Direct Physical Interaction Research Articles

Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome.

Mutations in the human MECP2 gene cause Rett syndrome (RTT), a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies.

Antiviral activity of Lactobacillus reuteri Protectis against Coxsackievirus A and Enterovirus 71 infection in human skeletal muscle and colon cell lines.

BackgroundRecurrence of hand, foot and mouth disease (HFMD) pandemics continues to threaten public health. Despite increasing awareness and efforts, effective vaccine and drug treatment have yet to be available. Probiotics have gained recognition in the field of healthcare worldwide, and have been extensively prescribed to babies and young children to relieve gastrointestinal (GI) disturbances and diseases, associated or not with microbial infections. Since the faecal-oral axis represents the major route of HFMD transmission, transient persistence of probiotic bacteria in the GI tract may confer some protection against HFMD and limit transmission among children.MethodsIn this work, the antiviral activity of two commercially available probiotics, namely Lactobacillus reuteri Protectis (L. reuteri Protectis) and Lactobacillus casei Shirota (L. casei Shirota), was assayed against Coxsackieviruses and Enterovirus 71 (EV71), the main agents responsible for HFMD. In vitro infection set-ups using human skeletal muscle and colon cell lines were designed to assess the antiviral effect of the probiotic bacteria during entry and post-entry steps of the infection cycle.ResultsOur findings indicate that L. reuteri Protectis displays a significant dose-dependent antiviral activity against Coxsackievirus type A (CA) strain 6 (CA6), CA16 and EV71, but not against Coxsackievirus type B strain 2. Our data support that the antiviral effect is likely achieved through direct physical interaction between bacteria and virus particles, which impairs virus entry into its mammalian host cell. In contrast, no significant antiviral effect was observed with L. casei Shirota.ConclusionsShould the antiviral activity of L. reuteri Protectis observed in vitro be translated in vivo, such probiotics-based therapeutic approach may have the potential to address the urgent need for a safe and effective means to protect against HFMD and limit its transmission among children.

Penetrance of Congenital Heart Disease in a Mouse Model of Down Syndrome Depends on a Trisomic Potentiator of a Disomic Modifier.

Down syndrome (DS) is a significant risk factor for congenital heart disease (CHD), increasing the incidence 50 times over the general population. However, half of people with DS have a normal heart and thus trisomy 21 is not sufficient to cause CHD by itself. Ts65Dn mice are trisomic for orthologs of >100 Hsa21 genes, and their heart defect frequency is significantly higher than their euploid littermates. Introduction of a null allele of Creld1 into Ts65Dn increases the penetrance of heart defects significantly. However, this increase was not seen when the Creld1 null allele was introduced into Ts1Cje, a mouse that is trisomic for about two thirds of the Hsa21 orthologs that are triplicated in Ts65Dn. Among the 23 genes present in three copies in Ts65Dn but not Ts1Cje, we identified Jam2 as necessary for the increased penetrance of Creld1-mediated septal defects in Ts65Dn. Thus, overexpression of the trisomic gene, Jam2, is a necessary potentiator of the disomic genetic modifier, Creld1 No direct physical interaction between Jam2 and Creld1 was identified by several methods. Regions of Hsa21 containing genes that are risk factors of CHD have been identified, but Jam2 (and its environs) has not been linked to heart formation previously. The complexity of this interaction may be more representative of the clinical situation in people than consideration of simple single-gene models.

Mining Dual Networks

Finding the densest subgraph in a single graph is a fundamental problem that has been extensively studied. In many emerging applications, there exist dual networks. For example, in genetics, it is important to use protein interactions to interpret genetic interactions. In this application, one network represents physical interactions among nodes, for example, protein--protein interactions, and another network represents conceptual interactions, for example, genetic interactions. Edges in the conceptual network are usually derived based on certain correlation measure or statistical test measuring the strength of the interaction. Two nodes with strong conceptual interaction may not have direct physical interaction. In this article, we propose the novel dual-network model and investigate the problem of finding the densest connected subgraph (DCS), which has the largest density in the conceptual network and is also connected in the physical network. Density in the conceptual network represents the average strength of the measured interacting signals among the set of nodes. Connectivity in the physical network shows how they interact physically. Such pattern cannot be identified using the existing algorithms for a single network. We show that even though finding the densest subgraph in a single network is polynomial time solvable, the DCS problem is NP-hard. We develop a two-step approach to solve the DCS problem. In the first step, we effectively prune the dual networks, while guarantee that the optimal solution is contained in the remaining networks. For the second step, we develop two efficient greedy methods based on different search strategies to find the DCS. Different variations of the DCS problem are also studied. We perform extensive experiments on a variety of real and synthetic dual networks to evaluate the effectiveness and efficiency of the developed methods.

Development of potent small-molecule inhibitors to drug the undruggable steroid receptor coactivator-3

Protein-protein interactions (PPIs) play a central role in most biological processes, and therefore represent an important class of targets for therapeutic development. However, disrupting PPIs using small-molecule inhibitors (SMIs) is challenging and often deemed as "undruggable." We developed a cell-based functional assay for high-throughput screening to identify SMIs for steroid receptor coactivator-3 (SRC-3 or AIB1), a large and mostly unstructured nuclear protein. Without any SRC-3 structural information, we identified SI-2 as a highly promising SMI for SRC-3. SI-2 meets all of the criteria of Lipinski's rule [Lipinski et al. (2001) Adv Drug Deliv Rev 46(1-3):3-26] for a drug-like molecule and has a half-life of 1 h in a pharmacokinetics study and a reasonable oral availability in mice. As a SRC-3 SMI, SI-2 can selectively reduce the transcriptional activities and the protein concentrations of SRC-3 in cells through direct physical interactions with SRC-3, and selectively induce breast cancer cell death with IC50 values in the low nanomolar range (3-20 nM), but not affect normal cell viability. Furthermore, SI-2 can significantly inhibit primary tumor growth and reduce SRC-3 protein levels in a breast cancer mouse model. In a toxicology study, SI-2 caused minimal acute cardiotoxicity based on a hERG channel blocking assay and an unappreciable chronic toxicity to major organs based on histological analyses. We believe that this work could significantly improve breast cancer treatment through the development of "first-in-class" drugs that target oncogenic coactivators.

OC-0238: Akt1 facilitates DNA double-strand breaks repair through a direct physical interaction with DNA-PKcs

OC-0238: Akt1 facilitates DNA double-strand breaks repair through a direct physical interaction with DNA-PKcs

Diaphragm Assessment in Mice Overexpressing Phospholamban in Slow‐Twitch Type I Muscle Fibers

Phospholamban (PLN) is a small transmembrane protein that regulates the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump through direct physical interactions. Recent work published by our laboratory revealed that when PLN was transgenetically overexpressed in mice (PlnOE), the postural hind limb muscles presented a phenotype for human Centronuclear Myopathy (CNM). Specifically, PlnOE soleus and gluteus minimus muscles displayed: centrally located oxidative activity, a predominance of hypotrophied type I fibers, and an abundance of central nuclei. To further assess this novel model for CNM, we examined the diaphragm muscles of PlnOE mice, since studies assessing respiratory or diaphragm function in animal models of CNM were limited. Interestingly, our unpublished results suggest that unlike the soleus and gluteus minimus, diaphragm muscles did not display the main characteristics of CNM. Rather than exhibiting type I fiber predominance, the diaphragm muscles exhibited lower type I and greater type II fiber counts, which is of importance since the PlnOE mice were designed to overexpress PLN specifically in their type I fibers. Here, we sought to determine why the PlnOE diaphragm failed to exhibit type I fiber predominance similar to that seen in the soleus and gluteus minimus muscles. Specifically, we focused on calciuneurin (CnA), a Ca2+ ‐dependent phosphatase, and its downstream effector target, nuclear factor of activating T‐cell (NFAT), that upon dephosphorylation by CnA, translocates to the nucleus and acts as a transcription factor for myosin heavy chain I (MHCI). We found that CnA content was significantly higher in both soleus (p=0.02) and gluteus minimus (p=0.05) muscles of PlnOE mice compared to wild type animals. Corresponding with this, nuclear NFAT levels were significantly increased in the gluteus minimus (p=0.01) and trending towards a significant increase in soleus muscle (p=0.07). In contrast, there were no differences between WT and PlnOE in CnA (p=0.23) or nuclear NFAT (p=0.42) content in diaphragm, which suggests the differences in fiber type transition and the CNM phenotype across muscles may be associated with the CnA‐NFAT pathway and calcium signaling. Together these data suggest that the diaphragm of the PlnOE mice may have some undetermined protective mechanism for better regulating cytosolic calcium levels and fiber type expression. Uncovering the mechanisms behind this resistance within the diaphragm of PLNOE mice may aid in exploring new therapeutic strategies for CNM.

