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Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain 1–4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage 5–8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL 9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage 3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES +KI67 + unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.

The identification of cancer maintenance genes—driver genes essential to tumor survival—is fundamental for developing effective cancer therapy. Transposon-based insertional mutagenesis screens can identify cancer driver genes broadly but not discriminate maintenance from progression or initiation drivers, which contribute to cancer phenotypes and tumorigenesis, respectively. We engineered a nested, double-jumping transposon system to first dysregulate gene expression during tumorigenesis and then restore gene expression following tumor induction, allowing for genome-wide screening of maintenance essentialityin vivo. In a mouse model of medulloblastoma, the most common pediatric malignancy, insertion and remobilization of this nested transposon uncovers potassium channel genes as recurrent maintenance drivers. In human medulloblastoma, KCNB2 is the most overexpressed potassium channel across Group 3, Group 4, and SHH subgroups, andKcnb2knockout in mice diminishes the replicative potential of medulloblastoma-propagating cells to mitigate tumor growth. Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives proliferative expansion of medulloblastoma-propagating cells. Thus, our novel transposon system reveals potassium homeostasis as essential to tumor maintenance through biomechanical modulation of membrane signaling.

Spatially resolved ambient mass spectrometry imaging methods have gained popularity to characterize cancer sites and their borders using molecular changes in the lipidome. This utility, however, is predicated on metabolic homogeneity at the border, which would create a sharp molecular transition at the morphometric borders. We subjected murine models of human medulloblastoma brain cancer to mass spectrometry imaging, a technique that provides a direct readout of tissue molecular content in a spatially resolved manner. We discovered a distance-dependent gradient of cancer-like lipid molecule profiles in the brain tissue within 1.2 mm of the cancer border, suggesting that a cancer-like state progresses beyond the histologic border, into the healthy tissue. The results were further corroborated using orthogonal liquid chromatography and mass spectrometry (LC-MS) analysis of selected tissue regions subjected to laser capture microdissection. LC-MS/MS analysis for robust identification of the affected molecules implied changes in a number of different lipid classes, some of which are metabolized from the essential docosahexaenoic fatty acid (DHA) present in the interstitial fluid. Metabolic molecular borders are thus not as sharp as morphometric borders, and mass spectrometry imaging can reveal molecular nuances not observed with microscopy. Caution must be exercised in interpreting multimodal imaging results stipulated on a coincidental relationship between metabolic and morphometric borders of cancer, at least within animal models used in preclinical research.

Posterior Fossa Group A (PFA) ependymomas are pediatric brain tumors with extremely poor survival outcomes. As protein-coding mutations in PFA are exceedingly rare, the underlying etiology of these tumors remains elusive. Elevated CpG island methylation and depletion of H3K27me3 have been described in PFA, leading to the hypothesis that PFA may be driven by a dysregulated epigenetic state. In this study, we sought to determine how three-dimensional (3D) genome features (such as DNA loops, domains, and compartments) differ between pediatric brain tumors. We performed Hi-C sequencing on a collection of 64 patient specimens and patient-derived primary cultures that collectively span multiple subgroups of ependymoma, medulloblastoma, high-grade glioma, and non-neoplastic brain. For certain samples, we further performed RNA-seq, histone modification ChIP-seq, or whole-genome bisulfite sequencing to allow multiomic data integration. Overall, the 3D genome organization of PFA samples appeared distinct from other tumor types. We identified and defined TULIPs: a subset of type B compartments, separated by genomic distances greater than 10 Mbp, that exhibit a striking fivefold increase in reciprocal interaction strength. These TULIPs recurred at the same genomic positions across the vast majority of PFA samples with minimal representation among other tumor or non-tumor samples. TULIPs displayed enrichment for heterochromatic features such as H3K9me3 and late replication timing and were depleted of euchromatic features such as H3K27ac and protein-coding genes. By using immuno-fluorescence for H3K9me3 and oligo-FISH to label TULIP regions, we demonstrated that TULIP regions are more compact in PFA than other tumors. Finally, by applying inhibitors of H3K9 lysine methylation to PFA cultures we showed that TULIPs become more diffuse and cell viability is reduced. Altogether, this work defines TULIPs as highly recurrent epigenetic features of PFA tumors.

Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention., Sonic Hedgehog medulloblastoma (Shh-MB) comprises four subtypes each with distinct clinical traits. Here the authors characterize the genome, transcriptome, and methylome of Shh-MB subtypes, revealing a complex fusion landscape and the molecular convergence of MYCN and cAMP signaling pathways.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma. Hypoxia reprograms the cellular metabolome and epigenome to promote growth of the most lethal ependymomas.

After more than a decade of genomic studies in medulloblastoma, the time has come to capitalize on the knowledge gained and use it to directly improve patient care. Although metastatic and relapsed disease remain poorly understood, much has changed in how we define medulloblastoma, and it has become evident that with conventional therapies, specific groups of patients are currently under- or overtreated. In this review, we summarize the latest insights into medulloblastoma biology, focusing on how genomics is affecting patient stratification, informing preclinical studies of targeted therapies, and shaping the new generation of clinical trials.

microRNAs (miRNAs) have wide-ranging effects on large-scale gene regulation. As such, they play a vital role in dictating normal development, and their aberrant expression has been implicated in cancer. There has been a large body of research on the role of miRNAs in medulloblastoma, the most common malignant brain tumor of childhood. The identification of the 4 molecular subgroups with distinct biological, genetic, and transcriptional features has revolutionized the field of medulloblastoma research over the past 5 years. Despite this, the growing body of research on miRNAs in medulloblastoma has largely focused on the clinical entity of a single disease rather than the molecular subgroups. This review begins by highlighting the role of miRNAs in development and progresses to explore their myriad of implications in cancer. Medulloblastoma is characterized by increased proliferation, inhibition of apoptosis, and maintenance of stemness programs—features that are inadvertently regulated by altered expression patterns in miRNAs. This review aims to contextualize the large body of work on miRNAs within the framework of medulloblastoma subgroups. The goal of this review is to stimulate new areas of research, including potential therapeutics, within a rapidly growing field.

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

Medulloblastoma, a childhood brain cancer of the cerebellum, has one of the highest mortality rates of pediatric cancers. For over a century, scientists believed that medulloblastoma metastasized exclusively through cerebrospinal fluid; however, novel research reveals that it can also spread through the circulatory system. Understanding this new mechanism is critical for the development of therapies and to help affected families understand their child’s disease. An educational 3D animation was developed to effectively disseminate this research to the scientific community and to educate physicians, researchers, and affected families about this disease. A story-driven narrative and emphasis on visual metaphor put the research in context by establishing why this research matters, what the future implications are, and how it is relevant to the audience. Additionally, various visual design strategies were implemented in order to increase memory retention and audience engagement. The final animation is the first visual resource that educates a lay audience on the two different mechanisms of medulloblastoma metastasis.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood–brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.

© The Author(s), under exclusive licence to Springer Nature Limited 2019, In cancer, recurrent somatic single-nucleotide variants-which are rare in most paediatric cancers-are confined largely to protein-coding genes1-3. Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only

Ependymomas are a lethal central nervous system (CNS) tumor found in both adults and children. Recent efforts have focused on risk stratification by classifying the molecular variants of CNS ependymoma. Despite this increased knowledge of molecular drivers, much less is known about the metabolism of these subgroups. Disruption of cellular metabolism can drive the transition of normal neuronal cells to tumor cells. A shift from anaerobic to aerobic metabolism as the primary energy source is a hallmark of cancer, promoting cancer cell proliferation, and avoidance of cellular apoptotic cues. This review aims to discuss the current knowledge regarding metabolism in ependymoma cells compared to normal brain cells and the implications of metabolic changes with regard to tumorigenesis, the tumor microenvironment, and possible targets for treatment.

The ductility of dual-phase (DP) 980 and transformation-induced plasticity (TRIP) assisted bainitic ferritic (TBF) 980 steels was studied in the presence of a sheared edge. Specimens were tested in uniaxial tension in a standard test frame as well as in situ in the scanning electron microscope (SEM). Incremental tensile straining was done in the SEM with images taken at each strain increment. Then digital image correlation (DIC) was used to compute the effective strain at the level of the individual phases in the microstructure. Shear banding across multiple phases was seen in strained TBF specimens, while the DP specimens exhibited more of a patchwork strain pattern, with high strains concentrated in ferrite and low strains observed in the martensite. Two-point statistics were applied to the strain data from the DIC work and the corresponding microstructure images to evaluate the effect of phase hardness on localization and fracture. It was observed that the DP 980 material had a greater tendency for localization around hard phases compared to the TBF 980. This at least partially explains the greater ductility of the TBF material, especially in specimens where a sheared edge was present.

International audience; Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

Selected solute atoms can strongly interact with and slow down the diffusion of point defects in alloy systems. While such additions can be beneficial, for instance to promote microstructural stability during thermal annealing or during irradiation by energetic particles, they create significant computational challenges when simulating these evolutions using atomistic techniques such as kinetic Monte Carlo (KMC) simulations. Point defect trapping in energy basins created by clusters of solute atoms leads to frequent re-visiting of states with short residence times, which dramatically reduces the efficiency of traditional KMC algorithms. We introduce here a hybrid algorithm that combines and expand on two prior KMC algorithms, the Chain KMC and the equilibrating basin KMC. This hybrid algorithm, referred to as the Equilibrating Chain algorithm, utilizes Chain KMC as previously reported, but leverages the data-handling framework to build an occupation distribution of the basin, allowing the equilibrating basin assumption to be statistically tested and applied. For a model A-B trapping alloy system on a face-centered cubic lattice, statistical comparisons of basin exit and cluster dissolution kinetics between traditional and accelerated KMC algorithms are presented to demonstrate the accuracy and the efficiency of the new algorithm. We also discuss our algorithm in the context of other accelerated KMC algorithms.

Study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. Here we use single-cell transcriptomics to study more than 60,000 cells from the developing mouse cerebellum and show that different molecular subgroups of childhood cerebellar tumours mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. The Sonic Hedgehog medulloblastoma subgroup transcriptionally mirrors the granule cell hierarchy as expected, while group 3 medulloblastoma resembles Nestin+ stem cells, group 4 medulloblastoma resembles unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumours demonstrates that many bulk tumours contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumours as a disorder of early brain development and provide a proximate explanation for the peak incidence of cerebellar tumours in early childhood. Sequencing data from the developing cerebellum are compared with bulk sequencing data from paediatric tumours, providing insights into their potential origins and suggesting that many cerebellar tumours have their origins early in utero.

SummaryThe study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. We used single-cell transcriptomics to study >60,000 cells from the developing murine cerebellum, and show that different molecular subgroups of childhood cerebellar tumors mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. Sonic Hedgehog medulloblastoma transcriptionally mirrors the granule cell hierarchy as expected, whereas Group 3 medulloblastoma resemble Nestin+ve stem cells, Group 4 medulloblastomas resemble unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumors demonstrates that many bulk tumors contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumors as a disorder of early brain development, and provide a proximate explanation for the peak incidence of cerebellar tumors in early childhood.

Ependymoma is the third most common malignant tumor of the posterior fossa and is a major cause of neurological morbidity and mortality in children. Current treatments, particularly surgery and external beam irradiation result in relatively poor outcomes with significant neurological and cognitive sequelae from treatment. Historical approaches have considered all ependymomas as similar entities based on their morphological appearance.Recent advances in genomics and epigenetics have revealed, however, that ependymomas from different CNS locations represent distinct entities. Moreover, ependymoma of the posterior fossa, the most common location in children, is actually comprised of two distinct molecular variants. These two variants have marked differences in demographics, transcriptomes, structure, methylation patterns, and clinical outcomes. This allows for the development of new biology-based clinical risk stratification, which can both prioritize patients for de-escalation of therapy and identify those who will benefit from novel therapeutic strategies. Indeed, the identification of these two variants allows an opportunity for robust preclinical modeling for development of novel therapeutic strategies.Herein, we have summarized our current clinical approach to diagnosis and treatment of posterior fossa ependymoma, recent advances in understanding the biology of posterior fossa ependymoma and how these new insights can be translated into the clinic to form the basis of the next generation of clinical trials.

