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Highlights • Endo- and exogenous stimuli activate inflammasomes. • Inflammasome activation contributes to innate and adaptive immune responses. • Inflammasome activation induces IL-1β secretion. • IL-1β promotes Th17 cell differentiation via IL-1/IL-1R signaling. • Th17 responses are associated with inflammasome activation. Abstract Immune sensing of exogenous molecules from microbes (e.g., pathogen-associated molecular patterns) and nonmicrobial molecules (e.g., asbestos, alum, and silica), as well as endogenous damage-associated molecular patterns (e.g., ATP, uric acid crystals, and amyloid A) activates innate immunity by inducing immune-related genes, including proinflammatory cytokines, which further facilitate the development of adaptive immunity. The roles of transcriptional responses downstream of immune sensing have been widely characterized in informing adaptive immunity; however, few studies focus on the effect of post-translational responses on the modulation of adaptive immune responses. Inflammasomes activated by the previously described endo- and exogenous stimuli autocatalytically induce intracellular pro-caspase-1, which cleaves the inactive precursors of interleukin-1β (IL-1β) and IL-18 into bioactive proinflammatory cytokines. IL-1β and IL-18 not only contribute to the host defense against infections by activating phagocytes, such as monocytes, macrophages, dendritic cells, and neutrophils, but also induce T-helper 17 (Th17)- and Th1-mediated adaptive immune responses. In synergy with IL-6 and IL-23, IL-1β activates IL-1 receptor (IL-1R) signaling to drive the differentiation of IL-17-producing Th17 cells, which not only play critical roles in host protective immunity to infections of bacteria, fungi, and certain viruses but also participate in the pathology of inflammatory disorders and tumorigenesis. Consequently, targeting inflammasomes and IL-1/IL-1R signaling may effectively improve the treatment of Th17-associated disorders, such as autoinflammatory diseases and cancers, thereby providing novel insights into drug development. [ABSTRACT FROM AUTHOR]

To improve the processing efficiency on batch query for MapReduce, a multiple query optimization approach based on Hive+ is proposed to reduce the number of MapReduce tasks on multiple query, decrease the start time of MapReduce task and the overhead of fault tolerance, improve the query efficiency. TPC-H benchmark test set is selected as the use cases to experiment on Hive-0.12. The experiment shows that the processing efficiency of batch query is effectively improved. [ABSTRACT FROM AUTHOR]

Our previous research showed that immulectin-2 (IML-2), a C-type lectin from the tobacco hornworn, Manduca sexta, is a pattern recognition receptor (PRR) that can bind to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PG) and β-1,3-glucan, and IML-2 plays an important role in cellular encapsulation, melanization, phagocytosis, and prophenoloxidase (proPO) activation. Unlike most mammalian C-type lectins that contain a single carbohydrate-recognition domain (CRD), IML-2 is composed of tandem CRDs, and the C-terminal CRD2 contains an extended loop, which is not present in most C-type CRDs. We hypothesize that the extended loop may participate in ligand binding, encapsulation, melanization, phagocytosis and/or proPO activation in M. sexta. To test this hypothesis, two deletion mutant proteins (IML-2 and IML-2), in which the extended loop of the CRD2 was partially or completely deleted, were expressed and purified. By comparing the characteristics of recombinant IML-2, IML-2 and IML-2, we found that deletion of the extended loop in CRD2 impaired the ability of IML-2 to bind microbial PAMPs and to stimulate proPO activation, indicating that the extended loop of IML-2 plays an important role in ligand binding and biological functions. [ABSTRACT FROM AUTHOR]

Abstract: Activation of prophenoloxidase and synthesis of antimicrobial peptides (AMPs) are two important innate immune mechanisms in insects. In the current study, we investigated immune responses activated by three major bacterial components, lipopolysaccharide (LPS) (including rough mutants of LPS), lipoteichoic acid (LTA), and peptidoglycan (PG), in the larvae of a lepidopteran insect, Manduca sexta. We found that two DAP (diaminopimelic acid)-type PGs from Escherichia coli and Bacillus subtilis were much more potent than LPS and LTA from the respective bacteria as well as a Lysine-type PG in activation of prophenoloxidase in M. sexta larval plasma in vitro. Transcription levels of AMP genes, such as Attacin, Lebocin and Moricin genes, in the hemocytes and fat body of larvae were significantly induced by smooth LPS (TLR4grade) and rough mutants of LPS (TLRgrade™), synthetic lipid A, LTA, and PG. LPS from E. coli and LTA from B. subtilis activated AMP expression to significantly higher levels than PGs from the respective bacterial strains, and smooth LPS were more potent than lipid A and rough mutants of LPS in activation of AMP expression. Our results demonstrated for the first time that LTA can activate AMP expression, and different moieties of LPS may synergistically activate AMP expression in M. sexta. [ABSTRACT FROM AUTHOR]

Abstract: The unprecedented, stereospecific interconversion of cis- and trans-γ,δ-epoxy α,β-unsaturated ester systems has been realized, which involves the palladium-catalyzed stereospecific alkoxy or hydroxy substitution reaction with double inversion of configuration at the γ-position as the key step. The new methodology is not only applicable to various disubstituted and trisubstituted epoxy unsaturated esters, but also these interconversions proceed with an extremely high degree of stereoselectivity and efficiency. [Copyright &y& Elsevier]

Abstract: Mammalian C-type lectins are calcium-dependent carbohydrate-binding proteins. They serve as cell adhesion molecules in cell–cell interactions, or function as pattern-recognition receptors in innate immunity. Calcium is a direct ligand for carbohydrate binding in mammalian C-type lectins such as mannose-binding proteins and macrophage mannose receptor. In the tobacco hornworm Manduca sexta, a group of lectins named immulectins have been discovered. Each immulectin contains dual carbohydrate-recognition domains. Previously, we showed that immulectin-2 (IML-2) binds to a bacterial lipopolysaccharide, and agglutination of Escherichia coli cells by IML-2 is calcium dependent. In this study, we demonstrated that IML-2 bound to bacterial lipid A, smooth and rough mutants of lipopolysaccharide, lipoteichoic acid and peptidoglycan, as well as to fungal mannan and β-1, 3-glucan (laminarin and curdlan). Binding of IML-2 to microbial components was calcium independent, and was increased by addition of spermine, a polyamine. In addition, plasma IML-2 bound to mannan-agarose independent of calcium. But trypsin digestion of IML-2 was inhibited in the presence of calcium. Our results suggest that calcium is not required for IML-2 binding but protects IML-2 from trypsin digestion. [Copyright &y& Elsevier]

Abstract: Phagocytosis is an important innate immune response against microbial infections and an effective mechanism to eliminate apoptotic cells. In vertebrates, phagocytes such as macrophages and dendritic cells are involved in phagocytosis. We demonstrate here that insect hemocytes have distinct functions in phagocytosis of foreign particles and self dead cells. Plasmatocytes from the tobacco hornworm Manduca sexta were major hemocytes involved in phagocytosis of non-self microsphere beads, whereas granulocytes were apparently the only hemocytes that phagocytose self dead cells. We also showed that M. sexta immulectin-2, a pattern recognition receptor that protects larvae from bacterial infection, has an opsonic activity in phagocytosis. Immulectin-2 bound to the surface of granulocytes from the naïve larvae, but more immulectin-2 bound to plasmatocytes when larvae were injected with microsphere beads. Coupling of immulectin-2 onto microsphere beads enhanced in vitro phagocytosis of the beads. Our results suggest that insect hemocytes can have specialized functions similar to vertebrate phagocytes in phagocytosis, and pattern recognition receptors may function as opsonins to enhance phagocytosis. [Copyright &y& Elsevier]

Abstract: An effective innate immune response against parasites in insects is encapsulation followed by melanization. In cellular encapsulation, formation of capsules requires plasmatocytes and granulocytes, the two most abundant hemocytes in lepidopteran insects. We have developed an in vitro encapsulation assay to elucidate functions of immulectins in encapsulation. Immulectins are members of the C-type (calcium-dependent) lectin superfamily, and they function as humoral pattern recognition receptors in innate immunity of the tobacco hornworm Manduca sexta. We demonstrate that coating of immulectins to agarose beads enhanced cellular encapsulation in vitro. Immulectin-1 enhanced encapsulation but not melanization. Encapsulation occurred with isolated hemocytes that lack plasmatocytes when agarose beads were coated with immulectins. However, plasmatocytes were required for aggregation of encapsulated beads. We also showed that inhibitors of serine proteinases such as para-aminophenylmethylsulfonyl fluoride (p-APMSF) inhibited cellular encapsulation of immulectin-coated agarose beads. Our results suggest that humoral pattern recognition receptors may enhance cellular encapsulation and melanization. [Copyright &y& Elsevier]

Abstract: In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12h post-injection, but dropped to the normal level at 24h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules. [Copyright &y& Elsevier]

Abstract: A new two-photon polymerization initiator, 4-vinylpyridine-4′-(N,N-Diphenylamino)benene (abbreviate:VPDB) has been synthesized with inexpensive reagents. Its nonlinear optical properties were investigated in detail. The experimental results confirm that VPDB is a good two-photon absorbing chromophore and can successfully initiate two-photon photopolymerization of ethoxylated trimethylolpropane. The two-photon absorption cross-section of VPDB has been measured using the two-photon-induced fluorescence method, whose value is 14.6×10−50 cm4 s photon−1 molecule−1. A microstructure has been fabricated by using VPDB as initiator. [Copyright &y& Elsevier]

Abstract: Lectins interact with carbohydrates. They can function as pattern recognition receptors and play an important role in the innate immune system of animals. Previously, we have isolated two calcium-dependent (C-type) lectins, named immulectin-1 and -2, from the tobacco hornworm Manduca sexta. Both immulectin-1 and -2 stimulate prophenoloxidase activation in plasma. Here, we describe isolation and cDNA cloning of a novel member of immulectins, immulectin-3 (IML-3). IML-3, like immulectin-1 and -2, contains tandem carbohydrate-recognition domains (CRDs). The cDNA clone encoding IML-3 is 3802bp long, with an open reading frame of 930bp. This cDNA clone has an extremely long noncoding region at the 3′ end that contains eight polyadenylation signal sequences. Northern analysis showed that a 5.0kb IML-3 transcript was present in the fat body of control larvae (injected with saline) but not in the fat body of larvae injected with bacteria. However, a much more abundant 3.1kb transcript was induced in the fat body of bacteria-injected larvae. IML-3 mRNA was not detected in hemocytes of control or bacteria-injected larvae. Recombinant IML-3 was expressed in bacteria and purified. It specifically bound to immobilized lipopolysaccharide (LPS) and lipoteichoic acid from bacteria, and to laminarin, a -1, 3-glucan. Binding of IML-3 to immobilized LPS was competed by excess free LPS. More importantly, IML-3 contains an anti-death-like motif in the carboxyl-terminal CRD. Endogenous IML-3 was detected in the cytoplasm of hemocytes, and FITC-labeled recombinant IML-3 was translocated from hemolymph into hemocytes. Coating of IML-3 onto agarose beads enhanced encapsulation of the beads. [Copyright &y& Elsevier]

In insects, encapsulation followed by melanization is a major defense mechanism against metazoan parasites. However, insects must recognize and differentiate nonself before they mount an immune response. Recognition of pathogens in insects is accomplished by a set of pattern recognition receptors (PRRs). Binding of PRRs to pathogens is linked to a variety of immune responses including phagocytosis, nodule formation, encapsulation, and prophenoloxidase activation. So far, little is known about how recognition of pathogens by PRRs triggers different immune responses. In this article, we report that immulectin-2, a C-type lectin, enhances encapsulation and melanization processes in the tobacco hornworm, Manduca sexta. Coating of agarose beads with recombinant carboxyl-terminal carbohydrate-recognition domain (CRD2-II) of immulectin-2 enhanced encapsulation of the beads in vitro by hemocytes and melanization of the beads in vivo in M. sexta larvae. Recombinant CRD2-II also directly bound to granular cells and oenocytoids, but not to plasmatocytes or spherule cells. Immulectin-2 in hemolymph of M. sexta larvae bound to the surface of a nematode, Caenorhabditis elegans, and recombinant CRD2-II directly bound to C. elegans and a human filarial nematode, Brugia malayi. Binding of CRD2-II to C. elegans enhanced melanization of the nematode in vivo. Our results suggest that binding of immulectin-2 to the surface of parasites can trigger encapsulation and melanization responses in M. sexta. [Copyright &y& Elsevier]

We previously reported the isolation of a lipopolysaccharide (LPS)-specific immulectin-2 from the tobacco hornworm, Manduca sexta [J. Biol. Chem. 275 (2000) 37373]. Immulectin-2 is a C-type lectin that is present at a constitutively low level in hemolymph of naive larvae, and its synthesis is induced after injection of Gram-negative bacteria or LPS. Immulectin-2 contains two carbohydrate recognition domains. It binds to LPS and stimulates prophenoloxidase activation in plasma. In this paper, we focus on properties of carbohydrate recognition domain-2 of immulectin-2 and the biological functions of immulectin-2 in immune responses. The carboxyl-terminal carbohydrate recognition domain (CRD2) of immulectin-2 was able to bind bacterial LPS. Binding of recombinant CRD2 to LPS stimulated activation of prophenoloxidase in plasma. Injection of antiserum against immulectin-2 into M. sexta larvae inhibited clearance of a Gram-negative bacterial pathogen, Serratia marcescens, and decreased survival of infection. These results suggest that immulectin-2 plays an important role in the immune system of M. sexta, and helps to protect the animal from Gram-negative bacterial infections. [Copyright &y& Elsevier]

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection. [Copyright &y& Elsevier]

Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis is induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin was demonstrated to bind to lipopolysaccharide (LPS) from Gram-negative bacteria and to lipoteichoic acid from Gram-positive bacteria. Binding of hemolin to smooth-type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and Rc) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS was partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate. Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O-specific antigen and the outer-core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern-recognition protein with broad specificity in the defense against micro-organisms. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are critical components of innate immunity in diverse organisms, including plants, vertebrates, and insects. This study identified and characterized a novel Lepidoptera‐specific AMP, named lepidoptin, from the invasive pest Spodoptera frugiperda (Lepidoptera: Noctuidae). Lepidoptin is a 116‐amino acid protein containing a signal peptide and a novel β‐sandwich domain that is distinct from previously reported AMPs. Temporal and spatial expression analyses revealed a significant upregulation of the lepidoptin gene in vivo and in cultured SF9 cells in response to pathogens. Molecular docking analysis identified a specific binding cavity. Enzyme‐linked immunosorbent assay and binding assays confirmed that lepidoptin can bind to pathogen‐associated molecular patterns, bacteria, and fungi. Recombinant lepidoptin exhibited potent antibacterial activity by inducing bacterial agglutination, inhibiting bacterial growth, increasing bacterial membrane permeability, and preventing biofilm formation. Lepidoptin also showed antifungal activity against the entomopathogenic fungus Beauveria bassiana by inhibiting spore germination, increasing fungal cell permeability, and increasing reactive oxygen species. Injection of recombinant lepidoptin into S. frugiperda larvae increased survival after B. bassiana infection, whereas knockdown of lepidoptin by RNA interference decreased larval survival. In addition, lepidoptin showed antimicrobial activity against the plant pathogen Fusarium graminearum by inhibiting spore germination and alleviating disease symptoms in wheat seedlings and cherry tomatoes. This study demonstrates the remarkable dual functionality of lepidoptin in enhancing S. frugiperda immunity and controlling plant pathogens, making it a promising candidate for biocontrol strategies in both pest management and plant disease prevention. [ABSTRACT FROM AUTHOR]

The diamondback moth, Plutella xylostella , is the first insect to develop resistance to Bacillus thuringiensis (Bt) in the field. To date, little is known about the molecular mechanism of the interaction between Bt and midgut immunity in P. xylostella. Here, we report immune responses in the P. xylostella midgut to Bt strain Bt8010 using a combined approach of transcriptomics and quantitative proteomics. Many genes in the Toll, IMD, JNK and JAK-STAT pathways and antimicrobial peptide genes were activated at 18 h post-infection. In the prophenoloxidase (PPO) cascade, four serpin genes were activated, and the PPO1 gene was suppressed by Bt8010. Inhibition of the two PPO proteins was observed at 18 h post-infection. Feeding Bt8010-infected larvae recombinant PPOs enhanced their survival. These results revealed that the Toll, IMD, JNK and JAK-STAT pathways were triggered and participated in the immune defence of the midgut against Bt8010, while the PPO cascade was inhibited and played an important role in this process. • The Toll, IMD, JNK and JAK-STAT pathways were activated in midgut by Bt8010. • Midgut attempted to trigger the PPO cascade, which was eventually inhibited by Bt8010. • Two recombinant PPOs improved the survival of Bt8010-infected larvae. • The PPO cascade plays an important role in the defence of midgut against Bt8010. [ABSTRACT FROM AUTHOR]

Simple Summary: Spermatogenesis, which is regulated by many different genes, is a conserved process across species to produce mature sperm for animal reproduction. Fox transcription factors can bind to DNA sequences in the promoters to regulate gene expression. FoxD subfamily members are mainly involved in metabolism and early organ development. In Drosophila melanogaster, FoxD subfamily member Fd59a may regulate the development of the nervous system and control the egg-laying behavior of females. However, the functions of insect FoxD members are still largely unknown. In this study, we investigated the role of Fd59a in the spermatogenesis of Drosophila. We found that mutations in Fd59a caused swelling of the apical region in the testis, resulting in fewer mature sperm in the seminal vesicle and significantly lower fertility of Fd59a mutant males compared to the control flies. We also found that the homeostasis of the testis stem cell niche in Fd59a mutant and RNAi flies was disrupted, causing increased apoptosis of sperm bundles. RNA sequencing and qRT-PCR results suggested that Fd59a can regulate the expression of genes related to reproductive process and cell death. Our collective results indicated that Fd59a plays a key role in Drosophila spermatogenesis, which will help to understand the role of FoxD members in insect spermatogenesis. Spermatogenesis is critical for insect reproduction and is regulated by many different genes. In this study, we found that Forkhead transcription factor Fd59a functions as a key factor in the spermatogenesis of Drosophila melanogaster. Fd59a contains a conversed Forkhead domain, and it is clustered to the FoxD subfamily with other FoxD members from some insect and vertebrate species. Mutations in Fd59a caused swelling in the apical region of the testis. More importantly, fewer mature sperm were present in the seminal vesicle of Fd59a mutant flies compared to the control flies, and the fertility of Fd59a2/2 mutant males was significantly lower than that of the control flies. Immunofluorescence staining showed that the homeostasis of the testis stem cell niche in Fd59a2/2 mutant and Fd59a RNAi flies was disrupted and the apoptosis of sperm bundles was increased. Furthermore, results from RNA sequencing and qRT-PCR suggested that Fd59a can regulate the expression of genes related to reproductive process and cell death. Taken together, our results indicated that Fd59a plays a key role in the spermatogenesis of Drosophila. [ABSTRACT FROM AUTHOR]

Spermatogenesis is critical for insect reproduction and the process is regulated by multiple genes. Glycosyltransferases have been shown to participate in the development of Drosophila melanogaster; however, their role in spermatogenesis is still unclear. In this study, we found that α1,4‐galactosyltransferase 1 (α4GT1) was expressed at a significantly higher level in the testis than in the ovary of Drosophila. Importantly, the hatching rate was significantly decreased when α4GT1 RNA interference (RNAi) males were crossed with w1118 females, with only a few mature sperm being present in the seminal vesicle of α4GT1 RNAi flies. Immunofluorescence staining further revealed that the individualization complex (IC) in the testes from α4GT1 RNAi flies was scattered and did not move synchronically, compared with the clustered IC observed in the control flies. Terminal deoxyribonucleotide transferase (TdT)‐mediated dUTP nick end labeling (TUNEL) assay showed that apoptosis signals in the sperm bundles of α4GT1 RNAi flies were significantly increased. Moreover, the expression of several individualization‐related genes, such as Shrub, Obp44a and Hanabi, was significantly decreased, whereas the expression of several apoptosis‐related genes, including Dronc and Drice, was significantly increased in the testes of α4GT1 RNAi flies. Together, these results suggest that α4GT1 may play dual roles in Drosophila spermatogenesis by regulating the sperm individualization process and maintaining the survival of sperm bundles. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis‐specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2H2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin‐protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587‐Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK‐STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila. [ABSTRACT FROM AUTHOR]

In invertebrates, both fibrinogen-related proteins (FREPs) and C-type lectins are acknowledged to act as pattern recognition receptors (PRRs) to participate particularly in an innate immunity. Hereby, a unique C-type lectin designated as FmLFd was isolated from the hemocytes of Fenneropenaeus merguiensis. FmLFd contained one open reading frame which encoding a peptide of 312 amino acid residues and a signal peptide of 18 amino acids. The primary sequence of FmLFd was composed of a fibrinogen-like domain (Fd) with a Ca2+-binding site and possessing specificity to bind N-acetyl glucosamine (GlcNAc). The FmLFd transcripts were detected mainly in hemocytes of healthy shrimp. The expression of FmLFd was significantly up-regulated upon challenge shrimp with Vibrio parahaemolyticus and Vibrio harveyi which more potent than by white spot syndrome virus (WSSV). The knocking down shrimp with FmLFd double-stranded RNA caused dramatical gene down-regulation. The gene silencing with co-injection of pathogens resulted in reduction of the shrimp survival rate. Recombinant protein of FmLFd (rFmLFd) could agglutinate and bind directly to both Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner and showed the sugar specificity to GlcNAc and bacterial saccharides; peptidoglycan (PGN), lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Recombinant protein of Fd domain (rFd) displayed the lower activity and specificity only to PGN. The binding between recombinant proteins of FmLFd and its domain confirming by ELISA demonstrated that both rFmLFd and rFd could bind to PGN, LPS and LTA with the highest affinity respected to PGN including a less extent of rFd. Besides, rFmLFd but not rFd could bind to WSSV proteins with the highest binding affinity to capsid VP15 and decreasing in order to envelope VP28 and tegument VP39A, respectively. It was presumed that entire molecule of FmLFd exhibited the antimicrobial ability by inhibiting the growth of pathogenic V. parahaemolyticus and this action was not affected by GlcNAc. Otherwise, FmLFd, a lectin containing fibrinogen-like domain, was firstly reported to be capable of promoting encapsulation by hemocytes. Altogether, we concluded that FmLFd belonged to a FREP family indentified by the existence of a conserved fibrinogen-like domain with possessing an ability to bind GlcNAc. It was a new C-type lectin existed in F. merguiensis and might presumably act as a kind of PRRs to participate in the shrimp immune defense towards bacterial and viral pathogens. • FmLFd expression is extremely up-regulated upon Vibrio parahaemolyticus challenge. • Gene silencing by dsRNA inhibits severely FmLFd transcription and raises mortality. • rFmLFd has activities of agglutination, direct binding and antibacterial ability. • rFmLFd but not its domain displays firstly to promote encapsulation by hemocytes. • FmLFd containing fibrinogen-like domain serves as PRR against pathogens. [ABSTRACT FROM AUTHOR]

Testicular fusion of Spodoptera litura occures during metamorphosis, which benefits sperms development. Previous research identified involvement of ECM-integrin interaction pathways, MMPs in testicular fusion, but the regulatory mechanism remains unclear. RNA-seq was performed to analyze long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in testes, aiming to uncover potential regulatory mechanisms of testicular fusion. 2150 lncRNAs, 2742 targeted mRNAs, and 347 miRNAs were identified in testes at three different developmental stages. Up-regulated DElncRNAs and DEmRNAs, as well as down-regulated DEmiRNAs, were observed during testicular fusion, while the opposite expression pattern was observed after fusion. Enrichment analysis of DEmRNAs revealed that cAMP signal pathway, ECM remodeling enzymes, ECM-integrin interaction pathways, and cell adhesion molecules were potentially associated with testicular fusion. The identified DElncRNA-DEmiRNA-DEmRNA regulatory network related to cAMP signal pathway, ECM remodeling enzymes suggests their roles during testicular fusion. Our research will provide new targets for studying the mechanism of testicular fusion. • 2150 lncRNAs, 2742 targeted mRNAs and 347 miRNAs were identified in the testis of S. litura during testicular fusion. • A large number of DElncRNAs, targeted DEmRNAs and a few of DEmiRNAs were up-regulated when the testes began to fuse. • The remodeling enzymes, ECM-integrin interaction pathway, cAMP signaling pathway may associate with the testicular fusion. • The lncRNA-miRNA-mRNA regulatory network related to ECM remodeling enzymes may play important roles in the testicular fusion. [ABSTRACT FROM AUTHOR]

Insect gut immunity plays a key role in defense against microorganism infection. The knowledge of insect gut immunity has been obtained mostly from Drosophila melanogaster . Little is known about gut immunity in the diamondback moth, Plutella xylostella (L.), a pest destroying cruciferous crops worldwide. In this study, expressions of the immune-related genes in the midgut of P . xylostella orally infected with Staphylococcus aureus , Escherichia coli and Pichia pastoris were profiled by RNA-seq and qRT-PCR approaches. The results revealed that the Toll, IMD, JNK and JAK-STAT pathways and possibly the prophenoloxidase activation system in P . xylostella could be activated by oral infections, and moricins, gloverins and lysozyme2 might act as important effectors against microorganisms. Subsequent knock-down of IMD showed that this gene was involved in regulating the expression of down-stream genes in the IMD pathway. Our work indicates that the Toll, IMD, JNK and JAK-STAT pathways may synergistically modulate immune responses in the P . xylostella midgut, implying a complex and diverse immune system in the midgut of insects. [ABSTRACT FROM AUTHOR]

N6‐methyladenosine (m6A) RNA is the most abundant modification of mRNA, and has been demonstrated in regulating various post‐transcriptional processes. Many studies have shown that m6A methylation plays key roles in sex determination, neuronal functions, and embryonic development in Drosophila and mammals. Here, we analyzed transcriptome‐wide profile of m6A modification in the embryonic development of the destructive agricultural pest Spodoptera frugiperda. We found that the 2 key mRNA m6A methyltransferases SfrMETTL3 and SfrMETTL14 have high homologies with other insects and mammals, suggesting that SfrMETTL3 and SfrMETTL14 may have conserved function among different species. From methylated RNA immunoprecipitation sequencing analysis, we obtained 46 869 m6A peaks representing 8 587 transcripts in the 2‐h embryos after oviposition, and 41 389 m6A peaks representing 9 230 transcripts in the 24‐h embryos. In addition, 5 995 m6A peaks were differentially expressed including 3 752 upregulated and 2243 downregulated peaks. Functional analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes suggested that differentially expressed m6A peak‐modified genes were enriched in cell and organ development between the 2‐ and 24‐h embryos. By conjoint analysis of methylated RNA immunoprecipitation‐seq and RNA‐seq data, we found that RNA m6A methylation may regulate the transcriptional levels of genes related to tissue and organ development from 2‐ to 24‐h embryos. Our study reveals the role of RNA m6A epigenetic regulation in the embryonic development of S. frugiperda, and provides new insights for the embryonic development of insects. [ABSTRACT FROM AUTHOR]

Insecticides are anthropogenic environmental stressors and also a common stressor for mosquito vectors. However, the use of insecticides is often guided by short‐term efficacy, and the sublethal effect on their target or nontarget species has long been ignored. Here, we analyzed how sublethal exposure of the promising vector‐control bioinsecticide spinetoram to Aedes aegypti larvae alter adult performance and susceptibility to dengue virus (DENV) infection. We found that the surviving adult mosquitoes were significantly smaller and exhibited weaker blood‐feeding capacity than control females, apart from the extended immature development period. In terms of reproductive potential, although the F0 generation produced a similar number of eggs and offspring during the first gonotrophic cycle, the survival rates of the F1 generations were significantly lower as compared to the control group, suggesting transgenerational sublethal effects on the F1 generation. Notably, surviving adult females had higher DENV‐2 viral loads than the control group after spinetoram sublethal exposure. Mechanistically, transcriptomic analysis showed that inhibition of oxidative phosphorylation may function in stimulating DENV production in adult Ae. aegypti. In Aag2 cells, significant accumulation of apoptosis, mitochondrial reactive oxygen species production, and DENV‐2 replication by spinetoram exposure consistently support our conclusion. Our study highlights the threat of sublethal spinetoram exposure on outbreaks of mosquito‐borne viruses. [ABSTRACT FROM AUTHOR]

20E‐hydroxyecdysone (20E) plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response. Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens. 20E regulates a series of antimicrobial peptides (AMPs) through the immunodeficiency (IMD) pathway activation in Drosophila; nevertheless, whether other immune pathways are involved in 20E‐regulated insect immunity is unknown. Our previous studies showed that BmMD‐2A is a member of the MD‐2‐related lipid recognition (ML) family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway. In this study, we further demonstrate that BmMD‐2A is also positively regulated by 20E, and the BmMD‐2A neutralization experiment suggested that 20E activates some downstream immune effect factors, the AMP genes against Escherichia coli and Staphylococcus aureus, through the regulation of BmMD‐2A in larval metamorphosis, implying that B. mori may use the Toll‐ML signaling pathway to maintain innate immune balance in the larval‐pupal metamorphosis stage, which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila. [ABSTRACT FROM AUTHOR]

Drosophila NF-κB transcription factor Dif has been well known for its function in innate immunity, and recent study also reveals its role in neuronal cells. However, the underlying mechanisms of Dif in the brain remain elusive. In this study, we aim to investigate the function of Dif in Drosophila brain development and how Dif regulates structure and plasticity of the brain to affect aging and behaviors. Based on the analysis of differentially expressed genes, we identified key genes associated with cell division, development and aging in the brain of Dif 1 loss of function mutant. In Dif 1 larvae, we found that the metamorphosis and brain development were delayed, and cell division was decreased. In Dif 1 adults, the number of neuron cells was reduced in the brain, the lifespan and locomotor activity were decreased, protein markers associated with aging-related neurodegenerative diseases in the brain were altered in abundance or activity. Our results indicated that Dif plays a crucial role in brain plasticity and neurogenesis, dysfunction of Dif delays larval brain development and impacts proliferation of neuronal cells, resulting in aging adult brain by regulating expression of key genes in multiple signaling pathways involved in cell division, neurogenesis and aging. • Loss of function in NF-κB transcription factor Dif delays brain development and impact neurogenesis in larvae. • Loss of function in Dif decreases cell division in larval brain, resulting in fewer neurons and aging brain in adults. • Dif regulates expression of key genes in the Hippo, Insulin/FoxO, MAPK and Wnt signaling pathways in the brain. • Dif expression in the brain is crucial for brain plasticity and neurogenesis. [ABSTRACT FROM AUTHOR]

Basic helix‐loop‐helix (bHLH) transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes, and bHLH proteins also participate in immune responses, especially in plants. However, their roles in insects upon entomopathogen infection are unknown. In this study, 54 bHLH genes in 41 families were identified in a polyphagous pest, Spodoptera litura, including a new bHLH gene in group B, which is specifically present in Lepidoptera and was thus named Lep. The conserved amino acids in the bHLH domain, structural architecture, and chromosomal distribution of bHLH genes in S. litura were analyzed. The bHLH genes in Plutella xylostella and Apis mellifera were also updated, and genome‐wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed. The expression profiles of S. litura bHLH (SlbHLH) genes in 3 tissues at different developmental stages and their responses to S. litura nucleopolyhedrovirus (SpltNPV), Nomuraea rileyi (Nr), and Bacillus thuringiensis (Bt) infection were investigated. More SlbHLHs in group B were expressed and differentially expressed during pathogen infections, and SlbHLHs tended to be downregulated in the midgut of S. litura larvae after B. thuringiensis treatment. Our study provides an overview of bHLH family members in S. litura and their responses to different pathogens used for pest biocontrol. These findings on bHLH members may contribute to uncovering the mechanism of host–pathogen interaction. [ABSTRACT FROM AUTHOR]

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the entomopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects of destruxins on changes in free calcium and hydrogen ions in the hemocytes of Exolontha serrulata, Bombyx mori and the Spodoptera litura SL-1 cell line were detected using laser scanning confocal microscopy (LSCM). An instant Ca2+ influx of hemocytes induced by destruxins A and B (DA and DB) was recorded. The DA/DB-dependent Ca2+ influx was not influenced by the Ca2+ channel inhibitors 2-aminoethoxydiphenyl borane (2-APB) and U73122. It also had an apparently different LSCM profile from that of the ionomycin-dependent Ca2+ influx. However, the instant Ca2+ influx was not seen in the SL-1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2+ was observed. Meanwhile, an instant intracellular free H+ decrease aroused by DA and DB was found. DB at 20 μmol/L and DA at 690 μmol/L significantly reduced intracellular free H+ levels. Furthermore, the vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 had obvious effects on the decreases of intracellular free H+ in hemocytes. These results suggest that the mechanism of DA/DB-dependent Ca2+ influx is perhaps not related to Ca2+ channels and ionophores; rather, the intracellular free H+ decrease might be due to V-ATPase inhibition. [ABSTRACT FROM AUTHOR]

Cadherin, aminopeptidase N (APN) and alkaline phosphatase (ALP) have been characterized as Cry receptors. In this study, comparative genomic analysis of the 3 receptor families was performed in 7 insects. ALPs and APNs are divided into three and eight clades in phylogenetic trees, respectively. ALPs in clade 3 and APNs in clade 1 contain multiple paralogs within each species and most paralogs are located closely in chromosomes. Drosophila melanogaster has expanded APNs in clade 5 and were lowly expressed in midgut. Cadherins are divided into 16 clades; they may diverge before holometabolous insect speciation except for BtR and Cad89D‐like clades. Eight insects from different orders containing BtR orthologs are sensitive to Cry1A or Cry3A, while five species without BtR are insensitive to both toxins. Most APNs in clade 1, several ALPs in clade 3, BtR and Cad89D‐like genes were highly or moderately expressed in larval midgut of Spodoptera litura and the other six species, and several members in these clades have been identified as Cry receptors. Expressions of putative S. litura Cry receptors in the midgut after exposing to Bt toxins were also analyzed. [ABSTRACT FROM AUTHOR]

Abstract: ML (MD-2 (myeloid differentiation factor-2)-related Lipid-recognition) is a conserved domain identified in MD-2, MD-1, NPC2 (Niemann-Pick disease type C2), and mite major allergen protein from animals, plants, and fungi. Vertebrate members of the ML family proteins, such as NPC2 and MD-2, play important roles in lipid metabolism and immune signaling pathway. MD-2 is an essential co-receptor in the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) signaling pathway. Insects contain multiple ML genes, arbitrarily named md-2- or npc2-like genes. However, whether insect ML genes have functions similar to vertebrate md-2 is unknown. In Drosophila melanogaster, there are eight npc2 genes (npc2a–h), and they can be further divided into three subgroups based on the numbers of cysteine residues (6, 7 and 8 Cys) in the mature proteins. The purpose of this study is to investigate whether any Drosophila npc2 genes may have functions in immune signaling pathways. We chose npc2a, npc2e and npc2h genes representing the three subgroups for this study. We showed that recombinant NPC2a, NPC2e and NPC2h not only bound to LPS and lipid A, but also bound to peptidoglycan (PG) and lipoteichoic acid (LTA), a property that has not been reported previously for vertebrate NPC2 or MD-2. More importantly, we showed that over-expression of NPC2a and NPC2e activated diptericin promoter reporter in S2 cells stimulated by PG, suggesting that NPC2e and NPC2a may play a role in the immune deficiency (Imd) pathway. This is the first in vitro study about NPC2 proteins in innate immunity of D. melanogaster. [Copyright &y& Elsevier]

First we present a theoretical analysis of classical noise in ghost imaging system based on the coherent-mode representation theory. The classical noise depends crucially on the distribution of the eigenvalues of the coherent-mode representation of the source and the decomposition coefficients of the object imaged. We show that both decreasing the distribution of the decomposition coefficients and increasing the distribution of the eigenvalues can lead to the decrease of classical noise. [ABSTRACT FROM AUTHOR]

Abstract: Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1–4 that are located close to the C-termini. In addition, we found that synthetic Leb-B22–48 peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects. [Copyright &y& Elsevier]

Abstract: Insects produce a variety of antimicrobial peptides (AMPs). Induction of insect AMP genes is regulated by the Toll and IMD (immune deficiency) pathways via NF-κB and GATA factors. Little is known about species-specific regulation of AMP genes. In this report, we showed that activities of most Manduca sexta and Drosophila melanogaster AMP gene promoters were regulated in a species-specific manner in Drosophila (Dipteran) S2 cells and Spodoptera frugiperda (Lepidopteran) Sf9 cells. A κB-GATA element (22 bp) from M. sexta moricin (MsMoricin) promoter could significantly increase activities of Drosophila AMP gene promoters in S2 cells, and an MsMoricin promoter activating element (MPAE) (140 bp) could increase activity of drosomycin promoter specifically in Sf9 cells. However, κB and GATA factors alone were not sufficient for MsMoricin gene activation, suggesting that other co-regulators may be required to fully activate AMP genes. Our results suggest that induction of insect AMP genes may require a transcription complex composed of common nuclear factors (such as NF-κB and GATA factors) and species-related co-regulators, and it is the co-regulators that may confer species-specific regulation of AMP genes. In addition, we showed that activity of Drosophila drosomycin promoter could be activated cooperatively by the inserted exogenous κB-GATA element and the endogenous κB element. These findings revealed an approach of engineering AMP genes with enhanced activities, which may lead to broad applications. [Copyright &y& Elsevier]

Abstract: Activation of the prophenoloxidase (proPO) system and synthesis of antimicrobial peptides (including lysozyme) are two key defense mechanisms in arthropods. Activation of proPO involves a cascade of serine proteinases that eventually converts proPO to active phenoloxidase (PO). However, a trade-off between lysozyme/antibacterial activity and PO activity has been observed in some insects, and a mosquito lysozyme can inhibit melanization. It is not clear whether lysozyme can inhibit PO activity and/or proPO activation. In this study, we used in vitro assays to investigate the role of lysozyme in proPO activation in the tobacco hornworm Manduca sexta. We showed that lysozymes from M. sexta, human milk and hen egg white did not inhibit PO activity in the pre-activated naïve plasma of M. sexta larvae, but significantly inhibited proPO activation in the naïve plasma. Western blot analysis showed that direct incubation of M. sexta lysozyme with the naïve plasma prevented conversion of proPO to PO, but stimulated degradation of precursor proteins for serine proteinase homolog-2 (SPH2) and proPO-activating proteinase-1 (PAP1), two key components required for proPO activation. Far-western blot analysis showed that M. sexta lysozyme and proPO interacted with each other. Altogether, our results suggest that lysozymes may inhibit the proPO activation system by preventing conversion of proPO to PO via direct protein interaction with proPO. [Copyright &y& Elsevier]

Abstract: Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Muts strain was much higher than that by Mut+ strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210mgl−1. With the (His)6-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin–magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin–magainin fusion protein and cleave it at the carboxyl terminus of ubquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH. [Copyright &y& Elsevier]

Abstract: Hybrid antibacterial peptide CecropinAD (CAD) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach to express the hybrid peptide CAD in the methylotrophic yeast Pichia pastoris, the cDNA sequence encoding CAD was obtained by recursive PCR (rPCR) and cloned into the vector pPICZα-A. The Sac I-linearized recombinant plasmid pPICZα-CAD was transformed into P. pastoris GS115 by electroporation. Expression of recombinant CAD was induced for 96h with 1.0% methanol at 28°C, pH 5.0. The recombinant CAD was purified by two steps of reversed-phase HPLC and 1.8mg pure active CAD was obtained from 100ml culture. Tricine-SDS-PAGE and mass spectrometry analyses demonstrated that the molecular weight of the purified CAD was 3.8kDa. Analysis of circular dichroism (CD) revealed that CAD mainly has α-helixes in the presence of 10mM phosphate buffer (pH 7.2), 50% TFE/water solution (pH 2.0), or 30mM SDS (pH 10.8). FACScan analysis showed that the antibacterial mechanism of CAD is to act on the cell membrane to disrupt bacterial cell structure. Antimicrobial assays demonstrated that recombinant CAD has a broad spectrum of anti-microbial property against fungi, as well as Gram-positive and Gram-negative bacteria, but does not have hemolytic activity against human erythrocytes. Our results suggest that recombinant antimicrobial peptide CAD may serve as an attractive candidate for the development of therapeutic antimicrobial drugs. [Copyright &y& Elsevier]

Abstract: Immulectin-3 (IML-3) is a C-type lectin from the tobacco hornworm Manduca sexta that contains a motif (NWGV) similar to the BH1 motif (NWGR) of the mammalian galectin-3. IML-3 is synthesized in fat body and secreted into hemolymph, but can be translocated into hemocytes. In this study, we showed that IML-3 was predominantly localized to the nucleus of hemocytes and some metaphase, anaphase and telophase hemocytes from M. sexta larvae injected with bacterial lipopolysaccharide (LPS). IML-3 was detected in the membrane and soluble extracts of hemocytes, suggesting that it may be translocated into hemocytes via receptor-mediated endocytosis. To investigate the role of IML-3 translocation to the nucleus, we expressed recombinant wild-type IML-3 and a deletion mutant ΔIML-3 that has the NWGV motif deleted in Drosophila S2 cells. We found that recombinant wild-type IML-3, but not ΔIML-3, was localized to the nucleus of some S2 cells and also detected in the nuclear extract. Expression of recombinant wild-type IML-3, but not ΔIML-3 or GFP, increased the number of proliferating S2 cells. Our results suggest that nuclear translocation of IML-3 may stimulate hemocyte proliferation. [Copyright &y& Elsevier]

Abstract: Genomic and cDNA sequences of a Toll receptor were cloned from the tobacco hornworm, Manduca sexta. M. sexta Toll (MsToll) gene contains six introns and seven exons. The full-length cDNA of MsToll is 3495bp with an open reading frame of 2892bp, which encodes a protein of 963 amino acids. MsToll is a typical single-pass transmembrane protein containing characteristic domain architecture of Toll and Toll-like receptors, including an extracellular domain composing of leucine-rich repeats (LRRs) and a cytoplasmic TIR domain. The deduced amino acid sequence of MsToll is most similar to Apis mellifera Toll (27% identity). Reverse-transcription polymerase chain reaction (RT-PCR) showed that MsToll was expressed in hemocytes, fat body, Malpighian tubule, midgut and epidermis. Real-time PCR analysis showed that MsToll mRNA in hemocytes was significantly induced by Escherichia coli, as well as by yeast (Saccharomyces cerevisiae) and Micrococcus lysodeikticus. However, MsToll transcript in fat body was not induced by these microorganisms. Immunohistochemistry assay showed that MsToll was detected on hemocytes. The TIR domain of MsToll also has high similarity to those of vertebrate TLR4 and zebra fish TLR (35–39% identity). Our results suggest that MsToll may play a role in innate signaling in response to E. coli infections. [Copyright &y& Elsevier]

Abstract: For the waveguide grating photocoupler with novel structure [Yu XQ, Zhang X, Wong KS, Xu GB, Xu XG, Ren Y, et al. A fabrication of coupling grating in the polymeric waveguide by using two-photon initiated photopolymerization. Mater Lett 2004;58:3879–83. ; Yu XQ, Zhang X, Xu GB, Zhao HP, He W, Shao ZS, et al. Fabrication of grating waveguide and coupling grating using two-photon initiated photopolymerization. Chem J Chin Univ 2004;25(10):1931–3 (in Chinese). ], the electric fields of the TE guided wave and the TE radiating wave are obtained by solving the Helmholtz equation in the spatial rectangular coordinates. And then the relations between the loss coefficient and the different structure parameters of the waveguide and grating are analyzed by using the mode coupling theories, and their corresponding numerical simulation results are given. In the end the result obtained for this novel structure and that for conventional rectangle structure are compared, and the difference and the sameness are obtained. [Copyright &y& Elsevier]

Abstract: C-type lectins are calcium-dependent carbohydrate binding proteins, and animal C-type lectins participate in innate immunity and cell–cell interactions. In the fruit fly Drosophila melanogaster, more than 30 genes encode C-type lectin domains. However, functions of Drosophila C-type lectins in innate immunity are not well understood. This study is to investigate whether two Drosophila C-type lectins, CG33532 and CG33533 (designated as DL2 and DL3, respectively), are involved in innate immune responses. Recombinant DL2 and DL3 were expressed and purified. Both DL2 and DL3 agglutinated Gram-negative Escherichia coli in a calcium-dependent manner. Though DL2 and DL3 are predicted to be secreted proteins, they were detected on the surface of Drosophila hemocytes, and recombinant DL2 and DL3 also directly bound to hemocytes. Coating of agarose beads with recombinant DL2 and DL3 enhanced their encapsulation and melanization by Drosophila hemocytes in vitro. However, hemocyte encapsulation was blocked when the lectin-coated beads were pre-incubated with rat polyclonal antibody specific for DL2 or DL3. Our results suggest that DL2 and DL3 may act as pattern recognition receptors to mediate hemocyte encapsulation and melanization by directly recruiting hemocytes to the lectin-coated surface. [Copyright &y& Elsevier]

Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes. [Copyright &y& Elsevier]

Wolbachia are Gram‐negative endosymbionts that are known to cause embryonic lethality when infected male insects mate with uninfected females or with females carrying a different strain of Wolbachia, a situation characterized as cytoplasmic incompatibility (CI). However, the mechanism of CI is not yet fully understood, although recent studies on Drosophila melanogaster have achieved great progress. Here, we found that Wolbachia infection caused changes in the expressions of several immunity‐related genes, including significant upregulation of kenny (key), in the testes of D. melanogaster. Overexpression of key in fly testes led to a significant decrease in egg hatch rates when these flies mate with wild‐type females. Wolbachia‐infected females could rescue this embryonic lethality. Furthermore, in key overexpressing testes terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labeling signal was significantly stronger than in the control testes, and the level of reactive oxygen species was significantly increased. Overexpression of key also resulted in alterations of some other immunity‐related gene expressions, including the downregulation of Zn72D. Knockdown of Zn72D in fly testes also led to a significant decrease in egg hatch rates. These results suggest that Wolbachia might induce the defect in male host fertility by immunity‐related pathways and thus cause an oxidative damage and cell death in male testes. [ABSTRACT FROM AUTHOR]

Introduction: Lung cancer is the leading cause of cancer-associated mortality worldwide. Recently, long non-coding RNAs (lncRNAs) have been studied as key regulators in some biological processes. Of note, the molecular mechanism and prognostic value of lncRNAs in non-small cell lung cancer (NSCLC) have largely remained unclear.Material and Methods: In this study, we compared the PTTG3P expression levels between lung cancer and normal lung samples by analyzing 5 public datasets (GSE18842, GSE19804, GSE27262, GSE30219, and GSE19188). Next, pentose phosphate pathway and co-expression networks were constructed to identify key targets of lncRNA PTTG3P. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to explore the potential roles of lncRNA PTTG3P. Moreover, we constructed PTTG3P-mediated ceRNA networks in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC).Results: In the present study, our analysis showed that PTTG3P expression was higher in high T stage LUAD and LUSC samples, as well as high N stage NSCLC tissues. Of note, we found that higher PTTG3P expression is correlated with shorter survival time in NSCLC patients by analyzing Kaplan-Meier plotter datasets. We found that PTTG3P was significantly associated with NSCLC cell proliferation regulation by affecting a series of cell cycle related biological processes.Conclusions: Bioinformatics analysis showed that PTTG3P was associated with NSCLC cell proliferation. These results suggested that PTTG3P could serve as a new therapeutic and prognostic target for NSCLC. [ABSTRACT FROM AUTHOR]

Galectins are a family of β-galactoside binding proteins, and insect galectins play a role in immune responses and may also affect Cry toxin activity. In this study, we aimed to further understand the function and molecular mechanism of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity. A. aegypti galectin-6 was cloned, and the recombinant galectin-6 was expressed and purified. Bioassays indicated that galectin-6 could reduce the toxicity of Cry11Aa, protecting A. aegypti larvae. To determine interactions among galectin-6, Cry11Aa and putative toxin receptors, Octet Red System, western blotting, far-western blotting and ELISA assays were performed. Octet Red System showed that galectin-6 bound to BBMVs of A. aegypti larvae with lower affinity than that of Cry11Aa. Western blotting and far-western blotting analyses demonstrated that galectin-6 bound to A. aegypti ALP1 and APN2 as well as to BBMVs, consistent with the results of ELISA and protein docking simulations. However, galectin-6 did not bind to Cadherin in far-western blotting or ELISA assay, though the protein docking simulations suggested their binding potential. These findings support the conclusion that galectin-6 may block Cry11Aa from binding to ALP1 and APN2 due to structural similarity, which might decrease the mosquitocidal toxicity of Cry11Aa. Unlabelled Image • Aedes aegypti galectin-6 was cloned, recombinant galectin-6 was expressed and purified in this study. • Bioassays indicated that galectin-6 could inhibit the toxicity of Cry11Aa, protecting Ae. aegypti larvae. • Far-western blotting and ELISA analyses showed galectin-6 bound to Ae. aegypti ALP1 and APN2, but not Cadherin. • Results showed that galectin-6 may block Cry11Aa from binding to ALP1 and APN2, which might decrease Cry11Aa toxicity. [ABSTRACT FROM AUTHOR]

Background: Bacillus thuringiensis israelensis (Bti) is a widely used mosquitocidal microbial pesticide due to its high toxicity. ATP-binding proteins (ABP) are prevalently detected in insects and are related to reaction against Bti toxins. However, the function of ABP in mosquito biocontrol is little known, especially in Aedes aegypti. Therefore, this study aimed to clarify the function of ABP in Ae. aegypti against Bti toxin. Results: Aedes aegypti ABP (GenBank: XM_001661856.2) was cloned, expressed and purified in this study. Far-western blotting and ELISA were also carried out to confirm the interaction between ABP and Cry11Aa. A bioassay of Cry11Aa was performed both in the presence and absence of ABP, which showed that the mortality of Ae. aegypti is increased with an increase in ABP. Conclusions: Our results suggest that ABP in Ae. aegypti can modulate the toxicity of Cry11Aa toxin to mosquitoes by binding to Bti toxin. This could not only enrich the mechanism of Bt toxin, but also provide more data for the biocontrol of this transmission vector. [ABSTRACT FROM AUTHOR]

