Single-cell RNA sequencing combined with proteomics of infected macrophages reveals prothymosin-α as a target for treatment of apical periodontitis.

INTRODUCTION: Chronic apical periodontitis (CAP) is a common infectious disease of the oral cavity. Immune responses and osteoclastogenesis of monocytes/macrophages play a crucial role in CAP progression, and this study want to clarify role of monocytes/macrophages in CAP, which will contribute to treatment of CAP. OBJECTIVES: We aim to explore the heterogeneity of monocyte populations in periapical lesion of CAP tissues and healthy control (HC) periodontal tissues by single-cell RNA sequencing (scRNA-seq), search novel targets for alleviating CAP, and further validate it by proteomics and in vitro and in vivo evaluations. METHODS: ScRNA-seq was used to analyze the heterogeneity of monocyte populations in CAP, and proteomics of THP-1-derived macrophages with porphyromonas gingivalis infection were intersected with the differentially expressed genes (DEGs) of macrophages between CAP and HC tissues. The upregulated PTMA (prothymosin-α) were validated by immunofluorescence staining and quantitative real time polymerase chain reaction. We evaluated the effect of thymosin α1 (an amino-terminal proteolytic cleavage product of PTMA protein) on inflammatory factors and osteoclast differentiation of macrophages infected by P. gingivalis. Furthermore, we constructed mouse and rat mandibular bone lesions caused by apical periodontitis, and estimated treatment of systemic and topical administration of PTMA for CAP. Statistical analyses were performed using GraphPad Prism software (v9.2) RESULTS: Monocytes were divided into seven sub-clusters comprising monocyte-macrophage-osteoclast (MMO) differentiation in CAP. 14 up-regulated and 21 down-regulated genes and proteins were intersected between the DEGs of scRNA-seq data and proteomics, including the high expression of PTMA. Thymosin α1 may decrease several inflammatory cytokine expressions and osteoclastogenesis of THP-1-derived macrophages. Both systemic administration in mice and topical administration in the pulp chamber of rats alleviated periapical lesions. CONCLUSIONS: PTMA upregulation in CAP moderates the inflammatory response and prevents the osteoclastogenesis of macrophages, which provides a basis for targeted therapeutic strategies for CAP.

Metabolic reprogramming of renal epithelial cells contributes to lithium-induced nephrogenic diabetes insipidus.

Lithium, mainstay treatment for bipolar disorder, frequently causes nephrogenic diabetes insipidus (NDI) and renal injury. However, the detailed mechanism remains unclear. Here we used the analysis of metabolomics and transcriptomics and metabolic intervention in a lithium-induced NDI model. Mice were treated with lithium chloride (40 mmol/kg chow) and rotenone (ROT, 100 ppm) in diet for 28 days. Transmission electron microscopy showed extensive mitochondrial structural abnormalities in whole nephron. ROT treatment markedly ameliorated lithium-induced NDI and mitochondrial structural abnormalities. Moreover, ROT attenuated the decrease of mitochondrial membrane potential in line with the upregulation of mitochondrial genes in kidney. Metabolomics and transcriptomics data demonstrated that lithium activated galactose metabolism, glycolysis, and amino sugar and nucleotide sugar metabolism. All these events were indicative of metabolic reprogramming in kidney cells. Importantly, ROT ameliorated metabolic reprogramming in NDI model. Based on transcriptomics analysis, we also found the activation of MAPK, mTOR and PI3K-Akt signaling pathways and impaired focal adhesion, ECM-receptor interaction and actin cytoskeleton in Li-NDI model were inhibited or attenuated by ROT treatment. Meanwhile, ROT administration inhibited the increase of Reactive Oxygen Species (ROS) in NDI kidneys along with enhanced SOD2 expression. Finally, we observed that ROT partially restored the reduced AQP2 and enhanced urinary sodium excretion along with the blockade of increased PGE2 output. Taken together, the current study demonstrates that mitochondrial abnormalities and metabolic reprogramming play a key role in lithium-induced NDI, as well as the dysregulated signaling pathways, thereby serving as a novel therapeutic target.

Hypoxia preconditioned DPSC-derived exosomes regulate angiogenesis via transferring LOXL2.

Hypoxia was proved to enhance the angiogenesis of stem cells. However, the mechanism of the angiogenic potential in hypoxia-pretreated dental pulp stem cells (DPSCs) is poorly understood. We previously confirmed that hypoxia enhances the angiogenic potential of DPSC-derived exosomes with upregulation of lysyl oxidase-like 2 (LOXL2). Therefore, our study aimed to illuminate whether these exosomes promote angiogenesis via transfer of LOXL2. Exosomes were generated from hypoxia-pretreated DPSCs (Hypo-Exos) stably silencing LOXL2 after lentiviral transfection and characterized with transmission electron microscopy, nanosight and Western blot. The efficiency of silencing was verified using quantitative real-time PCR (qRT-PCR) and Western blot. CCK-8, scratch and transwell assays were conducted to explore the effects of LOXL2 silencing on DPSCs proliferation and migration. Human umbilical vein endothelial cells (HUVECs) were co-incubated with exosomes to assess the migration and angiogenic capacity through transwell and matrigel tube formation assays. The relative expression of angiogenesis-associated genes was characterized by qRT-PCR and Western blot. LOXL2 was successfully silenced in DPSCs and inhibited DPSC proliferation and migration. LOXL2 silencing in Hypo-Exos partially reduced promotion of HUVEC migration and tube formation and inhibited the expression of angiogenesis-associated genes. Thus, LOXL2 is one of various factors mediating the angiogenic effects of Hypo-Exos.

Micro-CT Evaluation of Different Root Canal Irrigation Protocols on the Removal of Accumulated Hard Tissue Debris: A Systematic Review and Meta-Analysis.

Accumulated hard tissue debris (AHTD) is an inevitable by-product during endodontic treatment and is difficult to remove completely using traditional syringe and needle irrigation (SNI). Adjunctive irrigation is proposed to assist the clean-up of AHTD. This systematic review and meta-analysis aimed to evaluate the AHTD removal efficacy of different root canal irrigation devices using micro-computed tomography (Micro-CT). A literature search was carried out within the main scientific databases until 20 June 2022. All results were screened with detailed eligibility criteria. Eleven studies were included for analysis. SNI, passive ultrasonic irrigation (PUI), negative pressure systems, sonically activated irrigation (SAI), mechanical-activated system and laser-activated irrigation (LAI) were assessed. PUI is superior to SNI for debris removal and LAI has better AHTD removal performance than PUI. The negative pressure system and mechanical-activated system were proved to be less effective. Registration: PROSPERO (CRD42021273892).

Hypoxia Alters the Proteome Profile and Enhances the Angiogenic Potential of Dental Pulp Stem Cell-Derived Exosomes.

Dental pulp stem cells (DPSCs) and their exosomes (Exos) are effective treatments for regenerative medicine. Hypoxia was confirmed to improve the angiogenic potential of stem cells. However, the angiogenic effect and mechanism of hypoxia-preconditioned DPSC-Exos are poorly understood. We isolated exosomes from DPSCs under normoxia (Nor-Exos) and hypoxia (Hypo-Exos) and added them to human umbilical vein endothelial cells (HUVECs). HUVEC proliferation, migration and angiogenic capacity were assessed by CCK-8, transwell, tube formation assays, qRT-PCR and Western blot. iTRAQ-based proteomics and bioinformatic analysis were performed to investigate proteome profile differences between Nor-Exos and Hypo-Exos. Western blot, immunofluorescence and immunohistochemistry were used to detect the expression of lysyl oxidase-like 2 (LOXL2) in vitro and in vivo. Finally, we silenced LOXL2 in HUVECs and rescued tube formation with Hypo-Exos. Hypo-Exos enhanced HUVEC proliferation, migration and tube formation in vitro superior to Nor-Exos. The proteomics analysis identified 79 proteins with significantly different expression in Hypo-Exos, among which LOXL2 was verified as being upregulated in hypoxia-preconditioned DPSCs, Hypo-Exos, and inflamed dental pulp. Hypo-Exos partially rescued the inhibitory influence of LOXL2 silence on HUVEC tube formation. In conclusion, hypoxia enhanced the angiogenic potential of DPSCs-Exos and partially altered their proteome profile. LOXL2 is likely involved in Hypo-Exos mediated angiogenesis.

Simulations of a multi-wavelength differential absorption lidar method for CO2 measurement.

The increase of greenhouse gas is one of the most important factors leading to global climate change. Differential absorption lidar (DIAL) is considered to be the tool with the most potential to measure CO2 remotely. However, it is difficult to obtain accurate CO2 retrievals and determine carbon fluxes with the traditional dual-wavelength differential absorption inversion method, which is characterized by a low signal-to-noise ratio (SNR). Therefore, a multi-wavelength differential absorption inversion framework is proposed in this work. Based on the measurement of the absorption optical depths (ODs) of a single line at 30 different wavelengths, we tried to minimize the differences between the simulated and measured absorption ODs through an iterative process, which consisted of forward simulation and reverse inversion processes. The retrievals are determined when an optimal solution is obtained. The simulation experiments show that the precision of the nonuniform sampling wavelength method is better than that of the dual-wavelength method. The precision of the proposed method was improved by more than 60% for an SNR of 30-40 dB compared to the traditional method. Furthermore, the average error of this method is about 1/9th the traditional method.

Method for wavelength stabilization of pulsed difference frequency laser at 1572 nm for CO(2) detection lidar.

High-accuracy on-line wavelength stabilization is required for differential absorption lidar (DIAL), which is ideal for precisely measuring atmospheric CO(2) concentration. Using a difference-frequency laser, we developed a ground-based 1.57-µm pulsed DIAL for performing atmospheric CO(2) measurements. Owing to the system complexity, lacking phase, and intensity instability, the stabilization method was divided into two parts-wavelength calibration and locking-based on saturated absorption. After obtaining the on-line laser position, accuracy verification using statistical theory and locking stabilization using a one-dimensional template matching method, namely least-squares matching (LSM), were adopted to achieve wavelength locking. The resulting system is capable of generating a stable wavelength.

Expression of proliferating cell nuclear antigen and CD44 variant exon 6 in primary tumors and corresponding lymph node metastases of colorectal carcinoma with Dukes' stage C or D.

AIM: To study changes in characteristics of colorectal carcinoma during the metastatic process and to investigate the correlation between cell proliferation activity and metastatic ability of patients with Dukes' stage C or D. METHODS: Formalin fixed and paraffin embedded materials of primary tumors and corresponding lymph node metastases resected from 56 patients with Dukes' stage C or D of colorectal carcinoma were stained immunohistochemically with proliferating cell nuclear antigen (PCNA) and CD44 variant exon 6 (CD44v6). RESULTS: Thirty-one of 56 patients (55.4 %) expressed PCNA in the primary sites and 36 of 56 patients (64.3 %) expressed PCNA in the metastatic lymph nodes. A significant relation in PCNA expression was observed between the primary site and the metastatic lymph node (0.010