Expanding the soil antibiotic resistome: exploring environmental diversity

Antibiotic resistance has largely been studied in the context of failure of the drugs in clinical settings. There is now growing evidence that bacteria that live in the environment (e.g. the soil) are multi-drug-resistant. Recent functional screens and the growing accumulation of metagenomic databases are revealing an unexpected density of resistance genes in the environment: the antibiotic resistome. This challenges our current understanding of antibiotic resistance and provides both barriers and opportunities for antimicrobial drug discovery.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

Improved induction of antibodies against key neutralizing epitopes by human immunodeficiency virus type 1 gp120 DNA prime-protein boost vaccination compared to gp120 protein-only vaccination

A major challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development is to elicit potent and broadly neutralizing antibodies that are effective against primary viral isolates. Previously, we showed that DNA prime-protein boost vaccination using HIV-1 gp120 antigens was more effective in eliciting neutralizing antibodies against primary HIV-1 isolates than was a recombinant gp120 protein-only vaccination approach. In the current study, we analyzed the difference in antibody specificities in rabbit sera elicited by these two immunization regimens using peptide enzyme-linked immunosorbent assay and a competitive virus capture assay. Our results indicate that a DNA prime-protein boost regimen is more effective than a protein-alone vaccination approach in inducing antibodies that target two key neutralizing domains: the V3 loop and the CD4 binding site. In particular, positive antibodies targeting several peptides that overlap with the known CD4 binding area were detected only in DNA-primed sera. Different profiles of antibody specificities provide insight into the mechanisms behind the elicitation of better neutralizing antibodies with the DNA prime-protein boost approach, and our results support the use of this approach to further optimize Env formulations for HIV vaccine development.     

In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry

Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.     

Diurnal variation in temperature, mental and physical performance, and tasks specifically related to football (soccer)

Football (soccer) training and matches are scheduled at different times throughout the day. Association football involves a variety of fitness components as well as psychomotor and game-related cognitive skills. The purpose of the present research, consisting of two separate studies, was to determine whether game-related skills varied with time of day in phase with global markers of both performance and the body clock. In the first study, eight diurnally active male association football players (19.1+/-1.9 yrs of age; mean+/-SD) with 10.8+/-2.1 yrs playing experience participated. Measurements were made on different days at 08:00, 12:00, 16:00, and 20:00 h in a counterbalanced manner. Time-of-day changes in intra-aural temperature (used as a marker of the body clock), grip strength, reaction times, flexibility (markers of aspects of performance), juggling and dribbling tasks, and wall-volley test (football-specific skills) were compared. Significant (repeated measures analysis of variance, ANOVA) diurnal variations were found for body temperature (p<0.0005), choice reaction time (p<0.05), self-rated alertness (p<0.0005), fatigue (p<0.05), forward (sit-and-reach) flexibility (p<0.02), and right-hand grip strength (p<0.02), but not left-hand grip strength (p=0.40) nor whole-body (stand-and-reach) flexibility (p=0.07). Alertness was highest and fatigue lowest at 20:00 h. Football-specific skills of juggling performance showed significant diurnal variation (p<0.05, peak at 16:00 h), whereas performance on the wall-volley test tended to peak at 20:00 h and dribbling showed no time-of-day effect (p=0.55). In a second study, eight diurnally active subjects (23.0+/-0.7 yrs of age) completed five test sessions, at the same times as in the first study but with a second session at 08:00 h. Test-re-test comparisons at 08:00 h for all components indicated good reliability. Intra-aural temperature showed a significant time-of-day effect (p<0.001) with mean temperature at 16:00 h (36.4 degrees C) higher than at 08:00 h (35.4 degrees C). There was no significant effect of chronotype on the temperature acrophase (peak time) (p>0.05). Diurnal variation was found for performance tests, including sit-and-reach flexibility (p<0.01) and spinal hyper-extension (p<0.05). Peaks occurred between 16:00 and 20:00 h and the daytime changes paralleled the temperature rhythm. Diurnal variation was also found for football-specific tests, including dribbling time (p<0.001, peak at 20:00 h) and chip test performance (p<0.01), being more accurate at 16:00 h (mean error=0.75 m) than at 08:00 h (mean error=1.01 m). Results indicate football players perform at an optimum between 16:00 and 20:00 h when not only football-specific skills but also measures of physical performance are at their peak. Body temperature peaked at a similar time, but positive mood states seemed to peak slightly earlier. While causal links cannot be established in these experiments, the results indicate that the diurnal variation of some aspects of football performance is affected by factor(s) other than body temperature alone.     

Spinal cord injury reveals multilineage differentiation of ependymal cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.     

The power of data synthesis to shape the future of the restoration community and capacity

Restoration efforts will be taking place over the next decade(s) in the largest scope and capacity ever seen. Immense commitments, goals, and budgets are set, with impactful wide‐reaching potential benefits for people and the environment. These are ambitious aims for a relatively new branch of science and practice. It is time for restoration action to scale up, the legacy of which could impact over 350 million hectares targeted for the U.N. Decade on Ecosystem Restoration. However, restoration still proceeds on a case‐by‐case, trial by error basis and restoration outcomes can be variable even under similar conditions. The ability to put each case into context—what about it worked, what did not, and why—is something that the synthesis of data across studies can facilitate. The link between data synthesis and predictive capacity is strong. There are examples of extremely ambitious and successful efforts to compile data in structured, standardized databases which have led to valuable insights across regional and global scales in other branches of science. There is opportunity and challenge in compiling, standardizing, and synthesizing restoration monitoring data to inform the future of restoration practice and science. Through global collation of restoration data, knowledge gaps can be addressed and data synthesized to advance toward a more predictive science to inform more consistent success. The interdisciplinary potential of restoration ecology sits just over the horizon of this decade. Through truly collaborative synthesis across foci within the restoration community, we have the opportunity to rapidly reach that potential and achieve extraordinary outcomes together.     

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

QTLs for Tolerance of Drought and Breeding for Tolerance of Abiotic and Biotic Stress: An Integrated Approach

Background

The coupling of biotic and abiotic stresses leads to high yield losses in rainfed rice (Oryza sativa L.) growing areas. While several studies target these stresses independently, breeding strategies to combat multiple stresses seldom exist. This study reports an integrated strategy that combines QTL mapping and phenotypic selection to develop rice lines with high grain yield (GY) under drought stress and non-stress conditions, and tolerance of rice blast.

Methodology

A blast-tolerant BC₂F₃-derived population was developed from the cross of tropical japonica cultivar Moroberekan (blast- and drought-tolerant) and high-yielding indica variety Swarna (blast- and drought-susceptible) through phenotypic selection for blast tolerance at the BC₂F₂ generation. The population was studied for segregation distortion patterns and QTLs for GY under drought were identified along with study of epistatic interactions for the trait.

Results

Segregation distortion, in favour of Moroberekan, was observed at 50 of the 59 loci. Majority of these marker loci co-localized with known QTLs for blast tolerance or NBS-LRR disease resistance genes. Despite the presence of segregation distortion, high variation for DTF, PH and GY was observed and several QTLs were identified under drought stress and non-stress conditions for the three traits. Epistatic interactions were also detected for GY which explained a large proportion of phenotypic variance observed in the population.

Conclusions

This strategy allowed us to identify QTLs for GY along with rapid development of high-yielding purelines tolerant to blast and drought with considerably reduced efforts. Apart from this, it also allowed us to study the effects of the selection cycle for blast tolerance. The developed lines were screened at IRRI and in the target environment, and drought and blast tolerant lines with high yield were identified. With tolerance to two major stresses and high yield potential, these lines may provide yield stability in rainfed rice areas.