Transposition burst of mariner-like elements in the sequenced genome of Rhodnius prolixus

Transposable elements (TEs) are widespread in insect's genomes. However, there are wide differences in the proportion of the total DNA content occupied by these repetitive sequences in different species. We have analyzed the TEs present in R. prolixus (vector of the Chagas disease) and showed that 3.0% of this genome is occupied by Class II TEs, belonging mainly to the Tc1-mariner superfamily (1.65%) and MITEs (1.84%). Interestingly, most of this genomic content is due to the expansion of two subfamilies belonging to: irritans himar, a well characterized subfamily of mariners, and prolixus1, one of the two novel subfamilies here described. The high amount of sequences in these subfamilies suggests that bursts of transposition occurred during the life cycle of this family. In an attempt to characterize these elements, we performed an in silico analysis of the sequences corresponding to the DDD/E domain of the transposase gene. We performed an evolutionary analysis including network and Bayesian coalescent-based methods in order to infer the dynamics of the amplification, as well as to estimate the time of the bursts identified in these subfamilies. Given our data, we hypothesized that the TE expansions occurred around the time of speciation of R. prolixus around 1.4 mya. This suggestion lays on the “Transposon Model” of TE evolution, in which the members of a TE population that are replicative active are present at multiple loci in the genome, but their replicative potential varies, and of the “Life Cycle Model” that states that when present-day TEs have been involved in amplification bursts, they share an ancestral copy that dates back to this initial amplification.     

Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the “3R” principles of replacing, reducing and refining animal tests, the “binding and cleavage” (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics.Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit.In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.     

When physical oceanography meets population genetics: The case study of the genetic/evolutionary discontinuity in the endangered goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species

Epinephelus itajara is one of the marine fish species most threatened for extinction and it is considered to be “critically endangered” by the IUCN. The present study evaluated the genetic diversity of the species and the genetic/evolutionary relationships of its populations along the Atlantic coast of South America. The results indicate relatively reduced genetic variation, re-emphasizing the low adaptive potential of the species. One of the populations presented relatively high degrees of genetic diversity and it is evolutionary isolated from the all other populations. The evidences indicate the existence of two Evolutionarily Significant Units comprising E. itajara in the Atlantic coast of South America and the conservation prospects for the species must take these evidences into account.     

Cuticular wax variation in the tomato (Solanum lycopersicum L.), related wild species and their interspecific hybrids

The tomato (Solanum lycopersicum L.) is one of the world's most important vegetable crop species. Among the many tomato accessions available, only a few are tolerant to abiotic stresses, which are responsible for the majority of the crop losses worldwide. Wild tomato species are then secondary gene pool in the breeding of more resistant tomato cultivars. In the current study, the composition of leaf cuticular waxes from fourteen tomato accessions, including S. lycopersicum, Solanum pennellii, Solanum pimpinellifolium, and their interspecific hybrids was studied in order to select the most adequate chemotaxonomic markers. Total cuticular wax load of S. pennellii plants was much higher than in the other plant species. Hydrocarbons were usually the most abundant wax components, followed by minor quantities of triterpenes and other compounds. Interspecific hybrids showed intermediate wax characteristics. The amount and composition of surface waxes were not correlated with the abiotic stress tolerance in S. lycopersicum. The composition of the hydrocarbon fraction was the least variable both within a single accession and between all the plants studied. Based on the results, cuticular hydrocarbons are proposed as potential chemotaxonomic markers in the classification of tomato and related species.     

Characterization of three serotonin receptors from the small white butterfly,Pieris rapae

Serotonin (5-hydroxytryptamine, 5-HT) plays a key role in modulating diverse physiological processes and behaviors in both protostomes and deuterostomes. These functions are mediated through the binding of serotonin to its receptors, which are recognized as potential insecticide targets. We investigated the sequence, pharmacology and tissue distribution of three 5-HT receptors (Piera5-HT_1A, Piera5-HT_1B, Piera5-HT₇) from the small white butterfly Pieris rapae, an important pest of cultivated cabbages and other mustard family crops. Activation of Piera5-HT_1A or Piera5-HT_1B by 5-HT inhibited the production of cAMP in a dose-dependent manner. Stimulation of Piera5-HT₇ with 5-HT increased cAMP level significantly. Surprisingly, with the exception of 5-methoxytryptamine, agonists including α-methylserotonin, 8-Hydroxy-DPAT and 5-carboxamidotryptamine activated these receptors poorly. The results are consistent with previous findings in Manduca sexta. All three receptors were blocked by methiothepin, but ketanserin and yohimbine were not effective. The selective mammalian 5-HT receptor antagonists SB 216641 and SB 269970 displayed potent inhibition effects on Piera5-HT_1B and Piera5-HT₇ respectively. The results we achieved here indicate that the pharmacological properties of Lepidoptera 5-HT receptors are quite different from those in other insects and vertebrates and may contribute to development of new selective pesticides. This study offers important information on three 5-HT receptors from P. rapae that will facilitate further analysis of the functions of 5-HT receptors in insects.     

