The Case for GNMT as a Biomarker and a Therapeutic Target in Pancreatic Cancer.

The high mortality rate for pancreatic cancer (PC) is due to the lack of specific symptoms at early tumor stages and a high biological aggressiveness. Reliable biomarkers and new therapeutic targets would help to improve outlook in PC. In this study, we analyzed the expression of GNMT in a panel of pancreatic cancer cell lines and in early-stage paired patient tissue samples (normal and diseased) by quantitative reverse transcription-PCR (qRT-PCR). We also investigated the effect of 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranoside (PGG) as a therapeutic agent for PC. We find that GNMT is markedly downregulated (p < 0.05), in a majority of PC cell lines. Similar results are observed in early-stage patient tissue samples, where GNMT expression can be reduced by a 100-fold or more. We also show that PGG is a strong inhibitor of PC cell proliferation, with an IC50 value of 12 ng/mL, and PGG upregulates GNMT expression in a dose-dependent manner. In conclusion, our data show that GNMT has promise as a biomarker and as a therapeutic target for PC.

Archaeal assemblages inhabiting temperate mixed forest soil fluctuate in taxon composition and spatial distribution over time.

This study explored the persistence and spatial distribution of a diverse Archaeal assemblage inhabiting a temperate mixed forest ecosystem. Persistence under native conditions was measured from 2001 to 2010, 2011, and 2012 by comparison of 16S rRNA gene clone libraries. The Archaeal assemblages at each of these time points were found to be significantly different (AMOVA, P < 0.01), and the nature of this difference was dependent on taxonomic rank. For example, the cosmopolitan genus g_Ca. Nitrososphaera (I.1b) was detected at all time points, but within this taxon the abundance of s_SCA1145, s_SCA1170, and s_Ca. N. gargensis fluctuated over time. In addition, spatial heterogeneity (patchiness) was measured at these time points using 1D TRFLP-SSCP fingerprinting to screen soil samples covering multiple spatial scales. This included soil collected from small volumes of 3 cubic centimeters to larger scales-over a surface area of 50 m(2), plots located 1.3 km apart, and a separate locality 23 km away. The spatial distribution of Archaea in these samples changed over time, and while g_Ca. Nitrososphaera (I.1b) was dominant over larger scales, patches were found at smaller scales that were dominated by other taxa. This study measured the degree of change for Archaeal taxon composition and patchiness over time in temperate mixed forest soil.

One-dimensional TRFLP-SSCP is an effective DNA fingerprinting strategy for soil Archaea that is able to simultaneously differentiate broad taxonomic clades based on terminal fragment length polymorphisms and closely related sequences based on single stranded conformation polymorphisms.

DNA fingerprinting methods provide a means to rapidly compare microbial assemblages from environmental samples without the need to first cultivate species in the laboratory. The profiles generated by these techniques are able to identify statistically significant temporal and spatial patterns, correlations to environmental gradients, and biological variability to estimate the number of replicates for clone libraries or next generation sequencing (NGS) surveys. Here we describe an improved DNA fingerprinting technique that combines terminal restriction fragment length polymorphisms (TRFLP) and single stranded conformation polymorphisms (SSCP) so that both can be used to profile a sample simultaneously rather than requiring two sequential steps as in traditional two-dimensional (2-D) gel electrophoresis. For the purpose of profiling Archaeal 16S rRNA genes from soil, the dynamic range of this combined 1-D TRFLP-SSCP approach was superior to TRFLP and SSCP. 1-D TRFLP-SSCP was able to distinguish broad taxonomic clades with genetic distances greater than 10%, such as Euryarchaeota and the Thaumarchaeal clades g_Ca. Nitrososphaera (formerly 1.1b) and o_NRP-J (formerly 1.1c) better than SSCP. In addition, 1-D TRFLP-SSCP was able to simultaneously distinguish closely related clades within a genus such as s_SCA1145 and s_SCA1170 better than TRFLP. We also tested the utility of 1-D TRFLP-SSCP fingerprinting of environmental assemblages by comparing this method to the generation of a 16S rRNA clone library of soil Archaea from a restored Tallgrass prairie. This study shows 1-D TRFLP-SSCP fingerprinting provides a rapid and phylogenetically informative screen of Archaeal 16S rRNA genes in soil samples.

Possible contributions of TERMINAL FLOWER 1 to the evolution of rosette flowering in Leavenworthia (Brassicaceae).

Leavenworthia crassa is a rosette flowering species that differs from inflorescence flowering species, such as Arabidopsis thaliana, in having elongated pedicels and shortened interfloral internodes on the main axis. Based on previous experiments, we hypothesized that changes to the L. crassa TFL1 ortholog, LcrTFL1, were important in the evolution of rosette flowering. We isolated LcrTFL1 and introduced a genomic construct into tfl1 mutant A. thaliana plants. We also generated and analyzed EGFP-LcrTFL1 reporter-fusion lines, and LcrTFL1/LcrLFY doubly transgenic lines. The transgene rescued the mutant defects, but manifested gain-of-function phenotypes. However, LcrTFL1 lines differed from 35S:TFL1 lines in several regards. Defects in floral meristem identity establishment were observed, as was the production of flowers with extra petals. We also noted features that resemble rosette flowering: LcrTFL1 lines produced significantly shorter interfloral internodes and significantly longer pedicels than either wild-type or 35S:TFL1 plants. Our data show that there are substantive differences in the regulation and/or function of TFL1 orthologs between A. thaliana and L. crassa. These may reflect changes that occurred during the evolution of rosette flowering in Leavenworthia, but, if so, our results show that additional, as-yet-unidentified genes were involved in this instance of architectural evolution.

