The obtained 76 selected phosphotyrosine signature peptides were subsequently analysed in terms of follow-up treatments and outcomes recorded in the patient files. For tumour, node, metastasis (TNM) stage 1 LuAdCa patients, we noticed a larger tyrosine kinase inhibitor-induced decrease in tyrosine phosphorylation for long-term as opposed to short-term disease survivors, for which 26 of 76 selected peptides were significantly  more inhibited in the long-term survivors. Using statistical class prediction analysis, we obtained a 'prognostic-signature' for long- versus short-term disease survivors and correctly predicted the survival status of 73% of our patients. Our translational approach may assist clinical disease management after surgical resection and may help to direct patients for an optimal treatment strategy.Despite constant improvement in existing therapeutic efforts, the overall survival rate of lung cancer patients remains low. Enzyme activities may identify new therapeutically targetable biomarkers and overcome the marked lack of correlation between cellular abundance of translated proteins and corresponding mRNA expression levels. We analysed tyrosine kinase activities to classify lung adenocarcinoma (LuAdCa) resection specimens based on their underlying changes in cellular processes and pathways that are agents of or result from malignant transformation. We characterised 71 same-patient pairs of early-stage LuAdCa and non-neoplastic LuAdCa resection specimen lysates in the presence or absence of a tyrosine kinase inhibitor. We performed  Adenocarcinoma, squamous cell carcinoma and large cell carcinoma represent the main subtypes of non-small cell lung cancer (NSCLC), the leading cause of lung cancer-related deaths . At presDespite tremendous efforts, the search for such reliable lung cancer-specific molecular biomarkers for local stage NSCLC has remained unsuccessful, which may be attributed to a lack of correlation between mRNA expression levels and the cellular abundance of their corresponding protein , 10. Morin vivo, the triggering of relevant signalling pathway [In both eukaryotic and prokaryotic biological systems, protein phosphorylation is a ubiquitous and reversible post-translational modification. More than 500 protein kinase-coding motifs have been identified in humans . Ninety  pathway . Since t pathway , pharmac pathway . PTKI pr pathway  is due b pathway  and to aBy employing a same-patient set of lung resection specimens, we measured the tyrosine kinase activities in non-neoplastic and adenocarcinoma lysates, in addition to adenocarcinoma lysates containing gefitinib with a multiplex profiling approach using well-characterised tyrosine kinase substrates . The gefWe have summarized the clinical parameters of 49 TNM stage 1 and 22 TNM stage 2 LuAdCa patients enrolled in this study  or 5 long-term survivors with a median DSS of 36.9 months . The TNM stage 2 validation cohort included 6 short- and 1 long-term disease-specific survivors with a median DSS of 29.6 months . To evaluate the class prediction model for TNM stage 2, we omitted 5 patients from the validation cohort that were still alive at the time of the analysis but with a follow up time shorter than the cut-off time set at 24 months of survival .inhS” values as described in the Material and Methods section. For the training cohort, a total of 95 out of the 144 peptides available were selected and were present in at least 70% of the LuAdCa resection specimens analysed . The “inhS” values of the 95 peptides were combined with the patient survivor status in the form of a heatmap . Supervised classification performance was examined using partial least squares discriminant analysis (PLS-DA) based on the 95 peptide inhibition profiles of the LuAdCa resection specimens. The prediction accuracy was estimated using leave-one-out cross-validation (LOOCV), and we obtained an accuracy of 14/20 (70%) correctly segregated patients as short- or long-term survivors .norS” value in both supervised and unsupervised clustering analysis produced no correlation of substrate phosphorylation patterns with known clinical parameters  involved in lung cancer aetiology (data not shown). In particular, the patient survival status was not correlated with phosphorylation of the 95 selected peptides  out of the 95 peptides with significant differences between long- and short-term survivors . In addition, the supervised PLS-DA classification analysis did not discriminate between long- versus short-term survivors.In all LuAdCa protein lysates tested, we noticed a conspicuous and TNM stage-independent tyrosine phosphorylation of substrates. Lower levels of kinase substrate phosphorylation were detected for protein lysates of non-neoplastic tissues. Nonetheless, despite large differences in substrate phosphotyrosine levels, the calculated “peptides . For thenorS” ratio for LuAdCa TNM stage 2 also did not yield any useful classification model. Other clinical parameters  were not correlated with any characteristic substrate tyrosine phosphorylation patterns (data not shown).The same analysis of “We noticed a reduced effect of added gefitinib during the phosphorylation inhibition assay in recently collected malignant and