Central nervous system immune interactome is a function of cancer lineage, tumor microenvironment, and STAT3 expression.

BACKGROUNDImmune cell profiling of primary and metastatic CNS tumors has been focused on the tumor, not the tumor microenvironment (TME), or has been analyzed via biopsies.METHODSEn bloc resections of gliomas (n = 10) and lung metastases (n = 10) were analyzed via tissue segmentation and high-dimension Opal 7-color multiplex imaging. Single-cell RNA analyses were used to infer immune cell functionality.RESULTSWithin gliomas, T cells were localized in the infiltrating edge and perivascular space of tumors, while residing mostly in the stroma of metastatic tumors. CD163+ macrophages were evident throughout the TME of metastatic tumors, whereas in gliomas, CD68+, CD11c+CD68+, and CD11c+CD68+CD163+ cell subtypes were commonly observed. In lung metastases, T cells interacted with CD163+ macrophages as dyads and clusters at the brain-tumor interface and within the tumor itself and as clusters within the necrotic core. In contrast, gliomas typically lacked dyad and cluster interactions, except for T cell CD68+ cell dyads within the tumor. Analysis of transcriptomic data in glioblastomas revealed that innate immune cells expressed both proinflammatory and immunosuppressive gene signatures.CONCLUSIONOur results show that immunosuppressive macrophages are abundant within the TME and that the immune cell interactome between cancer lineages is distinct. Further, these data provide information for evaluating the role of different immune cell populations in brain tumor growth and therapeutic responses.FUNDINGThis study was supported by the NIH (NS120547), a Developmental research project award (P50CA221747), ReMission Alliance, institutional funding from Northwestern University and the Lurie Comprehensive Cancer Center, and gifts from the Mosky family and Perry McKay. Performed in the Flow Cytometry & Cellular Imaging Core Facility at MD Anderson Cancer Center, this study received support in part from the NIH (CA016672) and the National Cancer Institute (NCI) Research Specialist award 1 (R50 CA243707). Additional support was provided by CCSG Bioinformatics Shared Resource 5 (P30 CA046592), a gift from Agilent Technologies, a Research Scholar Grant from the American Cancer Society (RSG-16-005-01), a Precision Health Investigator Award from University of Michigan (U-M) Precision Health, the NCI (R37-CA214955), startup institutional research funds from U-M, and a Biomedical Informatics & Data Science Training Grant (T32GM141746)

Ground-State of Charged Bosons Confined in a Harmonic Trap

We study a system composed of N identical charged bosons confined in a harmonic trap. Upper and lower energy bounds are given. It is shown in the large N limit that the ground-state energy is determined within an accuracy of $\pm 8$ and that the mean field theory provides a reasonable result with relative error of less than 16% for the binding energy .Comment: 15 page

Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis

Abstract Background The indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease. Results We monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA. Conclusions The microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation.http://deepblue.lib.umich.edu/bitstream/2027.42/112442/1/40168_2012_Article_10.pd

Comparing the Bacterial Diversity of Acute and Chronic Dental Root Canal Infections

This study performed barcoded multiplex pyrosequencing with a 454 FLX instrument to compare the microbiota of dental root canal infections associated with acute (symptomatic) or chronic (asymptomatic) apical periodontitis. Analysis of samples from 9 acute abscesses and 8 chronic infections yielded partial 16S rRNA gene sequences that were taxonomically classified into 916 bacterial species-level operational taxonomic units (OTUs) (at 3% divergence) belonging to 67 genera and 13 phyla. The most abundant phyla in acute infections were Firmicutes (52%), Fusobacteria (17%) and Bacteroidetes (13%), while in chronic infections the dominant were Firmicutes (59%), Bacteroidetes (14%) and Actinobacteria (10%). Members of Fusobacteria were much more prevalent in acute (89%) than in chronic cases (50%). The most abundant/prevalent genera in acute infections were Fusobacterium and Parvimonas. Twenty genera were exclusively detected in acute infections and 18 in chronic infections. Only 18% (n = 165) of the OTUs at 3% divergence were shared by acute and chronic infections. Diversity and richness estimators revealed that acute infections were significantly more diverse than chronic infections. Although a high interindividual variation in bacterial communities was observed, many samples tended to group together according to the type of infection (acute or chronic). This study is one of the most comprehensive in-deep comparisons of the microbiota associated with acute and chronic dental root canal infections and highlights the role of diverse polymicrobial communities as the unit of pathogenicity in acute infections. The overall diversity of endodontic infections as revealed by the pyrosequencing technique was much higher than previously reported for endodontic infections

Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells.

LCK is a tyrosine kinase that is essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active-site lysine is replaced by a photocaged equivalent, using genetic code expansion. This strategy enabled fine temporal and spatial control over kinase activity, thus allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that autophosphorylation of the LCK active-site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, T-cell coreceptors, can enhance LCK activity, thereby helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics

JAK2 V617F Constitutive Activation Requires JH2 Residue F595: A Pseudokinase Domain Target for Specific Inhibitors

The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 αC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the αC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type

Analysis of jak2 catalytic function by peptide microarrays: The role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Kmfor ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Kmfor ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches

Factors That Affect Large Subunit Ribosomal DNA Amplicon Sequencing Studies of Fungal Communities: Classification Method, Primer Choice, and Error

Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project naïve Bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50–100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys

TMEFF2 Is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma

BACKGROUND: TMEFF2 is a protein containing a single EGF-like domain and two follistatin-like modules. The biological function of TMEFF2 remains unclear with conflicting reports suggesting both a positive and a negative association between TMEFF2 expression and human cancers. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that the extracellular domain of TMEFF2 interacts with PDGF-AA. This interaction requires the amino terminal region of the extracellular domain containing the follistatin modules and cannot be mediated by the EGF-like domain alone. Furthermore, the extracellular domain of TMEFF2 interferes with PDGF-AA-stimulated fibroblast proliferation in a dose-dependent manner. TMEFF2 expression is downregulated in human brain cancers and is negatively correlated with PDGF-AA expression. Suppressed expression of TMEFF2 is associated with its hypermethylation in several human tumor types, including glioblastoma and cancers of ovarian, rectal, colon and lung origins. Analysis of glioma subtypes indicates that TMEFF2 hypermethylation and decreased expression are associated with a subset of non-Proneural gliomas that do not display CpG island methylator phentoype. CONCLUSIONS/SIGNIFICANCE: These data provide the first evidence that TMEFF2 can function to regulate PDGF signaling and that it is hypermethylated and downregulated in glioma and several other cancers, thereby suggesting an important role for this protein in the etiology of human cancers