human mitochondrial dna monitoring primer  (TaKaRa)

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet:

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: The BRD4 tandem bromodomains stably associate with H4 poly-acetylated nucleosomes in biolayer interferometry. ( A-D ) Biolayer interferometry binding assays of ( A ) BD1-2 with H4 poly-acetylated nucleosomes ([H4K5acK8acK12acK16ac] 2 ), ( B ) BD1-2 with unacetylated nucleosomes, ( C ) BD1 alone with H4 poly-acetylated nucleosomes, and ( D ) BD1 alone with unacetylated nucleosomes. Protein concentrations are indicated at the right of each plot. Note the difference in y-axis scale between ( A ) 1 to -0.5 nm and ( B-D ) 0.3 to -0.3 nm. Octet fitting of BD1-2 binding curves to H4 poly-acetylated nucleosomes (12.5, 6.25, and 3.12 μM) using a 2:1 heterogeneous ligand model produced R 2 values >0.98 (Fig. S2). Experiments were performed in triplicate.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Stable Transfection, Binding Assay, Produced

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: AlphaFold model suggesting a plausible mode of BRD4-S engagement with an H4 K12 acetylated nucleosome ([H4K12ac] 2 ). ( A ) Top-ranked AlphaFold model prediction of BRD4-S (blue) in complex with an H4K12ac nucleosome (H2A, golden; H2B, tomato; H3, green; H4, violet, Widom 601 DNA, grey). BD1 and BD2 are shaded in darker blue for clarity. ( B ) Rotated (45°) view of the modeled BRD4-S-H4K12ac nucleosome complex. ( C ) Zoom-in of the BD1 acetyl-lysine binding pocket highlighting residues Y97 and N140 positioned near H4K12ac. ( D ) Zoom-in of the BD2 acetyl-lysine binding pocket highlighting residues Y390 and N433 positioned near H4K12ac. This view also shows the relevant placement of the C-terminal BRD4-S region including the BID, ET, and phosphoregulatory sites, in the model.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: Bromodomain binding pocket mutations in the BRD4-S disrupt breast cancer growth and migration phenotypes. ( A ) Cell growth assay following BRD4-S depletion and complementation with the indicated 3xFLAG-BRD4-S constructs (mean ± SEM; n≥5 independent experiments). Statistical analysis as described in Methods. ( B ) Transwell migration assay following BRD4-S depletion and complementation with the indicated 3xFLAG-BRD4-S constructs (mean ± SEM; n ≥ 3 independent experiments). Statistical significance was calculated by two-tailed t-tests (***p<0.0005, ****p<0.0001). ( C ) Western blots of endogenous BRD4-S depletion and rescue by 3xFLAG-tagged BRD4-S (1-719) constructs in doxycycline-treated MDA-MB-231 cells. Blots were probed with an antibody raised for the N-terminus of BRD4 (BRD4-S; for endogenous ∼100 kDa), M2 FLAG (FLAG), and β-actin (Actin) ( D ) Graphical illustration of putative model for BRD4-S chromatin engagement and regulation of cancer cell growth and migration (BioRender, https://BioRender.com/7dfa8yg ). This model shows multiple possible binding modes of BRD4-S on poly-acetylated nucleosomes that would be difficult to distinguish or determine in biochemical and structural studies, yet could all contribute to recruitment of regulatory complexes and transcriptional control of nearby genes including those for cancer growth and migration.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay, Migration, Growth Assay, Construct, Transwell Migration Assay, Two Tailed Test, Western Blot, Control

Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B induces mitochondrial damage in PDAC cells. (A) Results of label-free quantitative proteomics after Agrimol B treatment for 24 h. (B) Western blot analysis of HADHA, TIM23, and SOD2 in PANC-1 and AsPC-1 cells. (C) Flow cytometric analysis of Fluo-4 AM accumulation in cells treated with or without 45 μmol/l Agrimol B. (D) Representative images of Fluo-4 AM accumulation in PANC-1 and AsPC-1 cells treated with or without 45 μmol/l Agrimol B for 24 h. Scale bars, 10 μm. (E) Flow cytometric analysis of mitochondrial ROS accumulation in cells treated with or without 45 μmol/l Agrimol B. (F) Representative images of mitochondrial morphology stained with Annexin V-FITC and MitoTracker Red CMXRos in PANC-1 and AsPC-1 cells treated with or without 45 μmol/l Agrimol B for 24 h. Scale bars, 10 μm. (G) Quantitative RT-PCR analysis of mtDNA copies. (H) ATP levels in PANC-1 and AsPC-1 cells treated with or without 45 μmol/l Agrimol B for 24 h. (I) Mitochondrial morphology was observed via transmission electron microscopy after treatment with or without Agrimol B for 24 h. Scale bars, 500 nm.

Article Snippet: After different transfections, PANC-1 and AsPC-1 cells were seeded in 6-well plates and maintained for 24 h. DNA was extracted according to the manufacturer’s instructions (NucleoSpin Tissue) and mtDNA was detected using the Human Mitochondrial DNA Monitoring Primer Set (Takara).

