[Clinicopathological features of olfactory carcinoma].

Objective: To investigate the clinicopathological features and differential diagnosis of olfactory carcinoma (OC). Methods: Twenty-one cases of sinonasal tumors, including those initially diagnosed as olfactory neuroblastoma (ONB) and those with uncertain diagnosis, were collected from the Department of Pathology, the First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) from January 2016 to August 2022, among which 3 cases were reclassified as OC. The clinicopathological features were investigated, and the remaining 18 cases were used as control. Results: Of the three OC patients, 2 were male and 1 was female, with an average age of 57 years ranging from 35 to 74 years. Microscopically, the tumor cells were arranged in solid, nested or lobulated patterns with occasional palisading around the solid nests. The stroma was highly vascular with focal neurofibrillary areas. There were prominent rosettes or pseudorosettes formation. The tumor cells were mainly ovoid to spindly with scant to moderate amount of cytoplasm, one or several small nucleoli, and fine chromatin content. Brisk mitotic figures were seen. In all 3 cases of OC, there were scanty atypical glands and some were ciliated. Immunohistochemically, at least one epithelial marker and neuroendocrine marker were diffusely expressed in the tumor. Some of the tumor cells were positive for p40 and p63, and the sustentacular cells showed the expression of S-100 protein. All cases tested were negative for NUT, CD99 and desmin, with intact expression of SMARCA4 (BRG1) and SMARCB1 (INI-1). Ki-67 proliferation index varied from 20% to 80%. Follow-up after 16-18 months showed no mortality with tumor recurrence from 1 patient after 16 months. Conclusion: OC is a rare sinonasal tumor with neuroepithelial differentiation, its histomorphology is diverse, and the combination of immunohistochemical markers is essential for appropriate diagnosis.

[Precise minimally invasive treatment of mandibular fracture in children assisted by digital surgery technology].

Objective: To explore the role of digital three-dimensional printing technology and multifunctional board in the treatment of mandibular fracture in children. Methods: From January 2006 to January 2022, 42 children with mandibular fracture were treated by Department of Stomatology, in The First Affiliated Hospital of Bengbu Medical College, including 25 males and 17 females. The patients, aged from 4 to 12 years, with the median age was 10 years old, were divided into observation group (22 cases) and routine group (20 cases) according to the treatment methods. In the observation group, the multifunctional board was made before operation, and the CT data of the children were imported into Mimics software in".dicom"format, and the displaced mandible was virtually reset. The jaw reduction model was made by three-dimensional printing, and the surgical operation was simulated on the reduction model to determine the model and position of the internal fixation device and shape it. During the operation, the fracture was reduced and fixed according to the preoperative design; The conventional group was treated with open reduction and internal fixation of mandibular fracture by traditional methods, and the clinical application value was compared and analyzed through the intraoperative situation, occlusal relationship, and follow-up of the two groups. Results: The total intraoperative bleeding volume [(30.25±4.02) ml] and surgical time [(64.3±9.2) min] in the observation group were significantly lower than those in the conventional group [(35.13±5.69) ml and (84.6±13.9) min, respectively] (F=6.18, P=0.003; F=1.32, P=0.001). The excellent and good rate of occlusal relationship in the observation group [96% (21/22)] was significantly higher than that in the conventional group [85% (17/20)] (F=4.27, P=0.039). The incidence of complications, the observation group, 1 case of poor occlusion, 1 case of postoperative infection; In the routine group, there were 3 cases with poor occlusion, 1 case with nerve injury, 1 case with root injury and 1 case with tooth germ injury. Conclusions: The application of digitization three-dimensional printing technology combined with multifunctional occlusal plate in children's mandibular fracture is minimally invasive, safe, efficient and accurate, and the clinical effect is good.

[Prickle planar cell polarity protein 1 involved in the pathogenesis of skeletal class â ¢ malocclusion].

