Loss of SIL1 Affects Actin Dynamics and Leads to Abnormal Neural Migration.

SIL1 is a nucleotide exchange factor for the molecular chaperone protein Bip in the endoplasmic reticulum that plays a crucial role in protein folding. The Sil1 gene is currently the only known causative gene of Marinesco-Sjögren syndrome (MSS). Intellectual developmental disability is the main symptom of MSS, and its mechanism has not been fully elucidated. Studies have shown that mutations in the Sil1 gene can delay neuronal migration during cortical development, but the underlying molecular mechanisms remain unclear. To further identify potential molecules involved in the regulation of central nervous system development by SIL1, we established a cortical neuron model with SIL1 protein deficiency and used proteomic analysis to screen for differentially expressed proteins after Sil1 silencing, followed by GO functional enrichment and protein‒protein interaction (PPI) network analysis. We identified 68 upregulated and 137 downregulated proteins in total, and among them, 10 upregulated and 3 downregulated proteins were mainly related to actin cytoskeleton dynamics. We further validated the differential changes in actin-related molecules using qRT‒PCR and Western blotting of a Sil1 gene knockout (Sil1-/-) mouse model. The results showed that the protein levels of ACTN1 and VIM decreased, while their mRNA levels increased as a compensatory response to protein deficiency. The mRNA and protein levels of IQGAP1 both showed a secondary increase. In conclusion, we identified ACTN1 and VIM as the key molecules regulated by SIL1 that are involved in neuronal migration during cortical development.

Accumulation of ß-aminoisobutyric acid mediates hyperalgesia in ovariectomized mice through Mas-related G protein-coupled receptor D signaling.

Hyperalgesia is typified by reduced pain thresholds and heightened responses to painful stimuli, with a notable prevalence in menopausal women, but the underlying mechanisms are far from understood. ß-Aminoisobutyric acid (BAIBA), a product of valine and thymine catabolism, has been reported to be a novel ligand of the Mas-related G protein coupled receptor D (MrgprD), which mediates pain and hyperalgesia. Here, we established a hyperalgesia model in 8-week-old female mice through ovariectomy (OVX). A significant increase in BAIBA plasma level was observed and was associated with decline of mechanical withdrawal threshold, thermal and cold withdrawal latency in mice after 6 weeks of OVX surgery. Increased expression of MrgprD in dorsal root ganglion (DRG) was shown in OVX mice compared to Sham mice. Interestingly, chronic loading with BAIBA not only exacerbated hyperalgesia in OVX mice, but also induced hyperalgesia in gonadally intact female mice. BAIBA supplementation also upregulated the MrgprD expression in DRG of both OVX and intact female mice, and enhanced the excitability of DRG neurons in vitro. Knockout of MrgprD markedly suppressed the effects of BAIBA on hyperalgesia and excitability of DRG neurons. Collectively, our data suggest the involvement of BAIBA in the development of hyperalgesia via MrgprD-dependent pathway, and illuminate the mechanisms underlying hyperalgesia in menopausal women.

Age-related decline in melatonin contributes to enhanced osteoclastogenesis via disruption of redox homeostasis.

BACKGROUND: Increased oxidative stress contributes to enhanced osteoclastogenesis and age-related bone loss. Melatonin (MT) is an endogenous antioxidant and declines with aging. However, it was unclear whether the decline of MT was involved in the enhanced osteoclastogenesis during the aging process. METHODS: The plasma level of MT, oxidative stress status, bone mass, the number of bone marrow-derived monocytes (BMMs) and its osteoclastogenesis were analyzed in young (3-month old) and old (18-month old) mice (n = 6 per group). In vitro, BMMs isolated from aged mice were treated with or without MT, followed by detecting the change of osteoclastogenesis and intracellular reactive oxygen species (ROS) level. Furthermore, old mice were treated with MT for 2 months to investigate the therapeutic effect. RESULTS: The plasma level of MT was markedly lower in aged mice compared with young mice. Age-related decline in MT was accompanied by enhanced oxidative stress, osteoclastogenic potential and bone loss. MT intervention significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, decreased intracellular ROS and enhanced antioxidant capacity of BMMs from aged mice. MT supplementation significantly attenuated oxidative stress, osteoclastogenesis, bone loss and deterioration of bone microstructure in aged mice. CONCLUSIONS: These results suggest that age-related decline of MT enhanced osteoclastogenesis via disruption of redox homeostasis. MT may serve as a key regulator in osteoclastogenesis and bone homeostasis, thereby highlighting its potential as a preventive agent for age-related bone loss.

Tanshinone IIA attenuates osteoarthritis via inhibiting aberrant angiogenesis in subchondral bone.

Excessive angiogenesis in subchondral bone is a pathological feature of osteoarthritis (OA). Tanshinone IIA (TIIA), an active compound found in Salvia miltiorrhiza, demonstrates significant anti-angiogenic properties. However, the effect of TIIA on abnormal subchondral angiogenesis in OA is still unclear. This study aims to investigate the mechanism of TIIA in modulating subchondral bone angiogenesis during OA and assess its therapeutic potential in OA. Our findings demonstrate that TIIA attenuated articular cartilage degeneration, normalized subchondral bone remodeling, and effectively suppressed aberrant angiogenesis within subchondral bone in monosodium iodoacetate (MIA)-induced OA mice. Additionally, the angiogenesis capacity of primary CD31hiEmcnhi endothelial cells was observed to be significantly reduced after treatment with TIIA in vitro. Mechanically, TIIA diminished the proportion of hypertrophic chondrocytes, ultimately leading to a substantial reduction in the secretion of vascular endothelial growth factor A (VEGFA). The supernatant of hypertrophic chondrocytes promoted the tube formation of CD31hiEMCNhi endothelial cells, whereas TIIA inhibited this process. Furthermore, TIIA effectively suppressed the expression of vascular endothelial growth factor receptor 2 (VEGFR2) along with its downstream MAPK pathway in CD31hiEmcnhi endothelial cells. In conclusion, our data indicated that TIIA could effectively inhibit the abnormal angiogenesis in subchondral bone during the progression of OA by suppressing the VEGFA/VEFGR2/MAPK pathway. These findings significantly contribute to our understanding of the abnormal angiogenesis in OA and offer a promising therapeutic target for OA treatment.

A combination of ultrasound and contrast-enhanced ultrasound improves diagnostic accuracy for the differentiation of cervical tuberculous lymphadenitis from primary lymphoma.

OBJECTIVES: To present a method combining ultrasound (US) and contrast-enhanced ultrasound (CEUS) features for differential diagnosis of cervical tuberculous lymphadenitis (CTL) and primary lymphoma. METHODS: A total of 155 patients with CTL (n = 49) and lymphoma (n = 106) who underwent US and CEUS were retrospectively included. The features extracted from US and CEUS and the significant clinical data were created three models using the least absolute shrinkage and selection operator and logistic regression analysis. The diagnostic performance of the models was assessed using the area under the curve (AUC). RESULTS: The combined model outperformed US model and CEUS model in distinguish CTL from lymphoma achieved favorable performances in training set and validation set with AUCs of 0.958 and 0.946 as well as high accuracies (91.7% and 87.2%), sensitivities (95.9% and 84.4%) and specificities (82.4% and 93.3%). Delong's test showed that among the three models, combined model was significantly different from the other two models in training set (p = 0.011 and 0.029, respectively) and validation set (p = 0.018 and 0.001, respectively). CONCLUSIONS: A combination of US and CEUS achieved good diagnostic performance in differentiating lymphoma and CTL, which might aid in clinical decision-making.

A Soft-Reference Breast Ultrasound Image Quality Assessment Method That Considers the Local Lesion Area.

The quality of breast ultrasound images has a significant impact on the accuracy of disease diagnosis. Existing image quality assessment (IQA) methods usually use pixel-level feature statistical methods or end-to-end deep learning methods, which focus on the global image quality but ignore the image quality of the lesion region. However, in clinical practice, doctors' evaluation of ultrasound image quality relies more on the local area of the lesion, which determines the diagnostic value of ultrasound images. In this study, a global-local integrated IQA framework for breast ultrasound images was proposed to learn doctors' clinical evaluation standards. In this study, 1285 breast ultrasound images were collected and scored by experienced doctors. After being classified as either images with lesions or images without lesions, they were evaluated using soft-reference IQA or bilinear CNN IQA, respectively. Experiments showed that for ultrasound images with lesions, our proposed soft-reference IQA achieved PLCC 0.8418 with doctors' annotation, while the existing end-to-end deep learning method that did not consider the local lesion features only achieved PLCC 0.6606. Due to the accuracy improvement for the images with lesions, our proposed global-local integrated IQA framework had better performance in the IQA task than the existing end-to-end deep learning method, with PLCC improving from 0.8306 to 0.8851.

CircRBM23 regulates the switch between osteogenesis and adipogenesis of mesenchymal stem cells via sponging miR-338-3p.

BACKGROUND: The disruption of the balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) in bone marrow contributes to the adipocytes accumulation and bone loss, which leads to the development of osteoporosis (OP). The circular RNA (circRNA), circRBM23, was generated from the RNA binding motif protein 23 (RBM23) gene. It was reported that circRBM23 was down-regulated in OP patients, but it remains unknown whether its down-regulation is involved in the lineage switch of MSCs. OBJECTIVE: We aimed to explore the role and mechanism of circRBM23 in regulating the switch between osteogenic and adipogenic differentiation of MSCs. METHODS: The expression and function of circRBM23 in vitro were detected by qRT-PCR, alizarin red staining, and oil Red O staining. The interactions between circRBM23 and microRNA-338-3p (miR-338-3p) were analyzed by RNA pull-down assay, FISH, and dual-luciferase reporter assay. MSCs treated with lentivirus overexpression of circRBM23 was applied for both in vitro and in vivo experiments. RESULTS: CircRBM23 was expressed at lower levels in OP patients. Besides, circRBM23 was up-regulated during osteogenesis and down-regulated during adipogenesis of MSCs. CircRBM23 could promote the osteogenic differentiation but inhibit the adipogenic differentiation of MSCs. Mechanistically, circRBM23 acted as a sponge for microRNA-338-3p (miR-338-3p) to enhance the expression of RUNX family transcription factor 2 (RUNX2). CONCLUSIONS: Our research indicates that circRBM23 could promote the switch from adipogenic to osteogenic differentiation of MSCs via sponging miR-338-3p. It might improve the understanding of the lineage switch of MSCs and provide a potential target for diagnosing and treating OP.

N-acetyl-L-cysteine attenuates oxidative stress-induced bone marrow endothelial cells apoptosis by inhibiting BAX/caspase 3 pathway.

Bone marrow endothelial cells (BMECs) play a crucial role in the maintenance of bone homeostasis. The decline in BMECs is associated with abnormal bone development and loss. At present, the mechanism of age-related oxidative stress enhancement in BMEC dysfunction remains unclear. Our experiment explored injury caused by oxidative stress enhancement in BMECs both in vivo and in vitro. The BMECs, indicators of oxidative stress, bone mass, and apoptosis-related proteins were analyzed in different age groups. We also evaluated the ability of N-Acetyl-L-cysteine (NAC) attenuate oxidative stress injury in BMECs. NAC treatment attenuated reactive oxygen species (ROS) overgeneration and apoptosis in BMECs in vitro and alleviated the loss of BMECs and bone mass in vivo. In conclusion, this study could improve our understanding of the mechanism of oxidative stress-induced BMECs injury and whether NAC has therapeutic potential in senile osteoporosis.

PINK1/Parkin-mediated mitophagy inhibits osteoblast apoptosis induced by advanced oxidation protein products.