Investigating direct interaction between Escherichia coli topoisomerase I and RecA

Protein–protein interactions are of special importance in cellular processes, including replication, transcription, recombination, and repair. Escherichia coli topoisomerase I (EcTOP1) is primarily involved in the relaxation of negative DNA supercoiling. E. coli RecA, the key protein for homologous recombination and SOS DNA-damage response, has been shown to stimulate the relaxation activity of EcTOP1. The evidence for their direct protein–protein interaction has not been previously established. We report here the direct physical interaction between E. coli RecA and topoisomerase I. We demonstrated the RecA-topoisomerase I interaction via pull-down assays, and surface plasmon resonance measurements. Molecular docking supports the observation that the interaction involves the topoisomerase I N-terminal domains that form the active site. Our results from pull-down assays showed that ATP, although not required, enhances the RecA-EcTOP1 interaction. We propose that E. coli RecA physically interacts with topoisomerase I to modulate the chromosomal DNA supercoiling.

Epistasis in protein evolution.

The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions-called epistasis-within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage-specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis-in which one mutation influences the phenotypic effect of few other mutations-is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low-probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families.

Telomere-binding protein regulates the chromosome ends through the interaction with histone deacetylases in Arabidopsis thaliana.

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes. Many telomere-binding proteins bind to telomeric repeat sequences and further generate T-loops in animals. However, it is not clear if they regulate telomere organization using epigenetic mechanisms and how the epigenetic molecules are involved in regulating the telomeres. Here, we show direct interactions between the telomere-binding protein, AtTRB2 and histone deacetylases, HDT4 and HDA6, in vitro and in vivo. AtTRB2 mediates the associations of HDT4 and HDA6 with telomeric repeats. Telomere elongation is found in AtTRB2, HDT4 and HDA6 mutants over generations, but also in met1 and cmt3 DNA methyltransferases mutants. We also characterized HDT4 as an Arabidopsis H3K27 histone deacetylase. HDT4 binds to acetylated peptides at residue K27 of histone H3 in vitro, and deacetylates this residue in vivo. Our results suggest that AtTRB2 also has a role in the regulation of telomeric chromatin as a possible scaffold protein for recruiting the epigenetic regulators in Arabidopsis, in addition to its telomere binding and length regulation activity. Our data provide evidences that epigenetic molecules associate with telomeres by direct physical interaction with telomere-binding proteins and further regulate homeostasis of telomeres in Arabidopsis thaliana.

Receptor tyrosine kinase-like orphan receptor 1, a target of NKX2-1/TTF-1 lineage-survival oncogene, inhibits apoptosis signal-regulating kinase 1-mediated pro-apoptotic signaling in lung adenocarcinoma.

We previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a transcriptional target of the NKX2-1/TTF-1 lineage-survival oncogene in lung adenocarcinoma. ROR1 consequently sustains a favorable balance between pro-survival phosphatidylinositol 3-kinase-protein kinase B and pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1)-p38MAPK signaling. In contrast to recent advances in understanding how ROR1 sustains pro-survival signaling, the mechanism of ROR1 repression of pro-apoptotic signaling remains rather elusive. In the present study, we investigated the underlying mechanism of ROR1-mediated inhibition of the ASK1-p38MAPK signaling pathway. Growth inhibition mediated by siROR1 was partially but significantly alleviated by ASK1 co-knockdown in lung adenocarcinoma cell lines. Also, ASK1 phosphorylation at Thr845, which reflects its activated state, was clearly inhibited by ROR1 overexpression in both steady state and oxidative stress-elicited conditions in MSTO-211H cells. In addition, we found that ROR1 was physically associated with ASK1 at the C-terminal serine threonine-rich domain of ROR1. Furthermore, ROR1 kinase activity was shown to be required to repress the ASK1-p38 axis and oxidative stress-induced cell death. The present findings thus support our notion that ROR1 sustains lung adenocarcinoma survival, at least in part, through direct physical interaction with ASK1 and consequential repression of the pro-apoptotic ASK1-p38 axis in a ROR1 kinase activity-dependent manner.

Abstract PR06: Molecular and functional interactions between AKT and SOX2 in breast carcinoma

Abstract The pluripotency-associated transcription factor SOX2 (Sex determining region Y - Box 2) is a key regulator of early organogenesis and embryonic stem cell fate. Supporting the notion that embryonic pathways may reactivate during oncogenesis, SOX2 expression was more recently documented also in several cancers. In breast carcinoma (BC), SOX2 expression is linked to the disease-initiating clonogenic cancer stem cell (CSC) compartment and indicative of disease aggressiveness. However, the molecular regulation of SOX2 in BC remains largely unknown. Here, we describe a functional dependence of SOX2 protein expression on the Ser/Thr-kinase AKT, a central mediator of cell growth, proliferation and metabolism, whose deregulation is a common determinant in BC. Using qRT-PCR and immunoblot analyses, we confirm SOX2 expression in a series of human BC cell lines and primary patient-derived samples, and more importantly, observe co-induction of SOX2 protein expression and AKT kinase activity in cultivation conditions favoring the stem cell compartment. Suggesting functional coupling, lentiviral-induced knockdown of SOX2 and specific AKT kinase inhibition jointly impair BC cell clonogenicity, as deduced from in vitro tumor spheres assays. Verifying SOX2 as a functional downstream target of AKT, ectopic expression of a mCherry-SOX2 fusion protein rescues spheres formation from AKT-inhibited BC cells in vitro and restores tumorigenicity in xenotransplantation assays in vivo. Moreover, AKT inhibitors effectively impair the growth of putative CSC, as identified by an active SOX2 regulatory region (SRR) reporter construct, while conventional chemotherapeutic drugs (e.g. cisplatin or paclitaxel) suppress growth but allow selective survival and thereby enrichment of this cell population. On the molecular level, Western analyses indicate a stringent correlation between SOX2 protein expression and AKT kinase activity in all BC cell lines and patient-derived samples tested. Importantly, the inhibition of either AKT itself (MK-2206, Akti1/2) or of its up-stream regulator PI3K (Wortmannin, GDC-0941) impair SOX2 expression, whereas a modulation of de no protein synthesis by either rapamycin or cycloheximide has only minor impact on SOX2 protein levels on the same time-span. Cell fractionation studies further illustrate a preferential cytoplasmic clearance of SOX2 protein in AKT-inhibited cells, which is largely restored by co-treatment with the proteasomal inhibitor bortezomib. Taken together, these data indicate that AKT regulates SOX2 protein expression via post-translational mechanisms in BC. Supporting this notion, an AKT-phosphorylation motif was identified within the nuclear localization sequence of SOX2 and a direct physical interaction of both proteins verified by co-immunoprecipitation. Confirming a modulation of SOX2 nuclear entry, we observe a temporal cytoplasmic retention of SOX2 in BC cells of stalled AKT-kinase activity. This signal retention is fully restored by co-treatment with leptomycin B, indicating that the observed cytoplasmic accumulation results from nuclear exit of pre-formed SOX2 protein under compromised entry conditions (i.e. persisting AKT kinase inhibition). At extended incubation times, the SOX2 signal gradually disappears in AKT-inhibited cells but can be rescued by concomitant treatment with bortezomib. Importantly, the restored SOX2 protein mislocalizes to the cytosol in anti-AKT and bortezomib co-treated cells, demonstrating that in the absence of AKT activity the rescued SOX2 protein cannot perform nuclear entry. Taken together, our data indicate an unrecognized coupling of AKT and SOX2 proteins that regulates clonogenicity and in vivo tumorigenicity in human BC. Our investigations reveal important mechanistic details about the regulation of SOX2 subcellular distribution and may provide valuable insights on the potential and limitations of PI3K/AKT/mTOR inhibitors currently used in clinical trials of BC. Citation Format: Thorsten Schaefer, Hui Wang, Perihan Mir, Martina Konantz, Tamara C. Pereboom, Britta Merz, Tanja Fehm, Sven Perner, Oliver K. Rothfuss, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. Molecular and functional interactions between AKT and SOX2 in breast carcinoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR06.

