Belowground DNA-based techniques: untangling the network of plant root interactions

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Plant DNA Barcodes Can Accurately Estimate Species Richness in Poorly Known Floras

Extent: 9p.BACKGROUND: Widespread uptake of DNA barcoding technology for vascular plants has been slow due to the relatively poor resolution of species discrimination (~70%) and low sequencing and amplification success of one of the two official barcoding loci, matK. Studies to date have mostly focused on finding a solution to these intrinsic limitations of the markers, rather than posing questions that can maximize the utility of DNA barcodes for plants with the current technology. METHODOLOGY/PRINCIPAL FINDINGS: Here we test the ability of plant DNA barcodes using the two official barcoding loci, rbcLa and matK, plus an alternative barcoding locus, trnH-psbA, to estimate the species diversity of trees in a tropical rainforest plot. Species discrimination accuracy was similar to findings from previous studies but species richness estimation accuracy proved higher, up to 89%. All combinations which included the trnH-psbA locus performed better at both species discrimination and richness estimation than matK, which showed little enhanced species discriminatory power when concatenated with rbcLa. The utility of the trnH-psbA locus is limited however, by the occurrence of intraspecific variation observed in some angiosperm families to occur as an inversion that obscures the monophyly of species. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time, using a case study, the potential of plant DNA barcodes for the rapid estimation of species richness in taxonomically poorly known areas or cryptic populations revealing a powerful new tool for rapid biodiversity assessment. The combination of the rbcLa and trnH-psbA loci performed better for this purpose than any two-locus combination that included matK. We show that although DNA barcodes fail to discriminate all species of plants, new perspectives and methods on biodiversity value and quantification may overshadow some of these shortcomings by applying barcode data in new ways.Craig Costion, Andrew Ford, Hugh Cross, Darren Crayn, Mark Harrington and Andrew Low

MMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells

Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients

Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory

p-MMP-2 inhibits radiation-enhanced tumor culture medium-induced microtubule network formation in endothelial cells and downregulates expression of angiogenesis-associated molecules.

<p><b>A,</b> Human microvascular endothelial cells (5×10⁴) were seeded in 96-well plates and cultured with conditioned medium collected from U-251 and U-87 glioma cells transfected with mock, p-SV, and p-MMP-2, and irradiated as described earlier. 24 h after radiation treatment, the cells were washed, fixed and stained with Hema-3 and photographed. Percentages of branches were quantified by counting five fields in each condition. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock or irradiated controls. <b>B,</b> U-251 and U-87 transfection and radiation was carried out as described earlier. 24 h after radiation, whole cell lysates were prepared and analyzed by Western blotting for the angiogenic molecules VEGF, VEGFR-2 and p-VEGFR-2 as well as p-FAK, FAK, p-p38 and p38. GAPDH served as a loading control.</p

p-MMP-2 combined with radiation enhances apoptosis <i>in vivo</i>.

<p><b>A,</b> Immunohistochemical analysis of brain sections using anti-MMP-2, anti-VEGF and anti-pFAK antibodies. Sections were photographed (60×). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (insets). <b>B,</b> Tissue sections of mice were evaluated with the TUNEL assay according to manufacturer's instructions and photographed under fluorescent microscopy (60×). For the negative control, samples were incubated with label solution (without terminal transferase) instead of TUNEL reaction mixture (insets). <b>C,</b> siRNA against MMP-2 inhibits U251 tumor cell invasion <i>in vivo</i>. H&E staining was performed according to standard protocol, and representative pictures of tumor sections from mock, pSV, p-MMP-2-treated mice are shown (20× and 60×). <b>D</b>, Immunohistochemical analysis of brain sections using anti-human nuclei (HuNu) antibody, a histological marker for identification of human cells (a specific human nuclear antigen). Entire brain sections were photographed (4×; middle row); shown on the top row is a non-tumor region (40×; top row); and shown on the bottom row is tumor and non-tumor overlapping region (40×; bottom row). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (inset).</p

p-MMP-2 transfection inhibits radiation-enhanced MMP-2 activity and expression levels as well as cell viability.

<p><b>A,</b> U-251 and U-87 cells were transfected with mock (PBS), p-SV or p-MMP-2 (2 µg), and after 72 h of incubation, cells were irradiated with 0, 2, 4, 6 or 8 Gy and incubated for a further 24 h. Conditioned media was used to determine MMP-2 activity by gelatin zymography, and total cell lysates were used to determine MMP-2 levels by Western blotting. <b>B,</b> Total RNA was used to determine MMP-2 mRNA transcription levels by RT-PCR with gene-specific primers. GAPDH served as a loading control. <b>C,</b> U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2 and irradiated as described above. 24 h after radiation, the cells were fixed and processed to visualize MMP-2 expression. The cells were mounted using mounting media with DAPI to visualize the nucleus. <b>D,</b> U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated for 72 h after transfection. After a another 24 h of incubation, cell viability was analyzed by MTT assay (absorbance read at 550 nm). <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, significant difference from mock, p-SV or irradiated controls.</p