Journal: Molecular Therapy. Nucleic Acids

Article Title: Potential effects of endogenous RNA/DNA hybrids on CRISPR-Cas9-mediated homology-directed repair

doi: 10.1016/j.omtn.2026.102880

Figure Lengend Snippet: Modulation of HDR efficiency by R-loop resolution, cell-cycle perturbation, and locus context (A) ddPCR quantification of HDR events in HEPA1-6/SpCas9 cells transiently overexpressing human RNase H1 and transduced with 10,000 vg/cell of scAAVDJ-sgMid Alb + 10,000 vg/cell ssAAVDJ-donor Mid Alb, or 10,000 vg/cell of scAAVDJ-sg3′ Alb + 10,000 vg/cell ssAAVDJ-donor 3′ Alb. (B) Schematic representation of cell cycle profiles in HEPA1-6 cells and murine hepatocytes. (C) Flow cytometry analysis of HEPA1-6/SpCas9 cells treated for 24 h with rapamycin (1 μM, 5 μM) or palbociclib (PD; 5 μM, 10 μM). DMSO-treated cells served as controls. (D) DRIP-qPCR analysis of HEPA1-6/SpCas9 cells after 24 h treatment with 5 μM rapamycin; DMSO-treated cells served as controls. (E) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells pre-treated with 5 μM rapamycin for 24 h, followed by 72 h transduction with 10,000 vg/cell of scAAVDJ-sgMid Alb + 10,000 vg/cell ssAAVDJ-donor Mid Alb, or scAAVDJ-sg3′ Alb + ssAAVDJ-donor 3′ Alb. (F) IGV visualization of the Alb and Actb loci showing gRNA (sgActb) and donor (donor Actb) design targeting the 5′ region of Actb . (G) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells transduced for 72 h with 10,000 vg/cell of scAAVDJ-sgActb + 10,000 vg/cell ssAAVDJ-donor Actb. Scrambled gRNA with donor served as negative control. (H) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells pre-treated with 5 μM rapamycin for 24 h, followed by 72 h transduction with scAAVDJ-sgActb + ssAAVDJ-donor Actb. (I) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells transiently overexpressing human RNase H1 and transduced with scAAVDJ-sgActb + ssAAVDJ-donor Actb. (J) DRIP-qPCR analysis of CD4 + T cells from a healthy donor, pre- and post-activation with 10 μg/mL PHA, 50 IU/mL IL-2, 5 ng/mL IL-7, and 5 ng/mL IL-15. DRIP, immunoprecipitated samples treated with S9.6 antibody which are enriched in R loops; RNAseH + , samples treated with RNAseH1+S9.9 antibody which are depleted of R-loops. (K) Table summarizing the effects of R-loops levels on indels, HDR, and AAV integration. N/A, not affected; ↑ increase; ↓ = decrease. Statistical analysis: (A and E) multiple t test; (C) two-way ANOVA with Dunnett’s post hoc test; (G–I) Student’s t test. p < 0.05, ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001. Error bars represent mean ± SD.

Article Snippet: HEPA1-6/SpCas9 cells were treated with rapamycin (Selleck, S1039) or palbociclib (Selleck, S1116) for 24 h, fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry (NovoCyte Penteon, Agilent).

Techniques: Transduction, Flow Cytometry, Negative Control, Activation Assay, Immunoprecipitation

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 knockout alleviates stress-induced depressive-like behaviors and associated with mTOR Signaling (A) Western blot analysis of USP11 (110 kDa), p-mTOR (Ser2448, 289 kDa), total mTOR (289 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), PSD95 (95 kDa), and Tubulin (55 kDa) in mouse mPFC from wild-type (WT) and USP11 knockout (USP11 −/− ) male mice (n = 6, Tubulin as loading control). (B–E) Quantification of baseline protein band intensity in wild-type control (WT-CON) and USP11 knockout control (KO-CON) groups: (B) USP11 (relative to Tubulin, p < 0.0001), (C) p-GSK3β (relative to total GSK3β, p = 0.0072), (D) p-mTOR (relative to total mTOR, p = 0.0028), (E) PSD95 (relative to Tubulin, p = 0.0159). n = 6/group. (F–I) Behavioral results for four groups: WT-CON, KO-CON, WT-CUMS, and KO-CUMS (OFT, distance [cm], F [3, 28] = 8.234, p = 0.0004; OFT, velocity [cm/s], F [3, 28] = 8.233, p = 0.0004; FST, F [3, 28] = 8.721, p = 0.0003; TST, F [3, 29] = 5.378, p = 0.0046). n = 8/group. (J) Western blot analysis of USP11 (110 kDa), p-mTOR (Ser2448, 289 kDa), total mTOR (289 kDa), SYN (synaptophysin, 77 kDa), and Tubulin (55 kDa) in mPFC from all four groups (n = 3). (K-M) Quantification of (K) USP11 (relative to Tubulin, F (3, 8) = 139.5, p < 0.0001), (L) p-mTOR (relative to total mTOR, F (3, 8) = 8.298, p = 0.0077), (M) SYN (relative to Tubulin, F (3, 8) = 8.811, p = 0.0065). n = 3/group. (N) Schematic overview of the experimental design, including a 7-day acclimation period, a 28-day chronic unpredictable mild stress (CUMS) procedure, the rapamycin dosing regimen (3 mg/kg, i.p., three times per week; from day 14 of CUMS until 24 h before tissue collection), and the behavioral test battery in male USP11 −/− mice. (O-R) Behavioral results for three groups in USP11 −/− mice: CON + Veh, CUMS + Veh and CUMS + Rapa. (SPT, F (2, 18) = 7.019, p = 0.0056; OFT, center time [s], F [2, 18] = 8.788, p = 0.0022; OFT, velocity [cm/s], F [2, 18] = 0.09090, p = 0.9135; TST, F [2, 18] = 7.797, p = 0.0036). n = 7/group.) (T) Quantification of p-mTOR (relative to total mTOR, F (2, 6) = 38.49, p = 0.0004) Data are shown as mean ± SEM. Determined by t -test (baseline comparisons) or one-way ANOVA (multiple groups) unless otherwise indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (S) Representative immunoblots of p-mTOR (Ser2448, 289 kDa), total mTOR (289 kDa) in USP11 −/− mice under the indicated conditions. (n = 3, Tubulin as loading control).

Article Snippet: Rapamycin (MedChemExpress, Cat. No. 53123-88-9) was formulated in a mixed vehicle containing 10% DMSO, 40% PEG300, 5% Tween-80, and 45% sterile 0.9% saline and prepared fresh before injection( ).

Techniques: Knock-Out, Western Blot, Control, Battery

Journal: Neural Regeneration Research

Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01953

Figure Lengend Snippet: rtPA regulates the PI3K-AKT-mTOR pathway in the ICH in vitro cell model. (A, B) The DEGs between the control group and hemin group associated with the PI3K/AKT pathway (KEGG: mmu04151) and mTOR pathway (KEGG: mmu04150) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (C–F) Analysis of PI3K, p-AKT, AKT, p-mTOR, and mTOR of neurons ( n = 3 per group). **** P < 0.0001, vs . control group; ## P < 0.01, #### P < 0.0001, vs . hemin group. (G–J) Analysis of PI3K, p-AKT, AKT, p-mTOR, and mTOR of neurons ( n = 3 per group). (K, L) Analysis of apoptosis-associated proteins of neurons ( n = 3 per group). (M–P) Analysis of autophagy-associated proteins of neurons ( n = 3 per group). Data are shown as mean ± SEM and were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs. hemin group; & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001, vs . hemin + rtPA group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin + rtPA + DMSO group. AKT: RAC-alpha serine/threonine-protein kinase; DEGs: differential expression genes; DMSO: dimethyl sulfoxide; ICH: intracerebral hemorrhage; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; p62: sequestosome-1/ubiquitin-binding protein p62; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.

Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: In Vitro, Control, Quantitative Proteomics, Ubiquitin Proteomics, Binding Assay, Recombinant

Journal: Neural Regeneration Research

Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01953

Figure Lengend Snippet: The PI3K inhibitor LY294002 reverses the anti-ER stress effect of rtPA and the EGF domain of rtPA may mediate the PI3K/AKT pathway in the ICH in vitro cell model. (A–C) Analysis of ER stress–associated proteins ( n = 3 per group). (D) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 3 µm. (E) Immunofluorescence images of p-PERK (red, labeled by Cy3) in neurons after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 50 µm. (F–H) Analysis of PI3K p85 and p-AKT. Data are represented as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . hemin group; & P < 0.05, && P < 0.01, vs . hemin + rtPA group; # P < 0.05, vs . hemin + rtPA + DMSO group (one-way analysis of variance followed by Tukey’s post hoc test). (I) Transmission electron microscopy images of cells after rtPA and rtPA domain inhibitor treatment. Scale bar: 100 µm. 3D: Three-dimensional; AKT: RAC-alpha serine/threonine-protein kinase; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; EGF: epidermal growth factor; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; PERK: PRKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.

Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: In Vitro, Immunofluorescence, Labeling, Transmission Assay, Electron Microscopy, Recombinant

Journal: Molecular Medicine Reports

Article Title: Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats

doi: 10.3892/mmr.2026.13815

Figure Lengend Snippet: (A) Urethral tissue collection images from the control, model, model + rapamycin, and model + TDN group. (B) Body weight monitoring curves for rats in all groups over time. (C) Urethrographic imaging results for each group, with red arrows indicating the location of urethral injury model establishment. (D) Hematoxylin and eosin staining results for urethral tissues from each group. Red arrows indicate urethral lumen narrowing and associated fibrotic changes in the injured urethra. Scale bar, 500 µm (left) and 50 µm (right). TDN, tetrahedral DNA nanostructure.

Article Snippet: The rats were randomly divided into four groups: Control (n=6; intraperitoneal injection of an equal volume of saline every other day; injection volume, 0.2 ml per rat), model (n=6; urethral injury followed by intraperitoneal injection of saline every other day; injection volume, 0.2 ml per rat), model + rapamycin [n=6; 2.0 mg/kg of rapamycin (cat. no. HY-10219; MedChemExpress) injected intraperitoneally every other day after injury] and model + TDN (n=6; 10 nmol/day TDN administered via tail vein injection daily after injury; injection volume, 0.2 ml per rat).

Techniques: Control, Imaging, Staining

Journal: Molecular Medicine Reports

Article Title: Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats

doi: 10.3892/mmr.2026.13815

Figure Lengend Snippet: (A) PCA results. Different colors represent different treatment groups. (B) Sample correlation heatmap. The color intensity corresponds to correlation values. (C) Combined volcano plot showing the distribution of FCs in differentially expressed genes in the three group comparisons (model vs. control; model + rapamycin vs. model; model + TDN vs. model), with yellow dots representing upregulated genes and green dots representing downregulated genes. (D) Bar chart of differential gene counts showing the number of differential genes in the three group comparisons. (E) Venn diagram of differential genes displaying the distribution of differential genes in the three group comparisons. The numbers in different areas represent specific intersections or unique differential genes. (F) Heatmap showing the expression patterns of 25 common differentially expressed genes identified from three pairwise comparisons, displayed across four experimental groups (Control, Model, Model + rapamycin, and Model + TDN). Each row represents one gene and each column represents an individual sample. Color gradients indicate normalized gene expression levels. (G) KEGG pathway enrichment analysis of differentially expressed genes from the three pairwise comparisons (model vs. control; model + rapamycin vs. model; model + TDN vs. model). Enrichment results are presented as dot plots. The x-axis represents the GeneRatio, and the size of each dot reflects the proportion of genes enriched in the corresponding pathway. Dot color indicates the statistical significance expressed as -log10(P-value). KEGG pathways are displayed consistently across the three comparisons to facilitate direct visual comparison. KEGG, Kyoto Encyclopedia of Genes and Genomes; TDN, tetrahedral DNA nanostructure; FC, fold change; PCA, principal component analysis.

Article Snippet: The rats were randomly divided into four groups: Control (n=6; intraperitoneal injection of an equal volume of saline every other day; injection volume, 0.2 ml per rat), model (n=6; urethral injury followed by intraperitoneal injection of saline every other day; injection volume, 0.2 ml per rat), model + rapamycin [n=6; 2.0 mg/kg of rapamycin (cat. no. HY-10219; MedChemExpress) injected intraperitoneally every other day after injury] and model + TDN (n=6; 10 nmol/day TDN administered via tail vein injection daily after injury; injection volume, 0.2 ml per rat).

Techniques: Control, Expressing, Gene Expression, Comparison

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Benchmarking of chemogenetic BLAST against the RELEASE system. (a) Design of the chemogenetic BLAST (chemo-BLAST) and experimental timeline. Schematic of the plasmid configurations (upper) and the 4-day experimental schedule (lower). To enable a direct mechanistic comparison independent of light stimulation, the optogenetic modules (iLID/SspB) were replaced with the chemically inducible rapamycin-binding domains (FKBP/FRB). The cargo (A) contains varying repeats of FRB (1×, 2×, 4×, and 6×), and the regulator (B) consists of FKBP. Rapamycin (1 μM) was used as the inducer. (b) Optimization of FRB valency. Summary graph of SEAP secretion levels with varying FRB repeats. The construct containing 4× FRB repeats exhibited the most effective switching performance (4.2-fold induction). (c) Optimization of transfection ratios. Using the 4× FRB construct, SEAP levels were measured across different cargo (A) to regulator (B) ratios. The 1:3 ratio (62.5 ng : 187.5 ng) yielded the highest dynamic range (11.3-fold). (d) Head-to-head kinetic comparison with the RELEASE system. The optimized chemo-BLAST was compared against the RELEASE system, which employs split proteases (TEVp or TVMVp) to cleave a KKMP ER retention motif. Chemo-BLAST induced significant protein secretion starting at 2 h and achieved a higher fold change (7.4-fold at 24 h) compared to the RELEASE systems (3.2-fold for TEVp, 3.9-fold for TVMVp), demonstrating superior kinetics and signal-to-noise ratio. Open circles represent individual measurements from three biologically independent samples. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Rapamycin (Tocris) was prepared as a 3 mM stock solution in DMSO (Sigma-Aldrich).

Techniques: Plasmid Preparation, Comparison, Binding Assay, Construct, Transfection