Proteomic profiling of TBC1 domain family member 21-null sperms reveals the critical roles of TEKT 1 in their tail defects.

BACKGROUND: Approximately 7% of the males exhibit reduced fertility; however, the regulatory genes and pathways involved remain largely unknown. TBC1 domain family member 21 (TBC1D21) contains a conserved RabGAP catalytic domain that induces GDP/GTP exchange to inactivate Rabs by interacting with microtubules. We previously reported that Tbc1d21-null mice exhibit severe sperm tail defects with a disrupted axoneme, and that TBC1D21 interacts with RAB10. However, the pathological mechanisms underlying the Tbc1d21 loss-induced sperm tail defects remain unknown. RESULTS: Murine sperm from wild-type and Tbc1d21-null mice were comparatively analyzed using proteomic assays. Over 1600 proteins were identified, of which 15 were significantly up-regulated in Tbc1d21-null sperm. Notably, several tektin (TEKT) family proteins, belonging to a type of intermediate filament critical for stabilizing the microtubular structure of cilia and flagella, were significantly up-regulated in Tbc1d21-/- sperm. We also found that TBC1D21 interacts with TEKT1. In addition, TEKT1 co-localized with RAB10 during sperm tail formation. Finally, we found Tbc1d21-null sperm exhibited abnormal accumulation of TEKT1 in the midpiece region, accompanied by disrupted axonemal structures. CONCLUSIONS: These results reveal that TBC1D21 modulates TEKTs protein localization in the axonemal transport system during sperm tail formation.

Decreased Klotho Expression Causes Accelerated Decline of Male Fecundity through Oxidative Injury in Murine Testis.

Oxidative stress is the etiology for 30-80% of male patients affected by infertility, which is a major health problem worldwide. Klotho protein is an aging suppressor that functions as a humoral factor modulating various cellular processes including antioxidation and anti-inflammation, and its dysregulation leads to human pathologies. Male mice lacking Klotho are sterile, and decreased Klotho levels in the serum are observed in men suffering from infertility with lower sperm counts. However, the mechanism by which Klotho maintains healthy male fertility remains unclear. Klotho haplodeficiency (Kl+/-) accelerates fertility reduction by impairing sperm quality and spermatogenesis in Kl+/- mice. Testicular proteomic analysis revealed that loss of Klotho predominantly disturbed oxidation and the glutathione-related pathway. We further focused on the glutathione-S-transferase (GST) family which counteracts oxidative stress in most cell types and closely relates with fertility. Several GST proteins, including GSTP1, GSTO2, and GSTK1, were significantly downregulated, which subsequently resulted in increased levels of the lipid peroxidation product 4-hydroxynonenal and apoptosis in murine testis with low or no expression of Klotho. Taken together, the loss of one Kl allele accelerates male fecundity loss because diminished antioxidant capability induces oxidative injury in mice. This is the first study that highlights a connection between Klotho and GST proteins.

Potential prognostic and predictive value of UBE2N, IMPDH1, DYNC1LI1 and HRASLS2 in colorectal cancer stool specimens.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy worldwide. The poor specificity and sensitivity of the fecal occult blood test has prompted the development of CRC-related genetic markers for CRC screening and treatment. Gene expression profiles in stool specimens are effective, sensitive and clinically applicable. Herein, a novel advantage of using cells shed from the colon is presented for cost-effective CRC screening. Molecular panels were generated through a series of leave-one-out cross-validation and discriminant analyses. A logistic regression model following reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry was used to validate a specific panel for CRC prediction. The panel, consisting of ubiquitin-conjugating enzyme E2 N (UBE2N), inosine monophosphate dehydrogenase 1 (IMPDH1), dynein cytoplasmic 1 light intermediate chain 1 (DYNC1LI1) and phospholipase A and acyltransferase 2 (HRASLS2), accurately recognized patients with CRC and could thus be further investigated as a potential prognostic and predictive biomarker for CRC. UBE2N, IMPDH1 and DYNC1LI1 expression levels were upregulated and HRASLS2 expression was downregulated in CRC tissues. The predictive power of the panel was 96.6% [95% confidence interval (CI), 88.1-99.6%] sensitivity and 89.7% (95% CI, 72.6-97.8%) specificity at a predicted cut-off value at 0.540, suggesting that this four-gene panel testing of stool specimens can faithfully mirror the state of the colon. On the whole, the present study demonstrates that screening for CRC or cancer detection in stool specimens collected non-invasively does not require the inclusion of an excessive number of genes, and colonic defects can be identified via the detection of an aberrant protein in the mucosa or submucosa.

