Recognition of Gram-positive Intestinal Bacteria by Hybridoma- and Colostrum-derived Secretory Immunoglobulin A Is Mediated by Carbohydrates.

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with Gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of Gram-positive bacteria by control IgG was observed. The interaction with Gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for Gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.

Inferring the mode of origin of polyploid species from next-generation sequence data.

Many eukaryote organisms are polyploid. However, despite their importance, evolutionary inference of polyploid origins and modes of inheritance has been limited by a need for analyses of allele segregation at multiple loci using crosses. The increasing availability of sequence data for nonmodel species now allows the application of established approaches for the analysis of genomic data in polyploids. Here, we ask whether approximate Bayesian computation (ABC), applied to realistic traditional and next-generation sequence data, allows correct inference of the evolutionary and demographic history of polyploids. Using simulations, we evaluate the robustness of evolutionary inference by ABC for tetraploid species as a function of the number of individuals and loci sampled, and the presence or absence of an outgroup. We find that ABC adequately retrieves the recent evolutionary history of polyploid species on the basis of both old and new sequencing technologies. The application of ABC to sequence data from diploid and polyploid species of the plant genus Capsella confirms its utility. Our analysis strongly supports an allopolyploid origin of C. bursa-pastoris about 80 000 years ago. This conclusion runs contrary to previous findings based on the same data set but using an alternative approach and is in agreement with recent findings based on whole-genome sequencing. Our results indicate that ABC is a promising and powerful method for revealing the evolution of polyploid species, without the need to attribute alleles to a homeologous chromosome pair. The approach can readily be extended to more complex scenarios involving higher ploidy levels.

Molecular characterization of soybean cultivars by microsatellite markers with universal tail sequence

The objective of this work was to standardize a semiautomated method for genotyping soybean, based on universal tail sequence primers (UTSP), and to compare it with the conventional genotyping method that uses electrophoresis in polyacrylamide gels. Thirty soybean cultivars were genotypically characterized by both methods, using 13 microsatellite loci. For the UTSP method, the number of alleles (NA) was 50 (2-7 per marker) and the polymorphic information content (PIC) ranged from 0.40 to 0.74. For the conventional method, the NA was 38 (2-5 per marker) and the PIC varied from 0.39 to 0.67. The genetic dissimilarity matrices obtained by the two methods were highly correlated with each other (0.8026), and the formed groups were coherent with the phenotypic data used for varietal registration. The 13 markers allowed the distinction of all analyzed cultivars. The low cost of the UTSP method, associated with its high accuracy, makes it ideal for the characterization of soybean cultivars and for the determination of genetic purity.

Interpreting Breast International Group (BIG) 1-98: a randomized, double-blind, phase III trial comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive, early breast cancer.

The Breast International Group (BIG) 1-98 study is a four-arm trial comparing 5 years of monotherapy with tamoxifen or with letrozole or with sequences of 2 years of one followed by 3 years of the other for postmenopausal women with endocrine-responsive early invasive breast cancer. From 1998 to 2003, BIG -98 enrolled 8,010 women. The enhanced design f the trial enabled two complementary analyses of efficacy and safety. Collection of tumor specimens further enabled treatment comparisons based on tumor biology. Reports of BIG 1-98 should be interpreted in relation to each individual patient as she weighs the costs and benefits of available treatments. Clinicaltrials.gov ID: NCT00004205.

Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

C4 Photosynthesis evolved in grasses via parallel adaptive genetic changes.

Phenotypic convergence is a widespread and well-recognized evolutionary phenomenon. However, the responsible molecular mechanisms remain often unknown mainly because the genes involved are not identified. A well-known example of physiological convergence is the C4 photosynthetic pathway, which evolved independently more than 45 times [1]. Here, we address the question of the molecular bases of the C4 convergent phenotypes in grasses (Poaceae) by reconstructing the evolutionary history of genes encoding a C4 key enzyme, the phosphoenolpyruvate carboxylase (PEPC). PEPC genes belong to a multigene family encoding distinct isoforms of which only one is involved in C4 photosynthesis [2]. By using phylogenetic analyses, we showed that grass C4 PEPCs appeared at least eight times independently from the same non-C4 PEPC. Twenty-one amino acids evolved under positive selection and converged to similar or identical amino acids in most of the grass C4 PEPC lineages. This is the first record of such a high level of molecular convergent evolution, illustrating the repeatability of evolution. These amino acids were responsible for a strong phylogenetic bias grouping all C4 PEPCs together. The C4-specific amino acids detected must be essential for C4 PEPC enzymatic characteristics, and their identification opens new avenues for the engineering of the C4 pathway in crops.

