The Entamoeba histolytica transcription factor Upstream Regulatory Element 3-Binding Protein (URE3-BP) is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (Upstream Regulatory Element 3 (URE3)) in the promoter regions of hgl5 and fdx1. In this work, precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p<0.0001). Fifteen of the URE3-BP regulated genes were potential membrane proteins, suggesting that one function of URE3-BP is to remodel the surface of E. histolytica in response to a calcium signal. Induction of URE3-BP leads to an increase in tranwell migration, suggesting a possible role in the regulation of cellular motility.

Domino reactions were designed to allow the byproduct of an upstream reaction to be internally recycled to catalyze a downstream reaction in a one-pot tandem sequence. Nitroarene reduction by In(0) generates an amine and In (III) byproducts. Addition of aldehyde followed by Danishefsky's diene or silyl ketene acetal provides access to dihydropyridin-4-ones or beta-amino esters, respectively, in yields that are comparable or superior to the reported stepwise reactions.

The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. The sequencing of the E. histolytica genome combined with the development of an E. histolytica microarray has resulted in the identification of several distinct gene expression profiles associated with virulence. The function of many modulated transcripts is unknown and their role in pathogenicity unclear. They however represent a pool of potential virulence factors that could be targets for the development of novel therapeutics. Efficient tools and methods to characterize these novel virulence-associated genes and proteins would be beneficial. Here we report the use of the Gateway® cloning system to generate the E. histolytica expression vector pAHDEST. To test the usefulness of this system, the vector was used to construct a plasmid containing a recombinant version of the locus EHI_144490, which encoded a protein of unknown function. The recombinant gene was expressed and the recombinant protein, which was Strep-Myctagged, showed a cytoplasmic localization in transfected trophozoites. This expression vector with the Gateway® system should facilitate investigation into the functions of novel proteins in E. histolytica. Keywords Entamoeba histolytica; Gateway® Vector; EHI_144490Entamoeba histolytica is an enteric aerotolerant parasite that can colonize the large intestine, and invade through the intestinal epithelium to cause amebic colitis and liver abscess [1]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. fairly large data sets [2][3][4][5][6][7]. An efficient and high throughput method is needed for cloning and analyzing the function of the proteins encoded by these transcripts. NIH Public AccessThe Gateway® (Invitrogen) system has been used successfully to stably express open reading frames (ORFs) from P. falciparum by recombinational cloning, which like E. histolytica, contains an AT-rich genome [8][9][10]. The Gateway® system also has the potential to rapidly transfer cloned inserts from one vector to another [11]. To construct the E. histolytica 'Destination' plasmid we used the well-characterized plasmid pGir308 as the backbone of the new construct [12]. This vector uses the E. histolytica ferredoxin promoter, which drives strong expression of cloned genes in vivo and in vitro [2,13]. The vector also carries a hygromycin resistance gene, which permits the use of this vector in co-infection studies with other E. histolytica shuttle vectors that carry G418-selectable markers [14].The vector pGir308 was digested with Xb...

BACKGROUND: Health care reform may impact inpatient mental health services by increasing access and changing insurer incentives. We examined whether implementation of the 2014 Affordable Care Act (ACA) was associated with changes in psychiatric length of stay (LOS) and 30-day readmissions for pediatric patients. METHODS: We conducted an interrupted time-series analysis to evaluate LOS and 30-day readmissions during the 30 months before and 24 months after ACA implementation, with a 6-month wash-out period, on patients aged 4 to 17 years who were discharged from the psychiatry unit of a children's hospital. Differences by payer (Medicaid versus non-Medicaid) were examined in moderated interrupted time series. Logistic regression was used to examine the association between psychiatric LOS and 30-day readmissions. RESULTS: There were 1874 encounters in the pre-ACA period and 2186 encounters in the post-ACA period. Compared with pre-ACA implementation, post-ACA implementation was associated with LOS that was significantly decreasing over time (pre-ACA versus post-ACA slope difference: 20.10 days per encounter per month [95% confidence interval 20.17 to 20.02]; P 5 .01), especially for Medicaid-insured patients (pre-ACA versus post-ACA slope difference: 20.14 days per encounter per month [95% confidence interval 20.26 to 20.01]; P 5 .03). The overall proportion of 30-day readmissions increased significantly (pre-ACA 6%, post-ACA 10%; P , .05 for the difference). We found no association between LOS and 30-day readmissions. CONCLUSIONS: ACA implementation was associated with a decline in psychiatric inpatient LOS over time, especially for those on Medicaid, and an increase in 30-day readmissions. LOS was not associated with 30-day inpatient readmissions. Further investigation to understand the drivers of these patterns is warranted.