Developing a Sensitive Platform to Measure 5-Methyltetrahydrofolate in Subjects with MTHFR and PON1 Gene Polymorphisms.

Inadequate levels of 5-methyltetrahydrofolate (5-MTHF) and the T variant of MTHFR C677T have been suggested to be associated with an increased risk of developing mental illness, whereas the PON1 SNP variant provides a protective role. However, reports validating the methodology for plasma 5-MTHF levels in schizophrenia patients are limited. A sensitive LC−MS/MS system using an amide column and calibration curve was determined by dialyzed human plasma, and applied to schizophrenia patients and healthy controls in Taiwan, and the differences between the subgroups were discussed. This analysis system meets regulation criteria, and the lower limit of quantification for 5-MTHF levels was 4 nM from 200 µL plasma, within 7 min. The mean plasma 5-MTHF levels in schizophrenia patients (n = 34; 11.70 ± 10.37 nM) were lower than those in the healthy controls (n = 42; 22.67 ± 11.12 nM) significantly (p < 0.01). 5-MTHF concentrations were significantly lower in male carriers than in female carriers (18.30 ± 10.37 nM vs. 24.83 ± 11.01 nM, p < 0.05), especially in subjects who were MTHFR CT/PON1 Q allele carriers. In conclusion, this quantitative system, which employed sensitive and simple processing methods, was successfully applied, and identified that schizophrenic patients had significantly lower levels of 5-MTHF. Lower plasma 5-MTHF concentrations were observed in male subjects.

Suboptimal folic acid exposure rewires oncogenic metabolism and proteomics signatures to mediate human breast cancer malignancy.

Whether treatment with folic acid (FA) affects human breast cancer positively or negatively remains unclear. We subjected human Michigan Cancer Foundation-7 cells, a human breast cancer cell line, to suboptimal FA at low levels (10 nM; LF) and high levels (50 µM; HF) and investigated the molecular mechanisms underlying their effects through metabolic flux and systematic proteomics analyses. The data indicated that LF induced and HF aggravated 2-fold higher mitochondrial toxicity in terms of suppressed oxidative respiration, increased fermented glycolysis, and enhanced anchorage-independent oncospheroid formation. Quantitative proteomics and Gene Ontology enrichment analysis were used to profile LF- and HF-altered proteins involved in metabolism, apoptosis, and malignancy pathways. Through STRING analysis, we identified a connection network between LF- and HF-altered proteins with mammalian target of rapamycin (mTOR). Rapamycin-induced blockage of mTOR complex 1 (mTORC1) signaling, which regulates metabolism, differentially inhibited LF- and HF-modulated protein signatures of mitochondrial NADH dehydrogenase ubiquinone flavoprotein 2, mitochondrial glutathione peroxidase 4, kynureninase, and alpha-crystallin B chain as well as programmed cell death 5 in transcript levels; it subsequently diminished apoptosis and oncospheroid formation in LF/HF-exposed cells. Taken together, our data indicate that suboptimal FA treatment rewired oncogenic metabolism and mTORC1-mediated proteomics signatures to promote breast cancer development.

Butyrate supplementation regulates expression of chromosome segregation 1­like protein to reverse the genetic distortion caused by p53 mutations in colorectal cancer.

