Specific members of the TOPLESS family are susceptibility genes for Fusarium wilt in tomato and Arabidopsis.

Vascular wilt diseases caused by Fusarium oxysporum are a major threat to many agriculturally important crops. Genetic resistance is rare and inevitably overcome by the emergence of new races. To identify potentially durable and non-race-specific genetic resistance against Fusarium wilt diseases, we set out to identify effector targets in tomato that mediate susceptibility to the fungus. For this purpose, we used the SIX8 effector protein, an important and conserved virulence factor present in many pathogenic F. oxysporum isolates. Using protein pull-downs and yeast two-hybrid assays, SIX8 was found to interact specifically with two members of the tomato TOPLESS family: TPL1 and TPL2. Loss-of-function mutations in TPL1 strongly reduced disease susceptibility to Fusarium wilt and a tpl1;tpl2 double mutant exerted an even higher level of resistance. Similarly, Arabidopsis tpl;tpr1 mutants became significantly less diseased upon F. oxysporum inoculation as compared to wildtype plants. We conclude that TPLs encode susceptibility genes whose mutation can confer resistance to F. oxysporum.

Discovery of Three Bemisia tabaci Effectors and Their Effect on Gene Expression in Planta.

Bemisia tabaci (whitefly) is a polyphagous agroeconomic pest species complex. Two members of this species complex, Mediterranean (MED) and Middle-East-Asia Minor 1 (MEAM1), have a worldwide distribution and have been shown to manipulate plant defenses through effectors. In this study, we used three different strategies to identify three MEAM1 proteins that can act as effectors. Effector B1 was identified using a bioinformatics-driven effector-mining strategy, whereas effectors S1 and P1 were identified in the saliva of whiteflies collected from artificial diet and in phloem exudate of tomato on which nymphs were feeding, respectively. These three effectors were B. tabaci specific and able to increase whitefly fecundity when transiently expressed in tobacco plants (Nicotiana tabacum). Moreover, they reduced growth of Pseudomonas syringae pv. tabaci in Nicotiana benthamiana. All three effectors changed gene expression in planta, and B1 and S1 also changed phytohormone levels. Gene ontology and KEGG pathway enrichment analysis pinpointed plant-pathogen interaction and photosynthesis as the main enriched pathways for all three effectors. Our data thus show the discovery and validation of three new B. tabaci MEAM1 effectors that increase whitefly fecundity and modulate plant immunity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A salivary GMC oxidoreductase of Manduca sexta re-arranges the green leaf volatile profile of its host plant.

Green leaf volatiles (GLVs) are short-chain oxylipins that are emitted from plants in response to stress. Previous studies have shown that oral secretions (OS) of the tobacco hornworm Manduca sexta, introduced into plant wounds during feeding, catalyze the re-arrangement of GLVs from Z-3- to E-2-isomers. This change in the volatile signal however is bittersweet for the insect as it can be used by their natural enemies, as a prey location cue. Here we show that (3Z):(2E)-hexenal isomerase (Hi-1) in M. sexta's OS catalyzes the conversion of the GLV Z-3-hexenal to E-2-hexenal. Hi-1 mutants that were raised on a GLV-free diet showed developmental disorders, indicating that Hi-1 also metabolizes other substrates important for the insect's development. Phylogenetic analysis placed Hi-1 within the GMCß-subfamily and showed that Hi-1 homologs from other lepidopterans could catalyze similar reactions. Our results indicate that Hi-1 not only modulates the plant's GLV-bouquet but also functions in insect development.

Advancing wide implementation of precision oncology: A liquid nitrogen-free snap freezer preserves molecular profiles of biological samples.

PURPOSE: In precision oncology, tumor molecular profiles guide selection of therapy. Standardized snap freezing of tissue biospecimens is necessary to ensure reproducible, high-quality samples that preserve tumor biology for adequate molecular profiling. Quenching in liquid nitrogen (LN2 ) is the golden standard method, but LN2 has several limitations. We developed a LN2 -independent snap freezer with adjustable cold sink temperature. To benchmark this device against the golden standard, we compared molecular profiles of biospecimens. METHODS: Cancer cell lines and core needle normal tissue biopsies from five patients' liver resection specimens were used to compare mass spectrometry (MS)-based global phosphoproteomic and RNA sequencing profiles and RNA integrity obtained by both freezing methods. RESULTS: Unsupervised cluster analysis of phosphoproteomic and transcriptomic profiles of snap freezer versus LN2 -frozen K562 samples and liver biopsies showed no separation based on freezing method (with Pearson's r 0.96 (range 0.92-0.98) and >0.99 for K562 profiles, respectively), while samples with +2 h bench-time formed a separate cluster. RNA integrity was also similar for both snap freezing methods. Molecular profiles of liver biopsies were clearly identified per individual patient regardless of the applied freezing method. Two to 25 s freezing time variations did not induce profiling differences in HCT116 samples. CONCLUSION: The novel snap freezer preserves high-quality biospecimen and allows identification of individual patients' molecular profiles, while overcoming important limitations of the use of LN2 . This snap freezer may provide a useful tool in clinical cancer research and practice, enabling a wider implementation of (multi-)omics analyses for precision oncology.

High-Dose Intermittent Treatment with the Multikinase Inhibitor Sunitinib Leads to High Intra-Tumor Drug Exposure in Patients with Advanced Solid Tumors.

Patients with advanced cancer refractory to standard treatment were treated with sunitinib at a dose of 300 mg once every week (Q1W) or 700 mg once every two weeks (Q2W). Tumor, skin and plasma concentrations were measured and immunohistochemical staining for tumor cell proliferation (TCP), microvessel density (MVD) and T-cell infiltration was performed on tumor biopsies before and after 17 days of treatment. Oral administration of 300 mg sunitinib Q1W or 700 mg Q2W resulted in 19-fold (range 5-35×) and 37-fold higher (range 10-88×) tumor drug concentrations compared to parallel maximum plasma drug concentrations, respectively. Patients with higher tumor sunitinib concentrations had favorable progression-free and overall survival than those with lower concentrations (p = 0.046 and 0.024, respectively). In addition, immunohistochemistry of tumor biopsies revealed an induction of T-cell infiltration upon treatment. These findings provide pharmacological and biological insights in the clinical benefit from high-dose intermittent sunitinib treatment. It emphasizes the potential benefit from reaching higher tumor drug concentrations and the value of measuring TKI tumor- over plasma-concentrations. The finding that reaching higher tumor drug concentrations provides most clinical benefit in patients with treatment refractory malignancies indicates that the inhibitory potency of sunitinib may be enforced by a high-dose intermittent treatment schedule. These results provide proof of concept for testing other clinically available multitargeted tyrosine kinase inhibitors in a high-dose intermittent treatment schedule.

Changes in the Spore Proteome of Bacillus cereus in Response to Introduction of Plasmids.

Fluorescent fusion proteins were expressed in Bacillus cereus to visualize the germinosome by introducing a plasmid that carries fluorescent fusion proteins of germinant receptor GerR subunits or germinosome scaffold protein GerD. The effects of plasmid insertion and recombinant protein expression on the spore proteome were investigated. Proteomic analysis showed that overexpression of the target proteins had negligible effects on the spore proteome. However, plasmid-bearing spores displayed dramatic abundance changes in spore proteins involved in signaling and metabolism. Our findings indicate that the introduction of a plasmid alone alters the spore protein composition dramatically, with 993 proteins significantly down-regulated and 415 proteins significantly up-regulated among 3323 identified proteins. This shows that empty vector controls are more appropriate to compare proteome changes due to plasmid-encoded genes than is the wild-type strain, when using plasmid-based genetic tools. Therefore, researchers should keep in mind that molecular cloning techniques can alter more than their intended targets in a biological system, and interpret results with this in mind.

Between Protest and Counter-Expertise: User Knowledge, Activism, and the Making of Urban Cycling Networks in the Netherlands Since the 1970s.

Around 1970, high numbers of traffic casualties among cyclists led to the creation of numerous local protest movements in the Netherlands. While activists employed protest strategies, their main interest lie in the way they exemplify a highly successful instance of "lay expertise"; the idea that users of a technology have a fundamentally different and valuable perspective on a technology than experts or system-builders. Specifically, cyclists claimed to be more knowledgeable about cycling conditions and safety than the state-employed engineers and traffic experts who built the roads and cycling path network. A key actor in this story is the Dutch Cyclists' Union (Fietsersbond), a national platform of local action groups formed in 1975. These activists used the cycling experience of everyday utilitarian cyclists to compile maps and blacklists of locations where cycling was dangerous, unpleasant, uncomfortable, or otherwise discouraging. In doing so, they successfully claimed legitimacy as a valuable knowledge partner for local engineers and policymakers. As a result, they gained some level of influence within local governments, a relation which in the intervening years has only grown stronger. This case study shows how users can shape socio-technical systems bottom-up, and can therefore to an extent be seen as a successful example of co-construction of technology.

Tumor Drug Concentration and Phosphoproteomic Profiles After Two Weeks of Treatment With Sunitinib in Patients with Newly Diagnosed Glioblastoma.

PURPOSE: Tyrosine kinase inhibitors (TKI) have poor efficacy in patients with glioblastoma (GBM). Here, we studied whether this is predominantly due to restricted blood-brain barrier penetration or more to biological characteristics of GBM. PATIENTS AND METHODS: Tumor drug concentrations of the TKI sunitinib after 2 weeks of preoperative treatment was determined in 5 patients with GBM and compared with its in vitro inhibitory concentration (IC50) in GBM cell lines. In addition, phosphotyrosine (pTyr)-directed mass spectrometry (MS)-based proteomics was performed to evaluate sunitinib-treated versus control GBM tumors. RESULTS: The median tumor sunitinib concentration of 1.9 µmol/L (range 1.0-3.4) was 10-fold higher than in concurrent plasma, but three times lower than sunitinib IC50s in GBM cell lines (median 5.4 µmol/L, 3.0-8.5; P = 0.01). pTyr-phosphoproteomic profiles of tumor samples from 4 sunitinib-treated versus 7 control patients revealed 108 significantly up- and 23 downregulated (P < 0.05) phosphopeptides for sunitinib treatment, resulting in an EGFR-centered signaling network. Outlier analysis of kinase activities as a potential strategy to identify drug targets in individual tumors identified nine kinases, including MAPK10 and INSR/IGF1R. CONCLUSIONS: Achieved tumor sunitinib concentrations in patients with GBM are higher than in plasma, but lower than reported for other tumor types and insufficient to significantly inhibit tumor cell growth in vitro. Therefore, alternative TKI dosing to increase intratumoral sunitinib concentrations might improve clinical benefit for patients with GBM. In parallel, a complex profile of kinase activity in GBM was found, supporting the potential of (phospho)proteomic analysis for the identification of targets for (combination) treatment.

Characterization of Awp14, A Novel Cluster III Adhesin Identified in a High Biofilm-Forming Candida glabrata Isolate.

Candida glabrata is among the most prevalent causes of candidiasis. Unlike Candida albicans, it is not capable of changing morphology between yeast and hyphal forms but instead has developed other virulence factors. An important feature is its unprecedented large repertoire of predicted cell wall adhesins, which are thought to enable adherence to a variety of surfaces under different conditions. Here, we analyzed the wall proteome of PEU1221, a high biofilm-forming clinical strain isolated from an infected central venous catheter, under biofilm-forming conditions. This isolate shows increased incorporation of putative adhesins, including eight proteins that were not detected in walls of reference strain ATCC 2001, and of which Epa22, Awp14, and Awp2e were identified for the first time. The proteomics data suggest that cluster III adhesin Awp14 is relatively abundant in PEU1221. Phenotypic studies with awp14Δ deletion mutants showed that Awp14 is not responsible for the high biofilm formation of PEU1221 onto polystyrene. However, awp14Δ mutant cells in PEU1221 background showed a slightly diminished binding to chitin and seemed to sediment slightly slower than the parental strain suggesting implication in fungal cell-cell interactions. By structural modeling, we further demonstrate similarity between the ligand-binding domains of cluster III adhesin Awp14 and those of cluster V and VI adhesins. In conclusion, our work confirms the increased incorporation of putative adhesins, such as Awp14, in high biofilm-forming isolates, and contributes to decipher the precise role of these proteins in the establishment of C. glabrata infections.

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus.

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

High Resolution Analysis of Proteome Dynamics during Bacillus subtilis Sporulation.

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins, and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (termed blue, brown, green, and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue is negatively correlated with modules brown and green, containing ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green, and might also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation.

Effects of Cancer Presence and Therapy on the Platelet Proteome.