Techniques: Quantitative Proteomics, Western Blot, Staining, Quantitative RT-PCR, Transmission Assay, Electron Microscopy

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Quantitative analysis of hSMUG1-mediated uracil excision using nucleotide MALDI-TOF mass spectrometry. (A) Schematic representation of experimental design. A duplex DNA substrate was generated by annealing an 18-nucleotide lesion strand containing a signal uridine (U) with a complementary 19-nucleotide template strand (T), thereby introducing a U:G mispair at the centre of the duplex. Recombinant hSMUG1 catalysed the monofunctional glycosylase reaction, excising uracil to yield an abasic (AP site on the lesion strand called abasic primer (AP). The reaction products were subsequently profiled by nucleotide MALDI-TOF MS. Distinct m/z peaks corresponding to the U-, T-, and AP-containing strands enabled direct quantification of the excision reaction. Notably, uracil removal resulted in a 94 Da mass decrease, allowing precise discrimination between U and AP fragments. (B) In the absence of hSMUG1, the duplex exhibited two distinct peaks at 5789.8 Da (T strand) and 5541.6 Da (U strand) with high signal-to-noise ratios, confirming substrate integrity and mass resolution. (C) Upon hSMUG1 treatment, the uracil-containing (U) peak was predominantly converted into the AP peak at 5447.6 Da, indicating efficient excision of uracil from the U:G substate. Excision efficiency was determined from peak intensities using the formula: AP/(AP+U) × U x 100%.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Mass Spectrometry, Generated, Recombinant

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Evaluation of hSMUG1 concentration-dependent uracil excision activity. A 10 μL hSMUG1 glycosylase reaction mixture contained 50 pmol of uracil-containing duplex substrate (5 μM), 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding various concentrations of hSMUG1 (0.5–2 U, as indicated). (A) Representative MALDI-TOF mass spectra obtained from reactions at different enzyme concentrations and incubation times. Distinct peaks corresponding to the template strand (T), uracil-containing strand (U), and abasic primer (AP) enabled direct visualisation of uracil excision efficiency. (B) Quantitative analysis of AP primer formation. Each reaction was independently performed in triplicate for each concentration and time point. Data are presented as mean ± SD. The dashed line represents the best-fit linear regression curve describing the relationship between hSMUG1 concentration and uracil excision efficiency.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Concentration Assay, Activity Assay, Incubation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Evaluation of hSMUG1-mediated uracil excision efficiency in damaged partners duplex or single-stranded DNA. A 10 μL hSMUG1 glycosylase reaction mixture contained 1 U hSMUG1, 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding various 50 pmol (5 μM) of uracil-containing substrates, including U:G or U:A mispair in the duplex DNA, or uracil in single-stranded DNA (ss U). (A) Representative MALDI-TOF mass spectra of reactions with different substrates and incubation times. Distinct peaks corresponding to the template strand (T or T U:A ), uracil-containing strand (U), and abasic product (AP) allowed direct visualisation of uracil excision efficiency. (B) Quantitative analysis of AP product formation. Each reaction was independently performed in triplicate for each substrate and time point. Data are presented as mean ± SD. Statistical analyses were performed by two-way ANOVA with Geisser-Greenhouse correction. **** p < 0.0001.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Incubation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: hSMUG1-mediated excision efficiency of U:G mispair at different positions within duplex DNA. A10 μL hSMUG1 glycosylase reaction mixture contained 1 U hSMUG1, 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding various 50 pmol (5 μM) of substrates containing U:G mispair positioned at the 3′ end, middle, or 5′ end of duplex DNA. (A) Representative MALDI-TOF mass spectra of reactions with different substrates and incubation times. Distinct peaks corresponding to the template strand (T or T 3′ or T 5′ ), uracil-containing strand (U or U 3′ or U 5′ ), and abasic primer (AP) allowed direct visualisation of uracil excision efficiency. (B) Quantitative analysis of AP product formation. Each reaction was independently performed in triplicate for each substrate and time point. Data are presented as mean ± SD. Statistical analyses were performed by two-way ANOVA with Geisser-Greenhouse correction. *** p < 0.001; **** p < 0.0001.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Incubation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Effect of cadmium on hSMUG1-mediated uracil excision from DNA. A10 μL hSMUG1 glycosylase reaction mixture contained 1 U hSMUG1, 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding 20 pmol substrate DNA in the presence of cadmium at various concentrations (0–50 μM). (A) Representative MALDI-TOF mass spectra of reactions with different cadmium concentrations and incubation times. Distinct peaks corresponding to the template strand (T), uracil-containing strand (U), and abasic primer (AP) allowed direct visualisation of uracil excision efficiency. (B) Inhibition curve of hSMUG1 activity by cadmium. Triplicate reactions performed under each cadmium concentration were used to generate an inhibition curve. The IC 50 of cadmium for hSMUG1 inhibition was 4.6 μM. The red arrow indicates the peak corresponding to the AP product. Data are presented as mean ± SD.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Incubation, Inhibition, Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: Scaling Multiplex qPCR Primer Design to 1000-plex using the Degenerate Incomplete Multiplex Primer List Extension (DIMPLE) Algorithm

doi: 10.64898/2026.04.17.719221

Figure Lengend Snippet: Demonstration of DIMPLE multiple qPCR primer design for DNA methylation marker detection. (a) Illustration of the products of bisulfite/APOBEC conversion of DNA. For each DNA locus, up to 4 different sets of primers (8 primers) must be designed to maximize sensitivity to the insert sequence. Alternatively, primer specificity can be used to detect specific methylation states of the primer binding regions. (b) Demonstration of 60-plex qPCR amplification of DNA hypomethylation markers. Here, 120 primers were designed to amplify only the + strands of the converted unmethylated DNA molecules, because unmethylated DNA has the lower sequence diversity than converted methylated DNA molecules, and is more challenging for traditional PCR primer design algorithms.

Article Snippet: For methylation assays, the Human Methylated & Non-Methylated (WGA) DNA Set (Zymo Research) served as reference material.

Techniques: DNA Methylation Assay, Marker, Sequencing, Methylation, Binding Assay, Amplification