The chromosome segregation 1­like (CSE1L) protein, which regulates cellular mitosis and apoptosis, was previously found to be overexpressed in colorectal cancer (CRC) cells harboring mutations. Therefore, regulating CSE1L expression may confer chemotherapeutic effects against CRC. The gut microflora can regulate gene expression in colonic cells. In particular, metabolites produced by the gut microflora, including the short­chain fatty acid butyrate, have been shown to reduce CRC risk. Butyrates may exert antioncogenic potential in CRC cells by modulating p53 expression. The present study evaluated the association between CSE1L expression and butyrate treatment from two non­transformed colon cell lines (CCD­18Co and FHC) and six CRC cell lines (LS 174T, HCT116 p53+/+, HCT116 p53­/­, Caco­2, SW480 and SW620). Lentiviral knockdown of CSE1L and p53, reverse transcription­quantitative PCR (CSE1L, c­Myc and p53), western blotting [CSE1L, p53, cyclin (CCN) A2, CCNB2 and CCND1], wound healing assay (cell migration), flow cytometry (cell cycle analysis) and immunofluorescence staining (CSE1L and tubulin) were adopted to verify the effects of butyrate on CSE1L­expressing CRC cells. The butyrate­producing gut bacteria Butyricicoccus pullicaecorum was administered to mice with 1,2­dimethylhydrazine­induced colon tumors before the measurement of CSE1L expression. The effects of B. pullicaecorum on CSE1L expression were then assessed by immunohistochemical staining for CSE1L and p53 in tissues from CRC­bearing mice. Non­cancerous colon cells with the R273H p53 mutation or CRC cells haboring p53 mutations were found to exhibit significantly higher CSE1L expression levels. CSE1L knockdown in HCT116 p53­/­ cells resulted in G1­and G2/M­phase cell cycle arrest. Furthermore, in HCT116 p53­/­ cells, CSE1L expression was already high at interphase, increased at prophase, peaked during metaphase before declining at cytokinesis but remained relatively high compared with that in HCT116 expressing wild­type p53. Significantly decreased expression levels of CSE1L were also observed in HCT116 p53­/­ cells that were treated with butyrate for 24 h. In addition, the migration of HCT116 p53­/­ cells was significantly decreased after CSE1L knockdown or butyrate treatment. Tumors with more intense nuclear p53 staining and weaker CSE1L staining were found in mice bearing DMH/DSS­induced CRC that were administered with B. pullicaecorum. Taken together, the results indicated that butyrate can impair CSE1L­induced tumorigenic potential. In conclusion, butyrate­producing microbes, such as B. pullicaecorum, may reverse the genetic distortion caused by p53 mutations in CRC by regulating CSE1L expression levels.

Downregulated Calcium-Binding Protein S100A16 and HSP27 in Placenta-Derived Multipotent Cells Induce Functional Astrocyte Differentiation.

Little is known about genes that induce stem cells differentiation into astrocytes. We previously described that heat shock protein 27 (HSP27) downregulation is directly related to neural differentiation under chemical induction in placenta-derived multipotent stem cells (PDMCs). Using this neural differentiation cell model, we cross-compared transcriptomic and proteomic data and selected 26 candidate genes with the same expression trends in both omics analyses. Those genes were further compared with a transcriptomic database derived from Alzheimer's disease (AD). Eighteen out of 26 candidates showed opposite expression trends between our data and the AD database. The mRNA and protein expression levels of those candidates showed downregulation of HSP27, S100 calcium-binding protein A16 (S100A16) and two other genes in our neural differentiation cell model. Silencing these four genes with various combinations showed that co-silencing HSP27 and S100A16 has stronger effects than other combinations for astrocyte differentiation. The induced astrocyte showed typical astrocytic star-shape and developed with ramified, stringy and filamentous processes as well as differentiated endfoot structures. Also, some of them connected with each other and formed continuous network. Immunofluorescence quantification of various neural markers indicated that HSP27 and S100A16 downregulation mainly drive PDMCs differentiation into astrocytes. Immunofluorescence and confocal microscopic images showed the classical star-like shape morphology and co-expression of crucial astrocyte markers in induced astrocytes, while electrophysiology and Ca2+ influx examination further confirmed their functional characteristics. In conclusion, co-silencing of S100A16 and HSP27 without chemical induction leads to PDMCs differentiation into functional astrocytes.