Objective: To explore the mechanisms of prickle planar cell polarity protein 1 (PRICKLE1) involved in the occurrence of skeletal Class Ⅲ malocclusion. Methods: After extracting the genomic DNA of all family members of the skeletal Class Ⅲ malocclusion pedigree with maxillary hypoplasia collected in the Department of Orthodontics at the Affiliated Stomatological Hospital of Nanjing Medical University in October 2021, whole exome sequencing and Sanger sequencing were performed to screen pathogenic genes/mutation sites and validate the mutations. Jaw tissue was collected during the operation of orthognathic patients who were treated in the Department of Oral and Maxillofacial Surgery at the same hospital from October 2021 to December 2022. Following the extraction of human jaw bone marrow mesenchymal stem cells and transfection with overexpressing lentivirus (lentiviruses overexpressing the gene of interest served as the wild group, lentiviruses overexpressing mutation site served as the mutant group) and knockdown lentivirus (divided into knockdown group 1 and 2, with transfection interference negative lentiviruses as the control group). Various assays including real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, proliferation and Transwell assays, alkaline phosphatase staining and alizarin red staining were performed. Construction of zebrafish animal model, morpholino oligonucleotide (MO) were injected to knock down the expression of prickle1a and prickle1b in zebrafish (co-knocking group), and the control group was injected with standardized MO as a reference. Transcriptome sequencing, enrichment analysis and co-expression analysis were performed on the zebrafish craniofacial tissues of the two groups. Results: Two patients of this family carried this mutation PRICKLE1 c.113C>T. The transfection experiments showed that compared with the wild group (relative expression of PRICKLE1 was 21.97±0.60), the relative expression of mutant group (5.05±0.05) was significantly reduced (P<0.05), and cell proliferation and migration ability significantly enhanced (P<0.05), and osteogenic differentiation ability was significantly reduced (P<0.05). Compared with the control group, the proliferation and migration ability of cells in the two knockdown groups were significantly enhanced (P<0.05), and the osteogenic differentiation ability was significantly reduced (P<0.05). Zebrafish model experiments showed the width of the ethmoid plate was significantly reduced in the co-knocking group (282.50±61.77, t=5.29, P<0.001) compared with the control group (338.80±24.92). Transcriptome data and enrichment analysis showed that the differentially expressed genes were significantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway after the simultaneous knockdown of prickle1a and prickle1b in zebrafish. Conclusions: PRICKLE1 c.113C>T mutation might suppress the osteoblastic differentiation ability of jaw bone marrow mesenchymal stem cells by downregulating the MAPK signaling pathway, thereby involving the development of skeletal Class Ⅲ malocclusion.

MiR-376b-3p functions as a tumor suppressor by targeting KLF15 in non-small cell lung cancer.

OBJECTIVE: The aim of this study was to explore the regulatory effects of micro ribonucleic acid (miR)-376b-3p on proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells by targeting Kruppel-like factor 15 (KLF15) and its mechanism of action. PATIENTS AND METHODS: The expression of miR-376b-3p in NSCLC and para-carcinoma normal tissues, as well as NSCLC cell lines, was detected via quantitative Polymerase Chain Reaction (qPCR). The effects of miR-548-3p on the proliferation, cycle distribution, and apoptosis of NSCLC cells were detected via colony formation assay, flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. The interaction between miR-376b-3p and KLF15 was determined using Dual-Luciferase reporter gene assay. In vivo tumorigenic ability of NSCLC cells was studied using nude mouse tumorigenicity assay. Furthermore, the expression of Ki67 in tumor in nude mice was detected via immunohistochemistry. RESULTS: The expression of miR-376b-3p was significantly downregulated in NSCLC tissues when compared with para-carcinoma normal tissues (p<0.05). MiR-376b-3p was lowly expressed in NSCLC cells as well (p<0.05). After overexpression of miR-376b-3p, the proliferation ability of NSCLC cells remarkably declined (p<0.05). The apoptosis rate rose, and cell cycle was arrested in the G1/G0 phase. Dual-Luciferase reporter gene assay confirmed that miR-376b-3p could specifically bind to KLF15 3'UTR to regulate the expression activity of KLF15. After overexpression of miR-376b-3p, tumor volume and weight were significantly reduced in tumor-bearing mice (p<0.05). CONCLUSIONS: MiR-376b-3p plays an important role in the occurrence and development of NSCLC, which affects the proliferation and apoptosis of NSCLC cells by targeting KLF15.

Factor structure of 12 items in the Chinese Health Questionnaire among the elderly population in mainland China.

OBJECTIVES: The 12-item Chinese Health Questionnaire (CHQ-12) has been widely used for screening mental disorders. This study aims to examine the internal consistency and factor structure of CHQ-12 and its suitability for use in the elderly Chinese population. STUDY DESIGN: This is a cross-sectional study. METHODS: A total of 8526 elderly people aged ≥60 years from 11 cities in Shanxi Province were selected for participation in this study by stratified random cluster sampling. Cronbach's alpha was employed to assess internal consistency. An exploratory factor analysis (EFA) was performed to explore the underlying factor structure of the CHQ-12 in the elderly. A confirmatory factor analysis (CFA) was then conducted to test and compare the goodness-of-fit between possible factor structure obtained from the EFA and the unidimensional structure, which was originally recommended. RESULTS: The Cronbach's alpha for CHQ-12 was 0.838. The EFA extracted three factors, which explained 55.985% of the total variance of the data. The CFA of the three-factor model resulted in an acceptable model fit (Comparative Fit Index = 0.98, Tucker-Lewis Index = 0.97, Normed Fit Index = 0.98, Expected Cross-Validation Index = 0.28, Root-Mean-Square Error of Approximation = 0.071). The item loadings ranged from 0.58 to 0.82. Correlation coefficients among the three factors ranged from 0.40 to 0.75. CONCLUSIONS: The CHQ-12 presented satisfactory internal consistency and structural validity in the Chinese elderly population. The CFA of the three-factor structure expressed a preferred model fit in comparison to the unidimensional model. The three-factor structure of the CHQ-12 interpreted three different aspects of mental health: somatic symptoms, anxiety and worry, and depression/poor family relationship.

SPARCL1 suppresses osteosarcoma metastasis and recruits macrophages by activation of canonical WNT/ß-catenin signaling through stabilization of the WNT-receptor complex.

Metastasis significantly reduces the survival rate of osteosarcoma (OS) patients. Therefore, identification of novel targets remains extremely important to prevent metastasis and treat OS. In this report, we show that SPARCL1 is downregulated in OS by epigenetic methylation of promoter DNA. In vitro and in vivo experiments revealed that SPARCL1 inhibits OS metastasis. We further demonstrated that SPARCL1-activated WNT/ß-catenin signaling by physical interaction with various frizzled receptors and lipoprotein receptor-related protein 5/6, leading to WNT-receptor complex stabilization. Activation of WNT/ß-catenin signaling contributes to the SPARCL1-mediated inhibitory effects on OS metastasis. Furthermore, we uncovered a paracrine effect of SPARCL1 on macrophage recruitment through activated WNT/ß-catenin signaling-mediated secretion of chemokine ligand5 from OS cells. These findings suggest that the targeting of SPARCL1 as a new anti-metastatic strategy for OS patients.

[Exploration of the rational therapeutic regimen for advanced lung cancer patients with mild tumor enlargement].

