Discrepancies between subgeneric classification and molecular phylogeny of Ceratitis (Diptera: Tephritidae), can the evolution of host use provide some clues?

Using molecular data from three protein encoding genes and 49 taxa (98 specimens from 20 African countries), we provide an extended phylogeny of Ceratitis and investigate the evolution of stenophagy across clades. Bayesian tree reconstructions support previously proposed monophyletic lineages (Pardalaspis, Pterandrus section A, Pterandrus section B+Ceratitis sensu stricto) and reveal the occurrence of two new monophyletic groups including Ceratalaspis/Hoplolophomyia (viz. Cl(A), and Cl(B)+H). The reconstruction of ancestral character states shows that stenophagy evolved repeatedly and independently in five different clades (Podocarpus, Solanum, Strychnos, Tabernaemontana and Vepris feeders). The evolution of feeding preferences is closely related to the phylogenetic patterns of Ceratalaspis/Hoplolophomyia whose sections include either polyphagous species (Cl(A)) or stenophagous taxa (Cl(B)+H) that are further subdivided in Vepris and Solanum feeders. The evolution of stenophagy in the genus Ceratits appears as the result of a process leading to the exploitation of "unconventional" fruits (viz. toxic and/or not fleshy) and involving either metabolic adaptation to toxic plant compounds and/or the capability of penetrating fruits with thick cuticles.     

Influence of complex nutrient source on growth of and curvacin a production by sausage isolate Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of a complex nutrient source were investigated in vitro during laboratory fermentations with modified MRS medium. A modification of the nutrient depletion model was used to fit the data describing growth and bacteriocin production. Both cell growth and bacteriocin activity were influenced by changes in the complex nutrient source concentration. Standard MRS medium clearly limited the growth of L. curvatus LTH 1174. Higher nutrient concentrations, up to a certain degree, led to improved growth, a higher attainable biomass concentration, and a higher bacteriocin activity in the supernatant. A lower concentration of complex nutrient source caused severe growth inhibition, leading to a lower biomass concentration but a much higher specific bacteriocin production. When examining the separate components of the complex nutrient source, a stimulating effect of bacteriological peptone on growth was found without an adverse effect on bacteriocin production, resulting in increased curvacin A activity. Furthermore, specific depletion of the amino acids tyrosine, serine, and asparagine/aspartic acid was observed for this strain.     

New alkamide and ent-kaurane diterpenoid derivatives from Senecio erechtitoides (Asteraceae)

Fractionation of a MeOH/CH₂Cl₂ (1/1) extract of the aerial parts of Senecio erechtitoides led to the isolation of six compounds including the hitherto unknown N-phenethylamide derivative named N-(p-hydroxyphenethyl)pentacosanamide (1), and a kauranoid derivative named derivative named ent-7-oxo-16α,17-dihydroxykauran-19-oic acid (2), as well as four known compounds, ent-Kaur-16-en-19-oic acid (3), ent-7β-hydroxykaur-16-en-19-oic acid (4), ent-7-oxokaur-16-en-19-oic acid (5), steppogenin 4′-O-β-d-glucoside (6). Their structures and relative configurations were elucidated on the basis of spectroscopic methods, chemical reactions, and comparison with previously known analogs. All isolates were evaluated for their antimicrobial activity and only diterpenoids were found to possess a potent inhibitor effect against the range of microorganism.     

Label-free Quantitative Urinary Proteomics Identifies the Arginase Pathway as a New Player in Congenital Obstructive Nephropathy

Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets.Congenital obstructive nephropathy is the main cause of end stage renal disease (ESRD) in children (1). The most frequently found cause of congenital obstructive nephropathy is ureteropelvic junction (UPJ)¹ obstruction with an estimated incidence of 1 in 1000–1500 births. Milder forms of UPJ obstruction often progress to the spontaneous resolution of the pathology over time. This has led to a watchful waiting approach with surgical intervention only if renal deterioration is detected (2). Although this medical surveillance prevents unnecessary surgery, it mostly relies on invasive follow-up. Consequently with the aim to reduce this invasive follow-up, several groups have initiated research to identify noninvasive urinary biomarkers of UPJ obstruction using both targeted and nontargeted (e.g. proteome analysis based) strategies. Targeted strategies including urinary cytokine expression analyses failed to clearly determine the need for surgery in UPJ obstruction (3, 4). On the other hand, untargeted strategies have been more successful and by using urinary proteomics, biomarkers for renal and non-renal diseases have been identified (5–9). Using urinary peptidome analysis, we identified and validated a urinary peptide panel that predicted the clinical outcome of newborns with UPJ obstruction with 97% accuracy several months in advance (3, 10). An independent small-scale study confirmed the efficiency of this biomarker panel (7). These studies indicate the potential of urinary proteomics to predict the clinical fate of patients with UPJ obstruction. Although these endogenous urinary peptide biomarkers are of great potential clinical value, sequencing of these biomarkers mainly identified collagen fragments that are less informative on the pathophysiology of the disease. In contrast, studies of the high molecular weight urinary proteome (i.e. proteins) might be more informative on the pathophysiology of disease. Different approaches have been used in the past to characterize the urinary proteome, either by 2D-gel electrophoresis coupled to mass spectrometry (11, 12) or reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis (13–16). In-depth proteome analysis using extensive fractionation of the sample and high resolution, fast sequencing mass spectrometers have reported the identification of >2000 proteins in normal human urine (13, 15, 16). Here, we applied quantitative high-resolution label free LC-MS/MS analysis for the identification of urinary proteins associated to UPJ obstruction in newborns. Among a number of proteins uniquely associated with severe UPJ obstruction, we identified Arginase 1, not previously recognized in UPJ obstruction. Using an independent larger cohort, we further verified reduced urinary abundance of Arginase 1 using both Western blot and multiple reaction monitoring (MRM). Using the mouse model of obstructive nephropathy, we observed that the expression of arginases is modulated in situ in obstructed kidneys. Further gene expression analysis of the arginase pathway allowed us to hypothesize for a role of arginases in the development of fibrotic lesions in obstructive nephropathy.     

Guidelines to reach high-quality purified recombinant proteins

Applied Microbiology and Biotechnology - The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a...     

Nucleotide diversity of a genomic sequence similar to <Emphasis Type="Italic">SHATTERPROOF</Emphasis> (<Emphasis Type="Italic">PvSHP1</Emphasis>) in domesticated and wild common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Evolutionary studies in plant and animal breeding are aimed at understanding the structure and organization of genetic variations of species. We have identified and characterized a genomic sequence in Phaseolus vulgaris of 1,200 bp (PvSHP1) that is homologous to SHATTERPROOF-1 (SHP1), a gene involved in control of fruit shattering in Arabidopsis thaliana. The PvSHP1 fragment was mapped to chromosome Pv06 in P. vulgaris and is linked to the flower and seed color gene V. Amplification of the PvSHP1 sequence from the most agronomically important legume species showed a high degree of interspecies diversity in the introns within the Phaseoleae, while the coding region was conserved across distant taxa. Sequencing of the PvSHP1 sequence in a sample of 91 wild and domesticated genotypes that span the geographic distribution of this species in the centers of origin showed that PvSHP1 is highly polymorphic and, therefore, particularly useful to further investigate the origin and domestication history of P. vulgaris. Our data confirm the gene pool structure seen in P. vulgaris along with independent domestication processes in the Andes and Mesoamerica; they provide additional evidence for a single domestication event in Mesoamerica. Moreover, our results support the Mesoamerican origin of this species. Finally, we have developed three indel-spanning markers that will be very useful for bean germplasm characterization, and particularly to trace the distribution of the domesticated Andean and Mesoamerican gene pools.     

Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults

Human rotavirus-specific CD4(+) and CD8(+) T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8(+) and CD4(+) T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4(+) and CD8(+) T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4(+) and CD8(+) T cells and the frequencies of IL-13-secreting CD4(+) T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4(+) cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.