Cross correlation techniques applied to EBSD patterns have led to what has been termed “high-resolution EBSD” (HR-EBSD). The technique yields higher accuracy orientation and strain data which is obtained by comparing a given EBSD pattern with either a real or a simulated reference pattern. Real reference patterns are often taken from a “central” position in a given grain, where it is hoped that the material is “strain-free”, and they enable the determination of relative changes in orientation and strain from that present in the lattice at the reference position. Simulated patterns, on the other hand, enable comparison of the sample lattice with that of a perfect lattice, resulting in a measure of absolute strain and orientation. However, the simulated pattern method has several drawbacks, including the need to accurately specify microscope geometry, the lower fidelity/detail of simulated patterns compared with real patterns, and the potential for microscope-specific bias in the measured patterns (such as due to optical distortion). These drawbacks have led to much debate about the utility of the cross-correlation technique using simulated patterns. This paper is the first to assess the accuracy of the simulated pattern method relative to the real pattern approach in a setting where accuracy can be reasonably determined, thus providing a fair assessment of the potential of the simulated pattern technique. Based upon recent developments towards a standard material for assessing strain mapping techniques, this paper assesses the overall accuracy of the simulated pattern technique. Mismatch strains are calculated using both the real and simulated pattern techniques for a SiGe film deposited on a Si substrate. While the simulated pattern technique is not as accurate or precise as the real pattern technique for providing relative strains, it provides an estimate of absolute strain that is not available via the real pattern approach.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3. Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3. 10.1093/brain/awy039-video1 awy039media1 5742053534001

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two-hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. Indeed, mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not circadian KaiB1C1. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Bacteria of the genus Pseudomonas are motile Gram-negative bacteria characterized by high metabolic versatility, and aerobic respiration, although a few strains of different species are able to use nitrate as a final electron acceptor (Palleroni, 2010). Pseudomonads are ubiquitous soil and water microorganisms that colonize many different environments and, consequently, have diverse lifestyles. Strains of the species Pseudomonas putida are frequently soil inhabitants and are important in organic matter recycling in nature; they have a high bioremediation potential because they often carry genes to deal with natural and xenobiotic chemicals (Nelson et al., 2002; Caballero et al., 2005; van Dillewijn et al., 2007; Arias et al., 2008; Segura et al., 2009a,2009b). The key to the ubiquitous distribution of these bacteria is not only their metabolic potential, but also the arsenal of regulatory genes that allow them to adapt to changes in the environment (Sashidhar and Podile, 1998; Wu et al., 2011). A few P. putida strains, namely, S12, Idaho and DOT-T1E (Weber et al., 1993; Ramos et al., 1995; Pinkart et al., 1996), are able to thrive in the presence of toxic solvents (e.g. decanol, octanol, toluene, styrene), and these strains are considered extremophile microorganisms with great potential in bioremediation and in biocatalysis in biphasic systems (Ramos et al., 1995; Isken and de Bont, 1996; Molina et al., 2011; Ramos et al., 2011; Tao et al., 2011; Udaondo et al., 2012). Organic solvents are toxic to most microorganisms because they dissolve in the cell membranes, disorganize their structures and impair vital functions such as respiration, and the collapse in energy generation lead to cell death (Sikkema et al., 1995; Ramos et al., ,). Solvent tolerance in P. putida DOT-T1E is a multifactorial trait that involves chromosomal and plasmid encoded functions (Ramos et al., 2002; Segura et al., 2003; 2009b; Rodriguez-Herva et al., 2007; Garcia et al., 2006; Molina et al., 2011). The first barrier to solvents involves a reduction in the permeability of the cell membrane via a fast cis to trans isomerization of unsaturated fatty acids followed by a slower modification of phospholipid head groups (Keweloh and Heipieper, 1996; Junker and Ramos, 1999; Heipieper et al., ,; Bernal et al., 2007; Pini et al., 2009). However, this reduction in permeability does not prevent entry of the solvents, which results in unfolding of proteins and the consequential function of a number of chaperones (Segura et al., 2003; Dominguez-Cuevas et al., 2006; Volkers et al., 2006). The main mechanism underlying solvent tolerance lies in the action of RND (resistance-nodulation-cell division) efflux pumps encoded on the host chromosome and on the pGRT1 plasmid (Kieboom et al., 1998; Kim et al., 1998; Ramos et al., 1998; Mosqueda and Ramos, 2000; Rojas et al., 2001; Rodriguez-Herva et al., 2007; Godoy et al., 2010; Udaondo et al., 2012). Resistance to solvents is also modulated by the action of chromosomally encoded ABC efflux transporters that use energy to remove solvents from the cells to the outer medium (Kim et al., 1998; Garcia et al., 2006). Here, we present, the genome of the solvent tolerant P. putida DOT-T1E strain obtained using the 454 technology. This microorganism uses a wide range of carbon, nitrogen, sulfur and phosphorous sources due to its wide metabolic potential. In addition we summarize previous knowledge on the biotransformation potential of this strain and how the properties and genomic information can be used to design new biotechnological processes.