Metabolomic and proteomic characterization of sng and pain phenotypes in fibromyalgia.

BACKGROUND: Fibromyalgia (FM) is characterized by chronic widespread pain. Its pathophysiological mechanisms remain poorly understood, and effective diagnosis and treatments are lacking. This study aimed to identify significantly changed biosignatures in FM and propose a novel classification for FM based on pain and soreness (sng) symptoms. METHODS: Urine and serum samples from 30 FM patients and 25 controls underwent metabolomic and proteomic profiling. RESULTS: Compared with controls, FM patients showed significant differential expression of three metabolites in urine and five metabolites and eight proteins in serum. Of them, DETP, 4-guanidinobutanoic acid, SM(d18:1/18:0), PC(20:1(11Z)/18:0), S100A7, SERPINB3, galectin-7 and LYVE1 were first reported as potential biomarkers for FM. Furthermore, lactate, 2-methylmaleate and cotinine in urine and lactate, SM(d18:1/25:1), SM(d18:1/26:1) and prostaglandin D2 (PGD2) and PCYOX1, ITIH4, PFN1, LRG1, C8G, C8A, CP, CDH5 and DBH in serum could differentiate pain- (PG) and sng-dominant groups (SG). Lactate, 2-methylmaleate, cotinine, PCYOX1, ITIH4, PFN1 and DBH have a higher level in SG. SM(d18:1/25:1), SM(d18:1/26:1), PGD2, LRG1, C8G, C8A, CP and CDH5 in SG are lower than PG. The omics results indicated disordered free radical scavenging, and lipid and amino acid metabolism networks and resulting NF-κB-dependent cytokine generation in FM. Lactate level was altered simultaneously in urine and serum and significantly higher in sng-dominant patients than others. CONCLUSIONS: In this study, we identified potential biomarkers from FM patients. The selected biomarkers could discriminate sng and pain phenotypes in FM patients. These results could help elucidate the underlying pathological mechanisms for more effective diagnosis and therapy for FM.

Supplementation of Probiotic Butyricicoccus pullicaecorum Mediates Anticancer Effect on Bladder Urothelial Cells by Regulating Butyrate-Responsive Molecular Signatures.

In bladder cancer, urothelial carcinoma is the most common histologic subtype, accounting for more than 90% of cases. Pathogenic effects due to the dysbiosis of gut microbiota are localized not only in the colon, but also in regulating bladder cancer distally. Butyrate, a short-chain fatty acid produced by gut microbial metabolism, is mainly studied in colon diseases. Therefore, the resolution of the anti-cancer effects of butyrate-producing microbes on bladder urothelial cells and knowledge of the butyrate-responsive molecules must have clinical significance. Here, we demonstrate a correlation between urothelial cancer of the bladder and Butyricicoccus pullicaecorum. This butyrate-producing microbe or their metabolite, butyrate, mediated anti-cancer effects on bladder urothelial cells by regulating cell cycle, cell growth, apoptosis, and gene expression. For example, a tumor suppressor against urothelial cancer of the bladder, bladder cancer-associated protein, was induced in butyrate-treated HT1376 cells, a human urinary bladder cancer cell line. In conclusion, urothelial cancer of the bladder is a significant health problem. To improve the health of bladder urothelial cells, supplementation of B. pullicaecorum may be necessary and can further regulate butyrate-responsive molecular signatures.

Co-Occurrence of Differentiated Thyroid Cancer and Second Primary Malignancy: Correlation with Expression Profiles of Mismatch Repair Protein and Cell Cycle Regulators.

