861Q/S768I received afatinib treatment. The benefits of afatinib exhibited heterogeneity in different mutation genotypes. Notably, at baseline, NGS testing was performed in 59 patients, and TP53 was the most frequently coexisting mutation. Patients with TP53 mutations obtained fewer survival benefits than those with TP53 wild-type. A total of 68 patients ultimately experienced progression, and 27 patients received NGS testing to clarify the potential resistance mechanisms. EGFR-T790M, CDK4 amplification, FGFR1 amplification, PIK3CA, MET amplification, RET fusions, HER2, and BRAF mutations were identified in three (11.1%), three (11.1%), three (11.1%), three (11.1%), three (11.1%), one (3.7%), one (3.7%), and one (3.7%) of the cases, respectively. Five patients underwent ARMS-PCR testing for detecting EGFR-T790M mutation, and only one patient was T790M-positive. CONCLUSIONS: The present study elucidated the differential benefits of afatinib within different mutation genotypes and first revealed the spectrum of potential resistance mechanisms in patients with EGFR G719X/L861Q/S768I. The results of this study may provide practical clinical information that can guide optimal treatment in this setting.

One-Pot, One-Step Synthesis of Drug-Loaded Magnetic Multimicelle Aggregates.

Lipid-based formulations provide a nanotechnology platform that is widely used in a variety of biomedical applications because it has several advantageous properties including biocompatibility, reduced toxicity, relative ease of surface modifications, and the possibility for efficient loading of drugs, biologics, and nanoparticles. A combination of lipid-based formulations with magnetic nanoparticles such as iron oxide was shown to be highly advantageous in a growing number of applications including magnet-mediated drug delivery and image-guided therapy. Currently, lipid-based formulations are prepared by multistep protocols. Simplification of the current multistep procedures can lead to a number of important technological advantages including significantly decreased processing time, higher reaction yield, better product reproducibility, and improved quality. Here, we introduce a one-pot, single-step synthesis of drug-loaded magnetic multimicelle aggregates (MaMAs), which is based on controlled flow infusion of an iron oxide nanoparticle/lipid mixture into an aqueous drug solution under ultrasonication. Furthermore, we prepared molecular-targeted MaMAs by directional antibody conjugation through an Fc moiety using Cu-free click chemistry. Fluorescence imaging and quantification confirmed that antibody-conjugated MaMAs showed high cell-specific targeting that was enhanced by magnetic delivery.

Synthesis of metal ion-tolerant Mn-doped fluorescence silicon quantum dots with green emission and its application for selective imaging of ·OH in living cells.

Monitoring hydroxyl radical (·OH) in living cells remains a big challenge on account of its high reactivity and short half-life. In this work, we designed a fluorescent probe based on manganese-doped silicon quantum dots (Mn-SiQDs) for detecting and imaging of ·OH with good water solubility. The manganese was doped in its ethylene diamine tetra-acetic acid (EDTA) complex form and effectively improved the metal ion tolerance of fluorescence of SiQDs. And m-dihydroxybenzene was used as the reductant to extend the emission of SiQDs to the green region at 515 nm when the excitation wavelength was 424 nm. Basing on the fluorescence quenching of Mn-SiQDs, a linear response of ·OH was observed in the range 0.8-50 µM with a limit of detection (LOD) of 88.4 nM, which is lower than those reported with SiQDs. The interference from other ROS or RNS has been assessed and no impact was found. In fully aqueous systems, the Mn-SiQDs have been applied to monitor and image the endogenous ·OH in HeLa cells. Our work provided a new strategy for designing SiQDs with good biocompatibility, high selectivity and long monitoring wavelength. Synthesis of green-emitting silicon quantum dots with N-[3 -(trimethoxysilyl) propyl] ethylenediamine (DAMO), Ethylenediamine tetraacetic acid disodium salt dehydrate (EDTA-2Na·2H2O), manganese acetate tetrahydrate (Mn(CH3COO)2·4H20) and m-dihydroxybenzene. The green fluorescence of the silicon quantum dots can be selectively quenched by hydroxyl radicals.

Poly(adenosine diphosphate ribose) polymerase inhibitors induce autophagy-mediated drug resistance in ovarian cancer cells, xenografts, and patient-derived xenograft models.

BACKGROUND: Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors. METHODS: Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient-derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line. RESULTS: Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ-H2AX. Inhibition of autophagy also increased ROS and γ-H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy. CONCLUSIONS: PARP inhibitor-induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild-type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer.

Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition.

BACKGROUND: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. METHODS: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. RESULTS: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. CONCLUSIONS: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

The role of vascular endothelial growth factor, interleukin 8, and insulinlike growth factor in sustaining autophagic DIRAS3-induced dormant ovarian cancer xenografts.