non-neoplastic protein lysates. Moreover, this observation seemed to appear in parallel with the presence of a whitish background on images obtained from the top surface of the ceramic microarrays. We searched for causes explaining such an effect and ruled out the implication of either short versus long storage time after protein sample extraction or of heterogeneous batch processing of protein lysates (data not shown). Instead, we documented that the clear reduction in overall phosphorylation levels coincided with the year 2007 and the introduction of optimal cutting temperature (OCT) medium for improved embedding of resection specimens . To corrinhScor” applied to all our data, we pooled the TNM stage 1 patient training . Table p < 0.01, FDR < 3%). Among the 26 peptide substrates, members of the JAK/STAT-  the FER/FES- , and PDGFR cytoskeleton remodelling  but also EphrinA    signalliA7, LCK) .EGFR status (EGFR wild type/mutant and/or EGFR amplification), an important parameter that is used to help guide treatment decisions. As indicated in Figure EGFR or with amplified EGFR, in comparison to 70% who had the wild type form. The LuAdCa resection specimens with EGFR mutation/amplification were equally distributed between long- and short-term survivors.From the pooled cohorts with 37 patients, we also randomly assessed 13 LuAdCa resection specimens to determine the Protein phosphorylation represents an important and ubiquitous post-translational modification in eukaryotic biological systems and has a prominent role in cancer initiation and progression. The main role of kinases is to turn cellular processes “on” and “off”, and thus the kinome has been the focus of large efforts to understand cancer-modified signalling pathways . In thisUsing our approach, sets of activated versus inactivated (or gefitinib bound) protein tyrosine kinases present in the adenocarcinoma lysates were subjected to protein tyrosine kinase activity profiling on the PamChip®4 microarrays. We obtained inhibition profiles that were analysed for representative tumour signatures. Here, gefitinib was used as a tool to increase the discriminative power of the measurements by comparing activities with and without added inhibitor during the assay. The advantages of using PTKI inhibition over direct comparison with a non-neoplastic profile are both technical and biological. From a technical standpoint, quantitation and processing of the same malignant protein lysate, with or without presence of added PTKI, and from the exact same LuAdCa resection specimen tissue provides an advantage. This malignant sample can be measured either with PTKI or without PTKI as a control, thereby reducing differences in baseline signal between patients. From a biological perspective, the advantage lies in the ability to use specific inhibitors that may be efficient against a known signalling pathway important for tumour progression or treatment response. Moreover, drug targeting of a specific signalling pathway may help to better discriminate between sample phenotypes. In our study, gefitinib was selected because it is already used clinically as a treatment for distinct LuAdCa phenotypes . As appeIn TNM stage 1 LuAdCa, the tyrosine kinases responded more strongly to the inhibitory effect of gefitinib in long-term survivors compared with short-term survivors. Moreover, we used a set of 76 peptide inhibition profiles as input for PLS-DA supervised classification analysis, which resulted in correct prediction of survival status for 73% of the patients . Interestingly, we detected 26 peptide substrates that were significantly more inhibited in the protein lysates of the long- versus short-term survivors. Pathway analysis may provide hypotheses for further research and follow-up work. Among the 26 peptide substrates that significantly discriminated short- versus long-term survivors, we listed members of the EphrinA-signalling pathway  . InteresEGFR, which is similar to the prevalence observed in a randomly selected Caucasian population affected by LuAdCa [EGFR mutant or amplified status in all LuAdCa resection specimens and to analyse known frequent LuAdCa-specific mutations [A random screen of the LuAdCa resection specimens revealed that 30% of the total specimens analysed had a mutant or amplified y LuAdCa . To refiutations .inhScor” correction is unnecessary for future studies if a homogeneous type of embedding procedure is used for all the analysed samples. Clearly, the method should also be tested using a large validation set to overcome the effect of any optimistic biases in the current analysis. We were unable to perform such a new blinded analysis due to both the re-analysis of known samples after the application of the OCT corrective estimate and the scarcity of new untested LuAdCa resection specimens. A possible refinement could be achieved with an international validation study encompassing different human ethnic groups. In silico human genome analysis has described more than 500 types of DNA signatures with a kinase domain [When we applied the training model to the validation set, we identified assay interferences due to the presence of OCT in the samples embedded after 2007 , corresponding to the exact time we implemented our OCT-based embedding procedure to enhance the protection of our resection specimens . Nonethee domain . Ninety e domain  and, as e domain . The abie domain .in silico in the human genome and potentially involved in either lung cancer disease or the lung cancer drug-resistant state [et al [Nevertheless, we observed interesting and potentially clinically applicable differences in the kinomes of long- versus short-term survivors. Currently, molecularly targeted therapies based on small molecules and monoclonal antibodies directed against tyrosine kinases are already approved for lung cancer treatment. Approximately 20% of lung cancer patients can actually be stratified for treatment with EGFR targeting therapies that successfully suppress cell growth and promote cell death. New strategies are further needed to elucidate novel target signalling pathways among the numerous DNA signatures with a kinase domain described nt state . Kinase nt state . Our rese [et al  developee [et al  derived Our classifier achieved a rather high positive predictive value since a large proportion of the patients predicted to be long-term survivors were actually long-term survivors . In January 2003, the UHZ Division of Thoracic Surgery started a collection of same-patient fresh frozen LuAdCa and non-neoplastic tissues. In this study, all the mandatory informed consent approval forms with all clinical interventions and follow-up treatment decisions from every participating lung cancer patient were archived. All lung resection specimens collected until December 2006 were directly snap-frozen in liquid nitrogen and stored at −80°C. As of January 2007, this procedure was updated, and up to the censoring day of January 1, 2013, our lung resection specimens were processed in the following way: i) specimens were embedded in OCT , and the European standard for tumour banking was applied  ii) all EGFR mutation and amplification status as previously described [EGFR overexpression and gene amplification, we performed CEP7/EGFR dual colour FISH  with the Spectrum Orange probe specific for the EGFR locus (7p12) and the Spectrum green probe specific for the chromosome 7 centromere (7p11.1 to q11.1). A total of 50-100 non-overlapping tumour cell nuclei were counted. For exon 18 to 21 EGFR mutations, tissue areas from FFPE tissue were excised with a puncher and DNA was extracted and amplified by PCR. Finally, the PCR product were analysed by 2% agarose gel electrophoresis and subsequently purified and sequenced.A set of LuAdCa resection specimens were randomly tested for escribed . To asseth TNM classification of malignant lung tumours [We collected resection specimens with clinical characteristics of LuAdCa TNM stage 1 (1A and 1B) or 2 (2A or 2B) up to 12/2010 see . Startin tumours  and esti tumours . All theFinally, after thorough inspection of peptide tyrosine phosphorylation kinetics, the assay results for 4 patients were removed because two of our predefined inclusion criteria were unmet: 1) extensive image saturation as a result of either very low or highly saturated peptide tyrosine phosphorylation; 2) data points in the principal component analysis (PCA) that were clear outliers from the rest of the dataset (group 7).th edition textbook of the TNM classification stated a 5-year survival rate ranging from 73% for stage 1A to 52% for stage 1B and of 48% for stage 2A to 30% for stage 2B [The patient DSS time was computed either as the months elapsed from the day of surgery until death as a result of a relapse of their lung cancer or, for patients above our assigned cut-off values, as the total number of months elapsed until the last available follow-up. Briefly, we collected 83 TNM stage 1  and 35 TNM stage 2  LuAdCa resection specimens. We also had to allocate patients into training and validation cohorts, and we selected a 1 to 0.85 ratio for the stage 1 and a 1 to 0.7 ratio for the stage 2. We constructed both the TNM stage 1 and 2 training cohorts with the oldest resection specimens in our tumour collection. Data from the 6stage 2B .To distinguish short- versus long-term survivors and because of the incomplete 5-year follow-up time in our study population, we assigned shorter cut-off values in this study. In summary, and taking into account the high proportion of TNM B versus TNM A stages, the balancing of the short- versus long-term survivors training cohorts, the allocation in training and validation cohorts and the actual survival value, we selected 62% of our stage 1 and 45% of our stage 2 patient populations as cut-off values for survival. We then deduced the months cut-off value corresponding to the two above-mentioned survival percentages. For the 118 selected patients, the long- versus short-term survivor cut-off values were set at 53 months for stage 1 and 23.5 months for stage 2.3 of tissue) were collected from each of the LuAdCa and same-patient non-neoplastic resection specimens. Prior to protein extraction, the OCT-embedded cryosections were quickly thawed and briefly pelleted at 3000 rpm for 0.5 min in a microcentrifuge set at 4°C. The protein lysates were extracted and resuspended 30 times on ice using wide bore pipet tips in 100 μl of ice-cold Pierce mammalian extraction buffer  containing