Semi-Supervised Classification of PolSAR Images Based on Co-Training of CNN and SVM with Limited Labeled Samples.

Recently, convolutional neural networks (CNNs) have shown significant advantages in the tasks of image classification; however, these usually require a large number of labeled samples for training. In practice, it is difficult and costly to obtain sufficient labeled samples of polarimetric synthetic aperture radar (PolSAR) images. To address this problem, we propose a novel semi-supervised classification method for PolSAR images in this paper, using the co-training of CNN and a support vector machine (SVM). In our co-training method, an eight-layer CNN with residual network (ResNet) architecture is designed as the primary classifier, and an SVM is used as the auxiliary classifier. In particular, the SVM is used to enhance the performance of our algorithm in the case of limited labeled samples. In our method, more and more pseudo-labeled samples are iteratively yielded for training through a two-stage co-training of CNN and SVM, which gradually improves the performance of the two classifiers. The trained CNN is employed as the final classifier due to its strong classification capability with enough samples. We carried out experiments on two C-band airborne PolSAR images acquired by the AIRSAR systems and an L-band spaceborne PolSAR image acquired by the GaoFen-3 system. The experimental results demonstrate that the proposed method can effectively integrate the complementary advantages of SVM and CNN, providing overall classification accuracy of more than 97%, 96% and 93% with limited labeled samples (10 samples per class) for the above three images, respectively, which is superior to the state-of-the-art semi-supervised methods for PolSAR image classification.

Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion.

GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.

Differential activities and mechanisms of the four R-spondins in potentiating Wnt/ß-catenin signaling.

The four R-spondins (RSPO1-4) strongly potentiate Wnt signaling and play critical roles in normal development, adult stem cell survival, and cancer development and aggressiveness. All four RSPOs have been suggested to potentiate Wnt signaling by binding to three related receptors, i.e. leucine-rich repeat-containing, G protein-coupled receptors 4, 5, and 6 (LGR4/5/6), and then inducing the clearance of two E3 ubiquitin ligases (RNF43 and ZNRF3) that otherwise would ubiquitinate Wnt receptors for degradation. Here, we show that RSPO1-4 have differential dependence on LGRs in potentiating Wnt/ß-catenin signaling and that RSPO2 can enhance this pathway without any LGR. LGR4 knockout (LGR4KO) in HEK293 cells completely abrogated the Wnt/ß-catenin signaling response to RSPO1 and RSPO4 and strongly impaired the response to RSPO3. RSPO2, however, retained robust activity albeit with decreased potency. Complete rescue of RSPO1-4 activity in LGR4KO cells required the seven-transmembrane domain of LGR4. Furthermore, an RSPO2 mutant with normal binding affinity to ZNRF3 but no or little binding to LGR4 or LGR5 still potentiated Wnt/ß-catenin signaling in vitro, supported the growth of intestinal organoids ex vivo, and stimulated intestinal crypt growth in vivo Mechanistically, RSPO2 could increase Wnt receptor levels in the absence of any LGR without affecting ZNRF3 endocytosis and stability. These findings suggest that RSPO1-4 use distinct mechanisms in regulating Wnt and other signaling pathways, which have important implications for understanding the pleiotropic functions of RSPOs and LGRs in both normal and cancer development.

Evaluation of Anti-LGR5 Antibodies by ImmunoPET for Imaging Colorectal Tumors and Development of Antibody-Drug Conjugates.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis. Recently, we showed that LGR5 is a promising target for antibody-drug conjugate (ADC) therapy. However, it is important to identify LGR5-positive tumors that would respond to ADC treatment. Prior to drug conjugation, we evaluated two different anti-LGR5 monoclonal antibodies (mAbs), 8F2 and 9G5, using 89Zr-immunoPET to select the optimal mAb for ADC development and tumor imaging. Binding, specificity, and internalization were compared, and mAbs were prescreened as ADC candidates against colon cancer cells using secondary ADCs. Both mAbs demonstrated strong, specific binding in 293T-LGR5 cells but not 293T-vector cells. In DLD-1 colorectal cancer cells, which express high levels of LGR5, the mAbs rapidly internalized into lysosomes and promoted ADC-induced cytotoxicity, with 8F2 exhibiting slightly higher potency. No binding was detected in DLD-1-shLGR5 (LGR5 knockdown) cells. 89Zr-DFO-LGR5 mAbs were generated and shown to retain high affinity and LGR5-dependent uptake in vitro. PET/CT imaging of DLD-1 tumors was performed 5 days postinjection of 89Zr-DFO-LGR5 mAbs, and findings were consistent with biodistribution data, which showed significantly higher tumor uptake (%ID/g) for 89Zr-DFO-8F2 (17.9 ± 2.2) compared to 89Zr-DFO-9G5 (5.5 ± 1.2) and 89Zr-DFO-IgG (3.8 ± 1.0). No significant uptake was observed in DLD-1-shLGR5 tumors. This study identifies 8F2 as the optimal candidate for ADC development and provides initial evidence that 89Zr-DFO-LGR5 mAbs may be utilized to stratify tumors which would respond best to LGR5-targeted ADC therapy.

