Reading tea leaves worldwide: decoupled drivers of initial litter decomposition mass‐loss rate and stabilization

The breakdown of plant material fuels soil functioning and biodiversity. Currently, process understanding of global decomposition patterns and the drivers of such patterns are hampered by the lack of coherent large‐scale datasets. We buried 36,000 individual litterbags (tea bags) worldwide and found an overall negative correlation between initial mass‐loss rates and stabilization factors of plant‐derived carbon, using the Tea Bag Index (TBI). The stabilization factor quantifies the degree to which easy‐to‐degrade components accumulate during early‐stage decomposition (e.g. by environmental limitations). However, agriculture and an interaction between moisture and temperature led to a decoupling between initial mass‐loss rates and stabilization, notably in colder locations. Using TBI improved mass‐loss estimates of natural litter compared to models that ignored stabilization. Ignoring the transformation of dead plant material to more recalcitrant substances during early‐stage decomposition, and the environmental control of this transformation, could overestimate carbon losses during early decomposition in carbon cycle models

Leishmania major Abrogates Gamma Interferon-Induced Gene Expression in Human Macrophages from a Global Perspective▿ †

Infection with Leishmania major triggers several pathways in the host cell that are crucial to initial infection as well as those that are used by Leishmania to enhance its replication and virulence. To identify the molecular events of the host cell in response to Leishmania, the global gene expression of the human monocytic cell line THP-1 either infected with Leishmania major in the presence and absence of gamma interferon (IFN-γ) or in the presence of IFN-γ alone was analyzed using high-density human oligonucleotide microarrays, followed by statistical analysis. The persistence of the parasite despite an extensive response to IFN-γ, added 24 h after infection with L. major, suggests that L. major can survive in an IFN-γ-enriched environment in vitro. Results demonstrate that L. major counteracts the IFN-γ response in macrophages on a large scale. Expression of genes involved in the innate immune response, cell adhesion, proteasomal degradation, Toll-like receptor expression, a variety of signaling molecules, and matrix metalloproteinases was significantly modulated

Combined RNAi-mediated suppression of Rictor and EGFR resulted in complete tumor regression in an orthotopic glioblastoma tumor model.

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM

Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U251MG cell line.

<p>Optical density values shown are expressed relative to values obtained from untreated cells, which correspond to a value of 1. <b>a</b>) Representative immunoblots showing ILK, Rictor, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against ILK or Rictor or the negative control sequence (Ng ctrl). <b>b</b>) Representative immunoblots showing EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against EGFR or the negative control sequence (Neg ctrl). <b>c</b>) Representative immunoblots showing Rictor, EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96 hrs after transfection of the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). Optical density values shown under each band represent the average obtained from three independent experiments (±SEM) normalized to β-actin, and AKT in the case of p(473)AKT. <b>d</b>) Representative fluorescence photomicrograph (n = 3) of U251MG cells showing nuclei (Draq5; red), F-actin (Texas red phalloidin; Yellow), and p(473)-AKT (Alexa 488; blue) 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR, or the combination of two negative sequences (Ng2x).</p

Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U118MG and LN229 GBM lines.

<p>Histograms showing Rictor, p(473)AKT, AKT and β-actin protein levels relative to untreated cells. Optical density values were normalized to the β-actin value, and the AKT value in the case of p(473)AKT, and represent the average obtained from three independent experiments from <b>a</b>) U118MG and <b>b</b>) LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001.</p

Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.

<p>Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.</p

Induction of lentiviral shRNA-transduced cells results in downregulation of corresponding proteins <i>in vitro</i> and downstream effectors, and reduction in cell migration.

<p><b>a</b>) Representative immunoblots showing Rictor, EGFR and β-actin from parental U251MG cells, U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor in the absence (-) or presence (+) of doxycycline. Average of band optical density normalized to β-actin from three independent experiments (+/−SEM), and expressed as relative to values obtained from parental cells, is shown under each band. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to parental cells. <b>b</b>) Representative immunoblots showing Rictor, EGFR, p(473)-AKT, p(346)-NDRG1, p(422)-SGK, p(657)-PKCα and β-actin from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor exposed to doxycycline for 72hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x is shown under each band. <b>c</b>) Representative immunoblots showing Rictor, p(473)-AKT and β-actin from U251^Rictor in the absence (-) of doxycycline or exposed to doxycycline for 24–120 hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x in the absence of doxycycline is shown under each band. <b>d</b>) Scratch width scoring of U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor 18hrs after scratching in presence of doxycycline and after pre-incubation with doxycycline for 72 hrs.**p-value ≤0.01 compared to U251^Ng2x cells.</p

The combined silencing of Rictor and EGFR <i>in vivo</i> results in a complete inhibition of tumor growth.

<p>U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor cells were implanted into the right caudate nucleus-putamen of Rag2M mice (n = 6−8). Induction of shRNA expression in mice was initiated on day 21 by dissolving 2 mg/mL doxycyline and 5% sucrose in drinking water. <b>a</b>) On day 49, animals were imaged by Maestro™ fluorescence imaging unit for the expression of tRFP co-expressed with the shRNA sequences upon doxycycline-induced expression. Mice were then terminated and brains were harvested, sectioned and stained for nuclei, Rictor, EGFR and p(473)-AKT and imaged for all markers in addition to tRFP by robotic fluorescence microscopy. No tumor was detected in the U251^EGFR/Rictor group. <b>b</b>) A representative brain section from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor tumor groups is shown: tRFP (red) and Hoechst (blue). <b>c</b>) A representative tumor section from U251^Ng2x, U251^Rictor and U251^EGFR tumor groups is shown: nuclei (blue), rRFP (red), Rictor (yellow), EGFR (green) and p(473)-AKT (orange). <b>d</b>) The expression of EGFR (left axis), Rictor (right axis) and p(473)-AKT (right axis) in U251^Ng2x, U251^Rictor, U251^EGFR tumor sections were quantified (positive staining normalized to Hoechst nuclei staining). <b>e</b>) Tumor sizes were estimated by quantification of tumor areas in brain sections from all groups (left axis). The expression of the proliferation marker Ki67 in the tumor (proliferating fraction) was also quantified (right axis). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to control untreated cells. ‡: No tumor was detected in the U251^EGFR/Rictor group.</p