Development and validation of a deep learning algorithm for pattern-based classification system of cervical cancer from pathological sections.

Background: Multi-center research has demonstrated that adopting Silva's pattern-based classification system (SPBC) enhances the clinical prognosis and facilitates hierarchical management of patients with endocervical adenocarcinomas (EAC). However, inconsistencies in SPBC can arise due to variations in pathologists' experience levels. Thus, the implementation of standardized decision-making tools becomes crucial to enhance the practicality of SPBC in clinical diagnosis and treatment. Methods: We enrolled a total of 90 patients with EAC in this study, of which 63 were assigned to the training group, and the remaining 27 were allocated to the validation group. To create and validate the prediction models for SPBC, we utilized a deep learning system (DLS) and calculated the area under the receiver operating characteristic curve (AUC). Results: In Silva pattern classification, ResNet50 achieved an average accuracy of 74.36% (63.64% for pattern A, 55.56% for pattern B, and 89.47% for pattern C respectively). Moreover, in test set, ResNet50 achieved an AUC of 0.69 for pattern A, 0.58 for pattern B, and 0.91 for pattern C. Conclusions: We successfully established a DLS for SPBC, which holds the potential to aid pathologists in accurately classifying patients with EAC.

Morphology, immunohistochemistry characteristics, and clinical presentation of microcystic urothelial carcinoma: a series of 10 cases.

BACKGROUND: Microcystic urothelial carcinoma (MUC) is a rare variant of urothelial carcinoma with histological appearances similar to begin lesions. Thus far, approximately 50 cases have been reported. Here, we investigated the clinicopathological features of MUC. METHODS: Clinical data and paraffin-embedded tissue blocks were collected. Immunohistochemical staining and polymerase chain reaction-Sanger sequencing were performed to detect the phenotype and TERT mutation status of MUC, respectively. RESULTS: The mean patient age was 58.8 ± 14.5 years, with a male predominance (8:2). The pathological stage was T1 in one case, T2 in three cases, T3 in four cases, and T4 in two cases. Tumor metastases or death occurred in all five patients who were followed up within 1-3 years. Histological analyses revealed microcystic, tubular, cribriform, and occasionally cord-like structures, which generally lacked interstitial reactions. The lumens were empty, contained eosinophilic secretion, or were filled with mucin. The microcysts/tubules/cribriform patterns were lined by flat, cuboid, signet ring, or columnar types of epithelia. The cuboid, signet ring, and columnar types represented "glandular metaplasia" or glandular differentiation of urothelial carcinoma. Immunohistochemistry analyses revealed distinct co-expression patterns involving the luminal markers FOXA1 and GATA3, as well as the basal markers CK5/6 and CD44. All 10 cases exhibited a luminal phenotype according to the GATA3+/CK14- criterion, whereas nine cases exhibited a luminal phenotype according to the FOXA1+/CK14- criterion. The telomerase reverse transcriptase-C228T mutation was detected in seven cases. CONCLUSIONS: MUC is a rare variant with a deceptively benign form of urothelial carcinoma, which is generally identified as a late-stage tumor with a poor prognosis. It exhibits distinct co-expression of luminal and basal markers, along with the TERT-C228T mutation.

The protective role of Wnt3a in peroxynitrite-induced damage of cochlear hair cells in vitro.

OBJECTIVE: To investigate the effect of peroxynitrite on the cultured cochlear hair cells of C57BL/6 P3 mice in vitro as well as the role of Wnt3a, as an activator of the canonical Wnt signaling pathway, underlying the action of such an oxidative stress. METHODS: The in vitro primary cultured cochlear hair cells were subjected to l00 µM peroxynitrite and l00 µM peroxynitrite +25 ng/mL Wnt3a for 24 h, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy. RESULTS: The number of surviving hair cells was significantly reduced in the 100 µM peroxynitrite group, while it was significantly higher in the Wnt3a + peroxynitrite treated group compared with the peroxynitrite treated group. The transmission electron microscopy showed that exposure to peroxynitrite induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, while Wnt3a clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. CONCLUSION: These results indicated that peroxynitrite could cause oxidative damage to the cochlear hair cells, and low concentrations of Wnt3a has a protective effect against oxidative damage. LEVEL OF EVIDENCE: Level 2.

