Asymmetric cross-hemispheric connections link the rat anterior thalamic nuclei with the cortex and hippocampal formation

Dense reciprocal connections link the rat anterior thalamic nuclei with the prelimbic, anterior cingulate and retrosplenial cortices, as well as with the subiculum and postsubiculum. The present study compared the ipsilateral thalamic-cortical connections with the corresponding crossed, contralateral connections between these same sets of regions. All efferents from the anteromedial thalamic nucleus to the cortex, as well as those to the subiculum, remained ipsilateral. In contrast, all of these target sites provided reciprocal, bilateral projections to the anteromedial nucleus. While the anteroventral thalamic nucleus often shared this same asymmetric pattern of cortical connections, it received relatively fewer crossed inputs than the anteromedial nucleus. This difference was most marked for the anterior cingulate projections, as those to the anteroventral nucleus remained almost entirely ipsilateral. Unlike the anteromedial nucleus, the anteroventral nucleus also appeared to provide a restricted, crossed projection to the contralateral retrosplenial cortex. Meanwhile, the closely related laterodorsal thalamic nucleus had almost exclusively ipsilateral efferent and afferent cortical connections. Likewise, within the hippocampus, the postsubiculum seemingly had only ipsilateral efferent and afferent connections with the anterior thalamic and laterodorsal nuclei. While the bilateral cortical projections to the anterior thalamic nuclei originated predominantly from layer VI, the accompanying sparse projections from layer V largely gave rise to ipsilateral thalamic inputs. In testing a potentially unifying principle of anterior thalamic – cortical interactions, a slightly more individual pattern emerged that reinforces other evidence of functional differences within the anterior thalamic and also helps to explain the consequences of unilateral interventions involving these nuclei

Cytoplasmic Prep1 Interacts with 4EHP Inhibiting Hoxb4 Translation

embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function. development effect. mRNA to the 5′ cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development

Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.

Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA

Using a Training Video to Improve Agricultural Workers\u27 Knowledge of On-Farm Food Safety

A training video was produced and evaluated to assess its impact on the food safety knowledge of agricultural workers. Increasing food safety knowledge on the farm may help to improve the safety of fresh produce. Surveys were used to measure workers\u27 food safety knowledge before and after viewing the video. Focus groups were used to determine workers\u27 views of the video and identify areas that could be improved. Results indicated a high level of food safety knowledge, but some significant improvements were observed. The project provides a framework for assessing videos as training tools and suggestions for further research

Tumorigenesis by Meis1 overexpression is accompanied by a change of DNA target-sequence specificity which allows binding to the AP-1 element

Meis1 overexpression induces tumorigenicity but its activity is inhibited by Prep1 tumor suppressor. Why does overexpression of Meis1 cause cancer and how does Prep1 inhibit? Tumor profiling and ChIP-sequencing data in a genetically-defined set of cell lines show that: 1) The number of Meis1 and Prep1 DNA binding sites increases linearly with their concentration resulting in a strong increase of “extra” target genes. 2) At high concentration, Meis1 DNA target specificity changes such that the most enriched consensus becomes that of the AP-1 regulatory element, whereas the specific OCTA consensus is not enriched because diluted within the many extra binding sites. 3) Prep1 inhibits Meis1 tumorigenesis preventing the binding to many of the “extra” genes containing AP-1 sites. 4) The overexpression of Prep1, but not of Meis1, changes the functional genomic distribution of the binding sites, increasing seven fold the number of its “enhancer” and decreasing its “promoter” targets. 5) A specific Meis1 “oncogenic” and Prep1 “tumor suppressing” signature has been identified selecting from the pool of genes bound by each protein those whose expression was modified uniquely by the “tumor-inducing” Meis1 or tumor-inhibiting Prep1 overexpression. In both signatures, the enriched gene categories are the same and are involved in signal transduction. However, Meis1 targets stimulatory genes while Prep1 targets genes that inhibit the tumorigenic signaling pathways

Limited proteolysis analysis of recombinant PREP1 and PBX1.

<p>Full length PREP1 and PBX1 were subjected to limited proteolysis with trypsin. The reactions (total volume 100 μl) were performed at room temperature, 10 μl volumes were taken out at the indicated time points, supplemented with sample buffer and boiled prior to loading onto SDS PAGE. The gels were Coomassie stained. <b>A. Limited proteolysis of PREP1.</b> Lane M, Bio-Rad size standard; two bands of ~40 kDa (1) and ~28 kDa (2) were chosen for subsequent N-terminal sequencing. <b>B. Limited proteolysis of PBX1 with trypsin.</b> Lane M, Bio-Rad size standard; band 3 is the proteolysis fragment chosen for mass spectrometry analysis. <b>C and D. Identification of PREP1 and PBX1 fragments by MALDI-TOF mass spectrometry analysis.</b> Peptides of PREP1 and PBX1 were identified by MALDI-TOF analysis after digestion of fragments 1–3 with trypsin. Fragment 1 contained PREP1 and the matching peptides (red) covered 52.5% starting from the N-terminus and ending at residue 344. Fragment 2 contains the N-terminal part of PREP1 excluding the homeodomain. Fragment 3 contains PBX1, and the peptides (blue) covered 40.2% of the sequence, from residue 7 to 308.</p

Expression and purification of recombinant PREP1 and PBX1.

