Table_1_v1_Status Quo and Influencing Factors of Discharge Readiness of Patients with Bilateral Ureteral Stoma After Radical Cystectomy.docx

Bladder cancer is a common malignancy of the urinary system, which occurs mostly in elderly men, and the incidence is increasing year by year. To analyze the status quo and related factors of discharge readiness of patients with bilateral ureteral stoma after radical cystectomy, a retrospective, noncomparative was performed. 544 patients with bilateral ureteral stoma after radical cystectomy in our hospital from December 2018 to December 2020 were selected. The self-designed questionnaire, discharge readiness scale (RHDS) and discharge guidance quality scale (QDTS) were used to investigate the general data, and multiple linear regression was used to analyze the related influencing factors. The total score of RHDS was (72.57 ± 18.56) and the total score of QDTS was (105.63 ± 24.18); the total score of RHDS was positively correlated with the total score of QDTS (r = 0.882, p = 0.000); the results of multiple linear regression showed that age, discharge direction and care mode were the main factors influencing the discharge readiness of patients (p < 0.05). In conclusions, the discharge readiness of patients with bilateral ureteral stoma after radical cystectomy is in the medium level, and there is a large space for improvement. Nurses should strengthen the guidance and nursing of patients’ discharge preparation to reduce the incidence of postoperative complications and readmission rate.</p

Re-expression of RhoH does not affect the localization of NMHC IIA in neutrophil granules and mitochondria.

(A) The PNL from activated neutrophils expressing HA-RhoH or the EV was separated and collected equally into 1–10 fractions followed by immunoblot analysis. (B) Immunoblot analysis of NMHC IIA in cytosolic (C) and mitochondrial (M) fractions from activated neutrophils expressing HA-RhoH or EV. Data are representative of 3 independent experiments. The underlying data for S7A and S7B Fig can be found in S1 Raw images. EV, empty vector; NMHC IIA, non-muscle myosin heavy chain IIA; PNL, postnuclear lysate. (DOCX)</p

RhoH is not involved in microtubule rearrangement and does not interfere with Cdc42 and RhoA activity.

(A–D) The 5-day differentiated HoxB8 neutrophils expressing HA-RhoH or EV were treated as indicated. (A) Microtubule assembly in these cells was analyzed by confocal microscopy (left). Scale bars, 10 μm. Quantification of microtubule was performed by automated analysis of microscopic images using Imaris software (right). Values are means ± SD. Two-way ANOVA with Tukey’s multiple comparisons test was applied. (B) ROS activity was assessed by flow cytometry. (C, D) The levels of GTP-bound and total Cdc42 protein (C), GTP-bound and total RhoA protein (D), were compared using effector pulldown assay, followed by immunoblotting. GTPγS was used as a positive control, GDP as a negative control. All data are representative of 3 independent experiments. The underlying data for S9A and S9B Fig can be found in S1 Data. The underlying data for S9C and S9D Fig can be found in S1 Raw images. EV, empty vector. (DOCX)</p

RhoH inhibits NMHC IIA-mediated granules and mitochondria binding to F-actin.

(A) Enriched granules and mitochondria from mature HoxB8 neutrophils expressing HA-RhoH or the corresponding EV were incubated for 30 min in the presence or absence of F-actin. After centrifugation, the pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (B) Docking analysis for predicting the binding sites between RHOH and NMHC IIA (upper). Sequence alignment of RAC1, CDC42, RHOA, and RHOH (lower). (C) Immunoblot analysis of Co-IP with anti-HA antibody in cell lysates from activated HoxB8 neutrophils expressing WT or mutated HA-RhoH. Quantification of the precipitated NMHC IIA was shown below the representative images. (D) Enriched granules and mitochondria were incubated for 30 min in the presence or absence of F-actin. The pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (E–G) Mature HoxB8 neutrophils expressing WT or mutated HA-RhoH or EV were treated as indicated. (E) Neutrophil degranulation was determined by flow cytometry. (F) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (G) Quantification of dsDNA released into the supernatants. (A, C, D, F) Data are representative of 3 independent experiments. Values are means ± SD. Two-tailed Student t test (A, D); 1-way ANOVA with Dunnett’s multiple comparisons test (C); 2-way ANOVA with Tukey’s multiple comparisons test (E, G). The underlying numerical data for Fig 5A, 5C–E, and 5G can be found in S1 Data. The uncropped immunoblots for Fig 5A, 5C, and 5D can be found in S1 Raw images. Co-IP, co-immunoprecipitation; dsDNA, double-stranded DNA; EV, empty vector; NMHC IIA, non-muscle myosin heavy chain IIA; WT, wild-type.</p

The uncropped immunoblots for Figs 1B, 2E, 3A–3C, 3G, 4A, 4G, 5A, 5C, 5D, 6B–6D, 7A and 7H, S2A, S5A, S6B, S7A, S7B, S8B, S9C and S9D Figs.

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The underlying numerical data for Figs 1C–1E, 1G, 2B, 2D, 2F, 2H, 3B, 3C–3E, 3H, 3I, 4C, 4D, 4F, 4G, 5A, 5C–5E, 5G, 6A–6D, 7B–7G and 7I, S1A, S2B, S2C, S3B, S3D, S5B, S5D, S6A, S6C, S8A, S8B, S9A and S9B Figs.

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RhoH impairs actin rearrangement presumably by modulating Rac1 activity.

(A, B) Mature HoxB8 neutrophils expressing WT or mutated HA-RhoH or EV were treated as indicated. (A) F-actin distribution was analyzed by confocal microscopy (left). Scale bars, 10 μm. Quantification of F-actin fluorescence intensity was performed by automated analysis of microscopic images using Imaris software. The 25 images (each containing 8–12 cells) from 3 independent experiments were included for each condition (right). (B) Phosphorylation of myosin IIA was analyzed by immunoblotting. (C, D) The levels of GTP-bound and total Rac1 and Rac2 proteins in the indicated mouse bone marrow (BM) neutrophils (C) and HoxB8 neutrophils (D) were examined by immunoblotting following pulldown assay. All data are representative of 3 independent experiments. Values are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test was applied. The underlying numerical data for Fig 6A–D can be found in S1 Data. The uncropped immunoblots for Fig 6B–D can be found in S1 Raw images. EV, empty vector; WT, wild-type.</p

Reduced NMHC IIA expression by a second shRNA targeting <i>Myh9</i> decreases NET formation.

(A) Protein expression of NMHC IIA in mature HoxB8 neutrophils treated with control (Ctrl) or the second Myh9 shRNA was analyzed by immunoblotting. (B) Neutrophil degranulation was determined by flow cytometry. (C) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. At least 3 independent experiments were performed. (D) Quantification of released dsDNA in culture supernatants. (B, D) Values are means ± SD. (B, D) Two-way ANOVA with Šídák’s multiple comparisons test. The underlying data for S5B and S5D Fig can be found in S1 Data. The underlying data for S5A Fig can be found in S1 Raw images. (DOCX)</p

NMHC IIA associates with neutrophil organelles and mediates their binding to actin filaments.

(A) Phosphorylation of myosin IIA in human neutrophils treated as indicated was analyzed by immunoblotting. Threonine 18 (T18); Serine 19 (S19); Serine 1943 (S1943). (B–D) The PNL from unstimulated or GM-CSF primed and C5a-activated human neutrophils was isolated and collected equally into 1–10 fractions, from top to bottom. (B) Immunoblot analysis of density gradient fractions. (C) NE activity of density gradient fractions. (D) Bacteria killing mediated by density gradient fractions. (E) Immunoblot analysis of NMHC IIA in cytosolic and mitochondrial fractions from control and activated human neutrophils. C, cytosolic fraction. M, mitochondrial fraction. (F) Granular or mitochondrial localization of NMHC IIA in unstimulated and activated human neutrophils was analyzed by confocal microscopy. Scale bars, 10 μm. Numerical analysis was performed on 10 cells in each group and the Pearson’s coefficient in colocalized volume between NMHC IIA and CD63 or MTO was calculated using Imaris software. (G) Enriched granules and mitochondria from mature HoxB8 neutrophils treated with control (Ctrl) or Myh9 shRNA were incubated for 30 min in the presence or absence of F-actin. The pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (A, B, E–G) Data are representative of 3 independent experiments. (C, D, F, G) Values are means ± SD. (F, G) Unpaired 2-tailed Student t test was applied. The underlying numerical data for Fig 4C, 4D, 4F, and 4G can be found in S1 Data. The uncropped immunoblots for Fig 4A, 4B, 4E, and 4G can be found in S1 Raw images. GM-CSF, granulocyte/macrophage colony-stimulating factor; NE, neutrophil elastase; NMHC IIA, non-muscle myosin heavy chain IIA; PNL, postnuclear lysate.</p

Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection

G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H₂O₂ generation from O₂ and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or G-quadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications