TBP is not required for <i>IDE</i> transcription initiation.

<p>(<b>A</b>) and (<b>B</b>) TBP is not associated with the <i>IDE</i> promoter. Binding of TBP and RNA polymerase II to the <i>IDE</i> promoter in NIH-3T3 (<b>A</b>) and HeLa (<b>B</b>) cells was tested by ChIP assays. Promoters of <i>GAPDH</i> and <i>EF1α1</i> were used as positive controls for TBP and RNA polymerase II binding. Negative controls were also included. Data are represented as relative enrichment to the input. (<b>C</b>) <i>In vitro</i> transcription assays. DNA template for the <i>CMV</i>, mouse <i>IDE</i> or human <i>IDE</i> promoter was incubated with HeLa nuclear extracts and ribonucleotides. The resulting transcripts were purified, digested with DNase I to eliminate the DNA template and detected by RT-PCR. An internal control RNA was included to indicate the purification efficiency of different samples. Complete elimination of the DNA template was confirmed by PCR. Transcription from all the three DNA templates proceeded only when both the DNA template and HeLa nuclear extracts existed, and was inhibited by 1 µg/mL of α-amanitin. (<b>D</b>) Heat-inactivation of TBP does not block <i>IDE</i> transcription. HeLa nuclear extracts were heated at 47°C for 15 min before <i>in vitro</i> transcription assays.</p

Sequential mutation of the mouse <i>IDE</i> core promoter.

<p>The mouse <i>IDE</i> core promoter contains three CGGCG repeats and a NRF-1 binding motif. The core promoter region with sequential mutations (M1 to M11) was cloned into the pGL3-basic vector. Luciferase activity of the reporter plasmids in NIH-3T3 and HeLa cells is represented as a percentage of the wild-type (WT) reporter plasmid.</p

Mapping the core promoter region of mouse <i>IDE</i>.

<p>(<b>A</b>) and (<b>B</b>) The region between −23 and +9 of the mouse <i>IDE</i> promoter is essential for transcription initiation and behaves as the core promoter. Different truncations of the mouse <i>IDE</i> promoter were cloned into the pGL3-basic vector. NIH-3T3 or HeLa cells were transiently transfected with luciferase reporter plasmids of the mouse <i>IDE</i> promoter (0.8 µg) and <i>Renilla</i> luciferase reporter plasmid (pCMV-RL, 8 ng). Twenty-four hours after transfection, cells were lysed, and the luciferase activity was determined. Firefly luminescence signal was normalized based on the <i>Renilla</i> luminescence signal. Data are represented as fold of the firefly luciferase activity of the pGL3-basic vector.</p

Nuclear Respiratory Factor 1 Mediates the Transcription Initiation of <em>Insulin-Degrading Enzyme</em> in a TATA Box-Binding Protein-Independent Manner

<div><p>CpG island promoters often lack canonical core promoter elements such as the TATA box, and have dispersed transcription initiation sites. Despite the prevalence of CpG islands associated with mammalian genes, the mechanism of transcription initiation from CpG island promoters remains to be clarified. Here we investigate the mechanism of transcription initiation of the CpG island-associated gene, <em>insulin-degrading enzyme</em> (<em>IDE</em>). <em>IDE</em> is ubiquitously expressed, and has dispersed transcription initiation sites. The <em>IDE</em> core promoter locates within a 32-bp region, which contains three CGGCG repeats and a nuclear respiratory factor 1 (NRF-1) binding motif. Sequential mutation analysis indicates that the NRF-1 binding motif is critical for <em>IDE</em> transcription initiation. The NRF-1 binding motif is functional, because NRF-1 binds to this motif <em>in vivo</em> and this motif is required for the regulation of <em>IDE</em> promoter activity by NRF-1. Furthermore, the NRF-1 binding site in the <em>IDE</em> promoter is conserved among different species, and dominant negative NRF-1 represses endogenous IDE expression. Finally, TATA-box binding protein (TBP) is not associated with the <em>IDE</em> promoter, and inactivation of TBP does not abolish <em>IDE</em> transcription, suggesting that TBP is not essential for <em>IDE</em> transcription initiation. Our studies indicate that NRF-1 mediates <em>IDE</em> transcription initiation in a TBP-independent manner, and provide insights into the potential mechanism of transcription initiation for other CpG island-associated genes.</p> </div

The NRF-1 binding motif in the mouse <i>IDE</i> promoter is functional.

<p>(<b>A</b>) Representation of wild-type (−136/+139 WT) and the NRF-1 binding site-mutated (NRF-mut) luciferase reporter plasmids of the mouse <i>IDE</i> promoter. (<b>B</b>) and (<b>C</b>) Dominant negative NRF-1 represses <i>IDE</i> promoter activity. NIH-3T3 and HeLa cells were transiently co-transfected with wild-type (-136/+139 WT) or NRF-1 binding site-mutated (NRF-mut) <i>IDE</i> reporter plasmids (0.4 µg) and <i>Renilla</i> luciferase plasmid (4 ng) along with or without dominant negative (DN) NRF-1 expression plasmids (0.4 µg). Twenty-four hours after transfection, cells were lysed, and the luciferase activity was examined. Firefly luminescence signal was normalized based on the <i>Renilla</i> luminescence signal. (<b>D</b>) ChIP. NRF-1 binding to the <i>IDE</i> promoter in NIH-3T3 cells was determined by ChIP. The promoter of <i>cytochrome c</i> (<i>cyt c</i>) is used as a positive control for NRF-1 binding, while the promoter of <i>GAPDH</i> acts as a negative control.</p

The NRF-1 binding site in the <i>IDE</i> promoter is conserved among different species.

<p>(<b>A</b>) The NRF-1 binding site in the <i>IDE</i> promoter is conserved among different species. The underlined region indicates the conserved NRF-1 binding motif. (<b>B</b>) The NRF-1 binding motif is critical for human <i>IDE</i> transcription initiation. Different truncations of the human <i>IDE</i> promoter were cloned into the pGL3-basic vector. Luciferase activity of the reporter plasmids in HeLa cells is represented as fold of the pGL3-basic vector. (<b>C</b>) ChIP. NRF-1 binding to the <i>IDE</i> promoter in HeLa cells was determined by ChIP. The promoter of <i>cytochrome c</i> (<i>cyt c</i>) is used as a positive control for NRF-1 binding, while the promoter of <i>GAPDH</i> acts as a negative control. (<b>D</b>) The NRF-1 binding motif is essential for the effect of dominant negative NRF-1 on human <i>IDE</i> promoter activity. HeLa cells were transiently co-transfected with wild-type (−484/+173) or NRF-1 binding site-mutated (NRF-mut) human <i>IDE</i> reporter plasmids (0.4 µg) and <i>Renilla</i> luciferase plasmid (4 ng) along with or without dominant negative (DN) NRF-1 expression plasmids (0.4 µg). Twenty-four hours after transfection, cells were lysed, and the luciferase activity was examined. Firefly luminescence signal was normalized based on the <i>Renilla</i> luminescence signal.</p

The mouse <i>IDE</i> promoter contains a CpG island and has dispersed transcription initiation sites.

<p>(<b>A</b>) Representation of the mouse <i>IDE</i> promoter. The dashed line indicates the unknown region which was cloned and sequenced in this study. The mouse <i>IDE</i> promoter contains a CpG island with a length of approximately 1300 bp. (<b>B</b>) The transcription initiation sites of mouse <i>IDE</i>. The frequency of transcription initiation from different sites is shown. The mouse <i>IDE</i> promoter has dispersed transcription initiation sites located within a window of 62 bp. The first transcription initiation site is underlined.</p

Equipment-Free Quantitative Aptamer-Based Colorimetric Assay Based on Target-Mediated Viscosity Change

In this paper, we describe an aptamer-based colorimetric assay (ABCA), which integrates enzyme-loaded microparticles for signal amplification with distance measurement for equipment-free quantitative readout. The distance measurement readout is on the basis of target-induced selective reduction in viscosity of reaction solution. Its utility is well demonstrated with inexpensive, sensitive, and selective detection of adenosine (model analyte) in buffer samples and real samples of human serum and urine with the naked eye. This ABCA method just requires operators to simply count the number of colored distance-relevant marked bars on the calibrated glass microsyringes (testing containers) to provide quantitative results. It thus holds great promise for wide applications particularly in limited-resource settings

Additional file 1 of An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs

Additional file 1: Supplemental Fig. 1. Ovalbumin-EGFP fusion protein structures predicted by alphafold2 software. A. Structure of ovalbumin-EGFP fusion proteins with GS linker. B. Structure of ovalbumin-EGFP fusion proteins with (GS)3 linker. A. Structure of ovalbumin-EGFP fusion proteins with 32aa linker. A. Structure of ovalbumin-EGFP fusion proteins with (EAAAK)5 linker. Supplemental Fig. 2. Plasmids used in OVAL gene modified or CAG-EGFP chicken. A. Structure of CRISPR/Cas9 and donor plasmid used in ovalbumin locus site-specific gene integration. B. PiggayBac plasmid used in CAG-EGFP transgene chicken. Supplemental Fig. 3. EGFP fluorescence detection of PGCs derived from CAG-EGFP chicken. Supplemental Fig. 4. mCherry fluorescence detection of eggs from OVAL-E3-EGFP chicken. Supplemental Fig. 5. Immune fluorescence (IF) analysis of oviduct tissues from WT three-yellow chicken, CAG-EGFP chicken, and OVAL-E3-EGFP chicken. A. Ovalbumin antibody IF analysis of oviducts from WT three-yellow chicken, CAG-EGFP chicken, and OVAL-E3-EGFP chicken. B. EGFP antibody IF analysis of chicken oviduct from OVAL-E3-EGFP chicken. Supplemental Fig. 6. EGFP immune fluorescence analysis of oviduct tissues from wild type three-yellow chicken and CAG-EGFP chicken. Supplemental Fig. 7. FACS of EGFP+/EGFP- cells from OVAL-E3-EGFP chicken oviducts

An insight into the curdione biotransformation pathway by <i>Aspergillus niger</i>

<div><p>Curdione (<b>1</b>), a sesquiterpene with a germacrane skeleton from rhizomes of <i>Curcuma wenyujin</i>, has attracted attention due to its important pharmacological properties. Herein, we investigated the chemo-biotransformation of curdione (<b>1</b>) systematically using <i>Aspergillus niger</i> AS 3.739. Regio- and stereoselective hydroxylation of curdione with filamentous fungus <i>A. niger</i> AS 3.739 led to seven metabolites including four new compounds 3α-hydroxycurcumalactone, 2β-hydroxycurcumalactone, (10<i>S</i>)-9,10-dihydroxy-curcumalactone and (10<i>R</i>)-9,10-dihydroxy-curcumalactone. Their structures were determined by spectroscopic techniques including two-dimensional NMR and TOF-MS (Time of Flight Mass Spectrometry). Based upon the analysis of biological and chemical conversions of curdione, a tentative metabolic pathway via chemo-bio cascade reactions is proposed in <i>A. niger</i> system, which provides an insight into the corresponding metabolism of curdione in animal systems. In addition, experiments with selected monooxygenase inhibitors suggest that cytochrome P450 monooxygenase played a crucial role in the hydroxylation of curdione.</p></div