Measurement of <i>K</i>. <i>pneumoniae</i> cell size using India ink staining after co-culturing with <i>Acanthamoeba</i>.

(A). India ink staining images of K. pneumoniae at a magnification of 1000× after co-culturing with Acanthamoeba. Bacterial cells were screened after co-culturing with Acanthamoeba at 24, 72, and 168 h. Scale bar = 10 μm. (B). Comparison of average cell size between co-cultured and cultured alone K. pneumoniae at 24, 72, and 168 h. The average cell size was calculated for 50 bacterial cells observed under a magnification of 1000×. Statistical significance was determined by Student’s t-test. ***P < 0.001.</p

India ink staining of various serotypes <i>K</i>. <i>pneumoniae</i> environmental isolates treated with <i>Acanthamoeba</i>.

The images of Acanthamoeba co-cultured K. pneumoniae environmental isolates or isolates cultured alone after staining with India ink at a magnification of 1000×. The serotypes of the environmental isolates are as follows: CMU-A belongs to serotype K1, CMU-B belongs to serotype K2, CMU-C belongs to serotype K5, and CMU-D belongs to serotype K20. Scale bar = 10 μm.</p

Images of <i>K</i>. <i>pneumoniae</i> cells passaged two times after culturing alone or co-culturing with <i>Acanthamoeba</i>.

Acanthamoeba co-cultured K. pneumoniae or K. pneumoniae cultured alone was passaged two times in Luria-Bertani broth. The third generation of K. pneumoniae cells were harvested using India ink staining. The images were captured from three independent experiments.</p

India ink staining of <i>K</i>. <i>pneumoniae</i> cells from overnight co-culturing at serial density.

(A).Acanthamoeba cells at a density of 4 × 104, 8 × 104, and 1.6 × 105 were overnight co-cultured with K. pneumoniae adjusted at OD600 value of 0.2. (B). K. pneumoniae cells at OD600 of 0.2, 0.4, 0.8, and 1.6 were overnight co-cultured with Acanthamoeba cells at a density of 5 × 104 for India ink staining. Scale bar = 10 μm.</p

<i>K</i>. <i>pneumoniae</i> capsular viscosity examination by sedimentation assay and string test.

(A). The cells from K. pneumoniae cultured alone or Acanthamoeba co-cultured K. pneumoniae were incubated overnight in Luria-Bertani broth. The broth was centrifuged at 3000 rpm for 10 min. The absorbance of the supernatants was measured at 600 nm. Statistical significance was determined by Student’s t-test. ***P (B). Comparison of stretched colonies between K. pneumoniae cultured alone and Acanthamoeba co-cultured K. pneumoniae. A positive string and hypermucoviscous phenotype were defined by strings 5 mm in length or longer. The black arrows indicate the top of the string; however, no stretched string was observed for K. pneumoniae cultured alone.</p

Phenol-sulfuric acid assay for capsular polysaccharide of <i>K</i>. <i>pneumoniae</i>.

The cells from K. pneumoniae cultured alone or Acanthamoeba co-cultured K. pneumoniae were treated with sulfuric acid. Uronic acid from K. pneumoniae capsular polysaccharide was identified as an orange complex at a wavelength of 520 nm after phenol treatment. Statistical significance was determined by Student’s t-test. ***P < 0.001.</p

DataSheet_1_Overexpression of Pyruvate Dehydrogenase Kinase-3 Predicts Poor Prognosis in Urothelial Carcinoma.docx

BackgroundThe mitochondrial pyruvate dehydrogenase complex (PDC) link glycolysis to the tricarboxylic acid cycle by decarboxylating pyruvate to acetyl coenzyme A irreversibly. Cancer cells are characterized by a shift in cellular metabolism from mitochondrial respiration to glycolysis. PDC activity inhibition mediated by phosphorylation via pyruvate dehydrogenase kinase (PDK) has been linked to cancer. However, the clinical significance of PDKs in urothelial cancer prognosis is not clear. We investigated the role and prognostic value of PDK3 expression in patients with upper urinary tract urothelial carcinoma (UTUC) and urinary bladder urothelial carcinoma (UBUC).Patients and MethodsWe retrospectively analyzed clinical data and pathological features. Formalin-fixed urothelial carcinoma (UC) tissues were collected and embedded in paraffin. The correlation of PDK3 expression with clinical characteristics, pathological findings and patient outcomes, including metastasis-free survival (MFS) and disease-specific survival (DSS) were analyzed by Pearson’s chi-square test, Kaplan–Meier analysis, and the multivariate Cox proportional hazards model.ResultsData from 295 patients with UBUC and 340 patients with UTUC were evaluated. High PDK3 expression significantly correlated with several pathologic variables such as high T stage, lymph node metastases, high tumor grade, vascular invasion, and high mitotic rate (all P ConclusionHigh PDK3 expression has been linked to negative pathologic characteristics and poor oncological outcomes, suggesting that it could be used as a predictive biomarker for UC. PDK3 mRNA levels and its co-upregulated genes were strongly associated with DNA replication and repair. These results suggest that PDK3 may play a key role in tumor proliferation and development.</p

Image_2_High Level of Aristolochic Acid Detected With a Unique Genomic Landscape Predicts Early UTUC Onset After Renal Transplantation in Taiwan.tif

BackgroundThe unusual high dialysis prevalence and upper urinary tract urothelial carcinoma (UTUC) incidence in Taiwan may attribute to aristolochic acid (AA), which is nephrotoxic and carcinogenic, exposure. AA can cause a unique mutagenic pattern showing A:T to T:A transversions (mutational Signature 22) analyzed by whole exome sequencing (WES). However, a fast and cost-effective tool is still lacking for clinical practice. To address this issue, we developed an efficient and quantitative platform for the quantitation of AA and tried to link AA detection with clinical outcomes and decipher the genomic landscape of UTUC in Taiwan.Patients and MethodsWe recruited 61 patients with de novo onset of UTUC after kidney transplantation who underwent radical nephroureterectomy. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform was developed for the quantitation of AA. Pearson’s chi-square test, Kaplan–Meier method, and Cox proportional hazard model were utilized to assess the correlations among AA detection, clinicopathological characteristics, and clinical outcomes. Seven tumors and seven paired normal tissues were sequenced using WES (approximately 800x sequencing depth) and analyzed by bioinformatic tool.ResultsWe found that high level of 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) detected in paired normal tissues was significantly correlated with fast UTUC initiation times after renal transplantation (p = 0.035) and with no use of sirolimus (p = 0.046). Using WES analysis, we further observed that all tumor samples were featured by Signature 22 mutations, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC)-associated gene mutations, p53 mutations, no fibroblast growth factor receptor 3 (FGFR3) mutation, and high tumor mutation burden (TMB). Especially, mammalian target of rapamycin (mTOR) activation predominated in dA-AL-I-detected samples compared with those without dA-AL-I detection and might be associated with UTUC initiation through cell proliferation and suppression of UTUC progression via autophagy inhibition.ConclusionAccordingly, dA-AL-I detection can provide more direct evidence to AA exposure and serve as a more specific predictive and prognostic biomarker for patients with de novo onset of UTUC after kidney transplantation.</p

Image_1_High Level of Aristolochic Acid Detected With a Unique Genomic Landscape Predicts Early UTUC Onset After Renal Transplantation in Taiwan.tif

BackgroundThe unusual high dialysis prevalence and upper urinary tract urothelial carcinoma (UTUC) incidence in Taiwan may attribute to aristolochic acid (AA), which is nephrotoxic and carcinogenic, exposure. AA can cause a unique mutagenic pattern showing A:T to T:A transversions (mutational Signature 22) analyzed by whole exome sequencing (WES). However, a fast and cost-effective tool is still lacking for clinical practice. To address this issue, we developed an efficient and quantitative platform for the quantitation of AA and tried to link AA detection with clinical outcomes and decipher the genomic landscape of UTUC in Taiwan.Patients and MethodsWe recruited 61 patients with de novo onset of UTUC after kidney transplantation who underwent radical nephroureterectomy. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform was developed for the quantitation of AA. Pearson’s chi-square test, Kaplan–Meier method, and Cox proportional hazard model were utilized to assess the correlations among AA detection, clinicopathological characteristics, and clinical outcomes. Seven tumors and seven paired normal tissues were sequenced using WES (approximately 800x sequencing depth) and analyzed by bioinformatic tool.ResultsWe found that high level of 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) detected in paired normal tissues was significantly correlated with fast UTUC initiation times after renal transplantation (p = 0.035) and with no use of sirolimus (p = 0.046). Using WES analysis, we further observed that all tumor samples were featured by Signature 22 mutations, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC)-associated gene mutations, p53 mutations, no fibroblast growth factor receptor 3 (FGFR3) mutation, and high tumor mutation burden (TMB). Especially, mammalian target of rapamycin (mTOR) activation predominated in dA-AL-I-detected samples compared with those without dA-AL-I detection and might be associated with UTUC initiation through cell proliferation and suppression of UTUC progression via autophagy inhibition.ConclusionAccordingly, dA-AL-I detection can provide more direct evidence to AA exposure and serve as a more specific predictive and prognostic biomarker for patients with de novo onset of UTUC after kidney transplantation.</p

Supplementary Figure 1-6 from HMDB and 5-AzadC Combination Reverses Tumor Suppressor CCAAT/Enhancer-Binding Protein Delta to Strengthen the Death of Liver Cancer Cells

Supplemental Figure 1. Anti-cancer drugs induce CEBPD expression in liver cancer cells. Supplemental Figure 2. Scheme of depression of epigenetic effects to enhance HMDB-induced CEBPD expression and apoptosis. Supplemental Figure 3. (Related to Figure 5) The p38/CREB pathway mediates HMDB-induced CEBPD transcriptional activation and CEBPD participates in HMDB-induced apoptosis in liver cancer cells. Supplemental Figure 4. (Related to Figure 5) Combinatorial treatment of HMDB and 5-AzadC can additively affect cell death. Supplemental Figure 5. (Related to Figure 5) Enhanced cytotoxic effects of HMDB combined with 5-AzadC on Huh7 and HepG2 cells. Supplemental Figure 6. (Related to Figure 5) Loss of CEBPD attenuated dual treatment-induced tumor killing in liver cancer cells.</p