Clinical Genetic Features and Neoadjuvant Chemotherapy Response in HER2-Low Breast Cancers: A Retrospective, Multicenter Cohort Study.

BACKGROUND: The objective of this work is to reveal differences in clinical and genetic features, as well as neoadjuvant chemotherapy (NAC) response, between HER2-low and HER2-zero or HER2-positive breast cancers. PATIENTS AND METHODS: A total of 245 female patients with breast cancer were retrospectively enrolled from seven hospitals. Core needle biopsy (CNB) samples were collected before NAC and used for next-generation sequencing by a commercial gene panel. Clinical and genetic features, as well as NAC response, were compared between HER2-low and HER2-zero or HER2-positive breast cancers. The nonnegative matrix factorization (NMF) method was applied to cluster the C-Score of enrolled cases to reveal the intrinsic features of each HER2 subgroup. RESULTS: A total of 68 (27.8%) cases are HER2-positive, 117 (47.8%) cases are HER2-low, and 60 (24.5%) cases are HER2-zero. HER2-low breast cancers have a significantly lower pathologic complete response (pCR) rate than HER2-positive and HER2-zero breast cancers (p < 0.050 for all comparisons). Compared with HER2-low breast cancers, HER2-positive cases have higher rates of TP53 mutation, TOP2A amplification, and ERBB2 amplification, as well as lower rates of MAP2K4 mutation, ESR1 amplification, FGFR1 amplification, and MAPK pathway alteration (p < 0.050 for all comparisons). After clustering HER2-low cases by the NMF method, 56/117 (47.9%) are in cluster 1, 51/117 (43.6%) are in cluster 2, and 10/117 (8.5%) are in cluster 3. HER2-low cases in cluster 2 have the lowest pCR rate among the three clusters (p < 0.050). CONCLUSIONS: HER2-low breast cancers have significant genetic differences from HER2-positive cases. Genetic heterogeneity exists in HER2-low breast cancers and impacts on NAC response in this subgroup.

Novel Multifunctional Stimuli-Responsive Nanoparticles for Synergetic Chemo-Photothermal Therapy of Tumors.

In this study, a novel class of multifunctional responsive nanoparticles is designed and fabricated as drug nanocarriers for synergetic chemo-photothermal therapy of tumors. The proposed nanoparticles are composed of a thermo-/pH-responsive poly(N-isopropylacrylamide-co-acrylic acid) (PNA) nanogel core, a polydopamine (PDA) layer for photothermal conversion, and an outer folic acid (FA) layer as a targeting agent for the folate receptors on tumor cells. The fabricated nanoparticles show good biocompatibility and outstanding photothermal conversion efficiency. The proposed nanoparticles loaded with doxorubicin (DOX) drug molecules are stable under physiological conditions with low leakage of drugs, while rapidly release drugs in environments with low pH conditions and at high temperature. The experimental results show that the drug release process is mainly governed by Fickian diffusion. In vitro cell experimental results demonstrate that the PNA-DOX@PDA-FA nanoparticles can be phagocytized by 4T1 tumor cells and release drugs in tumor cell acidic environments, and confirm that the combined chemo and photothermal therapeutic efficacy of PNA-DOX@PDA-FA nanoparticles is higher than the photothermal therapeutic efficacy or the chemotherapeutic efficacy alone. The proposed multifunctional responsive nanoparticles in this study provide a novel class of drug nanocarriers as a promising tool for synergetic chemo-photothermal therapy of tumors.

Adalimumab ameliorates memory impairments and neuroinflammation in chronic cerebral hypoperfusion rats.

Vascular dementia (VaD) is the second most common type of dementia worldwide. Although there are five FDA-approved drugs for the treatment of Alzheimer's disease (AD), none of them have been applied to treat VaD. Adalimumab is a TNF-α inhibitor that is used for the treatment of autoimmune diseases such as rheumatoid arthritis. In a recent retrospective case-control study, the application of adalimumab for rheumatoid or psoriasis was shown to decrease the risk of AD. However, whether adalimumab can be used for the treatment of VaD is not clear. In this study, we used 2VO surgery to generate a VaD rat model and treated the rats with adalimumab or vehicle. We demonstrated that VaD rats treated with adalimumab exhibited significant improvements in memory. In addition, adalimumab treatment significantly alleviated neuronal loss in the hippocampi of VaD rats. Moreover, adalimumab significantly reduced microglial activation and reversed M1/M2 polarization in VaD rats. Furthermore, adalimumab treatment suppressed the activity of NF-κB, an important neuroinflammatory transcription factor. Finally, adalimumab displayed a protective role against oxidative stress in VaD rats. Our results indicate that adalimumab may be applied for the treatment of human patients with VaD.

