Automated identification of single and clustered chromosomes for metaphase image analysis.

Background: Chromosome analysis is laborious and time-consuming. Automated methods can significantly increase the efficiency of chromosome analysis. For the automated analysis of chromosome images, single and clustered chromosomes must be identified. Herein, we propose a feature-based method for distinguishing between single chromosomes and clustered chromosome. Method: The proposed method comprises three main steps. In the first step, chromosome objects are segmented from metaphase chromosome images in advance. In the second step, seven features are extracted from each segmented object, i.e., the normalized area, area/boundary ratio, side branch index, exhaustive thresholding index, normalized minimum width, minimum concave angle, and maximum boundary shift. Finally, the segmented objects are classified as a single chromosome or chromosome cluster using a combination of the seven features. Results: In total, 43,391 segmented objects, including 39,892 single chromosomes and 3,499 chromosome clusters, are used to evaluate the proposed method. The results show that the proposed method achieves an accuracy of 98.92% by combining the seven features using support vector machine. Conclusions: The proposed method is highly effective in distinguishing between single and clustered chromosomes and can be used as a preprocessing procedure for automated chromosome image analysis.

ABCB1 and ABCG2 Overexpression Mediates Resistance to the Phosphatidylinositol 3-Kinase Inhibitor HS-173 in Cancer Cell Lines.

Constitutive activation of the phosphoinositide-3-kinase (PI3K)/Akt signaling pathway is crucial for tumor growth and progression. As such, this pathway has been an enticing target for drug discovery. Although HS-173 is a potent PI3K inhibitor that halts cancer cell proliferation via G2/M cell cycle arrest, the resistance mechanisms to HS-173 have not been investigated. In this study, we investigated the susceptibility of HS-173 to efflux mediated by the multidrug efflux transporters ABCB1 and ABCG2, which are two of the most well-known ATP-binding cassette (ABC) transporters associated with the development of cancer multidrug resistance (MDR). We found that the overexpression of ABCB1 or ABCG2 significantly reduced the efficacy of HS-173 in human cancer cells. Our data show that the intracellular accumulation of HS-173 was substantially reduced by ABCB1 and ABCG2, affecting G2/M arrest and apoptosis induced by HS-173. More importantly, the efficacy of HS-173 in multidrug-resistant cancer cells could be recovered by inhibiting the drug-efflux function of ABCB1 and ABCG2. Taken together, our study has demonstrated that HS-173 is a substrate for both ABCB1 and ABCG2, resulting in decreased intracellular concentration of this drug, which may have implications for its clinical use.

Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe.

Hepatitis C virus (HCV) NS3/4A protease is an attractive target for direct-acting antiviral agents. Real-time tracking of the NS3/4A protease distribution and activity is useful for clinical diagnosis and disease management. However, no approach has been developed that can systemically detect NS3/4A protease activity or distribution. We designed a protease-activatable retention probe for tracking HCV NS3/4A protease activity via positron emission topography (PET) imaging. A cell-penetrating probe was designed that consisted of a cell-penetrating Tat peptide, HCV NS3/4A protease substrate, and a hydrophilic domain. The probe was labeled by fluorescein isothiocyanate (FITC) and 124I in the hydrophilic domain to form a TAT-ΔNS3/4A-124I-FITC probe. Upon cleavage at NS3/4A substrate, the non-penetrating hydrophilic domain is released and accumulated in the cytoplasm allowing PET or optical imaging. The TAT-ΔNS3/4A-FITC probe selectively accumulated in NS3/4A-expressing HCC36 (NS3/4A-HCC36) cells/tumors and HCV-infected HCC36 cells. PET imaging showed that the TAT-ΔNS3/4A-124I-FITC probe selectively accumulated in the NS3/4A-HCC36 xenograft tumors and liver-implanted NS3/4A-HCC36 tumors, but not in the control HCC36 tumors. The TAT-ΔNS3/4A-124I-FITC probe can be used to represent NS3/4 protease activity and distribution via a clinical PET imaging system allowing. This strategy may be extended to detect any cellular protease activity for optimization the protease-based therapies.

The WD repeat-containing protein 5 (WDR5) antagonist WDR5-0103 restores the efficacy of cytotoxic drugs in multidrug-resistant cancer cells overexpressing ABCB1 or ABCG2.

The development of multidrug resistance (MDR) is one of the major challenges in the treatment of cancer which is caused by the overexpression of the ATP-binding cassette (ABC) transporters ABCB1 (P-glycoprotein) and/or ABCG2 (BCRP/MXR/ABCP) in cancer cells. These transporters are capable of reducing the efficacy of cytotoxic drugs by actively effluxing them out of cancer cells. Since there is currently no approved treatment for patients with multidrug-resistant tumors, the drug repurposing approach provides an alternative route to identify agents to reverse MDR mediated by ABCB1 and/or ABCG2 in multidrug-resistant cancer cells. WDR5-0103 is a histone H3 lysine 4 (H3K4) methyltransferase inhibitor that disrupts the interaction between the WD repeat-containing protein 5 (WDR5) and mixed-lineage leukemia (MLL) protein. In this study, the effect of WDR5-0103 on MDR mediated by ABCB1 and ABCG2 was determined. We found that in a concentration-dependent manner, WDR5-0103 could sensitize ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to conventional cytotoxic drugs. Our results showed that WDR5-0103 reverses MDR and improves drug-induced apoptosis in multidrug-resistant cancer cells by inhibiting the drug-efflux function of ABCB1 and ABCG2, without altering the protein expression of ABCB1 or ABCG2. The potential sites of interactions of WDR5-0103 with the drug-binding pockets of ABCB1 and ABCG2 were predicted by molecular docking. In conclusion, the MDR reversal activity of WDR5-0103 demonstrated here indicates that it could be used in combination therapy to provide benefits to a subset of patients with tumor expressing high levels of ABCB1 or ABCG2.

Activities of imipenem-relebactam combination against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

BACKGROUND: Imipenem-relebactam is a new ß-lactam and ß-lactamase inhibitor combination to treat carbapenem-resistant gram-negative bacteria infections. However, difference in carbapenem resistant mechanisms existed with geographic variations. OBJECTIVE: To evaluate the susceptibility of imipenem-relebactam to 660 carbapenem-nonsusceptible Enterobacteriaceae isolates in Taiwan and to identify the in vivo efficacy with a Caenorhabditis elegans model. METHODS: 188 carbapenem-nonsusceptible Escherichia coli isolates and 472 carbapenem-nonsusceptible Klebsiella pneumoniae isolates were collected from a national surveillance study in Taiwan. The antimicrobial susceptibility profiles and carbapenemase distributions were determined. An agar dilution method was performed to evaluate the in vitro activities of imipenem monotherapy and imipenem-relebactam combination. Contributions of metallo-carbapenemase to imipenem-relebactam susceptibility was investigated via EDTA treatment. A C. elegans model was used to evaluate the in vivo efficacy of imipenem-relebactam combination. RESULTS: 87.8% and 82.2% susceptibility to imipenem-relebactam was observed for 188 carbapenem-nonsusceptible E. coli and 472 carbapenem-nonsusceptible K. pneumoniae, respectively. However, poor activities of imipenem-relebactam was observed against 23 metallo-carbapenemase producers tested in this study. In the in vivo C. elegans model, imipenem-relebactam significantly rescued nematodes from the infection of a blaKPC-producing K. pneumoniae isolate. CONCLUSION: Our study supports that imipenem-relebactam is a potential therapy against carbapenem-nonsusceptible Enterobacteriaceae, and to our knowledge, this is the first report of evaluation for imipenem-relebactam efficacy against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

Development of a method for dansylation of metabolites using organic solvent-compatible buffer systems for amine/phenol submetabolome analysis.

Metabolomics, which serves as a readout of biological processes and diseases monitoring, is an informative research area for disease biomarker discovery and systems biology studies. In particular, reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) has become a powerful and popular tool for metabolomics analysis, enabling the detection of most metabolites. Very polar and ionic metabolites, however, are less easily detected because of their poor retention in RP columns. Dansylation of metabolites simplifies the sub-metabolome analysis by reducing its complexity and increasing both hydrophobicity and ionization ability. However, the various metabolite concentrations in clinical samples have a wide dynamic range with highly individual variation in total metabolite amount, such as in saliva. The bicarbonate buffer typically used in dansylation labeling reactions induces solvent stratification, resulting in poor reproducibility, selective sample loss and an increase in false-determined metabolite peaks. In this study, we optimized the dansylation protocol for samples with wide concentration range of metabolites, utilizing diisopropylethylamine (DIPEA) or tri-ethylamine (TEA) in place of bicarbonate buffer, and presented the results of a systemic investigation of the influences of individual processes involved on the overall performance of the protocol. In addition to achieving high reproducibility, substitution of DIPEA or TEA buffer resulted in similar labeling efficiency of most metabolites and more efficient labeling of some metabolites with a higher pKa. With this improvement, compounds that are only present in samples in trace amounts can be detected, and more comprehensive metabolomics profiles can be acquired for biomarker discovery or pathway analysis, making it possible to analyze clinical samples with limited amounts of metabolites.

Multiplexed Molecular Imaging Strategy Integrated with RNA Sequencing in the Assessment of the Therapeutic Effect of Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles for Osteoporosis.

INTRODUCTION: Osteoporosis is a result of an imbalance in bone remodeling. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been considered as a potentially promising treatment for osteoporosis. However, the therapeutic effect, genetic alterations, and in vivo behavior of exogenous EVs for osteoporosis in mice models remain poorly understood. METHODS: A multiplexed molecular imaging strategy was constructed by micro-positron emission tomography (µPET)/computed tomography (CT), µCT, and optical imaging modality which reflected the osteoblastic activity, microstructure, and in vivo behavior of EVs, respectively. RNA sequencing was used to analyze the cargo of EVs, and the bone tissues of ovariectomized (OVX) mice post EV treatment. RESULTS: The result of [18F]NaF µPET showed an increase in osteoblastic activity in the distal femur of EV-treated mice, and the bone structural parameters derived from µCT were also improved. In terms of in vivo behavior of exogenous EVs, fluorescent dye-labeled EVs could target the distal femur of mice, whereas the uptakes of bone tissues were not significantly different between OVX mice and healthy mice. RNA sequencing demonstrated upregulation of ECM-related genes, which might associate with the PI3K/AKT signaling pathway, in line with the results of microRNA analysis showing that mir-21, mir-29, mir-221, and let-7a were enriched in Wharton's jelly-MSC-EVs and correlated to the BMP and PI3K/AKT signaling pathways. CONCLUSION: The therapeutic effect of exogenous WJ-MSC-EVs in the treatment of osteoporosis was successfully assessed by a multiplexed molecular imaging strategy. The RNA sequencing demonstrated the possible molecular targets in the regulation of bone remodeling. The results highlight the novelty of diagnostic and therapeutic strategies of EV-based treatment for osteoporosis.

Evaluation of the Organotellurium Compound AS101 for Treating Colistin- and Carbapenem-Resistant Klebsiella pneumoniae.

Colistin- and carbapenem-resistant Enterobacteriaceae cases are increasing at alarming rates worldwide. Drug repurposing is receiving greater attention as an alternative approach in light of economic and technical barriers in antibiotics research. The immunomodulation agent ammonium trichloro(dioxoethylene-O,O'-)tellurate (AS101) was repurposed as an antimicrobial agent against colistin- and carbapenem-resistant Klebsiella pneumoniae (CRKP). 134 CRKP isolates were collected between 2012 and 2015 in Taiwan. The in vitro antibacterial activities of AS101 was observed through broth microdilution, time-kill assay, and electron microscopy. Pharmaceutical manipulation and RNA microarray were applied to investigate these antimicrobial mechanisms. Caenorhabditis elegans, a nematode animal model, and the Institute for Cancer Research (ICR) mouse model was employed for the evaluation of in vivo efficacy. The in vitro antibacterial results were found for AS101 against colistin- and CRKP isolates, with minimum inhibitory concentration (MIC) values ranging from <0.5 to 32 µg/mL. ROS-mediated antibacterial activity eliminated 99.9% of bacteria within 2-4 h. AS101 also extended the median survival time in a C. elegans animal model infected with a colistin-resistant CRKP isolate and rescued lethally infected animals in a separate mouse model of mono-bacterial sepsis by eliminating bacterial organ loads. These findings support the use of AS101 as an antimicrobial agent for addressing the colistin and carbapenem resistance crisis.

Enhancement of Anticancer Potential of Pterostilbene Derivative by Chalcone Hybridization.

Pterostilbene, a natural metabolite of resveratrol, has been indicated as a potent anticancer molecule. Recently, several pterostilbene derivatives have been reported to exhibit better anticancer activities than that of the parent pterostilbene molecule. In the present study, a series of pterostilbene derivatives were designed and synthesized by the hybridization of pterostilbene, chalcone, and cinnamic acid. The cytotoxic effect of these hybrid molecules was determined using two oral cancer cell lines, HSC-3 and OECM-1. (E)-3-(2-((E)-4-Hydroxystyryl)-4,6-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (4d), with IC50 of 16.38 and 18.06 µM against OECM-1 and HSC-3, respectively, was selected for further anticancer mechanism studies. Results indicated that compound 4d effectively inhibited cell proliferation and induced G2/M cell cycle arrest via modulating p21, cyclin B1, and cyclin A2. Compound 4d ultimately induced cell apoptosis by reducing the expression of Bcl-2 and surviving. In addition, cleavage of PARP and caspase-3 were enhanced following the treatment of compound 4d with increased dose. To conclude, a number of pterostilbene derivatives were discovered to possess potent anticancer potentials. Among them, compound 4d was the most active, more active than the parent pterostilbene.

Clinical significance of olfactory dysfunction in patients of COVID-19.

BACKGROUND: Currently, as the coronavirus disease (COVID-19) has become a pandemic, rapidly obtaining accurate information of patient symptoms and their progression is crucial and vital. Although the early studies in China have illustrated that the representative symptoms of COVID-19 include (dry) cough, fever, headache, fatigue, gastrointestinal discomfort, dyspnea, and muscle pain, there is increasing evidence to suggest that olfactory and taste disorder are related to the COVID-19 pandemic. Therefore, we conduct this study to review the present literature about the correlation between anosmia or dysgeusia and COVID-19. METHODS: A comprehensive literature search in 2020 of the electronic journal databases, mainly PubMed or Web of Science, was performed using the keywords COVID-19 or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with hyposmia, anosmia, dysgeusia, olfactory disorder, or olfactory dysfunction. The country, study period, case number, inpatient or outpatient medical visit, evaluation method (subjective complaints of dysfunction or objective evaluation), and occurrence rate of olfactory or gustatory function were reviewed. RESULTS: Many studies reported that the recoverable olfactory or gustatory dysfunction may play an important role as the early clinical symptom of COVID-19. It is associated with better prognosis, although further investigation and validation should be carried out. CONCLUSION: Studies have shown that smell and taste disturbances may represent an early symptom of COVID-19 and healthcare professionals must be very vigilant when managing patients with these symptoms. In the pandemic era, this implies testing for COVID-19 by healthcare workers with full personal protective equipment.

Association of urinary ketamine and APOA1 levels with bladder dysfunction in ketamine abusers revealed via proteomics and targeted metabolite analyses.

Chronic ketamine abuse is associated with bladder dysfunction and cystitis. However, the effects of ketamine abuse on the urinary proteome profile and the correlations among urinary proteins, urinary ketamine (and metabolites) and clinicopathological features of ketamine-induced bladder dysfunction remain to be established. Here, we recruited 56 ketamine abusers (KA) and 40 age-matched healthy controls (HC) and applied the iTRAQ-based proteomics approach to unravel quantitative changes in the urine proteome profile between the two groups. Many of the differentially regulated proteins are involved in the complement and coagulation cascades and/or fibrotic disease. Among them, a significant increase in APOA1 levels in KA relative to control samples (392.1 ± 59.9 ng/ml vs. 13.7 ± 32.6 ng/ml, p < 0.0001) was detected via ELISA. Moreover, urinary ketamine, norketamine and dehydronorketamine contents (measured via LC-SRM-MS) were found to be positively correlated with overactive bladder syndrome score (OABSS) and APOA1 levels with urinary RBC, WBC, OABSS and numeric pain rating scale in KA. Collectively, our results may aid in developing new molecular tool(s) for management of ketamine-induced bladder dysfunction. Moreover, information regarding the differentially regulated proteins in urine of KA provides valuable clues to establish the molecular mechanisms underlying ketamine-induced cystitis.

In vitro and in vivo assessments of inspired Ag/80S bioactive nanocomposites against carbapenem-resistant Klebsiella pneumoniae.

In 2017 the World Health Organization listed carbapenem-resistant K. pneumoniae as a critical priority for developing a novel antimicrobial agent. Here we report on our investigation of the antibacterial efficacy of silver nanoparticles (AgNPs), confined to a mesostructured material and designated as an Ag/80S bioactive nanocomposite, against carbapenem-resistant K. pneumoniae. Results from a textural analysis indicate a 7.5 nm mesopore size and 307.6 m2/g surface area for Ag/80S. UV-Vis spectrum and transmission electron microscope images of Ag/80S revealed a uniform AgNP size distribution with an approximately 3.5 nm average. ICP-MS analysis demonstrated a significantly higher silver content in TSB (a protein-rich environment) compared to ultrapure water, suggesting a controllable release of Ag/80S and thus designated as the inspired Ag/80S. Minimum inhibitory concentration (MIC) values against 16 K. pneumoniae isolates ranged from 0.25 to 0.5% (2.5 to 5.0 mg/ml). NIH 3T3 fibroblast viability at 0.25% exceeded 80% and at 0.5% just under 70%, suggesting low cytotoxicity. Mechanistic study results indicate that the inspired Ag/80S attached to and deformed bacterial cells and induced a time-dependent accumulation of reactive oxygen species, leading to bacterial death. Further, inspired Ag/80S significantly extended median survival time in a Caenorhabditis elegans animal model infected with carbapenem-resistant K. pneumoniae ATCC BAA-1705. Combined, we found a novel Ag/80S which could prevent aggregation of AgNP and control its release via a specific environment for medical use against carbapenem-resistant K. pneumoniae.

Preclinical Characterization and In Vivo Imaging of 111In-Labeled Mesenchymal Stem Cell-Derived Extracellular Vesicles.

PURPOSE: Mesenchymal stem cell-derived EVs (MSC-EVs) are demonstrated to have similar therapeutic effect as their cells of origin and represent an attractive cell-free stem cell therapy. With the potential to be the future medical regimen, the information of fate and behavior of MSC-EVs in the living subject should be urgently gathered. This study aimed to track MSC-EVs by 111In-labeling and µSPECT/CT imaging. PROCEDURES: Wharton's jelly-MSC-EVs (WJ-MSC-EVs) were isolated using Exo-Prep kit followed by characterization of expressing markers and size. After labeled by 111In-oxine, 111In-EVs were injected into C57BL/6 mice followed by µSPECT/CT imaging. Organs were then taken out for ex vivo biodistribution analysis. RESULTS: The radiochemical purity of 111In-EVs was > 90 % and remained stable up to 24 h. The image results showed that with injection of 111In-EVs, the signal mainly accumulated in the liver, spleen, and kidney, compared to that in lung and kidney after 111In-oxine injection. The ex vivo biodistribution showed the similar pattern to that of imaging. Chelation of free 111In with EDTA was found necessary to reduce the nonspecific accumulation of signal. CONCLUSION: This study demonstrated the feasibility of radiolabeling WJ-MSC-EVs with 111In-oxine for in vivo imaging and quantitative analysis in a mouse model. This simple and quick labeling method preserves the characteristics of WJ-MSC-EVs. The results in this study provide a thorough and objective basis for future clinical study.

Combination of Colistin and Azidothymidine Demonstrates Synergistic Activity against Colistin-Resistant, Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Enterobacteriaceae (CRE) is listed as an urgent threat by the World Health Organization because of the limited therapeutic options, rapid evolution of resistance mechanisms, and worldwide dissemination. Colistin is a common backbone agent among the "last-resort" antibiotics for CRE; however, its emerging resistance among CRE has taken the present dilemma to the next level. Azidothymidine (AZT), a thymidine analog used to treat human immunodeficiency virus/acquired immunodeficiency syndrome, has been known to possess antibacterial effects against Enterobacteriaceae. In this study, we investigated the combined effects of AZT and colistin in 40 clinical isolates of colistin-resistant, carbapenem-resistant K. pneumoniae (CCRKP). Eleven of the 40 isolates harbored Klebsiella pneumoniae carbapenemase. The in vitro checkerboard method and in vivo nematode killing assay both revealed synergistic activity between the two agents, with fractional inhibitory concentration indexes of ≤0.5 in every strain. Additionally, a significantly lower hazard ratio was observed for the nematodes treated with combination therapy (0.288; p < 0.0001) compared with either AZT or colistin treatment. Toxicity testing indicated potentially low toxicity of the combination therapy. Thus, the AZT-colistin combination could be a potentially favorable therapeutic option for treating CCRKP.

In Vitro and In Vivo Evaluations of ß-Lactam/ß-Lactamase Mono- and Combined Therapies against Carbapenem-Nonsusceptible Enterobacteriaceae in Taiwan.

Increasing carbapenem resistance rates worldwide underscored the urgent need of novel antimicrobials. Ceftazidime-avibactam and aztreonam-avibactam combinations are developed to combat carbapenem resistance, but biological and geographic variations must be considered for antibiotic susceptibility patterns varied. Thus, we sought to assess the susceptibilities of ceftazidime-avibactam and aztreonam-avibactam against 660 carbapenem-nonsusceptible Enterobacteriaceae isolates (472 Klebsiella pneumoniae and 188 Escherichia coli) collected during an earlier Taiwan surveillance study. Agar dilution method was used to determine ceftazidime-avibactam and aztreonam-avibactam susceptibility. Metallo-carbapenemase's contribution to resistance were investigated with EDTA addition. The in vivo efficacies were evaluated using a Caenorhabditis elegans model. High susceptibility rates were observed for ceftazidime-avibactam and aztreonam-avibactam against the 472 carbapenem-nonsusceptible K. pneumoniae (CnsKP) (85.2% and 95.3%, respectively) and 188 carbapenem-nonsusceptible E. coli (CnsEC) isolates (91.5% and 94.1%, respectively). For non-metallo-carbapenemase producers, the susceptibility rates for ceftazidime-avibactam were 93.6% for CnsKP and 97.7% for CnsEC, whereas only 7.1% CnsKP and 11.1% CnsEC in metallo-carbapenemase producers were susceptible to ceftazidime-avibactam. Of all isolates, 95.3% CnsKP and 94.1% CnsEC were susceptible to aztreonam-avibactam. In C. elegans model, ceftazidime-avibactam and aztreonam-avibactam revealed effective against a blaKPC-producing K. pneumoniae isolate in vivo. Our results propose a positive therapeutic approach for both combinations against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network.

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

Correlation of 18F-FDG uptake and thyroid cancer stem cells.

BACKGROUND: 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography (18F-FDG PET) has the potential to detect various types of cancers, including thyroid cancer (TC), at a potentially curable stage. Increased uptake of 18F-FDG was observed in anaplastic and poorly differentiated thyroid cancer cells, and PET-positive tumors are more likely to be resistant to 131I treatment. As cancer stem cells (CSCs) possess a dedifferentiated phenotype and are resistant to many anticancer therapies, we hypothesized that the expression of CSC-related markers is correlated with the ability of tumor cells in TC to uptake FDG. METHODS: The present study cohort included 12 patients with TC, who underwent 18F-FDG PET/CT imaging before surgery. Quantitative polymerase chain reaction (QPCR) and immunohistochemical (IHC) staining were performed to analyze the expression patterns of gene markers related to embryonic stem (ES) cells and CSCs in TC. RESULTS: The mRNA expression levels of CSC- (CD133 and CD44) and ES-related genes (Oct4 and Nanog) were higher in TC tissue than in normal thyroid tissue, whereas the mRNA expression levels of thyroid-specific genes (Tg, TSHR, and TTF1) were higher in normal thyroid tissue than in TC tissue. There was a positive and statistically significant correlation between FDG uptake (SUVmax) of tumor and relative mRNA levels of CD133, CD44, Oct4, and Nanog. The IHC results demonstrated that CD133 and Nanog were expressed in TC tissue but not in normal thyroid tissue, however, CD44 expression was observed in both TC and normal thyroid tissue. Comparisons of the clinicopathological parameters between TC tissues with low and high SUVmax demonstrated significant differences in protein level of CD133 but not in that of Nanog. CONCLUSIONS: The pre-therapeutic tumor SUVmax obtained from 18F-FDG PET/CT may be a potential predictor for evaluating the proportion of CSC population in individual patients with TC.

Targeting amine- and phenol-containing metabolites in urine by dansylation isotope labeling and liquid chromatography mass spectrometry for evaluation of bladder cancer biomarkers.

Metabolomics is considered an effective approach for understanding metabolic responses in complex biological systems. Accordingly, it has attracted increasing attention for biomarker discovery, especially in cancer. In this study, we used a non-invasive method to evaluate four urine metabolite biomarker candidates-o-phosphoethanolamine, 3-amio-2-piperidone, uridine and 5-hydroxyindoleactic acid-for their potential as bladder cancer diagnostic biomarkers. To analyze these targeted amine- and phenol-containing metabolites, we used differential 12C2-/13C2-dansylation labeling coupled with liquid chromatography/tandem mass spectrometry, which has previously been demonstrated to exhibit high sensitivity and reproducibility. Specifically, we used ultra-performance liquid chromatography (UPLC) coupled with high-resolution Fourier transform ion-cyclotron resonance MS system (LC-FT/MS) and an ion trap MS with MRM function (LC-HCT/MS) for targeted quantification. The urinary metabolites of interest were well separated and quantified using this approach. To apply this approach to clinical urine specimens, we spiked samples with 13C2-dansylatedsynthetic compounds, which served as standards for targeted quantification of 12C2-dansylated urinary endogenous metabolites using LC-FT/MS as well as LC-HCT/MS with MRM mode. These analyses revealed significant differences in two of the four metabolites of interest-o-phosphoethanolamine and uridine-between bladder cancer and non-cancer groups. O-phosphoethanolamine was the most promising single biomarker, with an area-under-the-curve (AUC) value of 0.709 for bladder cancer diagnosis. Diagnostic performance was improved by combining uridine and o-phosphoethanolamine in a marker panel, yielding an AUC value of 0.726. This study confirmed discovery-phase features of the urine metabolome of bladder cancer patients and verified their importance for further study.

Integrated analyses utilizing metabolomics and transcriptomics reveal perturbation of the polyamine pathway in oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC), the most common malignancy of the oral cavity, is associated with poor prognosis and high mortality worldwide. Moreover, knowledge of the metabolic alterations that occur in OSCC is still limited. In the present study, we used a quantitative metabolomic approach with chemical isotope labeling (CIL) to analyze alterations in the metabolite levels in paired cancerous (T) and adjacent noncancerous (AN) tissues from 31 OSCC patients. Using volcano plot and orthogonal projections to latent structure-discriminant analysis (OPLS-DA), we uncovered 99 dysregulated metabolites in OSCC and verified the identities of seven metabolites via comparison with authenticated standards. From these seven metabolites, we constructed a 3-marker panel, consisting of putrescine, glycyl-leucine, and phenylalanine, using a support vector machine (SVM) model that can discriminate T from AN with high sensitivity and specificity based on receiver operator characteristic (ROC) analysis. Furthermore, by integrating the metabolomics profiles with transcriptomics data obtained from the same sample set, we revealed the dysregulation of the polyamine pathway in OSCC. Our findings provide insights into the metabolic perturbations present in OSCC and have uncovered potential metabolic biomarkers and therapeutic targets for use in the treatment of OSCC.

Bretschneider solution-induced alterations in the urine metabolome in cardiac surgery patients.

The development of Bretschneider's histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution represented a major advancement in cardiac surgery, offering significant myocardial protection. However, metabolic changes induced by this additive in the whole body have not been systematically investigated. Using an untargeted mass spectrometry-based method to deeply explore the urine metabolome, we sought to provide a holistic and systematic view of metabolic perturbations occurred in patients receiving HTK. Prospective urine samples were collected from 100 patients who had undergone cardiac surgery, and metabolomic changes were profiled using a high-performance chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method. A total of 14,642 peak pairs or metabolites were quantified using differential 13C-/12C-dansyl labeling LC-MS, which targets the amine/phenol submetabolome from urine specimens. We identified 223 metabolites that showed significant concentration change (fold change > 5) and assembled several potential metabolic pathway maps derived from these dysregulated metabolites. Our data indicated upregulated histidine metabolism with subsequently increased glutamine/glutamate metabolism, altered purine and pyrimidine metabolism, and enhanced vitamin B6 metabolism in patients receiving HTK. Our findings provide solid evidence that HTK solution causes significant perturbations in several metabolic pathways and establish a basis for further study of key mechanisms underlying its organ-protective or potential harmful effects.