Pyrrole-mediated peptide cyclization identified through genetically reprogrammed peptide synthesis

Flexible in vitro translation (FIT) was used as a screening method to uncover a new methodology for peptide constraining based on the attack of a nucleophilic side-chain functionality onto an oxidized furylalanine side chain. A set of template peptides, each containing furylalanine as furan-modified amino acid and a nucleophilic residue (Cys, His, Lys, Arg, Ser, or Tyr), was produced through FIT. The translation mixtures were treated with N-bromosuccinimide (NBS) to achieve selective furan oxidation and subsequent MALDI analysis demonstrated Lys and Ser as promising residues for cyclisation. Solid-phase peptide synthesis (SPPS) was used to synthesize suitable amounts of material for further in-depth analysis and characterisation. It was found that in the case of the peptide containing lysine next to a furylalanine residue, a one-pot oxidation and reduction reaction leads to the generation of a cyclic peptide featuring a pyrrole moiety as cyclisation motif, resulting from the attack of the lysine side chain onto the oxidized furylalanine side chain. Structural evidence was provided via NMR and the generality of the methodology was explored. We hereby expand the scope of our previously developed furan-based peptide labeling and crosslinking strategy

Into the fourth dimension

The influence of time on the drought response of Brassica rapa, an agriculturally important species of plant, has been clarified

Root hairs enable high transpiration rates in drying soils

What processes facilitate the ability of roots to take up water from the soil? Are root hairs advantageous for water uptake? Despite the well documented role of root hairs in phosphate uptake, their role in water extraction is controversial and the experimental data contradictory. We proposed a novel experimental method to address this question. We grew barley (Hordeum vulgare L. cv. Pallas) and its root-hairless mutant in a pressure chamber whereby the transpiration rate could be varied while monitoring the suction in the xylem. We monitored xylem water potential as function of different transpiration rate during a drying cycle. The relationship between transpiration rate and xylem suction linearly increased in wet soils and did not differ between genotypes. The slope of this increase was equal to the plant hydraulic resistance. When the soil dried the xylem water suction rapidly increased, particularly at high transpiration rates. The root-hairless mutant showed a more marked increase in the xylem suction, indicative of a lower capacity to take up water. Interestingly, the high rise in xylem suction at high transpiration rates did not quickly decrease as the transpiration rate was reduced. To quantitatively understand the relationship between transpiration rate and xylem suction and the role of root hairs, we employed a 3D root architectural model coupled with water flow in soils. The model was parametrized based on measured root architecture and soil hydraulic properties. The role of the root hairs was simulated by extending the root radius in presence of root hairs. This implicitly simulates the ability of root hairs to take up water from their tips, potential softening the drops in water potential across the rhizosphere. The simulations predicted that that as the soil dries a bigger drop in water potential develop around the roots of the root-hairless mutant. Extension of the root radius by 0.7 mm (to simulate the uptake of root hairs) reduced the drop in water potential around the roots and softened the decrease in the xylem water potential, particularly at high transpiration rates. We conclude that the root-soil interface plays a key role in root water uptake and that root hairs reduce the gradient in water potential around the roots and enable plants to sustain high transpiration rates in drying soils

A tillering inhibition gene influences root-shoot carbon partitioning and pattern of water use to improve wheat productivity in rainfed environments

Genetic modification of shoot and root morphology has potential to improve water and nutrient 19 uptake of wheat crops in rainfed environments. Near-isogenic lines (NILs) varying for a tillering 20 inhibition (tin) gene and representing multiple genetic backgrounds were investigated in contrasting 21 controlled environments for shoot and root growth. Leaf area, shoot and root biomass were similar 22 until tillering whereupon reduced tillering in tin-containing NILs produced reductions of up to 60% in 23 total leaf area and biomass, and increases in total root length of up to 120% and root biomass to 24 145%. Together, root-to-shoot ratio increased two-fold with the tin gene. The influence of tin on shoot 25 and root growth was greatest in the cv. Banks genetic background, particularly in the biculm-selected 26 NIL, and was typically strongest in cooler environments. A separate de-tillering study confirmed 27 greater root-to-shoot ratios with regular tiller removal in non-tin containing genotypes. In validating 28 these observations in a rainfed field study, the tin allele had a negligible effect on seedling growth but 29 was associated with significantly (P<0.05) reduced tiller number (-37%), leaf area index (-26%) and 30 spike number (-35%) to reduce plant biomass (-19%) at anthesis. Root biomass, root-to-shoot ratio at 31 early stem elongation and root depth at maturity were increased in tin-containing NILs. Soil water use 32 was slowed in tin-containing NILs resulting in greater water availability, greater stomatal 33 conductance, cooler canopy temperatures and maintenance of green leaf area during grain-filling. 34 Together these effects contributed to increases in harvest index and grain yield. In both the controlled 35 and field environments, the tin gene was commonly associated with increased root length and biomass 36 but the significant influence of genetic background and environment suggests careful assessment of 37 tin-containing progeny in selection for genotypic increases in root growth

De Novo Discovery of Nonstandard Macrocyclic Peptides as Noncompetitive Inhibitors of the Zika Virus NS2B-NS3 Protease

The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.Open AccessFinancial support by the Australian Research Council, including a Laureate Fellowship for G.O. is gratefully acknowledged. This work was also partially supported by CREST for Molecular Technologies, JST, and JSPS KAKENHI (16H06444 and 26220204) to H.S. C.K. acknowledges support by the Deutsche Forschungsgemeinschaft (KL-1356/3-2). We thank Mrs. Natascha Stefan for technical support

Interfering ribonucleic acids that suppress expression of multiple unrelated genes

<p>Abstract</p> <p>Background</p> <p>Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of <it>VEGF-A </it>and <it>ICAM-1</it>; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy.</p> <p>Results</p> <p>Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated.</p> <p>Conclusion</p> <p>Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach.</p

Spatial inequities of mental health nurses in rural and remote Australia

Abstract Despite an increased burden from chronic mental health conditions, access to effective mental health services in rural and remote areas is limited, and these services remain spatially undefined. We examine the spatial availability of mental health nurses across local government areas in Australia and identify gaps in mental health service delivery capacity in a finer-grained level than the state/territory data. A spatial distribution of mental health nurses was conducted. We utilized the 2017 National Health Workforce Dataset which was aggregated to LGA level based on the 2018 Australian Bureau Statistics (ABS) Data. The availability of mental health nurses was measured using the full time equivalent (FTE) rates per 100 000 population. We calculated the proportion of LGAs with zero total FTE rates based on remoteness categories. We also compared the mean of total FTE rates based on remoteness categories using analysis of variance. A spatial distribution of mental health nurses was visualized using GIS software for total FTE rates. Our analysis included 544 LGA across Australia, with 24.8% being defined as remote and very remote. The mean total FTE for mental health nurses per 100 000 populations is 56.6 (±132.2) with a median of 17.4 (IQR: 61.8). A wide standard deviation reflects unequal distribution of mental health nurses across LGAs. The availability of total FTE rates for mental health nurses per 100 000 populations is significantly lower in remote and very remote LGAs in comparison with major cities. As many as 35.1% of LGAs across Australia have no FTE for mental health nurses with 46% are remote and very remote. Our study reflects the existing unequal distribution of mental health nurses between metropolitan/urban setting and rural and remote areas. We suggest three broad strategies to address these spatial inequities: improving supply and data information systems; revisiting task-shifting strategies, retraining the existing health workforce to develop skills necessary for mental health care to rural and remote communities; and incorporating the provision of mental health services within expanding innovative delivery models including consumer-led, telemedicine and community-based groups

Variations in water use by a mature mangrove of Avicennia germinans, French Guiana

