Hair cell α9α10 nicotinic acetylcholine receptor functional expression regulated by ligand binding and deafness gene products.

Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10 nicotinic acetylcholine receptors (nAChRs), which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Here, genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Collectively, this study provides a mechanism to link cholinergic input with α9α10 assembly, identifies unexpected functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders.

Insulin-Like Growth Factor Binding Protein 5: an Important Regulator of Early Osteogenic Differentiation of hMSCs.

Insulin-like growth factor binding protein 5 (IGFBP5) is broadly bioactive, but its role in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) remains to be clarified. Here, we demonstrated that IGFBP5 expression was markedly increased during the early osteogenic differentiation of hMSCs. We then over-expressed and knocked down this gene in hMSCs and evaluated the impact of manipulation of IGFBP5 expression on osteogenic differentiation based upon functional assays, ALP staining, and expression of osteogenic markers. Together, these analyses revealed that IGFBP5 over-expression enhanced early osteogenic differentiation, as evidenced by increased ALP staining and osteogenic marker induction, whereas knocking down this gene impaired the osteogenic process. Over-expression of IGFBP5 also markedly bolstered the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation level, while IGFBP5 knockdown suppressed this signalling activity. We additionally compared the impact of simultaneous IGFBP5 overexpression and ERK1/2 inhibitor treatment to the effect of IGFBP5 over-expression alone in these hMSCs, revealing that small molecule-mediated EKR1/2 inhibition was sufficient to impair osteogenic differentiation in the context of elevated IGFBP5 levels. These findings indicated that IGFBP5 drives the early osteogenic differentiation of hMSCs via the ERK1/2 signalling pathway. Our results offer value as a foundation for future efforts to study and treat serious bone-related diseases including osteoporosis.

[Clinical cohort study of total hysterectomy via transvaginal natural orifice transluminal endoscopic surgery and transumbilical laparoendoscopic single site surgery].

Objective: To study the safety and feasibility of total hysterectomy by transvaginal natural orifice transluminal endoscopic surgery (vNOTES) and transumbilical laparoendoscopic single site surgery (TU-LESS). Methods: The study was a retrospective cohort analysis. In West China Second University Hospital, from October 1, 2018 to June 30, 2019, the patients with an indication for hysterectomy due to benign uterine diseases were enrolled, and were grouped by the surgical procedure. Various post-operative surgical outcomes, including the success rate of operation, uterine volume, operative time, blood loss, intraoperative complications, postoperative complications, the first time of flatus, the preserved time of catheter after operation, and the length of hospitalization were measured. The data were assessed by the software of SPSS. Results: Totally 30 patients were included in the vNOTES group, and 77 patients were included in the TU-LESS group, all of the procedures were completed successfully without conversion to traditional multiport laparoscopy or laparotomy. Non differences were observed between two groups, including uterine volume [(165±36) vs (235±38) cm3, P=0.243], operation time [(150±41) vs (169±48) minutes, P=0.063], blood loss [(54±15) vs (54±14) ml, P=0.985], the rate of intraoperative complications [3% (1/30) vs 4% (2/77), P=1.000], the rate of postoperative complications [13% (4/30) vs 4% (3/77), P=0.095] and the rate of bilateral salpingo-oophorectomy [50% (15/30) vs 31% (24/77), P=0.069]. Patients in the vNOTES group had significantly less recovery time of intestine function [(1.7±0.5) vs (2.3±0.6) days, P=0.001], shorter time of indwelling catheter [(1.9±0.4) vs (2.3±0.6) days, P=0.004], and duration of hospitalization [(3.2±0.8) vs (3.6±0.9) days, P=0.045]. Conclusions: vNOTES and TU-LESS are both safe and feasible for total hysterectomy. Compared with TU-LESS, vNOTES may be a promising approach with earlier recovery, less injury and better cosmetic.

[Situation analysis of off-label drug use in China].

To introduce current literature reporting situations of the off-label drug use(OLDU) by analyzing relevant literatures published in China, this study comprehensively focused on literatures about OLDU in China in seven Chinese and English databases, then extracted and analyzed the data by different literature types. A total of 667 papers were analyzed. The number of literatures about OLDU data analyzed in hospitals was 325, and the number of clinical studies relating to OLDU was 329, in which case series and case reports were the majority(69.91%). In addition, there were 13 expert consensuses of OLDU and another 56 studies about drug use based on the real-world data characteristics and influencing factors. The number of OLDU data studies has increased year by year. Based on the existing studies, there were more western medicine reports than traditional Chinese medicines, and OLDU types were mainly for over-dosage use. The literatures from OLDU data in hospital were mostly limited to one or several tertiary hospitals in a certain area, and the OLDU types were not uniform. Clinical studies were mainly clinical control trials and case series/reports, but with contradictory reporting results. There were fewer OLDU consensus, and the recommended classification was not uniform. The characteristics and analysis of influencing factors of drug using data in real-world focused on traditional Chinese medicine injections, and the results were not the same. In the future, we shall pay more attention to and strengthen reporting and analysis of OLDU, define study objectives, and unify the content and reporting standards, so as to promote the integrated utilization of OLDU data and reflect real situations in our country.

Effect of developmental stage of embryos at freezing on live birth outcomes after frozen embryo transfer.

　　Purpose Our objective was to evaluate the birth outcomes of FET from different developmental stage of embryo at freezing (Day 3, Day 5 and Day 6). Methods All vitrified-warmed day 3 (D3) and day 5/6 (D5, D6) embryo transfers during the time period from January 2013 until December 2015 were included in the study. The Birthweight, Low birthweight (LBW), sexual distinction, gestation and for singleton births and twins conceived by FET were compared retrospectively between three cryopreservation strategies utilizing either D3, D5 or D6 embryo freezing. All embryos were vitrified by Dimethylsulfoxide (DMSO), Ethylene glycol (EG) and Sucrose used as Cryoprotectants and using Cryoleaf as the receptacles. Results A total of 993 infants from 771 women. The length of embryos storage was 3-38 months, with a median of 8 months. For singleton, birthweight from D5 and D6 blastocysts transfers were significantly heavier than for D3 cleavage-stage embryos transfers (P =0.0065 P =0.0006). For twins, birthweight from D6 blastocysts transfers were significantly heavier than for D5 blastocysts transfers (P =0.0044), and children born after D3 were at a signigicantly increased risk of being born a LBW. Conclusions Birthweights from FET are influenced by developmental stage of embryos at freezing, which were lower for cleavage-stage embryos transfer than blastocysts transfer after FET in singletons. The birth rate of LBW infants was higher in the twins. The study was small so there may be other factors than cryopreservation which affected outcomes. A higher sample size or a multi-centre prospective randomized design could be used in future studies to corroborate the current findings.

Downregulation of guanine nucleotide-binding protein beta 1 (GNB1) is associated with worsened prognosis of clearcell renal cell carcinoma and is related to VEGF signaling pathway.

PURPOSE: Clear-cell renal cell carcinoma (ccRCC) is characterized by genetic abnormalities, while the role of Guanine Nucleotide-Binding Protein Beta 1 (GNB1) in ccRCC has not been studied. We thus aimed to evaluate the expression and prognostic value of GNB1 in ccRCC. METHODS: A two-stage study (exploration and validation) was conducted using in silico and immunohistochemical (IHC) scoring of ccRCC samples from our institute, to evaluate the association between GNB1 expression and clinicopathological parameters of ccRCC patients. Pathway analyses were performed for genes coexpressed with GNB1 using the KOBAS platform to profile the function of GNB1 and IHC validation. RESULTS: In the exploration stage, data from TCGA ccRCC dataset were reproduced, which contained 537 patients with ccRCC and found that downregulation of GNB1 was significantly associated with worse prognosis. IHC staining from the Human Protein Atlas showed significantly downregulation of GNB1 in ccRCC tissue compared with normal kidney. Pathway analysis showed significantly altered vascular endothelial growth factor (VEGF) signaling pathways among which expressions of 3 genes (WASF2, NRP1, and HIP1) were significantly associated with GNB1 expression, respectively. In the validation stage, included were 80 ccRCC samples and GNB1 expression was scored using IHC positivity. GNB1 expression was negatively associated with tumor stage, lymph node invasion, metastasis, older age, and increased tumor grade. Female gender and receiving neoadjuvant therapy were also associated with decreased GNB1 expression. The expressions of WASF2, NRP1 and HIP1 were also studied and found that they were significantly associated with GNB1. CONCLUSION: GNB1 was downregulated in ccRCC. Decreased GNB1 expression was associated with worsened disease characteristics and prognosis. GNB1 was related with VEGF signaling in ccRCC, implying a therapeutic potential of this factor.

