Optimization of Ustilago nuda Inoculum Concentration for Screening Un8-Mediated Loose Smut Resistance in Barley Reveals a Resistance Reaction that Disrupts Seed Germination and Suggests a Role for Abscisic Acid in Disease Development.

Barley loose smut has been effectively controlled for decades through resistance conferred by the Un8 gene. However, evaluation of loose smut reaction using floret inoculation at the standard inoculum concentration is associated with the production of small, discolored seeds in Un8 carriers and susceptible genotypes. Interestingly, Un8 carriers also displayed significantly poorer germination than susceptible genotypes and produce short-lived seedlings following inoculation. To understand these observations, a Un8 carrier (TR11698) and susceptible non-Un8 carrier (CDC Austenson) were assessed for seed traits, Ustilago nuda biomass in the seed, infection rate, and phytohormone profile across a range of lower inoculum concentrations. At lower inoculum concentrations, seed appearance and weight improved in both genotypes, and infection rate increased in CDC Austenson. Pathogen load in the seed was similar in both genotypes and was positively correlated with the CDC Austenson infection rate. No infection was ever observed in TR11698. Significantly, germination rate improved in CDC Austenson, whereas the very low germination rate and short-lived seedlings remained associated with TR11698. It appears that poor seed appearance in both genotypes and low germination rate in the susceptible genotype can be improved by lowering the inoculum concentration. However, the very low germination rates and seedling death associated with the Un8 carrier TR11698 are indicative of Un8-mediated resistance to loose smut. Finally, profiling of 38 phytohormones revealed that larger seeds observed at some inoculum concentrations compared with mock inoculation had higher abscisic acid concentrations. This could represent a pathogen survival strategy by ensuring better growth of the host.

Topical steroid therapy induces pro-tolerogenic changes in Langerhans cells in human skin.

We have investigated the efficacy of conditioning skin Langerhans cells (LCs) with agents to promote tolerance and reduce inflammation, with the goal of improving the outcomes of antigen-specific immunotherapy. Topical treatments were assessed ex vivo, using excised human breast skin maintained in organ bath cultures, and in vivo in healthy volunteers by analysing skin biopsies and epidermal blister roof samples. Following topical treatment with a corticosteroid, tumour necrosis factor-α levels were reduced in skin biopsy studies and blister fluid samples. Blister fluid concentrations of monocyte chemoattractant protein-1, macrophage inflammatory proteins -1α and 1ß and interferon-γ inducible protein-10 were also reduced, while preserving levels of interleukin-1α (IL-1α), IL-6, IL-8 and IL-10. Steroid pre-treatment of the skin reduced the ability of LCs to induce proliferation, while supernatants showed an increase in the IL-10/interferon-γ ratio. Phenotypic changes following topical steroid treatment were also observed, including reduced expression of CD83 and CD86 in blister-derived LCs, but preservation of the tolerogenic signalling molecules immunoglobulin-like transcript 3 and programmed death-1. Reduced expression of HLA-DR, CD80 and CD86 were also apparent in LCs derived from excised human skin. Topical therapy with a vitamin D analogue (calcipotriol) and steroid, calcipotriol alone or vitamin A elicited no significant changes in the parameters studied. These experiments suggest that pre-conditioning the skin with topical corticosteroid can modulate LCs by blunting their pro-inflammatory signals and potentially enhancing tolerance. We suggest that such modulation before antigen-specific immunotherapy might provide an inexpensive and safe adjunct to current approaches to treat autoimmune diseases.

Fine mapping and identification of a candidate gene for the barley Un8 true loose smut resistance gene.

KEY MESSAGE: The candidate gene for the barley Un8 true loose smut resistance gene encodes a deduced protein containing two tandem protein kinase domains. In North America, durable resistance against all known isolates of barley true loose smut, caused by the basidiomycete pathogen Ustilago nuda (Jens.) Rostr. (U. nuda), is under the control of the Un8 resistance gene. Previous genetic studies mapped Un8 to the long arm of chromosome 5 (1HL). Here, a population of 4625 lines segregating for Un8 was used to delimit the Un8 gene to a 0.108 cM interval on chromosome arm 1HL, and assign it to fingerprinted contig 546 of the barley physical map. The minimal tilling path was identified for the Un8 locus using two flanking markers and consisted of two overlapping bacterial artificial chromosomes. One gene located close to a marker co-segregating with Un8 showed high sequence identity to a disease resistance gene containing two kinase domains. Sequence of the candidate gene from the parents of the segregating population, and in an additional 19 barley lines representing a broader spectrum of diversity, showed there was no intron in alleles present in either resistant or susceptible lines, and fifteen amino acid variations unique to the deduced protein sequence in resistant lines differentiated it from the deduced protein sequences in susceptible lines. Some of these variations were present within putative functional domains which may cause a loss of function in the deduced protein sequences within susceptible lines.

Designing a combined liothyronine (LT3), L- thyroxine (LT4) trial in symptomatic hypothyroid subjects on LT4 - the importance of patient selection, choice of LT3 and trial design.

Approximately 10%-15% of subjects with hypothyroidism on L-thyroxine (LT4) alone have persistent symptoms affecting their quality of life (QoL). Although the cause is unclear, there is evidence that "tissue T3 lack" may be responsible. If so, combining liothyronine (LT3) with LT4 would be helpful. However, randomized controlled trials (RCT), have not established greater efficacy for the LT3 + LT4 combination in these subjects than for LT4 alone. While the trial design may have been responsible, the use of unphysiological, short-acting LT3 preparations and non-thyroid-specific patient-reported outcome measures (PROMs) may have contributed. We recommend attention to the following aspects of trial design for future RCTs of LT3 + LT4 compared to LT4 alone: (a) Subject selection-(i) measurable symptoms (disadvantages should be recognized); (ii) using a validated thyroid specific PROM such as ThyPRO39 or the Composite scale derived from it; (iii) those taking over 1.2 µg/day or 100 µg/day (for pragmatic reasons) of LT4 defining a population likely without intrinsic thyroid activity who depend on exogenous LT4; (iv) recruiting a preponderance of subjects with autoimmune thyroiditis increasing generalisability; and (v) those with a high symptom load with a greater response to combination therapy e.g. those with the deiodinase 2 polymorphism. (b) The use of physiological LT3 preparations producing pharmacokinetic similarities to T3 profiles in unaffected subjects: two long-acting LT3 preparations are currently available and must be tested in phase 2b/3 RCTs. (c) The superiority of a crossover design in limiting numbers and costs while maintaining statistical power and ensuring that all subjects experienced the investigative medication.

Growth and chemical defense in willow seedlings: trade-offs are transient.

Many studies have failed to detect costs of defense and some have even found a positive correlation between growth and the concentrations of chemical defenses. These studies contradict the theoretical assumption that anti-herbivore defenses are costly-produced at the expense of growth and/or reproduction. Costs, however, may be transient and therefore difficult to detect. Here we tested the hypothesis that costs of defense would be pronounced early in development when root growth is prioritized (high percent root allocation), but not later in development. To test this hypothesis, we grew F(2) hybrid willow seedlings from five different families, and harvested cohorts of even-aged seedlings after 6, 7, 8 and 9 weeks of growth. Seedlings were divided into root and shoot tissue and shoots were analyzed for phenolics (condensed tannins and phenolic glycosides). We found evidence for transient costs of defense. The concentrations of phenolics were negatively correlated with total biomass, shoot biomass, and the proportion of biomass allocated to roots in week 6. After week 6, however, the concentrations of phenolics were positively correlated with shoot biomass and total biomass, while phenolics were uncorrelated with the proportion of biomass allocated to roots. These results, the first ever, to our knowledge, with woody plants, suggest that costs of defense were transient; specifically, costs were found in early development, when root establishment was a priority. Our findings suggest that studies should focus more on trade-offs early in plant development.