A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE.

BACKGROUND: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. RESULTS: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 µM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 µg/mL) than classical Coomassie brilliant blue (14.11 µg/mL). SIGNIFICANCE: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

De Novo Designed Ru(II) Metallacycle as a Microenvironment-Adaptive Sonosensitizer and Sonocatalyst for Multidrug-Resistant Biofilms Eradication.

Albeit sonodynamic therapy (SDT) has achieved encouraging progress in microbial sterilization, the scarcity of guidelines for designing highly effective sonosensitizers and the intricate biofilm microenvironment (BME), substantially hamper the therapeutic efficacy against biofilm infections. To address the bottlenecks, we innovatively design a Ru(II) metallacycle-based sonosensitizer/sonocatalyst (named Ru-A3-TTD) to enhance the potency of sonotherapy by employing molecular engineering strategies tailored to BME. Our approach involves augmenting Ru-A3-TTD's production of ultrasonic-triggered reactive oxygen species (ROS), surpassing the performance of commercial sonosensitizers, through a straightforward but potent π-expansion approach. Within the BME, Ru-A3-TTD synergistically amplifies sonotherapeutic efficacy via triple-modulated approaches: (i) effective alleviation of hypoxia, leading to increased ROS generation, (ii) disruption of the antioxidant defense system, which shields ROS from glutathione consumption, and (iii) enhanced biofilm penetration, enabling ROS production in deep sites. Notably, Ru-A3-TTD sono-catalytically oxidizes NADPH, a critical coenzyme involved in antioxidant defenses. Consequently, Ru-A3-TTD demonstrates superior biofilm eradication potency against multidrug-resistant Escherichia coli compared to conventional clinical antibiotics, both in vitro and in vivo. To our knowledge, this study represents the pioneering instance of a supramolecular sonosensitizer/sonocatalyst. It provides valuable insights into the structure-activity relationship of sonosensitizers and paves a promising pathway for the treatment of biofilm infections.

HDAC1: An Essential and Conserved Member of the Diverse Zn2+-Dependent HDAC Family Driven by Divergent Selection Pressure.

Zn2+-dependent histone deacetylases (HDACs) are enzymes that regulate gene expression by removing acetyl groups from histone proteins. These enzymes are essential in all living systems, playing key roles in cancer treatment and as potential pesticide targets. Previous phylogenetic analyses of HDAC in certain species have been published. However, their classification and evolutionary origins across biological kingdoms remain unclear, which limits our understanding of them. In this study, we collected the HDAC sequences from 1451 organisms and performed analyses. The HDACs are found to diverge into three classes and seven subclasses under divergent selection pressure. Most subclasses show species specificity, indicating that HDACs have evolved with high plasticity and diversification to adapt to different environmental conditions in different species. In contrast, HDAC1 and HDAC3, belonging to the oldest class, are conserved and crucial in major kingdoms of life, especially HDAC1. These findings lay the groundwork for the future application of HDACs.

Acceptor engineering of metallacycles with high phototoxicity indices for safe and effective photodynamic therapy.

Although metallacycle-based photosensitizers have attracted increasing attention in biomedicine, their clinical application has been hindered by their inherent dark toxicity and unsatisfactory phototherapeutic efficiency. Herein, we employ a π-expansion strategy for ruthenium acceptors to develop a series of Ru(ii) metallacycles (Ru1-Ru4), while simultaneously reducing dark toxicity and enhancing phototoxicity, thus obtaining a high phototoxicity index (PI). These metallacycles enable deep-tissue (∼7 mm) fluorescence imaging and reactive oxygen species (ROS) production and exhibit remarkable anti-tumor activity even under hypoxic conditions. Notably, Ru4 has the lowest dark toxicity, highest ROS generation ability and an optimal PI (∼146). Theoretical calculations verify that Ru4 exhibits the largest steric bulk and the lowest singlet-triplet energy gap (ΔE ST, 0.62 eV). In vivo studies confirm that Ru4 allows for effective and safe phototherapy against A549 tumors. This work thus is expected to open a new avenue for the design of high-performance metal-based photosensitizers for potential clinical applications.

Streptococcus humanilactis sp.nov., isolated from healthy nursing mother's breast milk.

Two bacterial strains were isolated from the breast milk of two healthy nursing mothers. The isolates were Gram-positive, catalase-negative, coccus-shaped, chain-forming organisms. Analysis of the 16S rRNA gene sequences of strain IMAU99125T shared 99.7 and 99.6% similarity with Streptococcus mitis ATCC 49456 T and Streptococcus pseudopneumoniae ATCC BAA-960 T, respectively. The nearly complete 16S rRNA gene sequences of IMAU99125T and IMAU99674 strains were very closely related (with only 0.06% difference between them). Sequence analysis of the gyrB and rpoB genes also indicated that IMAU99125T was closely related to S. mitis ATCC 49456 T (94.7% and 97.1%, respectively) and S. pseudopneumoniae ATCC BAA-960 T (94.4% and 97.1%, respectively). Average nucleotide identity (ANI) values between strain IMAU99125T and S. mitis ATCC 49456 T and S. pseudopneumoniae ATCC BAA-960 T, were 93.3% and 92.7%, respectively. Genome-to-genome distance (GGD) values between strain IMAU99125T and S. mitis ATCC 99125 T and S. pseudopneumoniae ATCC BAA-960 T were 53.4% (50.7-56.0) and 50.4% (47.7-53.0), respectively. The major fatty acids of the strain were C16:0 (51.4%). On the basis of the results of phenotypic and phylogenetic analyses, we propose that the two strains be classified as representing a novel species of the genus Streptococcus, namely Streptococcus humanilactis sp.nov. The type strain is IMAU99125T (= GDMCC 1.1876 T = KCTC 21157 T). The genome of Streptococcus humanilactis sp. nov. is comprised of 2,027,143 bp. The DNA G + C content of the strain is 40.0 mol%.

Genome-wide comparison and in silico analysis of splicing factor SYF2/NTC31/p29 in eukaryotes: Special focus on vertebrates.

The gene SYF2-an RNA splicing factor-can interact with Cyclin D-type binding protein 1 (GICP) in many biological processes, including splicing regulation, cell cycle regulation, and DNA damage repair. In our previous study we performed genome-wide identification and functional analysis of SYF2 in plant species. The phylogenetic relationships and expression profiles of SYF2 have not been systematically studied in animals, however. To this end, the gene structure, genes, and protein conserved motifs of 102 SYF2 homologous genes from 91 different animal species were systematically analyzed, along with conserved splicing sites in 45 representative vertebrate species. A differential comparative analysis of expression patterns in humans and mice was made. Molecular bioinformatics analysis of SYF2 showed the gene was conserved and functional in different animal species. In addition, expression pattern analysis found that SYF2 was highly expressed in hematopoietic stem cells, T cells, and lymphoid progenitor cells; in ovary, lung, and spleen; and in other cells and organs. This suggests that changes in SYF2 expression may be associated with disease development in these cells, tissues, or organs. In conclusion, our study analyzes the SYF2 disease resistance genes of different animal species through bioinformatics, reveals the relationship between the SYF2 genotype and the occurrence of certain diseases, and provides a theoretical basis for follow-up study of the relationship between the SYF2 gene and animal diseases.

Rationally designed Ru(II) metallacycles with tunable imidazole ligands for synergistical chemo-phototherapy of cancer.

Herein, we construct a series of Ru(II) metallacycles with multimodal chemo-phototherapeutic properties, which exhibited much higher anticancer activity and better cancer-cell selectivity than cisplatin. The antitumor mechanism could be ascribed to the activation of caspase 3/7 and the resulting apoptosis. These results open new possibilities for Ru(II) metallacycles in biomedicine.

NIR-II Emissive Ru(II) Metallacycle Assisting Fluorescence Imaging and Cancer Therapy.

Despite the success of emissive Ruthenium (Ru) agents in biomedicine, problems such as the visible-light excitation/emission and single chemo- or phototherapy modality still hamper their applications in deep-tissue imaging and efficient cancer therapy. Herein, an second nearinfrared window (NIR-II) emissive Ru(II) metallacycle (Ru1000, λem  = 1000 nm) via coordination-driven self-assembly is reported, which holds remarkable deep-tissue imaging capability (≈6 mm) and satisfactory chemo-phototherapeutic performance. In vitro results indicate Ru1000 displays promising cellular uptake, good cancer-cell selectivity, attractive anti-metastasis properties, and remarkable anticancer activity against various cancer cells, including cisplatin-resistant A549 cells (IC50  = 3.4 × 10-6  m vs 92.8 × 10-6  m for cisplatin). The antitumor mechanism could be attributed to Ru1000-induced lysosomal membrane damage and mitochondrial-mediated apoptotic cell death. Furthermore, Ru1000 also allows the high-performance in vivo NIR-II fluorescence imaging-guided chemo-phototherapy against A549 tumors. This work may provide a paradigm for the development of long-wavelength emissive metallacycle-based agents for future biomedicine.

Phylogenetic Comparison and Splicing Analysis of the U1 snRNP-specific Protein U1C in Eukaryotes.

As a pivotal regulator of 5' splice site recognition, U1 small nuclear ribonucleoprotein (U1 snRNP)-specific protein C (U1C) regulates pre-mRNA splicing by interacting with other components of the U1 snRNP complex. Previous studies have shown that U1 snRNP and its components are linked to a variety of diseases, including cancer. However, the phylogenetic relationships and expression profiles of U1C have not been studied systematically. To this end, we identified a total of 110 animal U1C genes and compared them to homologues from yeast and plants. Bioinformatics analysis shows that the structure and function of U1C proteins is relatively conserved and is found in multiple copies in a few members of the U1C gene family. Furthermore, the expression patterns reveal that U1Cs have potential roles in cancer progression and human development. In summary, our study presents a comprehensive overview of the animal U1C gene family, which can provide fundamental data and potential cues for further research in deciphering the molecular function of this splicing regulator.

Phylogenetic comparison and splice site conservation of eukaryotic U1 snRNP-specific U1-70K gene family.

Eukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.

Synthesis and Herbicidal Activity of Triketone-Aminopyridines as Potent p-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

Exploring novel p-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) inhibitors has become one of the most promising research directions in herbicide innovation. On the basis of our tremendous interest in exploiting more powerful HPPD inhibitors, we designed a family of benzyl-containing triketone-aminopyridines via a structure-based drug design (SBDD) strategy and then synthesized them. Among these prepared derivatives, the best active 3-hydroxy-2-(3,5,6-trichloro-4-((4-isopropylbenzyl)amino)picolinoyl)cyclohex-2-en-1-one (23, IC50 = 0.047 µM) exhibited a 5.8-fold enhancement in inhibiting Arabidopsis thaliana (At) HPPD activity over that of commercial mesotrione (IC50 = 0.273 µM). The predicted docking models and calculated energy contributions of the key residues for small molecules suggested that an additional π-π stacking interaction with Phe-392 and hydrophobic contacts with Met-335 and Pro-384 were detected in AtHPPD upon the binding of the best active compound 23 compared with that of the reference mesotrione. Such a molecular mechanism and the resulting binding affinities coincide with the proposed design scheme and experimental values. It is noteworthy that inhibitors 16 (3-hydroxy-2-(3,5,6-trichloro-4-((4-chlorobenzyl)amino)picolinoyl)cyclohex-2-en-1-one), 22 (3-hydroxy-2-(3,5,6-trichloro-4-((4-methylbenzyl)amino)picolinoyl)cyclohex-2-en-1-one), and 23 displayed excellent greenhouse herbicidal effects at 150 g of active ingredient (ai)/ha after postemergence treatment. Furthermore, compound 16 showed superior weed-controlling efficacy against Setaria viridis (S. viridis) versus that of the positive control mesotrione at multiple test dosages (120, 60, and 30 g ai/ha). These findings imply that compound 16, as a novel lead of HPPD inhibitors, possesses great potential for application in specifically combating the malignant weed S. viridis.

Diaryl Ether: A Privileged Scaffold for Drug and Agrochemical Discovery.

Diaryl ether (DE) is a functional scaffold existing widely both in natural products (NPs) and synthetic organic compounds. Statistically, DE is the second most popular and enduring scaffold within the numerous medicinal chemistry and agrochemical reports. Given its unique physicochemical properties and potential biological activities, DE nucleus is recognized as a fundamental element of medicinal and agrochemical agents aimed at different biological targets. Its drug-like derivatives have been extensively synthesized with interesting biological features including anticancer, anti-inflammatory, antiviral, antibacterial, antimalarial, herbicidal, fungicidal, insecticidal, and so on. In this review, we highlight the medicinal and agrochemical versatility of the DE motif according to the published information in the past decade and comprehensively give a summary of the target recognition, structure-activity relationship (SAR), and mechanism of action of its analogues. It is expected that this profile may provide valuable guidance for the discovery of new active ingredients both in drug and pesticide research.

LARMD: integration of bioinformatic resources to profile ligand-driven protein dynamics with a case on the activation of estrogen receptor.

Protein dynamics is central to all biological processes, including signal transduction, cellular regulation and biological catalysis. Among them, in-depth exploration of ligand-driven protein dynamics contributes to an optimal understanding of protein function, which is particularly relevant to drug discovery. Hence, a wide range of computational tools have been designed to investigate the important dynamic information in proteins. However, performing and analyzing protein dynamics is still challenging due to the complicated operation steps, giving rise to great difficulty, especially for nonexperts. Moreover, there is a lack of web protocol to provide online facility to investigate and visualize ligand-driven protein dynamics. To this end, in this study, we integrated several bioinformatic tools to develop a protocol, named Ligand and Receptor Molecular Dynamics (LARMD, http://chemyang.ccnu.edu.cn/ccb/server/LARMD/ and http://agroda.gzu.edu.cn:9999/ccb/server/LARMD/), for profiling ligand-driven protein dynamics. To be specific, estrogen receptor (ER) was used as a case to reveal ERß-selective mechanism, which plays a vital role in the treatment of inflammatory diseases and many types of cancers in clinical practice. Two different residues (Ile373/Met421 and Met336/Leu384) in the pocket of ERß/ERα were the significant determinants for selectivity, especially Met336 of ERß. The helix H8, helix H11 and H7-H8 loop influenced the migration of selective agonist (WAY-244). These computational results were consistent with the experimental results. Therefore, LARMD provides a user-friendly online protocol to study the dynamic property of protein and to design new ligand or site-directed mutagenesis.

Genome-wide identification and functional analysis of the splicing component SYF2/NTC31/p29 across different plant species.

MAIN CONCLUSION: This study systematically identifies plant SYF2/NTC31/p29 genes from 62 plant species by a combinatory bioinformatics approach, revealing the importance of this gene family in phylogenetics, duplication, transcriptional, and post-transcriptional regulation. Alternative splicing is a post-transcriptional regulatory mechanism, which is critical for plant development and stress responses. The entire process is strictly attenuated by a complex of splicing-related proteins, designated splicing factors. Human p29, also referred to as synthetic lethal with cdc forty 2 (SYF2) or the NineTeen complex 31 (NTC31), is a core protein found in the NTC complex of humans and yeast. This splicing factor participates in a variety of biological processes, including DNA damage repair, control of the cell cycle, splicing, and tumorigenesis. However, its function in plants has been seldom reported. Thus, we have systematically identified 89 putative plant SYF2s from 62 plant species among the deposited entries in the Phytozome database. The phylogenetic relationships and evolutionary history among these plant SYF2s were carefully examined. The results revealed that plant SYF2s exhibited distinct patterns regarding their gene structure, promoter sequences, and expression levels, suggesting their functional diversity in response to developmental cues or stress treatments. Although local duplication events, such as tandem duplication and retrotransposition, were found among several plant species, most of the plant species contained only one copy of SYF2, suggesting the existence of additional mechanisms to confer duplication resistance. Further investigation using the model dicot and monocot representatives Arabidopsis and rice SYF2s indicated that the splicing pattern and resulting protein isoforms might play an alternative role in the functional diversity.

Low expression of microRNA-126 is associated with poor prognosis in colorectal cancer.

MicroRNA-126 (miR-126) has been reported to be a tumor suppressor that targets CXCR4 in colorectal cancer (CRC) cells. This study investigated whether miR-126 has any prognostic impact in patients with CRC. MiR-126 and CXCR4 mRNA expression in 92 pairs of CRC and adjacent nontumorous tissues was examined using quantitative real-time PCR, and CXCR4 protein expression was assessed by immunohistochemistry (IHC) and Western blotting. The correlation between miR-126 and CXCR4 protein expression and clinicopathological features and overall survival rate was determined. MiR-126 was downregulated in CRC tissues that expressed high levels of CXCR4 mRNA. IHC and Western blotting detected high expression of CXCR4 protein in CRC tissues. An inverse correlation was observed between miR-126 and CXCR4 protein expression in CRC tissues. Moreover, low miR-126 and high CXCR4 protein expression was associated with distant metastasis, clinical TNM stage, and poor survival. Multivariate analysis indicated that miR-126 was an independent prognostic factor for overall survival, suggesting its clinical significance as a prognostic predictor in CRC patients.

MicroRNA-135b regulates metastasis suppressor 1 expression and promotes migration and invasion in colorectal cancer.

MicroRNAs (miRNAs, miRs) play important roles in pathogenesis and development of human diseases, including malignancy. Some may affect tumor progression through targeting tumor suppressor genes. MiR-135b has been shown to be upregulated in CRC. In this study, we evaluated the role of miR-135b in colorectal cancer (CRC) and its regulatory role for metastasis suppressor-1 (MTSS1) and its mechanisms. The levels of miR-135b and MTSS1 gene expression in 35 CRC and corresponding cancer-adjacent tissues, 27 colorectal adenoma, and 16 normal tissue samples were quantified using qRT-PCR and western blot analysis. The effect of miR-135b on MTSS1 expression was assessed by miR-135b mimics or inhibitor transfection to deregulate miR-135b expression. The direct interaction between them was verified by 3'-UTR dual-luciferase reporter assay. Furthermore, the roles of miR-135b in regulating CRC cells migration and invasion properties were analyzed with miR-135b mimics or inhibitor-transfected cells and silenced expression of MTSS1 in miR-135b inhibitor transfected cells. CRC tissues showed significantly upregulated miR-135b expression and reduced MTSS1 expression. High miR-135b levels were significantly associated with lymph node and distant metastasis. The miR-135b inhibitor decreased miR-135b expression and caused MTSS1 upregulation at the post-transcriptional level. However, overexpression of miR-135b caused MTSS1 protein downregulation. The 3'-UTR of MTSS1 harbored a binding site for miR-135b. Finally, miR-135b inhibitor-transfected cells exhibited markedly reduced cell migration and invasive abilities, and this effect could be reversed by MTSS1-siRNA. Our results demonstrated that miR-135b downregulated MTSS1 expression and contributed to CRC cell invasion, indicating its involvement in CRC progression.

The relationship between and clinical significance of MicroRNA-32 and phosphatase and tensin homologue expression in colorectal cancer.

MicroRNAs (miRNAs, miRs) are suspected to play important roles in carcinogenesis. MiR-32 has altered expression in colorectal cancer (CRC); however, the clinical significance of miR-32 expression in the process of carcinogenesis is poorly understood. In this study, we determined the levels of, the correlation between, and the clinical significance of the expression of miR-32 and phosphatase and tensin homologue (PTEN), a tumor suppressor targeted by miR-32, in CRC. The levels of miR-32 and PTEN gene expression in 35 colorectal carcinoma samples, 35 corresponding cancer-adjacent tissue samples, 27 colorectal adenoma samples, and 16 normal tissue samples were quantified using real-time quantitative reverse transcriptase-polymerase chain reaction. PTEN protein expression was determined using western blot and immunohistochemistry (IHC). The relationship between the miR-32 and PTEN protein expression and clinicopathological factors was analyzed. Significant upregulation of miR-32 expression and reduction of PTEN were identified in CRC tissues. High miR-32 levels were significantly associated with lymph node and distant metastasis, and Kaplan-Meier analysis indicated that patients with high miR-32 expression had a poor overall survival. Low PTEN protein expression was also significantly correlated with distant metastasis. An inverse relationship between miR-32 and PTEN protein expression was identified. In addition, IHC analysis revealed weak or indiscernible PTEN staining in tumor tissue. MiR-32 overexpression was correlated with specific CRC clinicopathological features and may be a marker of poor prognosis in CRC patients. MiR-32 and PTEN expression were inversely correlated, and miR-32 may be associated with the development of CRC.

MicroRNA-32 (miR-32) regulates phosphatase and tensin homologue (PTEN) expression and promotes growth, migration, and invasion in colorectal carcinoma cells.

BACKGROUND: Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-32 has been shown to be upregulated in CRC. In this study, we identified the potential effects of miR-32 on some important biological properties of CRC cells, and clarified the regulation of PTEN by miR-32. METHODS: The effect of miR-32 on PTEN expression was assessed in CRC cell lines with miR-32 mimics/inhibitor to increase/decrease miR-32 expression. Furthermore, the roles of miR-32 in regulating CRC cells biological properties were analyzed with miR-32 mimics/inhibitor-transfected cells. The 3'-untranslated region (3'-UTR) of PTEN combined with miR-32 was verified by dual-luciferase reporter assay. RESULTS: Gain-of-function and loss-of-function studies showed that overexpression of miR-32 promoted SW480 cell proliferation, migration, and invasion, reduced apoptosis, and resulted in downregulation of PTEN at a posttranscriptional level. However, miR-32 knock-down inhibited these processes in HCT-116 cells and enhanced the expression of PTEN protein. In addition, we further identified PTEN as the functional downstream target of miR-32 by directly targeting the 3'-UTR of PTEN. CONCLUSIONS: Our results demonstrated that miR-32 was involved in tumorigenesis of CRC at least in part by suppression of PTEN.