Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O(6)-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system

Genotypic Diversity Is Associated with Clinical Outcome and Phenotype in Cryptococcal Meningitis across Southern Africa.

Cryptococcal meningitis is a major cause of mortality throughout the developing world, yet little is known about the genetic markers underlying Cryptococcal virulence and patient outcome. We studied a cohort of 230 Cryptococcus neoformans (Cn) isolates from HIV-positive South African clinical trial patients with detailed clinical follow-up using multi-locus sequence typing and in vitro phenotypic virulence assays, correlating these data with clinical and fungal markers of disease in the patient. South African Cn displayed high levels of genetic diversity and locus variability compared to globally distributed types, and we identified 50 sequence types grouped within the main molecular types VNI, VNII and VNB, with 72% of isolates typed into one of seven 'high frequency' sequence types. Spatial analysis of patients' cryptococcal genotype was not shown to be clustered geographically, which might argue against recent local acquisition and in favour of reactivation of latent infection. Through comparison of MLST genotyping data with clinical parameters, we found a relationship between genetic lineage and clinical outcome, with patients infected with the VNB lineage having significantly worse survival (n=8, HR 3.35, CI 1.51-7.20, p=0.003), and this was maintained even after adjustment for known prognostic indicators and treatment regimen. Comparison of fungal genotype with in vitro phenotype (phagocytosis, laccase activity and CSF survival) performed on a subset of 89 isolates revealed evidence of lineage-associated virulence phenotype, with the VNII lineage displaying increased laccase activity (p=0.001) and ex vivo CSF survival (p=0.0001). These findings show that Cryptococcus neoformans is a phenotypically heterogeneous pathogen, and that lineage plays an important role in cryptococcal virulence during human infection. Furthermore, a detailed understanding of the genetic diversity in Southern Africa will support further investigation into how genetic diversity is structured across African environments, allowing assessment of the risks different ecotypes pose to infection

The value of plantation forests for plant, invertebrate and bird diversity and the potential for cross-taxon surrogacy

As the area of plantation forest expands worldwide and natural, unmanaged forests decline there is much interest in the potential for planted forests to provide habitat for biodiversity. In regions where little semi-natural woodland remains, the biodiversity supported by forest plantations, typically non-native conifers, may be particularly important. Few studies provide detailed comparisons between the species diversity of native woodlands which are being depleted and non-native plantation forests, which are now expanding, based on data collected from multiple taxa in the same study sites. Here we compare the species diversity and community composition of plants, invertebrates and birds in Sitka spruce- (Picea sitchensis-) dominated and Norway spruce- (Picea abies-) dominated plantations, which have expanded significantly in recent decades in the study area in Ireland, with that of oak- and ash-dominated semi-natural woodlands in the same area. The results show that species richness in spruce plantations can be as high as semi-natural woodlands, but that the two forest types support different assemblages of species. In areas where non-native conifer plantations are the principle forest type, their role in the provision of habitat for biodiversity conservation should not be overlooked. Appropriate management should target the introduction of semi-natural woodland characteristics, and on the extension of existing semi-natural woodlands to maintain and enhance forest species diversity. Our data show that although some relatively easily surveyed groups, such as vascular plants and birds, were congruent with many of the other taxa when looking across all study sites, the similarities in response were not strong enough to warrant use of these taxa as surrogates of the others. In order to capture a wide range of biotic variation, assessments of forest biodiversity should either encompass several taxonomic groups, or rely on the use of indicators of diversity that are not species based

Method for estimation of apoptotic cell fraction of cytotherapy using in vivo fluorine-19 magnetic resonance: pilot study in a patient with head and neck carcinoma receiving tumor-infiltrating lymphocytes labeled with perfluorocarbon nanoemulsion

