Multiple paternity and its consequences in the white campion " Silene latifolia "

Summary Several studies have demonstrated that the number of pollen donors siring seeds of individual fruits is frequently greater than one and, consequently, that plants have multiple mates. Multiple paternity can have important consequences at the population level. It influences the genetic variability of a population, the reproductive success of males and the fitness of females and future generations. It also influences male-male interactions for fertilization and it is fundamental in providing opportunity of female choice. I investigated the occurrence and the importance of multiple paternity within fruits in natural populations of the dioecious Silene latifolia using microsatellite DNA markers, especially developed for this study. I found that multiple paternity occurs in all populations investigated in the European range of the species, varying from one to nine sires per fruit with a mean of three, suggesting that multiple paternity is highly prevalent in natural populations. In the presence of multiple paternity I investigated if there was a female genotype influence on siring success of the males. I used the same pollen mixture from two males and applied it to three replicate females of different relatedness (two full sisters and one unrelated). I found female genotype influence in one of the two populations investigated, which might reflect different population history. Since these results suggested some degree of female choice, we investigated whether the occurrence of multiple paternity and post-pollination selection could provide opportunity for inbreeding avoidance. First, I measured inbreeding depression at different life-cycle stages for offspring obtained by single-donor crosses with brothers or unrelated males replicated on distinct flowers on the same female plant. To address inbreeding avoidance, I determined paternity in crosses using mixed pollen loads of the two males. I found significant inbreeding depression in the studied population, even under benign experimental conditions, and although the unrelated male did not sire significantly more offspring, there was an effect of genetic dissimilarity on paternity. This suggests that paternity is affected by relatedness among mates, but maybe additionally affected by other factors such as pollen competitive ability or male-female interactions. Using inbred and outbred crosses, I further investigated sex ratio bias inheritance in this species, and found that sex ratio bias of the parental generation was significantly correlated to pollen germination success of the F2 generation, which suggests that sex ratio bias in this species results from the specific X/Y combination and not only on Y performance. An effect of X and Y is consistent with sex chromosome meiotic drive. In conclusion, I found multiple paternity to be widespread in the study species and that females of similar genotype produce similar paternity shares. I found that inbreeding depression is substantial, therefore receiving pollen from several donors might lead to fewer inbred offspring, I also found an effect of genetic dissimilarity on paternity shares, which indicates that there is some ability to discriminate against related pollen, although this seems not to be the only determinant of paternity outcome. Finally I found sex ratio bias to be dependent on both X and Y chromosomes as predicted by sex chromosome meiotic drive. Résumé Plusieurs études ont démontré qu'il n'était pas rare que les graines contenues dans un même fruit soient issues de la fécondation par plusieurs pollens provenant de mâles différents, ce qui sous-entend que les plantes peuvent avoir plusieurs partenaires sexuels. La paternité multiple peut avoir d'importantes conséquences au niveau populationnel dans la mesure où elle peut influencer le degré de variabilité génétique de la population, le succès reproducteur des mâles, la fitness des femelles et des futures générations. La paternité multiple peut également avoir un impact sur les interactions mâle-mâle lors de la fertilisation et peut être considérée comme fondamentale vis-à-vis de la femelle, qui y trouve alors une opportunité de choisir son ou ses partenaires. Dans le cadre de ce travail de thèse j'ai cherché à déterminer si la paternité multiple était un phénomène observable et important dans les populations naturelles de l'espèce dioïque, Silene latifolia. Pour ce faire, j'ai utilisé des marqueurs microsatellites, spécialement développés pour cette étude. J'ai observé des phénomènes de paternité multiple dans toutes les populations de l'étude, réparties dans l'aire de distribution européenne de l'espèce. Le nombre de pères par fruit varie de un à neuf, avec un nombre moyen de trois, ce qui signifie que la paternité multiple est très répandue dans les populations naturelles. En raison de ces résultats, je me suis demandée si le génotype de la femelle influence le succès de paternité des mâles. J'ai alors réalisé des pollinisations manuelles sur la base d'un mélange de pollens issus de deux mâles, que j'ai appliqué sur trois femelles (réplicats) présentant différents degrés d'apparentement (deux soeurs. et une femelle étrangère). Il ressort de cette expérience que le génotype de la femelle peut influencer la paternité dans l'une des deux populations étudiées, ce qui pourrait refléter des différences en terme d'histoire des populations. Dans la mesure où ces résultats suggèrent un certain degré de choix chez la femelle, j'ai cherché à savoir si la paternité multiple et la sélection post-pollinisation pouvaient être des moyens d'éviter les croisements consanguins. Dans un premier temps, j'ai évalué la dépression de consanguinité à différentes étapes du cycle de vie chez des descendants issus de croisements à un seul donneur, celui-ci étant alternativement un frère ou un étranger, répliqués sur plusieurs fleurs d'une même plante femelle. Afin d'estimer l'évitement de croisements consanguins, j'ai effectué des croisements dont le pollen était un mélange des deux mâles (frère et étranger), puis j'ai déterminé la paternité dans les fruits obtenus. J'ai pu mettre en évidence un effet de dépression de consanguinité- significatif dans les populations étudiées, même dans des conditions expérimentales moins rudes qu'à l'extérieur. Bien que le mâle étranger n'ait pas engendré un nombre significativement plus important de graines, il y avait un effet de dissimilarité génétique sur la paternité. Ceci suggère que la paternité est affectée par le degré d'apparentement entre les partenaires, mais qu'elle peut aussi être affectée par d'autres facteurs tels que la compétitivité du pollen ou encore par les interactions mâles-femelles. L'utilisation de croisements consanguins et hybrides m'a également permis d'étudier l'héritabilité du biais de sex ratio chez cette espèce. Il s'est avéré que le biais de sex ratio de la génération parentale était significativement corrélé au succès de germination du pollen de la génération F2, ce qui signifie que, chez cette espèce, le biais de sex ratio résulte d'une combinaison spécifique de X/Y et non uniquement de la performance de Y. Un effet de X et Y est compatible avec l'hypothèse de distorsion de ségrégation méiotique des chromosomes sexuels. En conclusion, il ressort de mes résultats que la paternité multiple est un phénomène largement répandu chez S. latifolia et la paternité accomplie par un mâle est plus similaire entre soeurs qu'avec une femelle étrangère J'ai également mis en évidence que la dépression de consanguinité a un impact considérable; aussi, recevoir du pollen de plusieurs donneurs différents pourrait permettre à la femelle de produire moins de descendants consanguins. J'ai aussi trouvé un effet de la dissimilarité génétique sur le partage de paternité, ce qui indique que la discrimination contre le pollen d'apparentés est possible, bien que cela ne semble pas être le seul facteur déterminant dans le résultat de la paternité. Enfin, j'ai trouvé que le biais de sex ratio est dépendant des deux chromosomes X et Y, conformément à la théorie de distorsion de ségrégation méiotique des chromosomes sexuels.