Objective Asthma is a common disease which places significant burden on the US healthcare system and which can be associated with significant patient morbidity and mortality. Current medical therapies are costly and not curative. A new approach for a more permanent asthma treatment is the use of radiofrequency ablation. However, this radiofrequency approach is thermal-based and can result in deleterious effects to the airways, such as stenosis or ulceration. We describe a novel, improved therapeutic approach for smooth muscle ablation using non-thermal DC electroporation ablation. Methods We developed and tested prototype electroporation ablation devices that access the airways both endoscopically and via a bronchoscope. We tested the feasibility of this approach and demonstrated proof-of-concept in 2 mongrel dogs. In order to assess for smooth muscle function, we performed functional studies pre and post ablation with methacholine challenge to assess for airway reversibility. We also evaluated bronchial lesions via direct vision with bronchoscopy. Results We developed novel electroporation catheters to delivery energy to the bronchial smooth muscle through an endoscopic approach. We tested these catheters in 2 acute canine studies and successfully demonstrated the ability to destroy smooth muscle tissue via novel prototypes and saline irrigation for widespread non-thermal electroporation ablation. Our functional studies demonstrate the efficacy of this approach. Conclusion We report a novel method for non-thermal bronchial smooth muscle ablation using novel prototypes and electroporation with normal saline. These early findings require further evaluation in larger, chronic canine studies to assess for use as a potential curative therapy.

2009).Biological detectors are usually cheaper and more sensi-tive than chemical detection methodologies, but they maypresent additional problems because contaminants canprejudicegrowthand/orbescarceandnotveryaccessibleto cells. Both the positive and the negative sides of biore-porters are particularly true when we are dealing withmarine oil spills, where clean up plus total recovery of theaffected zones are enduring, difficult and multifactorialprocesses. For this purpose, Kumari and colleagues(2011) present in

on enzymes from cold places; they start indi-cating that the temperature of the bulk of the Earth’sbiosphere is below 6°C (e.g. 90% of the ocean’s waters),and that this cold water sustains a broad diversity ofmicrobial life. Permanently cold environments are alsofound on land, high mountains and polar regions. Theexploration of those environments is revealing an unex-pected microbial biodiversity (Campanaro

a Planta, 14071, Cordoba, Spain. apart from the time saving facet of this methodology, this protocol can also avoid bias as a result of multiple steps and the limitations of cloning mutants by traditional methods (restriction enzymes and ligation); thus broaden- ing the range of potential candidates. Bacteria from the genus Streptomyces produce more than 70% of commercially available antibiotics, plus many other metabolites of therapeutic interest such as anti- tumour agents or immunosuppressive drugs. The produc- tion of these compounds is tightly controlled by means of g-butyrolactones; microbial hormones that are also involved in the regulation of other processes such as morphological differentiation or pathogenesis. The article presented by D'Alia and co-workers focuses on the regu- lation of these hormones by the ScbA regulator, a homo- logue of the A-factor biosynthesis afsA gene from Streptomyces coelicolor (D'Alia et al., 2011). For this purpose, they studied the differences in transcript levels between the wild-type strain and a DscbA mutant using global microarray analysis, and the results were con- firmed by more accurate qRT-PCR. The authors show that ScbA not only controls antibiotic production but also inter- feres with intracellular iron levels modulating siderophore desferrioxamine E biosynthesis. Interestingly, this study also shows that in the scbA mutant genes coding for enzymes related to primary metabolism are upregulated just before antibiotic production, placing butanolides upfront of general metabolic regulation in S. coelicolor. When S. coelicolor A3(2) grows under nutrient limited conditions, it triggers a gamut of nutrient-stress responses, mediated by a number of global regulators including PhoP, GlnR, AfsR and others. The action of these regulators is also integrated at the molecular level to control secondary metabolite biosynthesis and differen- tiation. As a result of the potential uses of these microbes in biotechnology, nutritional control has received relevant attention in the field and, in this issue, Martin and col- leagues (2011) describe how phosphate control of primary and secondary metabolism in Streptomyces species is mediated by the two-component regulatory system PhoR- PhoP. PhoP controls secondary metabolism by binding to the PHO box in the afsS promoter. They illustrate that this PHO Box overlaps with the AfsR binding site (D'Alia et al.

