NALIRIFOX versus nab-paclitaxel and gemcitabine in treatment-naive patients with metastatic pancreatic ductal adenocarcinoma (NAPOLI 3): a randomised, open-label, phase 3 trial.

BACKGROUND: Pancreatic ductal adenocarcinoma remains one of the most lethal malignancies, with few treatment options. NAPOLI 3 aimed to compare the efficacy and safety of NALIRIFOX versus nab-paclitaxel and gemcitabine as first-line therapy for metastatic pancreatic ductal adenocarcinoma (mPDAC). METHODS: NAPOLI 3 was a randomised, open-label, phase 3 study conducted at 187 community and academic sites in 18 countries worldwide across Europe, North America, South America, Asia, and Australia. Patients with mPDAC and Eastern Cooperative Oncology Group performance status score 0 or 1 were randomly assigned (1:1) to receive NALIRIFOX (liposomal irinotecan 50 mg/m2, oxaliplatin 60 mg/m2, leucovorin 400 mg/m2, and fluorouracil 2400 mg/m2, administered sequentially as a continuous intravenous infusion over 46 h) on days 1 and 15 of a 28-day cycle or nab-paclitaxel 125 mg/m2 and gemcitabine 1000 mg/m2, administered intravenously, on days 1, 8, and 15 of a 28-day cycle. Balanced block randomisation was stratified by geographical region, performance status, and liver metastases, managed through an interactive web response system. The primary endpoint was overall survival in the intention-to-treat population, evaluated when at least 543 events were observed across the two treatment groups. Safety was evaluated in all patients who received at least one dose of study treatment. This completed trial is registered with ClinicalTrials.gov, NCT04083235. FINDINGS: Between Feb 19, 2020 and Aug 17, 2021, 770 patients were randomly assigned (NALIRIFOX, 383; nab-paclitaxel-gemcitabine, 387; median follow-up 16·1 months [IQR 13·4-19·1]). Median overall survival was 11·1 months (95% CI 10·0-12·1) with NALIRIFOX versus 9·2 months (8·3-10·6) with nab-paclitaxel-gemcitabine (hazard ratio 0·83; 95% CI 0·70-0·99; p=0·036). Grade 3 or higher treatment-emergent adverse events occurred in 322 (87%) of 370 patients receiving NALIRIFOX and 326 (86%) of 379 patients receiving nab-paclitaxel-gemcitabine; treatment-related deaths occurred in six (2%) patients in the NALIRIFOX group and eight (2%) patients in the nab-paclitaxel-gemcitabine group. INTERPRETATION: Our findings support use of the NALIRIFOX regimen as a possible reference regimen for first-line treatment of mPDAC. FUNDING: Ipsen. TRANSLATION: For the plain language summary see Supplementary Materials section.

Intracluster correlation coefficients from school-based cluster randomized trials of interventions for improving health outcomes in pupils.

BACKGROUND AND OBJECTIVES: To summarize intracluster correlation coefficient (ICC) estimates for pupil health outcomes from school-based cluster randomized trials (CRTs) across world regions and describe their relationship with study design characteristics and context. METHODS: School-based CRTs reporting ICCs for pupil health outcomes were identified through a literature search of MEDLINE (via Ovid). ICC estimates were summarized both overall and for different categories of study characteristics. RESULTS: Two hundred and forty-six articles reporting ICC estimates were identified. The median (interquartile range) ICC was 0.031 (0.011 to 0.08) at the school level (N = 210) and 0.063 (0.024 to 0.1) at the class level (N = 46). The distribution of ICCs at the school level was well described by the beta and exponential distributions. Besides larger ICCs in definitive trials than feasibility studies, there were no clear associations between study characteristics and ICC estimates. CONCLUSION: The distribution of school-level ICCs worldwide was similar to previous summaries from studies in the United States. The description of the distribution of ICCs will help to inform sample size calculations and assess their sensitivity when designing future school-based CRTs of health interventions.

Systematic review of the characteristics of school-based feasibility cluster randomised trials of interventions for improving the health of pupils in the UK.