BACKGROUND: Re-expression of the imprinted tumor suppressor gene DIRAS family GTPase 3 (DIRAS3) (aplysia ras homology member I [ARHI]) induces autophagy and tumor dormancy in ovarian cancer xenografts, but drives autophagic cancer cell death in cell culture. The current study explored the tumor and host factors required to prevent autophagic cancer cell death in xenografts and the use of antibodies against those factors or their receptors to eliminate dormant autophagic ovarian cancer cells. METHODS: Survival factors (insulinlike growth factor 1 [IGF-1], vascular endothelial growth factor [VEGF], and interleukin 8 [IL-8]) were detected with growth factor arrays and measured using enzyme-linked immunoadsorbent assay analysis. Phosphorylation of protein kinase B (AKT), phosphorylation of extracellular signal-regulated kinase (ERK), nuclear localization of translocation factor EB (TFEB) or forkhead box O3a (FOXo3a), and expression of microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B; LC3B) were examined using Western blot analysis. The effect of treatment with antibodies against survival factors or their receptors was studied using DIRAS3-induced dormant xenograft models. RESULTS: Ovarian cancer cells grown subcutaneously in nude mice exhibited higher levels of phosphorylated ERK/AKT activity and lower levels of nuclear TFEB/FOXo3a, MAPLC3B, and autophagy compared with cells grown in culture. Induction of autophagy and dormancy with DIRAS3 was associated with decreased ERK/AKT signaling. The addition of VEGF, IGF-1, and IL-8 weakened the inhibitory effect of DIRAS3 on ERK/AKT activity and reduced DIRAS3-mediated TFEB or FOXo3a nuclear localization and MAPLC3B expression in ovarian cancer cells. Treatment with antibodies against VEGF, IL-8, and IGF receptor inhibited the growth of dormant xenografts, thereby prolonging survival from 99 to >220 days (P < .05) and curing a percentage of mice. CONCLUSIONS: Treatment with a combination of anti-VEGF, anti-IL-8, and anti-IGF receptor antibodies prevented the outgrowth of dormant cells and prolonged survival in a preclinical model.

Red emissive boron and nitrogen co-doped "on-off-on" carbon dots for detecting and imaging of mercury(II) and biothiols.

Red emissive B,N co-doped carbon dots (BN-CDs) were hydrothermally synthesized from cresyl violet and boric acid. The BN-CDs exhibited excellent photostability, low cytotoxicity, excitation/emission maxima at 520/616 nm, and a relatively high quantum yield of 18%. The BN-CDs can binded to mercury(II), and this results in quenching of the red-colored fluorescence. However, on subsequent addition of the biothiol (such as cysteine, homocysteine or glutathione), fluorescence recovers. Therefore, the BN-CDs can be used as a multifunctional probe based on "on-off-on" fluorescence response for the detection of Hg(II) and biothiols. The following detection limits were accomplished: (a) Hg(II): 2.8 µM; (b) glutathione: 1.7 µM; (c) cysteine: 2.3 µM; (d) homocysteine: 3.0 µM. The BN-CDs also have been successfully applied for the imaging of Hg(II) and biothiols in HepG2 cells with excellent bio-compatibility. Graphical abstract Red emissive B,N co-doped carbon dots (BN-CDs) were synthesized through hydrothermal treatment of cresyl violet and boric acid. The BN-CDs can be used as a multifunctional probe based on "on-off-on" fluorescence response for detecting mercury(II) and biothiols in aqueous solution and living cells.

Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell Responses and Host Resistance to an Attenuated West Nile Virus Infection in Old Mice.

UNLABELLED: The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6- to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21- to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection but were protected from subsequent lethal wild-type WNV challenge. Compared to responses in young mice, the NS4B-P38G mutant triggered higher inflammatory cytokine and interleukin-10 (IL-10) production, a delayed γδ T cell expansion, and lower antibody and WNV-specific T cell responses in old mice. Toll-like receptor 7 (TLR7) is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen-presenting function compromise and a reduced γδ T cell and regulatory T cell (Treg) expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist, decreased host vulnerability in NS4B-P38G-infected old mice by enhancing γδ T cell and Treg expansion and the antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with the WNV NS4B-P38G mutant. IMPORTANCE: The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available for human use. Here, we used an aged mouse model to investigate the protective efficacy of an attenuated WNV, the nonstructural 4B-P38G mutant, which was previously shown to induce no lethality but strong immune responses in young adult mice. Studies that contribute to a mechanistic understanding of immune defects in the elderly will allow the development of strategies to improve responses to infectious diseases and to increase vaccine efficacy and safety in aging individuals.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

OBJECTIVE AND DESIGN: Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. MATERIAL OR SUBJECTS: Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. TREATMENT: A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. METHODS: Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. RESULTS: Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. CONCLUSIONS: Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.

