Deciphering structure-activity relationships in a series of Tat/TAR inhibitors

A series of pentameric “Polyamide Amino Acids” (PAAs) compounds derived from the same trimeric precursor have been synthesized and investigated as HIV TAR RNA ligands, in the absence and in the presence of a Tat fragment. All PAAs bind TAR with similar sub-micromolar affinities but their ability to compete efficiently with the Tat fragment strongly differs, IC₅₀ ranging from 35 nM to >2 μM. While NMR and CD studies reveal that all PAA interact with TAR at the same site and induce globally the same RNA conformational change upon binding, a comparative thermodynamic study of PAA/TAR equilibria highlights distinct TAR binding modes for Tat competitor and non-competitor PAAs. This led us to suggest two distinct interaction modes that have been further validated by molecular modeling studies. While the binding of Tat competitor PAAs induces a contraction at the TAR bulge region, the binding of non-competitor ones widens it. This could account for the distinct PAA ability to compete with Tat fragment. Our work illustrates how comparative thermodynamic studies of a series of RNA ligands of same chemical family are of value for understanding their binding modes and for rationalizing structure-activity relationships.     

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.     

The impact of species concept on biodiversity studies

Species are defined using a variety of different operational techniques. While discussion of the various methodologies has previously been restricted mostly to taxonomists, the demarcation of species is also crucial for conservation biology. Unfortunately, different methods of diagnosing species can arrive at different entities. Most prominently, it is widely thought that use of a phylogenetic species concept may lead to recognition of a far greater number of much less inclusive units. As a result, studies of the same group of organisms can produce not only different species identities but also different species range and number of individuals. To assess the impact of different definitions on conservation issues, we collected instances from the literature where a group of organisms was categorized both under phylogenetic and nonphylogenetic concepts. Our results show a marked difference, with surveys based on a phylogenetic species concept showing more species (48%) and an associated decrease in population size and range. We discuss the serious consequences of this trend for conservation, including an apparent change in the number of endangered species, potential political fallout, and the difficulty of deciding what should be conserved.     

Virtual identification of essential proteins within the protein interaction network of yeast

Topological analysis of large scale protein-protein interaction networks (PINs) is important for understanding the organizational and functional principles of individual proteins. The number of interactions that a protein has in a PIN has been observed to be correlated with its indispensability. Essential proteins generally have more interactions than the nonessential ones. We show here that the lethality associated with removal of a protein from the yeast proteome correlates with different centrality measures of the nodes in the PIN, such as the closeness of a protein to many other proteins, or the number of pairs of proteins which need a specific protein as an intermediary in their communications, or the participation of a protein in different protein clusters in the PIN. These measures are significantly better than random selection in identifying essential proteins in a PIN. Centrality measures based on graph spectral properties of the network, in particular the subgraph centrality, show the best performance in identifying essential proteins in the yeast PIN. Subgraph centrality gives important structural information about the role of individual proteins, and permits the selection of possible targets for rational drug discovery through the identification of essential proteins in the PIN.     

A comprehensive synthesis of liana removal experiments in tropical forests

Lianas are a quintessential feature of tropical forests and are often perceived as being poorly studied. However, liana removal studies may be one of the most common experimental manipulations in tropical forest ecology. In this review, we synthesize data from 64 tropical liana removal experiments conducted over the past 90 yr. We explore the direction and magnitude of the effects of lianas on tree establishment, growth, survival, reproduction, biomass accretion, and plant and animal diversity in ecological and forestry studies. We discuss the geographical biases of liana removal studies and compare the various methods used to manipulate lianas. Overall, we found that lianas have a clear negative effect on trees, and trees benefitted from removing lianas in nearly every study across all forest types. Liana cutting significantly increased light and water availability, and trees responded with vastly greater reproduction, growth, survival, and biomass accumulation compared to controls where lianas were present. Removing lianas during logging significantly reduced damage of future merchantable trees and improved timber production. Our review demonstrates that lianas have an unequivocally detrimental effect on every metric of tree performance measured, regardless of forest type, forest age, or geographic location. However, lianas also appear to have a positive contribution to overall forest plant diversity and to different animal groups. Therefore, managing lianas reduces logging damage and improves timber production; however, the removal lianas may also have a negative effect on the faunal community, which could ultimately harm the plant community.     

Exploring macrophage cell therapy on Diabetic Kidney Disease

Alternatively activated macrophages (M2) have regenerative properties and shown promise as cell therapy in chronic kidney disease. However, M2 plasticity is one of the major hurdles to overcome. Our previous studies showed that genetically modified macrophages stabilized by neutrophil gelatinase‐associated lipocalin (NGAL) were able to preserve their M2 phenotype. Nowadays, little is known about M2 macrophage effects in diabetic kidney disease (DKD). The aim of the study was to investigate the therapeutic effect of both bone marrow‐derived M2 (BM‐фM2) and ф‐NGAL macrophages in the db/db mice. Seventeen‐week‐old mice with established DKD were divided into five treatment groups with their controls: D+BM‐фM2; D+ф‐BM; D+ф‐NGAL; D+ф‐RAW; D+SHAM and non‐diabetic (ND) (db/‐ and C57bl/6J) animals. We infused 1 × 10⁶ macrophages twice, at baseline and 2 weeks thereafter. BM‐фM2 did not show any therapeutic effect whereas ф‐NGAL significantly reduced albuminuria and renal fibrosis. The ф‐NGAL therapy increased the anti‐inflammatory IL‐10 and reduced some pro‐inflammatory cytokines, reduced the proportion of M1 glomerular macrophages and podocyte loss and was associated with a significant decrease of renal TGF‐β1. Overall, our study provides evidence that ф‐NGAL macrophage cell therapy has a therapeutic effect on DKD probably by modulation of the renal inflammatory response caused by the diabetic milieu.     

Epitope mapping of pathogenic Leptospira LipL32

Aims:  To identify LipL32 epitopes and to evaluate their capability to recognize specific antibodies using ELISA.
Methods and Results:  Epitope mapping by means of a library of overlapping peptide fragments prepared by simultaneous and parallel solid phase peptide synthesis on derivatized cellulose membranes (SPOT synthesis) was carried out. Eighty-seven overlapping decapentapeptides corresponding to the complete sequence of LipL32 were synthesized. According to spot-image intensities, the most reactive sequences were localized in regions 151–177 (sequence AAKAKPVQKLDDDDDGDDTYKEERHNK) and 181–204 (sequence LTRIKIPNPPKSFDDLKNIDTKKL). Two peptides (P1 and P2) corresponding to these sequences were synthesized, and their reactivity evaluated using ELISA test.
Conclusions:  Epitope identification and analysis suggested the existence of two antigenic regions within LipL32. These LipL32 reactive regions were highly conserved among antigenically variants of Leptospira spp. isolates. Peptides containing these regions (P1 and P2) showed a good capability for anti-leptospiral antibody recognition.
Significance and Impact of the Study:  This finding could have potential relevance not only for serodiagnosis but also as a starting point for the characterization of targets for vaccine design.     

Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer

Breast cancers can recur after removal of the primary tumor and treatment to eliminate remaining tumor cells. Recurrence may occur after long periods of time during which there are no clinical symptoms. Tumor cell dormancy may explain these prolonged periods of asymptomatic residual disease and treatment resistance. We generated a dormancy gene signature from published experimental models and applied it to both breast cancer cell line expression data as well as four published clinical studies of primary breast cancers. We found that estrogen receptor (ER) positive breast cell lines and primary tumors have significantly higher dormancy signature scores (P<0.0000001) than ER- cell lines and tumors. In addition, a stratified analysis combining all ER+ tumors in four studies indicated 2.1 times higher hazard of recurrence among patients whose tumors had low dormancy scores (LDS) compared to those whose tumors had high dormancy scores (HDS) (p<0.000005). The trend was shown in all four individual studies. Suppression of two dormancy genes, BHLHE41 and NR2F1, resulted in increased in vivo growth of ER positive MCF7 cells. The patient data analysis suggests that disseminated ER positive tumor cells carrying a dormancy signature are more likely to undergo prolonged dormancy before resuming metastatic growth. Furthermore, genes identified with this approach might provide insight into the mechanisms of dormancy onset and maintenance as well as dormancy models using human breast cancer cell lines.     

Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes

This transformation procedure generates, with high efficiency (70-90%), hairy roots in cultivars, landraces and accessions of Phaseolus vulgaris (common bean) and other Phaseolus spp. Hairy roots rapidly develop after wounding young plantlets with Agrobacterium rhizogenes, at the cotyledon node, and keeping the plants in high-humidity conditions. Callogenesis always precedes hairy-root formation, and after 15 days, when roots develop at wounded sites, the stem with the normal root is cleaved below the hairy root zone. Transgenic roots and nodules co-transformed with a binary vector can be easily identified using a reporter gene. This procedure, in addition to inducing robust transgenic hairy roots that are susceptible to being nodulated by rhizobia and to fixing nitrogen efficiently, sets the foundation for a high-throughput functional genomics approach on the study of root biology and root-microbe interactions. This protocol can be completed within 30 days.