The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in Arabidopsis and are required for plant development.

Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.

An exploration of LEAFY expression in independent evolutionary origins of rosette flowering in Brassicaceae.

Whereas most Brassicaceae produce flowers on an elongated inflorescence, a few lineages produce flowers directly from the vegetative rosette on elongated pedicels. Knowing the extent to which independent origins of rosette flowering involve the same developmental and genetic mechanisms could clarify the constraints acting on plant architectural evolution. Prior work in Idahoa, Ionopsidium, and Leavenworthia suggested that changes in the activity or expression of the flower meristem identity gene, LEAFY (LFY), played a role in all three origins of rosette flowering. Here we studied the developmental morphology of L. crassa and immunolocalization of LFY protein in Leavenworthia and Ionopsidium to further compare independent origins of rosette flowering. Leavenworthia crassa differs from Ionopsidium and Idahoa in producing ebracteate flowers. Flowers are, however, associated with "squamules," here interpreted as stipules of a cryptic bract. LFY was detected in L. crassa flower primordia but not in inflorescence meristems. In contrast, the rosette flowering Io. acaule accumulated LFY protein in the inflorescence meristem, whereas its inflorescence-flowering close relative, Io. prolongoi, did not. Thus, although different cases of rosette flowering likely entailed modifications of the same meristem identity program, distinct developmental genetic mechanisms appear to be involved in each case.

The role of two LEAFY paralogs from Idahoa scapigera (Brassicaceae) in the evolution of a derived plant architecture.

Idahoa scapigera produces solitary flowers in the axils of rosette leaves without elongation of the shoot axis, a rosette-flowering architecture. Previous work with one of the two I. scapigera LFY paralogs, IscLFY1, showed that this gene caused aerial flowering rosettes in Arabidopsis thaliana. In this paper, we report that after three generations IscLFY1 transgenic lines are phenotypically indistinguishable from wild-type Arabidopsis, indicating that IscLFY1 protein is able to replace normal LFY function. Additionally, we found that ectopic LFY expression late in development can phenocopy aspects of the aerial rosette phenotype, suggesting that shoot compression caused by IscLFY1 could be caused by localized overexpression of a functional IscLFY protein. We also characterized the expression and function of the second I. scapigera LFY paralog, IscLFY2, in A. thaliana. In contrast to IscLFY1, this paralog was expressed in floral meristems and the shoot apical meristem (SAM). In I. scapigera, LFY-specific antibodies detected high protein levels in developing flowers but not in the apex, suggesting trans-regulatory differences between I. scapigera and A. thaliana. Most IscLFY2 transgenic A. thaliana plants were indistinguishable from wild type, but in a minority of lines the SAM was converted to a terminal flower as would be expected from the reporter-expression pattern. Taken together these results show that both I. scapigera paralogs have conserved LFY function, both proteins can rescue lfy and both can modify inflorescence architecture in an A. thaliana background: either by affecting internode elongation (IscLFY1) or by causing homeotic conversion of shoots into flowers (IscLFY2).

The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5.

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K(+) TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1(E232Q), an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression of SKD1(E232Q) in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.

Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots.

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.

Spatial heterogeneity of crenarchaeal assemblages within mesophilic soil ecosystems as revealed by PCR-single-stranded conformation polymorphism profiling.

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division Crenarchaeota (domain Archaea). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [P < 0.0001] and least-significant-difference analysis [alpha = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b.

Comparison of crenarchaeal consortia inhabiting the rhizosphere of diverse terrestrial plants with those in bulk soil in native environments.

To explore whether the crenarchaeal consortium found in the rhizosphere is distinct from the assemblage of crenarchaeotes inhabiting bulk soil, PCR-single-stranded-conformation polymorphism (PCR-SSCP) profiles were generated for 76 plant samples collected from native environments. Divergent terrestrial plant groups including bryophytes (mosses), lycopods (club mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (seed plants) were collected for this study. Statistical analysis revealed significant differences between rhizosphere and bulk soil PCR-SSCP profiles (Hotelling paired T(2) test, P < 0.0001), suggesting that a distinct crenarchaeal consortium is associated with plants. In general, phylotype richness increased in the rhizosphere compared to the corresponding bulk soil, although the range of this increase was variable. Examples of a major change in rhizosphere (versus bulk soil) PCR-SSCP profiles were detected for all plant groups, suggesting that crenarchaeotes form associations with phylogenetically diverse plants in native environments. In addition, examples of minor to no detectable difference were found for all terrestrial plant groups, suggesting that crenarchaeal associations with plants are mediated by environmental conditions.