both Pierce Halt Phosphatase Inhibitor Cocktail and Pierce Halt Protease Inhibitor Cocktail EDTA free . All samples were pelleted at 3000 rpm for 0.5 min in a microcentrifuge set at 4°C, processed again 30 times using wide bore pipet tips and finally lysed for 15 mins on ice. After centrifugation at 10000 rpm for 15 mins and 4°C, each protein lysate supernatant was aliquoted into 10 tubes and quickly frozen before storage at −80°C. The protein concentration was estimated with the Pierce micro BCA kit  using bovine serum albumin (BSA) as a standard. Additionally, malignant samples were also analysed with a 10 μM final concentration of gefitinib . The 40 μl final volume of kinase master mix containing the kinase assay buffer  was prepared according to the instructions provided by the manufacturer  and assayed with the following modifications: i) we tested 5 μg of the extracted protein lysate in a 1 to 5 μl final volume of M-Per lysis buffer and, ii) we included either 2 μl of a 200 μM stock of gefitinib in DMSO or 2 μl DMSO. Finally, to synchronise the twelve kinase reactions, we added 4 μl of a 4 mM ATP stock solution to reach a 0.4 mM final ATP concentration.Two cryosections with a thickness of 30 microns and covering an area of approximately 5×5 mm  dotted with 13-15 amino acid consensus tyrosine phosphorylation peptide sequences that were identified in both the SwissProt and PhosphoBase databases [Multiplexed atabases . The lisDuplicate multiplex kinomic profiles of same-patient LuAdCa or non-neoplastic resection specimens were obtained. The software package BioNavigatoR  was used for inspection of the recorded images for quality control investigations and for quantitation of the tyrosine phosphorylation signals. This process included standard procedures for the automatic edge detection of microarrayed spots. For each spot, the tyrosine phosphorylation signal was used as the median spot pixel value minus the median local background pixel value. Measurements were carefully inspected and repeated for one or both replicates in the case of evident mechanical or technical problems during data acquisition, and for replicates with measurement inconsistencies. For further analysis, the signal obtained after a final washing step at the end of the incubation was used. This signal was recorded using 3 different CCD camera exposure times. A standard procedure was applied to combine these readings into a single signal value with a maximum dynamic range. Only peptides with clear growth of the tyrosine phosphorylation signal during the assay for at least 70% of non-neoplastic and malignant samples (without gefitinib) were selected for further analysis.norS” between same-patient non-neoplastic and malignant resection specimens was calculated as the Log2 ratio of the signal obtained for the malignant (Sm) or non-neoplastic (Snn) resection specimens: norS = Log2 (Sm/Snn)The ratio of peptide phosphorylation “inhS” was calculated as the Log2 ratio of the peptide phosphorylation signal obtained with (Si) or without (Sm) addition of gefitinib in malignant resection specimen lysates: inhS = Log2 (Si/Sm)The inhibition value “First, a Log2 ratio per run was calculated, and duplicated ratios were subsequently averaged. We performed a comparative analysis of ratios from between two groups of samples , which included heatmap visualisation of the data and per-peptide two-sample two-tailed Student’s t-tests to identify peptide that were significantly different between the groups.Unsupervised multivariate clustering of samples was inspected in low-dimensional score plots of the measurements obtained by PCA see . ClassifnorS” and “inhS” values. We designed and applied a correction in which median centring was performed on the “inhS” values of each peptide, separately for the samples with or without OCT. We obtained corrected values named “norScor” and “inhScor”. Statistical analyses were performed using BioNavigatoR  interfaced with R  and MAT


Ice adhesion is mainly dictated by surface properties, and water wettability is frequently correlated with ice adhesion strength. However, these established correlations are limited to high ice adhesion and become invalid when the ice adhesion strength is low. Here we carried out an experimental study to explore the relationships between low ice adhesion strength and room temperature surface properties. A variety of room temperature properties of 22 polymer-based hydrophilic and hydrophobic samples consisting of both low and high ice adhesion surfaces were analysed. The properties investigated include water adhesion force, water wettability, roughness, elastic modulus and hardness. Our results show that low ice adhesion strength does not correlate well with water contact angle and its variants, surface roughness and hardness. Low elastic modulus does not guarantee low ice adhesion, however, surfaces with low ice adhesion always show low elastic modulus. Low ice adhesion (below 60 kPa) of tested surfaces may be determinative of small water adhesion force (from 180 to 270 μN). Therefore, measurement of water adhesion force may provide an effective strategy for screening anti-icing or icephobic surfaces, and surfaces within specific values of water adhesion force will possibly lead to a low ice adhesion. A surface with a higher water contact angle (WCA) is considered to have a lower ice adhesion strength because of the low surface energy for both water and iceθe is the equilibrium contact angle, θrec is the receding contact angle, and γLV is the liquid-vapor interfacial tension. Meuler et al. showed a strong linear relationship between the value of [1 + cos θrec] and ice adhesion strength (larger than 160 kPa) for both hydrophilic and hydrophobic surfaces3536where et al.et al.2542et al. and Farhadi et al. demonstrated that a decrease in roughness led to an increase in ice adhesion strength44Roughness is also associated with ice adhesion strength. It was reported that reduced roughness tends to reduce ice adhesion630353839406Water adhesion force is a surface characteristic not yet explored which might demonstrate a correlating factor to ice adhesion strength. Water adhesion force is measured from the detaching process of a water droplet from the substrate which is similar to the ice detaching process. Water adhesion reflects both the surface chemical composition and the contact area46Although low ice adhesion surfaces play a significant role in various industries, high development cost and the difficulty of low temperature evaluation of these surfaces hinder their development. Effective correlations between low ice adhesion strength and room temperature surface characteristics would be extremely valuable. Herein, we examine relationships between low ice adhesion strength and various room temperature properties for 22 polymer-based hydrophilic and hydrophobic coatings, such as water adhesion force, roughness, elastic modulus, hardness, and water contact angle as well as its variants. Our results show that low ice adhesion of tested surfaces may be determinative of small water adhesion force (from 180 to 270 μN), which may provide an effective strategy for screening anti-icing or icephobic surfaces by evaluation of water adhesion force at room temperature.492θe), and the θadv and θrec, by using the following equation252In order to examine the relationship between ice adhesion strength and water wettability, the ice adhesion strength is plotted against two values of WCA and two values of CAH as shown in θadv  and 2 = 0.90) when the ice adhesion strength is larger than 60 kPa. This result indicates that ice adhesion strength decreases as the WCA increases, which is in agreement with previous studies1724415455θadv − θrec] and [cos θrec − cos θadv] is used to correlate with ice adhesion strength. For the coatings with high ice adhesion strength, R2 for [θadv − θrec] and [cos θre − cos θadv] are 0.90 and 0.74. These results show that [θadv − θrec] correlates better with ice adhesion strength than [cos θrec − cos θadv], similar to previous results302 for [θadv − θrec] and [cos θrec − cos θadv] are 0.64 and 0.59 as shown in the inserted sub-figures of θadv − θrec] and [cos θrec − cos θadv] show a weak linear relationship with low ice adhesion strength. The above results indicate that the linear trends for high and low ice adhesion strength are different.θrec) for all samples tested including the bare Al and steel. The correlations with [1 + cos 2 = 0.88) and [1 + cos θrec] (R2 = 0.86) are similar for samples with high ice adhesion strength. Furthermore, low ice adhesion strengths of the 17 polymer-based coatings do not correlate well with [1 + cos θrec] , ice adhesion strength even varies from 7.7 kPa to 282 kPa for different samples. Low ice adhesion strength is shown to be independent of the values of [1 + cos θrec] , and does not correlate well with other room temperature characteristics. Similar to the ice detachment tests, water adhesion forces may be able to characterize the potential surface deformation process. Water adhesion force could be therefore used to estimate low ice adhesion at room temperature, which greatly facilitates the design and fabrication of low-cost anti-icing or icephobic surfaces.−1 was used for the measurements of contact angles and water adhesion forces.Five hydrophobic  and seventeen hydrophilic  polymer based coatings, aluminum (S23), and steel (S24) were received from four commercial companies as described in two Master Theses49θadv) and receding (θrec) angles were measured as water was supplied through a syringe into or out of sessile droplets (~5 μL) and repeated 5 times. The water adhesion forces were investigated by Dynamic Contact Angle Tensiometer (dataphysics DCAT11®) at room temperature and each substrate was tested and repeated 10 times58® 950, Hysitron, Inc.) and repeated 9 times.The water contact angles of different samples were measured by using a CAM 200 contact-angle system at room temperature. Advancing  to minimize torque on the ice sample. Moreover, a schematic illustration of the working principle for ice detachments, the details of ice formation molds and the design of the sample holder are provided in the The ice adhesion test was modified from a laboratory ice adhesion testHow to cite this article: He, Z. et al. Room Temperature Characteristics of Polymer-Based Low Ice Adhesion Surfaces. Sci. Rep.7, 42181; doi: 10.1038/srep42181 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.