Summary Bacteria of the species Pseudomonas putida are ubiquitous soil inhabitants, and a few strains are able to thrive in the presence of extremely high concentrations of toxic solvents such as toluene and related aromatic hydrocarbons. Toluene tolerance is multifactorial in the sense that bacteria use a wide range of physiological and genetic changes to overcome solvent damage. This includes enhanced membrane impermeabilization through cis to trans isomerization of unsaturated fatty acids, activation of a stress response programme, and induction of efflux pumps that expulse toxic hydrocarbons to the outer medium. The most relevant element in this toluene tolerance arsenal is the TtgGHI efflux pump controlled by the TtgV regulator. We discuss here how TtgV controls expression of this efflux pump in response to solvents.

Pseudomonas putida DOT-T1E was used as a model to develop a “phenomics” platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Δ crc , Δ crp , Δ cyoB , and Δ ptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d -fructose is influenced by CyoB, and l - arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l -valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d -amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N -acetyl- l -cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival.

In the following highlight we refer to a number of new advances in the field of Biotechnology that address issues relating to the synthesis of new antibiotics, new biocatalysts and matrices in biofilms.

Summary Pseudomonas putida DOT-T1E is a highly solvent-tolerant strain. Although the main mechanism that confers solvent tolerance to the strain is the TtgGHI efflux pump, a number of other proteins are also involved in the response to toluene. Previous proteomic and transcriptomic analysis carried out in our lab with P. putida DOT-T1E, and the solvent-sensitive strain, P. putida KT2440, revealed several transporters that were induced in the presence of toluene. We prepared five mutants of the corresponding genes in P. putida DOT-T1E and analysed their phenotypes with respect to solvent tolerance, stress endurance and growth with different carbon, nitrogen and sulfur sources. The data clearly demonstrated that two transporters (Ttg2ABC and TtgK) are involved in multidrug resistance and toluene tolerance, whereas another (homologous to PP0219 of P. putida KT2440) is a sulfate/sulfite transporter. No clear function could be assigned to the other two transporters. Of the transporters shown to be involved in toluene tolerance, one (ttg2ABC) belongs to the ATP-Binding Cassette (ABC) family, and is involved in multidrug resistance in P. putida DOT-T1E, while the other belongs to the Major Facilitator Superfamily and exhibits homology to a putative transporter of the Bcr/CflA family that has not previously been reported to be involved in toluene tolerance.

a Planta, 14071, Cordoba, Spain. In this special issue a series of articles appear detailing methods of energy generation under different conditions; processes which are related to the future vision of microbes as a source of electric power (Lovley, 2009). Methanogenesis is identified as an important part of the biogeochemical carbon cycle in diverse anaerobic envi- ronments. Methane is an important fuel that can be gen- erated from several wastes in these anoxic environments and therefore, understanding how it is produced, which organisms produce it and the reactions required to achieve high yields is of major biotechnological interest. Alves and colleagues (2009) present a mini-review on the use of lipids as substrates for methane production. Conversion of lipids into methane is achieved by the syntrophic consortia of acetogenic bacteria and methano- genic archeae. The microbes involved are influenced by the nature of the incoming lipids and examples of different microbial communities when feeding reactors with oleate or palmitate are reviewed. Alves and colleagues (2009) point out that the number of acetogenic microorganisms that degrade butyrate or higher fatty acids is very low and includes microbes of the Syntrophaceae and Syntroph- omonadaceae families. These microbes live together with hydrogen-consuming methanogenic archeae and sulfate- reducing bacteria. The syntrophic cooperation seems to be optimal when microbes are organized as microcolo- nies, which is considered to favour interspecies hydrogen transfer. As in many biotechnological processes, lipid conversion into methane requires both the appropriate microbes and their performance optimization in the reac- tors, each of which still require some improvement. The authors note that previous failures to deal with high load lipids were related mainly to sludge flotation and biomass washout. To overcome these limitations the authors describe a new reactor concept which involves primary biomass retention through flotation and secondary biomass retention via settling. These reactors are being tested in scale and if, as expected, they solve the problem may soon come into industrial use. The seminal review of Bernhard Schinck (Schink, 1997)

In the March Microbial Biotechnology special issue on bioremediation, a new feature called ‘Crystal Ball 2009’ was published. In this section leading researchers in the field of microbial biotechnology looked into their crystal balls and speculated on the technical and conceptual developments that will drive innovative research and open new vistas over the next few years. Twenty one separate articles were presented, which contained information and ideas that every microbiologist should know.

In recent issues of Microbial Biotechnology there have been several exceptional articles ranging in topic from the antagonism of biofilm formation in pathogenic bacteria, and prebiotic selection of positive bacterial fermentations in the colon to the discovery of novel biotechnologically important enzymes encoded by bacteria of the deep‐sea floor.

Transcriptional responses of microorganisms to environmental perturbations are broad and complex, and often involve regulatory cascades in which an interplay of regulatory factors trigger a specific expression program. Here, we describe how Pseudomonas responds to challenges from toxic chemicals, for which they use a dedicated program that subsequently confers resistance to these compounds. A mosaic of domains has been recruited to sense and metabolize these chemicals in order to obtain energy and carbon.

Beta-lactamases, which evolved from bacterial penicillin-binding proteins (PBPs) involved in peptidoglycan (PG) synthesis, confer resistance to beta-lactam antibiotics. While investigating the genetic basis of biofilm development by Pseudomonas aeruginosa, we noted that plasmid vectors encoding the common beta-lactamase marker TEM-1 caused defects in twitching motility (mediated by type IV pili), adherence and biofilm formation without affecting growth rates. Similarly, strains of Escherichia coli carrying TEM-1-encoding vectors grew normally but showed reduced adherence and biofilm formation, showing this effect was not species-specific. Introduction of otherwise identical plasmid vectors carrying tetracycline or gentamicin resistance markers had no effect on biofilm formation or twitching motility. The effect is restricted to class A and D enzymes, because expression of the class D Oxa-3 beta-lactamase, but not class B or C beta-lactamases, impaired biofilm formation by E. coli and P. aeruginosa. Site-directed mutagenesis of the catalytic Ser of TEM-1, but not Oxa-3, abolished the biofilm defect, while disruption of either TEM-1 or Oxa-3 expression restored wild-type levels of biofilm formation. We hypothesized that the A and D classes of beta-lactamases, which are related to low molecular weight (LMW) PBPs, may sequester or alter the PG substrates of such enzymes and interfere with normal cell wall turnover. In support of this hypothesis, deletion of the E. coli LMW PBPs 4, 5 and 7 or combinations thereof, resulted in cumulative defects in biofilm formation, similar to those seen in beta-lactamase-expressing transformants. Our results imply that horizontal acquisition of beta-lactamase resistance enzymes can have a phenotypic cost to bacteria by reducing their ability to form biofilms. Beta-lactamases likely affect PG remodelling, manifesting as perturbation of structures involved in bacterial adhesion that are required to initiate biofilm formation.

Summary Di- N -acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella . WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3- N -acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy- D -mannuronic acid (UDP- D -Man(2NAc3NAc)A), a precursor for the D - Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long-chain B-band O antigen ( ≥ ≥ ≥ 2 repeating units) or semi-rough LPS (lipid A-core + one repeat). Adding wbpD in trans restored LPS production to the wild-type level; this indicates that wbpD is important for biosynthe- sis of individual B-band O-antigen repeating units. WbpD contains left-handed beta-helical (L b H) struc- ture as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N- terminally histidine-tagged fusion protein (His 6 - WbpD) and purified to >95% purity. The protein was subjected to Far-UV circular dichroism spectroscopy, and the data revealed that WbpD contains left- handed helical structure, which substantiated in silico predictions made earlier. Results from SDS- PAGE, matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) mass spectrometry (MS), and gel filtration analyses indicated that His 6 -WbpD has trimeric organization, consistent with the quater- nary structure of HexATs. The binding of acetyl-CoA by WbpD was demonstrated by MALDI-TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct-transfer reaction mechanism. Incuba- tion of WbpD with acetyl-CoA significantly enhanced the stability of the protein and prevented precipita- tion over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavail- able at present, a structure-based model for the L b H domain of WbpD was generated. Comparisons between this model and the L b H domains of known HexATs suggested that Lys136 plays a role in acetyl- CoA binding. A K136A site-directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl-CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl-CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP- D - Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP- 2-acetamido-3-amino-2,3-dideoxy- D -glucuronic acid 3- N -acetyltransferase.