Some patients with thyroid cancer develop a second primary cancer. Defining the characteristics of patients with double primary cancers (DPCs) is crucial and needs to be followed. In this study, we examine molecular profiles in DPC. We enrolled 71 patients who received thyroid cancer surgery, 26 with single thyroid cancer (STC), and 45 with DPC. A retrograde cohort was used to develop immunohistochemical expressions of mismatch repair (MMR) proteins and cell-cycle-related markers from tissue microarrays to produce an equation for predicting the occurrence of DPC. The multivariate logistic model of 67 randomly selected patients (24 with STC and 43 with DPC) identified that the expression of deficient MMR (dMMR) (odds ratio (OR), 10.34; 95% confidence interval (CI), 2.17-49.21) and pRb (OR, 62.71; 95% CI, 4.83-814.22) were significantly associated with a higher risk of DPC. In contrast, the expression of CDK4 (OR, 0.19; 95% CI, 0.04-0.99) and CDK6 (OR, 0.03; 95% CI, 0.002-0.44) was significantly associated with a lower risk of DPC. Collectively, dMMR, pRb, CDK4, and CDK6 have a sensitivity of 88.9% (95% CI, 75.1-95.8) and a specificity of 69.2% (95% CI, 48.1-84.9) for occurrence of DPC in all 71 patients. This is the first report to demonstrate the molecular differentiation of STC and DPC. Overall, the integral molecular profile performed excellent discrimination and denoted an exponential function to predict the probability of DPC.

Therapeutic Drug Monitoring of Busulfan in Patients Undergoing Hematopoietic Cell Transplantation: A Pilot Single-Center Study in Taiwan.

(1) Background: Busulfan has been used as a conditioning regimen in allogeneic hematopoietic cell stem transplantation (HSCT). Owing to a large inter-individual variation in pharmacokinetics, therapeutic drug monitoring (TDM)-guided busulfan dosing is necessary to reduce graft failure and relapse rate. As there exists no TDM of busulfan administration for HCT in Taiwan, we conducted a pilot study to assess the TDM-dosing of busulfan in the Taiwanese population; (2) Methods: Seven patients with HCT from The Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan, received conditioning regimens consisting of intravenous busulfan and other chemotherapies. After the initial busulfan dose, blood samples were collected for busulfan TDM at 5 min, 1 h, 2 h, and 3 h. Busulfan was extracted and detected by performing stable-isotope dilution LC-MS/MS. Plasma busulfan concentration was quantified and used for dose adjustment. Potential adverse effects of busulfan, such as mucositis and hepatic veno-occlusive disease (VOD), were also evaluated; (3) Results: The LC-MS/MS method was validated with an analyte recovery of 88-99%, within-run and between-run precision of <15%, and linearity ranging from 10 to 10,000 ng/mL. Using TDM-guided busulfan dosing, dose adjustment was necessary and performed in six out of seven patients (86%) with successful engraftments in all patients (100%). Mild mucositis was observed, and VOD was diagnosed in only one patient; (4) Conclusions: This single-center study in Taiwan demonstrated the importance of busulfan TDM in increasing the success rate of HCT transplantation. It is also necessary to further investigate the optimal busulfan target value in the Taiwanese population in the future.

G-Protein-coupled Estrogen Receptor 1 Agonist G-1 Perturbs Sunitinib Resistance-related Phosphoproteomic Signatures in Renal Cell Carcinoma.

BACKGROUND: Metastatic renal cell carcinoma (RCC) often develops resistance to first-line targeted therapy such as sunitinib. G-Protein-coupled estrogen receptor 1 (GPER1) agonist G-1 was recently reported to regulate RCC physiology but the role of G-1 in RCC tumorigenesis and sunitinib resistance remains largely unknown. MATERIALS AND METHODS: Parental and sunitinib-resistant 786-O cells were treated with GPER1 agonist G-1, and quantitative phosphoproteomics was performed. Bioinformatic analyses and validations, including immunoblotting, cell migration, and cell cycle distribution, were performed. RESULTS: G-1 repressed cell proliferation and migration in both parental and sunitinib-resistant 786-O cells. Phosphoproteomic signatures, including phosphoinositide 3-kinase and protein kinase B (PI3K-AKT) as well as other pathways, were up-regulated in sunitinib-resistant cells but application of G-1 reversed this effect. Among phosphoprotein candidates, activating transcription factor 2 (ATF2) Thr69/71 phosphorylation was antagonistically regulated by sunitinib resistance and G-1. CONCLUSION: Our results open up the possibility for managing RCC and sunitinib resistance by GPER1 agonist G-1 and its regulated pathways.