Impact of diabetes on clinical presentation and treatment outcome of pulmonary tuberculosis in Beijing.

Diabetes mellitus (DM) is currently known to be one of the risk factors for pulmonary tuberculosis (PTB) and the proportion of DM in PTB is rising along with the increased prevalence of DM in countries with high PTB burden. This study was designed to explore the impact of DM on clinical presentation and treatment outcome of PTB in China. In an urban setting in Beijing, 1126 PTB patients, 30·6% with positive sputum smear, registered in two PTB dispensaries from January 2010 to December 2011 were screened for DM and were followed up prospectively during PTB treatment. DM was observed in 16·2% of patients with PTB. PTB with DM appeared to be associated with older age and a higher proportion of re-treatment. On presentation, DM was associated with more severe PTB signs with higher proportions of smear positivity [odds ratio (OR) 2·533, 95% confidence interval (CI) 1·779-3·606], cavity (OR 2·253, 95% CI 1·549-3·276) and more symptoms (OR 1·779, 95% CI 1·176-2·690). DM was also associated with non-TB deaths (OR 5·580, 95% CI 2·182-14·270, P < 0·001) and treatment failure (OR 6·696, 95% CI 2·019-22·200, P = 0·002). In Beijing, the findings of this study underlined the need to perform early bi-directional screening programmes and explore the underlying mechanism for different treatment outcomes for PTB with DM.

Alternative splicing isoform of T cell factor 4K suppresses the proliferation and metastasis of non-small cell lung cancer cells.

The Wnt pathway has been implicated in the initiation, progression, and metastasis of lung cancer. T cell factor 4, a member of TCF/LEF family, acts as a transcriptional factor for Wnt pathways in lung cancer. Increasing amounts of evidence have shown that TCF-4 has multiple alternative splicing isoforms with transactivation or transrepression activity toward the Wnt pathway. Here, we found the presence of multiple TCF-4 isoforms in lung cancer cell lines and in normal bronchial epithelial cells. TCF-4K isoform expression was significantly decreased in lung cancer cells compared with normal bronchial epithelial cells and was identified as a transcriptional suppressor of the Wnt pathway in non-small cell lung carcinoma (NSCLC). Overexpression of TCF-4K significantly inhibited the proliferation and migration of NSCLC cells. Collectively, our data indicate that TCF-4K functions as a tumor suppressor in NSCLC by down-regulating the Wnt pathway.

Distally based saphenous neurocutaneous perforator flap combined with vac therapy for soft tissue reconstruction and hardware salvage in the lower extremities.

The complex wound with the exposed hardware and infection is one of the common complications after the internal fixation of the tibia fracture. The salvage of hardware and reconstruction of soft tissue defect remain challenging. In this report, we presented our experience on the use of the distally based saphenous neurocutaneous perforator flap combined with vacuum-assisted closure (VAC) therapy for the coverage of the soft tissue defect and the exposed hardware in the lower extremity with fracture. Between January 2008 and July 2010, seven patients underwent the VAC therapy followed by transferring a reversed saphenous neurocutaneous perforator flap for reconstruction of the wound with exposed hardware around the distal tibia. The sizes of the flaps ranged from 6 × 3 cm to 15 × 6 cm. Six flaps survived completely. Partial necrosis occurred in one patient. There were no other complications of repair and donor sites. Bone healing was achieved in all patients. In conclusion, the reversed saphenous neurocutaneous perfortor flaps combined with the VAC therapy might be one of the options to cover the complex wound with exposed hardware in the lower extremities.

A single infusion of intravenous lidocaine for primary headaches and trigeminal neuralgia: a retrospective analysis.

Introduction: Intravenous (IV) lidocaine has been used as a transitional treatment in headache and facial pain conditions, typically as an inpatient infusion over several days, which is costly and may increase the risk of adverse effects. Here we report on our experience using a single one-hour IV lidocaine infusion in an outpatient day-case setting for the management of refractory primary headache disorders with facial pain and trigeminal neuralgia. Methods: This is a retrospective, single-center analysis on patients with medically refractory headache with facial pain and trigeminal neuralgia who were treated with IV lidocaine between March 2018 and July 2022. Lidocaine 5 mg.kg-1 in 60 mL saline was administered over 1 h, followed by an observation period of 30 min. Patients were considered responders if they reported reduction in pain intensity and/or headache frequency of 50% or greater. Duration of response was defined as short-term (< 2 weeks), medium-term (2-4 weeks) and long-term (> 4 weeks). Results: Forty infusions were administered to 15 patients with trigeminal autonomic cephalalgias (n = 9), chronic migraine (n = 3) and trigeminal neuralgia (n = 3). Twelve patients were considered responders (80%), eight of whom were complete responders (100% pain freedom). The average duration of the treatment effect for each participant was 9.5 weeks (range 1-22 weeks). Six out of 15 patients reported mild and self-limiting side effects (40%). Conclusion: A single infusion of IV lidocaine might be an effective and safe transitional treatment in refractory headache conditions with facial pain and trigeminal neuralgia. The sustained effect of repeated treatment cycles in some patients may suggest a role as long-term preventive therapy in some patients.

Transgastric endoscopic gastroenterostomy using a partially covered occluder: a canine feasibility study.

BACKGROUND AND STUDY AIMS: The use of natural orifice transluminal endoscopic surgery (NOTES) for gastroenterostomy has been previously reported, but it remains technically challenging and additional assistance is often needed. The aim of this study was to develop and evaluate a novel method for the creation of a gastroenterostomy using NOTES with an occluder. METHODS: Transgastric endoscopic gastroenterostomy was performed in 12 healthy female dogs using a therapeutic upper gastrointestinal endoscope and a partially covered occluder. The occluder was removed with a snare 1 week later. The patency of the gastroenterostomy was confirmed by endoscopy, contrast radiological study, necropsy, and histological examination after 2 weeks. RESULTS: NOTES gastroenterostomy with an occluder was successful in all 12 dogs. The mean operative time was 32.3 ± 10.3 min (range 20.3 - 53.5). One dog (the first; 8.3 %) died 4 days after the operation of severe intra-abdominal infection due to incorrect deployment of the occluder and poor bowel preparation. Minor bleeding occurred at the anastomosis after removal of the occluder in two of the remaining dogs (18.2 %). Necropsy revealed postoperative adhesions that had developed at the anastomotic site in one dog (9.1 %). No anastomotic leakage or intestinal obstruction was observed. Complete healing of the anastomosis was confirmed on histological evaluation. CONCLUSION: Gastroenterostomy performed entirely by NOTES using an occluder was technically feasible in this survival animal model.

Bayesian analysis for genetic architecture of dynamic traits.

The dissection of the genetic architecture of quantitative traits, including the number and locations of quantitative trait loci (QTL) and their main and epistatic effects, has been an important topic in current QTL mapping. We extend the Bayesian model selection framework for mapping multiple epistatic QTL affecting continuous traits to dynamic traits in experimental crosses. The extension inherits the efficiency of Bayesian model selection and the flexibility of the Legendre polynomial model fitting to the change in genetic and environmental effects with time. We illustrate the proposed method by simultaneously detecting the main and epistatic QTLs for the growth of leaf age in a doubled-haploid population of rice. The behavior and performance of the method are also shown by computer simulation experiments. The results show that our method can more quickly identify interacting QTLs for dynamic traits in the models with many numbers of genetic effects, enhancing our understanding of genetic architecture for dynamic traits. Our proposed method can be treated as a general form of mapping QTL for continuous quantitative traits, being easier to extend to multiple traits and to a single trait with repeat records.

Spin-valley locking and bulk quantum Hall effect in a noncentrosymmetric Dirac semimetal BaMnSb2.

Spin-valley locking in monolayer transition metal dichalcogenides has attracted enormous interest, since it offers potential for valleytronic and optoelectronic applications. Such an exotic electronic state has sparsely been seen in bulk materials. Here, we report spin-valley locking in a Dirac semimetal BaMnSb2. This is revealed by comprehensive studies using first principles calculations, tight-binding and effective model analyses, angle-resolved photoemission spectroscopy measurements. Moreover, this material also exhibits a stacked quantum Hall effect (QHE). The spin-valley degeneracy extracted from the QHE is close to 2. This result, together with the Landau level spin splitting, further confirms the spin-valley locking picture. In the extreme quantum limit, we also observed a plateau in the z-axis resistance, suggestive of a two-dimensional chiral surface state present in the quantum Hall state. These findings establish BaMnSb2 as a rare platform for exploring coupled spin and valley physics in bulk single crystals and accessing 3D interacting topological states.

Study on acute toxicity and structure-activity relationship of zebrafish (Danio rerio) exposed to naphthoquinones.

In this paper, we evaluated the acute toxicity of five naphthoquinone compounds, expressed as median lethal concentration at 96 h (LC(50)) using zebrafish as a model. The five compounds were classified as harmful or very toxic. The Gaussian method was used to calculate the quantitative-chemical parameters of each compound. Based on the structure-activity relationship analysis, the interaction between the naphthoquinones and the target is likely mediated by 1-carbonyl and the hydrophobic fraction substituted in alpha -position of naphthoquinone via hydrogen bonding and hydrophobic interactions. Our results provide a foundation for evaluating the aquatic ecological risk and the mode of action of naphthoquinones in the environment.

Oxidative stress and structure-activity relationship in the zebrafish (Danio rerio) under exposure to paclobutrazol.

The acute toxicity and changes in the activity of antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) in the zebrafish Danio rerio were measured when the fish was exposed to paclobutrazol (PBZ) under laboratory conditions. The median lethal concentration value at 96 h post-exposure (LC(50) (96 h)) was determined to be 20.55 mg/L. In addition, the SOD activities in D. rerio were significantly higher at 96 h post-exposure among the various concentrations of the pollutant tested. The change of CAT activity could be considered as a sensitive biomarker of the oxidative stress induced by PBZ. The structure-activity relationship (SAR) analysis identified the potential active points for the pollutant and that the cavum may be the site of interaction with the receptor.

Effects of ${\rm \small L}$-methionine on growth performance, carcass quality, feather traits, and small intestinal morphology of Pekin ducks compared with conventional ${\rm \small {DL}}$-methionine.

The research studied the effects of l-methionine (l-Met) on growth performance, carcass quality, feather traits, and small intestinal morphology of Pekin ducks compared with conventional dl-methionine (dl-Met). A total of 1080, 1-day-old male Pekin ducks were randomly allotted to 9 groups with 6 replicate pens of 20 birds each. During the starter phase (1 to 14 d), ducks were fed a basal diet (Met, 0.30%) or that supplemented with dl-Met or l-Met at 0.05, 0.10, 0.15, or 0.20% of feed. During the grower phase (15 to 35 d), ducks were fed a basal diet (Met, 0.24%) or that supplemented with dl-Met or l-Met at 0.04, 0.08, 0.12, or 0.16% of feed. Compared with ducks fed the basal diet, supplementation with either dl-Met or l-Met increased the body weight (BW) of ducks at days 14 and 35, increased average daily gain (ADG) and average daily feed intake (ADFI), decreased F:G at the starter phase, and increased ADG over the whole 35-d period (P < 0.05). The efficacy of l-Met compared to dl-Met was 140.1% for 14-d BW, 137.6% for ADG and 121.0% for F:G for days 1 to 14. Ducks fed diets supplemented with l-Met had greater proportion of leg muscle, higher than in ducks provided with dl-Met (P < 0.05). The breast muscle proportion was enhanced with dl-Met rather than l-Met supplementation (P < 0.01). The back feathers score and fourth primary wing feather length were increased with dl-Met or l-Met supplementation (P < 0.01), and there was increased efficacy of l-Met relative to dl-Met for back feathers score (153.1%). Dietary dl-Met or l-Met supplementation increased villus height of ileal mucosa of ducks at days 14 and 35 (P < 0.01). Overall, dietary l-Met or dl-Met supplementation affected the growth performance of ducks during the starter phase, and improved the feather traits and small intestinal morphology. The efficacy of l-Met to dl-Met ranged from 120 to 140% for growth performance of young ducks (1 to 14 d) and was 153% for the feather traits of ducks (35 d).

