Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing

Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single‐nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild‐type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype.     

Antibody responses to NY-ESO-1 in primary breast cancer identify a subtype target for immunotherapy

The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)⁻ invasive ductal carcinomas of high grade, including both HER2⁻ and HER2⁺ tumors. In line with these results, we detected ESO expression in 20% of primary HR⁻ BC, including both ESO Ab⁺ and Ab⁻ patients, but not in HR⁺ BC. Interestingly, whereas expression levels in ESO⁺ BC were not significantly different between ESO Ab⁺ and Ab⁻ patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR⁻ (HER2⁻ or HER2⁺) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy.     

Abnormal electrophoretic mobility of spectrin tetramers in hereditary elliptocytosis

Summary Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, and . In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp ^I/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (₂ ^I/65-₂) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.     

Functional Diversity of Plant–Pollinator Interaction Webs Enhances the Persistence of Plant Communities

Pollination is exclusively or mainly animal mediated for 70% to 90% of angiosperm species. Thus, pollinators provide an essential ecosystem service to humankind. However, the impact of human-induced biodiversity loss on the functioning of plant–pollinator interactions has not been tested experimentally. To understand how plant communities respond to diversity changes in their pollinating fauna, we manipulated the functional diversity of both plants and pollinators under natural conditions. Increasing the functional diversity of both plants and pollinators led to the recruitment of more diverse plant communities. After two years the plant communities pollinated by the most functionally diverse pollinator assemblage contained about 50% more plant species than did plant communities pollinated by less-diverse pollinator assemblages. Moreover, the positive effect of functional diversity was explained by a complementarity between functional groups of pollinators and plants. Thus, the functional diversity of pollination networks may be critical to ecosystem sustainability.     

The basolateral sorting signals of the thyrotropin and luteinizing hormone receptors: an unusual family of signals sharing an unusual distal intracellular localization, but unrelated in their structures

The mechanisms of the basolateral targeting of G protein-coupled receptors remain largely unknown. Mutagenesis experiments have allowed us to identify the basolateral sorting signals of the TSH and LH receptors expressed in Madin-Darby canine kidney cells and thyroid follicular FRT cells. Unexpectedly these signals (amino acids 731-746 and 672-689, respectively) share an unusual localization in the distal part of the intracellular domain of the receptors at a marked distance from the membrane. When grafted onto the p75-neurotropin receptor, these signals redirect this normally apically expressed protein to the basolateral cell surface. They are independent of the endocytosis signal. The basolateral sorting signals of TSH, LH, and FSH receptors do not exhibit primary sequence homology with each other or with any other known signal. Furthermore, circular dichroism studies show that the three signals exhibit distinct secondary structures. The TSH receptor has a stable helical structure, the LH receptor has both helix and beta-sheet structures, and the FSH receptor sorting signal has a main random coil structure. This means that even in closely-related receptors different secondary structures can be found for basolateral signals unrelated to internalization signals. This observation contrasts with what is known about basolateral signals related to internalization signals for which a common beta-turn structure has been described. Deletion of the basolateral sorting signals results in apical targeting of the receptors, suggesting the existence of apical sorting information. However, a soluble form of the TSH receptor, which harbors all N- and putative O-linked oligosaccharides, is secreted in a nonpolarized fashion. This implies that apical sorting information must be located elsewhere, either in the transmembrane or in the intracellular domains of the receptor.     

Culicoides Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.     

Modifications of mesenteric adipose tissue during moderate experimental colitis in mice

Aims

Adipose tissue secretes various proteins referred to as adipokines, being involved in inflammation. It was recognized that mesenteric adipose tissue (MAT) is altered by inflammation, and pathologies such as inflammatory bowel disease (IBD). The aim of this study was to investigate the alterations of the mesenteric adipose tissue in two experimental colitis models in mice adapted to obtain moderate colonic inflammation.

Main methods

Colonic inflammation was obtained using two models, either DSS dissolved in drinking water or intra-colonic instillation of DNBS. The expression of adipokines (leptin and adiponectin) and inflammatory markers (IL-6, MCP-1, F4/80) was studied by qRT-PCR in the MAT of treated and control mice.

Key findings

Observations of the colon and IL-6 plasma level determination demonstrated that DNBS treatment led to stronger inflammation. Colitis induced a decrease of mRNA encoding to leptin and adiponectin in MAT. In contrast, colonic inflammation led to an increase of mRNA encoding to IL-6, MCP-1 and F4/80, a specific marker of macrophages.

Significance

The mesenteric adipose tissue, in two models of moderate colitis, shows a loss of adipose profile and a strong increase of inflammatory pattern, close to the observations made in MAT of IBD patients. These data suggest that these pro-inflammatory modifications of MAT have to be taken into account in the pathophysiology of IBD.     

The molecular targets of ivermectin and lotilaner in the human louse Pediculus humanus humanus: New prospects for the treatment of pediculosis

Control of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of parasites resistant to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanisms of action remain largely unknown. The macrocyclic lactone ivermectin is active against a broad range of insects including human lice. Isoxazolines are a new chemical class exhibiting a strong insecticidal potential. They preferentially act on the γ-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and, to a lesser extent on glutamate-gated chloride channels (GluCls) in some species. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Using toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and GluCl (Phh-GluCl) subunits were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC₅₀ values of 16.0 μM and 9.3 μM. Importantly, ivermectin displayed a super agonist action on Phh-GluCl, whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC₅₀ value of 40.7 nM, whereas it had no effect on Phh-GluCl. We report here for the first time the insecticidal activity of isoxazolines on human ectoparasites and reveal the mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize an expected extension of the use of the isoxazoline drug class as new pediculicidal agents to tackle resistant-louse infestations in humans.