With the advance of the combinatorial chemistry, a large number of synthetic compounds have surged. However, we have limited knowledge about them. On the other hand, the speed of designing new drugs is very slow. One of the key causes is the unacceptable toxicities of chemicals. If one can correctly identify the toxicity of chemicals, the unsuitable chemicals can be discarded in early stage, thereby accelerating the study of new drugs and reducing the R&D costs. In this study, a new prediction method was built for identification of chemical toxicities, which was based on ontology information of chemicals. By comparing to a previous method, our method is quite effective. We hope that the proposed method may give new insights to study chemical toxicity and other attributes of chemicals.

The non-crystallographic phase problem arises in numerous scientific and technological fields. An important application is coherent diffractive imaging. Recent advances in X-ray free-electron lasers allow capturing of the diffraction pattern from a single nanoparticle before it disintegrates, in so-called 'diffraction before destruction' experiments. Presently, the phase is reconstructed by iterative algorithms, imposing a non-convex computational challenge, or by Fourier holography, requiring a well-characterized reference field. Here we present a convex scheme for single-shot phase retrieval for two (or more) sufficiently separated objects, demonstrated in two dimensions. In our approach, the objects serve as unknown references to one another, reducing the phase problem to a solvable set of linear equations. We establish our method numerically and experimentally in the optical domain and demonstrate a proof-of-principle single-shot coherent diffractive imaging using X-ray free-electron lasers pulses. Our scheme alleviates several limitations of current methods, offering a new pathway towards direct reconstruction of complex objects.

Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

We applied and optimized the sparse representation (SR) approaches in the computer-aided diagnosis (CAD) to classify normal tissues and five kinds of diffuse lung disease (DLD) patterns: consolidation, ground-glass opacity, honeycombing, emphysema, and nodule. By using the K-SVD which is based on the singular value decomposition (SVD) and orthogonal matching pursuit (OMP), it can achieve a satisfied recognition rate, but too much time was spent in the experiment. To reduce the runtime of the method, the K-Means algorithm was substituted for the K-SVD, and the OMP was simplified by searching the desired atoms at one time (OMP1). We proposed three SR based methods for evaluation: SR1 (K-SVD+OMP), SR2 (K-Means+OMP), and SR3 (K-Means+OMP1). 1161 volumes of interest (VOIs) were used to optimize the parameters and train each method, and 1049 VOIs were adopted to evaluate the performances of the methods. The SR based methods were powerful to recognize the DLD patterns (SR1: 96.1%, SR2: 95.6%, SR3: 96.4%) and significantly better than the baseline methods. Furthermore, when the K-Means and OMP1 were applied, the runtime of the SR based methods can be reduced by 98.2% and 55.2%, respectively. Therefore, we thought that the method using the K-Means and OMP1 (SR3) was efficient for the CAD of the DLDs.

Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. Importance: Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.

Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was recently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability of Δact1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducible Δact1 parasites as long as 5 days after ACT1 deletion. We further found that the longer Δact1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained in Δact1 parasites being responsible for their invasive ability. Furthermore, invasion by the Δact1 parasites was also sensitive to the actin polymerization inhibitor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation by Δact1 parasites. However, Δact1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducible Δact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion.

This paper demonstrates the application of aerial multispectral images in cotton plant phenotyping. Four phenotypic traits (plant height, canopy cover, vegetation index, and flower) were measured from multispectral images captured by a multispectral camera on an unmanned aerial system. Data were collected on eight different days from two fields. Ortho-mosaic and digital elevation models (DEM) were constructed from the raw images using the structure from motion (SfM) algorithm. A data processing pipeline was developed to calculate plant height using the ortho-mosaic and DEM. Six ground calibration targets (GCTs) were used to correct the error of the calculated plant height caused by the georeferencing error of the DEM. Plant heights were measured manually to validate the heights predicted from the imaging method. The error in estimation of the maximum height of each plot ranged from -40.4 to 13.5 cm among six datasets, all of which showed strong linear relationships with the manual measurement (R2 > 0.89). Plot canopy was separated from the soil based on the DEM and normalized differential vegetation index (NDVI). Canopy cover and mean canopy NDVI were calculated to show canopy growth over time and the correlation between the two indices was investigated. The spectral responses of the ground, leaves, cotton flower, and ground shade were analyzed and detection of cotton flowers was satisfactory using a support vector machine (SVM). This study demonstrated the potential of using aerial multispectral images for high throughput phenotyping of important cotton phenotypic traits in the field.