The protective role of Wnt3a in peroxynitrite-induced damage of cochlear hair cells in vitro

Abstract Objective To investigate the effect of peroxynitrite on the cultured cochlear hair cells of C57BL/6 P3 mice in vitro as well as the role of Wnt3a, as an activator of the canonical Wnt signaling pathway, underlying the action of such an oxidative stress. Methods The in vitro primary cultured cochlear hair cells were subjected to l00 μM peroxynitrite and l00 μM peroxynitrite +25 ng/mL Wnt3a for 24 h, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy. Results The number of surviving hair cells was significantly reduced in the 100 μM peroxynitrite group, while it was significantly higher in the Wnt3a + peroxynitrite treated group compared with the peroxynitrite treated group. The transmission electron microscopy showed that exposure to peroxynitrite induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, while Wnt3a clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. Conclusion These results indicated that peroxynitrite could cause oxidative damage to the cochlear hair cells, and low concentrations of Wnt3a has a protective effect against oxidative damage. Level of evidence: Level 2.

The lncRNA XIST promotes the progression of breast cancer by sponging miR-125b-5p to modulate NLRC5.

X-inactivation-specific transcript (XIST) is a long noncoding RNA (lncRNA) that functions as an indicator of various human tumors, including those of breast cancer. This study was conducted to characterize a novel regulatory network involving XIST in breast cancer cells. The mRNAs of XIST, miR-125b-5p, and NOD-like receptor family CARD domain containing 5 (NLRC5) in breast cancer cells and tissues were analyzed using quantitative real-time polymerase chain reaction. Cell proliferation, apoptosis, migration, and invasion were separately detected via cell counting kit-8, flow cytometry, and Transwell assays. The relationships between XIST, miR-125b-5p, and NLRC5 were predicted and then confirmed using the dual-luciferase reporter assay. NLRC5 protein expression was quantitated using western blot assays. XIST was found to be overexpressed in breast cancer tissues and cells, which was accompanied by miR-125b-5p downregulation and NLRC5 upregulation. XIST knockdown significantly repressed cell proliferation, anti-apoptosis, migration, and invasion activities in breast cancer cells, and the loss of miR-125b-5p had a similar effect. XIST was shown to sponge miR-125b-5p, which in turn targeted NLRC5. NLRC5, a breast cancer promotor, is negatively regulated by miR-125b-5p. Moreover, the downregulation of NLRC5 induced by the loss of XIST was significantly reversed by miR-125b-5p knockdown. In conclusion, the lncRNA XIST promotes the malignancy of breast cancer cells partly by competitively binding to miR-125b-5p, which then led to increased NLRC5 expression. Our study suggests that targeting XIST may be a possible treatment for breast cancer.

[Determination of cinnamaldehyde residues in fresh fruits and meats by modified QuEChERS combined with gas chromatography-tandem mass spectrometry].

A method for the determination of cinnamaldehyde residues in fresh fruits and meats by gas chromatography-tandem mass spectrometry (GC-MS) coupled with modified QuEChERS was developed. The samples were extracted with ethyl acetate. Solid phase extraction agents such as graphitized carbon black (GCB), primary-secondary amine (PSA) and octadecyl bonded silica (C18) were used for adsorption and purification. The extracts were detected by electron impact ionization in selected ion monitoring (SIM) mode and quantified using external standard method. The results revealed correlation coefficients (R2) greater than 0.999 in the range of 0.01-0.5 mg/L. The limits of quantification (LOQs) were 0.05 to 0.1 mg/kg. The recoveries at the three spiked levels ranged from 81.9% to 104.5% with the relative standard deviations (RSDs) ranging from 1.1% to 8.6%. The proposed method is efficient, sensitive, reliable, and is suitable for the trace determination of the cinnamaldehyde residues in fresh fruits and meats.

[Simultaneous determination of 18 food-borne stimulant drug residues in beef using ultra-high performance liquid chromatography-tandem mass spectrometry].

A method for the simultaneous determination of 18 food-borne stimulant drug residues in beef was developed based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The beef sample was extracted with acidified acetonitrile, cleaned up by Captiva filtration cartridge. The extract was dried with anhydrous magnesium sulfate, and then concentrated by nitrogen flow. The obtained residue was re-dissolved in methanol-water (7:3, v/v). The separation was performed on an Agilent Zorbax Phenyl-Hexyl column with 5 mmol/L ammonium acetate solution (containing 0.01% (v/v) acetic acid) and methanol-acetonitrile (7:3, v/v) as mobile phases with gradient elution. The analyte was detected in positive and negative ion modes and the multiple reaction monitoring (MRM) mode. The quantification analysis was performed by external standard method using matrix-matched calibration curves. The method was linear with the correlation coefficients (R2) ≥ 0.9950 in the range of 0.10-50 µg/L. At the spiked levels of 0.4, 1.0 and 2.0 µg/kg, the recoveries of all compounds ranged from 57.3% to 117.5%, with RSDs in range of 3.1%-15.6% (n=5). The limits of detection and limits of quantification were in the range of 0.0006-0.0900 µg/kg and 0.0020-0.3000 µg/kg, respectively. The method is simple, rapid, accurate and sensitive, and can meet the requirement for the determination of the 18 food-borne stimulant drug residues in beef.

B Subunit of Human Chorionic Gonadotropin Promotes Tumor Invasion and Predicts Poor Prognosis of Early-Stage Colorectal Cancer.

BACKGROUND/AIMS: It is well established that many non-trophoblastic tumors secrete HCG (human chorionic gonadotropin) and that such secretion is correlated with the poor prognosis of tumor patients. This study aims to analyze the correlation between ß-HCG expression and outcome of colorectal cancer (CRC) and understand its role in CRC pathology Methods: We detected the mRNA and protein expression of ß-HCG in human CRC tissues with RT-qPCR and immunohistochemistry, and we compared the clinical-pathological characteristics, prognosis and progression between the ß-HCG positive and negative groups. We also generated CRC cell lines with ß-HCG over-expression as well as ß-HCG stable knockout, and evaluated cell function and mechanism in vitro and in vivo. RESULTS: Fifty out of 136 CRC patients (37%) expressed ß-HCG at the invasive front. Clinical-pathological data showed that ß-HCG was positively correlated with Dukes staging (P=0.031) and lymph node metastasis (P=0.012). Survival analysis suggested that the patients with high expression of ß-HCG had poorer prognosis than those with low ß-HCG expression (P=0.0289). ß-HCG expression level was also positively correlated with tumor invasion in early-stage CRC patient tissues (P=0.0227). Additionally ß-HCG promoted the migration and invasion of CRC in vitro and in vivo but had no effect on the proliferation of tumor cells. CONCLUSION: Our study demonstrated that ß-HCG was ectopically expressed in the CRC patients and its high expression correlated with poor prognosis of early-stage CRC. Additionally it worked as an oncogene that promotes the migration and invasion of CRC by epithelial-mesenchymal transition (EMT).

MiR-650 represses high-risk non-metastatic colorectal cancer progression via inhibition of AKT2/GSK3ß/E-cadherin pathway.

Although 5-year survival rate of non-metastatic colorectal cancer (CRC) is high, about 10% of patients in stage I and II still develop into metastatic CRC and eventually die after resection. Currently, there is no effective biomarker for predicting the prognosis of non-metastatic CRC in clinical practice. In this study, we identified miR-650 as a biomarker for prognosis prediction. We observed that the expression of miR-650 in tumor tissues had a positive association with overall survival. MiR-650 inhibited cell growth and invasion in vitro and in vivo. Furthermore, miR-650 targeted AKT2 and repressed the activation of the AKT pathway (AKT2/GSK3ß/E-cadherin). Thus it induced the translocation of E-cadherin and ß-catenin in cancer cells. Our results highlight the potential of miR-650 as a prognostic prediction biomarker and therapeutic target in non-metastatic CRC via inhibition of the AKT2/GSK3ß/E-cadherin pathway.

miR-375 inhibits the invasion and metastasis of colorectal cancer via targeting SP1 and regulating EMT-associated genes.

Accumulating evidence has shown that aberrantly expressed microRNAs (miRNAs) are associated with tumor development and progression. Our previous study found that microRNA-375 (miR-375) was downregulated in colorectal cancer (CRC), but little is known concerning the role of miR-375 and the related mechanism in CRC development. The proliferation, invasion and migration effects were investigated by Cell Counting Kit-8 (CCK-8), colony formation and Transwell assays with or without Matrigel. In addition, candidate target genes were screened and validated by luciferase reporter and western blot assays. In addition, western blot analysis was performed to explore the molecular mechanisms associated with epithelial­mesenchymal transition (EMT). It was found that miR-375 inhibited proliferation, invasion and migration in DLD1 and HCT8 cells. In addition, miR-375 negatively regulated Sp1 transcription factor (SP1) protein by directly binding to the 3'-untranslated region (3'-UTR). Furthermore, it was found that miR-375 regulated matrix metalloproteinase 2 (MMP2) and EMT-associated genes, E-cadherin, vimentin, snail, N-cadherin and ß-catenin. In conclusion, miR-375 inhibited the proliferation, invasion and migration by directly targeting SP1 and regulating MMP2 and EMT-associated genes.

Quantitation of microRNA-92a in colorectal adenocarcinoma and its precancerous lesions: Co-utilization of in situ hybridization and spectral imaging.

The expression level of microRNA (miR)-92a has been proven to increase during the development of colorectal adenocarcinoma (CA) and has been verified at the cellular, plasma and fecal levels by various quantitative methods. However, a method to quantitate the expression level using tissue sections has not been established. To do this, in situ hybridization (ISH) and multispectral imaging microscopy (MSI) were introduced to quantitate miR-92a expression on the microscopic level. ISH of miR-92a was first performed on 34 tissue samples of CA and adenomas with high-grade and low-grade intraepithelial neoplasms, while 31 paralesional normal tissue samples were defined as the control. Subsequently, a MSI technique was applied to quantitate the hybridization signal in terms of optical density (OD) at the visible wavelength. A t-test with unequal variance was used to examine the statistical significance between the groups. Despite all 34 tissue sections demonstrating at least partial positivity of miR-92a expression following ISH, visual grading was inconclusive. As such, the signal of ISH was transformed in terms of OD and further analyzed by employing the MSI system. A statistically significant difference was observed between the expression levels of miR-92a in CA and the paralesional normal controls. By contrast, a poor correlation was revealed between visual and spectral grading. The co-utilization of ISH and MSI generated a legible observation in the expression level of miR-92a, revealing the dynamic change in miR-92a expression in the progression of the disease and providing important information for further functional investigation.

Quantitative assessment of short amplicons in FFPE-derived long-chain RNA.

Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60 bp) and long (~200 bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues.

Symmetry breaking of α-[H2W12O40](6-) depends on the transformation of isopolyoxotungstates.

Two enantiotopic 1D chain compounds, [Cu3(L1)3(H2O)2(H2W12O40)]·4H2O (1a,b; L1 = 2-(4,6-bis(pyridin-2-yl)pyridin-2-yl)pyridine), crystallizing in the chiral space group P212121 were prepared and spontaneously resolved in the absence of any chiral source. Interestingly, compounds 1a,b can be prepared from a [W7O24](6-) aqueous solution, [(n-C4H9)4N]4[W10O32], or Na10[H2W12O42], but when [H2W12O40](6-) aqueous solution was the starting material, the achiral compound [CuL1]2[H4W12O40]·5H2O (2) was obtained. When a terpyridine ligand (L2) having a coordination mode similar to that of L1 was used, the mesomeric dimer [Cu3(L2)3(H2O)(H2W12O40)]2·4H2O (3) was obtained from [W7O24](6-) aqueous solution or Na10[H2W12O42], but from [H2W12O40](6-) aqueous solution only compound [Cu2(L2)2Cl2]2[W10O32] (4) was isolated. It is notable that in compounds 1a,b and 3 the symmetry of the α-[H2W12O40](6-) cluster is broken by asymmetric coordination with metal-organic units in a similar mode. As the asymmetric subunit based on a tridecorated [H2W12O40](6-) cluster can be obtained from several isopolyoxotungstate sources except for [H2W12O40](6-), we speculate that the symmetry breaking of α-[H2W12O40](6-) depends on the transformation of isopolyoxotungstates. Furthermore, during the transformation a possible reaction intermediate as the precursor for 1a,b, compound [Cu3(L1)3(H2O)3(H4W11O38)] (5), has been presented and characterized by density functional theory (DFT) calculations.

Lack of association between IL-4 -588C>T polymorphism and NHL susceptibility.

The relationship of non-Hodgkin's lymphoma (NHL) with the presence of interleukin-4 genetic polymorphism -588C>T has been reported with inconsistent results. The objective of this study was to quantitatively evaluate the association between -588C>T polymorphism and NHL susceptibility. Two investigators independently searched Medline and the Cochrane Library up to September 20, 2013. Pooled odds ratio and 95% confidence interval were calculated using a fixed or random effects model. Statistical analysis was performed with Review Manage 5.0 and Stata 11. Of the six case-control studies selected for this meta-analysis, a total of 1,909 NHL cases and 1,834 controls were included. The combined results based on all studies suggested that -588C>T was not associated with NHL risk under all genetic models. When stratifying for race, no noteworthy associations were observed in mixed populations or Caucasians. This meta-analysis suggests that IL-4 -588C>T polymorphism might not be a risk factor for NHL risk. However, further well-designed studies are required to confirm our findings.

[Determination of ribavirin and amantadine in chicken by ultra performance liquid chromatography-tandem mass spectrometry].

A method for the determination of ribavirin and amantadine in chicken has been developed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS). After extracted by 1% (volume percentage) trichloroacetic acid solution-acetonitrile (1:1, v/v) and purified by a Supelco LC-SCX cartridge, the samples were loaded onto an Acquity UPLC BEH Hillic column (150 mm x 2.1 mm, 1.7 microm) and separated with gradient elution. The electrospray was operated in the positive mode and the samples were monitored by the multiple reaction monitoring (MRM) mode. The limits of quantification (LOQs, S/N = 10) of ribavirin and amantadine were 4.0 microg/kg. The calibration curves showed good linearity in the range of 10.0 - 100.0 microg/L, and the correlation coefficients (r(2)) were not lower than 0.99. When the spiked levels were 4.0, 8.0 and 20.0 microg/kg, the recoveries of ribavirin and amantadine in chicken ranged from 78% to 102.5%, with the relative standard deviations (RSDs) of 2.2% - 7.6%. The results indicate that the method is simple, rapid, sensitive and suitable for the qualitative and quantitative analyses of ribavirin and amantadine in chicken samples.

Determination of amantadine and rimantadine in chicken muscle by QuEChERS pretreatment method and UHPLC coupled with LTQ Orbitrap mass spectrometry.

A novel sample pretreatment method was developed for the quantitative determination of amantadine and rimantadine in chicken muscle tissues by ultra high performance liquid chromatography coupled with high resolution LTQ Orbitrap mass spectrometry (UHPLC-LTQ Orbitrap MS). The samples were pretreated by modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method, using acetonitrile (1% acetic acid, v/v) as extraction solution and C18 sorbent for clean-up. The separation was carried out on a Waters ACQUITY UPLC HSS T3 column (150mm×2.1mm, 1.8µm particle), using a mobile phase of acetonitrile and 0.1% aqueous formic acid solution. LTQ Orbitrap MS with resolving power of 60000 was applied for analysis of the samples. Amantadine and rimantadine were identified from their accurate mass (within 5ppm) and retention times from the acquired full-scan chromatogram and quantified by their peak areas. The linear range for the determination of the analytes was 1-100µg/kg. Limits of detection (LODs) for amantadine and rimantadine were 1.02µg/kg and 0.67µg/kg, respectively. The intra- and inter-day accuracy ranged from 87.5% to 102.4%, and 82.5% to 105.8% for amantadine, and 95.3% to 97.4%, and 89.4% to 93.2% for rimantadine, respectively. The precision of intra- and inter-day was between 3.9-6.3% and 5.95-13.9% for amantadine, 6.0-7.45% and 7.8-12.4% for rimantadine, respectively. Finally, the method was applied for the determination of these antiviral agents in routine samples, and amantadine residue was detected in some cases.

Optimized pregelatinized starch technique for cell block preparation in cell cultures.