BACKGROUND: The last 20 years have seen a marked increase in the use of cluster randomised trials (CRTs) in schools to evaluate interventions for improving pupil health outcomes. Schools have limited resources and participating in full-scale trials can be challenging and costly, given their main purpose is education. Feasibility studies can be used to identify challenges with implementing interventions and delivering trials. This systematic review summarises methodological characteristics and objectives of school-based cluster randomised feasibility studies in the United Kingdom (UK). METHODS: We systematically searched MEDLINE from inception to 31 December 2020. Eligible papers were school-based feasibility CRTs that included health outcomes measured on pupils. RESULTS: Of 3285 articles identified, 24 were included. School-based feasibility CRTs have been increasingly used in the UK since the first publication in 2008. Five (21%) studies provided justification for the use of the CRT design. Three (13%) studies provided details of a formal sample size calculation, with only one of these allowing for clustering. The median (IQR; range) recruited sample size was 7.5 (4.5 to 9; 2 to 37) schools and 274 (179 to 557; 29 to 1567) pupils. The most common feasibility objectives were to estimate the potential effectiveness of the intervention (n = 17; 71%), assess acceptability of the intervention (n = 16; 67%), and estimate the recruitment/retention rates (n = 15; 63%). Only one study was used to assess whether cluster randomisation was appropriate, and none of the studies that randomised clusters before recruiting pupils assessed the possibility of recruitment bias. Besides potential effectiveness, cost-effectiveness, and the intra-cluster correlation coefficient, no studies quantified the precision of the feasibility parameter estimates. CONCLUSIONS: Feasibility CRTs are increasingly used in schools prior to definitive trials of interventions for improving health in pupils. The average sample size of studies included in this review would be large enough to estimate pupil-level feasibility parameters (e.g., percentage followed up) with reasonable precision. The review highlights the need for clearer sample size justification and better reporting of the precision with which feasibility parameters are estimated. Better use could be made of feasibility CRTs to assess challenges that are specific to the cluster design. TRIAL REGISTRATION: PROSPERO: CRD42020218993.

Of Young People and Internet Cafés.

This study examines how adolescent experience in Internet cafés (known as wangba in Chinese) relates to academic attainment in urban, rural, and Tibetan schools of China. By documenting the frustrations teenagers express in their negotiations with adults surrounding access to and use of wangba and, by comparing self-reported academic standing of students from similar backgrounds with how they differ in their experience in wangba, the study finds that visiting wangba is not strongly correlated with the probability of students reporting either high- or under-achievement. While students without any experience in wangba are substantially less likely to report academic underperformance, the association disappears after matching when the logit regression model is less model-dependent and vulnerable to the problems associated with missing data. The paper concludes that visiting wangba alone is not systematically correlated with academic attainment, and that much adult anxiety concerning adolescent visit to wangba represents moral-technological panic and, offers a simplified explanation for educational problems that have deep macrosocial roots.

Characteristics and practices of school-based cluster randomised controlled trials for improving health outcomes in pupils in the United Kingdom: a methodological systematic review.

BACKGROUND: Cluster randomised trials (CRTs) are increasingly used to evaluate non-pharmacological interventions for improving child health. Although methodological challenges of CRTs are well documented, the characteristics of school-based CRTs with pupil health outcomes have not been systematically described. Our objective was to describe methodological characteristics of these studies in the United Kingdom (UK). METHODS: MEDLINE was systematically searched from inception to 30th June 2020. Included studies used the CRT design in schools and measured primary outcomes on pupils. Study characteristics were described using descriptive statistics. RESULTS: Of 3138 articles identified, 64 were included. CRTs with pupil health outcomes have been increasingly used in the UK school setting since the earliest included paper was published in 1993; 37 (58%) studies were published after 2010. Of the 44 studies that reported information, 93% included state-funded schools. Thirty six (56%) were exclusively in primary schools and 24 (38%) exclusively in secondary schools. Schools were randomised in 56 studies, classrooms in 6 studies, and year groups in 2 studies. Eighty percent of studies used restricted randomisation to balance cluster-level characteristics between trial arms, but few provided justification for their choice of balancing factors. Interventions covered 11 different health areas; 53 (83%) included components that were necessarily administered to entire clusters. The median (interquartile range) number of clusters and pupils recruited was 31.5 (21 to 50) and 1308 (604 to 3201), respectively. In half the studies, at least one cluster dropped out. Only 26 (41%) studies reported the intra-cluster correlation coefficient (ICC) of the primary outcome from the analysis; this was often markedly different to the assumed ICC in the sample size calculation. The median (range) ICC for school clusters was 0.028 (0.0005 to 0.21). CONCLUSIONS: The increasing pool of school-based CRTs examining pupil health outcomes provides methodological knowledge and highlights design challenges. Data from these studies should be used to identify the best school-level characteristics for balancing the randomisation. Better information on the ICC of pupil health outcomes is required to aid the planning of future CRTs. Improved reporting of the recruitment process will help to identify barriers to obtaining representative samples of schools.

Characteristics and practices of school-based cluster randomised controlled trials for improving health outcomes in pupils in the UK: a systematic review protocol.

INTRODUCTION: Cluster randomised trials (CRTs) are studies in which groups (clusters) of participants rather than the individuals themselves are randomised to trial arms. CRTs are becoming increasingly relevant for evaluating interventions delivered in school settings for improving the health of children. Schools are a convenient setting for health interventions targeted at children and the CRT design respects the clustered structure in schools (ie, pupils within classrooms/teachers within schools). Some of the methodological challenges of CRTs, such as ethical considerations for enrolment of children into trials and how best to handle the analysis of data from participants (pupils) that change clusters (schools), may be more salient for the school setting. A better understanding of the characteristics and methodological considerations of school-based CRTs of health interventions would inform the design of future similar studies. To our knowledge, this is the only systematic review to focus specifically on the characteristics and methodological practices of CRTs delivered in schools to evaluate interventions for improving health outcomes in pupils in the UK. METHODS AND ANALYSIS: We will search for CRTs published from inception to 30 June 2020 inclusively indexed in MEDLINE (Ovid). We will identify relevant articles through title and abstract screening, and subsequent full-text screening for eligibility against predefined inclusion criteria. Disagreements will be resolved through discussion. Two independent reviewers will extract data for each study using a prepiloted data extraction form. Findings will be summarised using descriptive statistics and graphs. ETHICS AND DISSEMINATION: This methodological systematic review does not require ethical approval as only secondary data extracted from papers will be analysed and the data are not linked to individual participants. After completion of the systematic review, the data will be analysed, and the findings disseminated through peer-reviewed publications and scientific meetings. PROSPERO REGISTRATION NUMBER: CRD42020201792.

Harmine inhibits the proliferation and migration of glioblastoma cells via the FAK/AKT pathway.

AIMS: Glioblastoma is one of the most invasive tumors of the central nervous system, and has a high degree of malignancy and poor prognosis. Harmine, an active ingredient extracted from perennial herbs, has been reported to have obvious antitumor effects on various tumors. However, the effects of harmine on glioblastoma growth remain unknown. We here explored the effects of harmine on glioblastoma and its underlying molecular mechanisms related to tumorigenesis. MATERIALS AND METHODS: CCK-8 and immunofluorescent assay were performed to measure anti-proliferative effect of harmine on U251-MG and U373-MG cells. Wound healing assay was performed to measure the effects of harmine on cell migration. qRT-PCR and western blot were performed to detect the protein/gene expression. BALB/c nude mice bearing U251-MG xenografts was used to measure the effects of harmine on the growth of glioblastoma in vivo. KEY FINDINGS: Harmine treatment significantly suppressed the proliferation of U251-MG and U373-MG cells in a dose and time-dependent way. Mechanistically, harmine reduced the basal and EGF-enhanced the phosphorylation level of FAK and AKT. Moreover, harmine inhibited the cell viability of U251-MG and U373-MG cells by downregulating the phosphorylation of the FAK/AKT pathway. Besides, harmine significantly suppressed the migration of U251-MG cells by suppressing the expression of MMP2, MMP9 and VEGF. Subsequently, orthotopic xenograft models revealed that harmine treatment dramatically inhibited the growth of glioblastoma in vivo. SIGNIFICANCE: In conclusion, these results suggest that harmine suppresses the proliferation and migration of U251-MG and U373-MG cells by inhibiting the FAK/AKT signaling pathway. Our findings elucidate harmine could be a promising drug for glioblastoma therapy.