99mTc-sestamibi imaging and bone marrow karyotyping in the assessment of multiple myeloma and MGUS

AIM: The first pathogenetic step in multiple myeloma is the emergence of a limited number of clonal plasma cells, clinically known as monoclonal gammopathy of undetermined significance (MGUS). Patients with MGUS do not have symptoms or end-organ damage but they do have a 1% annual risk of progression to multiple myeloma or related malignant disorders. With progression of MGUS to multiple myeloma, complex genetic events occur in the neoplastic plasma cell. Karyotyping and fluorescence in-situ hybridization (FISH) were shown to be of prognostic value in patients with multiple myeloma. Tc-sestamibi imaging reflects myeloma disease activity in bone marrow with very high sensitivity and specificity predicting disease evolution. This study was undertaken to evaluate the role of Tc-sestamibi imaging and cytogenetic analysis in prognosis prediction of MGUS and multiple myeloma. METHODS: We enrolled 30 consecutive patients with a confirmed diagnosis of multiple myeloma or MGUS. Bone marrow biopsy and biochemical staging according to the International Staging System (ISS) were performed in all cases. Karyotype analysis and FISH were performed in 11 of 12 patients with MGUS and in 17 of 18 patients with multiple myeloma having adequate metaphases. RESULTS: The karyotype was abnormal in four of 11 MGUS and in six of 17 multiple myeloma. Abnormalities of chromosome 13 were present in one case of MGUS and in six cases of multiple myeloma whereas the involvement of immunoglobulin was observed in one case of multiple myeloma. An abnormal FISH panel was found in four MGUS and nine multiple myeloma patients. All patients with MGUS showed a normal MIBI scan (score 0). Among patients with multiple myeloma only three, all with ISS stage I, showed a normal scan while a positive scan was obtained in others (score range, 1-7). The MIBI uptake was strongly related to the bone marrow plasma cell infiltration and to cytogenetic abnormalities. Particularly, a MIBI uptake score above 5 identified patients with poor prognosis encompassing all stage III multiple myeloma and three of seven stage II multiple myeloma. On the other hand all stage I and II patients having a MIBI score less than 5 showed a good prognosis. CONCLUSION: Both cytogenetic analysis and a MIBI scan add no relevant prognostic information to the ISS in patients with stage I and III multiple myeloma. The MIBI scan was of prognostic value in stage II multiple myeloma patients. Additionally, MIBI imaging may be useful to guide bone marrow biopsy in order to obtain adequate samples for cytogenetic analysis.

Vitamin D has a direct immunomodulatory effect on CD8+ T cells of patients with early multiple sclerosis and healthy control subjects.

Little is known on a putative effect of vitamin D on CD8+ T cells. Yet, these cells are involved in the immmunopathogenesis of MS. We assessed the cytokine profile of EBV-specific CD8+ T cells of 10 early MS patients and 10 healthy control subjects with or without 1,25(OH)(2)D(3) and found that, with 1,25(OH)(2)D(3), these cells secreted less IFN-γ and TNF-α and more IL-5 and TGF-β. CD4+ T cell depletion or even culture with CD8+ T cells only did not abolish the immunomodulatory effect of 1,25(OH)(2)D(3) on CD8+ T cells, suggesting that 1,25(OH)(2)D(3) can act directly on CD8+ T cells.

Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Multiple refugia and barriers explain the phylogeography of the Valais shrew, Sorex antinorii (Mammalia: Soricomorpha), Sorex antinorii (Mammalia: Soricomorpha)

The aim of the present study was to investigate the genetic structure of the Valais shrew (Sorex antinorii) by a combined phylogeographical and landscape genetic approach, and thereby to infer the locations of glacial refugia and establish the influence of geographical barriers. We sequenced part of the mitochondrial cytochrome b (cyt b) gene of 179 individuals of S. antinorii sampled across the entire species' range. Six specimens attributed to S. arunchi were included in the analysis. The phylogeographical pattern was assessed by Bayesian molecular phylogenetic reconstruction, population genetic analyses, and a species distribution modelling (SDM)-based hindcasting approach. We also used landscape genetics (including isolation-by-resistance) to infer the determinants of current intra-specific genetic structure. The phylogeographical analysis revealed shallow divergence among haplotypes and no clear substructure within S. antinorii. The starlike structure of the median-joining network is consistent with population expansion from a single refugium, probably located in the Apennines. Long branches observed on the same network also suggest that another refugium may have existed in the north-eastern part of Italy. This result is consistent with SDM, which also suggests several habitable areas for S. antinorii in the Italian peninsula during the LGM. Therefore S. antinorii appears to have occupied disconnected glacial refugia in the Italian peninsula, supporting previous data for other species showing multiple refugia within southern refugial areas. By coupling genetic analyses and SDM, we were able to infer how past climatic suitability contributed to genetic divergence of populations. The genetic differentiation shown in the present study does not support the specific status of S. arunchi.

MOBP-specific cellular immune responses are weaker than MOG-specific cellular immune responses in patients with multiple sclerosis and healthy subjects.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Myelin oligodendrocyte glycoprotein (MOG) and myelin oligodendrocyte basic protein (MOBP) were both shown to be highly encephalitogenic in animal models of MS. In contrast, the association of MOG- and MOBP-specific humoral or cellular immune responses and MS in humans is far less established. In this study, we sought to analyse MOG- and MOBP-specific T-cell responses in a large cohort of patients with various stages of the disease. Patients with other neurological diseases and healthy subjects were enrolled to serve as control study subjects. We determined the proliferation and the secretion of IFN-γ secretion in our cohort. We found that MOG-specific T-cell responses were higher and more frequent as compared to MOBP-specific ones. However, both MS patients and control study subjects had similar myelin-specific T-cell responses at the periphery, thus calling for more precise studies at CNS level.

In vitro effect of malachite green on Candida albicans involves multiple pathways and transcriptional regulators UPC2 and STP2.

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC(50)], 100 ng ml(-1)) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.

Traitement de tla sclérose en plaques: nouveautés et complications [New treatment options in multiple sclerosis and their complications].