Background Adoptive transfer of T cells is a burgeoning cancer therapeutic approach. However, the fate of the cells, once transferred, is most often unknown. We describe the first clinical experience with a non-invasive biomarker to assay the apoptotic cell fraction (ACF) after cell therapy infusion, tested in the setting of head and neck squamous cell carcinoma (HNSCC). A patient with HNSCC received autologous tumor-infiltrating lymphocytes (TILs) labeled with a perfluorocarbon (PFC) nanoemulsion cell tracer. Nanoemulsion, released from apoptotic cells, clears through the reticuloendothelial system, particularly the Kupffer cells of the liver, and fluorine-19 (19F) magnetic resonance spectroscopy (MRS) of the liver was used to non-invasively infer the ACF.Methods Autologous TILs were isolated from a patient in their late 50s with relapsed, refractory human papillomavirus-mediated squamous cell carcinoma of the right tonsil, metastatic to the lung. A lung metastasis was resected for T cell harvest and expansion using a rapid expansion protocol. The expanded TILs were intracellularly labeled with PFC nanoemulsion tracer by coincubation in the final 24 hours of culture, followed by a wash step. At 22 days after intravenous infusion of TILs, quantitative single-voxel liver 19F MRS was performed in vivo using a 3T MRI system. From these data, we model the apparent ACF of the initial cell inoculant.Results We show that it is feasible to PFC-label ~70×1010 TILs (F-TILs) in a single batch in a clinical cell processing facility, while maintaining >90% cell viability and standard flow cytometry-based release criteria for phenotype and function. Based on quantitative in vivo 19F MRS measurements in the liver, we estimate that ~30% cell equivalents of adoptively transferred F-TILs have become apoptotic by 22 days post-transfer.Conclusions Survival of the primary cell therapy product is likely to vary per patient. A non-invasive assay of ACF over time could potentially provide insight into the mechanisms of response and non-response, informing future clinical studies. This information may be useful to developers of cytotherapies and clinicians as it opens an avenue to quantify cellular product survival and engraftment

<i>In vitro</i> phenotyping results ordered by molecular type, VNI subtype, and high frequency MLST type.

<p>* CSF survival was not performed for one of the eight VNB isolates.</p><p><i>In vitro</i> phenotyping results ordered by molecular type, VNI subtype, and high frequency MLST type.</p

Median <i>in vitro</i> phenotyping values ordered by molecular type, VNI Subtype and high frequency MLST.

<p>Kruskal Wallis analysis performed on groups, and p values shown. Overall median is plotted in red. (A) Survival in <i>ex vivo</i> CSF, (B) Laccase Activity normalised to H99 reference strain, (C) <i>In vitro</i> phagocytosis of isolates by J774 cells (per 1 μl lysate).</p

A Consensus-Based Set of Measures for Oral Health Care

Increasingly more responsive and accountable health care systems are demanded, which is characterized by transparency and explicit demonstration of competence by health care providers and the systems in which they work. This study aimed to establish measures of oral health for transparent and explicit reporting of routine data to facilitate more patient-centered and prevention-oriented oral health care. To accomplish this, an intermediate objective was to develop a comprehensive list of topics that a range of stakeholders would perceive as valid, important, and relevant for describing oral health and oral health care. A 4-stage approach was used to develop the list of topics: 1) scoping of literature and its appraisal, 2) a meeting of experts, 3) a 2-stage Delphi process (online), and 4) a World Café discussion. The aim was to create consensus through structured conversations via a range of stakeholders (general dental practitioners, patients, insurers, and policy makers) from the Netherlands, Germany, the United Kingdom, Ireland, Hungary, and Denmark. The study was part of the ADVOCATE project, and it resulted in a list of 48 topics grouped into 6 clusters: 1) access to dental care, 2) symptoms and diagnosis, 3) health behaviors, 4) oral treatments, 5) oral prevention, and 6) patient perception. All topics can be measured, as they all have a data source with defined numerators and denominators. This study is the first to establish a comprehensive and multiple-stakeholder consented topic list designed for guiding the implementation of transparent and explicit measurement of routine data of oral health and oral health care. Successful measurement within oral health care systems is essential to facilitate learning from variation in practice and outcomes within and among systems, and it potentiates improvement toward more patient-centered and prevention-oriented oral health care

Phylogenetic and Bayesian analysis of concatenated nucleotide sequences.

<p>(A) SplitsTree neighbour network shows diverse VNII and VNB, and clonal VNI clades. (B) Analysis of VNI clustering using StructureHARVESTER and ΔK shows optimal number of K clusters is 3. (C) Use of STRUCTURE allows VNI sequences to be subdivided into 3 distinct populations: VNI(a), VNI(b), VNI(c).</p

Clinical characteristics and survival of patients infected with different lineages of <i>C neoformans</i>.

<p>(A) Patient survival by molecular type; VNB isolates (n = 8) were associated with significantly worse outcome compared to VNI and VNII isolates at 70 days (p = 0.01) and 365 days (p = 0.003) follow-up in Cox’s proportional hazard survival analysis. (B) Patient survival by high frequency MLST type. (C) Number of patients with and without altered mental status by molecular group (% with altered mental status indicated above each bar). (D) Baseline fungal burden, showing variation by high frequency MLST type.</p