Real-World Comparative Effectiveness and Cost Comparison of Thromboprophylactic Use of Enoxaparin versus Unfractionated Heparin in 376,858 Medically Ill Hospitalized US Patients.

BACKGROUND: Venous thromboembolism (VTE) is a serious complication in medically ill inpatients. Enoxaparin or unfractionated heparin (UFH) thromboprophylaxis has been shown to reduce VTE in clinical trials; however, comparative effectiveness and differences in hospital costs are unknown in US hospital practice. OBJECTIVE: This study compared clinical and economic outcomes between enoxaparin and UFH thromboprophylaxis in medically ill inpatients. METHODS: A retrospective cohort study was conducted using the Premier Healthcare Database between 1 January 2010 and 30 September 2016. Inpatients aged ≥ 18 years with a ≥ 6-day hospital stay for serious medical conditions were included. Two patient groups receiving thromboprophylaxis were identified during hospitalization: one receiving enoxaparin and other receiving UFH. Regression models were constructed to compare VTE events, in-hospital mortality, pulmonary embolism (PE)-related mortality, major bleeding, and total hospital costs during both the index hospitalization and the 90-day readmission period between the two groups. RESULTS: A total of 242,474 and 134,384 inpatients received enoxaparin or UFH for thromboprophylaxis, respectively. Compared with UFH prophylaxis, enoxaparin was significantly associated with 15%, 9%, 33%, and 41% reduced odds of VTE, in-hospital mortality, PE-related mortality, and major bleeding, respectively, during index hospitalization, and 10% and 19% reduced odds of VTE and bleeding, respectively, during the readmission period. Mean total hospital costs were significantly lower in patients receiving enoxaparin prophylaxis than in those given UFH. CONCLUSIONS: Thromboprophylaxis with enoxaparin was associated with significantly reduced in-hospital VTE events, death, and major bleeding and lower hospital costs compared with UFH in hospitalized medically ill patients.

Severe Acute Localized Reactions Following Intra-Articular Hyaluronic Acid Injections in Knee Osteoarthritis.

OBJECTIVE: Concerns have been raised about severe acute localized reactions (SALR) following intra-articular (IA) hyaluronic acid (HA) injections for knee osteoarthritis (OA). We compared surrogate SALR measures between hylan G-F 20 and non-hylan G-F 20 HA patients and evaluated corresponding SALR risk factors for hylan G-F 20 patients. DESIGN: Knee OA patients were identified from the Optum Clinformatics dataset (January 2006 to June 2016), stratified into hylan G-F 20 and non-hylan G-F 20 HA users. Occurrences of surrogate SALR measures including inflammation/infection, intra-articular corticosteroid (CS) injections, arthrocentesis/aspiration, and office visits were evaluated within 3 days of HA use. Risk factors were evaluated using logistic regression. RESULTS: The cohort involved 748,428 HA patients (23.2% in the hylan G-F 20 group). Inflammation/infection rate was 0.001% for hylan G-F 20 and 0.002% for non-hylan G-F 20 HA groups. Risk of CS injection (any diagnosis) was greater for hylan G-F 20 patients by 28% (P < 0.001). Combined rates of CS injection and arthrocentesis/aspiration (any diagnosis) were comparable for both groups (hylan G-F 20, 2.2%; non-hylan G-F 20 HA, 2.6%). The risk of any visit or studied responses was lower for the hylan G-F 20 cohort by 12% (P < 0.001). Clinical characteristics, such as CS injections within 1 week before HA and fluoroscopic imaging, were associated with the outcomes. CONCLUSIONS: The diagnosis of inflammations or infections within 3 days of the HA injection was extremely rare. The overall risk of surrogate SALR measures was similar for hylan G-F 20 and non-hylan G-F 20 HA patients.

Comparative Effectiveness and Costs of Enoxaparin Monotherapy Versus Unfractionated Heparin Monotherapy in Treating Acute Coronary Syndrome.

BACKGROUND: Enoxaparin and unfractionated heparin (UFH) are guideline-recommended anticoagulants for patients with acute coronary syndrome (ACS), including unstable angina (UA) and myocardial infarction with (STEMI) or without ST-segment elevation (NSTEMI). Prior efficacy and safety evidence are mainly from clinical trials. Economic data are insufficient. This study examined the differences in utilization, effectiveness, safety, and costs in treating ACS between enoxaparin and UFH monotherapy using real-world data. METHODS: Using data from 859 US hospitals, inpatients ≥ 18 years of age with a diagnosis of an initial episode of ACS between 2010 and 2016 were identified. Outcomes included 30-day risk of non-fatal myocardial infarction (MI), recurrent angina, in-hospital mortality, composite ischemic complication (having MI/recurrent angina/death), major bleeding, and costs. Multivariable regression was used to compare outcomes between enoxaparin and UFH monotherapy. RESULTS: Among 1,048,053 eligible patients (UA: 219,259; NSTEMI: 582,134; STEMI: 246,660), the prevalence of enoxaparin monotherapy was 12.0%, 13.9%, and 5.1%, and the prevalence of UFH monotherapy was 45.1%, 43.1% and 59.8%, for UA, NSTEMI, and STEMI patients, respectively. Enoxaparin was associated with a lower risk of ischemic complications and death among NSTEMI, but not in UA or STEMI patients, and with a lower risk of major bleeding in all patients. Cost savings per patient during index admission and 30-day follow-up for enoxaparin over UFH was $2972 for UA, $2475 for NSTEMI, and $3050 for STEMI. CONCLUSIONS: Enoxaparin was associated with a lower risk of ischemic complications (including death), lower costs, and better safety than UFH among NSTEMI patients. Improving upstream selection of anticoagulants in appropriate populations may help optimize clinical outcomes and costs.

C6 Glioma-Secreted NGF and FGF2 Regulate Neuronal APP Processing Through Up-Regulation of ADAM10 and Down-Regulation of BACE1, Respectively.

Excessive accumulation of amyloid-ß (Aß) caused by cleavage of amyloid precursor protein (APP) is thought to be the primary cause of Alzheimer's disease (AD). Two key enzymes ADAM10 and BACE1 are involved in the initial cleavage of APP, resulting in the onset of two pathways, the amyloidogenic pathway and the non-amyloidogenic pathway, respectively. Altering APP metabolism towards the non-amyloidogenic pathway is thought to reduce Aß production. It has been reported that, in vivo, exogenous neurotrophic factors make APP apt to entering the non-amyloidogenic pathway. Since astrocytes secrete a battery of neurotrophic factors, we investigated the role of astrocyte-derived factors in the dynamics of Aß generation in neural cells. Results show that C6 glioma cell-conditioned medium (GCM), obtained from cultured astrocyte-derived C6 glioma cells, inhibit Aß1-42 production and shift APP processing towards the non-amyloidogenic pathway in APPswe-HEK293 cells. Such effect is attributed to two key APP cleavage enzymes, ADAM10 and BACE1. Two neurotrophic factors in the GCM, nerve growth factor and fibroblast growth factor 2, are responsible for the up-regulation of ADAM10 and down-regulation of BACE1, respectively. Our findings enhance our understanding of the relationship between astrocytes and Aß generation, indicating that stimulation of astrocytic neurotrophic factors could slow AD progression.

Cytogenetic and molecular identification of small-segment chromosome translocation lines from wheat-rye substitution lines to create wheat germplasm with beneficial traits.

