Downregulation of otulin induces inflammasome activation in neutrophilic asthma.

BACKGROUND: Neutrophilic asthma (NA) is a severe asthma phenotype associated with steroid resistance and IL-1ß overproduction; however, the exact mechanism remains unclear. Moreover, the dysfunction of TNF-α signaling pathway, a regulator of IL-1ß production, was associated with the deficiency of ovarian tumor protease deubiquitinase with linear linkage specificity (otulin) in autoimmune patients. OBJECTIVE: We hypothesized that otulin downregulation in macrophages (Mφ) could trigger Mφ activation via the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome signaling pathway. METHODS: We assessed the expressions of otulin in blood monocyte subsets from NA patients and in alveolar Mφ from NA mice. Additionally, we evaluated the functional consequences of otulin deficiency in bone marrow-derived Mφ. The effects of inhibiting receptor-interacting protein kinase (RIPK)-1 and RIPK-3 on neutrophils and group 3 innate lymphoid cells (ILC3s) were assessed in vitro and in vivo. RESULTS: When comparing nonclassical monocytes, a significant downregulation of otulin in the intracellular components was observed in NA patients compared to healthy controls (P = .005). Moreover, isolated alveolar Mφ from the NA mice exhibited lower otulin expression compared to those from control mice. After otulin knockdown in bone marrow-derived Mφ, we observed spontaneous IL-1ß production depending on NLRP3 inflammasome. Moreover, the infiltrated neutrophils and ILC3s were significantly decreased by combined treatment of RIPK-1 and RIPK-3 inhibitors through blocking IL-1ß release in NA. CONCLUSIONS: IL-1ß overproduction caused by a deficiency of otulin, an upstream triggering factor, could be a promising diagnostic and therapeutic target for NA.

Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Modulation of Nogo receptor 1 expression orchestrates myelin-associated infiltration of glioblastoma.

As the clinical failure of glioblastoma treatment is attributed by multiple components, including myelin-associated infiltration, assessment of the molecular mechanisms underlying such process and identification of the infiltrating cells have been the primary objectives in glioblastoma research. Here, we adopted radiogenomic analysis to screen for functionally relevant genes that orchestrate the process of glioma cell infiltration through myelin and promote glioblastoma aggressiveness. The receptor of the Nogo ligand (NgR1) was selected as the top candidate through Differentially Expressed Genes (DEG) and Gene Ontology (GO) enrichment analysis. Gain and loss of function studies on NgR1 elucidated its underlying molecular importance in suppressing myelin-associated infiltration in vitro and in vivo. The migratory ability of glioblastoma cells on myelin is reversibly modulated by NgR1 during differentiation and dedifferentiation process through deubiquitinating activity of USP1, which inhibits the degradation of ID1 to downregulate NgR1 expression. Furthermore, pimozide, a well-known antipsychotic drug, upregulates NgR1 by post-translational targeting of USP1, which sensitizes glioma stem cells to myelin inhibition and suppresses myelin-associated infiltration in vivo. In primary human glioblastoma, downregulation of NgR1 expression is associated with highly infiltrative characteristics and poor survival. Together, our findings reveal that loss of NgR1 drives myelin-associated infiltration of glioblastoma and suggest that novel therapeutic strategies aimed at reactivating expression of NgR1 will improve the clinical outcome of glioblastoma patients.

Correction: Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells.

[This corrects the article DOI: 10.1371/journal.pbio.1002152.].

Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells.

Epidermal growth factor receptor variant III (EGFRvIII) has been associated with glioma stemness, but the direct molecular mechanism linking the two is largely unknown. Here, we show that EGFRvIII induces the expression and secretion of pigment epithelium-derived factor (PEDF) via activation of signal transducer and activator of transcription 3 (STAT3), thereby promoting self-renewal and tumor progression of glioma stem cells (GSCs). Mechanistically, PEDF sustained GSC self-renewal by Notch1 cleavage, and the generated intracellular domain of Notch1 (NICD) induced the expression of Sox2 through interaction with its promoter region. Furthermore, a subpopulation with high levels of PEDF was capable of infiltration along corpus callosum. Inhibition of PEDF diminished GSC self-renewal and increased survival of orthotopic tumor-bearing mice. Together, these data indicate the novel role of PEDF as a key regulator of GSC and suggest clinical implications.

Does Botulinum Toxin Injection into Masseter Muscles Affect Subcutaneous Thickness?

BACKGROUND: Botulinum toxin (BoNT) is widely used to treat masseter muscle hypertrophy. Changes in the muscle thickness have been found in many studies, but there has been no report on changes in the thickness from the skin surface to the masseter muscle. OBJECTIVES: We aimed to use ultrasonography to measure not only changes in the muscle thickness but also changes in subcutaneous thickness. METHODS: This study enrolled 20 volunteer patients: 10 were assigned to an experimental group (injected with each side 25 U of botulinum toxin into both masseter muscles) and 10 to a control group (injected with normal saline). The thicknesses were measured before the injection and at 4, 8, and 12 weeks after the injection both at rest and during maximum muscle contraction. RESULTS: The subcutaneous thickness did not differ significantly over time either at rest (P = 0.063) or during maximal contraction (P = 0.392), or between the experimental and control groups at rest (P = 0.392) or during maximum contraction (P = 0.259). The muscle thickness in the experimental group differed significantly over time. CONCLUSIONS: Botulinum toxin injection only changes the muscle thickness and does not affect the subcutaneous thickness from the skin surface to the masseter muscle.

DEAD-box RNA helicase DDX23 modulates glioma malignancy via elevating miR-21 biogenesis.

Upregulation of microRNA-21 (miR-21) is known to be strongly associated with the proliferation, invasion, and radio-resistance of glioma cells. However, the regulatory mechanism that governs the biogenesis of miR-21 in glioma is still unclear. Here, we demonstrate that the DEAD-box RNA helicase, DDX23, promotes miR-21 biogenesis at the post-transcriptional level. The expression of DDX23 was enhanced in glioma tissues compared to normal brain, and expression level of DDX23 was highly associated with poor survival of glioma patients. Specific knockdown of DDX23 expression suppressed glioma cell proliferation and invasion in vitro and in vivo, which is similar to the function of miR-21. We found that DDX23 increased the level of miR-21 by promoting primary-to-precursor processing of miR-21 through an interaction with the Drosha microprocessor. Mutagenesis experiments critically demonstrated that the helicase activity of DDX23 was essential for the processing (cropping) of miR-21, and we further found that ivermectin, a RNA helicase inhibitor, decreased miR-21 levels by potentially inhibiting DDX23 activity and blocked invasion and cell proliferation. Moreover, treatment of ivermectin decreased glioma growth in mouse xenografts. Taken together, these results suggest that DDX23 plays an essential role in glioma progression, and might thus be a potential novel target for the therapeutic treatment of glioma.

Optimizing implant osseointegration, soft tissue responses, and bacterial inhibition: A comprehensive narrative review on the multifaceted approach of the UV photofunctionalization of titanium.

Titanium implants have revolutionized restorative and reconstructive therapy, yet achieving optimal osseointegration and ensuring long-term implant success remain persistent challenges. In this review, we explore a cutting-edge approach to enhancing implant properties: ultraviolet (UV) photofunctionalization. By harnessing UV energy, photofunctionalization rejuvenates aging implants, leveraging and often surpassing the intrinsic potential of titanium materials. The primary aim of this narrative review is to offer an updated perspective on the advancements made in the field, providing a comprehensive overview of recent findings and exploring the relationship between UV-induced physicochemical alterations and cellular responses. There is now compelling evidence of significant transformations in titanium surface chemistry induced by photofunctionalization, transitioning from hydrocarbon-rich to carbon pellicle-free surfaces, generating superhydrophilic surfaces, and modulating the electrostatic properties. These changes are closely associated with improved cellular attachment, spreading, proliferation, differentiation, and, ultimately, osseointegration. Additionally, we discuss clinical studies demonstrating the efficacy of UV photofunctionalization in accelerating and enhancing the osseointegration of dental implants. Furthermore, we delve into recent advancements, including the development of one-minute vacuum UV (VUV) photofunctionalization, which addresses the limitations of conventional UV methods as well as the newly discovered functions of photofunctionalization in modulating soft tissue and bacterial interfaces. By elucidating the intricate relationship between surface science and biology, this body of research lays the groundwork for innovative strategies aimed at enhancing the clinical performance of titanium implants, marking a new era in implantology.

Beyond microroughness: novel approaches to navigate osteoblast activity on implant surfaces.

Considering the biological activity of osteoblasts is crucial when devising new approaches to enhance the osseointegration of implant surfaces, as their behavior profoundly influences clinical outcomes. An established inverse correlation exists between osteoblast proliferation and their functional differentiation, which constrains the rapid generation of a significant amount of bone. Examining the surface morphology of implants reveals that roughened titanium surfaces facilitate rapid but thin bone formation, whereas smooth, machined surfaces promote greater volumes of bone formation albeit at a slower pace. Consequently, osteoblasts differentiate faster on roughened surfaces but at the expense of proliferation speed. Moreover, the attachment and initial spreading behavior of osteoblasts are notably compromised on microrough surfaces. This review delves into our current understanding and recent advances in nanonodular texturing, meso-scale texturing, and UV photofunctionalization as potential strategies to address the "biological dilemma" of osteoblast kinetics, aiming to improve the quality and quantity of osseointegration. We discuss how these topographical and physicochemical strategies effectively mitigate and even overcome the dichotomy of osteoblast behavior and the biological challenges posed by microrough surfaces. Indeed, surfaces modified with these strategies exhibit enhanced recruitment, attachment, spread, and proliferation of osteoblasts compared to smooth surfaces, while maintaining or amplifying the inherent advantage of cell differentiation. These technology platforms suggest promising avenues for the development of future implants.

Comparative Study of Thermal and Plasma-Enhanced Atomic Layer Deposition of Iron Oxide Using Bis(N,N'-di-butylacetamidinato)iron(II).

Only a few iron precursors that can be used in the atomic layer deposition (ALD) of iron oxides have been examined thus far. This study aimed to compare the various properties of FeOx thin films deposited using thermal ALD and plasma-enhanced ALD (PEALD) and to evaluate the advantages and disadvantages of using bis(N,N'-di-butylacetamidinato)iron(II) as an Fe precursor in FeOx ALD. The PEALD of FeOx films using iron bisamidinate has not yet been reported. Compared with thermal ALD films, PEALD films exhibited improved properties in terms of surface roughness, film density, and crystallinity after they were annealed in air at 500 °C. The annealed films, which had thicknesses exceeding ~ 9 nm, exhibited hematite crystal structures. Additionally, the conformality of the ALD-grown films was examined using trench-structured wafers with different aspect ratios.

Effects of Filler Functionalization on Filler-Embedded Natural Rubber/Ethylene-Propylene-Diene Monomer Composites.

Natural rubber (NR) presents a number of advantages over other types of rubber but has poor resistance to chemicals and aging. The incorporation of ethylene propylene diene monomer (EPDM) into the NR matrix may be able to address this issue. Mineral fillers, such as carbon black (CB) and silica are routinely incorporated into various elastomers owing to their low cost, enhanced processability, good functionality, and high resistance to chemicals and aging. Other fillers have been examined as potential alternatives to CB and silica. In this study, phlogopite was surface-modified using 10 phr of compatibilizers, such as aminopropyltriethoxysilane (A1S), aminoethylaminopropyltrimethoxysilane (A2S), or 3-glycidoxypropyltrimethoxysilane (ES), and mixed with NR/EPDM blends. The effects of untreated and surface-treated phlogopite on the mechanical properties of the rubber blend were then compared with those of common fillers (CB and silica) for rubbers. The incorporation of surface-modified phlogopite into NR/EPDM considerably enhanced various properties. The functionalization of the phlogopite surface using silane-based matters (amino- and epoxide-functionalized) led to excellent compatibility between the rubber matrix and phlogopite, thereby improving diverse properties of the elastomeric composites, with effects analogous to those of CB. The tensile strength and elongation at break of the phlogopite-embedded NR/EPDM composite were lower than those of the CB-incorporated NR/EPDM composite by 30% and 10%, respectively. Among the prepared samples, the ES-functionalized phlogopite showed the best compatibility with the rubber matrix, exhibiting a tensile strength and modulus of composites that were 35% and 18% higher, respectively, compared with those of the untreated phlogopite-incorporated NR/EPDM composite. The ES-functionalized phlogopite/NR/EPDM showed similar strength and higher modulus (by 18%) to the CB/NR/EPDM rubber composite, despite slightly lower elongation at break and toughness. The results of rebound resilience and compression set tests indicated that the elasticity of the surface-modified phlogopite/NR/EPDM rubber composite was higher than that of the silica- and CB-reinforced composites. These improvements could be attributed to enhancements in the physical and chemical interactions among the rubber matrix, stearic acid, and functionalized (compatibilized) phlogopite. Therefore, the functionalized phlogopite can be utilized in a wide range of applications for rubber compounding.

Human milk oligosaccharide 2'-fucosyllactose promotes melanin degradation via the autophagic AMPK-ULK1 signaling axis.

There is still an unmet need for development of safer antimelanogenic or melanin-degrading agents for skin hyperpigmentation, induced by intrinsic or extrinsic factors including aging or ultraviolet irradiation. Owing to the relatively low cytotoxicity compared with other chemical materials, several studies have explored the role of 2'-fucosyllactose (2'-FL), the most dominant component of human milk oligosaccharides. Here, we showed that 2'-FL reduced melanin levels in both melanocytic cells and a human skin equivalent three-dimensional in vitro model. Regarding the cellular and molecular mechanism, 2'-FL induced LC3I conversion into LC3II, an autophagy activation marker, followed by the formation of LC3II+/PMEL+ autophagosomes. Comparative transcriptome analysis provided a comprehensive understanding for the up- and downstream cellular processes and signaling pathways of the AMPK-ULK1 signaling axis triggered by 2'-FL treatment. Moreover, 2'-FL activated the phosphorylation of AMPK at Thr172 and of ULK1 at Ser555, which were readily reversed in the presence of dorsomorphin, a specific AMPK inhibitor, with consequent reduction of the 2'-FL-mediated hypopigmentation. Taken together, these findings demonstrate that 2'-FL promotes melanin degradation by inducing autophagy through the AMPK-ULK1 axis. Hence, 2'-FL may represent a new natural melanin-degrading agent for hyperpigmentation.

Uniqueness of gait kinematics in a cohort study.

Gait, the style of human walking, has been studied as a behavioral characteristic of an individual. Several studies have utilized gait to identify individuals with the aid of machine learning and computer vision techniques. However, there is a lack of studies on the nature of gait, such as the identification power or the uniqueness. This study aims to quantify the uniqueness of gait in a cohort. Three-dimensional full-body joint kinematics were obtained during normal walking trials from 488 subjects using a motion capture system. The joint angles of the gait cycle were converted into gait vectors. Four gait vectors were obtained from each subject, and all the gait vectors were pooled together. Two gait vectors were randomly selected from the pool and tested if they could be accurately classified if they were from the same person or not. The gait from the cohort was classified with an accuracy of 99.71% using the support vector machine with a radial basis function kernel as a classifier. Gait of a person is as unique as his/her facial motion and finger impedance, but not as unique as fingerprints.

Effects of Sling-Based Thoracic Active Exercise on Pain and Function and Quality of Life in Female Patients with Neck Pain: A Randomized Controlled Trial.

This study aimed to investigate the effects of sling-based thoracic active exercise on pain, function, and quality of life in female patients with neck pain. A total of 27 female patients with neck pain were divided into the sling-based thoracic active exercise group (n = 14) and the control group (n = 13). The study group performed a sling-based thoracic active exercise with cervical manual therapy for 50 min a day, twice a week for 4 weeks, whereas the control group performed a placebo exercise with cervical manual therapy in the same manner as the study group. Evaluation of the degree of pain before and after treatment was based on the pressure pain threshold and numeric pain rating scale scores. The craniovertebral angle and neck disability index (NDI) were used to evaluate neck function, and quality of life was measured using the Short Form-36. Afterwards, the patients' pressure pain thresholds were significantly increased, and the numeric pain rating scale score was significantly decreased in both groups (p < 0.05). In terms of function, the craniovertebral angle was significantly increased in both groups (p < 0.05), and neck dysfunction significantly decreased (p < 0.05). The quality of life significantly increased in both groups (p < 0.05). The pressure pain threshold, craniovertebral angle, neck dysfunction index, and quality of life scores (p < 0.05) were significantly different between groups, except the numeric pain scale score. Our results showed that sling-based thoracic active exercise is effective in reducing pain and improving function and quality of life in female patients with neck pain, thus emphasizing the need for thoracic treatment for such patients.

An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations.

Antibody-drug conjugates (ADCs) can selectively deliver cytotoxic agents to tumor cells and are frequently more potent than naked antibodies. However, optimization of the conjugation process between antibodies and cytotoxic agents and characterization of ADCs are laborious and time-consuming processes. Here, we describe a novel ADC platform using a tetravalent bispecific antibody that simultaneously binds to the tumor-associated antigen and a hapten conjugated to a cytotoxic agent. We selected cotinine as the hapten because it is not present in biological systems and is inert and nontoxic. We prepared an anti-epidermal growth factor receptor (EGFR) × cotinine bispecific antibody and mixed it with an equimolar amount of cotinine-conjugated duocarmycin to form the ADC. This ADC showed significant in vitro and in vivo antitumor activity against EGFR-positive, cetuximab-refractory lung adenocarcinoma cells with KRAS mutations.