Positive time-frequency distributions based on joint marginal constraints

This correspondence studies the formulation of members ofthe Cohen-Posch class of positive time-frequency energy distributions.Minimization of cross-entropy measures with respect to different priorsand the case of no prior or maximum entropy were considered. It isconcluded that, in general, the information provided by the classicalmarginal constraints is very limited, and thus, the final distributionheavily depends on the prior distribution. To overcome this limitation,joint time and frequency marginals are derived based on a "directioninvariance" criterion on the time-frequency plane that are directly relatedto the fractional Fourier transform.

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers.

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.

Complete DNA sequence of Kuraishia capsulata illustrates novel genomic features among budding yeasts (Saccharomycotina)

The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993(T)), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ~13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts.

Can resistance to trastuzumab be reversed by endocrine therapy? Combination of trastuzumab and letrozole after resistance to sequential trastuzumab and aromatase inhibitor monotherapies in patients with ER-positive, HER-2 positive, advanced breast cancer: a proof-of-concept trial (SAKK 23/03)

Introduction: Trastuzumab (T) is a cornerstone in the treatment of patients with HER2-overexpressing advanced breast cancer and development of resistance to T is a major therapeutic problem. HER-2 is part of a highly interactive signaling network that may impair efficacy of endocrine therapy. A sequential treatment design was chosen in this trial to ensure complete resistance to single agent therapy before receiving both a non-steroidal aromatase inhibitor (AI) and T. Any kind of clinical activity with combined treatment of AI and T after progression of single agent treatments could indicate restoration of sensitivity as a consequence of cross-talking and networking between both pathways. Methods: Key eligibility criteria included postmenopausal patients (pts.) with advanced, measurable, HER-2 positive (assessed by FISH, ratio (≥2)), HR positive disease and progression on prior treatment with a non-steroidal AI, e.g. letrozole or anastrozole, either in an adjuvant or advanced setting. Pts. received standard dose T monotherapy either weekly or three-weekly in step 1 and upon disease progression, continued T in combination with letrozole in step 2. The primary endpoint was clinical benefit response (CBR: CR, PR or SD for at least 24 weeks (+/- 1 week) according to RECIST) in step 2. Results: Thirteen pts. were enrolled in five centers in Switzerland. In step 1, six pts. (46%) achieved CBR. Median time to progression (TTP) was 161 days (Range: 50 - 627). Based on data collected until the end of May 2010, CBR was observed in seven out of the eleven evaluable pts. (64%) in step 2, including one pt. with partial response. Four of the seven pts. within step 2 that achieved CBR also had CBR in step 1. Seven out of eleven pts. have documented tumor progression during step 2 treatment. Median TTP for all eleven pts. was 184 days (range 61 - 471). Mean time on study treatment (TTP in step 1 plus TTP in step 2) for pts. reaching step 2 was 380 days (range 174 - 864). Adverse events were generally mild. Conclusion: Results of this proof-of-principle trial suggest that complete resistance to both AI and T can be overcome in a proportion of pts. by combined treatment of AI and T, as all pts. served as their own control. Our results appear promising for a new treatment strategy which offers a chemotherapy-free and well-tolerated option for at least a subset of the pts. with HR positive, HER-2 positive breast cancer. Further trials will need to corroborate this finding.

Actinobaculum schaalii: clinical observation of 20 cases.

Actinobaculum schaalii is a new species that has so far been isolated from human blood, urine and pus. Its importance has probably been underestimated and other Actinobaculum spp. may also have been underdiagnosed. This retrospective study comprises all known cases of A. schaalii infections identified since 2004 in the canton of Neuchâtel (170,000 inhabitants), Switzerland. Strains were cultivated and isolated in the bacteriology laboratory using its routine procedure. Identification included a Rapid ID 32 A strip (bioMérieux) and 16S rRNA gene sequencing. Twenty-one positive samples were found in 19 patients (11 male, 8 female) of all ages (range 16-91 years): 10 from urine (50%), six from blood (30%), one from both blood and urine (5%), and three from pus (15%). Thirteen out of 17 (76%) cases with either blood or urine specimens had underlying genitourinary tract pathologies. When urine cultures were positive for A. schaalii, leucocytes were found in all samples (10/10, 100%) but all nitrite tests were negative (10/10, 100%). The onset of appropriate treatment was delayed due to the diminished sensitivity of A. schaalii to the antibiotics commonly used for UTIs (i.e. ciprofloxacin and trimethoprim/sulfamethoxazole) and to the delay in microbiological diagnosis. A. schaalii should specifically be searched in all cases of leukocyturia with a negative nitrite test but with Gram-positive rods in the Gram stain, in patients with underlying genitourinary tract pathology, instead of dismissing these findings as clinically irrelevant colonization by coryneform bacteria. This infection may be much more common than previously thought.

Targeted sequence capture and Ultra High Throughput sequencing for gene discovery in inherited diseases

L'introduction des technologies de séquençage de nouvelle génération est en vue de révolutionner la médecine moderne. L'impact de ces nouveaux outils a déjà contribué à la découverte de nouveaux gènes et de voies cellulaires impliqués dans la pathologie de maladies génétiques rares ou communes. En revanche, l'énorme quantité de données générées par ces systèmes ainsi que la complexité des analyses bioinformatiques nécessaires, engendre un goulet d'étranglement pour résoudre les cas les plus difficiles. L'objectif de cette thèse a été d'identifier les causes génétiques de deux maladies héréditaires utilisant ces nouvelles techniques de séquençage, couplées à des technologies d'enrichissement de gènes. Dans ce cadre, nous avons développé notre propre méthode de travail (pipeline) pour l'alignement des fragments de séquence (reads). Suite à l'identification de gènes, nous avons réalisé une analyse fonctionnelle pour élucider leur rôle dans la maladie. Dans un premier temps, nous avons étudié et identifié des mutations impliquées dans une forme récessive de la rétinite pigmentaire qui est à ce jour la dégénérescence rétinienne héréditaire la plus fréquente. En particulier, nous avons constaté que des mutations faux-sens dans le gène FAM161A étaient la cause de la rétinite pigmentaire préalablement associé avec le locus RP28. De plus, nous avons démontré que ce gène avait des fonctions au niveau du cil du photorécepteur, complétant le large spectre des cilliopathies rétiniennes héréditaires. Dans un second temps, nous avons exploré la possibilité qu'un syndrome, relativement fréquent en pédiatrie de fièvre récurrente, appelé PFAPA (acronyme de fièvre périodique avec adénite stomatite, pharyngite et cervical aphteuse) puisse avoir une origine génétique. L'étiologie de cette maladie n'étant pas claire, nous avons tenté d'identifier le spectre génétique de patients PFAPA. Comme nous n'avons pas pu mettre à jour un nouveau gène unique muté et responsable de la maladie chez tous les individus dépistés, il semblerait qu'un modèle génétique plus complexe suggérant l'implication de plusieurs gènes dans la pathologie ait été identifié chez les patients touchés. Ces gènes seraient notamment impliqués dans des processus liés à l'inflammation ce qui élargirait l'impact de ces études à d'autres maladies auto-inflammatoires.

Rifaximin treatment for the irritable bowel syndrome with a positive lactulose hydrogen breath test improves symptoms for at least 3 months.

BACKGROUND: While rifaximin was able to improve symptoms in patients with irritable bowel syndrome (IBS) in phase III trials, these results are yet to be repeated in phase IV studies. AIM: To evaluate the treatment response to rifaximin in IBS patients in a phase IV trial. METHODS: IBS patients underwent lactulose hydrogen breath testing (LHBT). LHBT-positive patients were treated with rifaximin for 14 days. Prior to treatment as well as at week 4 and 14 following the start of rifaximin treatment, patients completed a questionnaire assessing symptom severity on a Likert scale from 0 to 10. RESULTS: One hundred and six of 150 IBS patients (71%) were LHBT-positive and treated with rifaximin. As assessed at week 4 following commencement of the therapy, rifaximin provided significant improvement of the following IBS-associated symptoms: bloating (5.5±2.6 before the start of the treatment vs. 3.6±2.7 at week 4, P<0.001), flatulence (5.0±2.7 vs. 4.0±2.7, P=0.015), diarrhoea (2.9±2.4 vs. 2.0±2.4, P=0.005) and abdominal pain (4.8±2.7 vs. 3.3±2.5, P<0.001). Overall well-being also significantly improved (3.9 ± 2.4 vs. 2.7 ± 2.3, P < 0.001). Similar improvements in IBS symptoms were obtained at week 14. Eighty-six per cent of patients undergoing repetitive LHBT (55/64) tested negative at week 4. CONCLUSIONS: We found a high percentage of LHBT-positive IBS patients. IBS-associated symptoms (bloating, flatulence, diarrhoea, pain) were improved for a period of 3 months following 2 weeks of treatment with rifaximin. We conclude that rifaximin treatment alleviates symptoms in LHBT-positive IBS patients.

Characterization of Fas (Apo-1, CD95)-Fas ligand interaction.

The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.