Platelets are involved in tumor angiogenesis and cancer progression. Previous studies indicated that cancer could affect platelet content. In the current study, we investigated whether cancer-associated proteins can be discerned in the platelets of cancer patients, and whether antitumor treatment may affect the platelet proteome. Platelets were isolated from nine patients with different cancer types and ten healthy volunteers. From three patients, platelets were isolated before and after the start of antitumor treatment. Mass spectrometry-based proteomics of gel-fractionated platelet proteins were used to compare patients versus controls and before and after treatment initiation. A total of 4059 proteins were detected, of which 50 were significantly more abundant in patients, and 36 more in healthy volunteers. Eight of these proteins overlapped with our previous cancer platelet proteomics study. From these data, we selected potential biomarkers of cancer including six upregulated proteins (RNF213, CTSG, PGLYRP1, RPL8, S100A8, S100A9) and two downregulated proteins (GPX1, TNS1). Antitumor treatment resulted in increased levels of 432 proteins and decreased levels of 189 proteins. In conclusion, the platelet proteome may be affected in cancer patients and platelets are a potential source of cancer biomarkers. In addition, we found in a small group of patients that anticancer treatment significantly changes the platelet proteome.

An updated checklist of marine gastropods of the Persian Gulf and Gulf of Oman.

Here we review and evaluate the marine gastropods of the Persian Gulf and Gulf of Oman based on published accounts. A total of 850 species belonging to 129 families have records in the Persian Gulf (585) and Gulf of Oman (648), of which 383 species occurred in both regions. We updated the taxonomy and deleted records with dubious identifications. The resultant checklist documents the currently known diversity of marine gastropods from the Persian Gulf and Gulf of Oman and provides a foundation for future studies of the biodiversity of these areas.

High Biofilm Formation of Non-Smooth Candida parapsilosis Correlates with Increased Incorporation of GPI-Modified Wall Adhesins.

Candida parapsilosis is among the most frequent causes of candidiasis. Clinical isolates of this species show large variations in colony morphotype, ranging from round and smooth to a variety of non-smooth irregular colony shapes. A non-smooth appearance is related to increased formation of pseudohyphae, higher capacity to form biofilms on abiotic surfaces, and invading agar. Here, we present a comprehensive study of the cell wall proteome of C. parapsilosis reference strain CDC317 and seven clinical isolates under planktonic and sessile conditions. This analysis resulted in the identification of 40 wall proteins, most of them homologs of known Candida albicans cell wall proteins, such as Gas, Crh, Bgl2, Cht2, Ecm33, Sap, Sod, Plb, Pir, Pga30, Pga59, and adhesin family members. Comparative analysis of exponentially growing and stationary phase planktonic cultures of CDC317 at 30 °C and 37 °C revealed only minor variations. However, comparison of smooth isolates to non-smooth isolates with high biofilm formation capacity showed an increase in abundance and diversity of putative wall adhesins from Als, Iff/Hyr, and Hwp families in the latter. This difference depended more strongly on strain phenotype than on the growth conditions, as it was observed in planktonic as well as biofilm cells. Thus, in the set of isolates analyzed, the high biofilm formation capacity of non-smooth C. parapsilosis isolates with elongated cellular phenotypes correlates with the increased surface expression of putative wall adhesins in accordance with their proposed cellular function.

Time dependent effect of cold ischemia on the phosphoproteome and protein kinase activity in fresh-frozen colorectal cancer tissue obtained from patients.

BACKGROUND: Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. METHODS: Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. RESULTS: Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p < 0.01 and fold change > 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p < 0.01). Only two phosphopeptides were significantly changed in all time points, including one peptide (PKP3) with a fold change > 2. CONCLUSIONS: The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

A specific microRNA profile as predictive biomarker for systemic treatment in patients with metastatic colorectal cancer.

BACKGROUND: Palliative systemic therapy is currently standard of care for patients with extensive metastatic colorectal cancer (mCRC). A biomarker predicting chemotherapy benefit which prevents toxicity from ineffective treatment is urgently needed. Therefore, a previously developed tissue-derived microRNA profile to predict clinical benefit from chemotherapy was evaluated in tissue biopsies and serum from patients with mCRC. METHODS: Samples were prospectively collected from patients (N = 132) who were treated with capecitabine or 5-FU/LV with oxaliplatin ± bevacizumab. Response evaluation was performed according to RECIST 1.1 after three or four cycles, respectively. Baseline tissue and serum miRNAs expression levels of miR-17-5p, miR-20a-5p, miR-30a-5p, miR-92a-3p, miR-92b-3p, and miR-98-5p were quantified with RT-qPCR and droplet digital PCR, respectively. Combined predictive performance of selected variables was tested using logistic regression analysis. RESULTS: From 132 patients, 81 fresh frozen tissue biopsies from metastases and 93 serum samples were available. Based on expression levels of miRNAs in tissue, progressive disease could be predicted with an AUC of 0.85 (95% CI:0.72-0.91) and response could be predicted with an AUC of 0.70 (95% CI:0.56-0.80). This did not outperform clinical parameters alone (respectively P = .14 and P = .27). Expression levels of miR-92a-3p and miR-98-5p in serum significantly improved the predictive value of clinical parameters for response to chemotherapy (AUC 0.74, 95% CI:0.64-0.84, P = .003) in this cohort. CONCLUSIONS: The additive predictive value to clinical parameters of the tissue-derived six miRNA profile for clinical benefit could not be validated in patients with mCRC treated with first-line systemic therapy. Although miR-92a-3p and miR-98-5p serum levels improved the predictive value of clinical parameters, it remained insufficient for clinical decision-making.

Kinase Inhibitor Treatment of Patients with Advanced Cancer Results in High Tumor Drug Concentrations and in Specific Alterations of the Tumor Phosphoproteome.

Identification of predictive biomarkers for targeted therapies requires information on drug exposure at the target site as well as its effect on the signaling context of a tumor. To obtain more insight in the clinical mechanism of action of protein kinase inhibitors (PKIs), we studied tumor drug concentrations of protein kinase inhibitors (PKIs) and their effect on the tyrosine-(pTyr)-phosphoproteome in patients with advanced cancer. Tumor biopsies were obtained from 31 patients with advanced cancer before and after 2 weeks of treatment with sorafenib (SOR), erlotinib (ERL), dasatinib (DAS), vemurafenib (VEM), sunitinib (SUN) or everolimus (EVE). Tumor concentrations were determined by LC-MS/MS. pTyr-phosphoproteomics was performed by pTyr-immunoprecipitation followed by LC-MS/MS. Median tumor concentrations were 2-10 µM for SOR, ERL, DAS, SUN, EVE and >1 mM for VEM. These were 2-178 × higher than median plasma concentrations. Unsupervised hierarchical clustering of pTyr-phosphopeptide intensities revealed patient-specific clustering of pre- and on-treatment profiles. Drug-specific alterations of peptide phosphorylation was demonstrated by marginal overlap of robustly up- and downregulated phosphopeptides. These findings demonstrate that tumor drug concentrations are higher than anticipated and result in drug specific alterations of the phosphoproteome. Further development of phosphoproteomics-based personalized medicine is warranted.

Imidazo[2,1-b] [1,3,4]thiadiazoles with antiproliferative activity against primary and gemcitabine-resistant pancreatic cancer cells.

A new series of eighteen imidazo [2,1-b] [1,3,4]thiadiazole derivatives was efficiently synthesized and screened for antiproliferative activity against the National Cancer Institute (NCI-60) cell lines panel. Two out of eighteen derivatives, compounds 12a and 12h, showed remarkably cytotoxic activity with the half maximal inhibitory concentration values (IC50) ranging from 0.23 to 11.4 µM, and 0.29-12.2 µM, respectively. However, two additional compounds, 12b and 13g, displayed remarkable in vitro antiproliferative activity against pancreatic ductal adenocarcinoma (PDAC) cell lines, including immortalized (SUIT-2, Capan-1, Panc-1), primary (PDAC-3) and gemcitabine-resistant (Panc-1R), eliciting IC50 values ranging from micromolar to sub-micromolar level, associated with significant reduction of cell-migration and spheroid shrinkage. These remarkable results might be explained by modulation of key regulators of epithelial-to-mesenchymal transition (EMT), including E-cadherin and vimentin, and inhibition of metalloproteinase-2/-9. High-throughput arrays revealed a significant inhibition of the phosphorylation of 45 tyrosine kinases substrates, whose visualization on Cytoscape highlighted PTK2/FAK as an important hub. Inhibition of phosphorylation of PTK2/FAK was validated as one of the possible mechanisms of action, using a specific ELISA. In conclusion, novel imidazothiadiazoles show potent antiproliferative activity, mediated by modulation of EMT and PTK2/FAK.

Crizotinib sensitizes the erlotinib resistant HCC827GR5 cell line by influencing lysosomal function.

In non-small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR-inhibitor erlotinib and the cMET-inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live-cell microscopy. We observed additive effects in EBC-1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR-inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live-cell microscopy with a pH-sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib-erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions.

SnRK2 Protein Kinases and mRNA Decapping Machinery Control Root Development and Response to Salt.

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis (Arabidopsis thaliana) SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the posttranscriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser-645 and Ser-1156 of VCS, whereas SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser-692 and Ser-680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5, and SnRK2.9 controlling root growth under nonstress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.