The pathological features of angioimmunoblastic T-cell lymphomas with IDH2R172 mutations.

Angioimmunoblastic T-cell lymphoma is a peripheral T-cell lymphoma derived from follicular T-helper cells. High-throughput genomic sequencing studies have shown that angioimmunoblastic T-cell lymphoma carries frequent mutations in RHOAG17V and IDH2R172 genes. The clinico-pathological features of angioimmunoblastic T-cell lymphoma cases with RHOAG17V mutations have been addressed; however, similar studies for IDH2 mutated cases are lacking. Therefore, the aim of the present study was to evaluate the pathological features of angioimmunoblastic T-cell lymphoma with IDH2 mutations. In order to identify cases with IDH2 mutations, 50 cases previously diagnosed as angioimmunoblastic T-cell lymphoma were subjected to next-generation sequencing analysis using a custom panel covering four genes frequently mutated in angioimmunoblastic T-cell lymphoma including DNMT3A, TET2, IDH2 and RHOA. All cases were analyzed for PD1, ICOS, CXCL13, CD10, BCL6, CD21, CD23 and EBER in situ hybridization. Mutational analysis recognized three groups. Group 1: IDH2R172 mutations were identified in 20 cases (40%). All cases carried RHOAG17V mutations. Group 2: RHOAG17V mutations without IDH2R172 mutation were identified in 16 cases (32%), and Group 3: 14 cases (28%) without RHOAG17V or IDH2R172 mutations. Morphologically, angioimmunoblastic T-cell lymphoma cases with IDH2R172 mutations were characterized by the presence of medium to large clear cells (p = 0.00001), and a follicular T-helper phenotype with the particular feature of strong CD10 (p = 0.0268) and CXCL13 expression (p = 0.0346). Interestingly, TET2 mutations were identified in 32 of 33 (97%) cases with IDH2R172 and/or RHOAG17V mutations whereas only 55% of angioimmunoblastic T-cell lymphoma cases wild-type for these two genes carried TET2 mutations (p = 0.0022). In contrast, DNMT3A mutations were found in 48% of the cases and were equally distributed in the three groups. In conclusion, our results support the results of gene expression profiling studies suggesting that IDH2R172 mutations define a unique subgroup within angioimmunoblastic T-cell lymphoma with strong follicular T-helper-like phenotype and characteristic morphological features.

MYCN-regulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differentiation in human neuroblastoma.

MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17 approximately 92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. Expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-alpha (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB.

How deviations in the elemental profile of Humulus lupulus grown throughout the U.S. and Germany influence hop and beer quality.

Recently studies based on limited sample sizes procured from minor hop growing regions have speculated that the elemental profile of hops can possibly be used to authenticate the origin of a hop because changes in hop elemental profiles were realted to growing region and that these changes might also be related to beer quality. To explore this further, 205 hop samples (i.e. 203 whole cone hops and 2 pelletized samples) compromised of 19 varieties were procured from the major hop growing regions (i.e. the U.S. and Germany). These hops were digested with microwave digestion and analyzed for 25 elements using ICP-MS. Hops from most of the U.S. regions (mainly WA) had vastly different elemental profiles than hops from Germany. German hops had significantly lower concentrations for most of the elements except for Cu and K. Interestingly, high alpha varieties had significantly different elemental profiles than varieties bred for aroma purposes. Dry-hopping trials were then performed in an ale and a lager with the hops that had significantly different elemental profiles. Although heavy metals were extracted from hops into beer, at the 5 g/L dry-hopping load used in this study, beer concentrations of these elements remained below regulated water quality standards set by Germany, the U.S., and Canada. Based on electron paramagnetic resonance, dry-hopping had an antioxidant impact on beer regardless of the original elemental profile of the hops which was correlated to hop polyphenol and α/ ß - acid concentrations. Overall these results highlight that many factors including location have the potential to influence the elemental profile of hops and that changes in the elemental profiles of hops can be related to beer quality.

A unifying hypothesis for PNMZL and PTFL: morphological variants with a common molecular profile.

Pediatric nodal marginal zone lymphoma (PNMZL) is an uncommon B-cell neoplasm affecting mainly male children and young adults. This indolent lymphoma has distinct characteristics that differ from those of conventional nodal marginal zone lymphoma (NMZL). Clinically, it exhibits overlapping features with pediatric-type follicular lymphoma (PTFL). To explore the differences between PNMZL and adult NMZL and its relationship to PTFL, a series of 45 PNMZL cases were characterized morphologically and genetically by using an integrated approach; this approach included whole-exome sequencing in a subset of cases, targeted next-generation sequencing, and copy number and DNA methylation arrays. Fourteen cases (31%) were diagnosed as PNMZL, and 31 cases (69%) showed overlapping histologic features between PNMZL and PTFL, including a minor component of residual serpiginous germinal centers reminiscent of PTFL and a dominant interfollicular B-cell component characteristic of PNMZL. All cases displayed low genomic complexity (1.2 alterations per case) with recurrent 1p36/TNFRSF14 copy number-neutral loss of heterozygosity alterations and copy number loss (11%). Similar to PTFL, the most frequently mutated genes in PNMZL were MAP2K1 (42%), TNFRSF14 (36%), and IRF8 (34%). DNA methylation analysis revealed no major differences between PTFL and PNMZL. Genetic alterations typically seen in conventional NMZL were absent in PNMZL. In summary, overlapping clinical, morphologic, and molecular findings (including low genetic complexity; recurrent alterations in MAP2K1, TNFRSF14, and IRF8; and similar methylation profiles) indicate that PNMZL and PTFL are likely part of a single disease with variation in the histologic spectrum. The term "pediatric-type follicular lymphoma with and without marginal zone differentiation" is suggested.

Follicular lymphoma t(14;18)-negative is genetically a heterogeneous disease.

Fifty-five cases of t(14;18)- follicular lymphoma (FL) were genetically characterized by targeted sequencing and copy number (CN) arrays. t(14;18)- FL predominated in women (M/F 1:2); patients often presented during early clinical stages (71%), and had excellent prognoses. Overall, t(14;18)- FL displayed CN alterations (CNAs) and gene mutations carried by conventional t(14;18)+ FL (cFL), but with different frequencies. The most frequently mutated gene was STAT6 (57%) followed by CREBBP (49%), TNFRSF14 (39%), and KMT2D (27%). t(14;18)- FL showed significantly more STAT6 mutations and lacked MYD88, NOTCH2, MEF2B, and MAP2K1 mutations compared with cFL, nodal marginal zone lymphoma (NMZL), and pediatric-type FL (PTFL). We identified 2 molecular clusters. Cluster A was characterized by TNFRSF14 mutations/1p36 alterations (96%) and frequent mutations in epigenetic regulators, with recurrent loss of 6q21-24 sharing many features with cFL. Cluster B showed few genetic alterations; however, a subgroup with STAT6 mutations concurrent with CREBBP mutations/16p alterations without TNFRSF14 and EZH2 mutations was noted (65%). These 2 molecular clusters did not distinguish cases by inguinal localization, growth pattern, or presence of STAT6 mutations. BCL6 rearrangements were demonstrated in 10 of 45 (22%) cases and did not cluster together. Cases with predominantly inguinal presentation (20 of 50; 40%) had a higher frequency of diffuse growth pattern, STAT6 mutations, CD23 expression, and a lower number of CNAs, in comparison with noninguinal cases (5.1 vs 9.1 alterations per case; P < .05). STAT6 mutations showed a positive correlation with CD23 expression (P < .001). In summary, t(14;18)- FL is genetically a heterogeneous disorder with features that differ from cFL, NMZL, and PTFL.

Impact of fluoride, milk and water rinsing on surface rehardening of acid softened enamel. An in situ study.

PURPOSE: To evaluate the impact of fluoride, milk and water rinsing on surface rehardening of acid softened enamel in situ. METHODS: Ten subjects performed six tests of4 hours each. In each test, three softened enamel samples were attached to intraoral appliances. For softening, samples were immersed extraorally in an acidic beverage for 120 seconds. Subsequently, specimens were worn intraorally for 5 minutes (Tests 1-3) or 30 minutes (Tests 4-6). Thereafter, the volunteers rinsed with a 250 ppm SnF2/Olaflur solution (Tests 1 and 4), milk (Tests 2 and 5) or non-carbonated mineral water (Tests 3 and 6) for 60 seconds. At each test, one sample was covered with tape during intraoral rinsing and thus, served as control. After rinsing, both test and the control samples were exposed to the oral cavity for up to 4 hours after demineralization. Surface microhardness (SMH) of the specimens was assessed at baseline, immediately after softening and 4 hours after softening. For each subject, the secretion rate of resting and stimulated saliva, buffering capacity and pH-value as well as calcium and phosphate concentration of saliva were measured. Statistical analysis was performed by ANCOVA followed by stratified analyses with Bonferroni correction. RESULTS: Baseline Knoop Hardness (mean +/- S.D.) amounted to 403.1 +/- 39.4. Immediately after softening, mean SMH was reduced to 214.4 +/- 24.1 KHN. Rinsing with 250 ppm fluoride, milk or water after 5 minutes or 30 minutes intraoral exposure of softened samples had a significant effect on rehardening. The increase of SMH (DeltaKHN) was highest after rinsing with fluoride (5 minutes: 95.0 +/- 18.3; 30 minutes: 94.2 +/- 24.3) followed by milk (5 minutes: 77.1 +/- 14.1; 30 minutes: 80.3 +/- 18.7) and water (5 minutes: 49.0 +/- 9.9; 30 minutes: 47.0 +/- 14.1), but did not achieve baseline values. It is concluded that a single rinse with a 250 ppm SnF2/Olaflur solution, milk or water increases rehardening of previously acid softened enamel.

