Prediction of myopia development among Chinese school-aged children using refraction data from electronic medical records: A retrospective, multicentre machine learning study.

BACKGROUND: Electronic medical records provide large-scale real-world clinical data for use in developing clinical decision systems. However, sophisticated methodology and analytical skills are required to handle the large-scale datasets necessary for the optimisation of prediction accuracy. Myopia is a common cause of vision loss. Current approaches to control myopia progression are effective but have significant side effects. Therefore, identifying those at greatest risk who should undergo targeted therapy is of great clinical importance. The objective of this study was to apply big data and machine learning technology to develop an algorithm that can predict the onset of high myopia, at specific future time points, among Chinese school-aged children. METHODS AND FINDINGS: Real-world clinical refraction data were derived from electronic medical record systems in 8 ophthalmic centres from January 1, 2005, to December 30, 2015. The variables of age, spherical equivalent (SE), and annual progression rate were used to develop an algorithm to predict SE and onset of high myopia (SE ≤ -6.0 dioptres) up to 10 years in the future. Random forest machine learning was used for algorithm training and validation. Electronic medical records from the Zhongshan Ophthalmic Centre (a major tertiary ophthalmic centre in China) were used as the training set. Ten-fold cross-validation and out-of-bag (OOB) methods were applied for internal validation. The remaining 7 independent datasets were used for external validation. Two population-based datasets, which had no participant overlap with the ophthalmic-centre-based datasets, were used for multi-resource validation testing. The main outcomes and measures were the area under the curve (AUC) values for predicting the onset of high myopia over 10 years and the presence of high myopia at 18 years of age. In total, 687,063 multiple visit records (≥3 records) of 129,242 individuals in the ophthalmic-centre-based electronic medical record databases and 17,113 follow-up records of 3,215 participants in population-based cohorts were included in the analysis. Our algorithm accurately predicted the presence of high myopia in internal validation (the AUC ranged from 0.903 to 0.986 for 3 years, 0.875 to 0.901 for 5 years, and 0.852 to 0.888 for 8 years), external validation (the AUC ranged from 0.874 to 0.976 for 3 years, 0.847 to 0.921 for 5 years, and 0.802 to 0.886 for 8 years), and multi-resource testing (the AUC ranged from 0.752 to 0.869 for 4 years). With respect to the prediction of high myopia development by 18 years of age, as a surrogate of high myopia in adulthood, the algorithm provided clinically acceptable accuracy over 3 years (the AUC ranged from 0.940 to 0.985), 5 years (the AUC ranged from 0.856 to 0.901), and even 8 years (the AUC ranged from 0.801 to 0.837). Meanwhile, our algorithm achieved clinically acceptable prediction of the actual refraction values at future time points, which is supported by the regressive performance and calibration curves. Although the algorithm achieved balanced and robust performance, concerns about the compromised quality of real-world clinical data and over-fitting issues should be cautiously considered. CONCLUSIONS: To our knowledge, this study, for the first time, used large-scale data collected from electronic health records to demonstrate the contribution of big data and machine learning approaches to improved prediction of myopia prognosis in Chinese school-aged children. This work provides evidence for transforming clinical practice, health policy-making, and precise individualised interventions regarding the practical control of school-aged myopia.

Early Enteral Nutrition is Associated with Faster Post-Esophagectomy Recovery in Chinese Esophageal Cancer Patients: A Retrospective Cohort Study.

We retrospectively examined a large cohort of esophageal carcinoma patients who received early enteral nutrition (EEN) to clarify the validity of EEN compared with total parenteral nutrition (TPN). Included were a total of 665 consecutive patients with histologically confirmed carcinoma of the esophagus or esophagogastric junction; and all patients underwent esophagectomy. The patients were divided into two groups: TPN (n = 262) and EEN (n = 403). The TPN group consisted of patients who only received intravenous nutrition support after operation. The postoperative length of hospital stay (PLOS), anastomotic leakage, mortality after surgery, and hospital charges were reviewed and analyzed. Compared with the TPN group, the EEN group had significantly shorter mean PLOS (15.6 days vs. 22.5 days; P < 0.01). Multivariable linear regression analysis revealed EEN to be associated with shorter PLOS even after adjustment for tumor histology, tumor location, type of esophagectomy, and postoperative albumin infusion. Hospital charges were also significantly less for those in the EEN group than the TPN group. There was no significant difference between the two groups regarding the complication of anastomotic leakage and clinical outcome after surgery. These findings suggest that EEN reduces PLOS and hospital charges of Chinese esophageal cancer patients who had an esophagectomy.

Construction and implications of structural equation modeling network for pediatric cataract: a data mining research of rare diseases.

BACKGROUND: The majority of rare diseases are complex diseases caused by a combination of multiple morbigenous factors. However, uncovering the complex etiology and pathogenesis of rare diseases is difficult due to limited clinical resources and conventional statistical methods. This study aims to investigate the interrelationship and the effectiveness of potential factors of pediatric cataract, for the exploration of data mining strategy in the scenarios of rare diseases. METHODS: We established a pilot rare disease specialized care center to systematically record all information and the entire treatment process of pediatric cataract patients. These clinical records contain the medical history, multiple structural indices, and comprehensive functional metrics. A two-layer structural equation model network was applied, and eight potential factors were filtered and included in the final modeling. RESULTS: Four risk factors (area, density, location, and abnormal pregnancy experience) and four beneficial factors (axis length, uncorrected visual acuity, intraocular pressure, and age at diagnosis) were identified. Quantifiable results suggested that abnormal pregnancy history may be the principle risk factor among medical history for pediatric cataracts. Moreover, axis length, density, uncorrected visual acuity and age at diagnosis served as the dominant factors and should be emphasized in regular clinical practice. CONCLUSIONS: This study proposes a generalized evidence-based pattern for rare and complex disease data mining, provides new insights and clinical implications on pediatric cataract, and promotes rare-disease research and prevention to benefit patients.

Objective method for evaluating orthodontic treatment from the lay perspective: An eye-tracking study.

INTRODUCTION: Currently, few methods are available to measure orthodontic treatment need and treatment outcome from the lay perspective. The objective of this study was to explore the function of an eye-tracking method to evaluate orthodontic treatment need and treatment outcome from the lay perspective as a novel and objective way when compared with traditional assessments. METHODS: The scanpaths of 88 laypersons observing the repose and smiling photographs of normal subjects and pretreatment and posttreatment malocclusion patients were recorded by an eye-tracking device. The total fixation time and the first fixation time on the areas of interest (eyes, nose, and mouth) for each group of faces were compared and analyzed using mixed-effects linear regression and a support vector machine. The aesthetic component of the Index of Orthodontic Treatment Need was used to categorize treatment need and outcome levels to determine the accuracy of the support vector machine in identifying these variables. RESULTS: Significant deviations in the scanpaths of laypersons viewing pretreatment smiling faces were noted, with less fixation time (P <0.05) and later attention capture (P <0.05) on the eyes, and more fixation time (P <0.05) and earlier attention capture (P <0.05) on the mouth than for the scanpaths of laypersons viewing normal smiling subjects. The same results were obtained when comparing posttreatment smiling patients, with less fixation time (P <0.05) and later attention capture on the eyes (P <0.05), and more fixation time (P <0.05) and earlier attention capture on the mouth (P <0.05). The pretreatment repose faces exhibited an earlier attention capture on the mouth than did the normal subjects (P <0.05) and posttreatment patients (P <0.05). Linear support vector machine classification showed accuracies of 97.2% and 93.4% in distinguishing pretreatment patients from normal subjects (treatment need), and pretreatment patients from posttreatment patients (treatment outcome), respectively. CONCLUSIONS: The eye-tracking device was able to objectively quantify the effect of malocclusion on facial perception and the impact of orthodontic treatment on malocclusion from the lay perspective. The support vector machine for classification of selected features achieved high accuracy of judging treatment need and treatment outcome. This approach may represent a new method for objectively evaluating orthodontic treatment need and treatment outcome from the perspective of laypersons.

Statistical modelling for thoracic surgery using a nomogram based on logistic regression.

A well-developed clinical nomogram is a popular decision-tool, which can be used to predict the outcome of an individual, bringing benefits to both clinicians and patients. With just a few steps on a user-friendly interface, the approximate clinical outcome of patients can easily be estimated based on their clinical and laboratory characteristics. Therefore, nomograms have recently been developed to predict the different outcomes or even the survival rate at a specific time point for patients with different diseases. However, on the establishment and application of nomograms, there is still a lot of confusion that may mislead researchers. The objective of this paper is to provide a brief introduction on the history, definition, and application of nomograms and then to illustrate simple procedures to develop a nomogram with an example based on a multivariate logistic regression model in thoracic surgery. In addition, validation strategies and common pitfalls have been highlighted.

Multiple P-TEFbs cooperatively regulate the release of promoter-proximally paused RNA polymerase II.

The association of DSIF and NELF with initiated RNA Polymerase II (Pol II) is the general mechanism for inducing promoter-proximal pausing of Pol II. However, it remains largely unclear how the paused Pol II is released in response to stimulation. Here, we show that the release of the paused Pol II is cooperatively regulated by multiple P-TEFbs which are recruited by bromodomain-containing protein Brd4 and super elongation complex (SEC) via different recruitment mechanisms. Upon stimulation, Brd4 recruits P-TEFb to Spt5/DSIF via a recruitment pathway consisting of Med1, Med23 and Tat-SF1, whereas SEC recruits P-TEFb to NELF-A and NELF-E via Paf1c and Med26, respectively. P-TEFb-mediated phosphorylation of Spt5, NELF-A and NELF-E results in the dissociation of NELF from Pol II, thereby transiting transcription from pausing to elongation. Additionally, we demonstrate that P-TEFb-mediated Ser2 phosphorylation of Pol II is dispensable for pause release. Therefore, our studies reveal a co-regulatory mechanism of Brd4 and SEC in modulating the transcriptional pause release by recruiting multiple P-TEFbs via a Mediator- and Paf1c-coordinated recruitment network.

The relationship between work stress and work ability among power supply workers in Guangdong, China: a cross-sectional study.

BACKGROUND: Faced with the challenge of population aging, a prolonged working life is increasingly important in today's society. Maintaining work ability of employees is one of the effective ways to cope with the challenges to sustainability of the workforce presented by population aging. Researchers have shown ongoing interest in exploring the determinants of restricted work ability. The aim of this study was to evaluate the effects of work stress on work ability among power supply workers in Guangdong, China. METHODS: A cross-sectional study was conducted among power supply workers during August 2014 to September 2014. A total of 805 subjects were enrolled in the study. Work stress was assessed by the Job Content Questionnaire and the Effort Reward Imbalance Questionnaire. Work ability was assessed by the Work Ability Index (WAI). The structural equation model was applied to test the relationship between different work stress components and work ability simultaneously using the Job Demands-Resources model as a framework. RESULTS: Job resources (measured by job control, reward and social support) were positively and directly associated with work ability (ß = 0.70, P < 0.001). The association between job demands and work ability was also statistically significant (ß = -0.09, P = 0.030). In addition, the findings also supported previous studies in that job demands were correlated with job resources (ß = -0.26, P < 0.001). CONCLUSIONS: Our findings suggest that decision makers and health care providers should consider increasing job resources available to power supply workers. Consideration of organizational changes related to the design of the job task also would be useful to improve the employees' work ability.

Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway.

Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKCε/PKD3/NF-κB signaling pathway.

Histone cross-talk connects protein phosphatase 1α (PP1α) and histone deacetylase (HDAC) pathways to regulate the functional transition of bromodomain-containing 4 (BRD4) for inducible gene expression.

Transcription elongation has been recognized as a rate-limiting step for the expression of signal-inducible genes. Through recruitment of positive transcription elongation factor P-TEFb, the bromodomain-containing protein BRD4 plays critical roles in regulating the transcription elongation of a vast array of inducible genes that are important for multiple cellular processes. The diverse biological roles of BRD4 have been proposed to rely on its functional transition between chromatin targeting and transcription regulation. The signaling pathways and the molecular mechanism for regulating this transition process, however, are largely unknown. Here, we report a novel role of phosphorylated Ser(10) of histone H3 (H3S10ph) in governing the functional transition of BRD4. We identified that the acetylated lysines 5 and 8 of nucleosomal histone H4 (H4K5ac/K8ac) is the BRD4 binding site, and the protein phosphatase PP1α and class I histone deacetylase (HDAC1/2/3) signaling pathways are essential for the stress-induced BRD4 release from chromatin. In the unstressed state, phosphorylated H3S10 prevents the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby locking up the majority of BRD4 onto chromatin. Upon stress, PP1α-mediated dephosphorylation of H3S10ph allows the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby leading to the release of chromatin-bound BRD4 for subsequent recruitment of P-TEFb to enhance the expression of inducible genes. Therefore, our study revealed a novel mechanism that the histone cross-talk between H3S10ph and H4K5ac/K8ac connects PP1α and HDACs to govern the functional transition of BRD4. Combined with previous studies on the regulation of P-TEFb activation, the intricate signaling network for the tight control of transcription elongation is established.

Abnormal response to the anorexic effect of GHS-R inhibitors and exenatide in male Snord116 deletion mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a genetic disease characterized by persistent hunger and hyperphagia. The lack of the Snord116 small nucleolar RNA cluster has been identified as the major contributor to PWS symptoms. The Snord116 deletion (Snord116del) mouse model manifested a subset of PWS symptoms including hyperphagia and hyperghrelinemia. In this study, male Snord116del mice were characterized and tested for their acute and chronic responses to anorexic substances related to the ghrelin pathway. In comparison with their wild-type littermates, the food intake rate of Snord116del mice was 14% higher when fed ad libitum, and 32% to 49% higher within 12 hours after fasting. Fasted Snord116del mice were less sensitive to the acute anorexic effect of competitive antagonist [d-Lys(3)]-GHRP6, YIL-781, and reverse agonist [d-Arg(1),d-Phe(5),d-Trp(7,9),Leu(11)]-substance P (SPA) of ghrelin receptor GHS-R. All 3 GHS-R inhibitors failed to inhibit chronic food intake of either Snord116del or wild-type mice due to rapid adaptation. Although fasted Snord116del mice had normal sensitivity to the acute anorexic effect of glucagon-like peptide 1 receptor agonist exenatide, those fed ad libitum required a higher dose and more frequent delivery to achieve ∼15% suppression of long-term food intake in comparison with wild-type mice. Ghrelin, however, is unlikely to be essential for the anorexic effect of exenatide in fed mice, as shown by the fact that exenatide did not reduce ghrelin levels in fed mice and food intake of ghrelin(-/-) mice fed ad libitum could be suppressed by exenatide. In conclusion, this study suggests that GHS-R may not be an effective therapeutic target, and in contrast, exenatide may produce anorexic effect in PWS individuals.

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

Divalent cation tolerance protein binds to ß-secretase and inhibits the processing of amyloid precursor protein.

The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by ß-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1-interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.

Signal-induced Brd4 release from chromatin is essential for its role transition from chromatin targeting to transcriptional regulation.

Bromodomain-containing protein Brd4 is shown to persistently associate with chromosomes during mitosis for transmitting epigenetic memory across cell divisions. During interphase, Brd4 also plays a key role in regulating the transcription of signal-inducible genes by recruiting positive transcription elongation factor b (P-TEFb) to promoters. How the chromatin-bound Brd4 transits into a transcriptional regulation mode in response to stimulation, however, is largely unknown. Here, by analyzing the dynamics of Brd4 during ultraviolet or hexamethylene bisacetamide treatment, we show that the signal-induced release of chromatin-bound Brd4 is essential for its functional transition. In untreated cells, almost all Brd4 is observed in association with interphase chromatin. Upon treatment, Brd4 is released from chromatin, mostly due to signal-triggered deacetylation of nucleosomal histone H4 at acetylated-lysine 5/8 (H4K5ac/K8ac). Through selective association with the transcriptional active form of P-TEFb that has been liberated from the inactive multi-subunit complex in response to treatment, the released Brd4 mediates the recruitment of this active P-TEFb to promoter, which enhances transcription at the stage of elongation. Thus, through signal-induced release from chromatin and selective association with the active form of P-TEFb, the chromatin-bound Brd4 switches its role to mediate the recruitment of P-TEFb for regulating the transcriptional elongation of signal-inducible genes.

An efficient approach for site-directed mutagenesis using central overlapping primers.

QuikChange is a popular method for site-directed mutagenesis in structural and functional studies of proteins and nucleic acids. However, the standard protocol is often inefficient in producing the desired mutations. Here we present a novel strategy for primer design, central overlapping primers (COP), which employs a pair of bipartite primers of different lengths, with the short primer complementary to the middle region of the long primer. The COP method is efficient and robust in generating approximately 90% mutation rate without supercompetent Escherichia coli cells or laborious screening for positive clones.

Presenilins regulate the cellular level of the tumor suppressor PTEN.

Alzheimer's Disease (AD) is characterized by amyloid plaques consisting of beta-amyloid (Abeta) peptides and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Abeta is proteolytically derived from its precursor protein through cleavages by beta-secretase and gamma-secretase complex comprising presenilins (PS, PS1/PS2), nicastrin, APH-1 and PEN-2. PS1 is also known to activate the PI3K/Akt cell survival pathway in a gamma-secretase-independent manner. The tumor suppressor PTEN, which antagonizes the PI3K/Akt pathway, has increasingly been recognized to play a key role in neural functions and its level found reduced in AD brains. Here, we demonstrate that the protein level of PTEN is dramatically reduced in cultured cells and embryonic tissues deficient in PS, and in the cortical neurons of PS1/PS2 conditional double knockout mice. Restoration of PS in PS-deficient cells reverses the reduction of PTEN. Regulation of PTEN by PS is independent of the PS/gamma-secretase activity since impaired gamma-secretase by the gamma-secretase inhibitor treatment or due to nicastrin deficiency has little effect on the protein level of PTEN. Our data suggest an important role for PS in signaling pathways involving PI3K/Akt and PTEN that are crucial for physiological functions and the pathogenesis of multiple diseases.

