Quantifiable peptide library bridges the gap for proteomics based biomarker discovery and validation on breast cancer.

Mass spectrometry (MS) based proteomics is widely used for biomarker discovery. However, often, most biomarker candidates from discovery are discarded during the validation processes. Such discrepancies between biomarker discovery and validation are caused by several factors, mainly due to the differences in analytical methodology and experimental conditions. Here, we generated a peptide library which allows discovery of biomarkers in the equal settings as the validation process, thereby making the transition from discovery to validation more robust and efficient. The peptide library initiated with a list of 3393 proteins detectable in the blood from public databases. For each protein, surrogate peptides favorable for detection in mass spectrometry was selected and synthesized. A total of 4683 synthesized peptides were spiked into neat serum and plasma samples to check their quantifiability in a 10 min liquid chromatography-MS/MS run time. This led to the PepQuant library, which is composed of 852 quantifiable peptides that cover 452 human blood proteins. Using the PepQuant library, we discovered 30 candidate biomarkers for breast cancer. Among the 30 candidates, nine biomarkers, FN1, VWF, PRG4, MMP9, CLU, PRDX6, PPBP, APOC1, and CHL1 were validated. By combining the quantification values of these markers, we generated a machine learning model predicting breast cancer, showing an average area under the curve of 0.9105 for the receiver operating characteristic curve.

Combined deletion of Bap1, Nf2, and Cdkn2ab causes rapid onset of malignant mesothelioma in mice.

We have generated mouse models of malignant mesothelioma (MM) based upon disruption of the Bap1, Nf2, and Cdkn2ab tumor suppressor loci in various combinations as also frequently observed in human MM. Inactivation of all three loci in the mesothelial lining of the thoracic cavity leads to a highly aggressive MM that recapitulates the histological features and gene expression profile observed in human patients. The tumors also show a similar inflammatory phenotype. Bap1 deletion alone does not cause MM but dramatically accelerates MM development when combined with Nf2 and Cdkn2ab (hereafter BNC) disruption. The accelerated tumor development is accompanied by increased Polycomb repression and EZH2-mediated redistribution of H3K27me3 toward promoter sites with concomitant activation of PI3K and MAPK pathways. Treatment of BNC tumor-bearing mice with cisplatin and pemetrexed, the current frontline treatment, prolongs survival. This makes the autochthonous mouse model described here very well suited to explore the pathogenesis of MM and validate new treatment regimens for MM, including immunotherapy.

Transcription Factor NFIB Is a Driver of Small Cell Lung Cancer Progression in Mice and Marks Metastatic Disease in Patients.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor, and no effective treatment is available to date. Mouse models of SCLC based on the inactivation of Rb1 and Trp53 show frequent amplifications of the Nfib and Mycl genes. Here, we report that, although overexpression of either transcription factor accelerates tumor growth, NFIB specifically promotes metastatic spread. High NFIB levels are associated with expansive growth of a poorly differentiated and almost exclusively E-cadherin (CDH1)-negative invasive tumor cell population. Consistent with the mouse data, we find that NFIB is overexpressed in almost all tested human metastatic high-grade neuroendocrine lung tumors, warranting further assessment of NFIB as a tumor progression marker in a clinical setting.

Paracrine signaling between tumor subclones of mouse SCLC: a critical role of ETS transcription factor Pea3 in facilitating metastasis.

Tumor heterogeneity can create a unique symbiotic tumor microenvironment. Earlier, we showed that clonal evolution in mouse small cell lung cancer (SCLC) can result in subclones that, upon cografting, endow the neuroendocrine tumor cells with metastatic potential. We now show that paracrine signaling between SCLC subclones is a critical requirement in the early steps of the metastatic process, such as local invasion and intravasation. We further show evidence that paracrine signaling via fibroblast growth factor 2 (Fgf2) and Mapk between these diverged tumor subclones causes enhanced expression of the Pea3 (polyomavirus enhancer activator 3) transcription factor, resulting in metastatic dissemination of the neuroendocrine tumor subclones. Our data reveal for the first time paracrine signaling between tumor cell subclones in SCLC that results in metastatic spread of SCLC.

Meteorin regulates mesendoderm development by enhancing nodal expression.

During gastrulation, distinct lineage specification into three germ layers, the mesoderm, endoderm and ectoderm, occurs through an elaborate harmony between signaling molecules along the embryonic proximo-distal and anterior-posterior axes, and Nodal signaling plays a key role in the early embryonic development governing embryonic axis formation, mesoderm and endoderm specification, and left-right asymmetry determination. However, the mechanism by which Nodal expression is regulated is largely unknown. Here, we show that Meteorin regulates Nodal expression and is required for mesendoderm development. It is highly expressed in the inner cell mass of blastocysts and further in the epiblast and extra-embryonic ectoderm during gastrulation. Genetic ablation of the Meteorin gene resulted in early embryonic lethality, presumably due to impaired lineage allocation and subsequent cell accumulation. Embryoid body culture using Meteorin-null embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification. Meteorin-null embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved. Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.

Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells.

Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

Electric double-layer capacitor based on an ionic clathrate hydrate.

Herein, we suggest a new approach to an electric double-layer capacitor (EDLC) that is based on a proton-conducting ionic clathrate hydrate (ICH). The ice-like structures of clathrate hydrates, which are comprised of host water molecules and guest ions, make them suitable for applications in EDLC electrolytes, owing to their high proton conductivities and thermal stabilities. The carbon materials in the ICH Me4NOH⋅5 H2O show a high specific capacitance, reversible charge-discharge behavior, and a long cycle life. The ionic-hydrate complex provides the following advantages in comparison with conventional aqueous and polymer electrolytes: 1) The ICH does not cause leakage problems under normal EDLC operating conditions. 2) The hydrate material can be utilized itself, without requiring any pre-treatments or activation for proton conduction, thus shortening the preparation procedure of the EDLC. 3) The crystallization of the ICH makes it possible to tailor practical EDLC dimensions because of its fluidity as a liquid hydrate. 4) The hydrate solid electrolyte exhibits more-favorable electrochemical stability than aqueous and polymer electrolytes. Therefore, ICH materials are expected to find practical applications in versatile energy devices that incorporate electrochemical systems.

Mouse models for lung cancer.

Lung cancer is a devastating disease and a major therapeutic burden with poor survival rates. It is responsible for 30% of all cancer deaths. Lung cancer is strongly associated with smoking, although some subtypes are also seen in non-smokers. Tumors in the latter group are mostly adenocarcinomas with many carrying mutations in the epidermal growth factor receptor (EGFR). Survival statistics of lung cancer are grim because of its late detection and frequent local and distal metastases. Although DNA sequence information from tumors has revealed a number of frequently occurring mutations, affecting well-known tumor suppressor genes and proto-oncogenes, many of the driver mutations remain ill defined. This is likely due to the involvement of numerous rather infrequently occurring driver mutations that are difficult to distinguish from the very large number of passenger mutations detected in smoking-related lung cancers. Therefore, experimental model systems are indispensable to validate putative driver lesions and to gain insight into their mechanisms of action. Whereas a large fraction of these analyzes can be performed in cell cultures in vitro, in many cases the consequences of the mutations have to be assessed in the context of an intact organism, as this is the context in which the Mendelian selection process of the tumorigenic process took place and the advantages of particular mutations become apparent. Current mouse models for cancer are very suitable for this as they permit mimicking many of the salient features of human tumors. The capacity to swiftly re-engineer complex sets of lesions found in human tumors in mice enables us to assess the contribution of defined combinations of lesions to distinct tumor characteristics such as metastatic behavior and response to therapy. In this review we will describe mouse models of lung cancer and how they are used to better understand the disease and how they are exploited to develop better intervention strategies.

Cardiomyocyte specific deletion of Crif1 causes mitochondrial cardiomyopathy in mice.

Mitochondria are key organelles dedicated to energy production. Crif1, which interacts with the large subunit of the mitochondrial ribosome, is indispensable for the mitochondrial translation and membrane insertion of respiratory subunits. To explore the physiological function of Crif1 in the heart, Crif1(f/f) mice were crossed with Myh6-cre/Esr1 transgenic mice, which harbor cardiomyocyte-specific Cre activity in a tamoxifen-dependent manner. The tamoxifen injections were given at six weeks postnatal, and the mutant mice survived only five months due to hypertrophic heart failure. In the mutant cardiac muscles, mitochondrial mass dramatically increased, while the inner structure was altered with lack of cristae. Mutant cardiac muscles showed decreased rates of oxygen consumption and ATP production, suggesting that Crif1 plays a critical role in the maintenance of both mitochondrial structure and respiration in cardiac muscles.

CRIF1 is essential for the synthesis and insertion of oxidative phosphorylation polypeptides in the mammalian mitochondrial membrane.

Although substantial progress has been made in understanding the mechanisms underlying the expression of mtDNA-encoded polypeptides, the regulatory factors involved in mitoribosome-mediated synthesis and simultaneous insertion of mitochondrial oxidative phosphorylation (OXPHOS) polypeptides into the inner membrane of mitochondria are still unclear. In the present study, disruption of the mouse Crif1 gene, which encodes a mitochondrial protein, resulted in a profound deficiency in OXPHOS caused by the disappearance of OXPHOS subunits and complexes in vivo. CRIF1 was associated with large mitoribosomal subunits that were located close to the polypeptide exit tunnel, and the elimination of CRIF1 led to both aberrant synthesis and defective insertion of mtDNA-encoded nascent OXPHOS polypeptides into the inner membrane. CRIF1 interacted with nascent OXPHOS polypeptides and molecular chaperones, e.g., Tid1. Taken together, these results suggest that CRIF1 plays a critical role in the integration of OXPHOS polypeptides into the mitochondrial membrane in mammals.

Abnormal thermal expansion of clathrate hydrates induced by asymmetric guest molecules.

We investigated for the first time the abnormal thermal expansion induced by an asymmetric guest structure using high-resolution neutron powder diffraction. Three dihydrogen molecules (H(2), D(2), and HD) were tested to explore the guest dynamics and thermal behavior of hydrogen-doped clathrate hydrates. We confirmed the restricted spatial distribution and doughnut-like motion of the HD guest in the center of anisotropic sII-S (sII-S=small cages of structure II hydrates). However, we failed to observe a mass-dependent relationship when comparing D(2) with HD. The use of asymmetric guest molecules can significantly contribute to tuning the cage dimension and thus can improve the stable inclusion of small gaseous molecules in confined cages.

Survival and differentiation of mammary epithelial cells in mammary gland development require nuclear retention of Id2 due to RANK signaling.

RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL(-/-) mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2(S5A)Tg mice, Id2(S5A) (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2(S5A) rescued increased apoptosis and defective differentiation of MECs in RANKL(-/-) mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.

Superoxide ions entrapped in water cages of ionic clathrate hydrates.

In the present work, we first described the stable entrapment of the superoxide ions in gamma-irradiated (Me(4)NOH + O(2)) clathrate hydrate. Owing to peculiar direct guest-guest ionic interaction, the lattice structure of gamma-irradiated (Me(4)NOH + O(2)) clathrate hydrate shows significant change of lattice contraction behavior even at relatively high temperature (120 K). Such findings are expected to provide useful information for a better understanding of unrevealed nature (such as icy nanoreactor concept, ice-based functional material synthesis and lattice tuning by specific ionic guests) of clathrate hydrate fields.

Essential role of CR6-interacting factor 1 (Crif1) in E74-like factor 3 (ELF3)-mediated intestinal development.

Although terminal differentiation of intestinal epithelium is essential for the efficient digestion and absorption of nutrients, little is known about the molecular mechanisms underlying this process. Recent studies have shown that Elf3 (E74-like factor 3), a member of the ETS transcription factor family, has an essential role in the terminal differentiation of absorptive enterocytes and mucus-secreting goblet cells. Here, we demonstrated that Crif1 (CR6-interacting factor 1) functions as transcriptional coactivator of Elf3 in intestinal epithelium differentiation. The intestinal epithelium-specific Crif1-deficient mice died soon after birth and displayed severe alterations of tissue architecture in the small intestine, including poor microvillus formation and abnormal differentiation of absorptive enterocytes. Strikingly, these phenotypes are largely similar to that of Elf3-deficient mice, suggesting that Elf3 signaling in the intestinal epithelium depends on the Crif1 expression. We dissected this relationship further and found that Crif1 indeed interacted with Elf3 through its ETS DNA binding domain and enhanced the transcriptional activity of Elf3 by regulating the DNA binding activity. Knockdown of Crif1 by RNA interference conversely attenuated the transcriptional activity of Elf3. Consistently, the expression level of Tgf-betaRII (transforming growth factor beta type II receptor), a critical target gene of Elf3, was dramatically reduced in the Crif1-deficient mice. Our results reveal that Crif1 is a novel and essential transcriptional coactivator of Elf3 for the terminal differentiation of absorptive enterocytes.

Mind bomb 1-expressing intermediate progenitors generate notch signaling to maintain radial glial cells.