Bupivacaine, a typical local anesthetic, induces neurotoxicity via reactive oxygen species regulation of apoptosis. High glucose could enhance bupivacaine-induced neurotoxicity through regulating oxidative stress, but the mechanism of it is not clear. Mitochondrial calcium uniporter (MCU), a key channel for regulating the mitochondrial Ca2+ (mCa2+) influx, is closely related to oxidative stress via disruption of mCa2+ homeostasis. Whether MCU is involved in high glucose-sensitized bupivacaine-induced neurotoxicity remains unknown. In this study, human neuroblastoma (SH-SY5Y) cells were cultured with high glucose and/or bupivacaine, and the data showed that high glucose enhanced bupivacaine-induced MCU expression elevation, mCa2+ accumulation, and oxidative damage. Next, Ru360, an inhibitor of MCU, was employed to pretreated SH-SY5Y cells, and the results showed that it could decrease high glucose and bupivacaine-induced mCa2+ accumulation, oxidative stress, and apoptosis. Further, with the knockdown of MCU with a specific small interfering RNA (siRNA) in SH-SY5Y cells, we found that it also could inhibit high glucose and bupivacaine-induced mCa2+ accumulation, oxidative stress, and apoptosis. We propose that downregulation expression or activity inhibition of the MCU channel might be useful for restoring the mitochondrial function and combating high glucose and bupivacaine-induced neurotoxicity. In conclusion, our study demonstrated the crucial role of MCU in high glucose-mediated enhancement of bupivacaine-induced neurotoxicity, suggesting the possible use of this channel as a target for curing bupivacaine-induced neurotoxicity in diabetic patients.

Medial knee joint osteoarthritis (OA) is a debilitating and prevalent condition. Surgical treatment consists of redistributing the forces from the medial to the lateral compartment through osteotomy, or replacing the joint surfaces. As the mediolateral load distribution is related to the action of the musculature around the knee, the aim of this study was to devise a technique to redistribute these forces non-surgically through changes in muscle excitation. Eight healthy subjects participated in the experiment, and neuromuscular electrical stimulation was used to change the muscle forces around the knee. A musculoskeletal model was used to quantify the loading on the medial compartment of the knee, and a novel algorithm devised and implemented to simulate neuromuscular electrical stimulation. The forces and moments at the knee, ground reaction forces, walking velocity and step length were quantified before and after stimulation. Stimulation of the biceps femoris resulted in a significant decrease in the second peak of the medial knee joint loading by up to 0.17 body weight (p = 0.016). Kinematic parameters were not significantly affected. Neuromuscular electrical stimulation can decrease the peak loads on the medial compartment of the knee, and thus offers a promising therapy for medial knee joint OA.

Phagocytic cells are the first line of innate defense against intracellular pathogens, and yet Toxoplasma gondii is renowned for its ability to survive in macrophages, although this paradigm is based on virulent type I parasites. Surprisingly, we find that avirulent type III parasites are preferentially cleared in naive macrophages, independent of gamma interferon (IFN-γ) activation. The ability of naive macrophages to clear type III parasites was dependent on enhanced activity of NADPH oxidase (Nox)-generated reactive oxygen species (ROS) and induction of guanylate binding protein 5 (Gbp5). Macrophages infected with type III parasites (CTG strain) showed a time-dependent increase in intracellular ROS generation that was higher than that induced by type I parasites (GT1 strain). The absence of Nox1 or Nox2, gp91 subunit isoforms of the Nox complex, reversed ROS-mediated clearance of CTG parasites. Consistent with this finding, both Nox1-/- and Nox2-/- mice showed higher susceptibility to CTG infection than wild-type mice. Additionally, Gbp5 expression was induced upon infection and the enhanced clearance of CTG strain parasites was reversed in Gbp5-/- macrophages. Expression of a type I ROP18 allele in CTG prevented clearance in naive macrophages, suggesting that it plays a role counteracting Gbp5. Although ROS and Gbp5 have been linked to activation of the NLRP3 inflammasome, clearance of CTG parasites did not rely on induction of pyroptosis. Collectively, these findings reveal that not all strains of T. gondii are adept at avoiding clearance in macrophages and define new roles for ROS and Gbps in controlling this important intracellular pathogen.IMPORTANCEToxoplasma infections in humans and other mammals are largely controlled by IFN-γ produced by the activated adaptive immune system. However, we still do not completely understand the role of cell-intrinsic functions in controlling Toxoplasma or other apicomplexan infections. The present work identifies intrinsic activities in naive macrophages in counteracting T. gondii infection. Using an avirulent strain of T. gondii, we highlight the importance of Nox complexes in conferring protection against parasite infection both in vitro and in vivo We also identify Gbp5 as a novel macrophage factor involved in limiting intracellular infection by avirulent strains of T. gondii The rarity of human infections caused by type III strains suggests that these mechanisms may also be important in controlling human toxoplasmosis. These findings further extend our understanding of host responses and defense mechanisms that act to control parasitic infections at the cellular level.

