What lies beneath: Hydra provides cnidarian perspectives into the evolution of FGFR docking proteins

Across the Bilateria, FGF/FGFR signaling is critical for normal development, and in both Drosophila and vertebrates, docking proteins are required to connect activated FGFRs with downstream pathways. While vertebrates use Frs2 to dock FGFR to the RAS/MAPK or PI3K pathways, the unrelated protein, downstream of FGFR (Dof/stumps/heartbroken), fulfills the corresponding function in Drosophila. To better understand the evolution of the signaling pathway downstream of FGFR, the available sequence databases were screened to identify Frs2, Dof, and other key pathway components in phyla that diverged early in animal evolution. While Frs2 homologues were detected only in members of the Bilateria, canonical Dof sequences (containing Dof, ankyrin, and SH2/SH3 domains) were present in cnidarians as well as bilaterians (but not in other animals or holozoans), correlating with the appearance of FGFR. Although these data suggested that Dof coupling might be ancestral, gene expression analysis in the cnidarian Hydra revealed that Dof is not upregulated in the zone of strong FGFRa and FGFRb expression at the bud base, where FGFR signaling controls detachment. In contrast, transcripts encoding other, known elements of FGFR signaling in Bilateria, namely the FGFR adaptors Grb2 and Crkl, which are acting downstream of Dof (and Frs2), as well as the guanyl nucleotide exchange factor Sos, and the tyrosine phosphatase Csw/Shp2, were strongly upregulated at the bud base. Our expression analysis, thus, identified transcriptional upregulation of known elements of FGFR signaling at the Hydra bud base indicating a highly conserved toolkit. Lack of transcriptional Dof upregulation raises the interesting question, whether Hydra FGFR signaling requires either of the docking proteins known from Bilateria

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Identification and characterization of downstream elements of Hydra FGFR signaling

Hydra polyps predominantly reproduce through budding in the lower half of the parent’s body column. FGFRa (Kringelchen), a member of FGF receptor tyrosine kinases, plays an essential role and controls bud detachment from the parent. Whether signal transduction through Hydra FGFR is comparable to FGFR signal-­ ing in vertebrate and fly is unknown. In both Bilateria, activated FGFRs recruit docking proteins to connect to downstream pathways and negative regulators. While vertebrates use FRS2 to dock FGFR to the Ras/MAPK or PI3K pathways, a completely unrelated protein, Downstream-­of-­FGFR (Dof/Stumps/Heartbro-­ ken), fulfills this function in Drosophila. In Drosophila, Dof couples FGFR to MAPK signaling and transcriptionally activates the negative regulator Sprouty (Spry). Spry proteins are necessary to modulate receptor tyrosine kinase activity by in-­ terfering with MAPK signaling downstream of RTK. To elucidate potential downstream signaling elements of ancestral FGFRs, I an-­ alyzed genomic and EST sequence databases and identified Spry, FRS2, and/or Dof proteins in phyla derived early from the main lineage of animals – including Hydra. Dof was found only within the Eumetazoa, while FRS2 proteins were also predicted in Metazoa and their sister taxon, the Choanoflagellata. For the known FRS2 proteins of Deuterostomia and Ecdysozoa an N-­terminal myristoylation site, a PTB domain and multiple C-­terminal Grb2 and Shp2 binding sites are typ-­ ical. This structure also applies to FRS2 in Choanoflagellata and sponges (Porif-­ era). A deviating domain structure of FRS2 proteins is predicted in Placozoa, Cnidaria, and Spiralia (Annelida, Mollusca). Their FRS2 proteins all carry an N-­ terminal PH, a PTB domain and few Grb2 and Shp2 binding sites. Phylogenetic analysis identified a novel protein family (PH-­FRS2). Expression analysis of Dof and FRS2 in Hydra revealed high levels of Dof transcripts in the upper body re-­ gion and the tentacle zone. FRS2 mRNA, in contrast, was detected only weakly at the tentacle bases. The presence of both putative docking proteins in Metazoa hints to an early toolkit for the transduction of FGFR signals. Their functional sig-­ nificance remains to be shown. I also identified four spry genes in Hydra, all positioned in the basal most position of the phylogenetic tree. They encode the typical features of Spry proteins namely a c-­Cbl TKB (Tyrosine kinase binding) site, a Raf1-­binding and a Spry domain. Transcripts of spry2 were detected at the bud base adjacent to Hydra FGFRa from mid to late stages. Since no spry-­encoding genes were found in the ge-­ nomes of Parazoa (Trichoplax), sponges (Oscarella, Amphimedon), or choanoflagellates (Salpingoeca), Sprouties might have occurred in the Cnidaria first -­ or been lost from the early derived taxa. Tissue dynamics and the spatiotemporal expression pattern of FGFRa, dof and spry2, reveals (a) spry2 and FGFRa are present in the same cells at the bud base, (b) co-­expression of FGFRa (weakly) and dof (strongly) in the head region. In summary, data suggest the existence of two FGFR pathways. The first path-­ way functions in bud detachment, as shown previously, and potentially links FGFRa to a negative feedback loop activating Spry2. A second pathway function for FGFR signaling in Hydra might be at the bud and at the adult head – in a zone, where the mRNA encoding the docking protein Dof is expressed at a high level, and where the two FGFRs are transcribed at a low level. Here, FGFR might func-­ tion to control or modulate cell migration towards the head and/or cell differentia-­ tion necessary to form and maintain a fully functional head. Further elucidation of these potential functions and the molecular network, in which Hydra FGFRs act, is a very interesting task for the future

Evaluating Soil Erosion through Geospatial Techniques: Difficulties and Prospects in the Context of the Central Indian Chambal River Basin

Soil erosion is the greatest threat to the ecosystem which gets accelerated due to environmental agents such as water and wind as well as anthropogenic activities. Effective estimation of soil degradation plays an important role in planning preventive measures and conserving the soil. This study was carried out to provide decision-makers with a picture of soil erosion in Madhya Pradesh's Chambal basin and to identify environmentally hot areas to assist in planning effective conservation measures. By using a few input parameters to create raster maps of the Rainfall erosivity factor (R), Soil erodibility factor (K), Topographic factor (LS), Cover and management factor (C), and Support practice factor (P), the Universal Soil Loss Equation (USLE) and Revised Universal Soil Loss Equation (RUSLE) models were applied. The classification of soil erosion and the area portion in each class was then acknowledged. According to the USLE and RUSLE models, the average soil loss for the entire basin is 2.00 t ha-1 yr-1 and 3.04 t ha-1 yr-1, respectively. According to the USLE and RUSLE models, the ranges under severe risk are 0.33% and 0.76%, while the ranges under extremely severe risk are 0.45% and 0.78%, respectively. The land use/land cover (LULC) map for the study area was acquired from satellite data in the USLE, and the Normalized Difference Vegetation Index (NDVI) map was incorporated into the RUSLE model to enhance the comprehension and identification of vegetation. This integration is crucial for capturing detailed information in the RUSLE model. Consequently, RUSLE yields superior results compared to the USLE model, underscoring the significance of incorporating finer details, especially those related to vegetation, for more accurate outcomes

Identification and characterization of downstream elements of Hydra FGFR signaling

Hydra polyps predominantly reproduce through budding in the lower half of the parent’s body column. FGFRa (Kringelchen), a member of FGF receptor tyrosine kinases, plays an essential role and controls bud detachment from the parent. Whether signal transduction through Hydra FGFR is comparable to FGFR signal-­ ing in vertebrate and fly is unknown. In both Bilateria, activated FGFRs recruit docking proteins to connect to downstream pathways and negative regulators. While vertebrates use FRS2 to dock FGFR to the Ras/MAPK or PI3K pathways, a completely unrelated protein, Downstream-­of-­FGFR (Dof/Stumps/Heartbro-­ ken), fulfills this function in Drosophila. In Drosophila, Dof couples FGFR to MAPK signaling and transcriptionally activates the negative regulator Sprouty (Spry). Spry proteins are necessary to modulate receptor tyrosine kinase activity by in-­ terfering with MAPK signaling downstream of RTK. To elucidate potential downstream signaling elements of ancestral FGFRs, I an-­ alyzed genomic and EST sequence databases and identified Spry, FRS2, and/or Dof proteins in phyla derived early from the main lineage of animals – including Hydra. Dof was found only within the Eumetazoa, while FRS2 proteins were also predicted in Metazoa and their sister taxon, the Choanoflagellata. For the known FRS2 proteins of Deuterostomia and Ecdysozoa an N-­terminal myristoylation site, a PTB domain and multiple C-­terminal Grb2 and Shp2 binding sites are typ-­ ical. This structure also applies to FRS2 in Choanoflagellata and sponges (Porif-­ era). A deviating domain structure of FRS2 proteins is predicted in Placozoa, Cnidaria, and Spiralia (Annelida, Mollusca). Their FRS2 proteins all carry an N-­ terminal PH, a PTB domain and few Grb2 and Shp2 binding sites. Phylogenetic analysis identified a novel protein family (PH-­FRS2). Expression analysis of Dof and FRS2 in Hydra revealed high levels of Dof transcripts in the upper body re-­ gion and the tentacle zone. FRS2 mRNA, in contrast, was detected only weakly at the tentacle bases. The presence of both putative docking proteins in Metazoa hints to an early toolkit for the transduction of FGFR signals. Their functional sig-­ nificance remains to be shown. I also identified four spry genes in Hydra, all positioned in the basal most position of the phylogenetic tree. They encode the typical features of Spry proteins namely a c-­Cbl TKB (Tyrosine kinase binding) site, a Raf1-­binding and a Spry domain. Transcripts of spry2 were detected at the bud base adjacent to Hydra FGFRa from mid to late stages. Since no spry-­encoding genes were found in the ge-­ nomes of Parazoa (Trichoplax), sponges (Oscarella, Amphimedon), or choanoflagellates (Salpingoeca), Sprouties might have occurred in the Cnidaria first -­ or been lost from the early derived taxa. Tissue dynamics and the spatiotemporal expression pattern of FGFRa, dof and spry2, reveals (a) spry2 and FGFRa are present in the same cells at the bud base, (b) co-­expression of FGFRa (weakly) and dof (strongly) in the head region. In summary, data suggest the existence of two FGFR pathways. The first path-­ way functions in bud detachment, as shown previously, and potentially links FGFRa to a negative feedback loop activating Spry2. A second pathway function for FGFR signaling in Hydra might be at the bud and at the adult head – in a zone, where the mRNA encoding the docking protein Dof is expressed at a high level, and where the two FGFRs are transcribed at a low level. Here, FGFR might func-­ tion to control or modulate cell migration towards the head and/or cell differentia-­ tion necessary to form and maintain a fully functional head. Further elucidation of these potential functions and the molecular network, in which Hydra FGFRs act, is a very interesting task for the future

Assessment of Eco-Environmental Vulnerability Using Remote Sensing and GIS Tools in Maharashtra Region, India

Maharashtra region is prone to various disasters such as drought, floods, cyclones and earthquake and has been exposed to extreme weather events like dry spells. Communities within these dry lands are poor and face extreme conditions of water stress. This study has been carried out to analyze and quantify climatic and anthropogenic effect on eco-environmental vulnerability dynamic change. To achieve that a numerical model is set up, consisting of eight factors that are elevation, land use, drought, slope, NDVI, soil-type, soil erosion (water), and population density index &amp; has been evaluated using the method of spatial principle component analysis (SPCA) on Remote Sensing and GIS platform. The integrated eco-environmental vulnerability index (EVI) of study area is estimated to analyse spatial-temporal dynamic vulnerability changes in the 11 years gap from 2000 and 2011. The results show that the study area has become eco-environmental vulnerable slightly (about 80% of the region) with an increased eco-environmental vulnerability integrated index (EVSI) value by more than 50% (i.e., about 74%) and the driving force of dynamic change is mainly caused by socio-economic activities. In addition the estimation has been regionalized into thirty-four districts to serve as a base for decision-making for eco-environmental recovering and rebuilding. It is found that the most vulnerable district in 2011 is Ratnagiri and the least one is Sangli. There are nine districts which shows more than 100% increase in EVSI value, with the highest increase in Hingoli(100.65%), indicating that the districts have become most environmental vulnerable in the study-period. The research concludes that the method, supported by G.I.S using SPCA can’t only represent distinctly the input spatial distribution of plain-mountain-belt feature, but also respect the whole river-valley as a single unit

Identification of Soil Erosion Prone Areas of Madhya Pradesh using USLE/RUSLE

Soil erosion is caused due to the dynamic action of erosive agents, mainly water, and is a major threat to the environment. Primary aim of the present study was to study the soil loss dynamics, and identify the environmental hotspots in Madhya Pradesh to aid decision-makers to plan and prioritize appropriate conservation measures. Universal Soil Loss Equation (USLE) and Revised Universal Soil Loss Equation (RUSLE) models were applied for erosion rate estimation by generating thematic maps of R (Rainfall erosivity factor), K (Soil erodibility factor), LS (Topographic factor), C (Cover and management factor), and P (Support practice factor) factors by using several input parameters in QGIS software. Subsequently, the different classes of soil erosion and percentage area under these classes were identified. The average annual soil erosion for the entire state as obtained from the USLE and RUSLE model were 5.80 t.ha-1.yr-1 and 6.64 t.ha-1.yr-1, respectively. The areas under severe risk were 1.09 % and 1.80 %, and very severe risk areas were 1.57 % and 1.83 % as estimated by USLE and RUSLE model, respectively. As compared to RUSLE model, USLE model underestimated rate of soil erosion for most river basins of the state as well as for the entire state