Enhancing the ligand efficiency of anti-HIV compounds targeting frameshift-stimulating RNA

Ribosomal frameshifting, a process whereby a translating ribosome is diverted from one reading frame to another on a contiguous mRNA, is an important regulatory mechanism in biology and an opportunity for therapeutic intervention in several human diseases. In HIV, ribosomal frameshifting controls the ratio of Gag and Gag-Pol, two polyproteins critical to the HIV life cycle. We have previously reported compounds able to selectively bind an RNA stemloop within the Gag-Pol mRNA; these compounds alter the production of Gag-Pol in a manner consistent with increased frameshifting. Importantly, they also display antiretroviral activity in human T-cells. Here, we describe new compounds with significantly reduced molecular weight, but with substantially maintained affinity and anti-HIV activity. These results suggest that development of more “ligand efficient” enhancers of ribosomal frameshifting is an achievable goal.     

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K_d value of 410?nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.     

Sodium‐powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan‐binding region

The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error‐prone PCR to introduce single point mutations into the sodium‐powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium‐channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C‐terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion‐channel blockers.     

Development of LCA benchmarks for Austrian torrent control structures

The International Journal of Life Cycle Assessment - This study emerged from a research project that aimed to develop a Life Cycle Assessment (LCA) model for torrent control structures. This...     

Seedling response to water stress in valley oak (Quercus lobata) is shaped by different gene networks across populations

Drought is a major stress for plants, creating a strong selection pressure for traits that enable plant growth and survival in dry environments. Many drought responses are conserved species‐wide responses, while others vary among populations distributed across heterogeneous environments. We tested how six populations of the widely distributed California valley oak (Quercus lobata) sampled from contrasting climates would differ in their response to soil drying relative to well‐watered controls in a common environment by measuring ecophysiological traits in 93 individuals and gene expression (RNA‐seq) in 42 individuals. Populations did not differ in their adjustment of turgor loss point during soil drying, suggesting a generalized species‐wide response. Differential expression analysis identified 689 genes with a common response to treatment across populations and 470 genes with population‐specific responses. Weighted gene co‐expression network analysis (WGCNA) identified groups of genes with similar expression patterns that may be regulated together (gene modules). Several gene modules responded differently to water stress among populations, suggesting regional differences in gene network regulation. Populations from sites with a high mean annual temperature responded to the imposed water stress with significantly greater changes in gene module expression, indicating that these populations may be locally adapted to respond to drought. We propose that this variation among valley oak populations provides a mechanism for differential tolerance to the increasingly frequent and severe droughts in California.     

Early‐breeding females experience greater telomere loss

Annual reproductive success is often highest in individuals that initiate breeding early, yet relatively few individuals start breeding during this apparently optimal time. This suggests that individuals, particularly females who ultimately dictate when offspring are born, incur costs by initiating reproduction early in the season. We hypothesized that increases in the ageing rate of somatic cells may be one such cost. Telomeres, the repetitive DNA sequences on the ends of chromosomes, may be good proxies of biological wear and tear as they shorten with age and in response to stress. Using historical data from a long‐term study population of dark‐eyed juncos (Junco hyemalis), we found that telomere loss between years was greater in earlier breeding females, regardless of chronological age. There was no relationship between telomere loss and the annual number of eggs laid or chicks that reached independence. However, telomere loss was greater when temperatures were cooler, and cooler temperatures generally occur early in the season. This suggests that environmental conditions could be the primary cause of accelerated telomere loss in early breeders.     

Willingness of rural communities to reforest with native tree species in central Chile

Economic development of rural people is not always feasible along with concomitant forest restoration, especially when meager reforestation incentives are oriented to poor rural people who probably are not willing to plant native species in their small plots of land. Forest restoration incentives have been created by the Chilean government to engage poor rural people in reforestation using native tree species to recover degraded lands. Our objective was to compare the willingness of people from rural communities to plant native species if they had to bear the costs or if the government did, and we related the answers to environmental and socioeconomic variables. Of the 217 respondents 53.9% were interested in planting native trees if subsidies became available. Interest decreased if the respondents had to pay for the cost, but only slightly. The willingness to reforest was significantly greater at lower distance from the community to the nearest native forest for those with lower income level, and was higher when there was use of nontimber forest products or wood by the respondents. However, in spite of the positive disposition to plant native trees, only 23% of the respondents were interested in planting on their own land, which is a requirement to receive the economic incentives. Most respondents were willing to plant in open sites and on degraded hillsides that surround their communities. We conclude that despite monetary incentives, benefits cannot reach most rural inhabitants because of their lack of interest in reforesting their own land.     

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of ¹³C-atoms from 1-[¹³C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.     

Canonical and noncanonical inflammasomes in intestinal epithelial cells

Inflammasomes are cytosolic, multimeric protein complexes capable of activating pro‐inflammatory cytokines such as IL‐1β and IL‐18, which play a key role in host defence. Inflammasome components are highly expressed in the intestinal epithelium. In recent years, studies have begun to demonstrate that epithelial‐intrinsic inflammasomes play a critical role in regulating epithelial homeostasis, both by defending the epithelium from pathogenic insult and through the regulation of the mucosal environment. However, the majority of research regarding inflammasome activation has focused on professional immune cells, such as macrophages. Here, we present an overview of the current understanding of inflammasome function in epithelial cells and at mucosal surfaces and, in particular, in the intestine.