Opioid-induced fragile-like regulatory T cells contribute to withdrawal.

Dysregulation of the immune system is a cardinal feature of opioid addiction. Here, we characterize the landscape of peripheral immune cells from patients with opioid use disorder and from healthy controls. Opioid-associated blood exhibited an abnormal distribution of immune cells characterized by a significant expansion of fragile-like regulatory T cells (Tregs), which was positively correlated with the withdrawal score. Analogously, opioid-treated mice also showed enhanced Treg-derived interferon-γ (IFN-γ) expression. IFN-γ signaling reshaped synaptic morphology in nucleus accumbens (NAc) neurons, modulating subsequent withdrawal symptoms. We demonstrate that opioids increase the expression of neuron-derived C-C motif chemokine ligand 2 (Ccl2) and disrupted blood-brain barrier (BBB) integrity through the downregulation of astrocyte-derived fatty-acid-binding protein 7 (Fabp7), which both triggered peripheral Treg infiltration into NAc. Our study demonstrates that opioids drive the expansion of fragile-like Tregs and favor peripheral Treg diapedesis across the BBB, which leads to IFN-γ-mediated synaptic instability and subsequent withdrawal symptoms.

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins.

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.

Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here, we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific α subunit α4 s/PSMA8 and/or the catalytic ß subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis.

A driven Kerr oscillator with two-fold degeneracies for qubit protection.

We present the experimental finding of multiple simultaneous two-fold degeneracies in the spectrum of a Kerr oscillator subjected to a squeezing drive. This squeezing drive resulting from a three-wave mixing process, in combination with the Kerr interaction, creates an effective static two-well potential in the phase space rotating at half the frequency of the sinusoidal drive generating the squeezing. Remarkably, these degeneracies can be turned on-and-off on demand, as well as their number by simply adjusting the frequency of the squeezing drive. We find that when the detuning Δ between the frequency of the oscillator and the second subharmonic of the drive equals an even multiple of the Kerr coefficient K, [Formula: see text], the oscillator displays [Formula: see text] exact, parity-protected, spectral degeneracies, insensitive to the drive amplitude. These degeneracies can be explained by the unusual destructive interference of tunnel paths in the classically forbidden region of the double well static effective potential that models our experiment. Exploiting this interference, we measure a peaked enhancement of the incoherent well-switching lifetime, thus creating a protected cat qubit in the ground state manifold of our oscillator. Our results illustrate the relationship between degeneracies and noise protection in a driven quantum system.

Unveiling the mysteries of operating voltages of lithium-carbon dioxide batteries.

Lithium-carbon dioxide (Li-CO2) batteries are regarded as a promising electrochemical system owing to their energy storage capability and CO2 utilization. However, the reported operating voltage of ~2.6 V is increasingly questioned as seemingly beyond the capability of the electrochemical carbon dioxide reduction reaction to carbon. Herein, the real operating voltage of a Li-CO2 battery is reacquainted, and the operating voltage and the equilibrium potential are clarified to be ~1.1 V and ~2.82 V, respectively. The products formed at low voltage are identified to be crystalline Li2CO3, amorphous C, and explicitly amorphous Li2CO3. Moreover, by decoupling small currents, 1% O2, and 500 ppm H2O, the operating voltage plateaus are stimulated to ~2.0 V. An ever-increasing plateau can be achieved up to the reported level of ~2.6 V activated by a minor air leak or residue in test environments. Conclusively, the operating voltages of Li-CO2 batteries are proposed to be deceptive and extremely sensitive to the surrounding environments. This work unveils the real operating voltage and provides the voltage regulation rules to advance next-generation Li-CO2 batteries.

Uncovering ghost introgression through genomic analysis of a distinct eastern Asian hickory species.

Ghost introgression, or the transfer of genetic material from extinct or unsampled lineages to sampled species, has attracted much attention. However, conclusive evidence for ghost introgression, especially in plant species, remains scarce. Here, we newly assembled chromosome-level genomes for both Carya sinensis and Carya cathayensis, and additionally re-sequenced the whole genomes of 43 C. sinensis individuals as well as 11 individuals representing 11 diploid hickory species. These genomic datasets were used to investigate the reticulation and bifurcation patterns within the genus Carya (Juglandaceae), with a particular focus on the beaked hickory C. sinensis. By combining the D-statistic and BPP methods, we obtained compelling evidence that supports the occurrence of ghost introgression in C. sinensis from an extinct ancestral hickory lineage. This conclusion was reinforced through the phylogenetic network analysis and a genome scan method VolcanoFinder, the latter of which can detect signatures of adaptive introgression from unknown donors. Our results not only dispel certain misconceptions about the phylogenetic history of C. sinensis but also further refine our understanding of Carya's biogeography via divergence estimates. Moreover, the successful integration of the D-statistic and BPP methods demonstrates their efficacy in facilitating a more precise identification of introgression types.

Detection of Ghost Introgression Requires Exploiting Topological and Branch Length Information.

In recent years, the study of hybridization and introgression has made significant progress, with ghost introgression-the transfer of genetic material from extinct or unsampled lineages to extant species-emerging as a key area for research. Accurately identifying ghost introgression, however, presents a challenge. To address this issue, we focused on simple cases involving 3 species with a known phylogenetic tree. Using mathematical analyses and simulations, we evaluated the performance of popular phylogenetic methods, including HyDe and PhyloNet/MPL, and the full-likelihood method, Bayesian Phylogenetics and Phylogeography (BPP), in detecting ghost introgression. Our findings suggest that heuristic approaches relying on site-pattern counts or gene-tree topologies struggle to differentiate ghost introgression from introgression between sampled non-sister species, frequently leading to incorrect identification of donor and recipient species. The full-likelihood method BPP uses multilocus sequence alignments directly-hence taking into account both gene-tree topologies and branch lengths, by contrast, is capable of detecting ghost introgression in phylogenomic datasets. We analyzed a real-world phylogenomic dataset of 14 species of Jaltomata (Solanaceae) to showcase the potential of full-likelihood methods for accurate inference of introgression.

Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Brap regulates liver morphology and hepatocyte turnover via modulation of the Hippo pathway.

Regulation of hepatocyte proliferation and liver morphology is of critical importance to tissue and whole-body homeostasis. However, the molecular mechanisms that underlie this complex process are incompletely understood. Here, we describe a role for the ubiquitin ligase BRCA1-associated protein (BRAP) in regulation of hepatocyte morphology and turnover via regulation of MST2, a protein kinase in the Hippo pathway. The Hippo pathway has been implicated in the control of liver morphology, inflammation, and fibrosis. We demonstrate here that liver-specific ablation of Brap in mice results in gross and cellular morphological alterations of the liver. Brap-deficient livers exhibit increased hepatocyte proliferation, cell death, and inflammation. We show that loss of BRAP protein alters Hippo pathway signaling, causing a reduction in phosphorylation of YAP and increased expression of YAP target genes, including those regulating cell growth and interactions with the extracellular environment. Finally, increased Hippo signaling in Brap knockout mice alters the pattern of liver lipid accumulation in dietary models of obesity. These studies identify a role for BRAP as a modulator of the hepatic Hippo pathway with relevance to human liver disease.

Complete chloroplast genomes of 13 species of sect. Tuberculata Chang (Camellia L.): genomic features, comparative analysis, and phylogenetic relationships.

BACKGROUND: Sect. Tuberculata belongs to Camellia, and its members are characterized by a wrinkled pericarp and united filaments. All the plants in this group, which are endemic to China, are highly valuable for exploring the evolution of Camellia and have great potential for use as an oil source. However, due to the complex and diverse phenotypes of these species and the difficulty of investigating them in the field, their complex evolutionary history and interspecific definitions have remained largely unelucidated. RESULTS: Therefore, we newly sequenced and annotated 12 chloroplast (cp) genomes and retrieved the published cp genome of Camellia anlungensis Chang in sect. Tuberculata. In this study, comparative analysis of the cp genomes of the thirteen sect. Tuberculata species revealed a typical quadripartite structure characterized by a total sequence length ranging from 156,587 bp to 157,068 bp. The cp.genome arrangement is highly conserved and moderately differentiated. A total of 130 to 136 genes specific to the three types were identified by annotation, including protein-coding genes (coding sequences (CDSs)) (87-91), tRNA genes (35-37), and rRNA genes (8). The total observed frequency ranged from 23,045 (C. lipingensis) to 26,557 (C. anlungensis). IR region boundaries were analyzed to show that the ycf1 gene of C. anlungensis is located in the IRb region, while the remaining species are present only in the IRa region. Sequence variation in the SSC region is greater than that in the IR region, and most protein-coding genes have high codon preferences. Comparative analyses revealed six hotspot regions (tRNA-Thr(GGT)-psbD, psbE-petL, ycf15-tRNA-Leu(CAA), ndhF-rpl32, ndhD, and trnL(CAA)-ycf15) in the cp genomes that could serve as potential molecular markers. In addition, the results of phylogenetic tree construction based on the cp genomes showed that the thirteen sect. Tuberculata species formed a monophyletic group and were divided into two evolutionarily independent clades, confirming the independence of the section. CONCLUSIONS: In summary, we obtained the cp genomes of thirteen sect. Tuberculata plants and performed the first comparative analysis of this group. These results will help us better characterize the plants in this section, deepen our understanding of their genetic characteristics and phylogenetic relationships, and lay the theoretical foundation for their accurate classification, elucidation of their evolutionary changes, and rational development and utilization of this section in the future.

Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry.

Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes.

Charge Carriers Localization Effect Revealed through Terahertz Spectroscopy of MXene: Ti3C2Tx.

The transport properties of charge carriers in MXene, a promising material, have been studied using terahertz time-domain spectroscopy (THz-TDS) to examine its potential applications in optical and electronic devices. However, previous studies have been limited by narrow frequency ranges, which have hindered the understanding of the intrinsic mechanisms of carrier transport in MXenes. To address this issue, ultrabroadband THz-TDS with frequencies of up to 15 THz to investigate the complex photoconductances of MXene (Ti3C2Tx) films with different thicknesses are employed. The findings indicate that the electronic localization is substrate-dependent, and this effect decreases with an increase in the number of layers. This is attributed to the screening effect of the high carrier density in Ti3C2Tx. Additionally, the layer-independent photocarrier relaxations revealed by optical pump THz probe spectroscopy (OPTP) provide evidence of the carrier heating-induced screening effect. These results are significant for practical applications in both scientific research and various industries.

The gut microbiota participates in the effect of linaclotide in patients with irritable bowel syndrome with constipation (IBS-C): a multicenter, prospective, pre-post study.

BACKGROUND: Interindividual variation characterizes the relief experienced by constipation-predominant irritable bowel syndrome (IBS-C) patients following linaclotide treatment. Complex bidirectional interactions occur between the gut microbiota and various clinical drugs. To date, no established evidence has elucidated the interactions between the gut microbiota and linaclotide. We aimed to explore the impact of linaclotide on the gut microbiota and identify critical bacterial genera that might participate in linaclotide efficacy. METHODS: IBS-C patients were administered a daily linaclotide dose of 290 µg over six weeks, and their symptoms were then recorded during a four-week posttreatment observational period. Pre- and posttreatment fecal samples were collected for 16S rRNA sequencing to assess alterations in the gut microbiota composition. Additionally, targeted metabolomics analysis was performed for the measurement of short-chain fatty acid (SCFA) concentrations. RESULTS: Approximately 43.3% of patients met the FDA responder endpoint after taking linaclotide for 6 weeks, and 85% of patients reported some relief from abdominal pain and constipation. Linaclotide considerably modified the gut microbiome and SCFA metabolism. Notably, the higher efficacy of linaclotide was associated with enrichment of the Blautia genus, and the abundance of Blautia after linaclotide treatment was higher than that in healthy volunteers. Intriguingly, a positive correlation was found for the Blautia abundance and SCFA concentrations with improvements in clinical symptoms among IBS-C patients. CONCLUSION: The gut microbiota, especially the genus Blautia, may serve as a significant predictive microbe for symptom relief in IBS-C patients receiving linaclotide treatment. TRIAL REGISTRATION: This trial was registered with the Chinese Clinical Trial Registry (Chictr.org.cn, ChiCTR1900027934).

A community challenge to predict clinical outcomes after immune checkpoint blockade in non-small cell lung cancer.