Intergeneric crop plant hybrid lines with small-segment chromosome translocations are very useful in plant genetic research and breeding. In this study, to create small-segment chromosome translocations with beneficial agronomic characters, the progeny of wheat-rye substitution lines 5R/5A and 6R/6A were selected from generations F2 to F5 for rye-specific characteristics. A PCR primer and specific simple sequence repeat marker for rye were used in F5 populations to detect rye chromatin and to amplify a specific chromosome band in six translocation lines (06-6-5, 06-6-6, 06-6-9, 6-26-1, 7-23, and 7-33). Fragment pSc119.1 cloned from 7-33 had 99% homology with the big ear gene sequence (GenBank AF512607.1) in wheat. The six lines were further characterized via pollen mother cell meiosis analysis for genetic stability, and chromosome C-banding and genomic in situ hybridization for rye chromatin. The results show that line 7-33 was still within the 5R/5A substitution lines and possessed the big ear gene. The other lines all contained small-segment rye chromosome translocations. The results indicated that substitution line hybridization is an effective method for creating small-segment chromosome translocations with useful agronomic traits. Trials for these six wheat-rye translocation lines are justified because they possess many important stably-inherited agronomic characters, including disease resistance and improved yield.

The neuroprotective effects of ipriflavone against H 2O 2 and amyloid beta induced toxicity in human neuroblastoma SH-SY5Y cells.

Estrogenic compounds have been shown to have great potential for the treatment of Alzheimer's disease as demonstrated by its ability to antagonize amyloid beta peptide (Aß) induced toxicity, the hallmark of Alzheimer's disease. Key mechanisms include the involvements of both the antioxidant and the anti-apoptotic pathways. However, side effects of estrogens, such as the increased incidence of breast cancer, are of concern for further clinical translation. Approaches to overcome such barriers include the structural modification of estrogenic compounds and the search of phytoestrogens, but these are a long way from being translated into the clinic. We identified a compound similar in structure to human estrogen-ipriflavone, an over-the-counter product marketed in the United States for the treatment of osteoporosis, efficiently antagonized Aß induced toxicity. Use of a model with SH-SY5Y neural cells, we first demonstrated that ipriflavone potently alleviated H2O2 induced cell death, reduced H2O2 induced elevations of both reactive oxygen species level and apoptosis. We extended our exploration of ipriflavone to Aß and observed similar effects. These protective effects were comparable to those produced by 17ß-estradiol at similar concentrations. Caspase-3 inhibition, PI3K and MAPK activation were shown to be responsible for such antagonism of ipriflavone on Aß. Our results suggest that ipriflavone, a previously characterized compound, has great potential for expedited clinical translation for the treatment of Alzheimer's disease.

Nuclear protein isoforms: implications for cancer diagnosis and therapy.

Post-translational modifications (PTMs) of nuclear proteins play essential roles in the regulation of gene transcription and signal transduction pathways. Numerous studies have demonstrated a correlation between specific nuclear protein isoforms and cellular malignant process. This communication reviews the impact of major PTM events such as phosphorylation, acetylation, methylation, ubiquitination, and sumoylation on several important nuclear proteins including p53, histones, proliferating cellular nuclear antigen (PCNA), and retinoblastoma protein (Rb) in the process. In addition, the implications of the PTMs as cancer biomarkers and therapeutic targets are considered.

Quality differences between NILs of wheat variety Long 97-586 possessing HMW-GS 7+8 and 7.

The high molecular weight glutenin subunits (HMW-GS) 7+8 were introduced into the Long 97-586 (1, 7, 2+12) wheat variety (Triticum aestivum) by 5 consecutive backcrosses with biochemical marker-assisted selection. Nearly isogenic lines (NILs) of HMW-GS 7 and 7+8 were obtained, and the NILs were planted in the experimental field at the Crop Breeding Institute of Heilongjiang Academy of Agricultural Science in 2004-2006. The field experiments were designed using the two-column contrast arrangement method with six replicates in 2004-2005 and four replicates in 2006. The result of three years experiments showed that the differences between NILs of Long 97-586 with subunit 7 and those with subunits 7+8 in the quality parameters of flour protein content and dry gluten content were negligible (P>0.1). However, the differences in some of the quality parameters were remarkably significant (P<0.01), including wet gluten content, ratio of wet gluten/dry gluten, gluten index, Zeleny sedimentation, ratio of sedimentation/dry gluten, and the farinogram parameters of water absorption, development time, stability, breakdown time and degree of softening. The difference between NILs with subunits 7+8 and subunit 7 was significant (P<0.05) on the alveogram W value and had a critical value (P=0.05) on the alveogram P value in 2006. The results show that HMW-GS 7+8 is far superior to HMW-GS 7 in terms of baking quality. The possibilities of using subunits 7+8 and subunit 7 in breeding strong and weak gluten wheat varieties are discussed in this paper.