<p><b>A. Expression and purification of full-length PREP1.</b> SDS PAGE analysis of proteins at different purification stages. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, pellet; Lane 4, cleared lysate; Lane 5,empty; Lane 6, first wash after incubation with glutathione beads (Tris-buffer with 0.5 M NaCl); Lane 7, second wash (Tris-buffer with 0.3 M NaCl); Lane 8, third wash (Tris-buffer with 0.3 M NaCl); Lane 9, glutathione beads before elution; Lane 10; glutathione beads after elution; Lane 11, supernatant from glutathione beads, containing the eluted protein. <b>B. Expression and purification of full-length PBX1.</b> Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, cleared lysate; Lane 4, flow-through after incubation with glutathione beads; Lane 5, first wash (Tris-buffer with 0.5 M NaCl); Lane 6, second wash (Tris-buffer with 0.3 M NaCl); Lane 7, glutathione beads before elution; Lane 8; glutathione beads after elution; Lane 9, empty; Lane 10, supernatant from glutathione beads, containing the eluted protein. <b>C. Expression and purification of PREP1</b>_{<b>1–344</b>}. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, cleared lysate; Lane 4, flow-through after incubation with glutathione beads; Lane 5, first wash (Tris-buffer with 0.5 M NaCl); Lane 6, second wash (Tris-buffer with 0.3 M NaCl); Lane 7, third wash (Tris-buffer with 0.3 M NaCl); Lane 8, empty; Lane 9; glutathione beads before elution; Lane 10, glutathione beads after elution; Lane 11, empty; Lane 12, supernatant from glutathione beads, containing the eluted protein. <b>D. Expression and purification of PBX1</b>_{<b>1–317</b>}. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, pellet; Lane 4, cleared lysate; Lane 5, flow-through after incubation with glutathione beads; Lane 6, first wash (Tris-buffer with 0.5 M NaCl); Lane 7, second wash (Tris-buffer with 0.3 M NaCl); lane 8, third wash (Tris-buffer with 0.3 M NaCl); Lane 9, glutathione beads before elution; Lane 10, glutathione beads after elution; Lane 11, empty; Lane 12, supernatant from glutathione beads, containing the eluted protein. <b>E. Co-expression and purification of GST-PBX1</b>_{<b>1–430</b>}<b>PREP1</b>_{<b>1–436</b>}. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, pellet; Lane 4, cleared lysate; Lane 5, flow-through after incubation with glutathione beads; Lane 6, first wash (Tris-buffer with 0.5 M NaCl); Lane 7, second wash (Tris-buffer with 0.3 M NaCl); Lane 8, third wash (Tris-buffer with 0.3 M NaCl); Lane 9, glutathione beads before elution; Lane 10, glutathione beads after elution; Lane 11, supernatant containing the eluted proteins.</p

Overview of PREP1 and PBX1 constructs.

<p>Overview of PREP1 and PBX1 constructs.</p

DNA binding of purified recombinant PBX1:PREP1 complexes.

<p><b>A and B.</b> EMSA of PBX1:PREP1 complex binding to O1 oligonucleotides. Co-purified full-length PBX1:PREP1 complex (2.5 μM) was incubated in the presence of 1 μg of poly(dIdC) at 1:1 molar ratio with a 22 bp DNA fragment (Panel A) or an 11 bp DNA fragment (Panel B) for 30 minutes at 4°C before loading on gel (Lanes 2). The sequences of DNA are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125789#pone.0125789.t002" target="_blank">Table 2</a>. As a reference the complex without DNA was also loaded on gel (Lanes 1). Note that with the 22 bp oligo (Panel B) at the highest ratios an additional upper band is visible, possibly corresponding to the binding of a second PREP1:PREP1 complex to the DNA. C and D. EMSA of PBX1_1–308:PREP1_1–344:DNA complex. Co-purified PBX1_1–308:PREP1_1–344 complex (2.5 μM) was incubated in the presence of 1 μg of poly(dIdC) at 1:1 molar ratio with the 22 bp (Panel C) or the 11 bp DNA fragment (Panel D) for 30 minutes at 4°C before loading on gels (Lanes 2). In Lanes 1 were loaded the protein complexes without DNA. E. Static light scattering analysis of the PBX1_1–308:PREP1_1–344 complex. The chromatograms show the UV absorbance in blue (scale on the left) and the calculated molecular mass in red (scale on the right). PBX1_1–308:PREP1_1–344 complex was analyzed in the absence (top) and in the presence (bottom) of the 11 bp DNA oligonucleotide</p

Primers used for cloning of PREP1 and PBX1 constructs into PGEX-6p-2rbs.

<p>Primers used for cloning of PREP1 and PBX1 constructs into PGEX-6p-2rbs.</p