Estimation of an in vivo fitness landscape experienced by HIV-1 under drug selective pressure useful for prediction of drug resistance evolution during treatment

MOTIVATION: HIV-1 antiviral resistance is a major cause of antiviral treatment failure. The in vivo fitness landscape experienced by the virus in presence of treatment could in principle be used to determine both the susceptibility of the virus to the treatment and the genetic barrier to resistance. We propose a method to estimate this fitness landscape from cross-sectional clinical genetic sequence data of different subtypes, by reverse engineering the required selective pressure for HIV-1 sequences obtained from treatment naive patients, to evolve towards sequences obtained from treated patients. The method was evaluated for recovering 10 random fictive selective pressures in simulation experiments, and for modeling the selective pressure under treatment with the protease inhibitor nelfinavir. RESULTS: The estimated fitness function under nelfinavir treatment considered fitness contributions of 114 mutations at 48 sites. Estimated fitness correlated significantly with the in vitro resistance phenotype in 519 matched genotype-phenotype pairs (R(2) = 0.47 (0.41 - 0.54)) and variation in predicted evolution under nelfinavir selective pressure correlated significantly with observed in vivo evolution during nelfinavir treatment for 39 mutations (with FDR = 0.05). AVAILABILITY: The software is available on request from the authors, and data sets are available from http://jose.med.kuleuven.be/~kdforc0/nfv-fitness-data/.     

Connexin Channels, Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃ elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Burkholderia phenoliruptrix sp. nov., to accommodate the 2,4,5-trichlorophenoxyacetic acid and halophenol-degrading strain AC1100

Strain AC1100 is well-known for its ability to degrade a variety of recalcitrant xenobiotics, including 2,4,5-trichlorophenoxyacetic acid. We performed a polyphasic-taxonomic study to determine its taxonomic position. The G+C content of strain AC1100 was 62.6 mol%. On the basis of 16S rRNA gene sequence similarity, strain AC1100 belonged to the b-Proteobacteria and was most closely related to Burkholderia fungorum (98.3% similarity). DNA-DNA hybridisations, comparison of protein profiles, cellular fatty acid analysis and biochemical tests allowed genotypic and phenotypic differentiation of strain AC1100 from other Burkholderia species. Our data show that strain AC1100 represents a novel species for which the name Burkholderia phenoliruptrix sp. nov. is proposed. The type strain is AC1100^T (= LMG 22037^T = CCUG 48558^T).     

Flocculation of microalgae using cationic starch

Due to their small size and low concentration in the culture medium, cost-efficient harvesting of microalgae is a major challenge. We evaluated the potential of cationic starch as a flocculant for harvesting microalgae using jar test experiments. Cationic starch was an efficient flocculant for freshwater (Parachlorella, Scenedesmus) but not for marine microalgae (Phaeodactylum, Nannochloropsis). At high cationic starch doses, dispersion restabilization was observed. The required cationic starch dose to induce flocculation increased linearly with the initial algal biomass concentration. Of the two commercial cationic starch flocculants tested, Greenfloc 120 (used in wastewater treatment) was more efficient than Cargill C*Bond HR 35.849 (used in paper manufacturing). For flocculation of Parachlorella using Greenfloc 120, the cationic starch to algal biomass ratio required to flocculate 80% of algal biomass was 0.1. For Scenedesmus, a lower dose was required (ratio 0.03). Flocculation of Parachlorella using Greenfloc 120 was independent of pH in the pH range of 5 to 10. Measurements of the maximum quantum yield of PSII suggest that Greenfloc 120 cationic starch was not toxic to Parachlorella. Cationic starch may be used as an efficient, nontoxic, cost-effective, and widely available flocculant for harvesting microalgal biomass.     

Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.

The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.     

Genome sequences and description of novel exopolysaccharides producing species Komagataeibacter pomaceti sp. nov. and reclassification of Komagataeibacter kombuchae (Dutta and Gachhui 2007) Yamada et al., 2013 as a later heterotypic synonym of Komagataeibacter hansenii (Gosselé et al. 1983) Yamada et al., 2013

Strains T5K1 and AV446 isolated from apple cider vinegars during a submerged vinegar production in two separate vinegar facilities showed 94% 16S rRNA gene similarity to its closest neighbors Komagataeibacter maltaceti LMG 1529^T and Gluconacetobacter entanii LTH 4560^T. Further phylogenetic and phenotypic characterizations indicated that the isolates belonged to a novel species of the Komagataeibacter genus. Comparison based on 16S–23S rRNA gene ITS sequences and concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, grouped both strains to a single phylogenetic cluster well separated from the other species of the Komagataeibacter genus. Average nucleotide identity of T5K1 and AV446 draft genome sequences compared to other Komagataeibacter type strains was below 94% and at the same time, in-silico DNA–DNA hybridization was below 70%. Both strains on the other hand showed approximately 98% (average nucleotide identity) and 87% (in silico DNA–DNA hybridization) similarity to each other. Strains T5K1 and AV446 can be differentiated from other Komagataeibacter type strains based on their ability to produce 2-keto-d-gluconic acid and at the same time inability to produce 5-keto-d-gluconic acid. Furthermore, strains of the new species do not grow on Asai medium supplemented with d-glucose or d-mannitol. The growth is also absent (T5K1) or weak (AV446) on Hoyer–Frateur medium supplemented with afore mentioned sugars. Both strains produce cellulose. In addition, draft genome analysis revealed that strains T5K1 and AV446 possess genes involved in the synthesis of acetan-like extracellular heteropolysaccharide. We propose the name Komagataeibacter pomaceti sp. nov. for the new species with LMG 30150^T [= CCM 8723^T = ZIM B1029^T] as the type strain. Data collected in this study and in a previous study also revealed that Komagataeibacter kombuchae is a later heterotypic synonym of Komagataeibacter hansenii.     

Complete nucleotide sequence of the new simian T-lymphotropic virus, STLV-PH969 from a Hamadryas baboon, and unusual features of its long terminal repeat.

A third type of primate T-lymphotropic virus, PTLV-L, with STLV-PH969 as a prototype, has recently been isolated from an African baboon (Papio hamadryas). Classification of this virus has been based on partial sequence analysis of cDNA from a virus-producing cell line, PH969. We obtained the complete nucleotide sequence of this virus with a proviral genome of 8,916 bp. All major genes, homologous in all human T-cell lymphotropic virus (HTLV)-related viruses, and their corresponding mRNAs, including appropriate splicing, were identified. One additional nonhomologous open reading frame in the proximal pX region is accessible for translation through alternative splicing. Sequence comparison shows that STLV-PH969 is equidistantly related to HTLV type 1 (HTLV-1) and HTLV-2. In all coding regions, the similarity tends to be the lowest between STLV-PH969 and HTLV-1. However, in the long terminal repeat (LTR) region, the lowest similarity was found between STLV-PH969 and HTLV-2. The U3-R and R-U5 boundaries of the STLV-PH969 LTR were experimentally determined at nucleotides 268 and 524, respectively. This 695-bp LTR is 60 and 73 bp shorter than the LTRs of HTLV-1 and HTLV-2, respectively, but its general organization is similar to the one found in the HTLV-bovine leukemia virus genus. In the long region between the polyadenylation signal and the poly(A) site, sequence similarity with the HTLV-1 Rex-responsive element (RexRE) core and secondary structure prediction suggest the presence of a RexRE. The presence of three 21-bp repeats is conserved within the U3 region of HTLV-1, HTLV-2, and BLV. Only two direct repeats with similarity to these Tax-responsive elements were found in the STLV-PH969 LTR, which might suggest differences in the Tax-mediated transactivation of this virus. We conclude that STLV-PH969 has all the genes and genomic regions to suggest a replication cycle comparable to that of HTLV-1 and HTLV-2.     

Seed weight affects shoot and root growth among and within soybean genotypes beyond the seedling stage: implications for low P tolerance screening

Background and aims

Invasive plants have been associated with alterations to soil properties, functions, and organisms, with the potential to impact ecosystem processes. An observational study was conducted to determine how the invasive plant Frangula alnus affects soil microbial communities and biogeochemical processes in Wisconsin forests.

Methods

Paired invaded/non-invaded sites (n = 10), including high (n = 5) and low (n = 5) density invasions, were sampled in spring, summer, and fall. Soil was analyzed for extractable and total nitrogen (N), N mineralization rate, total carbon, microbial biomass carbon and N, and microbial community structure using terminal restriction fragment length polymorphisms.

Results

Linear regression analysis with robust variance estimation revealed higher N mineralization rates in invaded sites than non-invaded sites in summer, and in high density invaded sites than non-invaded sites overall (p < 0.05). There was not a corresponding increase in extractable N. No differences between invaded and non-invaded sites were observed for other variables.

Conclusions

Nitrogen-rich F. alnus leaf litter (3.2 % of dry mass) may contribute to elevated N mineralization at these sites, though pre-existing conditions may be responsible. Results suggest that F. alnus alters N cycling but has little impact on soil carbon pools and microbial communities.

     

Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations

The production of Leuconostoc mesenteroides sucrose phosphorylase has been studied in 10- and 20-L batch fermentations. A fermentation medium was devised combining rapid growth, high cell yield, and high enzyme levels. Overall fermentation dynamics and enzyme fermentation patterns are elucidated here in detail. Sucrose is phosphorolyzed into fructose and glucose-1-phosphate (G-1-P) with G-1-P preferentially utilized (thus saving ATP). Subsequently, fructose is gradually metabolized and is also converted to mannitol. Invertase activity is absent. Sucrose phosphorylase is formed transitorily with peak levels toward the end of active growth; a sharp decline in enzyme activity occurs upon further fermentation. The moment of cell (enzyme) harvest is thus critical in view of obtaining active cell or enzyme preparations for sucrose phosphorolysis. Microaerophilic and strictly anaerobic fermentations displayed no appreciable difference in sucrose phosphorylase formation profile. The enzyme is intracellularly located. It is constitutively formed in the absence of sucrose, contrary to that of Pseudomonas species; other disaccharide phosphorylases are not formed.