Comparative analysis of the human gimap gene cluster encoding a novel GTPase family.

There is a growing family of novel GTPases conserved among higher plants and vertebrates, abbreviated as AIG1, IAP, IMAP, and IAN, respectively. Here, we comparatively analyze the human gene family encoding GTPases of the immunity-associated protein family recently re-termed GIMAP. Chromosome 7q36.1 contains, within 300 kb, a gimap gene cluster with seven functional genes and one pseudogene (hgimap3). The six genes hgimap1, hgimap2, hgimap4, hgimap5, hgimap6, and hgimap7 encode 33-46 kDa proteins with one GTP-binding domain, whereas hgimap8 encodes a very unusual 75-kDa protein with three GTP-binding domains. All hgimap genes except hgimap2 have orthologs in the mouse. Major expression sites of hgimap mRNAs are the spleen and lymph nodes, but also other organs such as muscle, heart, placenta, and digestive tract display detectable hgimap mRNA levels. The proteins hGIMAP4 and hGIMAP7 can be localized at ER and Golgi apparatus, but not in mitochondria, lysosomes and nuclei. All hgimap genes were expressed at very low levels-if at all-in diverse cancer cell lines. Our data support the view that the GIMAP proteins are involved in the control of cell survival not only in cells of the immune system as commonly anticipated.

Targeting of the MYCN protein with small molecule c-MYC inhibitors.

Members of the MYC family are the most frequently deregulated oncogenes in human cancer and are often correlated with aggressive disease and/or poorly differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a poor prognosis, targeting MYCN using small molecule inhibitors could represent a promising therapeutic approach. We have previously demonstrated that the small molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma tumor models with MYCN overexpression. Our previous work also revealed that MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of lipid droplets in neuroblastoma cells. To expand our understanding of how small molecules interfere with MYCN, we have now analyzed the direct binding of 10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon resonance. Using a proximity ligation assay, we found reduced interaction between MYCN and MAX after treatment with all molecules except for the 10058-F4 metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 10058-F4 were the most efficient in inducing neuronal differentiation and lipid accumulation in MYCN-amplified neuroblastoma cells. Together our data demonstrate MYCN-binding properties for a selection of small molecules, and provide functional information that could be of importance for future development of targeted therapies against MYCN-amplified neuroblastoma.

A unique mechanism for methyl ester formation via an amide intermediate found in myxobacteria.

Secondary metabolism involves a broad diversity of biochemical reactions that result in a wide variety of biologically active compounds. Terminal amide formation during the biosynthesis of the myxobacterial electron-transport inhibitor, myxothiazol, was analyzed by heterologous expression of the unique nonribosomal-peptide synthetase, MtaG, and incubation with a synthesized substrate mimic. These experiments provide evidence that the terminal amide is formed from a carrier protein-bound myxothiazol acid that is thioesterified to MtaF. This intermediate is transformed to an amide by extension with glycine and subsequent oxidative cleavage by MtaG. The final steps of melithiazol assembly involve a highly similar protein-bound intermediate (attached to MelF, a homologue of MtaF), which is transformed to an amide by MelG (homologue of MtaG). In this study, we also show that the amide moiety of myxothiazol A can be hydrolyzed in vivo to the formerly unknown free myxothiazol acid by heterologous expression of melJ in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The methyltransferase MelK can finally methylate the acid to give rise to the methyl ester, which is produced as the final product in the melithiazol A biosynthetic pathway. These experiments clarify the role of MelJ and MelK during melithiazol assembly.