Direct Interaction Between N and C Termini of α1C Subunit of CaV1.2 L-Type Calcium Channel

The modulation and regulation of voltage-gated Ca2+ channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca2+ channel CaV1.2, α1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca2+, presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca2+-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of CaV1.2 inactivation induced by Ca2+ entry into the cell.

A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone

BackgroundMultimeric naphthoquinones are redox-active compounds that exhibit antineoplastic, antiprotozoal, and antiviral activities. Due to their multimodal effect on perturbation of cellular oxidative state, these compounds hold great potential as therapeutic agents against highly proliferative neoplastic cells. In our previous work, we developed a series of novel dimeric naphthoquinones and showed that they were selectively cytotoxic to human acute myeloid leukemia (AML), breast and prostate cancer cell lines. We subsequently identified the oxidoreductase NAD(P)H dehydrogenase, quinone 1 (NQO1) as the major target of dimeric naphthoquinones and proposed a mechanism of action that entailed induction of a futile redox cycling.ResultsHere, for the first time, we describe a direct physical interaction between the bromohydroxy dimeric naphthoquinone E6a and NQO1. Moreover, our studies reveal an extensive binding interface between E6a and the isoalloxazine ring of the flavin adenine dinucleotide (FAD) cofactor of NQO1 in addition to interactions with protein side chains in the active site. We also present biochemical evidence that dimeric naphthoquinones affect the redox state of the FAD cofactor of NQO1. Comparison of the mode of binding of E6a with those of other chemotherapeutics reveals unique characteristics of the interaction that can be leveraged in future drug optimization efforts.ConclusionThe first structure of a dimeric naphthoquinone-NQO1 complex was reported, which can be used for design and synthesis of more potent next generation dimeric naphthoquinones to target NQO1 with higher affinity and specificity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-016-0052-x) contains supplementary material, which is available to authorized users.

CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors

Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors.

Down-regulation of the cyclin-dependent kinase inhibitor p57 is mediated by Jab1/Csn5 in hepatocarcinogenesis.

Down-regulation of p57 (KIP2) cyclin-dependent kinase inhibitors accelerates the growth and invasion of hepatocellular carcinoma (HCC), suggesting that p57 may play an important role in liver carcinogenesis. However, the mechanism or oncogenic signal leading to p57 down-regulation in HCC remains to be determined. Herein, we demonstrated that Jab1/Csn5 expression is negatively correlated with p57 levels in HCC tissues. Kaplan-Meier analysis of tumor samples revealed that high Jab1/Csn5 expression with concurrent low p57 expression is associated with poor overall survival. The inverse pattern of Jab1 and p57 expression was also observed during carcinogenesis in a chemically induced rat HCC model. We also found that mechanistically, Jab1-mediated p57 proteolysis in HCC cells is dependent on 26S-proteasome inhibitors. We further demonstrated that direct physical interaction between Jab1 and p57 triggers p57 down-regulation, independently of Skp2 and Akt pathways, in HCC cells. These data suggest that Jab1 is an important upstream negative regulator of p57 and that aberrant expression of Jab1 in HCC could lead to a significant decrease in p57 levels and contribute to tumor cell growth. Furthermore, restoration of p57 levels induced by loss of Jab1 inhibited tumor cell growth and further increased cell apoptosis in HCC cells. Moreover, silencing Jab1 expression further enhanced the antitumor effects of cisplatin-induced apoptosis in HCC cells. Jab1-p57 pathway confers resistance to chemotherapy and may represent a potential target for investigational therapy in HCC.