BACKGROUND: Predictive biomarkers of immune checkpoint inhibitor (ICI) efficacy are currently lacking for non-small cell lung cancer (NSCLC). Here, we describe the results from the Anti-PD-1 Response Prediction DREAM Challenge, a crowdsourced initiative that enabled the assessment of predictive models by using data from two randomized controlled clinical trials (RCTs) of ICIs in first-line metastatic NSCLC. METHODS: Participants developed and trained models using public resources. These were evaluated with data from the CheckMate 026 trial (NCT02041533), according to the model-to-data paradigm to maintain patient confidentiality. The generalizability of the models with the best predictive performance was assessed using data from the CheckMate 227 trial (NCT02477826). Both trials were phase III RCTs with a chemotherapy control arm, which supported the differentiation between predictive and prognostic models. Isolated model containers were evaluated using a bespoke strategy that considered the challenges of handling transcriptome data from clinical trials. RESULTS: A total of 59 teams participated, with 417 models submitted. Multiple predictive models, as opposed to a prognostic model, were generated for predicting overall survival, progression-free survival, and progressive disease status with ICIs. Variables within the models submitted by participants included tumor mutational burden (TMB), programmed death ligand 1 (PD-L1) expression, and gene-expression-based signatures. The best-performing models showed improved predictive power over reference variables, including TMB or PD-L1. CONCLUSIONS: This DREAM Challenge is the first successful attempt to use protected phase III clinical data for a crowdsourced effort towards generating predictive models for ICI clinical outcomes and could serve as a blueprint for similar efforts in other tumor types and disease states, setting a benchmark for future studies aiming to identify biomarkers predictive of ICI efficacy. TRIAL REGISTRATION: CheckMate 026; NCT02041533, registered January 22, 2014. CheckMate 227; NCT02477826, registered June 23, 2015.

Association of Valproic Acid and Its Main Metabolites' Plasma Concentrations with Clinical Outcomes among Epilepsy Patients: A 10-Year Retrospective Study Based on Therapeutic Drug Monitoring.

Valproic acid (VPA) is a first-line antiepileptic drug with broad efficacy. Due to significant individual differences in its metabolism, therapeutic drug monitoring is commonly used. However, the recommended therapeutic range (50-100 µg/mL) is inadequate for predicting clinical outcomes. Additionally, the relationship between VPA metabolites and clinical outcomes remains unclear. In this retrospective study, 485 Chinese Southern Han epilepsy patients receiving VPA monotherapy were analyzed after reaching steady-state levels. Plasma concentrations of VPA and its five main metabolites were determined by liquid chromatography-mass spectrometry (LC-MS). We assessed the relevance of the recommended therapeutic VPA range for clinical outcomes and explored the association between VPA/metabolites levels and treatment efficacy/adverse effects. Vitro experiments were conducted to assess 4-ene-VPA hepatotoxicity. The therapeutic range of VPA exhibited no significant correlation with clinical outcomes, and plasma concentrations of VPA failed to serve as predictive indicators for treatment response/adverse effects. Treatment responders had higher 2-PGA concentrations (median, 26.39 ng/mL versus 13.68 ng/mL), with a threshold of 36.5 ng/mL for optimal epilepsy treatment. Patients with abnormal liver function had a higher 4-ene-VPA median concentration (6.41 µg/mL versus 4.83 µg/mL), and the ratio of 4-ene-VPA to VPA better predicted VPA-induced hepatotoxicity (area under the curve, 0.718) than 4-ene-VPA concentration. Vitro experiments revealed that 4-ene-VPA was more hepatotoxic than VPA in HepaRG and L02 cell lines. Total plasma VPA concentration does not serve as a predictor of clinical outcomes. 2-PGA concentrations may be associated with efficacy, whereas the ratio of 4-ene-VPA to VPA may be considered a better biomarker (threshold 10.03%) for VPA-induced hepatotoxicity. SIGNIFICANCE STATEMENT: This was the first and largest observational cohort in China to explore the relationship between patients' parent and metabolites concentrations of VPA and clinical outcomes during the maintenance of VPA monotherapy in epileptic patients. This study provided feasible references of VPA for epilepsy clinical treatment with a larger sample of patients compared with previous studies for a more definitive conclusion based on real-world situations. We found two potential biomarkers in predicting efficacy and liver injury, respectively. This breakthrough has the potential to assist in the rational use of VPA.

Experimental Virtual Distillation of Entanglement and Coherence.

Noise is, in general, inevitable and detrimental to practical and useful quantum communication and computation. Under the resource theory framework, resource distillation serves as a generic tool to overcome the effect of noise. Yet, conventional resource distillation protocols generally require operations on multiple copies of resource states, and strong limitations exist that restrict their practical utilities. Recently, by relaxing the setting of resource distillation to only approximating the measurement statistics instead of the quantum state, a resource-frugal protocol, "virtual resource distillation," is proposed, which allows more effective distillation of noisy resources. Here, we report its experimental implementation on a photonic quantum system for the distillation of quantum coherence (up to dimension four) and bipartite entanglement. We show the virtual distillation of the maximal superposed state of dimension four from the state of dimension two, an impossible task in conventional coherence distillation. Furthermore, we demonstrate the virtual distillation of entanglement with operations acting only on a single copy of the noisy Einstein-Podolsky-Rosen (EPR) pair and showcase the quantum teleportation task using the virtually distilled EPR pair with a significantly improved fidelity of the teleported state. These results illustrate the feasibility of the virtual resource distillation method and pave the way for accurate manipulation of quantum resources with noisy quantum hardware.

Impact of Ghost Introgression on Coalescent-Based Species Tree Inference and Estimation of Divergence Time.

The species studied in any evolutionary investigation generally constitute a small proportion of all the species currently existing or that have gone extinct. It is therefore likely that introgression, which is widespread across the tree of life, involves "ghosts," that is, unsampled, unknown, or extinct lineages. However, the impact of ghost introgression on estimations of species trees has rarely been studied and is poorly understood. Here, we use mathematical analysis and simulations to examine the robustness of species tree methods based on the multispecies coalescent model to introgression from a ghost or extant lineage. We found that many results originally obtained for introgression between extant species can easily be extended to ghost introgression, such as the strongly interactive effects of incomplete lineage sorting (ILS) and introgression on the occurrence of anomalous gene trees. The relative performance of the summary species tree method (ASTRAL) and the full-likelihood method (*BEAST) varies under different introgression scenarios, with the former being more robust to gene flow between nonsister species, whereas the latter performing better under certain conditions of ghost introgression. When an outgroup ghost (defined as a lineage that diverged before the most basal species under investigation) acts as the donor of the introgressed genes, the time of root divergence among the investigated species generally was overestimated, whereas ingroup introgression, as commonly perceived, can only lead to underestimation. In many cases of ingroup introgression that may or may not involve ghost lineages, the stronger the ILS, the higher the accuracy achieved in estimating the time of root divergence, although the topology of the species tree is more prone to be biased by the effect of introgression. [Anomalous gene trees; divergence time; ghost introgression; multispecies coalescent; simulation; species tree.].

Therapeutic Effect of a Novel M1 Macrophage-Targeted Nanodrug in Chronic Periodontitis Mice.

Chronic periodontitis is a chronic, progressive, and destructive disease. Especially, the large accumulation of advanced glycation end products (AGEs) in a diseased body will aggravate the periodontal tissue damage, and AGEs induce M1 macrophages. In this project, the novel nanodrugs, glucose-PEG-PLGA@MCC950 (GLU@MCC), are designed to achieve active targeting with the help of glucose transporter 1 (GLUT1) which is highly expressed in M1 macrophages induced by AGEs. Then, the nanodrugs release MCC950, which is a kind of NLRP3 inhibitor. These nanodrugs not only can improve the water solubility of MCC950 but also exhibit superior characteristics, such as small size, stability, innocuity, etc. In vivo experiments showed that GLU@MCC could reduce periodontal tissue damage and inhibit cell apoptosis in periodontitis model mice. In vitro experiments verified that its mechanism of action might be closely related to the inhibition of the NLRP3 inflammatory factor in M1 macrophages. GLU@MCC could effectively reduce the damage to H400 cells caused by AGEs, decrease the expression of NLRP3, and also obviously reduce the M1-type macrophage pro-inflammatory factors such as IL-18, IL-1ß, caspase-1, and TNF-α. Meanwhile, the expression of anti-inflammatory factor Arg-1 in the M2 macrophage was increased. In brief, GLU@MCC would inhibit the expression of inflammatory factor NLRP3 and exert antiperiodontal tissue damage in chronic periodontitis via GLUT1 in the M1 macrophage as the gating target. This study provides a novel nanodrug for chronic periodontitis treatment.