Estrogen stimulates degradation of beta-amyloid peptide by up-regulating neprilysin.

Postmenopausal estrogen depletion is a characterized risk factor for Alzheimer disease (AD), a human disorder linked to high levels of beta-amyloid peptide (Abeta) in brain tissue. Previous studies suggest that estrogen negatively regulates the level of Abeta in the brain, but the molecular mechanism is unknown. Here, we provide evidence that estrogen promotes Abeta degradation mainly through a principal Abeta degrading enzyme, neprilysin, in neuroblastoma SH-SY5Y cells. We also demonstrate that up-regulation of neprilysin by estrogen is dependent on both estrogen receptor alpha and beta (ERalpha and ERbeta), and ligand-activated ER regulates expression of neprilysin through physical interactions between ER and estrogen response elements (EREs) identified in the neprilysin gene. These results were confirmed by in vitro gel shift and in vivo chromatin immunoprecipitation analyses, which demonstrate specific binding of ERalpha and ERbeta to two putative EREs in the neprilysin gene. The EREs also enhance ERalpha- and ERbeta-dependent reporter gene expression in a yeast model system. Therefore, the study described here provides a putative mechanism by which estrogen positively regulates expression of neprilysin to promote degradation of Abeta, reducing risk for AD. These results may lead to novel approaches to prevent or treat AD.

Estrogen regulation of the neprilysin gene through a hormone-responsive element.

Our previous data showed that neprilysin (NEP), a zinc metalloendopeptidase, which can degrade amyloid-beta peptide (Abeta) whose central nerve system accumulation is the primary cause of Alzheimer's disease (AD), responds to estrogen in the brain. Recently, it has been shown that the transcription of the neprilysin gene can be up regulated by progesterone, androgens, and glucocorticoids through two androgen response elements within the NEP gene--an androgen response region (ARR) and an androgen response element (ARE). Through a yeast report gene system, we now find that the ARR but not the ARE respond to estrogen. However, androgen could efficiently enhance the expression of the report gene mainly through ARE. Our results indicate that the decrease of NEP, caused by the decline of estrogen or androgen with aging, may be an important factor leading to Abeta accumulation and AD.

A bipartite recombinant yeast system for the identification of subtype-selective estrogen receptor ligands.

Since the estrogen receptor alpha (ERalpha) and beta (ERbeta) are thought to mediate different biological effects, there is intense interest in designing or screening subtype-selective ER ligands. Here, we constructed a biosensor identified as bipartite recombinant yeast system (BRYS) to screen and evaluate subtype-selective ER ligands. Uniform design and immunoblotting was used to determine an optimal dose of copper which control the expression of ERs through a copper inducible metallothionine promoter (CUP1). Some chemicals including natural estrogen (17beta-estradiol), specific estrogen receptor agonist (PPT and DPN), and phytoestrogens (genistein) were tested to validate this system. There was obvious and anticipative discrimination between the agonistic activities when these chemicals were identified and characterized. Furthermore, antagonist (ICI 182,780), which could antagonize the transactivation of estrogen, and chromatin immunoprecipitation (ChIP) were used to confirm that the agonistic effects were mediated through ERs. Comparative studies showed that this system was reproducible and sensitive. 4.7 pM (1.3 ng/l) estrogen was the lowest concentration that could be detected to ERalpha and 0.12 nM (33.5 ng/l) for ERbeta. The results from our study showed that in vitro screening for subtype-selective ER ligands could be conducted in a simple yeast system that is rapid, sensitive, reliable, and quantitative.