To characterize the microRNAs that contribute to the development of brace root, Solexa high-throughput sequencing of three libraries derived from tissues of node (N), nodes with just-emerged brace roots (NR), and nodes with just-emerged brace roots after IAA treatment (NRI) was performed. Total 650,793, 957,303 and 1,082,948 genome-matched unique reads were obtained in N, NR and NRI libraries, respectively. Further analysis confirmed the authenticity of 137 known miRNAs and the discovery of 159 novel miRNAs in maize. 14 conserved and 16 novel miRNAs differentially expressed in brace root, as well as 15 target genes, were identified and validated by qRT-PCR during maize brace root development. Moreover, we identified 9 miRNA precursor-matched novel sRNAs that may form miRNA clusters, as well as 24 nt siRNAs in the three libraries. In addition, we suggest that auxin represent a regulator in brace root development and can be regulated at the posttranscriptional level by miRNAs.

The aim of the present study was to investigate the effect of portal vein ligation (PVL) on the tumor growth rate and liver regeneration in rat cirrhotic liver lobes. A total of 45 male Wistar rats were randomly divided into PVL, hepatic tumor (HT) and HT + PVL groups (n=15 per group). Liver regeneration and tumor growth in ligated and non-ligated lobes were evaluated prior to and following PVL. In addition, serum alanine transaminase, total bilirubin levels and liver tissue samples were evaluated. The results indicated that PVL induced apparent hypertrophy in normal and HT rats. However, the ratio of non-ligated lobes to total liver weight or body weight in the HT + PVL group was significantly lower when compared with the PVL group (P<0.05). Compared with the HT group, the tumor growth rate in the ligated lobes of the HT + PVL group significantly increased (P<0.05). However, tumor growth in the non-ligated lobes exhibited no statistically significant difference between the HT and HT + PVL groups. In addition, Knodell scores indicated that fibrosis was more apparent in the non-ligated lobes of the HT + PVL group when compared with the HT group (P<0.05). Therefore, tumor growth was accelerated in ligated lobes following PVL, but not in non-ligated lobes. PVL also induced liver regeneration in cirrhotic liver lobes with lower efficiency than that in the non-cirrhotic lobes. However, hypertrophy in the contralateral cirrhotic lobes appeared to be non-functional.

Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens.

An ideal wound dressing can both promote wound healing and prevent bacterial infection. Here, we report a potential dressing prepared by incorporating an optimized concentration of silver nanoparticles (AgNPs) into the microfibers of a natural eggshell membrane (EM) using environmentally friendly and mussel-inspired dopamine. Briefly, acid-treated EM was used as a porous membrane for polydopamine-reduced AgNPs synthesis. To obtain the optimal cytocompatible silver concentration, cellular attachment and MTT assay were performed with different concentrations of AgNPs. The morphology of the EM and AgNPs was confirmed by scanning electronic microscopy, scanning transmission electronic microscopy and Fourier transform infrared spectroscopy. The synthesized EM/AgNPs exhibited steady and safe AgNPs release, which was further tested for antibacterial activity against Escherichia coli and Staphylococcus aureus by disc diffusion method and bacterial suspension assay. Finally, in a murine full-thickness skin wound model, we found that EM/AgNPs could promote re-epithelialization, granulation tissue formation and wound healing via enhancing cell proliferation, as demonstrated by the expression of proliferating cell nuclear antigen (PCNA), and controlling inflammation response, as demonstrated by the expression of interleukin-1β (IL-1β). These findings suggest that EM/AgNPs may have a promising application in wound management.

Lithium titanate and titanium dioxide are two best-known high-performance electrodes that can cycle around 10,000 times in aprotic lithium ion electrolytes. Here we show there exists more lithium titanate hydrates with superfast and stable cycling. That is, water promotes structural diversity and nanostructuring of compounds, but does not necessarily degrade electrochemical cycling stability or performance in aprotic electrolytes. As a lithium ion battery anode, our multi-phase lithium titanate hydrates show a specific capacity of about 130 mA h g-1 at ~35 C (fully charged within ~100 s) and sustain more than 10,000 cycles with capacity fade of only 0.001% per cycle. In situ synchrotron diffraction reveals no 2-phase transformations, but a single solid-solution behavior during battery cycling. So instead of just a nanostructured intermediate to be calcined, lithium titanate hydrates can be the desirable final destination.Water is usually not favorable in high-voltage window aprotic electrolytes. Here the authors discover some lithium titanate hydrates that allow superior power rate and ultralong cycle life in aprotic electrolytes.

IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of guanylate-binding proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1(-/-) cells, and decreased virulence of this mutant was compensated in Gbp1(-/-) mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this protein in host defense.

Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.

Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b-k) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites.

The small GTPases regulate many major biological processes in both tumorigenesis and tumor progression such as cell survival, actin cytoskeleton organization, cell polarity and movement. Wnt5a, a non-canonical Wnt family member, is implicated in the activation of small GTPases in breast cancer. We previously demonstrated that Wnt5a signaling stimulates the migration of breast cancer cells MDA-MB-231 via activating RhoA. However, we found here that RhoA activation was not enhanced by Wnt5a in breast cancer cells MCF-7. The conflicting results prompted us to further probe novel small GTPases in response to Wnt5a and investigate the mechanisms whereby cell migration is regulated. We showed here that Wnt5a dose dependently activated Dvl2, Rab35 and Rac1 and subsequently promoted the migration of MCF-7 cells, which was, however, abolished by knocking down Wnt5a expression via small interfering RNA (siRNA) transfection. Dvl2 siRNA significantly decreased background and Wnt5a-induced Rab35/Rac1 activation and, consequently, cell migration. Rab35 short hairpin RNA (shRNA) remarkably inhibited background and Wnt5a-induced Rac1 activation and cell migration. Additionally, blockade of Rac1 activation with Rac1 siRNA suppressed background and Wnt5a-induced cell migration. Co-immunoprecipitation and immunofluorescence assays showed that Dvl2 bound to Rab35 in mammalian cells. Taken together, we demonstrated that Wnt5a promotes breast cancer cell migration via the Dvl2/Rab35/Rac1 signaling pathway. These findings implicate Wnt5a signaling in regulating small GTPases, which could be targeted for manipulating breast cancer cell migration.

We demonstrated previously that the incorporation of a membrane-anchored form of flagellin into influenza virus-like particles (VLPs) improved the immunogenicity of VLPs significantly, inducing partially protective heterosubtypic immunity by intramuscular immunization. Because the efficacy of mucosal vaccination is highly dependent on an adjuvant, and is particularly effective for preventing mucosal infections such as influenza, we determined whether the membrane-anchored flagellin is an efficient adjuvant for VLP vaccines by a mucosal immunization route. We compared the adjuvant effect of membrane-anchored and soluble flagellins for immunization with influenza A/PR8 (H1N1) VLPs by the intranasal route in a mouse model. The results demonstrate that membrane-anchored flagellin is an effective adjuvant for intranasal (IN) immunization, inducing enhanced systemic and mucosal antibody responses. High cellular responses were also observed as shown by cytokine production in splenocyte cultures when stimulated with viral antigens. All mice immunized with flagellin-containing VLPs survived challenge with a high lethal dose of homologous virus as well as a high dose heterosubtypic virus challenge (40 LD(50) of A/Philippines/82, H3N2). In contrast, no protection was observed with a standard HA/M1 VLP group upon heterosubtypic challenge. Soluble flagellin exhibited a moderate adjuvant effect when co-administered with VLPs by the mucosal route, as indicated by enhanced systemic and mucosal responses and partial heterosubtypic protection. The membrane-anchored form of flagellin incorporated together with antigen into influenza VLPs is effective as an adjuvant by the mucosal route and unlike standard VLPs, immunization with such chimeric VLPs elicits protective immunity to challenge with a distantly related influenza A virus.