Descending GABAergic pathway links brain sugar-sensing to peripheral nociceptive gating in Drosophila.

Although painful stimuli elicit defensive responses including escape behavior for survival, starved animals often prioritize feeding over escape even in a noxious environment. This behavioral priority is typically mediated by suppression of noxious inputs through descending control in the brain, yet underlying molecular and cellular mechanisms are incompletely understood. Here we identify a cluster of GABAergic neurons in Drosophila larval brain, designated as SEZ-localized Descending GABAergic neurons (SDGs), that project descending axons onto the axon terminals of the peripheral nociceptive neurons and prevent presynaptic activity through GABAB receptors. Remarkably, glucose feeding to starved larvae causes sustained activation of SDGs through glucose-sensing neurons and subsequent insulin signaling in SDGs, which attenuates nociception and thereby suppresses escape behavior in response to multiple noxious stimuli. These findings illustrate a neural mechanism by which sugar sensing neurons in the brain engages descending GABAergic neurons in nociceptive gating to achieve hierarchical interaction between feeding and escape behavior.

Presynaptic Ube3a E3 ligase promotes synapse elimination through down-regulation of BMP signaling.

Inactivation of the ubiquitin ligase Ube3a causes the developmental disorder Angelman syndrome, whereas increased Ube3a dosage is associated with autism spectrum disorders. Despite the enriched localization of Ube3a in the axon terminals including presynapses, little is known about the presynaptic function of Ube3a and mechanisms underlying its presynaptic localization. We show that developmental synapse elimination requires presynaptic Ube3a activity in Drosophila neurons. We further identified the domain of Ube3a that is required for its interaction with the kinesin motor. Angelman syndrome-associated missense mutations in the interaction domain attenuate presynaptic targeting of Ube3a and prevent synapse elimination. Conversely, increased Ube3a activity in presynapses leads to precocious synapse elimination and impairs synaptic transmission. Our findings reveal the physiological role of Ube3a and suggest potential pathogenic mechanisms associated with Ube3a dysregulation.

A neurogenetic mechanism of experience-dependent suppression of aggression.

Aggression is an ethologically important social behavior, but excessive aggression can be detrimental to fitness. Social experiences among conspecific individuals reduce aggression in many species, the mechanism of which is largely unknown. We found that loss-of-function mutation of nervy (nvy), a Drosophila homolog of vertebrate myeloid translocation genes (MTGs), increased aggressiveness only in socially experienced flies and that this could be reversed by neuronal expression of human MTGs. A subpopulation of octopaminergic/tyraminergic neurons labeled by nvy was specifically required for such social experience-dependent suppression of aggression, in both males and females. Cell type-specific transcriptomic analysis of these neurons revealed aggression-controlling genes that are likely downstream of nvy. Our results illustrate both genetic and neuronal mechanisms by which the nervous system suppresses aggression in a social experience-dependent manner, a poorly understood process that is considered important for maintaining the fitness of animals.

The prognostic significance of apical lymph node metastasis in patients with high-risk stage III colon cancer.

The effect of apical lymph node (APN) metastasis on the prognosis of colon cancer is unknown. The present study investigated the impact of APN metastasis on the prognosis of the patients with high-risk stage III colon cancer. This retrospective multi-institutional study included patients with pathological high-risk stage III colon cancer who underwent surgery between April 2009 and December 2014. Clinicopathological factors were examined by univariate and multivariate analyses to clarify independent risk factors for overall survival (OS) and relapse-free survival (RFS). A total of 185 patients were collected. The 5-year OS rates of patients with and without APN metastasis were 35.0% and 72.1%, respectively (p = 0.0014). The 5-year RFS rates of patients with and without APN metastasis was 16.2% and 57.2%, respectively (p = 0.0002). The rate of distant metastasis in patients with APN metastasis was significantly higher than that in patients without APN metastasis (68.8% vs. 36.7%, p = 0.012). The univariate analysis revealed that the differentiation, lymph node ratio, and APN metastasis were significantly associated with 5-year OS, and the preoperative CEA and CA19-9 levels and APN metastasis were significantly associated with 5-year RFS. The multivariate analysis showed that APN metastasis was an independent risk factor for 5-year OS and RFS. APN metastasis may be independently associated with the prognosis of patients with high-risk Stage III colon cancer.

Serum apolipoprotein A-I potentiates the therapeutic efficacy of lysocin E against Staphylococcus aureus.

Lysocin E is a lipopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. For unclear reasons, the antibacterial activity of lysocin E in a mouse systemic infection model is higher than expected from in vitro results, and the in vitro activity is enhanced by addition of bovine serum. Here, we confirm that serum from various species, including humans, increases lysocin E antimicrobial activity, and identify apolipoprotein A-I (ApoA-I) as an enhancing factor. ApoA-I increases the antibacterial activity of lysocin E when added in vitro, and the antibiotic displays reduced activity in ApoA-I gene knockout mice. Binding of ApoA-I to lysocin E is enhanced by lipid II, a cell-wall synthesis precursor found in the bacterial membrane. Thus, the antimicrobial activity of lysocin E is potentiated through interactions with host serum proteins and microbial components.

Quantifying influence of human choice on the automated detection of Drosophila behavior by a supervised machine learning algorithm.

Automated quantification of behavior is increasingly prevalent in neuroscience research. Human judgments can influence machine-learning-based behavior classification at multiple steps in the process, for both supervised and unsupervised approaches. Such steps include the design of the algorithm for machine learning, the methods used for animal tracking, the choice of training images, and the benchmarking of classification outcomes. However, how these design choices contribute to the interpretation of automated behavioral classifications has not been extensively characterized. Here, we quantify the effects of experimenter choices on the outputs of automated classifiers of Drosophila social behaviors. Drosophila behaviors contain a considerable degree of variability, which was reflected in the confidence levels associated with both human and computer classifications. We found that a diversity of sex combinations and tracking features was important for robust performance of the automated classifiers. In particular, features concerning the relative position of flies contained useful information for training a machine-learning algorithm. These observations shed light on the importance of human influence on tracking algorithms, the selection of training images, and the quality of annotated sample images used to benchmark the performance of a classifier (the 'ground truth'). Evaluation of these factors is necessary for researchers to accurately interpret behavioral data quantified by a machine-learning algorithm and to further improve automated classifications.

Layered roles of fruitless isoforms in specification and function of male aggression-promoting neurons in Drosophila.

Inter-male aggressive behavior is a prominent sexually dimorphic behavior. Neural circuits that underlie aggressive behavior are therefore likely under the control of sex-determining genes. However, the neurogenetic mechanism that generates sex-specific aggressive behavior remains largely unknown. Here, we found that a neuronal class specified by one of the Drosophila sex determining genes, fruitless (fru), belongs to the neural circuit that generates male-type aggressive behavior. This neuronal class can promote aggressive behavior independent of another sex determining gene, doublesex (dsx), although dsx is involved in ensuring that aggressive behavior is performed only toward males. We also found that three fru isoforms with different DNA binding domains show a division of labor on male aggressive behaviors. A dominant role of fru in specifying sex-specific aggressive behavior may underscore a genetic mechanism that allows male-type aggressive behavior to evolve at least partially independently from courtship behavior, which is under different selective pressures.

Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons.

For successful mating, a male animal must execute effective courtship behaviors toward a receptive target sex, which is female. Whether the courtship execution capability and upregulation of courtship toward females are specified through separable sex-determining genetic pathways remains uncharacterized. Here, we found that one of the two Drosophila sex-determining genes, doublesex (dsx), specifies a male-specific neuronal component that serves as an execution mechanism for courtship behavior, whereas fruitless (fru) is required for enhancement of courtship behavior toward females. The dsx-dependent courtship execution mechanism includes a specific subclass within a neuronal cluster that co-express dsx and fru. This cluster contains at least another subclass that is specified cooperatively by both dsx and fru. Although these neuronal populations can also promote aggressive behavior toward male flies, this capacity requires fru-dependent mechanisms. Our results uncover how sex-determining genes specify execution capability and female-specific enhancement of courtship behavior through separable yet cooperative neurogenetic mechanisms.

Phenotypic and genomic comparisons of highly vancomycin-resistant Staphylococcus aureus strains developed from multiple clinical MRSA strains by in vitro mutagenesis.

The development of vancomycin (VCM) resistance in Staphylococcus aureus threatens global health. Studies of the VCM-resistance mechanism and alternative therapeutic strategies are urgently needed. We mutagenized S. aureus laboratory strains and methicillin-resistant S. aureus (MRSA) with ethyl methanesulfonate, and isolated mutants that exhibited high resistance to VCM (minimum inhibitory concentration = 32 µg/ml). These VCM-resistant strains were sensitive to linezolid and rifampicin, and partly to arbekacin and daptomycin. Beta-lactams had synergistic effects with VCM against these mutants. VCM-resistant strains exhibited a 2-fold increase in the cell wall thickness. Several genes were commonly mutated among the highly VCM-resistant mutants. These findings suggest that MRSA has a potential to develop high VCM resistance with cell wall thickening by the accumulation of mutations.

Antennal RNA-sequencing analysis reveals evolutionary aspects of chemosensory proteins in the carpenter ant, Camponotus japonicus.

Chemical communication is essential for the coordination of complex organisation in ant societies. Recent comparative genomic approaches have revealed that chemosensory genes are diversified in ant lineages, and suggest that this diversification is crucial for social organisation. However, how such diversified genes shape the peripheral chemosensory systems remains unknown. In this study, we annotated and analysed the gene expression profiles of chemosensory proteins (CSPs), which transport lipophilic compounds toward chemosensory receptors in the carpenter ant, Camponotus japonicus. Transcriptome analysis revealed 12 CSP genes and phylogenetic analysis showed that 3 of these are lineage-specifically expanded in the clade of ants. RNA sequencing and real-time quantitative polymerase chain reaction revealed that, among the ant specific CSP genes, two of them (CjapCSP12 and CjapCSP13) were specifically expressed in the chemosensory organs and differentially expressed amongst ant castes. Furthermore, CjapCSP12 and CjapCSP13 had a ratio of divergence at non-synonymous and synonymous sites (dN/dS) greater than 1, and they were co-expressed with CjapCSP1, which is known to bind cuticular hydrocarbons. Our results suggested that CjapCSP12 and CjapCSP13 were functionally differentiated for ant-specific chemosensory events, and that CjapCSP1, CjapCSP12, and CjapCSP13 work cooperatively in the antennal chemosensilla of worker ants.

Primed Immune Responses Triggered by Ingested Bacteria Lead to Systemic Infection Tolerance in Silkworms.

In the present study, we examined whether microorganisms collaterally ingested by insects with their food activate the innate immune system to confer systemic resistance against subsequent bacterial invasion. Silkworms orally administered heat-killed Pseudomonas aeruginosa cells showed resistance against intra-hemolymph infection by P. aeruginosa. Oral administration of peptidoglycans, cell wall components of P. aeruginosa, conferred protective effects against P. aeruginosa infection, whereas oral administration of lipopolysaccharides, bacterial surface components, did not. In silkworms orally administered heat-killed P. aeruginosa cells, P. aeruginosa growth was inhibited in the hemolymph, and mRNA amounts of the antimicrobial peptides cecropin A and moricin were increased in the hemocytes and fat body. Furthermore, the amount of paralytic peptide, an insect cytokine that activates innate immune reactions, was increased in the hemolymph of silkworms orally administered heat-killed P. aeruginosa cells. These findings suggest that insects sense bacteria present in their food by peptidoglycan recognition, which activates systemic immune reactions to defend the insects against a second round of infection.

Studies of host-pathogen interactions and immune-related drug development using the silkworm: interdisciplinary immunology, microbiology, and pharmacology studies.

Innate immunity acts as a front-line barrier against invading pathogens, and the majority of the components are widely conserved among species. Regulation of innate immunity is important for overcoming infections and preventing self-damaging sepsis. Using the silkworm (Bombyx mori) as an animal model, we elucidated the activation processes of innate immunity with emphasis on a multifunctional insect cytokine called paralytic peptide. Moreover, we established an ex vivo system using silkworm larval specimens to quantitatively evaluate the immunostimulatory activity of natural compounds. We observed that overactivation of innate immunity in silkworms induces tissue damage followed by host death, resembling sepsis-induced multi-organ failure in humans. Here, we summarize our recent findings and propose the usefulness of the silkworm as an animal model for studying immune regulation and for evaluating compounds with the potential to regulate innate immunity.

Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane.

To obtain therapeutically effective new antibiotics, we first searched for bacterial culture supernatants with antimicrobial activity in vitro and then performed a secondary screening using the silkworm infection model. Through further purification of the in vivo activity, we obtained a compound with a previously uncharacterized structure and named it 'lysocin E'. Lysocin E interacted with menaquinone in the bacterial membrane to achieve its potent bactericidal activity, a mode of action distinct from that of any other known antibiotic, indicating that lysocin E comprises a new class of antibiotic. This is to our knowledge the first report of a direct interaction between a small chemical compound and menaquinone that leads to bacterial killing. Furthermore, lysocin E decreased the mortality of infected mice. To our knowledge, lysocin E is the first compound identified and purified by quantitative measurement of therapeutic effects in an invertebrate infection model that exhibits robust in vivo effects in mammals.

Paralytic peptide: an insect cytokine that mediates innate immunity.

Host animals combat invading pathogens by activating various immune responses. Modulation of the immune pathways by cytokines is critical for efficient pathogen elimination. Insects and mammals possess common innate immune systems, and individual immune pathways have been intensively studied over the last two decades. Relatively less attention, however, has been focused on the functions of cytokines in insect innate immunity. Here, we summarize our recent findings from studies of the insect cytokine, paralytic peptide, in the silkworm Bombyx mori. The content of this report was presented at the First Asian Invertebrate Immunity Symposium. Acute activation of paralytic peptide occurs via proteolysis after stimulation with the cell wall components of pathogens, leading to the induction of a wide range of cellular and humoral immune responses. The pathogenic bacterium Serratia marcescens suppresses paralytic peptide-dependent immune activation, which impairs host resistance. Studies of insect cytokines will broaden our understanding of the basic mechanisms underlying the interaction between host innate immunity and pathogenic agents.

Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists.

Establishment of a bacterial infection model using the European honeybee, Apis mellifera L.

Injection of human pathogenic bacteria (Pseudomonas aeruginosa, Serratia marcescens, Salmonella enterica, Staphylococcus aureus, and Listeria monocytogenes) into the hemocoel of honeybee (Apis mellifera L.) workers kills the infected bees. The bee-killing effects of the pathogens were affected by temperature, and the LD50 values at 37°C were more than 100-fold lower than those at 15°C. Gene-disrupted S. aureus mutants of virulence genes such as agrA, saeS, arlR, srtA, hla, and hlb had attenuated bee-killing ability. Nurse bees were less susceptible than foragers and drones to S. aureus infection. Injection of antibiotics clinically used for humans had therapeutic effects against S. aureus infections of bees, and the ED50 values of these antibiotics were comparable with those determined in mammalian models. Moreover, the effectiveness of orally administered antibiotics was consistent between honeybees and mammals. These findings suggest that the honeybee could be a useful model for assessing the pathogenesis of human-infecting bacteria and the effectiveness of antibiotics.

Identification of a Serratia marcescens virulence factor that promotes hemolymph bleeding in the silkworm, Bombyx mori.

Injection of culture supernatant of Serratia marcescens, a Gram-negative bacterium pathogenic to a wide range of host animals including insects and mammals, into the hemolymph of silkworm (Bombyx mori) larvae led to continuous flow of the hemolymph (blood of insects) from the injection site. The amount of hemolymph lost within 60 min reached 15-20% of the total larval weight. Using a bioassay with live silkworms, we purified Serralysin, a metalloprotease that requires divalent cations for its activity, as the factor responsible for the promotion of hemolymph bleeding from the culture supernatant of S. marcescens. Recombinant protein also induced hemolymph bleeding in silkworms. Moreover, the culture supernatant of an S. marcescens disruption mutant of the ser gene showed attenuated ability to promote hemolymph bleeding. In addition, this bleeding-promoting activity of the S. marcescens culture supernatant was attenuated by disruption of the wecA gene, which is involved in the biosynthesis of the lipopolysaccharide O-antigen. These findings suggest that Serralysin metalloprotease contributes to the pathogenesis of S. marcescens by inhibiting wound healing, which leads to a massive loss of hemolymph from silkworm larvae.

Niemann-Pick disease type C2 protein induces triglyceride accumulation in silkworm and mammalian cell lines.

Silkworm haemolymph induced both the cessation of growth and an increase in triglyceride (triacylglycerol) storage in BmN4 cells. We purified the growth inhibitory factor from the silkworm haemolymph and identified this protein as the Bombyx mori PP (promoting protein), an orthologue of NPC2 (Niemann-Pick disease type C2) protein. Recombinant silkworm NPC2 inhibited cellular proliferation and increased triglyceride accumulation in BmN4 cells. Injection of either the recombinant protein or antiserum of NPC2 into living silkworms increased or decreased respectively triglyceride levels in the fat body. A mutation that depletes the cholesterol-binding capacity did not abolish the activity of NPC2. We further revealed that NPC2 induced the phosphorylation of AMPK (AMP-activated protein kinase) and that an AMPK inhibitor suppressed NPC2-dependent triglyceride accumulation. These findings suggest that NPC2 induces triglyceride accumulation via the activation of AMPK independently of its cholesterol-binding capacity in the silkworm.

Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.