The front and rear of collective cell migration.

Collective cell migration has a key role during morphogenesis and during wound healing and tissue renewal in the adult, and it is involved in cancer spreading. In addition to displaying a coordinated migratory behaviour, collectively migrating cells move more efficiently than if they migrated separately, which indicates that a cellular interplay occurs during collective cell migration. In recent years, evidence has accumulated confirming the importance of such intercellular communication and exploring the molecular mechanisms involved. These mechanisms are based both on direct physical interactions, which coordinate the cellular responses, and on the collective cell behaviour that generates an optimal environment for efficient directed migration. The recent studies have described how leader cells at the front of cell groups drive migration and have highlighted the importance of follower cells and cell-cell communication, both between followers and between follower and leader cells, to improve the efficiency of collective movement.

The role of OsMSH4 in male and female gamete development in rice meiosis.

Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4-green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex.

G9a orchestrates PCL3 and KDM7A to promote histone H3K27 methylation.

Methylation of histone H3-lysine 9 (H3K9) and H3K27 by the methyltransferase G9a and polycomb repressive complex 2 (PRC2) inhibits transcription of target genes. A crosstalk between G9a and PRC2 via direct physical interaction has been shown recently. Here, we demonstrate an alternative mechanism by which G9a promotes H3K27 methylation. Overexpression of G9a increases both H3K9 and H3K27 methylation, reduces E-cadherin expression, and induces epithelial-mesenchymal transition in PANC-1 pancreatic cancer cells. Conversely, the depletion of G9a or ectopic expression of methyltransferase-dead G9a in G9a-overexpressing gemcitabine-resistant PANC-1-R cells exhibits opposite effects. G9a promotes H3K27 methylation of the E-cadherin promoter by upregulating PCL3 to increase PRC2 promoter recruitment and by downregulating the H3K27 demethylase KDM7A to silence E-cadherin gene. The depletion of PCL3 or overexpression of KDM7A elevated expression of E-cadherin in PANC-1-R cells while ectopic expression of PCL3 or knockdown of KDM7A downregulated E-cadherin in PANC-1 cells. Collectively, we provide evidence that G9a orchestrates the dynamic balance within histone-modifying enzymes to regulate H3K27 methylation and gene expression.

Comparison of Interaction Modalities for Mobile Indoor Robot Guidance: Direct Physical Interaction, Person Following, and Pointing Control

Three advanced natural interaction modalities for mobile robot guidance in an indoor environment were developed and compared using two tasks and quantitative metrics to measure performance and workload. The first interaction modality is based on direct physical interaction requiring the human user to push the robot in order to displace it. The second and third interaction modalities exploit a 3-D vision-based human-skeleton tracking allowing the user to guide the robot by either walking in front of it or by pointing toward a desired location. In the first task, the participants were asked to guide the robot between different rooms in a simulated physical apartment requiring rough movement of the robot through designated areas. The second task evaluated robot guidance in the same environment through a set of waypoints, which required accurate movements. The three interaction modalities were implemented on a generic differential drive mobile platform equipped with a pan-tilt system and a Kinect camera. Task completion time and accuracy were used as metrics to assess the users’ performance, while the NASA-TLX questionnaire was used to evaluate the users’ workload. A study with 24 participants indicated that choice of interaction modality had significant effect on completion time ( $F(2,61) = 84.874$ , $p ), accuracy ( $F(2,29) = 4.937$ , $p = 0.016$ ), and workload ( $F(2,68) = 11.948$ , $p ). The direct physical interaction required less time, provided more accuracy and less workload than the two contactless interaction modalities. Between the two contactless interaction modalities, the person-following interaction modality was systematically better than the pointing-control one: The participants completed the tasks faster with less workload.