The aim of the present study was to optimize the pregelatinized starch technique for cell block preparation and apply this approach in cultured cells of all types of growing forms, suspension and adherent. In order to evenly mix the starch powder and the cell suspension, we crafted a special plastic dropper. To prove the effectiveness of this optimized technique we used different cell lines, NCI-H69, NCI-H345, HCT-116, SKBR3 and MDA-MB-231. The morphology features, immunocytochemistry (ICC) and fluorescent/chromogenic in-situ hybridization (FISH/CISH) on the cell block sections were evaluated. The morphology features, the ICC and ISH results of cell block sections prepared by the new method were satisfactory comparing with the results obtained in biopsies, the gold standard test for this kind of analysis. The most attractive advantage of our optimized pregelatinized starch technique is that this new method is based on cell suspensions instead of cell sediment, so with our technique every section will contain cells due to the even distribution of the starch powder and the cells forming a homogeneous cell block. To the authors' knowledge, this is the first description on cell block preparation based on cell suspension.

[Rapid screening and confirming carcinogenic banned azo colorants in textiles by high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry].

A method of high performance liquid chromatography-linear ion trap/orbitrap highresolution mass spectrometry (HPLC-LTP/Orbitrap MS) was ued to screen and confirm-banned azo colorants in textiles rapidly. The analytes were reduced to carcinogenic aromatic amines with sodium dithionite in citrate buffer solution. The reduced solution was extracted bydiatomite, and loadd onto an Acquity UPLC BEH C18 column (50 mm x 2.1 MM. 1.7 microm) with a gradient elution of methanol and 0.1% (v/v) methane acid aqueous solution, and finally detected by linear ion trap/orbitrap high-resolution mass spectrometry in positive ESI mode. In mass spectrometry method, the MS spectrum of high-resolution and the collision induced dissociation (CID) spectrum of data-dependent scan mode were used for screening analysis and conformation, respectively. The calibration curves showed a good linearity in the range of 0.05 -2.00 mg/b, and the correlation coefficients (r) were higher than 0.99. By detecting spiked samples, the limits of quantification were 0.08 mg/kg for all the residues and the recoveries were in the range of 65.5% - 111.5% with the relative standard deviations (RSDs) between 0.87% and 2.49%. The results indicate that the method is simple, rapid, sensitive and suitable for the qualitative and quantitative analysis of carcinogenic aromatic amines in textiles.

Intriguing role of a quaternary ammonium cation in the dissociation chemistry of Keggin polyoxometalate anions.

The gas-phase fragmentations of a series of Keggin polyoxometalate anions with molecular formula of TBA(n)[XM(12)O(40)] (X = P, Si; M = Mo, W) were studied by electrospray ionization tandem mass spectrometry. The bare polyoxoanions [XM(12)O(40)](n-) as well as the non-covalent complexes {TBA[XM(12)O(40)]}((n-1)-) and {TBA(m)[XM(12)O(40)](2)}(3-) displayed characteristic dissociation pathways. Fragmentation of [XM(12)O(40)](n-) led to pairs of complementary product anions whose total stoichiometry and charge matched those of the precursor anion, consistent with the previous study by Ma et al. The nature of the non-covalent interaction between [XM(12)O(40)](n-) and TBA(+) was addressed in detail via the example of {TBA[XM(12)O(40)]}((n-1)-). The non-covalent interaction [1] primarily dominated by the Coulombic attraction of the opposite charges completely changed the dissociation chemistry of [XM(12)O(40)](n-). The non-covalent complexes {TBA[XM(12)O(40)]}((n-1)-) and {TBA(m)[XM(12)O(40)](2)}(3-), formed by the charge reduction during the electrospray process, underwent distinct dissociation routes: {TBA[XM(12)O(40)]}((n-1)-) fragmented to give rise to its product ion {(C(4)H(9))[XM(12)O(40)]}((n-1)-) by cleaving the N-C covalent bond inside the TBA(+) cation whereas {TBA(m)[XM(12)O(40)](2)}(3-) dissociated into a pair of product ions, {TBA(i)[XM(12)O(40)]}(2-) and {TBA(m-i)[XM(12)O(40)]}(-), by breaking the non-covalent bond between [XM(12)O(40)](n-) and TBA(+). In addition, energy-variable CID was used to map the relative stabilities of the ion clusters in the gas phase, which was in excellent agreement with the relative orders of thermal stability in the condensed phase.