Osteoblast apoptosis plays an important role in age-related bone loss and osteoporosis. Our previous study revealed that advanced oxidation protein products (AOPPs) could induce nicotinamide adenine dinucleotide phosphate oxidase (NOX)-derived reactive oxygen species (ROS) production, cause mitochondrial membrane potential (ΔΨm) depolarization, trigger the mitochondria-dependent intrinsic apoptosis pathway, and lead to osteoblast apoptosis and ultimately osteopenia and bone microstructural destruction. In this study, we found that AOPPs also induced mitochondrial ROS (mtROS) generation in osteoblastic MC3T3-E1 cells, which was closely related to NOX-derived ROS, and aggravated the oxidative stress condition, thereby further promoting apoptosis. Removing excessive ROS and damaged mitochondria is the key factor in reversing AOPP-induced apoptosis. Here, by in vitro studies, we showed that rapamycin further activated PINK1/Parkin-mediated mitophagy in AOPP-stimulated MC3T3-E1 cells and significantly alleviated AOPP-induced cell apoptosis by eliminating ROS and damaged mitochondria. Our in vivo studies revealed that PINK1/Parkin-mediated mitophagy could decrease the plasma AOPP concentration and inhibit AOPP-induced osteoblast apoptosis, thus ameliorating AOPP accumulation-related bone loss, bone microstructural destruction and bone mineral density (BMD) loss. Together, our study indicated that therapeutic strategies aimed at upregulating osteoblast mitophagy and preserving mitochondrial function might have potential for treating age-related osteoporosis.

[Establishment of a high-velocity fragment-induced penetrating liver injury model in landrace pigs].

OBJECTIVE: To establish a stable fragment-induced penetrating liver injury model in landrace pigs and evaluate the characteristics of deep tissue injury. METHODS: According to the different positioning methods of aiming points, twelve healthy adult landrace pigs were divided into group A (the relative height "h" of the aiming point and the highest point of the body surface on the tracing line was set to 5 cm) and group B ("h" was set to 6 cm). Ultrasonography was used to determine the direction of fragment projection, and an experimental ballistic gun was used to project high-velocity fragments to cause injury to animals. The vital signs of the two groups were monitored, and whole blood cell count, blood gas analysis, and liver and renal function were tested. Damages to the liver and adjacent organs, as well as the amount of bleeding and survival time were analyzed. RESULTS: For the overall analysis of the two groups, the liver hit rate of fragment simulating projectiles was 100% (right anterior lobe and right lateral lobe injury), the hit rate of other organs in the abdominal cavity was 25% (3/12), and the incidence of hemothorax or pneumothorax was 8% (1/12). The wounds were mainly characterized by liver lacerations, with total or partial disconnection of the distal liver lobe. There was no significant difference in wound length and bleeding amount between groups A and B [wound length (cm): 9.8±1.7 vs. 11.2±3.8, bleeding amount (g): 597.0±477.1 vs. 1 032.0±390.3, both P > 0.05]. The depth of liver parenchymal laceration in group B with the aiming point closer to the anterior median line was significantly longer than that in group A (cm: 2.8±0.4 vs. 1.9±0.6, P = 0.015). Mean arterial pressure (MAP), pH value, residual arterial blood base (BE), hemoglobin (Hb) and hematocrit (HCT) levels decreased after the fragment-induced injury, and then reached a trough level [MAP (mmHg, 1 mmHg ≈ 0.133 kPa): 87.0±33.6, pH: 7.26±0.15, BE (mmol/L): -6.65±8.48, Hb (g/L): 9.86±1.10, HCT: 0.309±0.029, all P < 0.05] in the first hour. Blood lactate (Lac), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) levels increased over time, and reached a peak level [Lac (mmol/L): 10.21±4.40, LDH (U/L): 1 417.0±223.3, AST (U/L): 234.5 (162.5, 357.5), both P < 0.05] at 1 hour after injury. Pearson's correlation analysis showed that the total amount of bleeding was correlated with the depth of liver parenchyma laceration (r = 0.684,P = 0.014). The Kaplan-Meier survival curve showed that the 3 hours survival rate in group A was higher than that in group B, but the difference was not statistically significant [83.3% (5/6) vs. 33.3% (2/6), P > 0.05]. CONCLUSIONS: The high-velocity fragment-induced penetrating liver injury model established by striking landrace pigs closer to the anterior median line with fragment simulating projectiles is reproducible and the degree of damage is controllable, and the model is applicable to further relevant research of hepatic ballistic trauma.

Accumulation of advanced oxidation protein products contributes to age-related impairment of gap junction intercellular communication in osteocytes of male mice.

AIMS: Gap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes' GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes' GJIC in aged male mice and its mechanism. METHODS: Changes in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks. RESULTS: The AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes' GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo. CONCLUSION: Our study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing. Cite this article: Bone Joint Res 2022;11(7):413-425.

The Diagnostic Value of Ultrasound-Based Deep Learning in Differentiating Parotid Gland Tumors.

Objectives: Evidence suggests that about 80% of all salivary gland tumors involve the parotid glands, with approximately 20% of parotid gland tumors (PGTs) being malignant. Discriminating benign and malignant parotid gland lesions preoperatively is vital for selecting the appropriate treatment strategy. This study explored the diagnostic performance of deep learning system for discriminating benign and malignant PGTs in ultrasonography images and compared it with radiologists. Methods. A total of 251 consecutive patients with surgical resection and proven parotid gland malignant or benign tumors who underwent preoperative ultrasound examinations were enrolled in this study between January 2014 and November 2020. Next, we compared the diagnostic accuracy of deep learning methods (ViT-B\16, EfficientNetB3, DenseNet121, and ResNet50) and radiologists in parotid gland tumor. In addition, the area under the curve (AUC), specificity, sensitivity, positive predictive value, and negative predictive value were calculated. Results: Among the 251 patients, 176/251 were the training set, whereas 75/251 were the validation set. Results showed that 74/251 patients had malignant tumor. Deep learning models achieved good performance in differentiating benign from malignant tumors, with the diagnostic accuracy and AUCs of ViT-B\16, EfficientNetB3, DenseNet121, and ResNet50 model being 81% and 0.81, 80% and 0.82, 77% and 0.81, and 79% and 0.80, respectively. On the other hand, the diagnostic accuracy and AUCs of radiologists were 77%-81% and 0.68-0.75, respectively. It was evident that the diagnostic accuracy of deep learning methods was higher than that of inexperienced radiologists, but there was no significant difference between deep learning methods and experienced radiologists. Conclusions: This study shows that the deep learning system can be used for diagnosing parotid tumors. The findings also suggest that the deep learning system may improve the diagnosis performance of inexperienced radiologists.

Ferroptosis-associated DNA methylation signature predicts overall survival in patients with head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a common cancer characterized by late diagnosis and poor prognosis. The aim of this study was to identify a novel ferroptosis-related DNA methylation signature as an alternative diagnosis index for patients with HNSCC. METHODS: Methylome and transcriptome data of 499 HNSCC patients, including 275 oral squamous cell carcinoma (OSCC) samples, were obtained from The Cancer Genome Atlas (TCGA). An additional independent methylation dataset of 50 OSCC patients from the NCBI Gene Expression Omnibus (GEO) database was used for validation. As an index of ferroptosis activity, the ferroptosis score (FS) of each patient was inferred from the transcriptome data using single-sample gene set enrichment analysis. Univariate, multivariate, and LASSO Cox regression analyses were used to select CpG sites for the construction of a ferroptosis-related DNA methylation signature for diagnosis of patients. RESULTS: We initially inferred the FS of each TCGA HNSCC patient and divided the samples into high- and low-FS subgroups. Results showed that the high-FS subgroup displayed poor overall survival. Moreover, 378 differentially methylated CpG sites (DMCs) were identified between the two HNSCC subgroups, with 16 selected to construct a 16-DNA methylation signature for risk prediction in HNSCC patients using the LASSO and multivariate Cox regression models. Relative operating characteristic (ROC) curve analysis showed great predictive efficiency for 1-, 3-, and 5-year HNSCC survival using the 16-DNA methylation signature. Its predictive efficiency was also observed in OSCC patients from the TCGA and GEO databases. In addition, we found that the signature was associated with the fractions of immune types in the tumor immune microenvironment (TIME), suggesting potential interactions between ferroptosis and TIME in HNSCC progression. CONCLUSIONS: We established a novel ferroptosis-related 16-DNA methylation signature that could be applied as an alternative tool to predict prognosis outcome in patients with HNSCC, including OSCC.

Age-related accumulation of advanced oxidation protein products promotes osteoclastogenesis through disruption of redox homeostasis.

Enhanced osteoclastogenesis is one of the major causes of age-related bone loss. Aging is accompanied by accumulation of advanced oxidation protein products (AOPPs). However, whether AOPPs accumulation contributing to the osteoclastogenesis with aging remains unclear. Here, we showed that AOPPs accumulation was associated with the enhanced osteoclastogenesis and deterioration of bone microstructure in aged mice. In vitro, AOPPs directly induced osteoclastogenesis by interaction with receptor activator of nuclear factor κ B (RANK) and the receptor for advanced glycation end products (RAGE) in the primary bone marrow monocytes. Bindings of AOPPs to RANK and RAGE were able to activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, trigger generation of reactive oxygen species, then induce phosphorylation of mitogen-activated protein kinases and c-fos, upregulation of the nuclear factor of activated T cell c1, eventually induce bone marrow monocytes to differentiate into mature osteoclasts. Chronic exposure to AOPPs enhanced osteoclastogenesis and bone loss in mice, which could be alleviated by NADPH oxidase inhibitor apocynin. Local injection of AOPPs into subperiosteal area induced bone resorption at the site of administration, which was similar to the effect of RANK ligand. Together, these results suggested that AOPPs could serve as a novel regulator of osteoclastogenesis and AOPPs accumulation might play an important role in the development of age-related bone loss.

Identification of a Ferroptosis Gene Set That Mediates the Prognosis of Squamous Cell Carcinoma of the Head and Neck.

Squamous cell carcinoma of the head and neck (HNSCC) is one of the six most common malignancies. HNSCC has both a high incidence and poor prognosis, and its prognostic factors remain unclear. Ferroptosis is a newly discovered form of programmed cell death that is iron-dependent. Increasing evidence indicates that targeting ferroptosis may present a new form of anti-tumor treatment. However, the prognostic value of ferroptosis-related genes (FRGs) in HNSCC is unclear. This study was designed to identify molecular markers associated with ferroptosis that influence prognosis in patients with HNSCC. We used HNSCC tumor and normal data from The Cancer Genome Atlas (TCGA) to identify prognosis-related FRGs. An FRG-based prognostic risk score was constructed, and its prognostic value for patients with HNSCC was evaluated using receiver operating characteristic curve (ROC) and nomogram analyses. The model was validated using the Gene Expression Omnibus (GEO) database. Univariate Cox regression analysis in patients with HNSCC revealed 11 FRGs that were significantly associated with overall survival (OS). We constructed a ferroptosis risk score model based on five genes and divided the patients into different risk groups based on its median value. Kaplan-Meier curve analysis showed that patients with a higher ferroptosis risk score had shorter OS (TCGA training set: P < 0.001, TCGA validation set: P < 0.05,GEO validation set: P < 0.001), and Gene Expression Profiling Interactive Analysis (GEPIA) further verified the relationships between these five genes and prognosis in patients with HNSCC. Multivariate Cox regression analysis showed that the risk score remained an independent predictor of OS after the exclusion of clinical confounders (HR > 1, P < 0.01). Significant differences in gene function enrichment analysis and immune cell infiltration status were identified between the two groups. The prognostic model can be used to predict the prognosis of patients with HNSCC. Moreover, the five FRGs may affect ferroptosis in HNSCC and thereby represent potential treatment targets. These results provide new directions for HNSCC treatment.

Identification and validation of an immune-related prognostic signature and key gene in papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common pathological type of thyroid cancer. The effect of traditional anti-tumor therapy is not ideal for the patients with recurrence, metastasis and radioiodine resistance. The abnormal expression of immune-related genes (IRGs) has critical roles in the etiology of PTC. However, the effect of IRGs on PTC prognosis remains unclear. METHODS: Based on The Cancer Genome Atlas (TCGA) and ImmPort databases, we integrated IRG expression profiles and progression-free intervals (PFIs) of PTC patients. First, we identified the differentially expressed IRGs and transcription factors (TFs) in PTC. Subsequently, an IRG model that can predict the PFI was constructed by using univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analyses of the differentially expressed IRGs in the TCGA. Additionally, a protein-protein interaction (PPI) network showed the interactions between the differentially expressed genes (DEGs), and the top 30 genes with the highest degree were extracted from the network. Then, the key IRG was identified by the intersection analysis of the PPI network and univariate Cox regression, which was verified the differential expression of by western blotting and immunohistochemistry (IHC). ssGSEA was performed to understand the correlation between the key IRG expression level and immune activity. RESULTS: A total of 355 differentially expressed IRGs and 43 differentially expressed TFs were identified in PTC patients. Then, eight IRGs were finally utilized to construct an IRG model. The respective areas under the curve (AUCs) of the IRG model reached 0.948, 0.820, and 0.831 at 1, 3 and 5 years in the training set. In addition, lactotransferrin (LTF) was determined as the key IRG related to prognosis. The expression level of LTF in tumor tissues was significantly lower than that in normal tissues. And the results of ssGSEA showed the expression level of LTF is closely related to immune activity. CONCLUSIONS: These findings show that the prognostic model and key IRG may become promising molecular markers for the prognosis of PTC patients.

The combination of contrast-enhanced ultrasonography with blue dye for sentinel lymph node detection in clinically negative node breast cancer.

PURPOSE: The aim of this prospective study was to evaluate the value of the combination of contrast-enhanced ultrasonography (CEUS) and blue dye (BD) for SLN detection in patients with clinically negative node breast cancer. METHODS: Patients with clinically negative node breast cancer were randomized into two cohorts for SLN biopsy (SLNB): the combination method cohort using CEUS and BD together, and the single BD method cohort. Standard axillary lymph node dissection was performed if any of the SLNs confirmed positive by pathology. The identification rate, the number of SLNs removed and recurrence-free survival (RFS) rates were evaluated between two cohorts. In addition, we assessed the sensitivity, specificity, accuracy, false-negative rate of CEUS for diagnosis of SLNs based on patterns of CEUS enhancement. RESULTS: 144 consecutive patients with clinically negative node breast cancer were randomized into two cohorts. Each cohort consisted of 72 cases. In the combination method cohort, contrast-enhanced lymphatic vessels were clearly visualized and SLNs were accurately localized in 72 cases. The identification rate and the mean number of SLNs detected by the combination method were 100% (72/72) and 3.26 (1-9), respectively. In contrast, in the single BD method cohort, SLNs in 69 cases were successfully identified. The identification rate and the mean number of SLNs using BD alone were 95.8% (69/72) and 2.21 (1-4), respectively. According to patterns of CEUS enhancement, the sensitivity, specificity, accuracy, and the FNR of CEUS for SLN diagnosis were 69.2%, 96.6%, 91.7%, and 30.8%, respectively. After a median follow-up of 50 months for the combination method cohort and 51 months for the blue dye alone cohort, five patients in the combination method cohort and nine in the blue dye alone cohort had recurrence. RFS rates showed no significant difference (P = 0.26) between two cohorts. CONCLUSION: The combination of CEUS and BD is more effective than BD alone for SLNB in clinically negative node patients with an identification rate as high as 100%. Use of BD and CEUS in combination may provide the possibility of a non-radioactive alternative method for SLNB in centers without access to radioisotope.

Identification of Genes Related to Immune Infiltration in the Tumor Microenvironment of Cutaneous Melanoma.

