Regional Genetic Structure in the Aquatic Macrophyte Ruppia cirrhosa Suggests Dispersal by Waterbirds

The evolutionary history of the genus Ruppia has been shaped by hybridization, polyploidisation and vicariance that have resulted in a problematic taxonomy. Recent studies provided insight into species circumscription, organelle takeover by hybridization, and revealed the importance of verifying species identification to avoid distorting effects of mixing different species, when estimating population connectivity. In the present study, we use microsatellite markers to determine population diversity and connectivity patterns in Ruppia cirrhosa including two spatial scales: (1) from the Atlantic Iberian coastline in Portugal to the Siculo-Tunisian Strait in Sicily and (2) within the Iberian Peninsula comprising the Atlantic-Mediterranean transition. The higher diversity in the Mediterranean Sea suggests that populations have had longer persistence there, suggesting a possible origin and/or refugial area for the species. The high genotypic diversities highlight the importance of sexual reproduction for survival and maintenance of populations. Results revealed a regional population structure matching a continent-island model, with strong genetic isolation and low gene flow between populations. This population structure could be maintained by waterbirds, acting as occasional dispersal vectors. This information elucidates ecological strategies of brackish plant species in coastal lagoons, suggesting mechanisms used by this species to colonize new isolated habitats and dominate brackish aquatic macrophyte systems, yet maintaining strong genetic structure suggestive of very low dispersal.Fundacao para a Cincia e Tecnologia (FCT, Portugal) [PTDC/MAR/119363/2010, BIODIVERSA/0004/2015, UID/Multi/04326/2013]Pew FoundationSENECA FoundationMurcia Government, Spain [11881/PI/09]FCT Investigator Programme-Career Development [IF/00998/2014]Spanish Ministry of Education [AP2008-01209]European Community [00399/2012]info:eu-repo/semantics/publishedVersio

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα

Berkchaetoazaphilone B has antimicrobial activity and affects energy metabolism

Antimicrobial resistance has become one of the major threats to human health. Therefore, there is a strong need for novel antimicrobials with new mechanisms of action. The kingdom of fungi is an excellent source of antimicrobials for this purpose because it encompasses countless fungal species that harbor unusual metabolic pathways. Previously, we have established a library of secondary metabolites from 10,207 strains of fungi. Here, we screened for antimicrobial activity of the library against seven pathogenic bacterial strains and investigated the identity of the active compounds using ethyl acetate extraction, activity-directed purification using HPLC fractionation and chemical analyses. We initially found 280 antimicrobial strains and subsequently identified 17 structurally distinct compounds from 26 strains upon further analysis. All but one of these compounds, berkchaetoazaphilone B (BAB), were known to have antimicrobial activity. Here, we studied the antimicrobial properties of BAB, and found that BAB affected energy metabolism in both prokaryotic and eukaryotic cells. We conclude that fungi are a rich source of chemically diverse secondary metabolites with antimicrobial activity.Animal science

Gross tumour volume delineation in anal cancer on T2-weighted and diffusion-weighted MRI - Reproducibility between radiologists and radiation oncologists and impact of reader experience level and DWI image quality

Abstract Purpose To assess how gross tumour volume (GTV) delineation in anal cancer is affected by interobserver variations between radiologists and radiation oncologists, expertise level, and use of T2-weighted MRI (T2W-MRI) vs. diffusion-weighted imaging (DWI), and to explore effects of DWI quality. Methods and materials We retrospectively analyzed the MRIs (T2W-MRI and b800-DWI) of 25 anal cancer patients. Four readers (Senior and Junior Radiologist; Senior and Junior Radiation Oncologist) independently delineated GTVs, first on T2W-MRI only and then on DWI (with reference to T2W-MRI). Maximum Tumour Diameter (MTD) was calculated from each GTV. Mean GTVs/MTDs were compared between readers and between T2W-MRI vs. DWI. Interobserver agreement was calculated as Intraclass Correlation Coefficient (ICC), Dice Similarity Coefficient (DSC) and Hausdorff Distance (HD). DWI image quality was assessed using a 5-point artefact scale. Results Interobserver agreement between radiologists vs. radiation oncologists and between junior vs. senior readers was good–excellent, with similar agreement for T2W-MRI and DWI (e.g. ICCs 0.72–0.94 for T2W-MRI and 0.68–0.89 for DWI). There was a trend towards smaller GTVs on DWI, but only for the radiologists (P = 0.03–0.07). Moderate-severe DWI-artefacts were observed in 11/25 (44%) cases. Agreement tended to be lower in these cases. Conclusion Overall interobserver agreement for anal cancer GTV delineation on MRI is good for both radiologists and radiation oncologists, regardless of experience level. Use of DWI did not improve agreement. DWI artefacts affecting GTV delineation occurred in almost half of the patients, which may severely limit the use of DWI for radiotherapy planning if no steps are undertaken to avoid them

Outcomes and potential impact of a virtual hands-on training program on MRI staging confidence and performance in rectal cancer

Objectives: To explore the potential impact of a dedicated virtual training course on MRI staging confidence and performance in rectal cancer. // Methods: Forty-two radiologists completed a stepwise virtual training course on rectal cancer MRI staging composed of a pre-course (baseline) test with 7 test cases (5 staging, 2 restaging), a 1-day online workshop, 1 month of individual case readings (n = 70 cases with online feedback), a live online feedback session supervised by two expert faculty members, and a post-course test. The ESGAR structured reporting templates for (re)staging were used throughout the course. Results of the pre-course and post-course test were compared in terms of group interobserver agreement (Krippendorf’s alpha), staging confidence (perceived staging difficulty), and diagnostic accuracy (using an expert reference standard). // Results: Though results were largely not statistically significant, the majority of staging variables showed a mild increase in diagnostic accuracy after the course, ranging between + 2% and + 17%. A similar trend was observed for IOA which improved for nearly all variables when comparing the pre- and post-course. There was a significant decrease in the perceived difficulty level (p = 0.03), indicating an improved diagnostic confidence after completion of the course. // Conclusions: Though exploratory in nature, our study results suggest that use of a dedicated virtual training course and web platform has potential to enhance staging performance, confidence, and interobserver agreement to assess rectal cancer on MRI virtual training and could thus be a good alternative (or addition) to in-person training. // Clinical relevance statement: Rectal cancer MRI reporting quality is highly dependent on radiologists’ expertise, stressing the need for dedicated training/teaching. This study shows promising results for a virtual web-based training program, which could be a good alternative (or addition) to in-person training. // Key Points: • Rectal cancer MRI reporting quality is highly dependent on radiologists’ expertise, stressing the need for dedicated training and teaching. • Using a dedicated virtual training course and web-based platform, encouraging first results were achieved to improve staging accuracy, diagnostic confidence, and interobserver agreement. • These exploratory results suggest that virtual training could thus be a good alternative (or addition) to in-person training

Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels

Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhibitors in wild-type (wt) and mutant (mt) p53 CRC cells, Lovo and WiDr, respectively, transfected with mt and wt p53. Lovo 175X2 cells (transfected with mt p53) were more resistant to 5-fluorouracil (5-FU; 2-fold), nolatrexed (3-fold), raltitrexed (3-fold) and pemetrexed (10-fold) in comparison with the wt p53 parental cells Lovo 92. Resistance was associated with an increase in TS protein expression and catalytic activity, which might be caused by the loss of the inhibitory effect on the activity of TS promoter or by the lack of TS mRNA degradation, as suggested by the reversal of TS expression to the levels of Lovo 92 cells by adding actinomycin. In contrast, Lovo li cells, characterized by functionally inactive p53, were 3-13-fold more sensitive to nolatrexed, raltitrexed and pemetrexed, and had a lower TS mRNA, protein expression and catalytic activity than Lovo 92. However, MDM-2 expression was significantly higher in Lovo li, while no significant differences were observed in Lovo 175X2 cells with respect to Lovo 92. Finally, mt p53 WiDr transfected with wt p53 were not significantly different from mt p53 WiDr cells with respect to sensitivity to TS inhibitors or TS levels. Altogether, these results indicate that changes in the status of p53, can differently alter sensitivity to TS inhibitors by affecting TS levels, depending on activity or cell line, and might explain the lack of clear correlation between mutations in p53 and clinical outcome after chemotherapy with TS inhibitors

The impact of customer-specific marketing expenses on customer retention and customer profitability

We study the effects of customer-specific marketing expenses on customer retention and customer profitability in a business-to-business setting. Using data from a company providing hygiene services, we look at the impact of a hitherto unstudied type of expense targeted at individual customer relationships: the offering of free equipment to customers. The data allow tracking the activities performed in more than 4,500 customer relationships over a period of 4 years. Retention rates are higher for customers targeted with free equipment, but this effect results from an interaction with customer size. First-order dynamic panel data analyses show that the impact of targeted marketing expenses on customer dollar profit is positive for large customers, but there is no effect for smaller customers. Thus, targeted marketing expenses seem to be a tool for relationship maintenance rather than customer development: they help in retaining large customers that generate more profit, but they do not seem to work in developing new customers into larger, more profitable ones

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKCβ inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription–PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3β and Akt phosphorylation, which was additionally reduced by drug combination (−58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (>50% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients

Cellular pharmacology of multi- and duplex drugsconsisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2′deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK−. ETC-FdUrd was active (IC50 of 2.2 and 79 nM) in FM3A/0 and TK− cells, respectively. ETC-L-FdUrd was less active (IC50: 7 nM in FM3A/0 vs 4500 nM in FM3A/TK−). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC50:19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80–90%), but to a lower extent in FM3A/TK− (10–50%). FdUMP was hardly detected in FM3A/TK− cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation