PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

Prediction of clinically significant prostate cancer through urine metabolomic signatures: A large-scale validated study.

PURPOSE: Currently, there are no accurate markers for predicting potentially lethal prostate cancer (PC) before biopsy. This study aimed to develop urine tests to predict clinically significant PC (sPC) in men at risk. METHODS: Urine samples from 928 men, namely, 660 PC patients and 268 benign subjects, were analyzed by gas chromatography/quadrupole time-of-flight mass spectrophotometry (GC/Q-TOF MS) metabolomic profiling to construct four predictive models. Model I discriminated between PC and benign cases. Models II, III, and GS, respectively, predicted sPC in those classified as having favorable intermediate risk or higher, unfavorable intermediate risk or higher (according to the National Comprehensive Cancer Network risk groupings), and a Gleason sum (GS) of ≥ 7. Multivariable logistic regression was used to evaluate the area under the receiver operating characteristic curves (AUC). RESULTS: In Models I, II, III, and GS, the best AUCs (0.94, 0.85, 0.82, and 0.80, respectively; training cohort, N = 603) involved 26, 24, 26, and 22 metabolites, respectively. The addition of five clinical risk factors (serum prostate-specific antigen, patient age, previous negative biopsy, digital rectal examination, and family history) significantly improved the AUCs of the models (0.95, 0.92, 0.92, and 0.87, respectively). At 90% sensitivity, 48%, 47%, 50%, and 36% of unnecessary biopsies could be avoided. These models were successfully validated against an independent validation cohort (N = 325). Decision curve analysis showed a significant clinical net benefit with each combined model at low threshold probabilities. Models II and III were more robust and clinically relevant than Model GS. CONCLUSION: This urine test, which combines urine metabolic markers and clinical factors, may be used to predict sPC and thereby inform the necessity of biopsy in men with an elevated PC risk.

Rh(III)-Catalyzed Switchable [4 + 1] and [4 + 2] Annulation of N-Aryl Pyrazolones with Maleimides: An Access to Spiro Pyrazolo[1,2-a]indazole-pyrrolidine and Fused Pyrazolopyrrolo Cinnolines.

A rhodium(III)-catalyzed controllable [4 + 1] and [4 + 2] annulation of N-aryl pyrazolones with maleimides as C1 and C2 synthon has been explored for the synthesis of spiro[pyrazolo[1,2-a]indazole-pyrrolidines] and fused pyrazolopyrrolo cinnolines. The product selectivity was achieved through time-dependent annulation. The [4 + 1] annulation reaction involves sequential Rh(III)-catalyzed C-H alkenylation of N-aryl pyrazolone, followed by an intramolecular spirocyclization via aza-Michael-type addition to afford spiro[pyrazolo[1,2-a]indazole-pyrrolidine]. However, prolonged reaction time converts in situ formed spiro[pyrazolo[1,2-a]indazole-pyrrolidine] into fused pyrazolopyrrolocinnoline. This unique product formation switch proceeds via strain-driven ring expansion through a 1,2-shift of the C-C bond.

Heparan sulfate is essential for thymus growth.

Thymus organogenesis and T cell development are coordinated by various soluble and cell-bound molecules. Heparan sulfate (HS) proteoglycans can interact with and immobilize many soluble mediators, creating fields or gradients of secreted ligands. While the role of HS in the development of many organs has been studied extensively, little is known about its function in the thymus. Here, we examined the distribution of HS in the thymus and the effect of its absence on thymus organogenesis and T cell development. We found that HS was expressed most abundantly on the thymic fibroblasts and at lower levels on endothelial, epithelial, and hematopoietic cells. To study the function of HS in the thymus, we eliminated most of HS in this organ by genetically disrupting the glycosyltransferase Ext1 that is essential for its synthesis. The absence of HS greatly reduced the size of the thymus in fetal thymic organ cultures and in vivo, in mice, and decreased the production of T cells. However, no specific blocks in T cell development were observed. Wild-type thymic fibroblasts were able to physically bind the homeostatic chemokines CCL19, CCL21, and CXCL12 ex vivo. However, this binding was abolished upon HS degradation, disrupting the CCL19/CCL21 chemokine gradients and causing impaired migration of dendritic cells in thymic slices. Thus, our results show that HS plays an essential role in the development and growth of the thymus and in regulating interstitial cell migration.

CHK2 activation contributes to the development of oxaliplatin resistance in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC), the most common cancer type, causes high morbidity and mortality. Patients who develop drug resistance to oxaliplatin-based regimens have short overall survival. Thus, identifying molecules involved in the development of oxaliplatin resistance is critical for designing therapeutic strategies. METHODS: A proteomic screen was performed to reveal altered protein kinase phosphorylation in oxaliplatin-resistant (OR) CRC tumour spheroids. The function of CHK2 was characterised using several biochemical techniques and evident using in vitro cell and in vivo tumour models. RESULTS: We revealed that the level of phospho-CHK2(Thr68) was elevated in OR CRC cells and in ~30% of tumour samples from patients with OR CRC. We demonstrated that oxaliplatin activated several phosphatidylinositol 3-kinase-related kinases (PIKKs) and CHK2 downstream effectors and enhanced CHK2/PARP1 interaction to facilitate DNA repair. A phosphorylation mimicking CHK2 mutant, CHK2T68D, but not a kinase-dead CHK2 mutant, CHK2D347A, promoted DNA repair, the CHK2/PARP1 interaction, and cell growth in the presence of oxaliplatin. Finally, we showed that a CHK2 inhibitor, BML-277, reduced protein poly(ADP-ribosyl)ation (PARylation), FANCD2 monoubiquitination, homologous recombination and OR CRC cell growth in vitro and in vivo. CONCLUSION: Our findings suggest that CHK2 activity is critical for modulating oxaliplatin response and that CHK2 is a potential therapeutic target for OR CRC.

Spatial multi-omics analyses of the tumor immune microenvironment.

In the past decade, single-cell technologies have revealed the heterogeneity of the tumor-immune microenvironment at the genomic, transcriptomic, and proteomic levels and have furthered our understanding of the mechanisms of tumor development. Single-cell technologies have also been used to identify potential biomarkers. However, spatial information about the tumor-immune microenvironment such as cell locations and cell-cell interactomes is lost in these approaches. Recently, spatial multi-omics technologies have been used to study transcriptomes, proteomes, and metabolomes of tumor-immune microenvironments in several types of cancer, and the data obtained from these methods has been combined with immunohistochemistry and multiparameter analysis to yield markers of cancer progression. Here, we review numerous cutting-edge spatial 'omics techniques, their application to study of the tumor-immune microenvironment, and remaining technical challenges.

AUY922 induces retinal toxicity through attenuating TRPM1.

BACKGROUND: Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. METHODS: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)­based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. RESULTS: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. CONCLUSIONS: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Targeting triple-negative breast cancer with an aptamer-functionalized nanoformulation: a synergistic treatment that combines photodynamic and bioreductive therapies.

BACKGROUND: Areas of hypoxia are often found in triple-negative breast cancer (TNBC), it is thus more difficult to treat than other types of breast cancer, and may require combination therapies. A new strategy that combined bioreductive therapy with photodynamic therapy (PDT) was developed herein to improve the efficacy of cancer treatment. Our design utilized the characteristics of protoporphyrin IX (PpIX) molecules that reacted and consumed O2 at the tumor site, which led to the production of cytotoxic reactive oxygen species (ROS). The low microenvironmental oxygen levels enabled activation of a bioreductive prodrug, tirapazamine (TPZ), to become a toxic radical. The TPZ radical not only eradicated hypoxic tumor cells, but it also promoted therapeutic efficacy of PDT. RESULTS: To achieve the co-delivery of PpIX and TPZ for advanced breast cancer therapy, thin-shell hollow mesoporous Ia3d silica nanoparticles, designated as MMT-2, was employed herein. This nanocarrier designed to target the human breast cancer cell MDA-MB-231 was functionalized with PpIX and DNA aptamer (LXL-1), and loaded with TPZ, resulting in the formation of TPZ@LXL-1-PpIX-MMT-2 nanoVector. A series of studies confirmed that our nanoVectors (TPZ@LXL-1-PpIX-MMT-2) facilitated in vitro and in vivo targeting, and significantly reduced tumor volume in a xenograft mouse model. Histological analysis also revealed that this nanoVector killed tumor cells in hypoxic regions efficiently. CONCLUSIONS: Taken together, the synergism and efficacy of this new therapeutic design was confirmed. Therefore, we concluded that this new therapeutic strategy, which exploited a complementary combination of PpIX and TPZ, functioned well in both normoxia and hypoxia, and is a promising medical procedure for effective treatment of TNBC.

Molecular Iodine-Promoted [3 + 2] Oxidative Cyclization for the Synthesis of Heteroarene-Fused [1,2,4] Thiadiazoles/Selenadiazoles.

Two new classes of heteroarene-fused [1,2,4]thiadiazole and [1,2,4]selenadiazole are synthesized through the iodine-mediated [3 + 2] oxidative cyclization of 2-aminoheteroarenes and isothiocyanates/isoselenocyanates. This oxidative [3 + 2] annulation strategy is highly regiospecific to proceed a selective C-N bond formation at the endo-nitrogen of 2-aminoheteroarenes followed by an intramolecular oxidative N-S/N-Se bond formation. It is the first example of an I2-mediated oxidative nitrogen-selenium (N-Se) bond formation.

Roles of Textural and Surface Properties of Nanoparticles in Ultrasound-Responsive Systems.

Acoustic inertial cavitation (IC) is a crucial phenomenon for many ultrasound (US)-related applications. This study aimed to investigate the roles of textural and surface properties of NPs in IC generation by combining typical IC detection methods with various types of silica model NPs. Acoustic passive cavitation detection, optical high-speed photography, and US imaging have been used to quantify IC activities (referred to as the IC dose, ICD) and describe the physical characteristics of IC activities from NPs. The results showed that the ICDs from NPs were positively correlated to their surface hydrophobicity and that their external surface hydrophobicity plays a much more crucial role than do the textural properties. The high-speed photography revealed that the sizes of IC-generated bubbles from superhydrophobic NPs ranged from 20-40 µm at 4-6 MPa and collapsed in several microseconds. Bubble clouds monitored with US imaging showed that IC from NPs was consistent with the surface hydrophobicity. The simulation results based on the crevice model of cavitation nuclei correlated well with the experimental results. This study has demonstrated that the surface property, instead of the textural property, of NPs dominated the IC generation, and surface nanobubbles adsorbed on the NP surface have been proposed to be cavitation nuclei.

Ethylene-Regulated Glutamate Dehydrogenase Fine-Tunes Metabolism during Anoxia-Reoxygenation.

Ethylene is an essential hormone in plants that is involved in low-oxygen and reoxygenation responses. As a key transcription factor in ethylene signaling, ETHYLENE INSENSITIVE3 (EIN3) activates targets that trigger various responses. However, most of these targets are still poorly characterized. Through analyses of our microarray data and the published Arabidopsis (Arabidopsis thaliana) EIN3 chromatin immunoprecipitation sequencing data set, we inferred the putative targets of EIN3 during anoxia-reoxygenation. Among them, GDH2, which encodes one subunit of glutamate dehydrogenase (GDH), was chosen for further studies for its role in tricarboxylic acid cycle replenishment. We demonstrated that both GDH1 and GDH2 are induced during anoxia and reoxygenation and that this induction is mediated via ethylene signaling. In addition, the results of enzymatic assays showed that the level of GDH during anoxia-reoxygenation decreased in the ethylene-insensitive mutants ein2-5 and ein3eil1 Global metabolite analysis indicated that the deamination activity of GDH might regenerate 2-oxoglutarate, which is a cosubstrate that facilitates the breakdown of alanine by alanine aminotransferase when reoxygenation occurs. Moreover, ineffective tricarboxylic acid cycle replenishment, disturbed carbohydrate metabolism, reduced phytosterol biosynthesis, and delayed energy regeneration were found in gdh1gdh2 and ethylene mutants during reoxygenation. Taken together, these data illustrate the essential role of EIN3-regulated GDH activity in metabolic adjustment during anoxia-reoxygenation.

Simvastatin and a Plant Galactolipid Protect Animals from Septic Shock by Regulating Oxylipin Mediator Dynamics through the MAPK-cPLA2 Signaling Pathway.

Sepsis remains a major medical issue despite decades of research. Identification of important inflammatory cascades and key molecular mediators are crucial for developing intervention and prevention strategies. In this study, we conducted a comparative oxylipin metabolomics study to gain a comprehensive picture of lipid mediator dynamics during the initial hyperinflammatory phase of sepsis, and demonstrated, in parallel, the efficacy of simvastatin and plant galactolipid, 1,2-di-O-α-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG) in the homeostatic regulation of the oxylipin metabolome using a lipopolysaccharide (LPS)-induced sepsis C57BL/6J mouse model. LPS increased the systemic and organ levels of proinflammatory metabolites of linoleic acid including leukotoxin diols (9-,10-DHOME, 12-,13-DHOME) and octadecadienoic acids (9-HODE and 13-HODE) and arachidonic acid-derived prostanoid, PGE2, and hydroxyeicosatetraenoic acids (8-, 12- and 15-HETE). Treatment with either compound decreased the levels of proinflammatory metabolites and elevated proresolution lipoxin A4, 5(6)-EET, 11(12)-EET and 15-deoxy-PGJ2. dLGG and simvastatin ameliorated the effects of LPS-induced mitogen-activated protein kinase (MAPK)-dependent activation of cPLA2, cyclooxygenase-2, lipoxygenase, cytochrome P450 and/or epoxide hydrolase lowered systemic TNF-α and IL-6 levels and aminotransferase activities and decreased organ-specific infiltration of inflammatory leukocytes and macrophages, and septic shock-induced multiple organ damage. Furthermore, both dLGG and simvastatin increased the survival rates in the cecal ligation and puncture (CLP) sepsis model. This study provides new insights into the role of oxylipins in sepsis pathogenesis and highlights the potential of simvastatin and dLGG in sepsis therapy and prevention.

Health Knowledge Effects: An Integrated Community Health Promotion Platform.

The Taiwanese government subsidizes healthcare providers offering preventive medicine to patients to help reduce the threats of chronic sickness and halt skyrocketing medical expenditures. Usually, nurses are the primary workers who perform community health promotion; however, because of the chronic shortage of working nurses, many Taiwan hospitals have closed wards and deferred the responsibility of promoting primary prevention. With a community health promotion platform integrating interactive response features and Web sites for community patients and hospital staff, a case hospital efficiently sustained the community health services. The objective of this study was to assess the impact of the integrated community health promotion platform for conducting education. Fifty-four patients/residents were invited to join a quasi-experiment of health education, and a follow-up survey was conducted to assess the acceptance of the community health promotion platform from both the experimental group of learners/users and the hospital staff. The results showed that the community health promotion platform was effective in improving participant health awareness. The experimental group outperformed the control group, with higher posttest scores and longer knowledge retention. Furthermore, users indicated a high acceptance of the community health promotion platform.

Reliability of the Montreal Cognitive Assessment in people with stroke.

This study examined the relative and absolute reliability of the Taiwanese version of the MoCA (MoCA-T) in people with stroke. The study recruited 114 individuals who were at least 3 months after the onset of a first-ever unilateral stroke. The MoCA-T was administered twice, at a 6-week interval, to all participants. The relative reliability was assessed using the intraclass correlation coefficient (ICC), and the absolute reliability was assessed using standard error of measurement (SEM), the smallest real difference (SRD), the SRD percentage, and the Bland-Altman method. The ICC analysis showed the MoCA-T was highly reliable (ICC = 0.85). The absolute reliability was between an acceptable and excellent level, where the SEM and the SRD at the 95% confidence interval were 1.38 and 3.83, respectively. The Bland-Altman analyses showed no systematic bias between repeated measurements. The range of the 95% limits of agreement was narrow, indicating a high level of stability over time. These findings suggest that the MoCA-T has high agreement between repeated measurements without systematic bias. The threshold to detect real change stands between an acceptable and excellent level. The MoCA-T is a reliable tool for cognitive screening in stroke rehabilitation.

Glutathione determines chronic myeloid leukemia vulnerability to an inhibitor of CMPK and TMPK.

Bcr-Abl transformation leads to chronic myeloid leukemia (CML). The acquirement of T315I mutation causes tyrosine kinase inhibitors (TKI) resistance. This study develops a compound, JMF4073, inhibiting thymidylate (TMP) and cytidylate (CMP) kinases, aiming for a new therapy against TKI-resistant CML. In vitro and in vivo treatment of JMF4073 eliminates WT-Bcr-Abl-32D CML cells. However, T315I-Bcr-Abl-32D cells are less vulnerable to JMF4073. Evidence is presented that ATF4-mediated upregulation of GSH causes T315I-Bcr-Abl-32D cells to be less sensitive to JMF4073. Reducing GSH biosynthesis generates replication stress in T315I-Bcr-Abl-32D cells that require dTTP/dCTP synthesis for survival, thus enabling JMF4073 susceptibility. It further shows that the levels of ATF4 and GSH in several human CML blast-crisis cell lines are inversely correlated with JMF4073 sensitivity, and the combinatory treatment of JMF4073 with GSH reducing agent leads to synthetic lethality in these CML blast-crisis lines. Altogether, the investigation indicates an alternative option in CML therapy.

Predicting Clinically Significant Prostate Cancer Using Urine Metabolomics via Liquid Chromatography Mass Spectrometry.

PURPOSE: Biomarkers predicting clinically significant prostate cancer (sPC) before biopsy are currently lacking. This study aimed to develop a non-invasive urine test to predict sPC in at-risk men using urinary metabolomic profiles. MATERIALS AND METHODS: Urine samples from 934 at-risk subjects and 268 treatment-naïve PC patients were subjected to liquid chromatography/mass spectrophotometry (LC-MS)-based metabolomics profiling using both C18 and hydrophilic interaction liquid chromatography (HILIC) column analyses. Four models were constructed (training cohort [n=647]) and validated (validation cohort [n=344]) for different purposes. Model I differentiates PC from benign cases. Models II, III, and a Gleason score model (model GS) predict sPC that is defined as National Comprehensive Cancer Network (NCCN)-categorized favorable-intermediate risk group or higher (Model II), unfavorable-intermediate risk group or higher (Model III), and GS ≥7 PC (model GS), respectively. The metabolomic panels and predicting models were constructed using logistic regression and Akaike information criterion. RESULTS: The best metabolomic panels from the HILIC column include 25, 27, 28 and 26 metabolites in Models I, II, III, and GS, respectively, with area under the curve (AUC) values ranging between 0.82 and 0.91 in the training cohort and between 0.77 and 0.86 in the validation cohort. The combination of the metabolomic panels and five baseline clinical factors that include serum prostate-specific antigen, age, family history of PC, previously negative biopsy, and abnormal digital rectal examination results significantly increased AUCs (range 0.88-0.91). At 90% sensitivity (validation cohort), 33%, 34%, 41%, and 36% of unnecessary biopsies were avoided in Models I, II, III, and GS, respectively. The above results were successfully validated using LC-MS with the C18 column. CONCLUSIONS: Urinary metabolomic profiles with baseline clinical factors may accurately predict sPC in men with elevated risk before biopsy.

UniQua: a universal signal processor for MS-based qualitative and quantitative proteomics applications.

Recent developments in high resolution mass spectrometry (HR-MS) technology have ushered proteomics into a new era. However, the importance of using a common, open data platform for signal processing of HR-MS spectra has not been sufficiently addressed. In this study, a MS signal processor was developed to facilitate data integration from different instruments and different proteomics approaches into a unified platform without compromising protein identification and quantitation performance. This processor supports parallel processing capability which allows full utilization of computing resources to speed up signal processing performance to >1 gigabytes/min. The storage space occupied by the processed MS data can be reduced to ~10%, which helps the analysis and management of large quantities of data from comprehensive proteomics studies. For quantitation at the MS level, processing accuracy is improved and processing time for ASAPRatio is reduced to ~50%. For quantitation at the MS/MS level, accurate reporter ion ratios from different instruments can be directly determined by the processed MS/MS spectra and reported in the Mascot search result directly without using specialized iTRAQ software.

Comparative study of sialyl glycoprotein with multiple glycosylation sites using isotope labeling and capillary liquid chromatography/mass spectrometry.

RATIONALE: A comparative strategy has been demonstrated using RNase B, a single-site N-linked high-mannose glycoprotein. Glycoproteins are more common with multiple glycosylation sites and with complex glycans. A strategy capable of differentiating the changes caused by glycoprotein concentration, glycosylation site occupancy, and a glycoform profile of complex glycoproteins would be beneficial. METHODS: Tryptic-digested glycoproteins were labeled using 12 C,H-formaldehyde and 13 C, D-formaldehyde, purified, and then analyzed using capillary reversed-phase liquid chromatography/mass spectrometry (RPLC/MS). The relative intensity of non-glycosylated peptides provided information on glycoprotein concentration variation. A site-specific glycoform profile variation was obtained by comparing the glycoform profile of CH2 and 13 CD2 glycopeptides. Determining the protein concentration and glycoform profile variations allows the glycosylation site occupancy variation to be calculated. RESULTS: A strong correlation between the observed and prepared ratios for fetuin glycopeptides from 0.2 to 5 was obtained. Two fetuin samples with different glycoprotein concentrations (4-fold change), glycoform profiles (normal and modified), and glycosylation site occupancies (100% and 50%) were prepared, labeled, mixed, purified, and analyzed using RPLC/MS. The results of the comparative study had a strong correlation with the prepared values. CONCLUSIONS: In this report, we demonstrated a comparative analysis of fetuin, a glycoprotein with multiple glycosylation sites and complex sialyl glycans. Compared to our previous approach, we made several modifications including the use of RPLC, a larger mass difference isotope tag, and isotope overlapping correction. The modified approach is expected to be applicable to most glycoproteins. Copyright © 2013 John Wiley & Sons, Ltd.

Thiolated Mesoporous Silica Nanoparticles as an Immunoadjuvant to Enhance Efficacy of Intravesical Chemotherapy for Bladder Cancer.

The characteristics of global prevalence and high recurrence of bladder cancer has led numerous efforts to develop new treatments. The spontaneous voiding and degradation of the chemodrug hamper the efficacy and effectiveness of intravesical chemotherapy following tumor resection. Herein, the externally thiolated hollow mesoporous silica nanoparticles (MSN-SH(E)) is fabricated to serve as a platform for improved bladder intravesical therapy. Enhanced mucoadhesive effect of the thiolated nanovector is confirmed with porcine bladder. The permeation-enhancing effect is also verified, and a fragmented distribution pattern of a tight junction protein, claudin-4, indicates the opening of tight junction. Moreover, MSN-SH(E)-associated reprogramming of M2 macrophages to M1-like phenotype is observed in vitro. The antitumor activity of the mitomycin C (MMC)-loaded nanovector (MMC@MSN-SH(E)) is more effective than that of MMC alone in both in vitro and in vivo. In addition, IHC staining is used to analyze IFN-γ, TGF-ß1, and TNF-α. These observations substantiated the significance of MMC@MSN-SH(E) in promoting anticancer activity, holding the great potential for being used in intravesical therapy for non-muscle invasive bladder cancer (NMIBC) due to its mucoadhesivity, enhanced permeation, immunomodulation, and prolonged and very efficient drug exposure.