Cutaneous melanoma (CM) is the leading cause of skin cancer deaths and is typically diagnosed at an advanced stage, resulting in a poor prognosis. The tumor microenvironment (TME) plays a significant role in tumorigenesis and CM progression, but the dynamic regulation of immune and stromal components is not yet fully understood. In the present study, we quantified the ratio between immune and stromal components and the proportion of tumor-infiltrating immune cells (TICs), based on the ESTIMATE and CIBERSORT computational methods, in 471 cases of skin CM (SKCM) obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) were analyzed by univariate Cox regression analysis, least absolute shrinkage, and selection operator (LASSO) regression analysis, and multivariate Cox regression analysis to identify prognosis-related genes. The developed prognosis model contains ten genes, which are all vital for patient prognosis. The areas under the curve (AUC) values for the developed prognostic model at 1, 3, 5, and 10 years were 0.832, 0.831, 0.880, and 0.857 in the training dataset, respectively. The GSE54467 dataset was used as a validation set to determine the predictive ability of the prognostic signature. Protein-protein interaction (PPI) analysis and weighted gene co-expression network analysis (WGCNA) were used to verify "real" hub genes closely related to the TME. These hub genes were verified for differential expression by immunohistochemistry (IHC) analyses. In conclusion, this study might provide potential diagnostic and prognostic biomarkers for CM.

[Corrigendum] FNDC3B promotes epithelial­mesenchymal transition in tongue squamous cell carcinoma cells in a hypoxic microenvironment.

After the publication of the article, and also the publication of a Corrigendum (see doi: 10.3892/or.2020.7744), there are further errors in the published paper that the authors wish to correct in a subsequent corrigendum. In the printed version of Fig. 5, the "NC" images were mistakenly presented in the data panels showing the results of the TCA8113 and TSCCA invasion assay experiments. Furthermore, in Fig. 4A and 6A, the ß­actin control bands were erroneously selected for these figures. The corrected versions of Figs. 4, 5 and 6 are shown opposite and on the next page, incorporating the correct data for Figs. 4A, 5 and 6A. These further corrections do not affect the results and conclusions of this work. The authors all agree to this Corrigendum, and are grateful to the Editor of Oncology Reports for allowing them to have the opportunity to correct these additional errors. Lastly, the authors apologize to the readership for any inconvenience these errors may have caused. [the original article was published in Oncology Reports 39: 1853­1859, 2018; DOI: 10.3892/or.2018.6231].

Identification of an immune-related signature indicating the dedifferentiation of thyroid cells.

BACKGROUND: Immune cells account for a large proportion of the tumour microenvironment in anaplastic thyroid carcinomas (ATCs). However, the expression pattern of immune-related genes (IRGs) in ATCs is unclear. Our study aimed to identify an immune-related signature indicating the dedifferentiation of thyroid cells. METHODS: We compared the differences in thyroid differentiation score (TDS), infiltration of immune cells and enriched pathways between ATCs and papillary thyroid carcinomas (PTCs) or normal thyroid tissues in the Gene Expression Omnibus database. Univariate and multivariable Cox analyses were used to screen prognosis-associated IRGs in The Cancer Genome Atlas database. After constructing a risk score, we investigated its predictive value for differentiation and survival by applying receiver operating characteristic and Kaplan-Meier curves. We further explored its associations with important immune checkpoint molecules, infiltrating immune cells and response to immunotherapy. RESULTS: Compared with PTCs or normal thyroid tissues, ATCs exhibited lower TDS values and higher enrichment of immune cells and activation of the inflammatory response. The quantitative analyses and immunohistochemical staining validated that most ATC cell lines and ATC tissues had higher expression of MMP9 and lower expression of SDC2 than normal thyroid samples and PTC. Higher risk scores indicates dedifferentiation and a worse prognosis. Additionally, the risk score was positively correlated with the immune checkpoint molecules PDL1, CTLA4, IDO1, and HAVCR2 and infiltration of multiple immune cells. Importantly, we found that the samples with higher risk scores tended to have a better response to immunotherapy than those with lower scores. CONCLUSION: Our findings indicate that the risk score may not only contribute to the determination of differentiation and prognosis of thyroid carcinomas but also help the prediction of immune cells infiltration and immunotherapy response.