Cystic fibrosis (CF) airways are characterized by bacterial infections, excess mucus production, and robust neutrophil recruitment. The main CF airway pathogen is Pseudomonas aeruginosa. Neutrophils are not capable of clearing the infection. Neutrophil primary granule components, myeloperoxidase (MPO) and human neutrophil elastase (HNE), are inflammatory markers in CF airways, and their increased levels are associated with poor lung function. Identifying the mechanism of MPO and HNE release from neutrophils is of high clinical relevance for CF. In this article, we show that human neutrophils release large amounts of neutrophil extracellular traps (NETs) in the presence of P. aeruginosa. Bacteria are entangled in NETs and colocalize with extracellular DNA. MPO, HNE, and citrullinated histone H4 are all associated with DNA in Pseudomonas-triggered NETs. Both laboratory standard strains and CF isolates of P. aeruginosa induce DNA, MPO, and HNE release from human neutrophils. The increase in peroxidase activity of neutrophil supernatants after Pseudomonas exposure indicates that enzymatically active MPO is released. P. aeruginosa induces a robust respiratory burst in neutrophils that is required for extracellular DNA release. Inhibition of the cytoskeleton prevents Pseudomonas-initiated superoxide production and DNA release. NADPH oxidase inhibition suppresses Pseudomonas-induced release of active MPO and HNE. Blocking MEK/ERK signaling results in only minimal inhibition of DNA release induced by Pseudomonas. Our data describe in vitro details of DNA, MPO, and HNE release from neutrophils activated by P. aeruginosa. We propose that Pseudomonas-induced NET formation is an important mechanism contributing to inflammatory conditions characteristic of CF airways.

Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.

Pseudogout is an autoinflammatory condition triggered by calcium pyrophosphate dehydrate (CPPD) crystal deposition in the joints. The innate immune system is irritated by and responds to the presence of the crystals with an inflammatory response. The synovial fluid contains activated inflammatory macrophages and neutrophil granulocytes. Several details of crystal-induced macrophage activation were recently uncovered, but very little is known about interactions of CPPD crystals with neutrophils. In this study, we show that human neutrophils engulf CPPD crystals and form large amounts of neutrophil extracellular traps (NETs) in vitro. Released extracellular DNA binds myeloperoxidase and citrullinated histone H4. CPPD crystal-stimulated neutrophils and their nuclear DNA undergo morphological changes characteristic for NET formation. The ERK/MEK signaling pathway, heat shock protein 90, PI3K, and an intact cytoskeleton are required for CPPD-induced NET formation. Blocking crystal-activated respiratory burst has, however, no effect on NETs. Human neutrophils release IL-1ß and IL-8 in response to CPPD crystals, and blocking CXCR2, the main IL-8R, diminishes NET formation. Proinflammatory cytokines, TNF-α, GM-CSF, and IL-1ß, increase NET release by the crystals. Enhanced bacterial killing by CPPD-induced NETs demonstrates their ability to cause cellular damage. Our work documents and provides details about extracellular trap release in human neutrophils activated by CPPD microcrystals. We suggest that crystal-triggered NET formation can be a novel contributor to inflammatory conditions observed in CPPD crystal-driven synovitis.

Synthesis of non-modified near-infrared carbon dots for hypochlorite detection and cell membrane imaging.

Devising carbon dots with long wavelength emission (red light or near infrared), high selectivity and good bio-compatibility is critical in fluorescence detection and imaging, but achieving this goal remains a great challenge. Herein, near-infrared emissive carbon dots (NIR-CDs) with obvious emission characteristic of 653 nm were synthesized through hydrothermally treatment of toluidine bule and gallic acid. Noticeably, the NIR-CDs exhibited excellent selectivity and sensitivity to hypochlorite (ClO-), and the limit of detection is as low as 42.7 nM. The selective recognition reaction between ClO- and the surface functional groups of NIR-CDs inhibits the fluorescence from NIR-CDs. The quenching mechanism was confirmed by fluorescence lifetime decays, FT-IR spectroscopy and UV-vis absorption spectra. More remarkably, the NIR-CDs have rich hydrophilic groups showed lower cytotoxicity, excellent bio-compatibility and specific cell membrane localization ability. The established spectrofluorometric method based on NIR-CDs has been used to determination of ClO- level in tap water sample, the recoveries were 97.7 %-103.3 %. In addition, the NIR-CDs also has been successfully applied for the imaging of cell membrane. The study provides a novel idea for developing NIR ClO- probe as well as cell membrane localization probe based on CDs, which present bright prospects in real water samples monitoring and cell membrane imaging.