In the tropical intertidal zones, little is known on water uptake by mangroves. Transpiration rates are generally measured at leaf level, but few studies exist on water use at tree or stand levels. The objective of this study was to measure sap flow in trees of different sizes to appreciate the range of variation in water use that may exist in a site dominated by 80% mature Avicennia germinans. The results showed that from the dry to the wet season the mean water use increased from 3.2 to 5.3 dm3 d−1 in small trees (DBH ∼ 13 cm), from 11.5 to 30.8 dm3 d−1 in medium trees (∼24 cm) and from 40.8 to 64.1 dm3 d−1 in large ones (∼45 cm). Sapwood remained active up to a depth of 8 cm with radial variations within the stem. Weak correlations were obtained with VPD and net radiation. This study confirmed that transpiration was larger under low levels of salinity. Water use at stand level (∼1900 living stems ha−1) was estimated to be in the range of 5.8 to 11.8 m3 ha−1 d−1 according to the season

Additive effects of Na+ and Cl– ions on barley growth under salinity stress

Soil salinity affects large areas of the world’s cultivated land, causing significant reductions in crop yield. Despite the fact that most plants accumulate both sodium (Na+) and chloride (Cl–) ions in high concentrations in their shoot tissues when grown in saline soils, most research on salt tolerance in annual plants has focused on the toxic effects of Na+ accumulation. It has previously been suggested that Cl– toxicity may also be an important cause of growth reduction in barley plants. Here, the extent to which specific ion toxicities of Na+ and Cl– reduce the growth of barley grown in saline soils is shown under varying salinity treatments using four barley genotypes differing in their salt tolerance in solution and soil-based systems. High Na+, Cl–, and NaCl separately reduced the growth of barley, however, the reductions in growth and photosynthesis were greatest under NaCl stress and were mainly additive of the effects of Na+ and Cl– stress. The results demonstrated that Na+ and Cl– exclusion among barley genotypes are independent mechanisms and different genotypes expressed different combinations of the two mechanisms. High concentrations of Na+ reduced K+ and Ca2+ uptake and reduced photosynthesis mainly by reducing stomatal conductance. By comparison, high Cl– concentration reduced photosynthetic capacity due to non-stomatal effects: there was chlorophyll degradation, and a reduction in the actual quantum yield of PSII electron transport which was associated with both photochemical quenching and the efficiency of excitation energy capture. The results also showed that there are fundamental differences in salinity responses between soil and solution culture, and that the importance of the different mechanisms of salt damage varies according to the system under which the plants were grown

Different mechanisms of adaptation to cyclic water stress in two South Australian bread wheat cultivars

In the South Australian wheat belt, cyclic drought is a frequent event represented by intermittent periods of rainfall which can occur around anthesis and post-anthesis in wheat. Three South Australian bread wheat (Triticum aestivum L.) cultivars, Excalibur, Kukri, and RAC875, were evaluated in one greenhouse and two growth-room experiments. In the first growth-room experiment, where plants were subjected to severe cyclic water-limiting conditions, RAC875 and Excalibur (drought-tolerant) showed significantly higher grain yield under cyclic water availability compared to Kukri (drought-susceptible), producing 44% and 18% more grain compared to Kukri, respectively. In the second growth-room experiment, where plants were subjected to a milder drought stress, the differences between cultivars were less pronounced, with only RAC875 showing significantly higher grain yield under the cyclic water treatment. Grain number per spike and the percentage of aborted tillers were the major components that affected yield under cyclic water stress. Excalibur and RAC875 adopted different morpho-physiological traits and mechanisms to reduce water stress. Excalibur was most responsive to cyclic water availability and showed the highest level of osmotic adjustment (OA), high stomatal conductance, lowest ABA content, and rapid recovery from stress under cyclic water stress. RAC875 was more conservative and restrained, with moderate OA, high leaf waxiness, high chlorophyll content, and slower recovery from stress. Within this germplasm, the capacity for osmotic adjustment was the main physiological attribute associated with tolerance under cyclic water stress which enabled plants to recover from water deficit