Towards an eDNA metabarcode-based performance indicator for full-scale municipal wastewater treatment plants.

Knowledge-driven management for wastewater treatment plant (WWTP) performance becomes increasingly important considering the globally growing production of wastewater and the rising demand of clean water supply. Even though the potential of microbial organisms (bacteria and protists) as bioindicators for WWTP performance is well known, it is far from being fully exploited for routine monitoring programs. Therefore, we here used massive sequencing of environmental (e)DNA metabarcodes from bacterial (V3-V4 region of the SSU rRNA gene) and eukaryote (V9 region of the SSU rRNA gene) communities in 21 activated sludge samples obtained from full-scale municipal WWTPs in Germany. Microbial community patterns were compared to standard WWTP operating parameters and two traditionally used WWTP performance indicators (Sludge Biotic Index and Sludge Index). Both indices showed low concordance and hardly correlated with chemical WWTP performance parameters nor did they correlate with microbial community structures. In contrast, microbial community profiles significantly correlated with WWTP performance parameters and operating conditions of the plants under study. Therefore, eDNA metabarcode profiles of whole microbial communities indicate the performance of WWTP and can provide useful information for management strategies. We here suggest a strategy for the development of an eDNA metabarcode based bioindicator system, which can be implemented in future standard monitoring programs for WWTP performance and effluent quality.

CD44v6 coordinates tumor matrix-triggered motility and apoptosis resistance.

Tumor progression requires a crosstalk with the tumor surrounding, where the tumor matrix plays an essential role. We recently reported that only the matrix delivered by a CD44v6-competent (ASML(wt)), but not that of a CD44v6-deficient (ASML-CD44v(kd)) rat pancreatic adenocarcinoma line supports metastasis formation. We here describe that this matrix provides an important feedback toward the tumor cell and that CD44v6 accounts for orchestrating signals received from the matrix. ASML(wt) cells contain more hyaluronan synthase-3 and secrete higher amounts of >50 kDa HA than ASML-CD44v(kd) cells, which secrete more hyaluronidase. Only the ASML(wt)-matrix supports migration and apoptosis resistance, which both can be initiated via CD44v6, c-Met, and α6ß4 ligand binding and proceed via FAK, PI3K/Akt, and MAPK activation, respectively. However, c-Met- and α6ß4-initiated signaling are strongly augmented by the association with CD44v6 as only very weak effects are observed in CD44v6-deficient cells. The same CD44v6-dependent convergence of motility- and apoptosis resistance-related signals also accounts for human tumor lines. Thus, CD44v6 promotes motility and apoptosis resistance via its involvement in assembling a matrix that, in turn, triggers activation of signaling cascades, which proceeds, independent of the initiating receptor-ligand interaction, in a concerted action via CD44v6.

CD44v6 dependence of premetastatic niche preparation by exosomes.

The metastasizing capacity of the rat pancreatic adenocarcinoma BSp73ASML (ASML(wt)) is strikingly reduced by a knockdown of CD44v4-v7 (ASML(kd)). We used this model to analyze the role of the CD44 variant isoform (CD44v) in (pre)metastatic niche formation. Intrafootpad injections of ASML(wt)-, but not ASML(kd)-conditioned medium (CM), strongly promote settlement of ASML(kd) cells in lymph nodes and lung. Fractionation of CM revealed a contribution by a soluble matrix and exosomes, where the CD44v6-containing ASML(wt)-soluble fraction can complement ASML(kd)-exosomes, but not vice versa. This implies that exosomes are the final actors, are CD44v-independent, but require a soluble matrix, which depends on CD44v. Analyzing the composition revealed that only the ASML(wt)-matrix contains c-Met and urokinase-type plasminogen activator receptor. In vitro, mostly ASML(wt)-exosomes promote proliferation and induce gene expression in metastatic organ cells. However, in vivo corresponding changes in the (pre) metastatic organ are only observed when both, exosomes plus the soluble matrix, are provided. Thus, neither CD44v nor exosomes alone suffice for (pre)metastatic niche formation. Instead, CD44v suffices for assembling a soluble matrix, which allows exosomes, independent of their origin from poorly or highly metastatic cells, to modulate (pre) metastatic organ cells for tumor cell embedding and growth.

CD44 variant isoforms promote metastasis formation by a tumor cell-matrix cross-talk that supports adhesion and apoptosis resistance.

CD44 designates a large family of proteins with a considerable structural and functional diversity, which are generated from one gene by alternative splicing. As such, the overexpression of CD44 variant isoform (CD44v) has been causally related to the metastatic spread of cancer cells. To study the underlying mechanism, stable knockdown clones with deletion of exon v7 containing CD44 isoforms (CD44v(kd)) of the highly metastatic rat adenocarcinoma line BSp73ASML (ASML(wt)) were established. ASML-CD44v(kd) clones hardly form lung metastases after intrafootpad application and the metastatic load in lymph nodes is significantly reduced. Rescuing, albeit at a reduced level, CD44v expression in ASML-CD44v(kd) cells (ASML-CD44v(rsc)) restores the metastatic potential. The following major differences in ASML(wt), ASML-CD44v(kd), and ASML-CD44v(rsc) clones were observed: (a) ASML(wt) cells produce and assemble a matrix in a CD44v-dependent manner, which supports integrin-mediated adhesion and favors survival. This feature is lost in the ASML-CD44v(kd) cells. (b) CD44v cross-linking initiates phosphatidylinositol 3-kinase/Akt activation in ASML(wt) cells. Accordingly, apoptosis resistance is strikingly reduced in ASML-CD44v(kd) cells. The capacity to generate an adhesive matrix but not apoptosis resistance is restored in ASML-CD44v(rsc) cells. These data argue for a 2-fold effect of CD44v on metastasis formation: CD44v-mediated matrix formation is crucial for the settlement and growth at a secondary site, whereas apoptosis resistance supports the efficacy of metastasis formation.