Identification, validation and biological characterisation of novel glioblastoma tumour microenvironment subtypes: implications for precision immunotherapy.

BACKGROUND: New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles have failed to direct treatment strategies. We hypothesised that interrogation of the GBM tumour microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision immunotherapy treatment strategies. MATERIALS AND METHODS: A refined and validated microenvironment cell population (MCP) counter method was applied to >800 GBM patient tumours (GBM-MCP-counter). Specifically, partition around medoids (PAM) clustering of GBM-MCP-counter scores in the GLIOTRAIN discovery cohort identified three novel patient clusters, uniquely characterised by TME composition, functional orientation markers and immune checkpoint proteins. Validation was carried out in three independent GBM-RNA-seq datasets. Neoantigen, mutational and gene ontology analysis identified mutations and uniquely altered pathways across subtypes. The longitudinal Glioma Longitudinal AnalySiS (GLASS) cohort and three immunotherapy clinical trial cohorts [treatment with neoadjuvant/adjuvant anti-programmed cell death protein 1 (PD-1) or PSVRIPO] were further interrogated to assess subtype alterations between primary and recurrent tumours and to assess the utility of TME classifiers as immunotherapy biomarkers. RESULTS: TMEHigh tumours (30%) displayed elevated lymphocyte, myeloid cell immune checkpoint, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 transcripts. TMEHigh/mesenchymal+ patients featured tertiary lymphoid structures. TMEMed (46%) tumours were enriched for endothelial cell gene expression profiles and displayed heterogeneous immune populations. TMELow (24%) tumours were manifest as an 'immune-desert' group. TME subtype transitions upon recurrence were identified in the longitudinal GLASS cohort. Assessment of GBM immunotherapy trial datasets revealed that TMEHigh patients receiving neoadjuvant anti-PD-1 had significantly increased overall survival (P = 0.04). Moreover, TMEHigh patients treated with adjuvant anti-PD-1 or oncolytic virus (PVSRIPO) showed a trend towards improved survival. CONCLUSIONS: We have established a novel TME-based classification system for application in intracranial malignancies. TME subtypes represent canonical 'termini a quo' (starting points) to support an improved precision immunotherapy treatment approach.

Updated guidelines for gene nomenclature in wheat.

KEY MESSAGE: Here, we provide an updated set of guidelines for naming genes in wheat that has been endorsed by the wheat research community. The last decade has seen a proliferation in genomic resources for wheat, including reference- and pan-genome assemblies with gene annotations, which provide new opportunities to detect, characterise, and describe genes that influence traits of interest. The expansion of genetic information has supported growth of the wheat research community and catalysed strong interest in the genes that control agronomically important traits, such as yield, pathogen resistance, grain quality, and abiotic stress tolerance. To accommodate these developments, we present an updated set of guidelines for gene nomenclature in wheat. These guidelines can be used to describe loci identified based on morphological or phenotypic features or to name genes based on sequence information, such as similarity to genes characterised in other species or the biochemical properties of the encoded protein. The updated guidelines provide a flexible system that is not overly prescriptive but provides structure and a common framework for naming genes in wheat, which may be extended to related cereal species. We propose these guidelines be used henceforth by the wheat research community to facilitate integration of data from independent studies and allow broader and more efficient use of text and data mining approaches, which will ultimately help further accelerate wheat research and breeding.

Elective and Emergency Deep Brain Stimulation in Refractory Pediatric Monogenetic Movement Disorders Presenting with Dystonia: Current Practice Illustrated by Two Cases.

BACKGROUND: Dystonia is characterized by sustained or intermittent muscle contractions, leading to abnormal posturing and twisting movements. In pediatric patients, dystonia often negatively influences quality of life. Pharmacological treatment for dystonia is often inadequate and causes adverse effects. Deep brain stimulation (DBS) appears to be a valid therapeutic option for pharmacoresistant dystonia in children. METHODS: To illustrate the current clinical practice, we hereby describe two pediatric cases of monogenetic movement disorders presenting with dystonia and treated with DBS. We provide a literature review of similar previously described cases and on different clinical aspects of DBS in pediatric dystonia. RESULTS: The first patient, a 6-year-old girl with severe dystonia, chorea, and myoclonus due to an ADCY5 gene mutation, received DBS in an elective setting. The second patient, an 8-year-old boy with GNAO1-related dystonia and chorea, underwent emergency DBS due to a pharmacoresistant status dystonicus. A significant amelioration of motor symptoms (65% on the Burke-Fahn-Marsden Dystonia Rating Scale) was observed postoperatively in the first patient and her personal therapeutic goals were achieved. DBS was previously reported in five patients with ADCY5-related movement disorders, of which three showed objective improvement. Emergency DBS in our second patient resulted in the successful termination of his GNAO1-related status dystonicus, this being the eighth case reported in the literature. CONCLUSION: DBS can be effective in monogenetic pediatric dystonia and should be considered early in the disease course. To better evaluate the effects of DBS on patients' functioning, patient-centered therapeutic goals should be discussed in a multidisciplinary approach.

The effects of small-scale physical and social environmental interventions on walking behaviour among Dutch older adults living in deprived neighbourhoods: results from the quasi-experimental NEW.ROADS study.

PURPOSE: Improving the physical and social conditions of residential neighbourhoods may increase walking, especially among older people. Evidence on the effects of physical and social environmental interventions, and particularly the combination of both, on walking behaviour is scarce. We evaluated the effects of a small-scale physical environmental intervention (designated walking route), a social environmental intervention (neighbourhood walking group) and the combination of both on walking behaviour of older adults living in deprived neighbourhoods. METHODS: Survey data of 644 older adults residing in four deprived neighbourhoods of Rotterdam, the Netherlands, were used to compare changes in walking behaviour over time (weekly minutes spent recreational walking, utilitarian walking and total walking) of those exposed to 1) a designated walking route (physical condition), 2) walking groups (social condition), 3) walking routes and walking groups (combined condition), and 4) no intervention (control condition). Measurements took place at baseline (T0), and 3 months (T1) and 9 months (T2) after the intervention. Data were analysed on a multiple imputed dataset, using multi-level negative binomial regression models, adjusting for clustering of observations within individuals. All models were adjusted for demographic covariates. RESULTS: Total time spent walking per week increased between T0 and T1 for all conditions. The Incidence Rate Ratio (IRR) for the physical condition was 1.46 (95% CI:1.06;2.05) and for the social intervention 1.52 (95%CI:1.07;2.16). At T2, these differences remained significant for the physical condition, but not for the social condition and the combined condition. These findings were mirrored for utilitarian walking. No evidence was found for an effect on recreational walking. CONCLUSION: Implementing small scale, feasible, interventions in a residential neighbourhood may increase total and utilitarian walking behaviour among older adults.

Effects of urban motorways on physical activity and sedentary behaviour in local residents: a natural experimental study.

BACKGROUND: There is little evidence on how changing the physical environment changes health-related behaviours. We studied the effects of the new M74 motorway (freeway) - opened in 2011 - and the existing M8 motorway in Glasgow, Scotland, on physical activity and sedentary behaviour among local residents. METHODS: This natural experimental study used baseline (T1; 2005) and follow-up data (T2; 2013) from a longitudinal cohort (N = 365) and two cross-sectional samples (T1 N = 980; T2 N = 978). Adult participants were recruited from three study areas: one surrounding the new motorway, one surrounding the existing motorway, and a third, control, area without a motorway. The outcomes were self-reported time spent sitting, walking, and in moderate-to-vigorous physical activity (MVPA). Motorway exposure was defined in terms of (1) study area and (2) distance from home to the nearest motorway junction. Outcomes were regressed on exposures in two-part (walking and MVPA) or linear (sedentary behaviour) cohort and repeat cross-sectional models, adjusted for baseline behaviour and sociodemographic covariates. RESULTS: Cohort participants living in the M8 area were less likely to participate in MVPA at follow-up than those living in the area without a motorway (OR 0.37; 95%CI 0.15, 0.91). Within the M8 area, those living closer to the motorway were also less likely to do so (OR 0.30; 95%CI 0.09, 0.97). No other statistically significant results were found. CONCLUSIONS: We found some evidence of a negative association between exposure to an existing urban motorway and MVPA. However, the behavioural impacts of motorways are likely to be complex and evolve over time.