TRAF4, a new substrate of SIAH1, participates in chemotherapy resistance of breast cancer cell by counteracting SIAH1-mediated downregulation of ß-catenin.

PURPOSE: TRAF4 plays an important role in the development and progression of breast cancer, but its impact on chemotherapy resistance is as yet, however, poorly understood. METHODS: Western blotting, immunoprecipitation, and immunofluorescence staining were used to identify and verify that TRAF4 was a novel substrate of SIAH1 and prevented SIAH1-mediated ß-catenin degradation. Cell proliferation analysis and Flow cytometry analysis were utilized to detect TRAF4's function on the growth-inhibitory effect of etoposide. Immunohistochemistry was used to detect the expression of TRAF4, SIAH1, and ß-catenin. Statistical analysis was used to analyze the relationships between them with clinical parameters and curative effect of chemotherapy pathologically. RESULTS: Our results suggested that TRAF4 prevents SIAH1-mediated ß-catenin degradation. TRAF4 was a novel substrate of SIAH1 and the TRAF domain of TRAF4 was critical for binding to SIAH1. TRAF4 reduced the growth-inhibitory effect of etoposide via reducing the number of S-phase cells and suppressing cell apoptosis. Concordantly, we found that breast cancer patients with a low-TRAF4 expression benefited most from chemotherapy, who had higher tumor volume reduction rate and better pathological response, while, the high-TRAF4 expression group had lower tumor volume reduction rate and poor pathological response. CONCLUSIONS: TRAF4 was a novel substrate of SIAH1 and prevented SIAH1-mediated ß-catenin degradation, which explains the protective effect of TRAF4 on ß-catenin during cell stress and links TRAF4 to chemotherapy resistance in tumors. These findings implicated a novel pathway for the oncogenic function of TRAF4.

Exosomes Derived From MicroRNA-148b-3p-Overexpressing Human Umbilical Cord Mesenchymal Stem Cells Restrain Breast Cancer Progression.

Exosomes derived from human umbilical cord mesenchymal stem cells (HUCMSCs) expressing microRNAs (miRs) have been highlighted as important carriers for gene or drug therapy. Hence, this study aimed to explore the role of exosomal miR-148b-3p from HUCMSCs in breast cancer. Clinical samples subjected to RT-qPCR detection revealed that miR-148b-3p was poorly expressed, while tripartite motif 59 (TRIM59) was highly expressed in breast cancer tissues. Online analyses available at miRanda, TargetScan, and miRbase databases revealed that miR-148b-3p could bind to TRIM59, while dual-luciferase reporter gene assay further verified that TRIM59 was a target gene of miR-148b-3p. Next, miR-148b-3p mimic or inhibitor and siRNA against TRIM59 were delivered into the breast cancer cells (MDA-MB-231) to alter the expression of miR-148b-3p and TRIM59 so as to evaluate their respective effects on breast cancer cellular processes. Evidence was obtained demonstrating that miR-148b-3p inhibited cell proliferation, invasion, and migration, but promoted cell apoptosis in breast cancer by down-regulating TRIM59. Next, MDA-MB-231 cells were co-cultured with the exosomes derived from HUCMSCs expressing miR-148b-3p. The results of co-culture experiments demonstrated that HUCMSCs-derived exosomes carrying miR-148b-3p exerted inhibitory effects on MDA-MB-231 progression in vitro. In vivo experimentation further confirmed the anti-tumor effects of HUCMSCs-derived exosomes carrying miR-148b-3p. Taken together, HUCMSC-derived exosomes carrying miR-148b-3p might suppress breast cancer progression, which highlights the potential of exosomes containing miR-148b-3p as a promising therapeutic approach for breast cancer treatment.

Evaluation of Cardiovascular Risk Factors and Restless Legs Syndrome in Women and Men: A Preliminary Population-Based Study in China.

STUDY OBJECTIVES: Many studies have investigated the association between restless legs syndrome (RLS) and cardiovascular risk factors, leading to conflicting results. Therefore, the aim of the current study was to determine whether RLS is associated with cardiovascular risk factors and disease. METHODS: This cross-sectional study included 5,324 consecutive subjects who visited the Physical Examination Center of The First Affiliated Hospital of Zhengzhou University for their yearly routine physical examination. Participants underwent a face-to-face interview with a neurologist for the assessment of RLS, based on the International Restless Legs Study Group criteria. They also completed a questionnaire related to cardiovascular risk factors and other health-related and demographic information. Logistic regression was used to assess which of the demographic and cardiovascular risk factors increased the odds of RLS. Then, unadjusted and adjusted models were designed to determine whether RLS was associated with increased odds of cardiovascular disease, coronary artery disease, or hypertension. RESULTS: RLS was observed in 9.2% of the participants. Multivariable logistic regression models, which included the covariates age, sex, body mass index, smoking status, hypercholesterolemia, and Pittsburgh Sleep Quality Index score (dichotomized at 5), demonstrated that female sex (odds ratio [OR]: 2.42, 95% confidence interval [CI]: 1.99-2.95), smoking (OR: 1.96, 95% CI: 1.31-2.92), high cholesterol (OR: 1.30, 95% CI: 1.03-1.64), and PSQI score > 5 (OR: 5.61, 95% CI: 2.14-14.69) are significantly associated with RLS. Additionally, RLS was associated with hypertension, after adjusting for age, sex, body mass index, smoking, hypercholesterolemia, Pittsburgh Sleep Quality Index score > 5, diabetes, anemia, and decreased renal function. CONCLUSIONS: RLS is associated with the prevalence of hypertension but not with that of cardiovascular disease or coronary artery disease.

MicroRNA-16-5p overexpression suppresses proliferation and invasion as well as triggers apoptosis by targeting VEGFA expression in breast carcinoma.

MicroRNAs (miRNAs), a class of small noncoding RNA molecules, can manipulate the expressions of endogenous tumor-related genes, and are implicated in the development and progression of a wide type of tumors. In this study, the investigation from real-time quantitative PCR revealed that miRNA-16-5p was downregulated in breast carcinoma tissues and cells, coupled with the elevations of HIF-α and VEGFA protein expressions, compared with normal tissues. Lentiviral armed with miR-16-5p markedly increased the miR-16-5p levels in MCF-7 and MDA-MB-231 cells, compared to blank and NC groups, and miR-16-5p overexpression significantly inhibited the proliferation and colony formation in MCF-7 and MDA-MB-231 cells. Besides, miR-16-5p upregulation markedly induced apoptosis and reduced invasion ability in MCF-7 and MDA-MB-231 cells. Notably, VEGFA was direct target of miR-16-5p. Stepwise investigation from in vitro and in vivo experiments demonstrated that miR-16-5p overexpression suppressed tumor growth and reduced HIF-α and VEGFA expressions in breast carcinoma cells and nude mice tumor tissues. These findings provide novel insights into molecular mechanism involved in the roles of miR-16-5p in tumor development and progression of breast carcinoma, and thus manipulation of miR-16-5p may be a novel potential therapeutic target for future therapies of the patients with breast carcinoma.

Over-expression of microRNA-940 promotes cell proliferation by targeting GSK3ß and sFRP1 in human pancreatic carcinoma.

Increasing study reports that Wnt/ß-catenin signaling pathway plays an essential role in numerous cancers growth, progression and metastasis. Aberrant miR-940 expression has been studied in gastric and breast cancer. However, the molecular mechanism of miR-940 enhancing proliferation and metastatic ability in human pancreatic carcinoma is far from to know. Real-time PCR was used to quantify miR-940 expression. Luciferase reporter assays here were performed to verify the activity of Wnt/ß-catenin signaling pathway and targeting gene relationships, and immunofluorescence assay was applied to observe ß-catenin expressed intensity. Bioinformatics analysis together with in vivo and vitro functional analysis indicated the potential targeting genes of miR-940. Specimens from 15 pairs of patients with human pancreatic carcinoma were involoved to confirm the relationship between miR-940 expression and the GSK3ß/sFRP1 through real-time PCR and western blot assays. Bioinformatics combined with cell luciferase function researches determined the possible regulation of miR-940 on the 3'-UTR of the GSK3ß and sFRP1 genes, resulting in the Wnt/ß-catenin signaling activation. Further, miR-940 knockdown significantly recovered GSK3ß and sFRP1 expression and relieved Wnt/ß-catenin-mediated cell invasion, migration, metastasis and proliferation. The ectopic up-regulation of miR-940 significantly suppressed GSK3ß/sFRP1 expression and promoted pancreatic carcinoma proliferation and invasion. Our study suggested mechanistic relationship between miR-940 and Wnt/ß-catenin in the development and progression of pancreatic carcinoma through regulation of GSK3ß and sFRP1.

The effect of quercetin nanoparticle on cervical cancer progression by inducing apoptosis, autophagy and anti-proliferation via JAK2 suppression.

Cervical cancer is a cause of cancer death, making it as the one of the most common cause for death among women globally. Though many studies before have explored a lot for cervical cancer prevention and treatment, there are still a lot far from to know based on the molecular mechanisms. Janus kinase 2 (JAK2) has been reported to play an essential role in the progression of apoptosis, autophagy and proliferation for cells. We loaded gold-quercetin into poly (dl-lactide-co-glycolide) nanoparticles to cervical cancer cells due to the propertities of quercetin in ameliorating cellular processes and the easier absorbance of nanoparticles. Here, in our study, quercetin nanoparticles (NQ) were administrated to cells to investigate the underlying mechanism by which the cervical cancer was regulated. First, JAK2-inhibited carvical cancer cell lines were involved for our experiments in vitro and in vivo. Western blotting, quantitative RT-PCR (qRT-PCR), ELISA, Immunohistochemistry, and flow-cytometric analysis were used to determine the key signaling pathway regulated by JAK2 for cervical cancer progression. And the role of quercetin nanoparticles was determined during the process. Data here indicated that JAK2, indeed, expressed highly in cancer cell lines compared to the normal cervical cells. And apoptosis and autophagy were found in JAK2-inhibited cancer cells through activating Caspase-3, and suppressing Cyclin-D1 and mTOR regulated by Signal Transducer and Activator of Transcription (STAT) 3/5 and phosphatidylinositide 3-kinase/protein kinases (PI3K/AKT) signaling pathway. The cervical cancer cells proliferation was inhibited. Further, tumor size and weight were reduced by inhibition of JAK2 in vivo experiments. Notably, administration with quercetin nanoparticles displayed similar role with JAK2 suppression, which could inhibit cervical cancer cells proliferation, invasion and migration. In addition, autophogy and apoptosis were induced, promoting cervical cancer cell death. To our knowledge, it was the first time to evaluate the role of quercetin nanoparticles in improving cervical cancer from apoptosis, autophagy and proliferation, which could be a potential target for future therapeutic approach clinically.

Expression Profile of MiR-128 in the Astrocytoma Patients and Cell Lines.

Malignant astrocytomas are the most common primary brain tumors. The critical characterizes of astrocyomas are their aggressive and infiltrative in the brain, which leads to uncontrollable by conventional forms of therapy. MicroRNAs are small RNAs that had been found to regulate their targets by specific binding to the 3'-untranslated region (3'UTR) of mRNA. Recent advances in understanding the molecular biology of these tumors have revealed that microRNA (miRNA) disruption may play important roles in the pathogenesis of astrocytomas. And some of the miRNA alterations were found in the serum of astrocytoma patients. In this study, we studied the expression profile of miR-128, in the different stages of astrocytoma tissues and two human astrocytoma cell lines, A172 and T98G cells. We found that the levels of miR-128 are decreased in the A172 and T98G cells when compared to normal human astrocyte (NHA). Furthermore, the levels of miR-128 decreased gradually to the pathological stages of astrocytomas. We also identified that TROVE2 is a novel target of miR-128 by the luciferase reporter system. Furthermore, the expression levels of TROVE2 are dramatically increased with the pathological stages increasing. Finally, the levels of TROVE2 are negatively correlated with miR-128 in astrocytoma tissues. Our data provided novel evidence for the miR-128 and TROVE2 in the development of human astrocytomas.

Overexpression of AIB1 correlates inversely with E-cadherin expression in pancreatic adenocarcinoma and may promote lymph node metastasis.

BACKGROUND: It was reported that the nuclear receptor coactivator amplified in breast cancer1 (AIB1) could regulate cancer cell invasion and migration in a nuclear receptor signaling-independent manner. Meanwhile, the process of epithelial mesenchymal transition (EMT) is critical for tumor invasion and metastasis. The present study aimed to determine the role of AIB1 and EMT markers in human pancreatic adenocarcinoma. METHODS: AIB1, ZO-1, E-cadherin, vimentin, and N-cadherin protein expression in 76 pancreatic adenocarcinomas were assessed using immunohistochemistry and analyzed for clinicopathological significance. RESULTS: The frequency of AIB1 overexpression in pancreatic adenocarcinomas with lymph node metastasis is 68 % (19/28), which is significantly higher than in pancreatic adenocarcinomas without lymph node metastasis (42 %; 20/48). In addition, the frequency of low expression of E-cadherin in pancreatic carcinomas with lymph node metastasis (68 %; 19/28) was significantly higher than in tumors without lymph node metastasis (44 %; 21/48). Correlation analysis demonstrated that the overexpression of AIB1 was inversely correlated with low expression of E-cadherin in pancreatic adenocarcinomas. CONCLUSION: Overexpression of AIB1 might promote invasion and metastasis of cancer cells and is associated with down-regulation of E-cadherin in pancreatic adenocarcinomas.

[Hydroa vacciniforme-like lymphoma: two cases report and literatures review].

OBJECTIVE: Two children with hydroa vacciniforme-like lymphoma (HVLL) were reported for a better understanding of this disease. METHODS: The clinical manifestation, pathological characteristics, therapeutic outcomes of two children with HVLL were analyzed and presented by compared with what described in literatures. RESULTS: Two children were girls, who treated firstly in the hospital in May 2012, July 2012 and their duration were 1 years, more than 10 years respectively. Their clinical manifestations were both limbs and craniofacial polymorphous rashes. Pathological findings revealed that the dermis and subcutaneous tissue were profiled by atypical lymphocytic infiltration. Immunohistochemistry showed that the infiltration of cells from T/NK cell, and Epstein-Barr virus encoded small RNA (EBER)(+). Case 1 was treated with chemotherapy, but her condition continued to deteriorate. Case 2 just received symptomatic treatment, her skin lesions gradually reduced and rash disappeared completely 2 months later. CONCLUSION: HVLL is found with special clinical manifestation, its diagnosis mainly depend on skin biopsy and immunohistochemistry, there is no specific treatment method now, and its prognosis still needs further research.

Overexpression of phosphatase regenerating liver 3 in oesophageal squamous cell carcinoma associated with metastasis and its comparison with phosphatase regenerating liver 1.

Expression of PRL3 (phosphatase of regenerating liver 3) protein was examined with immunohistochemistry in 60 cases of ESCC (oesophageal squamous cell carcinoma) with matched lymph node metastasis (n  =  40) and 6 cases of oesophageal adenocarcinoma. Its associations with PRL1 and clinicopathological parameters were analysed. The results showed the frequency of PRL3 protein expression was significantly higher in ESCC (39/60, 65%) than in normal oesophageal mucosa (0/20, P < 0.001); higher in ESCC with lymph node metastasis (30/40, 75%) than in ESCC without lymph node metastasis (9/20, P = 0.022), as well as higher in metastatic ESCC in lymph node (38/40, 95%) than in the primary ESCC (39/60, 65%, P < 0.001). PRL3 was expressed in 1 out of 6 oesophageal adenocarcinomas, but showed no nuclear staining of PRL1. Expression of PRL3 protein was positively associated with the grade and partially with the stage of ESCC. These results suggest that expression of PRL3 protein may be involved in the metastasis of ESCC and serve as a biomarker for prediction of ESCC metastasis.

Knockdown of the Bmi-1 oncogene inhibits cell proliferation and induces cell apoptosis and is involved in the decrease of Akt phosphorylation in the human breast carcinoma cell line MCF-7.

It is well documented that B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), widely overexpressed in the vast majority of malignancies, plays an essential role in the occurrence and development of several different tumors. Here, we report Bmi-1 siRNA-mediated cell proliferation inhibition and cell apoptosis in vitro and in vivo in the human breast carcinoma cell line MCF-7. Our results demonstrated that Bmi-1 siRNA effectively down-regulated the expression of Bmi-1, inhibited cell proliferation in vitro and in vivo, evoked cell cycle arrest in the G0/G1 phase and induced cell apoptosis in MCF-7 cells, coupled with decrease in cyclin D1, cyclin E, cdk2, bcl-2 and Ki-67 expression and Akt phosphorylation levels and an increase of p21 and bax expression and activities of caspase-3/-9. Taken together, our results suggest that Bmi-1 may be a potential molecular target for the therapy of breast carcinoma.

[Effect of nanosize delivery system for ASODN against hTERT on the expression of telomerase in the esophageal cancer EC9706 cells].

OBJECTIVE: To investigate the inhibitory effect of nanoparticle-mediated antisense oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) on telomerase in the esophageal cancer EC9706 cells. METHODS: Line-polyethylenimine (L-PEI) was used to condense ASODN into nanoparticle and to couple NGR peptides into targeting nanoparticle, and the prepared L-PEI/ASODN complexes were transfected into the EC9706 cells. Cellular uptake of L-PEI/ASODN complexes was detected by laser confocal scanning microscopy. MTT assay was used to detect the inhibitory rate of EC9706 cell growth. The level of hTERT mRNA and its protein expression were measured by RT-PCR and immunohistochemistry, respectively. Annexin V FITC/PI double labeling was used to detect cell apoptosis. The distribution of drug in nude mice was observed by laser confocal scanning microscopy, and the growth and morphology of the tumor was examined. RESULTS: The L-PEI-mediated ASODN uptake was enhanced. After transfection, the inhibitory rate of EC9706 cells was time-dependant and there was a significant difference between control cell group and L-PEI/ASODN group (P < 0.05). At 48 h after transfection, the level of hTERT mRNA was decreased significantly compared with that of control cell group (P < 0.05), and the expression of hTERT protein was negative. There was apparent apoptosis in EC9706 cells after transfection with L-PEI/ASODN complexes. For the two NGR/L-PEI/ASODN groups, fluorescence was observed in the liver, kidney, lung and tumor tissues of nude mice, and their uptake intensity was time-dependent. The mean volume of tumors in the two NGR/L-PEI/ASODN groups was significantly smaller than those in blank control group and SODN group (P < 0.05). Apoptotic bodies were detected in the tumors of L-PEI/ASODN group. CONCLUSION: The NGR/L-PEI/ASODN nanoparticles can effectively reach into the human esophageal cancer xenograft and inhibit the tumor growth in nude mice, and this may provide a theoretical and experimental basis for gene therapy for human esophageal squamous cell carcinoma.

Expression of phosphatase of regenerating liver 1 and 3 mRNA in esophageal squamous cell carcinoma.

CONTEXT: Phosphatase of regenerating liver (PRL) 3 messenger RNA (mRNA) was reported to express in human colorectal, gastric, ovarian, breast, and hepatic cancers. OBJECTIVE: To examine the expression of PRL-1 and PRL-3 mRNAs in human esophageal squamous cell carcinoma (ESCC). DESIGN: Expression of PRL-1 and PRL-3 mRNA was examined with reverse transcriptase-polymerase chain reaction in fresh tissue collected from 40 cases of ESCC with matched lymph node metastasis in 21 cases. The association of expression of PRL-1 and PRL-3 mRNAs with clinicopathologic parameters was analyzed. RESULTS: The frequencies of PRL-1 and PRL-3 mRNA expression were significantly higher in ESCC than in normal esophageal tissue (P = .001; P = .01) and also significantly higher in ESCC with lymph node metastasis than in those without lymph node metastasis (P = .01; P = .03). The levels of PRL-1 and PRL-3 mRNA expression were significantly higher in ESCC with lymph node metastasis than in those without lymph node metastasis (P = .04; P = .04). The frequencies and levels of PRL-1 and PRL-3 mRNA expression were correlated with the later stages but not with tumor differentiation, tumor location in the esophagus, patient's sex, and age. CONCLUSIONS: PRL-1 and PRL-3 mRNAs may be involved in and used to predict the metastasis of ESCC. The possibility of using PRL-1 and PRL-3 as the therapeutical target is also discussed.