Notch signaling is critical for the stemness of radial glial cells (RGCs) during embryonic neurogenesis. Although Notch-signal-receiving events in RGCs have been well characterized, the signal-sending mechanism by the adjacent cells is poorly understood. Here, we report that conditional inactivation of mind bomb-1 (mib1), an essential component for Notch ligand endocytosis, in mice using the nestin and hGFAP promoters resulted in complete loss of Notch activation, which leads to depletion of RGCs, and premature differentiation into intermediate progenitors (IPs) and finally neurons, which were reverted by the introduction of active Notch1. Interestingly, Mib1 expression is restricted in the migrating IPs and newborn neurons, but not in RGCs. Moreover, sorted Mib1+ IPs and neurons can send the Notch signal to neighboring cells. Our results reveal that not only newborn neurons but also IPs are essential Notch-ligand-presenting cells for maintaining RGC stemness during both symmetric and asymmetric divisions.

Influence of the wastewater composition on denitrification and biological p-removal in the S-DN-P-process (b) Effect of acetate.

The effect of acetate was examined during the p-removal and denitrification of wastewater. The plant was operated based on the sequencing-batch-biofilm-reactor (SBBR) process. As microbial media, ca. 9 mm Bio-Flow granules made from polyethylene and polypropylene were used. Three preparations were made to compare the level of biological p-removal and denitrification. In comparison to the batch test, 42 mg/L (AC 30) and 84 mg/L (AC 60) of NaCH(3)COO were mixed with the 500 mL of raw wastewater and the effect of the acetate concentration on the level of p-removal was monitored. All samples were immediately filtered with 0.45 microm membrane filter, and PO(4)-P, NO(3)-N, NO(2)-N and acetate were analyzed using Ion Chromatography, whereas P(total) and chemical oxygen demand (COD) were measured by a spectrophotometer. The p-removals for the WW, WW+AC 30 and WW+AC 60 preparations were found to be 9.4, 9.1 and 13.1mg/L, respectively. The WW+AC 30 preparation did not show any significant effect on the p-removal, while p-removal in WW+AC 60 preparation was higher than that in the other two preparations. A comparison of the data revealed the COD: NO(3)-N:AC:P ratio of the WW, WW+AC 30 and WW+AC 60 preparations to be 18.07:2.90:6.87:1, 21.28:2.45:5.98:1 and 15.95:2.75:6.18:1, respectively. The experimental results showed that approximately 7 mg/L of acetate was consumed per 1mg/L of p-removal.

Crif1 is a novel transcriptional coactivator of STAT3.

Signal transducer and activator of transcription 3 (STAT3) is a transcriptional factor that performs a broad spectrum of biological functions in response to various stimuli. However, no specific coactivator that regulates the transcriptional activity of STAT3 has been identified. Here we report that CR6-interacting factor 1 (Crif1) is a specific transcriptional coactivator of STAT3, but not of STAT1 or STAT5a. Crif1 interacts with STAT3 and positively regulates its transcriptional activity. Crif1-/- embryos were lethal around embryonic day 6.5, and manifested developmental arrest accompanied with defective proliferation and massive apoptosis. The expression of STAT3 target genes was markedly reduced in a Crif1-/- blastocyst culture and in Oncostatin M-stimulated Crif1-deficient MEFs. Importantly, the key activities of constitutively active STAT3-C, such as transcription, DNA binding, and cellular transformation, were abolished in the Crif1-null MEFs, suggesting the essential role of Crif1 in the transcriptional activity of STAT3. Our results reveal that Crif1 is a novel and essential transcriptional coactivator of STAT3 that modulates its DNA binding ability, and shed light on the regulation of oncogenic STAT3.

Receptor activator of NF-kappaB ligand regulates the proliferation of mammary epithelial cells via Id2.

Receptor activator of NF-kappaB ligand (RANKL) is a key regulator for mammary gland development during pregnancy. RANKL-deficient mice display impaired development of lobulo-alveolar mammary structures. Similar mammary gland defects have been reported in mice lacking Id2. Here we report that RANKL induces the proliferation of mammary epithelial cells via Id2. RANKL triggers marked nuclear translocation of Id2 in mammary epithelial cells. In vivo studies further demonstrated the defective nuclear translocation of Id2, but the normal expression of cyclin D1, in the mammary epithelial cells of rankl-/- mice. In vitro studies with nuclear localization sequence-tagged Id2 revealed that the nuclear localization of Id2 itself is critical for the downregulation of p21 promoter activity. Moreover, RANKL stimulation failed to induce cell growth and to downregulate p21 expression in Id2-/- mammary epithelial cells. Our results indicate that the inhibitor of helix-loop-helix protein, Id2, is critical to control the proliferation of mammary epithelial cells in response to RANKL stimulation.

Mind bomb 1 is essential for generating functional Notch ligands to activate Notch.

The Delta-Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for cell fate specification. Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. We now report that mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulated in the plasma membrane, while it was localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. Our results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling.