CircRNAs regulate the crosstalk between inflammation and tumorigenesis: The bilateral association and molecular mechanisms.

Inflammation, a hallmark of cancer, has been associated with tumor progression, transition into malignant phenotype and efficacy of the chemotherapeutic agents in cancer. Chronic inflammation provides a favorable environment for tumorigenesis by inducing immunosuppression, whereas acute inflammation prompts tumor suppression by generating anti-tumor immune responses. Inflammatory factors derived from interstitial cells or tumor cells can stimulate cell proliferation and survival by modulating oncogenes and/or tumor suppressors. Recently, a new class of RNAs, i.e., circular RNAs (circRNAs), has been implicated in inflammatory diseases. Although there are reports on circRNAs imparting functions in inflammatory insults, whether these circularized transcripts hold the potential to regulate inflammation-induced cancer or tumor-related inflammation, and modulate the interactions between tumor microenvironment (TME) and the inflammatory stromal/immune cells, awaits further elucidation. Contextually, the current review describes the molecular association between inflammation and cancer, and spotlights the regulatory mechanisms by which circRNAs can moderate TME in response to inflammatory signals/triggers. We also present comprehensive information about the immune cell(s)-specific expression and functions of the circRNAs in TME, modulation of inflammatory signaling pathways to drive tumorigenesis, and their plausible roles in inflammasomes and tumor development. Moreover, the therapeutic potential of these circRNAs in harnessing inflammatory responses in cancer is also discussed.

Circular RNA translation: novel protein isoforms and clinical significance.

In recent years, significant attention has focused on circular RNA (circRNA) translation to determine its clinical significance. Cap-independent translation of circRNAs driven by an internal ribosome entry site (IRES) or an N6-methyladenosine (m6A)-containing short sequence is different from the canonical cap-dependent translation of linear mRNAs. New proteins or isoforms possessing novel physiological roles can be generated from translatable circRNAs. The present review describes the elements involved in circRNA translation, and the functions of the translated novel protein isoforms in human diseases. Bifunctional characteristics of translatable circRNAs exerted by the circRNAs and the translated proteins are also discussed. Furthermore, various molecular strategies that could be used as appropriate therapeutic options are proposed.

Divergence and correlated evolution of male wing spot and courtship display between Drosophila nepalensis and D. trilutea.

Male-specific wing spots are usually associated with wing displays in the courtship behavior of Drosophila and may play important roles in sexual selection. Two closely related species, D. nepalensis and D. trilutea, differ in wing spots and scissoring behavior. Here, we compare male morphological characters, pigmentation intensity of male wing spots, wing-scissoring behavior, courtship songs, and reproductive isolation between 2 species. F1 fertile females and sterile males result from the cross between females of D. nepalensis and males of D. trilutea. The pigmentation of wing spots is significantly weaker in D. trilutea than in D. nepalensis and the F1 hybrid. Males scissor both wings in front of the female during courtship, with a posture spreading wings more widely, and at a faster frequency in D. nepalensis than in D. trilutea and the F1s. Males of D. trilutea vibrate wings to produce 2 types (A and B) of pulse songs, whereas D. nepalensis and the F1s sing only type B songs. The incidence of wing vibration and scissoring during courtship suggests that wing vibration is essential but scissoring is a facultative courtship element for successful mating in both species. The association between the darker wing spots with more elaborate scissoring might be the consequence of correlated evolution of these traits in D. nepalensis; however, D. trilutea retains wing scissoring during courtship despite having weaker pigmentation of wing spots. The genetic architecture of 2 traits differs in the F1s, consistent with maternal or sex-linked effects for spots but nonadditive effects for scissoring.

Evolution of a neuromuscular sexual dimorphism in the Drosophila montium species group.