Proteomic approach for caudal trauma-induced acute phase proteins reveals that creatine kinase is a key acute phase protein in amphioxus humoral fluid.

Elevated creatine kinase (CK) in the circulation was generally regarded to be a passive release from muscle damage. We utilized proteomic methodologies to characterize amphioxus humoral fluid APPs in response to caudal trauma, and found several spots of CK alterations with up-regulation and pI shift. Its amount and enzyme activity showed a dynamic pattern of APP in humoral fluid accompanied with a reduction in enzyme activity of muscle, whereas there was no significant difference in CK amount of muscle and the other tissues and in CK enzyme activity of the other tissues between different time points of sample collection following caudal trauma. In addition, CK phosphorylation regulation during injury was not achieved by monoclonal antibodies separately against phosphothreonine, phosphotyrosine, and phosphoserine. These results suggested that the CK elevation of humoral fluid might be from muscle, being an active response to caudal trauma rather than a passive release from muscle damage. Therefore, CK ability in response to caudal trauma should be highly concerned.

Transcriptional regulation of APH-1A and increased gamma-secretase cleavage of APP and Notch by HIF-1 and hypoxia.

The proteolytic cleavage of Alzheimer beta-amyloid precursor protein (APP) and signaling receptor Notch is mediated by the PS/gamma-secretase complex, which consists of presenilins, nicastrin, APH-1, and PEN-2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. Here we characterized the 5'-flanking region of the human APH-1A gene and identified a 271-bp fragment containing the transcription initiation site for the promoter activity. Sequence analysis, mutagenesis, and gel shift studies revealed a binding of AP4 and HIF-1 to the promoter, which affects the promoter activity. Activation of HIF-1 by short-term NiCl2 treatments (a condition of chemical hypoxia) dramatically increased APH-1A mRNA and protein expression, accompanied by increased secretion of Abeta and decreased APP CTFs formation, indicative of an increase in gamma-secretase activity. NiCl2 treatments had little effect on APP and the other three components of the gamma-secretase complex. The cellular concentration of Notch intracellular domain (NICD) was also increased by the hypoxic treatment. Our results demonstrate that APH-1A expression and the gamma-secretase mediated Abeta and Notch NICD generation are regulated by HIF-1, and the specific control of APH-1A expression may imply physiological functions uniquely assigned to APH-1A.

Transcriptional regulation of PEN-2, a key component of the gamma-secretase complex, by CREB.

Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's beta-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APPalpha and Abeta without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2.

Establishment of persistent avian infectious bronchitis virus infection in antibody-free and antibody-positive chickens.

Avian infectious bronchitis virus (IBV) causes a highly contagious and economically significant disease in chickens. Establishment of a carrier state in IBV infection and the potential for the persistent virus to undergo mutations and recombination in chicken tissues have important consequences for disease management. Nevertheless, whether chickens can maintain persistent IBV infection in the absence of reinfection from exogenous sources or the presence of antibody in the host can modulate virus persistence remains unclear. Indeed, whether or not IBV genome can undergo genetic changes during in vivo infection has not been demonstrated experimentally. In the present study, IBV shedding and tissue persistence were monitored in individual chickens maintained under strict isolation that precluded reinfection from exogenous sources. In the first of two experiments, intranasal exposure of 6-wk-old antibody-free chickens to IBV vaccine virus resulted in intermittent shedding of the virus from both trachea and cloaca of individual birds for up to 63 days. Also, the virus was recovered from the internal organs (spleen, gonad, kidney, lung, cecal tonsil, and cloacal bursa) of six of eight birds killed at various intervals between 27 and 163 days postinoculation (DPI). In the second experiment, IBV exposure of 1-day-old maternal antibody-positive chicks led to periodic virus shedding from the trachea and cloaca in all chickens until 77 days; however, internal organs (lungs and kidneys) of only one of seven birds (killed at 175 DPI) were virus positive, suggesting that presence of antibody at the time of infection protects internal organs from IBV infection. When the lung and kidney isolates of IBV from the latter experiment were compared with the parent-vaccine virus, no changes in their antigenicity, tissue tropism, or the nucleotide sequence of the S1 glycoprotein gene were observed. These findings indicate that, unlike the mammalian coronaviruses, propensity for frequent genetic change may not be inherent in the IBV genome.