Engineering,Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.     

Relative DNA content in diploid,polyploid, and multiploid species of <Emphasis Type="Italic">Paspalum</Emphasis> (Poaceae) with relation to reproductive mode and taxonomy

It is generally accepted that polyploids have downsized basic genomes rather than additive values with respect to their related diploids. Changes in genome size have been reported in correlation with several biological characteristics. About 75 % of around 350 species recognized for Paspalum (Poaceae) are polyploid and most polyploids are apomictic. Multiploid species are common with most of them bearing sexual diploid and apomictic tetraploid or other ploidy levels. DNA content in the embryo and the endosperm was measured by flow cytometry in a seed-by-seed analysis of 47 species including 77 different entities. The relative DNA content of the embryo informed the genome size of the accession while the embryo:endosperm ratio of DNA content revealed its reproductive mode. The genome sizes (2C-value) varied from 0.5 to 6.5 pg and for 29 species were measured for the first time. Flow cytometry provided new information on the reproductive mode for 12 species and one botanical variety and supplied new data for 10 species concerning cytotypes reported for the first time. There was no significant difference between the mean basic genome sizes (1Cx-values) of 32 sexual and 45 apomictic entities. Seventeen entities were diploid and 60 were polyploids with different degrees. There were no clear patterns of changes in 1Cx-values due to polyploidy or reproductive systems, and the existing variations are in concordance with subgeneric taxonomical grouping.     

E‐cadherin expression is associated with somatostatin analogue response in acromegaly

Acromegaly is a rare disease resulting from hypersecretion of growth hormone (GH) and insulin‐like growth factor 1 (IGF1) typically caused by pituitary adenomas, which is associated with increased mortality and morbidity. Somatostatin analogues (SSAs) represent the primary medical therapy for acromegaly and are currently used as first‐line treatment or as second‐line therapy after unsuccessful pituitary surgery. However, a considerable proportion of patients do not adequately respond to SSAs treatment, and therefore, there is an urgent need to identify biomarkers predictors of response to SSAs. The aim of this study was to examine E‐cadherin expression by immunohistochemistry in fifty‐five GH‐producing pituitary tumours and determine the potential association with response to SSAs as well as other clinical and histopathological features. Acromegaly patients with tumours expressing low E‐cadherin levels exhibit a worse response to SSAs. E‐cadherin levels are associated with GH‐producing tumour histological subtypes. Our results indicate that the immunohistochemical detection of E‐cadherin might be useful in categorizing acromegaly patients based on the response to SSAs.     

Multiparametric magnetic resonance imaging-guided salvage radiotherapy in prostate cancer

AimTo analyse the efficacy and toxicity of postprostatectomy SRT in patients with a BCR evaluated with mpMRI.BackgroundMultiparametric magnetic resonance imaging (mpMRI) has the ability to detect the site of pelvic recurrence in patients with biochemical recurrence (BCR) after radical prostatectomy (RP). However, we do not know the oncological outcomes of mpMRI-guided savage radiotherapy (SRT).ResultsLocal, lymph node, and pelvic bone recurrence was observed in 13, 4 and 2 patients, respectively. PSA levels were significantly lower in patients with negative mpMRI (0.4 ng/mL [0.4]) vs. positive mpMRI (2.2 ng/mL [4.1], p = 0.003). Median planning target volume doses in patients with visible vs. non-visible recurrences were 76 Gy vs. 70 Gy. Overall, mean follow-up was 41 months (6–81). Biochemical relapse-free survival (bRFS) at 3 years was 82.3% and 82.5%, respectively, for the negative and positive mpMRI groups (p = 0.800). Three-year rates of late grade ≥2 urinary and rectal toxicity were 14.8% and 1.9%, respectively; all but one patient recovered without sequelae.ConclusionSRT to the macroscopic recurrence identified by mpMRI is a feasible and well-tolerated option. In this study, there were no differences in bRFS between MRI-positive and MRI-negative patients, indicating effective targeting of MRI-positive lesions.     

Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay

BackgroundIn a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides.AimsTo identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test.MethodsWe surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides.ResultsSeven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a “brute force” approach for peptide design.ConclusionsThe H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.     

Direct seeding and outplantings in drylands of Argentinean Patagonia: estimated costs,and prospects for large‐scale restoration and rehabilitation

In large areas of the world that are deeply scarred by desertification and hampered by low capacity for natural regeneration, the scaling up of ecological restoration and rehabilitation can be achieved only if it is low in cost with high return on investment, and shows promise of providing long‐lasting social‐economic as well as ecological benefits. In the Monte Austral region of Patagonia Argentina, concerted efforts are underway to facilitate scaling up of ecological restoration and rehabilitation practices. Here, we evaluate financial costs and preliminary results of direct seeding as compared to outplanting of nursery‐grown seedlings of three native species (Atriplex lampa, Senecio subulatus var. subulatus, and Hyalis argentea var. latisquama) considered to be high‐priority dryland framework species. Comparative success is expressed in terms of plant survival and in monetary terms. The three candidate species showed low survival rates, ranging from 4.3 to 22.3%, after the first summer following direct seeding. In contrast, survival rates for planted seedlings of the same three taxa varied between 84 and 91%, after the first summer following reintroduction. However, cost of direct seeding varied between 1,693 and 1,772 US$ less per hectare, that is, 64% less than the cost of outplanting nursery seedlings. Therefore, in the search for ways to scale up ecological restoration and rehabilitation in drylands, direct seeding should receive more attention. We discuss the social and ecological perspectives and the way forward for direct seeding techniques in Patagonia. We also consider how costs could be reduced and effectiveness improved in large‐scale efforts.     

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.     

A comparative assessment of plant flammability through a functional approach: The case of woody species from Argentine Chaco region

Recent changes to fire regimes in many regions of the world have led to renewed interest in plant flammability experiments to understand and predict the consequences of such changes. These experiments require the development of practical and standardised flammability testing protocols. The research aims were (i) to compare plant flammability assessments carried out using two different approaches, namely functional trait analysis and testing with a shoot‐level device; and (ii) to evaluate the effect of disturbances and seasonal variability on flammability. The study area was located in the Western Chaco region, Argentina, and 11 species were selected based on their representativeness in forests. We studied six functional traits related to flammability, growth habit and foliar persistence, in forests without disturbances over the three last decades as well as in disturbed forests. The seasonal variation of these functional traits was evaluated over two consecutive years. Functional trait flammability index (FI) and shoot‐level measurements followed standard protocols. Sixty per cent of the species measured presented a high to very high FI. The results of both assessment methods were significantly correlated. Both methods identified the same species as having medium flammability, but differed in regards to the most flammable species. Senegalia gilliesii was identified as the most flammable species when using functional trait analysis, whereas shoot‐level assessments found Larrea divaricata and Schinus johnstonii to be the most flammable. There were no disturbance effects on the FI but there was seasonal variation. Our results validate the use of functional traits as a predictive method of flammability testing and represent the first global effort comparing flammability obtained through functional trait analysis with empirical measurements. The significant correlation between both methods allows the selection of the one that is more appropriate for the size of the area to be evaluated and for the availability of technical resources. Abstract in Spanish is available with online material.