The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone.

BACKGROUND AND OBJECTIVES: A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity. MATERIALS AND METHODS: The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including (1)H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments. RESULTS: The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15.0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa. CONCLUSIONS: These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.

IL-6 stimulates Th2 type cytokine secretion and upregulates VEGF and NRP-1 expression in pancreatic cancer cells.

BACKGROUND: Although upregulation of interleukin-6 (IL-6) is associated with many solid tumors, its role in pancreatic cancer has not been well elucidated. In this study, we examined the expression of IL-6 in pancreatic cancer cells, and determined the effect of exogenous IL-6 on cytokine secretion, gene expression and signaling in human pancreatic cancer cells. METHODS: The mRNA levels of IL-6, VEGF165, neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2) were determined by real-time RT PCR. Phosphorylation of ERK2 in pancreatic cancer cells was determined by using Bio-Plex phosphoprotein assay. The expression of IL-6 and other cytokines in human pancreatic cancer cell lines was determined with Bio-Plex cytokine assay. RESULTS: Pancreatic cancer cell lines expressed higher levels of IL-6 than normal human pancreatic ductal epithelium (HPDE) cells. Exogenous IL-6 increased the secretion of multiple Th2 type of cytokines in Panc-1, MIA PaCa-2 and BxPC-3 cells. IL-6 also upregulated the expression of VEGF165, and NRP-1, and both IL-6 and VEGF165 were inducible by hypoxia. In addition, IL-6 activated ERK2 signaling pathways in pancreatic cancer cells. CONCLUSIONS: IL-6 may be involved in promoting human pancreatic cancer develop ment by furnishing Th2 type of cytokine environment and upregulating cell proliferation and angiogenesis related genes. Targeting IL-6 might be an effective treatment for pancreatic cancer.

Exonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors: measurement of activated NF-kappaB.

This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor kappaB (NF-kappaB). For EMEA, a special double-stranded DNA (dsDNA)-coupled plate was first prepared by immobilizing a DNA probe on an N-oxysuccinimide ester-coated plate. The immobilized DNA probe, which was internally labeled with digoxigenin (DIG)-dT contained a NF-kappaB binding consensus sequence for capturing activated NF- kappaB in analyzed samples. For measurement, the plate was first incubated with a protein sample and then treated with exonuclease III to eliminate the probes not bound by NF-kappaB. Finally, the probes protected by NF-kappaB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-kappaB without the need for NF-kappaB antibodies. EMEA may provide a general approach for assays of DNA sequence-specific transcription factors for which specific antibodies are unavailable, expensive, or of insufficient quality.

Assay of DNA-binding proteins with a dsDNA-coupled plate.

OBJECTIVES: This paper fabricated a cost-effective dsDNA-coupled plate (dcPlate) and applied it to measure the abundance and DNA-binding activity of a DNA-binding protein (DBP). DESIGN AND METHODS: The dcPlate was manufactured by covalently immobilizing an amino-modified oligonucleotide in wells of the plate coated with N-oxysuccinimide esters. The dcPlate was applied to measure the abundance of DNA-binding activity of a DBP in the same four steps, including protein incubation, primary antibody binding, enzyme-linked secondary antibody binding, and colorimetric development. RESULTS: The detections of three purified DBPs including NF-kappaB, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-kappaB p50 to five various DNA sequences demonstrated that dcPlate can be used to measure the abundance of DBPs quantitatively and assay DNA-binding activity of DBPs in high throughputs format. CONCLUSIONS: The homemade cost-effective dcPlate provides a simple and versatile platform for studying DBPs.