Game theory-based visual tracking approach focusing on color and texture features.

It is difficult for a single-feature tracking algorithm to achieve strong robustness under a complex environment. To solve this problem, we proposed a multifeature fusion tracking algorithm that is based on game theory. By focusing on color and texture features as two gamers, this algorithm accomplishes tracking by using a mean shift iterative formula to search for the Nash equilibrium of the game. The contribution of different features is always keeping the state of optical balance, so that the algorithm can fully take advantage of feature fusion. According to the experiment results, this algorithm proves to possess good performance, especially under the condition of scene variation, target occlusion, and similar interference.

LGR4 and LGR5 form distinct homodimers that only LGR4 complexes with RNF43/ZNRF3 to provide high affinity binding of R-spondin ligands.

LGR4 and LGR5 are two homologous receptors that potentiate Wnt/ß-catenin signaling in response to R-spondin (RSPO) ligands. The RSPO and LGR4 complex binds to and inhibits activities of two related E3 ubiquitin ligases, RNF43 and ZNRF3, and thus protects Wnt receptors from the E3 ligase-mediated degradation. The RSPO and LGR5 complex, however, does not interact with the E3 ligases, and the structural basis of this difference remained unknown. Here we examined the affinities of monovalent and bivalent RSPO ligands in binding to LGR4, RNF43/ZNRF3, and LGR5 in whole cells and found unique features among the receptors and E3 ligases. Monovalent RSPO2 furin domain had much lower affinity in binding to LGR4 or RNF43/ZNRF3 than the bivalent form. In contrast, monovalent and bivalent forms had nearly identical affinity in binding to LGR5. Co-expression of ZNRF3 with LGR4 led to much higher binding affinity of the monovalent form whereas co-expression of ZNRF3 with LGR5 had no effect on the affinity. These results suggest that LGR4 and RNF43/ZNRF3 form a 2:2 dimer that accommodates bivalent binding of RSPO whereas LGR5 forms a homodimer that does not. Structural models are proposed to illustrate how RSPOs bind to LGR4, RNF43/ZNRF3, and LGR5 in whole cells.

Drug Conjugates of Antagonistic R-Spondin 4 Mutant for Simultaneous Targeting of Leucine-Rich Repeat-Containing G Protein-Coupled Receptors 4/5/6 for Cancer Treatment.

LGR4-6 (leucine-rich repeat-containing G-protein-coupled receptors 4, 5, and 6) are three related receptors with an upregulated expression in gastrointestinal cancers to various extents, and LGR5 is enriched in cancer stem cells. Antibody-drug conjugates (ADCs) targeting LGR5 showed a robust antitumor effect in vivo but could not eradicate tumors due to plasticity of LGR5-positive cancer cells. As LGR5-negative cancer cells often express LGR4 or LGR6 or both, we reasoned that simultaneous targeting of all three LGRs may provide a more effective approach. R-spondins (RSPOs) bind to LGR4-6 with high affinity and potentiate Wnt signaling. We identified an RSPO4 furin domain mutant (Q65R) that retains potent LGR binding but no longer potentiates Wnt signaling. Drug conjugates of a peptibody comprising the RSPO4 mutant and IgG1-Fc showed potent cytotoxic effects on cancer cell lines expressing any LGR in vitro and suppressed tumor growth in vivo without inducing intestinal enlargement or other adverse effects.

Unlike LGR4, LGR5 potentiates Wnt-ß-catenin signaling without sequestering E3 ligases.

LGR4 and LGR5 encode two homologous receptors with critical, yet distinct, roles in organ development and adult stem cell survival. Both receptors are coexpressed in intestinal crypt stem cells, bind to R-spondins (RSPOs) with high affinity, and potentiate Wnt-ß-catenin signaling, presumably by the same mechanism: forming RSPO-bridged complexes with the E3 ligases RNF43 and ZNRF3 to inhibit ubiquitylation of Wnt receptors. However, direct evidence for RSPO-bound, full-length LGR5 interacting with these E3 ligases in whole cells has not been reported, and only LGR4 is essential for the self-renewal of intestinal stem cells. Here, we examined the mechanisms of action of LGR4 and LGR5 in parallel using coimmunoprecipitation, proximity ligation, competition binding, and time-resolved FRET assays in whole cells. Full-length LGR4 formed a tight complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 did not interact with either E3 ligase with or without RSPO. Domain-swapping experiments with LGR4 and LGR5 revealed that the seven-transmembrane domain of LGR4 conferred interaction with the E3 ligases. Native LGR4 and LGR5 existed as dimers on the cell surface, and LGR5 interacted with both FZD and LRP6 of the Wnt signalosome to enhance LRP6 phosphorylation and potentiate Wnt-ß-catenin signaling. These findings provide a molecular basis for the weaker activity of LGR5 in the potentiation of Wnt signaling that may underlie the distinct roles of LGR4 and LGR5 in organ development, as well as the self-renewal and fitness of adult stem cells.