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two α-helical turns downstream a stutter in the central coiled-coil helix - precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the “SOSIP” mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form. These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization.     

PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

Gene expression and molecular characterization of a novel C-type lectin,encapsulation promoting lectin (EPL), in the rice armyworm,Mythimna separata

Insect cellular immune reactions differ depending on the target species. Phagocytosis is activated to scavenge microorganisms such as bacteria and fungi. On the other hand, larger invaders such as parasitoid wasps are eliminated by activation of encapsulation. In this study, we hypothesized that novel determinants regulate cellular immunities independent of surface molecular pattern recognition involving pattern recognition receptors (PRRs). Immune-related genes differentially expressed depending on the treated material size were screened in larval hemocytes of the rice armyworm, Mythimna separata. Consequently, we identified a novel C-type lectin gene up-regulated by injection of large beads but not small beads of identical material. Examination of in vitro effect of the recombinant protein on the immune reactions clarified that the protein activated encapsulation reaction, while it suppressed phagocytosis. These results suggest that this novel C-type lectin designated “encapsulation promoting lectin (EPL)” regulates cellular immunity by a novel immune target size-recognition mechanism.     

Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG_2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.     

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach,Diploptera punctata

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca²⁺ and cAMP, which suggests that Dippu-AstR can act through the Gα_q and Gα_s protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.     

Increased potential for wound activated production of Prostaglandin E2 and related toxic compounds in non-native populations of Gracilaria vermiculophylla

The capacity of the East Asian seaweed Gracilaria vermiculophylla (“Ogonori”) for production of prostaglandin E₂ from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. Wounding of non-native specimens resulted on average in 390% more 15-keto-PGE₂, in 90% more PGE₂, in 37% more PGA₂ and in 96% more 7,8-di-hydroxy-eicosatetraenoic acid than wounding of native specimens. Not only PGE₂, but also PGA₂ and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.     

MitomiRs delineating the intracellular localization of microRNAs at mitochondria

Mitochondria play a crucial role in energetic metabolism, signaling pathways, and overall cell viability. Mitochondrial dysfunctions are known to cause a wide range of human diseases that affect tissues especially those with high energetic requirements, such as skeletal muscle, heart, kidney, and central nervous system, while being involved in cancer, aging, and metabolic processes. At the same time, the microRNA (miRNA) gene family has been demonstrated to be involved in most cellular processes through modulation of proteins critical for cellular homeostasis. Given the broad scope of reactivity profiles and the ability of miRNAs to modify numerous proteomic and genomic processes, new emphasis is being placed on the influence of miRNAs at the mitochondrial level. Recently, the localization of miRNAs in mitochondria was characterized in different species. This raises the idea that those miRNAs, noted “mitomiRs,” could act as “vectors” that sense and respond dynamically to the changing microenvironment of mitochondria at the cellular level. Reciprocally, we present the involvement of mitochondria in small RNA biogenesis. With the aim of deciphering the significance of this localization, we discuss the putative mechanism of import of miRNAs at mitochondria, their origin, and their hypothetical roles within the organelle.     

Sensitivity of human embryonic stem cells to different conditions during cryopreservation

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me₂SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.     

Examining the practical limits of batch effect-correction algorithms: When should you care about batch effects?

Batch effects are technical sources of variation and can confound analysis. While many performance ranking exercises have been conducted to establish the best batch effect-correction algorithm (BECA), we hold the viewpoint that the notion of best is context-dependent. Moreover, alternative questions beyond the simplistic notion of “best” are also interesting: are BECAs robust against various degrees of confounding and if so, what is the limit? Using two different methods for simulating class (phenotype) and batch effects and taking various representative datasets across both genomics (RNA-Seq) and proteomics platforms, we demonstrate that under situations where sample classes and batch factors are moderately confounded, most BECAs are remarkably robust and only weakly affected by upstream normalization procedures. This observation is consistently supported across the multitude of test datasets. BECAs do have limits: When sample classes and batch factors are strongly confounded, BECA performance declines, with variable performance in precision, recall and also batch correction. We also report that while conventional normalization methods have minimal impact on batch effect correction, they do not affect downstream statistical feature selection, and in strongly confounded scenarios, may even outperform BECAs. In other words, removing batch effects is no guarantee of optimal functional analysis. Overall, this study suggests that simplistic performance ranking exercises are quite trivial, and all BECAs are compromises in some context or another.     