Objective: To investigate the factors that impacts of therapeutic effect in advanced non-small cell lung cancer (NSCLC) patients with mild tumor enlargement and the rational therapeutic strategy for them. Methods: The clinicopathological features and prognostic data of advanced NSCLC patients whose sum of tumor longest diameters with 0 to 20% increase were retrospectively explored, and the Cox proportional hazards model was used to analyze the independent prognostic factors in patients. Results: The median progression-free survival (PFS) of 54 patients with the original regimen was 87 days, significantly less than 168 days of the median PFS of 49 patients with replacing regimen (P<0.001). The median PFS of other chemotherapeutic regiems (154 days) and the targeted therapy (287 days) were longer than the origional therapy (P<0.05 for all). The left 7 patients received radiotherapy. Receiver operating characteristic (ROC) curve indicated a significant difference in the PFS when the maximal cut-off value of tumor enlargement ratio was 7%. Univariate analysis of patients with targeted therapy after disease progression showed that gender, pathological type, clinical stage, lung metastasis and tumor enlargement ratio were the prognostic factors (all of P<0.05). Multivariate analysis showed that the tumor enlargement ratio was an independent prognostic factor (P=0.001). Single factor analysis showed that the chemotherapeutic regimens before and after disease progression were prognostic factors of patients received chemotherapy after disease progression (P<0.05). Cox multivariate analysis showed that the chemotherapeutic regimen after disease progression was an independent prognostic factor of patients (P=0.004). In the patients whose tumor enlargement ratio was 0 to 7%, Univariate analysis showed that chemotherapeutic regimen before tumor enlargement was a prognostic factor (P=0.030), while Cox multivariate analysis showed that it was not an independent prognostic factor (P=0.560). In the patients whose tumor enlargement ratio was 7.1% to 20%, single factor analysis showed that pathological type, bone metastasis and chemotherapeutic regimen after disease progression were prognostic factors (all of P<0.05), and Cox multivariate analysis showed that all of them were independent prognostic factors of these patients (all of P<0.05). Conclusions: To the advanced NSCLC patients whose tumor enlargement ratio is 0 to 20%, the PFS of patients receive replacing regimen is longer than that of patients receive original regimen. There is a significant difference in the PFS when the maximal cut-off value of tumor enlargement ratio is 7%. To patients undergo second-line chemotherapy before disease progression and the tumor enlargement ratio is 7.1% to 20%, the PFS of patients receive replacing regimen is significantly extended. Dual drug replacing regimen is especially benefit to the adenocarcinoma patients without bone metastasis.

Bone Morphogenetic Protein-2 regulates in vitro osteogenic differentiation of mouse adipose derived stem cells.

OBJECTIVE: The aim of this study is to investigate the feasibility and efficiency of Bone Morphogenetic Protein-2 (BMP-2) in regulating in vitro osteogenic differentiation of mouse adipose derived stem cells (ADSCs). MATERIALS AND METHODS: Mouse ADSCs were isolated from adipose tissues of C57/BL6 mice (age of 4-6 w) and cultured. Surface antigens of passage 3 (P3) ADSCs, including CD31, CD34, CD90, CD105 and CD133, were analyzed using flow cytometry. Overexpression of BMP-2 was achieved through gene transfection of ADSCs. In vitro osteogenic differentiation of transfected and non-transfected ADSCs cultured in specific induction media was evaluated by Alizarin Red staining. In addition, expression of osteoblast-specific gene, Runx2, was analyzed by quantitative RT-PCR (qRT-PCR). RESULTS: Abundant ADSCs could be isolated from adipose tissue. P3 ADSCs expressed stem cell-specific molecular markers, CD90 and CD105 but did not express CD31, CD34 or CD133. BMP-2 could efficiently transfect mouse ADSCs. Alizarin Red staining revealed that more calcified nodules were formed in BMP-2 transfected ADSCs. qRT-PCR further confirmed higher level of Runx2 expression in BMP-2 transfected ADSCs (p < 0.05). CONCLUSIONS: BMP-2 can promote in vitro osteogenic differentiation of mouse adipose stem cells.

Treatment of vertebral body compression fractures using percutaneous kyphoplasty guided by a combination of computed tomography and C-arm fluoroscopy with finger-touch guidance to determine the needle entry point.