The Effectiveness of Ferritin as a Contrast Agent for Cell Tracking MRI in Mouse Cancer Models.

PURPOSE: We aimed to investigate the effectiveness of ferritin as a contrast agent and a potential reporter gene for tracking tumor cells or macrophages in mouse cancer models. MATERIALS AND METHODS: Adenoviral human ferritin heavy chain (Ad-hFTH) was administrated to orthotopic glioma models and subcutaneous colon cancer mouse models using U87MG and HCT116 cells, respectively. Brain MR images were acquired before and daily for up to 6 days after the intracranial injection of Ad-hFTH. In the HCT116 tumor model, MR examinations were performed before and at 6, 24, and 48 h after intratumoral injection of Ad-hFTH, as well as before and every two days after intravenous injection of ferritin-labeled macrophages. The contrast effect of ferritin in vitro was measured by MR imaging of cell pellets. MRI examinations using a 7T MR scanner comprised a T1-weighted (T1w) spin-echo sequence, T2-weighted (T2w) relaxation enhancement sequence, and T2*-weighted (T2*w) fast low angle shot sequence. RESULTS: Cell pellet imaging of Ad-hFTH in vitro showed a strong negatively enhanced contrast in T2w and T2*w images, presenting with darker signal intensity in high concentrations of Fe. T2w images of glioma and subcutaneous HCT116 tumor models showed a dark signal intensity around or within the Ad-hFTH tumor, which was distinct with time and apparent in T2*w images. After injection of ferritin-labeled macrophages, negative contrast enhancement was identified within the tumor. CONCLUSION: Ferritin could be a good candidate as an endogenous MR contrast agent and a potential reporter gene that is capable of maintaining cell labeling stability and cellular safety.

Preclinical development of a humanized neutralizing antibody targeting HGF.

Hepatocyte growth factor (HGF) and its receptor, cMET, play critical roles in cell proliferation, angiogenesis and invasion in a wide variety of cancers. We therefore examined the anti-tumor activity of the humanized monoclonal anti-HGF antibody, YYB-101, in nude mice bearing human glioblastoma xenografts as a single agent or in combination with temozolomide. HGF neutralization, The extracellular signal-related kinases 1 and 2 (ERK1/2) phosphorylation, and HGF-induced scattering were assessed in HGF-expressing cell lines treated with YYB-101. To support clinical development, we also evaluated the preclinical pharmacokinetics and toxicokinetics in cynomolgus monkeys, and human and cynomolgus monkey tissue was stained with YYB-101 to test tissue cross-reactivity. We found that YYB-101 inhibited cMET activation in vitro and suppressed tumor growth in the orthotopic mouse model of human glioblastoma. Combination treatment with YYB-101 and temozolomide decreased tumor growth and increased overall survival compared with the effects of either agent alone. Five cancer-related genes (TMEM119, FST, RSPO3, ROS1 and NBL1) were overexpressed in YYB-101-treated mice that showed tumor regrowth. In the tissue cross-reactivity assay, critical cross-reactivity was not observed. The terminal elimination half-life was 21.7 days. Taken together, the in vitro and in vivo data demonstrated the anti-tumor efficacy of YYB-101, which appeared to be mediated by blocking the HGF/cMET interaction. The preclinical pharmacokinetics, toxicokinetics and tissue cross-reactivity data support the clinical development of YYB-101 for advanced cancer.

CPEB1 modulates differentiation of glioma stem cells via downregulation of HES1 and SIRT1 expression.

Glioma stemness has been recognized as the most important reason for glioma relapse and drug resistance. Differentiation of glioma stem cells (GSCs) has been implicated as a novel approach to target recurrent glioma. However, the detailed molecular mechanism involved in the differentiation of GSCs has not yet been elucidated. This study identified CPEB1 as the key modulator that induces the differentiation of GSCs at the post-transcriptional level. Gain and loss of function experiments showed that CPEB1 expression reduced sphere formation ability and the expression of stemness markers such as Nestin and Notch. To elucidate the detailed molecular mechanism underlying the action of CPEB1, we investigated the interacting ribonome of the CPEB1 complex using a Ribonomics approach. CPEB1 specifically suppressed the translation of HES1 and SIRT1 by interacting with a cytoplasmic polyadenylation element. The expression profile of CPEB1 negatively correlated with overall survival in glioma patients. Overexpression of CPEB1 decreased the number of GSCs in an orthotopically implanted glioma animal model. These results suggest that CPEB1-mediated translational control is essential for the differentiation of GSCs and provides novel therapeutic concepts for differentiation therapy.