Chemically induced degradation of CK2 by proteolysis targeting chimeras based on a ubiquitin–proteasome pathway

As a ubiquitous, highly pleiotropic and constitutively active serine/threonine protein kinase, casein kinase 2 (CK2) is closely associated with tumorigenesis by its overexpression in cancer cells. Here we report several proteolysis targeting chimeras (PROTACs) via “click reaction” to connect a CK2 inhibitor (CX-4945) and pomalidomide for degradation of CK2 protein. Among them, compound 2 degraded CK2 in a dose and time-dependent manner, and kept CK2 at a low basal level by recruiting ubiquitin-proteasome system. The degradation of CK2 resulted in the reduced phosphorylation of Akt and the up-regulation of p53. As a CK2 protein degrader, 2 showed the analogous cytotoxicity to CX-4945 but with a quite different mechanism of action from the CK2 inhibitor, hinting that degradation of CK2 proteins by PROTACs is a potential way for cancer treatments.     

Farnesyl biliverdins IXα are novel ligands of biliproteins from moths of the Noctuoidea superfamily: A chemosystematic view of the Lepidoptera

Bilins, derived from biliverdin IXα, are known from animals, plants and microorganisms, where they play vital roles as light-absorbing pigments. Bilins occur also in many insects. Recently, we discovered in insects a novel structural type of bilins with a farnesyl substituent at pyrrole ring A of biliverdin IXα. The first of these unusual bilins with a molecular mass of 852 (C₄₈H₆₀O₁₀N₄) was identified in Cerura vinula, subsequently in Spodoptera littoralis; both species are members of the Noctuoidea superfamily of moths. From an evolutionary point of view, it was of interest to examine other species and families of this monophyletic clade. Here, we show that other moths species in this clade (three Notodontidae species, one Erebidae species, and one Noctuidae species) have farnesylated biliverdins IXα that are present as a mixture of three bilins, differing by the number of oxygen atoms (O_8-10). These bilins are associated with typical hemolymph storage proteins, which were identified by mass spectroscopic sequencing of tryptic peptides as arylphorins (a class of 500-kDa hexamerins) in the Notodontidae and Erebidae families, and as 350-kDa very high-density lipoproteins in the Noctuidae family. Circular dichroism spectroscopy revealed that the bilins adopt opposite conformations in complex with the two different classes of proteins. At present, farnesylated biliverdins and IXα-isomers of bilins in general are known only from species of the Noctuoidea clade; the sister clades of Bombycoidea and Papilionoidea synthesise the IXγ-isomer of biliverdin and derivatives thereof.     

Ins and Outs of Interpreting Lipidomic Results

Membrane lipids are essential for life; however, research on how cells regulate cell lipid composition has been falling behind for quite some time. One reason was the difficulty in establishing analytical methods able to cope with the cell lipid repertoire. Development of a diversity of mass spectrometry-based technologies, including imaging mass spectrometry, has helped to demonstrate beyond doubt that the cell lipidome is not only greatly cell type dependent but also highly sensitive to any pathophysiological alteration such as differentiation or tumorigenesis. Interestingly, the current popularization of metabolomic studies among numerous disciplines has led many researchers to rediscover lipids. Hence, it is important to underscore the peculiarities of these metabolites and their metabolism, which are both radically different from protein and nucleic acid metabolism. Once differences in lipid composition have been established, researchers face a rather complex scenario, to investigate the signaling pathways and molecular mechanisms accounting for their results. Thus, a detail often overlooked, but of crucial relevance, is the complex networks of enzymes involved in controlling the level of each one of the lipid species present in the cell. In most cases, these enzymes are redundant and promiscuous, complicating any study on lipid metabolism, since the modification of one particular lipid enzyme impacts simultaneously on many species. Altogether, this review aims to describe the difficulties in delving into the regulatory mechanisms tailoring the lipidome at the activity, genetic, and epigenetic level, while conveying the numerous, stimulating, and sometimes unexpected research opportunities afforded by this type of studies.