Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells. Consistent with this, lem3Δ sfk1Δ double mutant cells exposed more PS/PE than the lem3Δ mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol-4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3Δ was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3Δ sfk1Δ double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3Δ sfk1Δ mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.

The vertebrate hedgehog pathway is organized in primary cilia, and hedgehog components relocate into or out of cilia during signaling. Defects in intraflagellar transport (IFT) typically disrupt ciliary assembly and attenuate hedgehog signaling. Determining whether IFT drives the movement of hedgehog components is difficult due to the requirement of IFT for building cilia. Unlike most IFT proteins, IFT27 is dispensable for cilia formation but affects hedgehog signaling similarly to other IFTs, allowing us to examine its role in the dynamics of signaling. Activating signaling at points along the pathway in Ift27 mutant cells showed that IFT is extensively involved in the pathway. Similar analysis of Bbs mutant cells showed that BBS proteins participate at many levels of signaling but are not needed to concentrate Gli transcription factors at the ciliary tip. Our analysis showed that smoothened delivery to cilia does not require IFT27, but the role of other IFTs is not known. Using a rapamycin-induced dimerization system to sequester IFT-B proteins at the mitochondria in cells with fully formed cilia did not affect the delivery of Smo to cilia, suggesting that this membrane protein may not require IFT-B for delivery.

Synthesizing a genetic network which generates stable Turing patterns is one of the great challenges of synthetic biology, but a significant obstacle is the disconnect between the mathematical theory and the biological reality. Current mathematical understanding of patterning is typically restricted to systems of two or three chemical species, for which equations are tractable. However, when models seek to combine descriptions of intercellular signal diffusion and intracellular biochemistry, plausible genetic networks can consist of dozens of interacting species. In this paper, we suggest a method for reducing large biochemical systems that relies on removing the non-diffusible species, leaving only the diffusibles in the model. Such model reduction enables analysis to be conducted on a smaller number of differential equations. We provide conditions to guarantee that the full system forms patterns if the reduced system does, and vice versa. We confirm our technique with three examples: the Brusselator, an example proposed by Turing, and a biochemically plausible patterning system consisting of 17 species. These examples show that our method significantly simplifies the study of pattern formation in large systems where several species can be considered immobile.

Neurophysiological data obtained in primates suggests that merely observing others' actions can modulate activity in the observer's motor cortices. In humans, it has been suggested that these multimodal vicarious responses extend well beyond the motor cortices, including somatosensory and insular brain regions, which seem to yield vicarious responses when witnessing others' actions, sensations, or emotions (Gazzola and Keysers, 2009). Despite the wealth of data with respect to shared action responses in the monkey motor system, whether the somatosensory and insular cortices also yield vicarious responses during observation of touch remains largely unknown. Using independent tactile and motor fMRI localizers, we first mapped the hand representations of two male monkeys' primary (SI) and secondary (SII) somatosensory cortices. In two subsequent visual experiments, we examined fMRI brain responses to (1) observing a conspecific's hand being touched or (2) observing a human hand grasping or mere touching an object or another human hand. Whereas functionally defined "tactile SI" and "tactile SII" showed little involvement in representing observed touch, vicarious responses for touch were found in parietal area PFG, consistent with recent observations in humans (Chan and Baker, 2015). Interestingly, a more anterior portion of SII, and posterior insular cortex, both of which responded when monkeys performed active grasping movements, also yielded visual responses during different instances of touch observation.SIGNIFICANCE STATEMENT Common coding of one's own and others' actions, sensations, and emotions seems to be widespread in the brain. Although it is currently unclear to what extent human somatosensory cortices yield vicarious responses when observing touch, even less is known about the presence of similar vicarious responses in monkey somatosensory cortex. We therefore localized monkey somatosensory hand representations using fMRI and investigated whether these regions yield vicarious responses while observing various instances of touch. Whereas "tactile SI and SII" did not elicit responses during touch observation, a more anterior portion of SII, in addition to area PFG and posterior insular cortex, all of which responded during monkeys' own grasping movements, yielded vicarious responses during observed touch.

Plant nitrogen (N) use is a key component of the N cycle in terrestrial ecosystems. The supply of N to plants affects community species composition and ecosystem processes such as photosynthesis and carbon (C) accumulation. However, the availabilities and relative importance of different N forms to plants are not well understood. While nitrate (NO3-) is a major N form used by plants worldwide, it is discounted as a N source for Arctic tundra plants because of extremely low NO3- concentrations in Arctic tundra soils, undetectable soil nitrification, and plant-tissue NO3- that is typically below detection limits. Here we reexamine NO3- use by tundra plants using a sensitive denitrifier method to analyze plant-tissue NO3- Soil-derived NO3- was detected in tundra plant tissues, and tundra plants took up soil NO3- at comparable rates to plants from relatively NO3--rich ecosystems in other biomes. Nitrate assimilation determined by 15N enrichments of leaf NO3- relative to soil NO3- accounted for 4 to 52% (as estimated by a Bayesian isotope-mixing model) of species-specific total leaf N of Alaskan tundra plants. Our finding that in situ soil NO3- availability for tundra plants is high has important implications for Arctic ecosystems, not only in determining species compositions, but also in determining the loss of N from soils via leaching and denitrification. Plant N uptake and soil N losses can strongly influence C uptake and accumulation in tundra soils. Accordingly, this evidence of NO3- availability in tundra soils is crucial for predicting C storage in tundra.

Current evidence supports a protective role for virus-neutralizing antibodies in immunity against hepatitis C virus (HCV) infection. Many cross-neutralizing monoclonal antibodies have been identified. These antibodies have been shown to provide protection or to clear infection in animal models. Previous clinical trials have shown that a gpE1/gpE2 vaccine can induce antibodies that neutralize the in vitro infectivity of all the major cell culture-derived HCV (HCVcc) genotypes around the world. However, cross-neutralization appeared to favor certain genotypes, with significant but lower neutralization against others. HCV may employ epitope masking to avoid antibody-mediated neutralization. Hypervariable region 1 (HVR1) at the amino terminus of glycoprotein E2 has been shown to restrict access to many neutralizing antibodies. Consistent with this, other groups have reported that recombinant viruses lacking HVR1 are hypersensitive to neutralization. It has been proposed that gpE1/gpE2 lacking this domain could be a better vaccine antigen to induce broadly neutralizing antibodies. In this study, we examined the immunogenicity of recombinant gpE1/gpE2 lacking HVR1 (ΔHVR1). Our results indicate that wild-type (WT) and ΔHVR1 gpE1/gpE2 antigens induced antibodies targeting many well-characterized cross-genotype-neutralizing epitopes. However, while the WT gpE1/gpE2 vaccine can induce cross-genotype protection against various genotypes of HCVcc and/or HCV-pseudotyped virus (HCVpp), antisera from ΔHVR1 gpE1/gpE2-immunized animals exhibited either reduced homologous neutralization activity compared to that of the WT or heterologous neutralization activity similar to that of the WT. These data suggest that ΔHVR1 gpE1/gpE2 is not a superior vaccine antigen. Based on previously reported chimpanzee protection data using WT gpE1/gpE2 and our current findings, we are preparing a combination vaccine including wild-type recombinant gpE1/gpE2 for clinical testing in the future.IMPORTANCE An HCV vaccine is an unmet medical need. Current evidence suggests that neutralizing antibodies play an important role in virus clearance, along with cellular immune responses. Previous clinical data showed that gpE1/gpE2 can effectively induce cross-neutralizing antibodies, although they favor certain genotypes. HCV employs HVR1 within gpE2 to evade host immune control. It has been hypothesized that the removal of this domain would improve the production of cross-neutralizing antibodies. In this study, we compared the immunogenicities of WT and ΔHVR1 gpE1/gpE2 antigens as vaccine candidates. Our results indicate that the ΔHVR1 gpE1/gpE2 antigen confers no advantages in the neutralization of HCV compared with the WT antigen. Previously, we showed that this WT antigen remains the only vaccine candidate to protect chimpanzees from chronic infection, contains multiple cross-neutralizing epitopes, and is well tolerated and immunogenic in humans. The current data support the further clinical development of this vaccine antigen component.

Object We identified the β-adrenoceptor (β-AR) subtypes responsible for the relaxant responses to adrenaline (AD) and noradrenaline (NA) in the rat thoracic aorta and examined the role of cAMP which is involved in these relaxant responses. Methods The effects of β-AR antagonists or the adenylyl cyclase inhibitor SQ 22,536 on AD- and NA-induced relaxant responses in phenylephrine-induced contraction and increases in cAMP levels were examined in isolated, endothelium-denuded rat thoracic aorta segments. Results AD-induced relaxation was completely suppressed by propranolol (10-7 M) or by ICI-118,551 (10-8 M) plus atenolol (10-6 M), and was also very strongly inhibited by ICI-118,551 (10-8 M) alone. AD (10-5 M) increased tissue cAMP levels by approximately 1.9-fold compared with that in non-stimulated aortic tissue, but did not significantly increase cAMP levels in the presence of ICI-118,551 (10-8 M) or SQ 22,536 (10-4 M). AD-induced relaxation was strongly suppressed by SQ 22,536 (10-4 M). NA-induced relaxation was almost completely suppressed by atenolol (10-6 M) plus ICI-118,551 (10-8 M) although it was hardly affected by ICI-118,551 (10-8 M) alone. NA (10-5 M) increased tissue cAMP levels by approximately 2.2-fold compared with that in non-stimulated aortic tissue, but did not significantly increase cAMP levels in the presence of atenolol (10-6 M) or SQ 22,536 (10-4 M). NA-induced relaxation was strongly suppressed by SQ 22,536 (10-4 M). Conclusion In rat thoracic aorta, AD- and NA-induced relaxations, which are both strongly dependent on increased tissue cAMP levels, are mainly mediated through β2- and β1-adrenoceptors respectively.

Endohyphal bacteria (EHB), dwelling within fungal hyphae, markedly affect the growth and metabolic potential of their hosts. To date, two EHB belonging to the family Burkholderiaceae have been isolated and characterized as new taxa, Burkholderia rhizoxinica (HKI 454T) and Mycoavidus cysteinexigens (B1-EBT), in Japan. Metagenome sequencing was recently reported for Mortierella elongata AG77 together with its endosymbiont M. cysteinexigens (Mc-AG77) from a soil/litter sample in the USA. In the present study, we elucidated the complete genome sequence of B1-EBT and compared it with those of Mc-AG77 and HKI 454T. The genomes of B1-EBT and Mc-AG77 contained a higher level of prophage sequences and were markedly smaller than that of HKI 454T. Although the B1-EBT and Mc-AG77 genomes lacked the chitinolytic enzyme genes responsible for invasion into fungal cells, they contained several predicted toxin-antitoxin systems including an insecticidal toxin complex and PIN domain imposing an addiction-like mechanism essential for endohyphal growth control during host colonization. Despite the different host fungi, the alignment of amino acid sequences showed that the HKI 454T genome consisted of 1,265 (32.6%) and 1,221 (31.5%) orthologous coding sequences (CDSs) with those of B1-EBT and Mc-AG77, respectively. This comparative study of three phylogenetically associated endosymbionts has provided insights into their origin and evolution, and suggests the later bacterial invasion and adaptation of B1-EBT to its host metabolism.

Olanzapine is an effective antipsychotic drug but since it causes significant weight gain, it is not well tolerated by psychosis patients. The prebiotic, B-GOS®, attenuates metabolic dysfunction in obese subjects, and in rodents, alters central NMDA receptors and may affect serotonin receptors that are relevant in psychosis. We have determined whether B-GOS® influenced olanzapine-associated weight gain and central NMDA and serotonin receptors. Circulating acetate, IL-1β, IL-8 and TNFα, liver acetyl-CoA carboxylase (ACC), white adipose tissue (WAT) acetate receptor GPR43, and specific faecal bacteria genera were also measured to provide mechanistic information. Adult female Sprague-Dawley rats were administered a B-GOS® (0.5 g/kg/day) solution or water for 21 days, and received a single, daily, intraperitoneal injection of olanzapine or saline on days 8-21. The intake of B-GOS® significantly attenuated olanzapine-induced weight gain without altering frontal cortex 5-HT2AR blockade. Cortical GluN1 levels were elevated by olanzapine in the presence of B-GOS®. Plasma acetate concentrations increased following B-GOS® or olanzapine administration alone, but reduced when prebiotic and drug were administered in combination. This pattern was paralleled by hepatic ACC mRNA expression. The abundance of WAT GPR43 mRNA was reduced by olanzapine, only in the absence of B-GOS®. Co-administration of B-GOS® and olanzapine also elevated plasma TNFα, which is reported to influence lipid metabolism. Finally, B-GOS® elevated faecal Bifidobacterium spp. and reduced some bacteria in the Firmicutes phylum, whilst olanzapine treatment either alone or with B-GOS®, was without effect. These data suggest that inclusion of B-GOS® as an adjunct to olanzapine treatment in schizophrenia may prevent weight gain and have benefits on cognitive function in psychosis. The role of acetate in these effects requires further investigation.

During mammalian heart development, restricted myocardial Bmp2 expression is a key patterning signal for atrioventricular canal specification and the epithelial-mesenchyme transition that gives rise to the valves. Using a mouse transgenic line conditionally expressing Bmp2, we show that widespread Bmp2 expression in the myocardium leads to valve and chamber dysmorphogenesis and embryonic death by E15.5. Transgenic embryos show thickened valves, ventricular septal defect, enlarged trabeculae and dilated ventricles, with an endocardium able to undergo EMT both in vivo and in vitro. Gene profiling and marker analysis indicate that cellular proliferation is increased in transgenic embryos, whereas chamber maturation and patterning are impaired. Similarly, forced Bmp2 expression stimulates proliferation and blocks cardiomyocyte differentiation of embryoid bodies. These data show that widespread myocardial Bmp2 expression directs ectopic valve primordium formation and maintains ventricular myocardium and cardiac progenitors in a primitive, proliferative state, identifying the potential of Bmp2 in the expansion of immature cardiomyocytes.

The role of arsenic trioxide (As2O3) in inhibiting immune rejection and prolonging islet allograft survival has been identified in islet allotransplantation. This study aims to explore the role of As2O3 in islet xenotransplantation and the action mechanism. The streptozotocin (STZ) was used in C57BL/6 mice to induce the type 1 diabetes mellitus (T1DM) for xenotransplantation models establishment. Donor islets were isolated by digesting. The flow cytometry (FCM) was used to analyze lymphocyte types. The blood sugar level was detected by using intraperitoneal glucose tolerance test (IPGTT). The serum level of cytokines was determined by the enzyme-linked immunosorbent assay (ELIZA). The cell proliferation was measured by MTT assay. The mRNA levels were quantified with qRT-PCR. As2O3 prolonged the survival of the recipient mice but had no influence on body weight. As2O3 protected the function of xenograft in insulin secretion and suppressed immune rejection of recipient. As2O3 inhibited proliferation of T lymphocyte and increased the proportion of Foxp3+ regulatory T cells in recipient mice. As2O3 inhibited activation and promoted clonal anergy of T lymphocyte. As2O3 decreased total number of B cells and reduced partial antibody levels in recipient mice. As2O3 and leflunomide showed a synergistic effect in suppressing islet xenotransplant rejection. As2O3 prolongs islet xenograft survival by inhibiting cellular immune response, and increasing Foxp3+ regulatory T cells, while decreasing partial antibody levels in serum.

The human genome is generally organized into stable chromosomes, and only tumor cells are known to accumulate kilobase (kb)-sized extrachromosomal circular DNA elements (eccDNAs). However, it must be expected that kb eccDNAs exist in normal cells as a result of mutations. Here, we purify and sequence eccDNAs from muscle and blood samples from 16 healthy men, detecting ~100,000 unique eccDNA types from 16 million nuclei. Half of these structures carry genes or gene fragments and the majority are smaller than 25 kb. Transcription from eccDNAs suggests that eccDNAs reside in nuclei and recurrence of certain eccDNAs in several individuals implies DNA circularization hotspots. Gene-rich chromosomes contribute to more eccDNAs per megabase and the most transcribed protein-coding gene in muscle, TTN (titin), provides the most eccDNAs per gene. Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of full-length or truncated genes.

Mammalian circadian clocks have a hierarchical organization, governed by the suprachiasmatic nucleus (SCN) in the hypothalamus. The brain itself contains multiple loci that maintain autonomous circadian rhythmicity, but the contribution of the non-SCN clocks to this hierarchy remains unclear. We examine circadian oscillations of clock gene expression in various brain loci and discovered that in mouse, robust, higher amplitude, relatively faster oscillations occur in the choroid plexus (CP) compared to the SCN. Our computational analysis and modeling show that the CP achieves these properties by synchronization of "twist" circadian oscillators via gap-junctional connections. Using an in vitro tissue coculture model and in vivo targeted deletion of the Bmal1 gene to silence the CP circadian clock, we demonstrate that the CP clock adjusts the SCN clock likely via circulation of cerebrospinal fluid, thus finely tuning behavioral circadian rhythms.

Oesophageal cancers (adenocarcinomas [AC] and squamous cell carcinomas [SCC]) are characterized by high incidence/mortality in many countries. We aimed to delineate its global incidence and mortality, and studied whether socioeconomic development and its incidence rate were correlated. The age-standardized rates (ASRs) of incidence and mortality of this medical condition in 2012 for 184 nations from the GLOBOCAN database; national databases capturing incidence rates, and the WHO mortality database were examined. Their correlations with two indicators of socioeconomic development were evaluated. Joinpoint regression analysis was used to generate trends. The ratio between the ASR of AC and SCC was strongly correlated with HDI (r = 0.535 [men]; r = 0.661 [women]) and GDP (r = 0.594 [men]; r = 0.550 [women], both p < 0.001). Countries that reported the largest reduction in incidence in male included Poland (Average Annual Percent Change [AAPC] = -7.1, 95%C.I. = -12,-1.9) and Singapore (AAPC = -5.8, 95%C.I. = -9.5,-1.9), whereas for women the greatest decline was seen in Singapore (AAPC = -12.3, 95%C.I. = -17.3,-6.9) and China (AAPC = -5.6, 95%C.I. = -7.6,-3.4). The Philippines (AAPC = 4.3, 95%C.I. = 2,6.6) and Bulgaria (AAPC = 2.8, 95%C.I. = 0.5,5.1) had a significant mortality increase in men; whilst Columbia (AAPC = -6.1, 95%C.I. = -7.5,-4.6) and Slovenia (AAPC = -4.6, 95%C.I. = -7.9,-1.3) reported mortality decline in women. These findings inform individuals at increased risk for primary prevention.

High-mobility group box 1 (HMGB1) facilitates neural stem cells (NSCs) proliferation and differentiation into neuronal linage. However, the effect of HMGB1 on NSCs migration is still elusive. The present study is to investigate the corelation between HMGB1 and NSCs migration and the potential mechanism. The results indicated that 1 ng/ml HMGB1 promoted NSCs proliferation using CCK8 assays. Moreover, data showed that 1 ng/ml HMGB1 facilitated NSCs migration via filopodia formation using phase-contrast and transwell assays. Furthermore, 1 ng/ml HMGB1 upregulated the expression of RAGE, one of the HMGB1 receptor, using western blotting assays and immunofluorescence staining. In addition, 1 ng/ml HMGB1 increased the percentage of filopodia formation using phalloidin staining. Meanwhile, the enhanced migration effect could be abrogated by 50 nM FPS-ZM1, one of the RAGE antagonist, and RAGE-specific siRNA through immunofluorescence and phalloidin staining. Together, our data demonstrate that HMGB1/RAGE axis facilitates NSCs migration via promoting filopodia formation, which might serve as a candidate for central nervous system (CNS) injury treatment and/or a preconditioning method for NSCs implantation.

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 Å X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells.

Chronic exposure to n-hexane, a widely used organic solvent in industry, induces central-peripheral neuropathy, which is mediated by its active metabolite, 2,5-hexanedione (HD). We recently reported that transplantation of bone marrow-mesenchymal stem cells (BMSC) significantly ameliorated HD-induced neuronal damage and motor deficits in rats. However, the mechanisms remain unclear. Here, we reported that inhibition of HD-induced autophagy contributed to BMSC-afforded protection. BMSC transplantation significantly reduced the levels of microtubule-associated protein 1 light chain 3-II (LC3-II) and the degradation of sequestosome-1 (p62) in the spinal cord and sciatic nerve of HD-intoxicated rats. Downregulation of autophagy by BMSC was also confirmed in VSC4.1 cells exposed to HD. Moreover, inhibition of autophagy by PIK III mitigated the neurotoxic effects of HD and, meanwhile, abolished BMSC-afforded neuroprotection. Furthermore, we found that BMSC failed to interfere with Beclin 1, but promoted activation of mammalian target of rapamycin (mTOR). Unc-like kinse 1 (ULK1) was further recognized as the downstream target of mTOR responsible for BMSC-mediated inhibition of autophagy. Altogether, BMSC transplantation potently ameliorated HD-induced autophagy through beclin 1-independent activation of mTOR pathway, providing a novel insight for the therapeutic effects of BMSC against n-hexane and other environmental toxicants-induced neurotoxicity.

Transection of the median nerve typically causes lifelong restriction of fine sensory and motor skills of the affected hand despite the best available surgical treatment. Inspired by recent findings on activity-dependent structural plasticity of the adult brain, we used voxel-based morphometry to analyze the brains of 16 right-handed adults who more than two years earlier had suffered injury to the left or right median nerve followed by microsurgical repair. Healthy individuals served as matched controls. Irrespective of side of injury, we observed gray matter reductions in left ventral and right dorsal premotor cortex, and white matter reductions in commissural pathways interconnecting those motor areas. Only left-side injured participants showed gray matter reduction in the hand area of the contralesional primary motor cortex. We interpret these effects as structural manifestations of reduced neural processing linked to restrictions in the diversity of the natural manual dexterity repertoire. Furthermore, irrespective of side of injury, we observed gray matter increases bilaterally in a motion-processing visual area. We interpret this finding as a consequence of increased neural processing linked to greater dependence on vision for control of manual dexterity after median nerve injury because of a compromised somatosensory innervation of the affected hand.

The role of genetic components in cancer development is an area of interest for cancer biologists in general. Intriguingly, some genes have both oncogenic and tumor-suppressor functions. In this study, we systematically identified these genes through database search and text mining. We find that most of them are transcription factors or kinases and exhibit dual biological functions, e.g., that they both positively and negatively regulate transcription in cells. Some cancer types such as leukemia are over-represented by them, whereas some common cancer types such as lung cancer are under-represented by them. Across 12 major cancer types, while their genomic mutation patterns are similar to that of oncogenes, their expression patterns are more similar to that of tumor-suppressor genes. Their expression profile in six human organs propose that they mainly function as tumor suppressor in normal tissue. Our network analyses further show they have higher network degrees than both oncogenes and tumor-suppressor genes and thus tend to be the hub genes in the protein-protein interaction network. Our mutation, expression spectrum, and network analyses might help explain why some cancer types are specifically associated with them. Finally, our results suggest that the functionally altering mutations in "double-agent" genes and oncogenes are the main driving force in cancer development, because non-silent mutations are biasedly distributed toward these two gene sets across all 12 major cancer types.

Ezrin is highly expressed in glomerular podocytes and is reported to form a multi-protein complex with scaffold protein Na+/H+ exchanger regulatory factor 2 (NHERF2) and podocalyxin, a major sialoprotein. Podocalyxin-knockout mice died within 24 h of birth with anuric renal failure, whereas NHERF2-knockout mice show no apparent changes in the glomerular functions. However, the physiological roles of ezrin in glomerular podocytes remain unclear. Here, we investigated the importance of ezrin in the regulation of glomerular podocyte function using ezrin-knockdown mice (Vil2 kd/kd ). The Vil2 kd/kd mice did not exhibit apparent glomerular dysfunction, morphological defects or abnormal localisation of podocalyxin and NHERF2 in podocytes. Thus, we investigated the influence of ezrin defects on Rho-GTPase activity, as ezrin interacts with the Rho-GTPase dissociation inhibitor (Rho-GDI), which plays a key role in the regulation of podocyte actin organisation. In Vil2 kd/kd glomeruli, Rac1 activity was significantly reduced compared to wildtype (WT) glomeruli at baseline. Furthermore, Vil2 kd/kd mice showed reduced susceptibility to glomerular injury. In WT glomeruli, Rac1 activity was enhanced in nephrotic conditions, but remained at baseline levels in Vil2 kd/kd glomeruli, suggesting that loss of ezrin protects podocytes from injury-induced morphological changes by suppressing Rac1 activation.