A new therapeutic era opened for multiple sclerosis (MS) with the appearance of molecules given p.o. and/or molecules with greater efficiency. Early diagnosis is critical, as the time and the choice of therapeutic intervention. The initiation of treatments must be personalized, including the risks associated with MS and those potentially related to the treatment chosen, answering the question <Who, when and how to treat?>. Monitoring tools that allow to objectively evaluate: I) MS activity and aggressiveness for each patient and 2) the safety of treatments and their risks of complications, must be further investigated.

The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa.

A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.

Phylogenomics of C(4) photosynthesis in sedges (Cyperaceae): multiple appearances and genetic convergence.

C(4) photosynthesis is an adaptive trait conferring an advantage in warm and open habitats. It originated multiple times and is currently reported in 18 plant families. It has been recently shown that phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the C(4) pathway, evolved through numerous independent but convergent genetic changes in grasses (Poaceae). To compare the genetics of multiple C(4) origins on a broader scale, we reconstructed the evolutionary history of the C(4) pathway in sedges (Cyperaceae), the second most species-rich C(4) family. A sedge phylogeny based on two plastome genes (rbcL and ndhF) has previously identified six fully C(4) clades. Here, a relaxed molecular clock was used to calibrate this tree and showed that the first C(4) acquisition occurred in this family between 19.6 and 10.1 Ma. According to analyses of PEPC-encoding genes (ppc), at least five distinct C(4) origins are present in sedges. Two C(4) Eleocharis species, which were unrelated in the plastid phylogeny, acquired their C(4)-specific PEPC genes from a single source, probably through reticulate evolution or a horizontal transfer event. Acquisitions of C(4) PEPC in sedges have been driven by positive selection on at least 16 codons (3.5% of the studied gene segment). These sites underwent parallel genetic changes across the five sedge C(4) origins. Five of these sites underwent identical changes also in grass and eudicot C(4) lineages, indicating that genetic convergence is most important within families but that identical genetic changes occurred even among distantly related taxa. These lines of evidence give new insights into the constraints that govern molecular evolution.

RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia.

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Effect of modafinil on subjective fatigue in multiple sclerosis and stroke patients.

BACKGROUND: Modafinil has anecdotal response to neurological fatigue, but such an effect may depend on the type and location of cerebral impairment. OBJECTIVES: It was the aim of this study to compare fatigue observed in different neurological pathologies, to evaluate the tolerability to modafinil, and to describe changes in subjective fatigue. METHODS: We enrolled 14 brainstem or diencephalic stroke (BDS) patients, 9 cortical stroke (CS) patients and 17 multiple sclerosis (MS) patients. The Fatigue Assessment Instrument severity scale was performed at baseline, after 3 months of modafinil and after 1 month of washout. Cognition, mood and somnolence were assessed. A subgroup of 14 patients underwent activity measures before and during treatment. RESULTS: Thirty-one patients completed the study (10 BDS, 9 CS, 12 MS). The responder profile is more frequent in MS than in CS (p = 0.04), and in BDS than in CS patients (p = 0.04). Actiwatch measures showed no changes in activity during, before and after therapy. CONCLUSION: Modafinil was tolerated in 75% of patients at small doses and seemed to improve the severity of fatigue in the MS and BDS groups but not in the CS group. There was no modification in measured physical activity.

Multiple mating and offspring quality in Lasius ants

Genetic diversity benefits for social insect colonies headed by polyandrous queens have received intense attention, whereas sexual selection remains little explored. Yet mates of the same queen may engage in sperm competition over the siring of offspring, and this could confer benefits on queens if the most successful sire in each colony (the majority sire) produces gynes (daughter queens) of higher quality. These benefits could be increased if high-quality sires make queens increase the percentage of eggs that they fertilize (unfertilized eggs develop into sons in social hymenopterans), or if daughters of better genetic quality are over-represented in the gyne versus worker class. Such effects would lead to female-biased sex ratios in colonies with high-quality majority gynes. I tested these ideas in field colonies of Lasius niger black garden ants, using body mass of gynes as a fitness trait as it is known to correlate with future fecundity. Also, I established the paternity of gynes through microsatellite DNA offspring analyses. Majority sires did not always produce heavier gynes in L. niger, but whenever they did do so colonies produced more females, numerically and in terms of the energetic investment in female versus male production. Better quality sires may be able to induce queens to fertilize more eggs or so-called caste shunting may occur wherever the daughters of better males are preferentially shunted to into the gyne caste. My study supports that integrating sexual selection and social evolutionary studies may bring a deeper understanding of mating system evolution in social insects.