Kaempferitrin-Treated HepG2 Differentially Expressed Exosomal Markers and Affect Extracellular Vesicle Sizes in the Secretome.

Kaempferitrin is extracted in significantly high quantities from the leaves of Cinnamomum osmophloeum, which belongs to a group of plant species that comes under the genus Cinnamomum, well-known for its established anti-diabetic property in Chinese medicine. Oral administration of kaempferitrin and Cinnamomum osmophloeum extract reduced blood sugar in alloxan-induced diabetic rats and improved the lipid profile in hamsters respectively. In this paper we studied the differential protein expression profile using mass spectrometry approach in the kaempferitrin-treated conditioned medium of liver cancer cell line HepG2. We discovered that 33 genes were up/down-regulated consistently between two biological samples. A slightly different version of the analysis software selected 28 genes, and the final 18 genes that appeared in both lists were selected. Interestingly, 5 proteins out of 18 were either exosomal markers or reported in high frequency of occurrence in exosome/secreted vesicles. We also examined the extracellular particles with atomic force microscopy (AFM), which showed that the conditioned medium of kaempferitrin treated had larger vesicles and fewer small vesicles. Expression of some lipid-regulating genes were also altered. Our data suggested that extracellular vesicle secretions may be regulated by kaempferitrin, and regulation of lipid profile by kampeferitrin involves multiple mechanisms.

ACTN4 Mediates SEPT14 Mutation-Induced Sperm Head Defects.

Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerized SEPTs participate in the modulation of various cellular processes, such as cytokinesis, cell polarity, and membrane dynamics, through their interactions with microtubules, actin, and other cellular components. The main objective of this study was to dissect the molecular pathological mechanism of SEPT14 mutation-induced sperm head defects. To identify SEPT14 interactors, co-immunoprecipitation (co-IP) and nano-liquid chromatography-mass spectrometry/mass spectrometry were applied. Immunostaining showed that SEPT14 was significantly localized to the manchette structure. The SEPT14 interactors were identified and classified as (1) SEPT-, (2) microtubule-, (3) actin-, and (4) sperm structure-related proteins. One interactor, ACTN4, an actin-holding protein, was selected for further study. Co-IP experiments showed that SEPT14 interacts with ACTN4 in a male germ cell line. SEPT14 also co-localized with ACTN4 in the perinuclear and manchette regions of the sperm head in early elongating spermatids. In the cell model, mutated SEPT14 disturbed the localization pattern of ACTN4. In a clinical aspect, sperm with mutant SEPT14, SEPT14A123T (p.Ala123Thr), and SEPT14I333T (p.Ile333Thr), have mislocalized and fragmented ACTN4 signals. Sperm head defects in donors with SEPT14 mutations are caused by disruption of the functions of ACTN4 and actin during sperm head formation.

Deficiency of the Tbc1d21 gene causes male infertility with morphological abnormalities of the sperm mitochondria and flagellum in mice.

Approximately 2-15% of couples experience infertility, and around half of these cases are attributed to male infertility. We previously identified TBC1D21 as a sterility-related RabGAP gene derived from infertile men. However, the in vivo function of TBC1D21 in male fertility remains unclear. Here, we show that loss of Tbc1d21 in mice resulted in male infertility, characterized by defects in sperm tail structure and diminished sperm motility. The mitochondria of the sperm-tail had an abnormal irregular arrangement, abnormal diameter, and structural defects. Moreover, the axoneme structure of sperm tails was severely disturbed. Several TBC1D21 interactors were selected via proteomic analysis and functional grouping. Two of the candidate interactors, a subunit protein of translocase in the outer membrane of mitochondria (TOMM20) and an inner arm component of the sperm tail axoneme (Dynein Heavy chain 7, DNAH7), confirmed in vivo physical co-localization with TBC1D21. In addition, TOMM20 and DNAH7 detached and dispersed outside the axoneme in Tbc1d21-deficient sperm, instead of aligning with the axoneme. From a clinical perspective, the transcript levels of TBC1D21 in sperm from teratozoospermia cases were significantly reduced when compared with those in normozoospermia. We concluded that TBC1D21 is critical for mitochondrial and axoneme development of mammalian sperm.

Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin ß4 axis.

The detachment of tumor cells from extracellular matrix and survival under anchorage-independence were recognized as the initial step of tumor metastasis. Previously we had demonstrated that anchorage-independence altered gene expressions and showed characteristics of cell invasiveness loss, enhanced chemosensitivity, and enhanced subcutaneous tumor formation. However, whether it affected histological phenotypes in tumor tissues remained unclear. Melanoma metastases were generated in nude mice using adherent or suspended melanoma cells. Examination of melanoma metastases revealed histological features of extensive vascular structures in adherent cell-derived tumors, while not seen in suspended cell-derived tumors. Quantitative proteomic analysis at adherent, suspended, and re-attached melanoma cells suggested that aminopeptidase N was potentially downregulated upon cell suspension or reattachment. Downregulation of aminopeptidase N by gene-specific shRNAs showed reduced cell invasiveness and enhanced subcutaneous tumor formation that was consistent with previous observations. Experiments by suppression or overexpression of aminopeptidase N expression demonstrated that aminopeptidase N regulated syndecan-1 and integrin ß4 expression through PKCδ pathway. Histological analysis at melanoma metastases further suggested that CD31+/aminopeptidase N+/syndecan-1+/integrin ß4+ phenotypes were associated with vascular structures. In summary, we suggested the expression axis of aminopeptidase N/syndecan-1/integrin ß4 in melanoma cells was suppressed by detachment stress, which diminished vascular phenotypes of melanoma metastases.

Using Differential Threshold Effects of Individual and Combined Periconceptional Methyl Donor Status on Maternal Genomic LINE-1 and Imprinted H19 DNA Methylation to Predict Birth Weight Variance in the Taiwan Pregnancy-Newborn Epigenetics (TPNE) Cohort Study.

BACKGROUND: Few studies have comprehensively examined the effect of methyl donor status on maternal DNA methylation and birth outcomes. OBJECTIVES: This study examined associations between periconceptional methyl donor status and genome-wide and specific imprinted gene methylation and fetal growth indices in the Taiwan Pregnancy-Newborn Epigenetics cohort. METHODS: Plasma folate, choline (free form), and betaine concentrations of the participants enrolled at 7-10 weeks of gestation were analyzed. DNA methylation at regulatory sequences of the imprinted H19 gene and genomic long interspersed nuclear element 1 (LINE-1) were measured in maternal lymphocytes using bisulfite/high-resolution melt polymerase chain reaction. Associations with birth weight (BW) were estimated through multiple regressions from 112 mother-newborn pairs. RESULTS: A nonlinear "L-shaped" relation and an inverse association between maternal plasma folate in T1 (mean ± SE: 17.6 ± 5.1 nmol/L) and lymphocytic LINE-1 methylation (ß: -0.49, P = 0.027) were characterized. After adjusting for LINE-1 methylation, individual maternal folate concentrations were positively associated with BW variance (ß = 0.24, P = 0.035), and the association was more pronounced in mothers with choline in T1 (mean ± SE: 5.4 ± 0.6 µmol/L; ß: 0.40, P = 0.039). Choline status of the mothers in T2 (mean ± SE: 7.2 ± 0.6 µmol/L) was inversely associated with LINE-1 methylation (ß: -0.43, P = 0.035), and a positive association was evident between T1 choline and H19 methylation (ß: 0.48, P = 0.011). After adjusting for epigenetic modification, maternal choline status predicted a positive association with BW (ß: 0.56, P = 0.005), but the effect was limited to mothers with high betaine concentrations in T3 (mean ± SE: 36.4 ± 8.8 µmol/L), depending on folate status. CONCLUSIONS: Our data highlight the differential threshold effects of periconceptional folate, choline, and betaine status on genomic LINE-1 and H19 DNA methylation and how their interplay has a long-term effect on BW variance.

ASIC3-dependent metabolomics profiling of serum and urine in a mouse model of fibromyalgia.

Fibromyalgia (FM) is characterized by chronic widespread pain. The pathogenesis of FM remains unclear. No specific biomarkers are available. Animal models of FM may provide an opportunity to explore potential biomarkers in a relative homogenous disease condition. Here, we probed the metabolomics profiles of serum and urine in a mouse model of FM induced by intermittent cold stress (ICS). We focused on the role of acid-sensing ion channel 3 (ASIC3) in the metabolomics profiling because ICS treatment induced chronic widespread muscle pain lasting for 1 month in wild-type (Asic3+/+) but not Asic3-knockout (Asic3-/-) mice. Serum and urine samples were collected from both genotypes at different ICS stages, including before ICS (basal level) and post-ICS at days 10 (middle phase, P10) and 40 (recovery phase, P40). Control naïve mice and ICS-induced FM mice differed in 1H-NMR- and LC-MS-based metabolomics profiling. On pathway analysis, the leading regulated pathways in Asic3+/+ mice were taurine and hypotaurine, cysteine and methionine, glycerophospholipid, and ascorbate and aldarate metabolisms, and the major pathways in Asic3-/- mice involved amino acid-related metabolism. Finally, we developed an algorithm for the impactful metabolites in the FM model including cis-aconitate, kynurenate, taurine, pyroglutamic acid, pyrrolidonecarboxylic acid, and 4-methoxyphenylacetic acid in urine as well as carnitine, deoxycholic acid, lysoPC(16:0), lysoPC(20:3), oleoyl-L-carnitine, and trimethylamine N-oxide in serum. Asic3-/- mice were impaired in only muscle allodynia development but not other pain symptoms in the ICS model, so the ASIC3-dependent metabolomics changes could be useful for developing diagnostic biomarkers specific to chronic widespread muscle pain, the core symptom of FM. Further pharmacological validations are needed to validate these metabolomics changes as potential biomarkers for FM diagnosis and/or treatment responses.

Comparison of different fractionation strategies for in-depth phosphoproteomics by liquid chromatography tandem mass spectrometry.

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. Graphical Abstract.

Combined proteomic and metabolomic analyses of cerebrospinal fluid from mice with ischemic stroke reveals the effects of a Buyang Huanwu decoction in neurodegenerative disease.

Ischemic stroke is one of the most common causes of death worldwide and is a major cause of acquired disability in adults. However, there is still a need for an effective drug for its treatment. Buyang Huanwu decoction (BHD), a traditional Chinese medicine (TCM) prescription, has long been used clinically to aid neurological recovery after stroke. To establish potential clinical indicators of BHD efficacy in stroke treatment and prognosis, we conducted a combined proteomic and metabolomic analysis of cerebrospinal fluid (CSF) samples in a mouse stroke model. CSF samples were obtained from male mice with acute ischemic stroke induced by middle cerebral ischemic/reperfusion (CI/R) injury, some of which were then treated with BHD. Label-free quantitative proteomics was conducted using nano-LC-MS/MS on an LTQ Orbitrap mass and metabolomic analysis was performed using nanoprobe NMR and UHPLC-QTOF-MS. The results showed that several proteins and metabolites were present at significantly different concentrations in the CSF samples from mice with CI/R alone and those treated with BHD. These belonged to pathways related to energy demand, inflammatory signaling, cytoskeletal regulation, Wnt signaling, and neuroprotection against neurodegenerative diseases. In conclusion, our in silico data suggest that BHD treatment is not only protective but can also ameliorate defects in pathways affected by neurological disorders. These data shed light on the mechanism whereby BHD may be effective in the treatment and prevention of stroke-related neurodegenerative disease.