The Therapeutic Potential of Neutrophil Extracellular Traps and NLRP3 Inflammasomes in Mycoplasma pneumoniae Pneumonia.

INTRODUCTION: Mycoplasma pneumoniae (MP) is the most common pathogen of community-acquired pneumonia in children. However, the role of neutrophil extracellular traps (NETs) in the pathogenesis of MP is unclear. METHODS: Both the level of NETs were detected between the 60 MP pneumonia patients and 20 healthy controls, whose the clinical characteristics were compared. Additionally, NETs formation induced by community-acquired respiratory distress syndrome (CARDS) toxin was also analyzed through transcriptome sequencing. RESULTS: The levels of cell-free DNA, Cit-H3, and MPO-DNA complexes were significantly increased in the patients with MP pneumonia. Importantly, both cell-free DNA and LDH were higher in hospitalized patients with severity than those without severity. In addition, CARDS toxin induced the NETs formation in vitro and in vivo. Transcriptomics GO and KEGG pathway analysis indicate that NOD like receptor signaling pathway and Toll-like receptor signaling pathway are significantly enriched. Finally, we found that DNase I significantly attenuated the higher levels of Cit-H3, and up-regulation of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) by down-regulating the expression of NLRP3 and Caspase1(p20) in the lung tissues. DISCUSSION: These results indicate that inhibiting excessive activation of NLRP3 inflammasomes, and NETs formation may alleviate MP pneumonia.

Influencing factors of low vision 2 years after vitrectomy for proliferative diabetic retinopathy: an observational study.

BACKGROUND: Proliferative diabetic retinopathy (PDR) can seriously affect the vision and quality of life of patients. The present study aimed to evaluate the clinical effect of vitrectomy for PDR by observing visual recovery and postoperative complications and to explore the factors influencing low vision. METHODS: This was a case series observational study. Consecutive eyes of patients with PDR who underwent 23G vitrectomy in our hospital within one year (2019.11-2020.11) were collected and followed up for more than 2 years. Patients' visual acuity, surgical complications and management were collected before the operation and during the follow-up. Decimal visual acuity was recorded and converted to the logarithm of the minimal angle of resolution (logMAR) for statistical analysis. Excel was used to establish a database, and SPSS 22.0 statistical software was used for data analysis. RESULTS: A total of 127 patients and 174 eyes were included in the study. The mean age was 57.8 years. The best corrected visual acuity (BCVA) was < 0.3 in 89.7% of eyes before surgery and ≥ 0.3 in 48.3% of eyes after surgery. Among 174 eyes, visual acuity improved in 83.3%. There was no change in 8.6% of eyes, while 8.1% of eyes had decreased visual acuity after surgery. The average logMAR visual acuity was 1.5 ± 0.7 before surgery and 0.7 ± 0.6 after surgery, indicating significant improvement (p < 0.05). Logistic regression analysis showed that intraoperative silicone oil filling and postoperative complication were significant risk factors for postoperative low vision, while preoperative pseudophakic lens and postoperative intra vitreal injection of anti-VEGF were protective factors for vision recovery (p < 0.05). The incidence of postoperative complications was 15.5%, top three of which were vitreous haemorrhage, neovascular glaucoma and traction retinal detachment. CONCLUSION: Vitrectomy is safe and effective in the treatment of PDR with few complication. Postoperative intra vitreal injection of anti-VEGF is a protective factor for vision recovery. TRIAL REGISTRATION: The trial registration number is ChiCRT2100051628, and the date of registration was September 28, 2021.

LINC00028 regulates the development of TGFß1-treated human tenon capsule fibroblasts by targeting miR-204-5p.

Glaucoma is a leading cause of blindness worldwide with complex pathogenesis. The excessive proliferation and fibrosis of human tenon capsule fibroblasts (HTFs) trigger the scar formation after glaucoma filtration surgery. The purpose was to investigate the role of long intergenic non-protein coding RNA 28 (LINC00028) and mechanism in transforming growth factor ß1 (TGFß1)-treated HTFs. The detection of LINC00028 and miR-204-5p expression was conducted using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay. Cell migration and invasion were monitored by transwell assay. The expression of Epithelial-mesenchymal transition (EMT)-related markers, including E-cadherin, α-Smooth muscle actin (α-SMA), fibronectin and ß-catenin, and autophagy-related markers, including Beclin 1 and light chain 3 (LC3-II and LC3-I) at the protein level was quantified using western blot. The prediction of the relationship between LINC00028 and miR-204-5p was performed by the online tool miRcode, and the verification of the relationship between them was conducted using dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay. The expression of LINC00028 was elevated in glaucoma tissues and TGFß1-treated HTFs. LINC00028 downregulation blocked proliferation, migration, invasion, EMT, fibrosis and autophagy of TGFß1-treated HTFs. MiR-204-5p was a target of LINC00028, and its reintroduction exerted a similar role of LINC00028 downregulation. The inhibition of miR-204-5p reversed the effects of LINC00028 downregulation in TGFß1-treated HTFs. LINC00028 regulated proliferation, migration, invasion, EMT, fibrosis and autophagy to induce the development of HTFs by competitively targeting miR-204-5p, and LINC00028 was regarded as a promising biomarker for glaucoma filtration treatment.

Correction to: Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional (3D) structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a mouse tumor model that has highly endogenous mTEM1 expression in the vasculature. Our data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here, we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a tumor mouse model that has highly endogenous mTEM1 expression in the vasculature. Our data indicated that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.

Inhibiting MiR-34α reduces retinal cell apoptosis and downstream NF-κB pathway in diabetic retinopathy rats through regulating HMGB1 expression.

BACKGROUND: This is a research aimed to study the effect of micro ribonucleic acid (miR)-34α on the retinal cell apoptosis in diabetic retinopathy (DR) rats and its key molecular mechanism. METHODS: Sprague-Dawley rats were randomly divided into healthy group (H group, N.=5), diabetes group (D group, N.=5), diabetes + negative control transfection group (N group, N.=5) and diabetes + miR-34α inhibitor transfection group (M group, N.=5). The rat model of diabetes was established via intraperitoneal injection of 2% streptozotocin solution (60 mg/kg). After 72 h, the urine glucose and blood glucose were detected, and the urine glucose above 3+ and the blood glucose concentration >16.7 mmol/L indicated the successful modeling. After the rats were normally fed for 4 months, the changes in expression of miR-34α in retinal tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR), the pathological changes in retinal tissues were observed via hematoxylin-eosin (HE) staining, and the retinal cell apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the changes in the number of cells containing active caspase-3 in retinal tissues were determined through immunohistochemistry, and the changes in expressions of caspase-3, high mobility group box 1 (HMGB1) and nuclear factor-κB (NF-κB) in retinal tissues were determined through Western blotting. RESULTS: Compared with those in H group, the cell density declined, and the cells were arranged disorderly with swelling in each retinal layer, the expression of miR-34α in retinal tissues was increased, the retinal cell apoptosis was enhanced, the number of cells containing active caspase-3 in retinal tissues rose, and the expressions of caspase-3, HMGB1 and NF-κB in retinal tissues were increased in D group, N group and M group (P<0.05). Compared with those in D group and N group, the cell density rose, and the cells were arranged less disorderly with milder swelling in each retinal layer, the expression of miR-34α in retinal tissues declined, the retinal cell apoptosis was weakened, the number of cells containing active caspase-3 in retinal tissues was decreased, and the expressions of caspase-3, HMGB1 and NF-κB in retinal tissues were reduced in M group (P<0.05). CONCLUSIONS: Inhibiting miR-34α reduces the retinal cell apoptosis in DR rats through regulating the HMGB1 expression and downstream NF-κB pathway.

Stem Cell-derived Extracellular Vesicles: A Promising Nano Delivery Platform to the Brain?

A very important cause of the frustration with drug therapy for central nervous system (CNS) diseases is the failure of drug delivery. The blood-brain barrier (BBB) prevents most therapeutic molecules from entering the brain while maintaining CNS homeostasis. Scientists are keen to develop new brain drug delivery systems to solve this dilemma. Extracellular vesicles (EVs), as a class of naturally derived nanoscale vesicles, have been extensively studied in drug delivery due to their superior properties. This review will briefly present current brain drug delivery strategies, including invasive and non-invasive techniques that target the brain, and the application of nanocarriers developed for brain drug delivery in recent years, especially EVs. The cellular origin of EVs affects the surface protein, size, yield, luminal composition, and other properties of EVs, which are also crucial in determining whether EVs are useful as drug carriers. Stem cell-derived EVs, which inherit the properties of parental cells and avoid the drawbacks of cell therapy, have always been favored by researchers. Thus, in this review, we will focus on the application of stem cell-derived EVs for drug delivery in the CNS. Various nucleic acids, proteins, and small-molecule drugs are loaded into EVs with or without modification and undergo targeted delivery to the brain to achieve their therapeutic effects. In addition, the challenges facing the clinical application of EVs as drug carriers will also be discussed. The directions of future efforts may be to improve drug loading efficiency and precise targeting.

Retrospective analysis of changes in the anterior corneal surface after Q value guided LASIK and LASEK in high myopic astigmatism for 3 years.

BACKGROUND: To compare the corneal high-order aberrations (HOAs), asphericity and regularity after Q-value guided laser in situ keratomileusis (LASIK) and laser epithelial keratomileusis (LASEK) in high myopic astigmatism. METHODS: In this retrospectively comparative study, we measured the corneal HOAs, asphericity indices (Q values) and corneal regularity indices preoperatively and 36 months postoperatively in 70 eyes (35 patients) with Q-value guided surgeries. All the patients with high myopic astigmatism were divided into two groups which included 34 eyes underwent LASIK and 36 eyes underwent LASEK procedures. The main impact factors of the high-order aberrations were also analyzed. RESULTS: In the two groups, the efficacy index was more than 1.00 and safety index approached 1.00 at year 3 postoperatively. Statistically significant (P < 0.05) increased in Q values and main corneal HOAs (spherical aberrations and coma) following Q-value guided LASIK and LASEK procedures. Spherical aberrations increased more in the LASEK group and there was statistically difference compared to the LASIK group (P < 0.05). LASEK had better effects in correcting corneal astigmatism (P < 0.05). All the corneal regularity indices after surgeries increased and there was no significant difference (P = 0.707, P = 0.8 and P = 0.224, respectively) between the two groups. The main impact factors of spherical aberration included the optic zone size, changes of Q value, surgical procedure and the corrected refraction. CONCLUSIONS: In high myopic astigmatism, Q-value guided ablation showed good safety, efficacy and predictability. Q value, regularity indices, spherical aberration and coma increased in both LASIK and LASEK procedures. Astigmatism could be corrected more effectively by LASEK but greater spherical aberration could be created. The difference might be related to the different healing mechanisms. Optic zone size and the corrected refraction might be the main influence factors on the anterior corneal high order aberrations.

Adipose-Derived Mesenchymal Stem Cell-Derived Exosomal miR-301a-3p Regulates Airway Smooth Muscle Cells During Asthma by Targeting STAT3.

BACKGROUND: Asthma is a chronic inflammatory disease featured by inflammation and remodeling of airway. Adipose-derived mesenchymal stem cell (ADSCs)-derived exosomal miRNAs have been suggested as promising therapeutic manners for diseases. METHODS: ADSCs and airway smooth muscle cells (ASMCs) were isolated from SD rats. Flow cytometry was conducted to detect the surface biomarkers of isolated cells. Exosomes were extracted by sequentially centrifuge method and identified by Western blotting and nanoparticle tracking analysis (NTA). Uptake of exosomes by ASMCs was detected by confocal assay. ASMCs were treated with platelet-derived growth factor-BB (PDGF-BB) to mimic cell remodeling and inflammation. Cell counting 8 (CCK-8), Transwell, and flow cytometry were performed to determine the viability, migration, and apoptosis of ASMCs. Release of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA). Levels of RNAs and proteins were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Interaction between miR-301a-3p and signal transducer and activator of transcription 3 (STAT3) was determined by luciferase reporter gene assay. The effect of Exosomal miR-301a-3p was analyzed in ovalbumin (OVA)-induced asthma mouse model. RESULTS: ADSCs-derived exosomes could be effectively internalized by ASMCs. Exosomal miR-301a-3p notably suppressed the PDGF-BB-stimulated proliferation and migration of ASMCs, and enhanced apoptosis, as well as decreased the secretion of inflammatory factors. MiR-301a-3p directly targeted the 3'UTR region of STAT3. STAT3 overexpression reversed the suppressive effects of exosomal miR-301a-3p on ASMCs under PDGF-BB stimulation. The expression of miR-301a-3p and STAT3 was negative correlation in specimen from patients with asthma. Exosomal miR-301a-3p inhibited OVA-induced lung injury by targeting STAT3 in mice. CONCLUSION: This study exposed that exosomal miR-301a-3p from ADSCs could effectively alleviate PDGF-BB-stimulated remodeling and inflammation of ASMCs via targeting STAT3, presented ADSCs-derived exosomal miR-301a-3p as a promising therapeutic approach for asthma.

The dual role of CD70 in B-cell lymphomagenesis.

BACKGROUND: CD70 is a costimulatory molecule that is transiently expressed on a small set of activated lymphocytes and is involved in T-cell-mediated immunity. However, the role of CD70 in B-cell malignancies remains controversial. METHODS: We investigated the clinical relevance of CD70 genetic alterations and its protein expression in two diffuse large B-cell lymphoma (DLBCL) cohorts with different ethnic backgrounds. We also performed transcriptomic analysis to explore the role of CD70 alterations in tumour microenvironment. We further tested the blockade of CD70 in combination with PD-L1 inhibitor in a murine lymphoma model. RESULTS: We showed that CD70 genetic aberrations occurred more frequently in the Chinese DLBCL cohort (56/233, 24.0%) than in the Swedish cohort (9/84, 10.8%), especially in those with concomitant hepatitis B virus (HBV) infection. The CD70 genetic changes in DLBCL resulted in a reduction/loss of protein expression and/or CD27 binding, which might impair T cell priming and were independently associated with poor overall survival. Paradoxically, we observed that over-expression of CD70 protein was also associated with a poor treatment response, as well as an advanced disease stage and EBV infection. More exhausted CD8+ T cells were furthermore identified in CD70 high-expression DLBCLs. Finally, in a murine lymphoma model, we demonstrated that blocking the CD70/CD27 and/or PD1/PD-L1 interactions could reduce CD70+ lymphoma growth in vivo, by directly impairing the tumour cell proliferation and rescuing the exhausted T cells. CONCLUSIONS: Our findings suggest that CD70 can play a role in either tumour suppression or oncogenesis in DLBCL, likely via distinct immune evasion mechanisms, that is, impairing T cell priming or inducing T cell exhaustion. Characterisation of specific dysfunction of CD70 in DLBCL may thus provide opportunities for the development of novel targeted immuno-therapeutic strategies.

Fibronectin 1: A Potential Biomarker for Ovarian Cancer.

METHODS: OVCAR3 and A2780 are the two common cell lines that are used for ovarian cancer studies. The different invasion and migration abilities were observed by scratch tests and transwell experiments in our preliminary study. Gene chip was used to screen the expression gene in these two different cell lines, and then, the differentially expressed genes (at least 2-fold difference, P value < 0.05) were analyzed using KEGG. RESULT: Fibronectin 1 (FN1) was found to be the most strongly correlated with the invasion and migration abilities of the OVCAR3 cells. Real-time PCR and FN1 knockout cell line was conducted and confirmed this finding. Based on the Oncomine database analysis, comparing with normal people, ovarian cancer patients exhibited high levels of FN1 expression. Additionally, higher FN1 expression was found in patients with higher FIGO stages of cancer. CONCLUSION: FN1 could be a new biomarker for ovarian cancer detection and progress indicator.

Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway.

Objective: CA125/MUC16 is an O-glycosylated protein that is expressed on the surfaces of ovarian epithelial cells. This molecule is a widely used tumor-associated marker for diagnosis of ovarian cancer. Recently, CA125 was shown to be involved in ovarian cancer metastasis. The purpose of this study was to investigate the mechanism of CA125 during ovarian cancer metastasis. Methods: We analyzed the Oncomine and CSIOVDB databases to determine the expression levels of DKK1 in ovarian cancer. DKK1 expression levels were upregulated or downregulated and applied with CA125 to Transwell and Western blot assays to ascertain the underlying mechanism by which CA125 stimulates cell migration via the SGK3/FOXO3 pathway. Anti-mesothelin antibodies (anti-MSLN) were used to block CA125 stimulation. Then the expression levels of DKK1were tested by enzyme-linked immunosorbent assay (ELISA) to eliminate the blocking effect of anti-MSLN to CA125 stimulation. Xenograft mouse models were used to detect the effects of CA125 and anti-MSLN on ovarian cancer metastasis in vivo. Results: DKK1 levels were downregulated in ovarian tumor tissues according to the analyses of two databases and significantly correlated with FIGO stage, grade and disease-free survival in ovarian cancer patients. DKK1 levels were downregulated by CA125 stimulation in vitro. Overexpression of DKK1 reversed the ability of exogenous CA125 to mediate cell migration by activating the SGK3/FOXO3 signaling pathway. Anti-MSLN abrogated the DKK1 reduction and increased the apoptosis of ovarian cancer cells. The use of anti-MSLN in xenograft mouse models significantly reduced tumor growth and metastasis accelerated by CA125. Conclusions: These experiments revealed that the SGK3/FOXO3 pathway was activated, wherein decreased expression of DKK1 was caused by CA125, which fuels ovarian cancer cell migration. Mesothelin is a potential therapeutic target for the treatment of ovarian cancer metastasis.

Evaluation of Diffusional Characteristics and Microstructure in Unilateral Vocal Fold Paralysis Using Diffusion Tensor Imaging.

OBJECTIVE: To investigate the value of diffusion tensor imaging (DTI) in the evaluation of vocal fold tissue microstructure after recurrent laryngeal nerve (RLN) injury. METHODS: Six canines were divided into 2 groups: a unilateral vocal fold paralysis group (n = 4) and a control group (n = 2). The RLN was cut in the unilateral vocal fold paralysis group, and no intervention was applied in the control group. After 4 months, the canines' larynges were removed and placed in a small animal magnetic resonance imaging (MRI) system (9.4T BioSpec MRI; Bruker, Germany). After scanning, the vocal folds were isolated, sectioned, and stained. The slides were then analyzed for the cross-sectional area and muscle fiber density through feature extraction technology. Pearson correlation analysis was performed on the DTI scan and histological section extraction results. RESULTS: In the vocal fold muscle layer, the fractional anisotropy (FA) of the unilateral RLN injury group was higher than that of the control group, and the Tensor Trace was lower than that of the control group. This difference was statistically significant, P < .05. In the lamina propria, the FA of the unilateral RLN injury group was lower than that of the control group, P > .05, and the Tensor Trace was lower than that of the control group, P < .05. The muscle fiber cross-sectional area of the RLN injury group was significantly smaller than the control group with statistical significance, P < .05, and the density of muscle fibers was lower, P < .05. The correlation coefficient between FA and the cross-sectional area was -0.838, P = .002, and .726; P = .017 between Tensor Trace and the cross-sectional area. CONCLUSION: Diffusion tensor imaging is an effective method to assess the changes in the microstructure of atrophic vocal fold muscle tissue after RLN injury.

Interleukin-23 derived from CD16+ monocytes drives IL-17 secretion by TLR4 pathway in children with mycoplasma pneumoniae pneumonia.

AIMS: The study aimed to investigate whether IL-23 is amplified in monocyte subsets of MP pneumonia and to determine its relevant pathway. MATERIALS AND METHODS: We firstly analyze the IL-23p19 expression in monocyte subgroups in MP pneumonia patients and healthy controls subjects by using flow cytometry. Then, we also analyzed the percentage of IL-17+γδT cells and Th17 cells in patients with MP pneumonia and controls subjects. At the same time, the relation between IL-23 and IL-17 were also assessed. Furthermore, we constructed the recombinant community-acquired respiratory distress syndrome (CARDS) toxin and intend to stimulate peripheral blood mononuclear cells and RAW264.7 cells in vitro. IL-23p19 was detected by flow cytometry and the mRNA levels were measured by real-time PCR. Finally, TLR4 pathway was also investigated by TAK242 inhibitor. KEY FINDINGS: It turned out that the expression of IL-23p19 was increased in CD14brightCD16+ monocyte of MP pneumonia patients than controls subjects. The patients with MP pneumonia had significantly higher the percentage of IL-17+γδT cells and Th17 cells than controls subjects. Interestingly, the levels of IL-23 were positively related to IL-17 in MP pneumonia patients. CD16+ monocytes and RAW264.7 cells, respectively can be induced by CARDS toxin to secrete IL-23 by TLR4 pathway in vitro. SIGNIFICANCE: These results indicated that IL-23-IL-17+γδT/Th17 axis may play a role in the pathogenesis of MP pneumonia, whereas IL-23 derived from CD16+ monocytes was expanded in MP pneumonia by TLR4 pathway.

Spatial Motion of Arytenoid Cartilage Using Dynamic Computed Tomography Combined with Euler Angles.

OBJECTIVE: To investigate the feasibility of dynamic computed tomography in recording and describing the spatial motion characteristics of the arytenoid cartilage. METHODS: Dynamic computed tomography recorded the real-time motion trajectory of the arytenoid cartilage during inspiration and phonation. A stationary coordinate system was established with the cricoid cartilage as a reference and a motion coordinate system was established using the movement of the arytenoid cartilage. The Euler angles of the arytenoid cartilage movement were calculated by transformation of the two coordinate systems, and the spatial motion characteristics of the arytenoid cartilage were quantitatively studied. RESULTS: Displacement of the cricoid cartilage was primarily inferior during inspiration. During phonation, the displacement was mainly superior. When the glottis closed, the superior displacement was about 5-8 mm within 0.56 s. During inspiration, the arytenoid cartilage was displaced superiorly approximately 1-2 mm each 0.56 s. The rotation angle was subtle with slight rotation around the XYZ axis, with a range of 5-10 degrees. During phonation, the displacement of the arytenoid cartilage was mainly inferior (about 4-6 mm), anterior (about 2-4 mm) and medial (about 1-2 mm). The motion of the arytenoid cartilage mainly consisted of medial rolling, and there was an alternating movement of anterior-posterior tilting. The arytenoid cartilage rolled medially (about 20-40 degrees within 0.56 s), accompanied by anterior-posterior tilting (about 15-20 degrees within 0.56 s). CONCLUSION: Dynamic computed tomography recordings of arytenoid cartilage movement can be combined with Euler transformations as a tool to study the spatial characteristics of laryngeal structures during phonation. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E646-E653, 2020.

Interaction of HCCR-1 and Bax in breast cancer.

PURPOSE: To evaluate the effect on breast cancer cell proliferation and apoptosis after silencing the HCCR-1 and to study its mechanism. METHODS: HCCR-1 siRNA was transfected into the breast cancer cell line MCF -7, and mRNA and protein level of HCCR-1 and Bax were evaluated by real-time quatitative PCR (qRT-PCR) and Western blotting, respectively. The cell proliferation and apoptosis were studied by MTT assay and flow cytometry. RESULTS: The apoptosis rate in the experimental, control and blank groups were 32.57±2.35%, 3.53±0.60% and 3.15±0.46% respectively. The apoptosis rate of MCF-7 cells was significantly increased in the experimental group, compared with the other two groups (p<0.05). The A490 value in the experimental, control and blank groups were: 24h: 0.78±0.06, 1.18±0.05, 1.24±0.05; 48h: 1.09±0.05, 1.48±0.02, 1.54±0.04; 72h: 1.29±0.01, 1.81±0.02, 1.84±0.04. The proliferation of MCF-7 cells was significantly decreased in the experimental group, compared with the other two groups (p<0.05). The mRNA levels of HCCR-1 and Bax in the experimental, control and blank groups were: HCCR-1 0.46±0.03, 1.01±0.11, 1.00; and Bax 4.40±0.99, 1.03±0.10, 1.00. The protein levels were: HCCR-1 0.62±0.07, 0.89±0.09, 0.94±0.17; and Bax 0.95±0.22, 0.67±0.19, 0.69±0.11. The expressions of mRNA and protein of HCCR-1 were significantly reduced, however the expressions of mRNA and protein of Bax were significantly increased in the experimental group compared with the other two groups (p<0.05). CONCLUSIONS: HCCR-1 siRNA transfection causes significant increase in the apoptosis and decreases in the proliferation of MCF-7 cells．These effects are related to the upregulation of the Bax expression in the MCF-7 cells.

Neutrophil-to-Lymphocyte Ratio in Ovarian Cancer Patients with Low CA125 Concentration.

Ovarian cancer cases with low CA125 concentration are problematic and increase the high false negative results ratio during routine physical examination testing. Unfortunately, patients without early discovery have very low survival rates. In our study, we investigated the possible role of differential leukocyte counts and the neutrophil-to-lymphocyte ratio (NLR) in ovarian cancer patients to identify an additional discriminative marker to avoid missing diagnoses in normal physical examinations. One hundred seventy-three patients with epithelial ovarian cancer and 70 healthy controls were involved in our study. Based on the results, compared with the healthy controls, NLR was significantly different both in the low CA125 concentration group and in the complete patient group, indicating that NLR could be an effective marker for ovarian cancer screening. According to ROC, sensitivity, specificity, and NPV results, CA125 >35 U/ml is a good indicator for cancer in routine physical examination. However, in patients with low CA125 concentration, the CA125>7.65 U/ml and NLR >1.72 group yielded increased sensitivity with appropriate specificity and higher NPV results than the CA125 >35 U/ml group. We believe CA125>7.65 U/ml and NLR >1.72 should be effective makers for patients with low CA125 concentration. As a more sensitive and cost-effective strategy, this method could be conducted during routine ovarian cancer screening.

Effects of Angle of Epiglottis on Aerodynamic and Acoustic Parameters in Excised Canine Larynges.

OBJECTIVES: The aim of this study is to explore the effects of the angle of epiglottis (Aepi) on phonation and resonance in excised canine larynges. METHODS: The anatomic Aepi was measured for 14 excised canine larynges as a control. Then, the Aepis were manually adjusted to 60° and 90° in each larynx. Aerodynamic and acoustic parameters, including mean flow rate, sound pressure level, jitter, shimmer, fundamental frequency (F0), and formants (F1'-F4'), were measured with a subglottal pressure of 1.5 kPa. Simple linear regression analysis between acoustic and aerodynamic parameters and the Aepi of the control was performed, and an analysis of variance comparing the acoustic and aerodynamic parameters of the three treatments was carried out. RESULTS: The results of the study are as follows: (1) the larynges with larger anatomic Aepi had significantly lower jitter, shimmer, formant 1, and formant 2; (2) phonation threshold flow was significantly different for the three treatments; and (3) mean flow rate and sound pressure level were significantly different between the 60° and the 90° treatments of the 14 larynges. CONCLUSIONS: The Aepi was proposed for the first time in this study. The Aepi plays an important role in phonation and resonance of excised canine larynges.

Strontium promotes osteogenic differentiation by activating autophagy via the the AMPK/mTOR signaling pathway in MC3T3­E1 cells.

Strontium (Sr) is an alkaline earth metal that exerts the dual effect of improving bone formation and suppressing bone resorption, resulting in increased bone apposition rates and bone mineral density. However, the mechanisms through which Sr exerts these beneficial effects on bone have yet to be fully elucidated. The present study aimed to reveal the underlying molecular mechanisms associated with Sr­induced osteogenic differentiation. The effects of Sr on cell proliferation and osteogenic differentiation were analyzed by MTT assay, RT­qPCR, western blot analysis, alkaline phosphatase (ALP) and Alizarin red staining assays. The extent of autophagy was determined by monodansylcadaverine (MDC) staining and western blot analysis of two markers of cellular autophagic activity, the steatosis­associated protein, sequestosome­1 (SQSTM1/p62), and the two isoforms of microtubule­associated protein 1 light chain 3 (LC3), LC­3­I/II. The expression levels of AMP­activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were also detected by western blot analysis. Sr at a concentration of 3 mM exerted the most pronounced effect on osteogenic differentiation, without any apparent cell toxicity. At the same time, cellular autophagy was active during this process. Subsequently, autophagy was blocked by 3­methyladenine, and the enhancement of osteogenic differentiation in response to Sr was abrogated. Additionally, the phosphorylation level of AMPK was significantly increased, whereas that of mTOR was significantly decreased, in the Sr­treated group. Taken together, the findings of the present study demonstrate that Sr stimulates AMPK­activated autophagy to induce the osteogenic differentiation of MC3T3­E1 cells.