Causal pathways linking environmental change with health behaviour change: Natural experimental study of new transport infrastructure and cycling to work.

BACKGROUND: Mechanisms linking changes to the environment with changes in physical activity are poorly understood. Insights into mechanisms of interventions can help strengthen causal attribution and improve understanding of divergent response patterns. We examined the causal pathways linking exposure to new transport infrastructure with changes in cycling to work. METHODS: We used baseline (2009) and follow-up (2012) data (N=469) from the Commuting and Health in Cambridge natural experimental study (Cambridge, UK). Exposure to new infrastructure in the form of the Cambridgeshire Guided Busway was defined using residential proximity. Mediators studied were changes in perceptions of the route to work, theory of planned behaviour constructs and self-reported use of the new infrastructure. Outcomes were modelled as an increase, decrease or no change in weekly cycle commuting time. We used regression analyses to identify combinations of mediators forming potential pathways between exposure and outcome. We then tested these pathways in a path model and stratified analyses by baseline level of active commuting. RESULTS: We identified changes in perceptions of the route to work, and use of the cycle path, as potential mediators. Of these potential mediators, only use of the path significantly explained (85%) the effect of the infrastructure in increasing cycling. Path use also explained a decrease in cycling among more active commuters. CONCLUSION: The findings strengthen the causal argument that changing the environment led to changes in health-related behaviour via use of the new infrastructure, but also show how some commuters may have spent less time cycling as a result.

Physical and social environmental changes to promote walking among Dutch older adults in deprived neighbourhoods: the NEW.ROADS study.

BACKGROUND: Physical activity is important for healthy ageing, and daily walking is seen as a feasible way to be active at older ages. Yet, many older persons, particularly in lower socioeconomic groups and residing in deprived neighbourhoods, are insufficiently active. Creating a physical and social neighbourhood environment that is more supportive for walking has the potential to improve walking behaviour. Current evidence of the impact of changes to the physical and/or social environmental on walking behaviour is scarce. The aim of the NEW.ROADS study is to design, implement and evaluate changes to the physical and social environment for the purpose of increasing walking behaviour among older residents of deprived neighbourhoods. METHODS: Physical and social environmental interventions were developed by matching scientific evidence on environmental determinants of walking, with input from the target population and stakeholders, and ongoing neighbourhood activities. Specifically, a neighbourhood walking route was designed and marked, and neighbourhood walking groups were organised. These environmental interventions were evaluated in a four-armed experimental study. In addition, the design of the study to evaluate the effect of these environmental changes on walking behaviour is described. DISCUSSION: Designing and implementing environmental interventions is a complex endeavour, challenged by limited available theory and evidence. Input from the target population and professional stakeholders is essential, but may also put constraints on the evaluation. TRIAL REGISTRATION: NTR3800 (registered 9/1/2013).

Development of a Greenhouse Screening Method for Adult Plant Response in Wheat to Stem Rust.