Protein kinase C betaII and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis.

Increasing evidence demonstrates that protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. We previously reported that colonic PKCbetaII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCbetaII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCbetaII represses transforming growth factor beta receptor type II (TGFbetaRII) expression and reduces sensitivity to TGF-beta-mediated growth inhibition in intestinal epithelial cells. Transgenic PKCbetaII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFbetaRII expression. Chemopreventive dietary omega-3 fatty acids inhibit colonic PKCbetaII activity in vivo and block PKCbetaII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFbetaRII expression in the colonic epithelium of transgenic PKCbetaII mice. These data indicate that dietary omega-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCbetaII signaling and restoration of TGF-beta responsiveness.

Protein kinase Ciota is required for Ras transformation and colon carcinogenesis in vivo.

Protein kinase C iota (PKCiota) has been implicated in Ras signaling, however, a role for PKCiota in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCiota is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCiota in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCiota (kdPKCiota) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCiota in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCiota. Our data demonstrate that PKCiota is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCiota, and Rac1.

Online Scale Adaptive Visual Tracking Based on Multilayer Convolutional Features.

Convolutional neural networks can efficiently exploit sophisticated hierarchical features which have different properties for visual tracking problem. In this paper, by using multilayer convolutional features jointly and constructing a scale pyramid, we propose an online scale adaptive tracking method. We construct two separate correlation filters for translation and scale estimations. The translation filters improve the accuracy of target localization by a weighted fusion of multiple convolutional layers. Meanwhile, the separate scale filters achieve the optimal and fast scale estimation by a scale pyramid. This design decreases the mutual errors of translation and scale estimations, and reduces computational complexity efficiently. Moreover, in order to solve the problem of tracking drifts due to the severe occlusion or serious appearance changes of the target, we present a new adaptive and selective update mechanism to update the translation filters effectively. Extensive experimental results show that our proposed method achieves the excellent overall performance compared with the state-of-the-art methods.

The most common RNF43 mutant G659Vfs*41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis.

RNF43 is an E3 ligase that inhibits Wnt signaling by ubiquitinating Wnt receptors for degradation. It is mutated in various cancer types with the most recurrent mutation being the frameshift G659Vfs*41 with frequencies of ~5-8% in colon, stomach and endometrial cancers. This mutation, a deletion of G in a 7-G repeat, has been assumed to encode an inactive enzyme that would lead to increased Wnt signaling and drive tumorigenesis, yet no functional characterization has been reported. We analyzed the distribution of G659Vfs*41 and its association with other cancer gene mutations, and found that the mutation occurred nearly exclusively in tumors with low expression of the DNA mismatch repair gene MLH1. Mutant RNF43-G659Vfs*41 was no different from wild type RNF43 in expression, stability, localization, R-spondin binding, and inhibition of Wnt signaling. No dominant negative activity of the mutant was observed. Colon tumors with RNF43-G659Vfs*41 had low Wnt/ß-catenin signaling and were frequently mutated in BRAF. A colon cancer cell line with RNF43-G659Vfs*41 and BRAF-V600E mutations was sensitive to activation of Wnt/ß-catenin signaling. These findings suggest that the frequent occurrence of RNF43-G659Vfs*41 may result from error-prone replication of the 7-G repeat in MLH1-deficient tumors and that the mutation itself does not inactivate enzyme.

Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract.

5-Hydroxytryptamine (serotonin) (5-HT) is a neurotransmitter with both central and peripheral functions, including the modulation of mood, appetite, hemodynamics, gastrointestinal (GI) sensation, secretion, and motility. Its synthesis is initiated by the enzyme tryptophan hydroxylase (TPH). Two isoforms of TPH have been discovered: TPH1, primarily expressed in the enterochromaffin cells of the gastrointestinal tract, and TPH2, expressed exclusively in neuronal cells. Mice lacking Tph1 contain little to no 5-HT in the blood and GI tract while maintaining normal levels in the brain. Because GI 5-HT is known to play important roles in normal and pathophysiology, we set out to discover and characterize novel compounds that selectively inhibit biosynthesis of GI 5-HT. Here, we describe two of a series of these inhibitors that are potent for TPH activity both in biochemical and cell-based assays. This class of compounds has unique properties with respect to pharmacokinetic and pharmacodynamic effects on GI serotonin production. Similar to the Tph1 knockout results, these TPH inhibitors have the ability to selectively reduce 5-HT levels in the murine GI tract without affecting brain 5-HT levels. In addition, administration of these compounds in a ferret model of chemotherapy-induced emesis caused modest reductions of intestinal serotonin levels and a decreased emetic response. These findings suggest that GI-specific TPH inhibitors may provide novel treatments for various gastrointestinal disorders associated with dysregulation of the GI serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.

A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease.

Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 Å resolution, respectively. A Wing domain, absent in other ßßα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic 'tape-measure'. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase γ.

Adult Tph2 knockout mice without brain serotonin have moderately elevated spine trabecular bone but moderately low cortical bone thickness.

Disruption of serotonin synthesis in neurons and the periphery by knockout (KO) of mouse genes for tryptophan hydroxylases (peripheral Tph1 and neuronal Tph2) has been claimed to decrease (Tph2 KO) and increase (Tph1 KO) bone mass. In this report, adult male and female Tph2 KO mice were observed to have elevated spine trabecular bone. Female Tph2 KO mice have reduced midshaft femur cortical bone thickness. Bone mass was normal in male and female Tph1 KO mice examined as part of a Tph1/Tph2 double knockout (DKO) mouse cohort.

Mechanism of Inhibition of Novel Tryptophan Hydroxylase Inhibitors Revealed by Co-crystal Structures and Kinetic Analysis.

Trytophan Hydroxylase Type I (TPH1), most abundantly expressed in the gastrointestinal tract, initiates the synthesis of serotonin by catalyzing hydroxylation of tryptophan in the presence of biopterin and oxygen. We have previously described three series of novel, periphery-specific TPH1 inhibitors that selectively deplete serotonin in the gastrointestinal tract. We have now determined co-crystal structures of TPH1 with three of these inhibitors at high resolution. Analysis of the structural data showed that each of the three inhibitors fills the tryptophan binding pocket of TPH1 without reaching into the binding site of the cofactor pterin, and induces major conformational changes of the enzyme. The enzyme-inhibitor complexes assume a compact conformation that is similar to the one in tryptophan complex. Kinetic analysis showed that all three inhibitors are competitive versus the substrate tryptophan, consistent with the structural data that the compounds occupy the tryptophan binding site. On the other hand, all three inhibitors appear to be uncompetitive versus the cofactor 6-methyltetrahydropterin, which is not only consistent with the structural data but also indicate that the hydroxylation reaction follows an ordered binding mechanism in which a productive complex is formed only if tryptophan binds only after pterin, similar to the kinetic mechanisms of tyrosine and phenylalanine hydroxylase.

Role of cyclooxygenase 2 in protein kinase C beta II-mediated colon carcinogenesis.

Elevated expression of protein kinase C beta II (PKC beta II) is an early promotive event in colon carcinogenesis (Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z., and Fields, A. P. (2001) Cancer Res. 61, 1375-1381). Expression of PKC beta II in the colon of transgenic mice leads to hyperproliferation and increased susceptibility to colon carcinogenesis due, at least in part, to repression of transforming growth factor beta type II receptor (TGF-beta RII) expression (Murray, N. R., Davidson, L. A., Chapkin, R. S., Gustafson, W. C., Schattenberg, D. G., and Fields, A. P. (1999) J. Cell Biol., 145, 699-711). Here we report that PKC beta II induces the expression of cyclooxygenase type 2 (Cox-2) in rat intestinal epithelial (RIE) cells in vitro and in transgenic PKC beta II mice in vivo. Cox-2 mRNA increases more than 10-fold with corresponding increases in Cox-2 protein and PGE2 production in RIE/PKC beta II cells. PKC beta II activates the Cox-2 promoter by 2- to 3-fold and stabilizes Cox-2 mRNA by at least 4-fold. The selective Cox-2 inhibitor Celecoxib restores expression of TGF-beta RII both in vitro and in vivo and restores TGF beta-mediated transcription in RIE/PKC beta II cells. Likewise, the omega-3 fatty acid eicosapentaenoic acid (EPA), which inhibits PKC beta II activity and colon carcinogenesis, causes inhibition of Cox-2 protein expression, re-expression of TGF-beta RII, and restoration of TGF-beta1-mediated transcription in RIE/PKC beta II cells. Our data demonstrate that PKC beta II promotes colon cancer, at least in part, through induction of Cox-2, suppression of TGF-beta signaling, and establishment of a TGF-beta-resistant, hyperproliferative state in the colonic epithelium. Our data define a procarcinogenic PKC beta II --> Cox-2 --> TGF-beta signaling axis within the colonic epithelium, and provide a molecular mechanism by which dietary omega-3 fatty acids and nonsteroidal antiinflammatory agents such as Celecoxib suppress colon carcinogenesis.