This study aimed to evaluate the results and complications of image-guided percutaneous kyphoplasty (PKP) using computed tomography (CT) and C-arm fluoroscopy, with finger-touch guidance to determine the needle entry point. Of the 86 patients (106 PKP) examined, 56 were treated for osteoporotic vertebral compression fractures and 30 for vertebral tumors. All patients underwent image-guided treatment using CT and conventional fluoroscopy, with finger-touch identification of a puncture point within a small incision (1.5 to 2 cm). Partial or complete pain relief was achieved in 98% of patients within 24 h of treatment. Moreover, a significant improvement in functional mobility and reduction in analgesic use was observed. CT allowed the detection of cement leakage in 20.7% of the interventions. No bone cement leakages with neurologic symptoms were noted. All work channels were made only once, and bone cement was distributed near the center of the vertebral body. Our study confirms the efficacy of PKP treatment in osteoporotic and oncological patients. The combination of CT and C-arm fluoroscopy with finger-touch guidance reduces the risk of complications compared with conventional fluoroscopy alone, facilitates the detection of minor cement leakage, improves the operative procedure, and results in a favorable bone cement distribution.

Experimental study for the mechanism of gastroesophageal-reflux-associated asthma.

Epidemiologic studies have shown a strong association between gastroesophageal reflux (GER) and asthma, especially in children. Diagnosing GER can be difficult in some patients when GER presents solely with asthma. The aim of this study was to explore the relationship between GER and asthma with animal model. Sixty rats were randomly divided into six equal groups, GER group, GER-associated-asthma group, allergic asthma group, and their control groups. The cytokine levels and concentration of inflammatory cells in bronchoalveolar lavage (BAL) were determined. The BAL of the rats with allergic asthma contained higher concentration of Interleukin-5 (IL-5) and more eosinophils than those of rats with GER-associated-asthma. This demonstrates that assaying the concentrations of IL-5 and inflammatory cells in BAL may be an effective method of distinguishing GER-associated asthma from allergic asthma.

Anatomical reconstruction of donor site after large iliac crest graft harvest with equivalent iliac crest allograft. A prospective controlled study.

BACKGROUND: Iliac bone graft harvesting is a common procedure in spinal surgery and trauma center for spinal fusion and nonunion of the extremities. AIM, PATIENTS AND METHODS: To compare the pain and cosmetic outcomes of patients undergoing iliac crest anatomical reconstruction using equivalent iliac crest allograft (R group) with those of patients without reconstruction of the iliac crest defect (NR group), a prospective nonrandomized controlled study was conducted. RESULTS: In R group, the intensity and prevalence of pain were significantly lower than those in NR group. Cosmetic outcome and satisfaction score were also significantly improved in R group. One patient of R group suffered from lipolysis and superficial infection which healed by regular dressing change for two weeks. There were no cases of allograft displacement, implant loosening, internal fixation breakage or immunological rejection. Seven patients in NR group had significant pain related to the tenting of skin over the defect. Radiologic incorporation of pelvis was documented in all patients except four having partial re-sorption of the allograft. Early fibrous healing and the late creeping substitution were noted in all patients of R group. CONCLUSIONS: Equivalent iliac crest allograft provides an effective alternative for iliac crest anatomical reconstruction, leading to reduced donor site pain and better cosmesis.

Biomaterial implants mediate autologous stem cell recruitment in mice.

Autologous stem cells, recognized as the best cells for stem cell therapy, are associated with difficult extraction procedures which often lead to more traumas for the patients and time-consuming laboratory work, which delays their subsequent application. To combat such challenges, it was recently uncovered that, shortly after biomaterial implantation, following the recruitment of inflammatory cells, substantial numbers of mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) were recruited to the implantation sites. These multipotent MSC could be differentiated into various lineages in vitro. Inflammatory signals may be responsible for the gathering of stem cells, since there is a good relationship between biomaterial-mediated inflammatory responses and stem cell accumulation in vivo. In addition, the treatment with the anti-inflammatory drug dexamethasone substantially reduced the recruitment of both MSC and HSC. The results from this work support that such strategies could be further developed towards localized recruitment and differentiation of progenitor cells. This may permit the future development of autologous stem cell therapies without the need for tedious cell isolation, culture and transplantation.

Selective targeting of the mTORC1/2 protein kinase complexes leads to antileukemic effects in vitro and in vivo.

The BCR/ABL tyrosine kinase promotes leukemogenesis through activation of several targets that include the phosphoinositide 3-kinase (PI3K). Tyrosine kinase inhibitors (TKIs), which target BCR/ABL, induce striking clinical responses. However, therapy with TKIs is associated with limitations such as drug intolerance, inability to universally eradicate the disease and emergence of BCR/ABL drug-resistant mutants. To overcome these limitations, we tested whether inhibition of the PI3K/target of rapamycin (mTOR) signaling pathway has antileukemic effect in primary hematopoietic stem cells and BA/F3 cells expressing the BCR/ABL oncoprotein. We determined that dual inhibition of PI3K/mTOR causes growth arrest and apoptosis leading to profound antileukemic effects both in vitro and in vivo. We also established that pharmacologic inhibition of the mTORC1/mTORC2 complexes is sufficient to cause these antileukemic effects. Our results support the development of inhibitors of the mTORC1/2 complexes for the therapy of leukemias that either express BCR/ABL or display deregulation of the PI3K/mTOR signaling pathway.

Down-regulation of 67LR reduces the migratory activity of human glioma cells in vitro.

OBJECTIVES: Glioma is the most common brain tumor in central nervous system. Traditional therapies are not effective to cure this disease. Experimental evidence indicates that the 67 kDa elastin-laminin receptor (67LR) subunit is a high-affinity non-integrin laminin-binding protein that is over-expressed on the tumor cell surface in a variety of human carcinomas, and directly correlates with a higher proliferation rate of malignant cells and tendency to metastasize. However, little is known of the expression and function of 67LR in glioma cells. METHODS: In this study, we estimated whether 67LR was constitutively over-expressed in high-grade astrocytomas by immunohistochemical staining and Western blotting, and investigated the role of a low level of 67LR expression in glioma cell line-U251 by constructing an interfering RNA expression plasmid. RESULTS: The results showed that the 67LR had an enhanced over-expression in high-grade astrocytomas against normal brain tissues samples, and that the migratory activity of glioma cells was reduced after the down-regulation of the 67LR gene by RNAi. DISCUSSION: It was hypothesized that a low level of 67LR expression could reduce migratory activity of glioma cells, which further proved that 67LR played an important role in glioma invasion by mediating tumor cell functions leading to sarcomata. This study provided a new alternative to gene therapy for glioma treatment.

Activation of the p38 mitogen-activated protein kinase pathway by estrogen or by 4-hydroxytamoxifen is coupled to estrogen receptor-induced apoptosis.

17beta-Estradiol (E(2)) or the antiestrogen, 4-hydroxytamoxifen (OHT), induce apoptosis in stably transfected estrogen receptor (ER)-positive HeLa-ER5 cells. p38 mitogen-activated protein kinase is implicated in cellular processes involving apoptosis. The p38 kinase inhibitor, SB203580, partially protects HeLa-ER5 cells against apoptosis induced by E(2) or by OHT. E(2) induces the p38 pathway 12-36-fold in ER-positive cell lines, while OHT induces p38 activity 2-5-fold. In an ER-positive cell line selected for resistance to E(2)-induced apoptosis, E(2) no longer induced p38, and the ER no longer bound to the estrogen response element, while OHT induced both p38 and apoptosis. In cells selected for resistance to OHT-induced apoptosis, OHT no longer induced p38, while E(2) induced p38 and apoptosis, and transactivated an estrogen response element-containing reporter gene. In MCF-7 cells, whose growth is stimulated by estrogen, E(2) did not induce p38 or apoptosis, while OHT induced both p38 and apoptosis, and SB203580 protected against OHT-induced apoptosis. This work shows that E(2) and OHT activate the p38 pathway, suggests that they use different pathways for p38 activation, and links activation of the p38 pathway to apoptosis induced by E(2) and by OHT.

High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP.

The expression of high levels of full-length human estrogen receptor alpha (hERalpha) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERalpha (fER) was expressed in E. coli at approximately 1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17beta-estradiol with the same affinity as hER expressed in human cells (K(D) approximately 0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.