Abstract An important innate immune response in Drosophila melanogaster is the production of antimicrobial peptides (AMPs). Expression of AMP genes is mediated by the Toll and immune deficiency (IMD) pathways via NF-κB transcription factors Dorsal, DIF and Relish. Dorsal and DIF act downstream of the Toll pathway, whereas Relish acts in the IMD pathway. Dorsal and DIF are held inactive in the cytoplasm by the IκB protein Cactus, while Relish contains an IκB-like inhibitory domain at the C-terminus. NF-κB factors normally form homodimers and heterodimers to regulate gene expression, but formation of heterodimers between Relish and DIF or Dorsal and the specificity and activity of the three NF-κB homodimers and heterodimers are not well understood. In this study, we compared the activity of Rel homology domains (RHDs) of Dorsal, DIF and Relish in activation of Drosophila AMP gene promoters, demonstrated that Relish-RHD (Rel-RHD) interacted with both Dorsal-RHD and DIF-RHD, Relish-N interacted with DIF and Dorsal, and overexpression of individual RHD and co-expression of any two RHDs activated the activity of AMP gene promoters to various levels, suggesting formation of homodimers and heterodimers among Dorsal, DIF and Relish. Rel-RHD homodimers were stronger activators than heterodimers of Rel-RHD with either DIF-RHD or Dorsal-RHD, while DIF-RHD-Dorsal-RHD heterodimers were stronger activators than either DIF-RHD or Dorsal-RHD homodimers in activation of AMP gene promoters. We also identified the nucleotides at the 6th and 8th positions of the 3' half-sites of the κB motifs that are important for the specificity and activity of NF-κB transcription factors. Highlights • Relish-N interacted with DIF and Dorsal, suggesting formation of NF-κB homodimers and heterodimers. • Homodimers and heterodimers of DIF, Dorsal and Relish RHDs activated antimicrobial peptide gene promoters. • Relish-RHD homodimers were stronger activators than heterodimers of Relish-RHD with DIF-RHD or Dorsal-RHD. • DIF-RHD-Dorsal-RHD heterodimers were stronger activators than DIF-RHD or Dorsal-RHD homodimers. • The nucleotides at the 6th and 8th positions of NF-κB motifs are important for specificity and activity of NF-κB factors. [ABSTRACT FROM AUTHOR]

Abstract Drosophila melanogaster possesses a sophisticated and effective immune system composed of humoral and cellular immune responses, and production of antimicrobial peptides (AMPs) is an important defense mechanism. Expression of AMPs is regulated by the Toll and IMD (immune deficiency) pathways. Production of AMPs can be systemic in the fat body or a local event in the midgut and epithelium. So far, most studies focus on systemic septic infection in adult flies and little is known about AMP gene activation after ingestion of killed bacteria. In this study, we investigated activation of AMP genes in the wild-type w 1118 , MyD88 and Imd mutant flies after ingestion of heat-killed Escherichia coli and Staphylococcus aureus. We showed that ingestion of E. coli activated most AMP genes, including drosomycin and diptericin , in the first to third instar larvae and pupae, while ingestion of S. aureus induced only some AMP genes in some larval stages or in pupae. In adult flies, ingestion of killed bacteria activated AMP genes differently in males and females. Interestingly, ingestion of killed E. coli and S. aureus in females conferred resistance to septic infection by both live pathogenic Enterococcus faecalis and Pseudomonas aeruginosa , and ingestion of E. coli in males conferred resistance to P. aeruginosa infection. Our results indicated that E. coli and S. aureus can activate both the Toll and IMD pathways, and systemic and local immune responses work together to provide Drosophila more effective protection against infection. Highlights • Feeding killed E. coli in Drosophila activated most AMP genes in larvae and pupae. • Feeding S. aureus activated only some AMP genes in some larval stages or in pupae. • Feeding killed bacteria activated AMP genes differently in adult females and males. • Feeding E. coli and S. aureus triggered both the Toll and IMD pathways. • Feeding bacteria protects flies from septic infection by E. faecalis and P. aeruginosa. [ABSTRACT FROM AUTHOR]

Abstract Wolbachia are endosymbiotic bacteria present in a wide range of invertebrates. Although their dramatic effects on host reproductive biology have been well studied, little is known about the effects of Wolbachia on the learning and memory capacity (LMC) of hosts, despite their distribution in the host nervous system, including brain. In this study, we found that Wolbachia infection significantly enhanced LMC in both Drosophila melanogaster and D. simulans. Expression of LMC-related genes was significantly increased in the head of D. melanogaster infected with the w Mel strain, and among these genes, crebA was up-regulated the most. Knockdown of crebA in Wolbachia -infected flies significantly decreased LMC, while overexpression of crebA in Wolbachia -free flies significantly enhanced the LMC of flies. More importantly, a microRNA (miRNA), dme-miR-92b , was identified to be complementary to the 3'UTR of crebA. Wolbachia infection was correlated with reduced expression of dme-miR-92b in D. melanogaster , and dme-miR-92b negatively regulated crebA through binding to its 3'UTR region. Overexpression of dme-miR-92b in Wolbachia -infected flies by microinjection of agomirs caused a significant decrease in crebA expression and LMC, while inhibition of dme-miR-92b in Wolbachia -free flies by microinjection of antagomirs resulted in a significant increase in crebA expression and LMC. These results suggest that Wolbachia may improve LMC in Drosophila by altering host gene expression through a miRNA-target pathway. Our findings help better understand the host-endosymbiont interactions and, in particular, the impact of Wolbachia on cognitive processes in invertebrate hosts. Graphical abstract Image 1 Highlights • Wolbachia infection improves the learning and memory capacity (LMC) of Drosophila. • Wolbachia infection increases the expression of crebA. • Wolbachia infection decreases the expression of dme-miR-92b. • CrebA is negatively regulated by dme-miR-92b. • Wolbachia improves LMC likely by altering crebA expression through dme-miR-92b. [ABSTRACT FROM AUTHOR]

Abstract Antimicrobial peptides (AMPs) are important effector molecules of insect humoral immunity, and expression of AMPs is mainly regulated by the Toll and immune deficiency (IMD) pathways. FoxO, a key downstream regulator of the insulin-like signaling (ILS) pathway, has been recently reported to be involved in the regulation of AMPs in Drosophila melanogaster. In the present study, we investigated AMP gene expression and the regulation pathway controlled by the starvation in the silkworm Bombyx mori. We discovered that antibacterial activity in the hemolymph of B. mori larvae was increased by starvation, and expression of AMP genes (BmCecB6 , BmAtta1 , BmLeb3 and BmDefB) as well as the ILS target genes (FoxO , InR and Brummer) were strongly activated in the fat body by starvation. Moreover, phosphorylation of Akt kinase was reduced in the Bm-12 cells after starvation, suggesting that the ILS pathway was inhibited by starvation. We then showed that more FoxO protein was present in the cytoplasm than in the nucleus of Bm-12 cells under normal conditions, but more FoxO was detected in the nucleus after cells were starved for 8 h, indicating that FoxO was activated by starvation. In summary, our results indicated that starvation can activate AMP gene expression in B. mori via the ILS/FoxO signaling pathway. Graphical abstract Image 1 Highlights • Transcription factor FoxO can be activated by starvation in Bombyx mori. • Overexpression of an active form FoxOCA can induce promoter activity of BmDefB. • RNAi of insulin receptor (InR) decreases the insulin-like signal (ILS) and activates BmDefB. • Starvation upregulates the expression of AMPs via the ILS/FoxO signaling pathway. [ABSTRACT FROM AUTHOR]