Screening for adult plant resistance in wheat to stem rust, caused by Puccinia graminis f. sp. tritici, is generally conducted in field plots. Although such evaluations are successful if managed properly, field ratings are time consuming, expensive, weather dependent, and open to inoculum of unwanted races or other confounding diseases. The objective of this study was to develop a dependable system of screening the response of adult plants to stem rust under greenhouse conditions. A comparison of inoculation methods and incubation environments showed that plants inoculated with urediniospores suspended in water, followed by a 24 h dew period in a plastic chamber constructed in a greenhouse, gave the most consistent results. Measurements of response type, stem rust severity, and frequency in follow-up experiments indicated that the most reliable infection was obtained when plants sprayed with 1.25 mg urediniospores per ml water were incubated in the plastic chamber. Using the optimized protocol, a Kariega × Avocet S doubled haploid population was inoculated with two P. graminis f. sp. tritici races. Depending on the race, composite interval mapping showed flag leaf infection type to be significantly influenced by regions on chromosomes 6A, 6D, and 7D. Stem rust severity and reaction type mapped to chromosomes 6D and/or 6A. The Lr34/Yr18/Sr57 gene derived from Kariega on chromosome 7D affected the rust response on flag leaves but not on stems of greenhouse-grown plants. This study showed that phenotyping and genetic analysis of especially major effect stem rust resistance in adult wheat plants is possible and reproducible under controlled conditions in a greenhouse.

Identification of adult plant resistance to stripe rust in the wheat cultivar Cappelle-Desprez.

Following the appearance of stripe rust in South Africa in 1996, efforts have been made to identify new sources of durable resistance. The French cultivar Cappelle-Desprez has long been considered a source of durable, adult plant resistance (APR) to stripe rust. As Cappelle-Desprez contains the seedling resistance genes Yr3a and Yr4a, wheat lines were developed from which Yr3a and Yr4a had been removed, while selecting for Cappelle-Desprez derived APR effective against South African pathotypes of the stripe rust fungus, Puccinia striiformis f. sp. tritici. Line Yr16DH70, adapted to South African wheat growing conditions, was selected and crossed to the stripe rust susceptible cultivar Palmiet to develop a segregating recombinant inbred line mapping population. A major effect QTL, QYr.ufs-2A was identified on the short arm of chromosome 2A derived from Cappelle-Desprez, along with three QTL of smaller effect, QYr.ufs-2D, QYr.ufs-5B and QYr.ufs-6D. QYr.ufs-2D was located within a region on the short arm of chromosome 2D believed to be the location of the stripe rust resistance gene Yr16. An additional minor effect QTL, QYr.ufs-4B, was identified in the cv. Palmiet. An examination of individual RILs carrying single or combinations of each QTL indicated significant resistance effects when QYr.ufs-2A was combined with the three minor QTL from Cappelle-Desprez, and between QYr.ufs-2D and QYr.ufs-5B.

Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration.

In this Phase I trial, patients' peripheral blood dendritic cells were pulsed with peptides eluted from the surface of autologous glioma cells. Three biweekly intradermal vaccinations of peptide-pulsed dendritic cells were administered to seven patients with glioblastoma multiforme and two patients with anaplastic astrocytoma. Dendritic cell vaccination elicited systemic cytotoxicity in four of seven tested patients. Robust intratumoral cytotoxic and memory T-cell infiltration was detected in two of four patients who underwent reoperation after vaccination. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous peptide-pulsed dendritic cell vaccine for patients with malignant glioma.

The hydrogenosomal enzyme hydrogenase from the anaerobic fungus Neocallimastix sp. L2 is recognized by antibodies, directed against the C-terminal microbody protein targeting signal SKL.

The question was addressed whether antibodies directed against the general microbody C-terminal protein targeting signal SKL recognized hydrogenosomal proteins from Neocallimastix sp. L2. Immunofluorescence, immunocytochemistry and Western blotting experiments using these antibodies indicated the presence of hydrogenosomal proteins containing SKL-COOH. One of these proteins, the hydrogenase, was purified to homogeneity. It has a native molecular mass of 87 kDa and consists of two subunits of approximately 30 and 60 kDa, both cross-reacting with anti-SKL antibodies. Its activity could be inhibited by CO, NO2-, and acetylene, suggesting a (Ni-Fe-Se) hydrogenase. Immunocytochemistry using polyclonal antibodies raised against the hydrogenase revealed the location of this protein in the hydrogenosomal matrix. The results described in this paper suggest that hydrogenosomes from Neocallimastix sp. L2 are related to microbodies from aerobic eukaryotes and support the idea of a common evolutionary origin for these organelles.

Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome.

The presence of hydrogenosomes in phylogenetically distinct anaerobic eukaryotes implies that they have been acquired independently, and previously reported differences in ultrastructure among taxa have suggested that some hydrogenosomes have different origins. Of particular interest are reports that Neocallimastix frontalis hydrogenosomes resemble microbodies in possessing a single membrane, in contrast to those in ciliates and trichomonads which have two and thus resemble mitochondria. In this investigation we have clearly demonstrated that N. frontalis hydrogenosomes possess two, rather than one, closely apposed membranes and in some preparations cristae-like structures were observed. These observations have led us to reject the microbody hypothesis and provide some indirect support for a possible mitochondrion origin as proposed for other hydrogenosomes. N. frontalis hydrogenosomes were shown to lack an associated genome as previously demonstrated for trichomonad hydrogenosomes. This might be explained by assuming that a mitochondrial genome encoding proteins for aerobic function is no longer necessary for either organelle.

Isolated coccidioidomycosis of the uterus.

Coccidioidomycosis rarely involves the female genital tract. This report describes a case of coccidioidomycosis that was incidentally discovered involving the uterus of an asymptomatic 70 year old woman who had squamous carcinoma of the cervix. Reports of the few previously documented examples of coccidioidomycosis of the female genitalia are reviewed. Although the uterine lesion probably resulted from a solitary focus of dissemination from a clinically inapparent and completely resolved primary pulmonary infection, evidence is presented that it may have been a primary uterine infection.

Vaginal embryogenesis, estrogens, and adenosis.

The recent outbreak of vaginal adenocarcinoma and the associated genital tract anomalies in women with histories of prenatal exposure to diethylstilbestrol (DES) has generated interest in vaginal embryogenesis and the influence of hormones on the developing genital tract. In this report the embryology of the vagina and the role of sex hormones in normal and abnormal sex duct development are presented and discussed in relation to the DES-induced anomalies. Although the teratogenic activity of DES in humans has been confirmed, the available evidence suggests that the clear cell carcinomas are initiated by endogenous estrogens and not the prenatally administered DES.

Cervical ripening with intravaginal prostaglandin E2 gel.

Ripening of the cervix by intravaginal application of a prostaglandin-containing gel is evaluated. Thirty patients with an unfavorable cervix needing labor induction were studied in a double-blind, prospective fashion. The prostaglandin gel proved superior to placebo in ripening the cervix (P less than .05), reducing induction failures (P less than .025), diminishing the oxytocin dosage necessary for induction (P less than .05), and lowering the rate of cesarean section (.05 less than P less than .1). One hundred fifty additional patients with varying Bishop scores and differing clinical situations were also studied. There were 35 cesarean sections (23.3%), nine failed inductions (6%), a spontaneous labor rate of 46%, and an average Bishop score change of 2.5. Twenty patients with premature rupture of the membranes and an unfavorable cervix received a modified gel containing 2.5 mg of prostaglandin E2. Average Bishop score change was 2.9, and there was a 55% incidence of spontaneous labor.

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria.

From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.

Functional-group-directed diastereoselective hydrogenation of aromatic compounds. 2(1)

Diastereoselective liquid-phase hydrogenation of a series of monosubstituted indan substrates was studied on supported rhodium catalysts. Predominantly the cis-cis diastereomer, obtained by hydrogenation from the diastereoface opposite the substituent at the stereogenic center, and the cis-trans diastereomer, obtained by hydrogenation from the diastereoface on the same side as the substituent, were formed. The diastereoselectivity depends on the balance between steric repulsion and electronic attraction of the substituent with the surface of the catalyst. For alkoxy and carboxyl groups (acid, methyl ester, and amide), the steric repulsion dominated and the cis-cis diastereomer was obtained with moderately high selectivity. The diastereoselectivity obtained in the hydrogenation was influenced by the addition of bases to the reaction mixture. Addition of triethylamine caused a small increase in the selectivity to the cis-cis diastereomer in some substrates, whereas the addition of NaOH significantly increased the selectivity toward the cis-trans isomer in all substrates.

Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens.

OBJECT: An approach toward the treatment of intracranial gliomas was developed in a rat experimental model. The authors investigated the ability of "professional" antigen-presenting cells (dendritic cells) to enhance host antitumor immune responses when injected as a vaccine into tumor-bearing animals. METHODS: Dendritic cells, the most potent antigen-presenting cells in the body, were isolated from rat bone marrow precursors stimulated in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Cultured cell populations were confirmed to be functional antigen-presenting cells on the basis of expressed major histocompatibility molecules, as analyzed by fluorescence-activated cell sorter cytofluorography. These dendritic cells were then pulsed (cocultured) ex vivo with acid-eluted tumor antigens from 9L glioma cells. Thirty-eight adult female Fischer 344 rats harboring 7-day-old intracranial 9L tumors were treated with three weekly subcutaneous injections of either control media (10 animals), unpulsed dendritic cells (six animals), dendritic cells pulsed with peptides extracted from normal rat astrocytes (10 animals), or 9L tumor antigen-pulsed dendritic cells (12 animals). The animals were followed for survival. At necropsy, the rat brains were removed and examined histologically, and spleens were harvested for cell-mediated cytotoxicity assays. The results indicate that tumor peptide-pulsed dendritic cell therapy led to prolonged survival in rats with established intracranial 9L tumors implanted 7 days prior to the initiation of vaccine therapy in vivo. Immunohistochemical analyses were used to document a significantly increased perilesional and intratumoral infiltration of CD8+ and CD4+ T cells in the groups treated with tumor antigen-pulsed dendritic cells compared with the control groups. In addition, the results of in vitro cytotoxicity assays suggest that vaccination with these peptide-pulsed dendritic cells can induce specific cytotoxic T lymphocytes against 9L tumor cells. CONCLUSIONS: Based on these results, dendritic antigen-presenting cells pulsed with acid-eluted peptides derived from autologous tumors represent a promising approach to the immunotherapy of established intracranial gliomas. which may serve as a basis for designing clinical trials in patients with brain tumors.

Role of DL-lactic acid as an intermediate in rumen metabolism of dairy cows.

The role of DL-lactic acid as an intermediate in the rumen of a Friesian X Holstein dairy cow adapted to a diet of hay ad libitum plus 12 kg of a concentrate mixture was studied in vitro and in vivo. Concentrations of soluble sugars in the rumen fluid became maximal at 30 min postfeeding, but at 90 min no sugars were detectable. The DL-lactate concentration increased very rapidly to about 30 mm at 30 min after feeding, whereas the maximum total VFA concentration was reached 15 min later. More than 80% of the DL-lactate fermented to VFA was converted by Megasphaera elsdenii. Whereas only 16% of L-lactate was fermented to propionate, 75% of the D-lactate was converted to propionic acid. When all soluble sugars had been fermented, the participation of M. elsdenii to lactate fermentation declined and fermentation patterns for D- and L-lactate became similar yielding mostly acetate. Except for a brief period immediately after feeding, DL-lactate did not appear to be an important precursor of VFA in the rumen of a cow adapted to concentrate feeding. DL-lactate may become a more important intermediate in rumen fermentation temporarily when dairy cows are gradually changed from a hay diet to a diet including concentrates. The first 30 d after parturition, when the changeover takes place, is an unstable period, during which the microbial population is changing to fit the new environment.