While epigamic traits likely evolve via sexual selection, the mechanism whereby internal sexual dimorphism arises remains less well understood. Seeking clues as to how the internal sexual dimorphism evolved, we compared the abdominal musculature of 41 Drosophila montium group species, to determine whether any of these species carry a male-specific muscle of Lawrence (MOL). Our quantitative analysis revealed that the size of a sexually dimorphic MOL analog found in 19 montium group species varied widely from species to species, suggesting the gradual evolution of this sexually dimorphic neuromuscular trait. We attempted the ancestral state reconstitution for the presence or absence of the neuromuscular sexual dimorphism in the A5 segment; the neuromuscular sexual dimorphism existed in an old ancestor of the montium group, which was lost in some of the most recent common ancestors of derived lineages, and subsequently some species regained it. This loss-and-gain history was not shared by evolutionary changes in the courtship song pattern, even though both traits were commonly regulated by the master regulator male-determinant protein FruM. It is envisaged that different sets of FruM target genes may serve for shaping the song and MOL characteristics, respectively, and, as a consequence, each phenotypic trait underwent a distinct evolutionary path.

Evolution and diversity of the courtship repertoire in the Drosophila montium species group (Diptera: Drosophilidae).

Changes in elements of courtship behaviour can influence sexual isolation between species. Large-scale analyses of changes, including loss and gain of courtship elements, across a relatively complete phylogenetic group are rare but needed to understand the significance of such changes, for example whether the gain and loss of courtship elements are essentially arbitrary or equally reversible. In most species of Drosophila, courtship, including singing, mainly occurs before mounting as premounting courtship. The Drosophila montium species group is unusual because loss of premounting courtship and gain of post-mounting one has been detected in this group. Here, we provide an extensive analysis on the courtship repertoire and songs of 42 species in this group. Synchronously captured video and audio recordings were analysed to describe courtship patterns and male courtship songs, and changes were analysed in a phylogenetic context. Ancestral state reconstruction suggests that a gain of post-mounting courtship singing at the ancestor of this species group has been accompanied by a concurrent decrease in the incidence of premounting courtship singing and has led to subsequent further decrease and eventually complete loss of premounting courtship song in several lineages. Alongside this evolutionary trend towards post-mounting courtship, sine song and a special type of "high pulse repetition song" have become more widely used for courtship during species diversification in the montium group. It is likely that the elaboration of post-mounting courtship behaviours is associated with changes in the relative importance of pre- and post-mounting components of mating systems, such as sperm competition or cryptic female choice.

Exploring the Caste-Specific Multi-Layer Defense Mechanism of Formosan Subterranean Termites, Coptotermes formosanus Shiraki.

The survival and foraging of Coptotermesformosanus Shiraki in a microbe-rich environment reflect the adaptation of an extraordinary, sophisticated defense mechanism by the nest-mates. We aimed to explore the host pathogen interaction by studying caste-specific volatile chemistry and genes encoding the antioxidant defense of winged imagoes, nymphs, soldiers and workers of Formosan subterranean termites. Qualitative analyses of C.formosanus Shiraki performed by HS-SPME/GC-MS showed considerable variations in the chemical composition of volatile organic compounds (VOCs) and their proportions among all the castes. Winged imagoes produced the most important compounds such as naphthalene and n-hexanoic acid. The antifungal activity of these compounds along with nonanal, n-pentadecane, n-tetradecane, n-heptadecane and methyl octanoate against the conidial suspensions of Metarhiziumanisopliae and Beauveriabassiana isolates enable us to suggest that the failure of natural fungal infection in the nest is due to the antiseptic environment of the nest, which is mainly controlled by the VOCs of nest-mates. In addition, conidial germination of M.anisopliae and B.bassiana isolates evaluated on the cuticle of each caste showed significant variations among isolates and different castes. Our results showed that the conidia of M.anisopliae 02049 exhibited the highest germination on the cuticle of all the inoculated castes. Moreover, we recorded the lowest germination of the conidia of B.bassiana 200436. Caste-specific germination variations enabled us to report for the first time that the cuticle of winged imagoes was found to be the most resistant cuticle. The analysis of the transcriptome of C.formosanus Shiraki revealed the identification of 17 genes directly involved in antioxidant defense. Expression patterns of the identified antioxidant genes by quantitative real-time PCR (qPCR) revealed the significantly highest upregulation of CAT, GST, PRXSL, Cu/Zn-SOD2, TXN1, TXN2, TXNL1, TXNL2, TXNL4A and TPx genes among winged imagoes upon infection with the most virulent isolate, M.anisopliae 02049. Furthermore, soldiers showed the least expression of genes encoding antioxidant defense. Our findings indicated that the volatile chemistry of nest-mates and genes encoding antioxidant defense greatly contribute to the survival and foraging of Formosan subterranean termites in a microbe-rich habitat.

Courtship patterns in the Drosophila montium species subgroup: repeated loss of precopulatory courtship?

During precopulatory courtship, male Drosophila typically produce wing vibration to generate species-specific songs before mounting females. Three species in the lini clade of the montium species subgroup have been found to produce species-specific sine song only after mounting and during copulation. Here we investigated and analyzed the courtship behavior of 29 species in the montium subgroup from video and song recordings and measured the duration of wing vibration. We describe a great diversity of courtship behavior in the montium subgroup. The courtship patterns can be categorized into four types in the montium subgroup: 1) type P/C, species with both precopulatory and copulatory courtship, such as D. parvula and D. nikananu, 2) type P-/C, species with sporadic precopulatory and mainly copulatory courtship, such as D. auraria and D. triauraria. 3) type C, species with only copulatory courtship, such as D. tani and D. pectinifera, 4) type C-, species with only very brief copulatory courtship, such as D. rufa and D. asahinai. According to a phylogenetic tree based on sequences of mitochondrial COI and COII, and the nuclear Adh, both precopulatory courtship and copulatory courtship were present in the most basal species D. parvula. Each of two branches in the montium subgroup contains four types of courtship behavior. Type C is present in each sub-branch. These results suggest that the courtship behavior initially involved both precopulatory and copulatory courtship, but that subsequently precopulatory courtship has gradually been lost in the montium subgroup. We suggest reasons why precopulatory behavior might come to be lost in the montium subgroup.

Immune-related transcriptome of Coptotermes formosanus Shiraki workers: the defense mechanism.

Formosan subterranean termites, Coptotermes formosanus Shiraki, live socially in microbial-rich habitats. To understand the molecular mechanism by which termites combat pathogenic microbes, a full-length normalized cDNA library and four Suppression Subtractive Hybridization (SSH) libraries were constructed from termite workers infected with entomopathogenic fungi (Metarhizium anisopliae and Beauveria bassiana), Gram-positive Bacillus thuringiensis and Gram-negative Escherichia coli, and the libraries were analyzed. From the high quality normalized cDNA library, 439 immune-related sequences were identified. These sequences were categorized as pattern recognition receptors (47 sequences), signal modulators (52 sequences), signal transducers (137 sequences), effectors (39 sequences) and others (164 sequences). From the SSH libraries, 27, 17, 22 and 15 immune-related genes were identified from each SSH library treated with M. anisopliae, B. bassiana, B. thuringiensis and E. coli, respectively. When the normalized cDNA library was compared with the SSH libraries, 37 immune-related clusters were found in common; 56 clusters were identified in the SSH libraries, and 259 were identified in the normalized cDNA library. The immune-related gene expression pattern was further investigated using quantitative real time PCR (qPCR). Important immune-related genes were characterized, and their potential functions were discussed based on the integrated analysis of the results. We suggest that normalized cDNA and SSH libraries enable us to discover functional genes transcriptome. The results remarkably expand our knowledge about immune-inducible genes in C. formosanus Shiraki and enable the future development of novel control strategies for the management of Formosan subterranean termites.

DNA Barcoding and Molecular Phylogeny of Drosophila lini and Its Sibling Species.

Drosophila lini and its two sibling species, D. ohnishii and D. ogumai, are hardly distinguishable from one another in morphology. These species are more or less reproductively isolated. The mitochondrial ND2 and COI-COII and the nuclear ITS1-ITS2 regions were sequenced to seek for the possibility of DNA barcoding and to reconstruct the phylogeny of them. The character-based approach for DNA barcoding detected some diagnostic nucleotides only for monophyletic D. ogumai, but no informative sites for the other two very closely species, D. lini and D. ohnishii, of which strains intermingled in the molecular phylogenetic trees. Thus, this study provides another case of limited applicability of DNA barcoding in species delineation, as in other cases of related Drosophila species. The molecular phylogenetic tree inferred from the concatenated sequences strongly supported the monophyly of the cluster of the three species, that is, the lini clade. We propose some hypotheses of evolutionary events in this clade.

Copulatory courtship behavior and sine song as a mate recognition cue in Drosophila lini and its sibling species.

Most Drosophila species sing species-specific pulse songs during their "precopulatory courtship." Three sibling species of the Drosophila montium species subgroup performed "copulatory courtship": males generated courtship songs by vibrating either wing only after mounting and during copulation. In these three species, strong sexual isolation was detected between D. ohnishii and D. lini and between D. ohnishii and D. ogumai, but not between D. lini and D. ogumai. Female showed strong repelling behavior when they were mounted by a heterospecific male in the species combinations including D. ohnishii, resulting in failure of the copulation attempt of the male. Acoustic analyses of courtship songs revealed that the pulse song was irregular, without any species-specific parameters, but that the frequency of the sine song was different among the three species in accordance with the modes of sexual isolation between them; it was significantly lower in D. ohnishii (mean ± SE = 193.0 ± 1.7 Hz) but higher in D. lini (253.4 ± 2.7 Hz) and D. ogumai (246.7 ± 5.3 Hz). We suggest that this difference in the sine song frequency is a sexual signal in the Specific Mate Recognition System (SMRS) among these three Drosophila species.

Gene expression divergence and evolutionary analysis of the drosomycin gene family in Drosophila melanogaster.

Drosomycin (Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes, Dro1, Dro2, Dro3, Dro4, Dro5, and Dro6, forming a multigene family on the 3L chromosome arm in Drosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the seven drosomycin genes, Drs, Dro2, Dro3, Dro4, and Dro5 showed constitutive expressions. Three out of five, Dro2, Dro3, and Dro5, were able to be upregulated by simple injury. Interestingly, Drs is an only gene strongly upregulated when Drosophila was infected with microbes. In contrast to these five genes, Dro1 and Dro6 were not transcribed at all in either noninfected or infected flies. Furthermore, by 5' rapid amplification of cDNA ends, two transcription start sites were identified in Drs and Dro2, and one in Dro3, Dro4, and Dro5. In addition, NF-kappaB binding sites were found in promoter regions of Drs, Dro2, Dro3, and Dro5, indicating the importance of NF-kappaB binding sites for the inducibility of drosomycin genes. Based on the analyses of flanking sequences of each gene in D. melanogaster and phylogenetic relationship of drosomycins in D. melanogaster species-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates of drosomycin genes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes.

Molecular and functional characterization of a c-type lysozyme from the Asian corn borer, Ostrinia furnacalis.

Some lepidopteran lysozymes have been reported to display activity against Gram-positive and Gram-negative bacteria, in contrast to most lysozymes that are active only against Gram-positive bacteria. OstrinLysC, a c-type lysozyme, was purified from the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae), and shows activity against Gram-positive and Gram-negative bacteria. The NH2-terminal amino acid sequence was determined by Edman degradation and used in a homology cloning strategy. The gene coding for OstrinLysC contains three exons and two introns. The expression profile of the OstrinlysC gene was examined by quantitative real-time PCR. Following injection of the larvae with bacteria, the OstrinlysC gene is strongly up-regulated in immune tissues. Transcripts were also detected in gut tissue. After feeding the larvae with bacteria, OstrinlysC transcripts increased in immune tissues. A very low level of transcript abundance was also detected in gut tissue. These results suggested that the OstrinlysC gene is involved in immune responses. The three dimensional structure of OstrinLysC was predicted. Based on comparison of the 3-D structure of OstrinLysC with that of silkworm lysozyme and chicken lysozyme, we hypothesize that the positive charge-rich surface and the short loop-2, which is close to the cluster of hydrophobic residues, may play important roles in the interaction with the outer membrane of Gram-negative bacterial cell walls.

Bactrocerin-1: a novel inducible antimicrobial peptide from pupae of oriental fruit fly Bactrocera dorsalis Hendel.

A novel antimicrobial peptide, Bactrocerin-1, was purified and characterized from an immunized dipteran insect, Bactrocera dorsalis. Bactrocerin-1 has 20 amino acid residues with a mass of 2,325.95 Da. The amino acid sequence of Bactrocerin-1 showed very high similarity to the active fragment (46V-65S-NH(2)) of Coleoptericin A. The composition of amino acid residues revealed that Bactrocerin-1 is a hydrophobic, positively charged, and Lys/Ile/Gly-rich peptide. Minimal growth inhibition concentration (MIC) measurements for synthesized Bactrocerin-1 showed a very broad spectrum of anti-microbial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. Bactrocerin-1 did not show hemolytic activity toward mouse red blood cells even at a concentration of 50 microM. Analysis of the Helical-wheel projection and the CD spectrum suggested that Bactrocerin-1 contains the amphipathic alpha-helix.

Functional divergence of six isoforms of antifungal peptide Drosomycin in Drosophila melanogaster.

Drosomycin (Drs) gene encodes a 44-residue inducible antifungal peptide, Drosomycin, in Drosophila melanogaster. Six genes, Drs-lC, Drs-lD, Drs-lE, Drs-lF, Drs-lG and Drs-lI, show homology to the Drs form in a multigene family on the 3rd chromosome of D. melanogaster. It is the first experimental demonstration that the six members in the Drs family act as functional genes. To further delineate the functional divergence of these six members, their cDNA sequences were cloned respectively into the pET-3C vector and expressed in the E. coli. The antifungal activity of the expression products was assayed using the Cerletti's method. The results showed a difference among the six isoforms in antifungal activity against the tested fungal strains: in which Drs was most effective and showed antifungal activity to all seven fungal strains, whereas isoform Drs-lC was effective to six strains, Drs-lD was effective to five strains, Drs-lG was effective to four strains, and Drs-lE and Drs-lF were effective to only three strains. Drs-lI had no activity against any tested fungal strains. By comparing the variable residue sites of these six isoforms to that of Drosomycin in the three-dimensional structure, we suggested that the reduction in the antifungal activity was due to the variable residues that were not in the alpha-helix. In addition, two inserted residues (RV) in Drs-lI may affect the dimensional structure and resulted in a functional change. These results may explain the evolution of the Drosomycin multigene family and its functional divergence.

Strong purifying selection on the Odysseus gene in two clades of sibling species of the Drosophila montium species subgroup.

The Odysseus (OdsH) gene was duplicated from its ancestral neuron-expressed gene, unc-4, and then evolved very rapidly under strong positive Darwinian selection as a speciation gene causing hybrid-male sterility between closely related species of the Drosophila simulans clade. Has OdsH also experienced similar positive selection between Drosophila sibling species other than those of the simulans clade? We cloned and sequenced OdsH and unc-4 from two clades of the Drosophila montium species subgroup, the Drosophila lini and the Drosophila kikkawai clades. The ratios of Ka/Ks for OdsH were remarkably low between sibling species of these two clades, suggesting that OdsH has been subjected to strong purifying selection in these two clades.