Two central dimensions in psychotherapeutic work are a therapist’s empathy with clients and challenging their judgments. We investigated how they influence psychophysiological responses in the participants. Data were from psychodynamic therapy sessions, 24 sessions from 5 dyads, from which 694 therapist’s interventions were coded. Heart rate and electrodermal activity (EDA) of the participants were used to index emotional arousal. Facial muscle activity (electromyography) was used to index positive and negative emotional facial expressions. Electrophysiological data were analyzed in two time frames: (a) during the therapists’ interventions and (b) across the whole psychotherapy session. Both empathy and challenge had an effect on psychophysiological responses in the participants. Therapists’ empathy decreased clients’ and increased their own EDA across the session. Therapists’ challenge increased their own EDA in response to the interventions, but not across the sessions. Clients, on the other hand, did not respond to challenges during interventions, but challenges tended to increase EDA across a session. Furthermore, there was an interaction effect between empathy and challenge. Heart rate decreased and positive facial expressions increased in sessions where empathy and challenge were coupled, i.e., the amount of both empathy and challenge was either high or low. This suggests that these two variables work together. The results highlight the therapeutic functions and interrelation of empathy and challenge, and in line with the dyadic system theory by  Psychotherapy is done through social interaction. While the form and content of therapists’ responses to their clients’ experience may vary greatly, among other possible distinctions, psychotherapist’s attitudes toward the client can be divided to two basic orientations or facets: empathy and challenge . To empaWe presume that the therapist’s empathetic and challenging behaviors are linked to the mental and bodily emotion systems of the participants. This paper will focus on that linkage. We ask how empathy and challenge in the therapists’ interventions connect to emotion-related physiological responses  in the participants during these interventions, and more globally during the therapy sessions.The dyadic systems theory by The structure of the paper is as follows: we will next discuss earlier research on psychophysiology and social interaction in psychotherapy, present our micro-analytical approach [conversation analysis (CA)], and hypotheses. In the method section we discuss the data collection, interaction variables, and the method of the analysis. We will then present our results from two time frames: (1) across the sessions and (2) during interventions. Finally, we will discuss the results with regard to earlier research.valence and arousal . Some take a different direction, focusing on shorter events within larger encounters: participants’ psychophysiological responses during interventions , during As whole, the picture provided by earlier research is not quite unified. For example, empathy has been variably connected to an increase and decrease in arousal, and in a divergence in arousal in the participants. In a recent systematic review of the literature, In the current study, we will investigate the interplay of empathy, challenge, and psychophysiological responses in therapists and clients, leaving aside the complex questions of synchrony. We draw our hypotheses from earlier research that suggests that empathy and challenge connect to dyadic internal regulation of emotion. More specifically, as in Besides autonomic arousal, we will analyze facial muscle activation (EMG) associated with the valence of emotional expression (smile and frown). Earlier experimental research using EMG has linked increase in frown activation to empathy  and mentIn his recent review,  suggesteOur micro-analytical approach on psychotherapy interaction is based on CA. CA is a social scientific and linguistic method for the study of the practices and processes of social interaction see . It giveformulations  during the interventions, and (2) during the whole therapy sessions(1)Empathy, viewed from the formulations by observers, decreases the clients’ and increases the therapists’ physiological arousal (as indexed by EDA and heart rate).(2)Empathy increases positive emotional facial expressions (as indexed by orbicularis oculi muscle activity) and decreases negative emotional expressions (as indexed by corrugator supercilii muscle activity) in both participants.(3)Challenge, viewed from the formulations by CA coders, increases physiological arousal (EDA and heart rate) in both participants.(4)Challenge decreases positive emotional expressions (orbicularis oculi muscle activity) and increases negative emotional expressions (corrugator supercilii muscle activity) in both participants.The data were recorded from psychodynamic psychotherapy sessions. The participants were five dyads. The therapists were experienced private psychotherapists in their fifties and sixties with advanced level psychotherapy training  and long experience in psychodynamic psychotherapy. They all had post-graduate degrees (from medicine or psychology) alongside their psychotherapy training. The participants were sitting during the sessions, the frequency of which was once or twice a week. Two clients suffered primarily from depression, one from anxiety disorder, one from eating disorder and one from obsessive-compulsive disorder. Four of the clients were in their twenties, one in her sixties. The data include one dyad of a male therapist and a male client, two dyads of a female therapist and a female client and two dyads of a male therapist and a female client.Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. They reviewed oral and written information about the research, and they were given the opportunity to cancel the consent for any reason. When the data was collected, there was no relevant ethics committee in Finland for this study, because the study was done at a social science department and did not involve patients in public health care . However, another study  that was part of the same project used identical procedure of data collection, data management and publication practices. This study involved patients in public health care, and was thus under the auspices of the Ethical Board of Helsinki University Hospital, which gave its permission to the study on 21.09.2011. From each dyad six subsequent sessions were recorded. The sessions were from different phases of the therapies: one from the early phase, three from the middle and one in the ending phase of the therapy. From these 30 sessions, six sessions were used in a preliminary data-driven analysis of the interaction, building of coding schemes, and in the training of coders. This training set was excluded from the final coding and analysis of the data. For building the coding scheme, we selected first two sessions from one of the dyads and then the first session from the other dyads. The rest of the sessions (24) were used in the actual coding and analysis.orbicularis oculi and corrugator supercilii muscle locations on the left side of the face. Physiological data were synchronized with the audiovisual recording using digital markers from the computer running the video collection.Data were recorded with two Nexus-10 amplifiers . EDA signal was recorded at 32 Hz with two electrodes affixed to middle phalanges of the non-dominant hand. ECG signal was recorded at 512 Hz using a modified Lead II electrode placement. Facial EMG data were recorded at 2048 Hz with bipolar electrodes placed at Data preprocessing was conducted with Matlab 8.5. The EDA data were smoothed with a low-pass filter at 1 Hz. ECG signal was filtered and R-peaks were detected using the ECGLAB  toolbox.Conversation analysis is mostly a qualitative research method, but it can be applied also in quantitative research, as in the current study. The method is context-sensitive in terms of the actual interaction, but independent from any theories of psychotherapy. The basic principle in CA is to observe the relation between adjacent turns at talk; the social actions are not identified as separated from their context, but in relation to what has been said before, most importantly in the just preceding turn. This was done in this study too. This kind of context-sensitivity to the previous utterance sets challenges to the reliability of coding the interaction, but on the other hand it leaves freedom for the coders to interpret the situation from the perspective of the participants see . We findApplying CA, we coded all interventions in which the therapists communicate to the client what they have heard the client said in his/her previous talk. The formulations can have declarative or interrogative forms but their primary function is not to elicit information but to offer a rephrased version of the client’s talk. Coders of the data (Voutilainen and Kahri) are conversation analysts with 5–10 years’ experience of the method. The coders did not have clinical background, and the coding scheme was independent from clinical reasoning.Challenge was coded from all formulations. We assessed whether a formulation is challenging or not. On the basis of qualitative analysis of the data, we built a coding scheme that identified three basic ways in which the psychodynamic therapists challenge the clients’ preceding talk: (1) therapist can formulate the problematic emotion as more intense than expressed by the client, (2) therapist can shift the focus from an external referent to an internal referent (experience) of the client, or (3) therapist can formulate the content of the clients’ experience as different than the client had described it as. If the therapist’s formulation performed one or more of these it was coded as challenging. The challenge can be very discreet and overlap with empathetic understanding of the client. Obviously, interpretation by the coders was needed. This interpretation, however, was based on CA , not clinical reasoning. Coders (Voutilainen and Kahri) were the same as in coding of formulations. First the coders coded sessions of the training set  and discussed cases of disagreement before coding independently the rest of the sessions.From the 24 coded sessions, 694 formulations were coded. Of the formulations, 455 were coded as non-challenging (“benign”) and 239 as challenging. Ten of the 24 sessions were double coded. Unlike the identification of formulations and challenge, the measurement of empathy was not based on CA coding but on the judgment of naïve raters. We considered rating, rather than coding, as apt for measuring empathy, because of the multimodality of empathetic displays e.g.,  and undeFor the analysis of physiological responses to the whole session (tonic responses), we calculated the mean skin conductance level (SCL), heart rate [beats per minute (bpm)], and root mean square (RMS) value of EMG activity for each session. In addition, we quantified changes in arousal (as indexed by SCL and heart rate) across the session. The EDA signal was downsampled (by factor of 8) to 4 Hz and cardiac  signal was interpolated to form a 4-Hz uniformly sampled time series and converted to heart rate (bpm). Coefficient of the least squares regression line  during session, multiplied by time and thus resulting in difference between the start and end point values in original units, was used as an index of change across a session. Logarithmic transformations were conducted for the data to normalize the distributions where appropriate. The interactional variables were the ratio of challenging formulations for a session and mean empathy of the formulations during a session.When analyzing physiological responses to interventions (phasic responses), as the length of the interventions differed, we focused on the last part of an intervention where the content of the intervention is apparent and the client begins to respond to the possible challenge. We calculated the mean physiological values for 10-s epochs ranging from 5 s before to 5 s after the end of an intervention. The interactional variables were the type of formulation  and empathy score of the formulation.Physiological data were analyzed by the linear mixed models (LMM) procedure in SPSS with restricted maximum likelihood estimation. When analyzing physiological responses to the whole session, role nested within dyad was specified as the subject variable and session number was specified as the repeated variable, and AR(1) was specified  as the covariance structure for the residuals. When analyzing physiological responses to interventions, role nested within dyad and session were specified as the subject variables and intervention number was specified as the repeated variable, and ARMA was specified as the covariance structure for the residuals. Role , challenge , therapist empathy (rating), and Role × Challenge, Role × Empathy, and Challenge × Empathy interactions were specified as fixed effects.The data supported many of our hypotheses but also provided unexpected results. Most importantly, the change in the participants’ physiological activation across a session was as we expected: the therapist’s empathy increased the therapist’s physiological activation and decreased the client’s physiological activation. We also found that the therapist’s arousal increased during the challenging interventions. Furthermore, there was an interaction effect of empathy and challenge, suggesting that these variables work together. Besides the statistically significant results, we also report trends in the data, considering also them of importance given the small database and the natural  setting of the research.F = 14.68, p < 0.01] and Role × Empathy  significantly predicted SCL change. Larger mean therapist empathy led to an increase in SC levels in therapists, whereas in patients it led to decrease of EDA. Figure 1 illustrates the effect of empathy on the change in SCL across a session for therapists and clients.Our hypothesis on empathy and physiological arousal was confirmed regarding the change in SCL across a session. Role of the participant . This appeared to be the case primarily for the clients (four of the five dyads as shows in the figure 2 below) rather than the therapists.Regarding our hypothesis on challenge, a higher ratio of challenging interventions tended to be associated with an increase in SCL across a session, but this effect failed to reach statistical significance . There was no significant effect of empathy or challenge on the mean SCL across a session .In addition, a higher ratio of challenging interventions tended to lead to a larger increase in SCL across the session especially when empathy was low, but the interaction between challenge and empathy failed to reach statistical significance  and high mean heart rate . Also empathy increased heart rate of the participants across a session ; thus, the hypothesis that empathy would decrease the client’s heart rate was not supported. Unexpectedly, empathy and challenge also interacted. In sessions with low empathy and a low ratio of challenging interventions, and sessions with high empathy and a high ratio of challenging interventions, the heart rate of the participants decreased across the sessions . On the other hand, in these sessions the mean heart rate of both participants was higher . This suggests that empathy and challenge are connected also at the physiological level. We will return to this in the discussion.As we hypothesized, challenge increased heart rate of the participants. Challenge was associated with both an increase in heart rate across a session , whereas challenge was negatively associated with it . Also the Challenge × Empathy interaction was significant . As was the case for heart rate, empathy was related to high orbicularis oculi activity across sessions with a high ratio of challenging responses, but the reverse was true for sessions with low challenge.As hypothesized, empathy was positively associated with orbicularis oculi EMG activity across sessions . Also empathy tended to be positively associated corrugator activity, but the effect was non-significant .Although challenge tended to be associated with higher corrugator EMG activity across sessions, the association was non-significant . Our hypothesis that challenge would increase SCL in both participants was partly supported. Challenge and role interacted significantly . A post hoc comparison showed that challenging interventions increased SCL in therapists (p < 0.001), but not in clients. Empathy was not related to SCL elicited by interventions.Therapists had a significantly lower SCL  compared to clients . The positive association of empathy with heart rate failed to reach statistical significance . However, the Empathy × Role interaction was significant , indicating that empathy increased heart rate of therapists, but not of clients. The main effect of challenge was a non-significant trend , but the Challenge × Role interaction was significant . Challenging interventions increased heart rate in therapists (p < 0.001) and decreased it in clients (p < 0.05). Thus, both empathy and challenge seem to increase the therapist’s, but not client’s, physiological arousal in response to interventions (formulations).Therapists had a significantly lower heart rate  = 9.86, p < 0.001]. Empathy tended to increase orbicularis oculi activity more in response to non-challenging interventions, but the Challenge × Empathy interaction failed to reach significance . The interaction of challenge and role was significant ; challenging interventions decreased orbicularis oculi activity only in clients. Thus, although empathy and challenge did not influence a client’s arousal during interventions, they appear to influence the client’s positive emotional expressions.Empathy was positively associated with orbicularis oculi EMG responses  to interventions . Also, empathy tended to increase corrugator activity in therapists, whereas the reverse tended to be the case in clients, although the Empathy × Role interaction failed to reach significance .Corrugator Supercilii activity indexing negative emotional expressions tended to lower in the therapists compared to clients, although the difference was not significant [Figure 5 shows these results on responses to interventions.empathy increased the therapist’s and decreased the client’s skin conductance over the session, (b) empathy increased orbicularis oculi EMG activity indexing positive emotional expressions  in both the therapists and the client across the sessions, (c) challenge increased heart rate of both the therapist and client across the sessions, and (d) challenge increased the therapist’s skin conductance during the very intervention. Although the hypotheses were not confirmed in all respects, the results support our general hypothesis that the therapist’s empathy is connected to positive valence and decrease in arousal in the client, and that the therapist’s challenge is connected to increase in physiological arousal in the participants. This is in line with how clinical literature describes the effect of these two basic orientations of therapeutic work (The results confirmed our hypotheses in four respects: (a) tic work . Our stuIn particular, the result on the effect of empathy at the session level suggests that the phenomenon of “sharing the emotional load” that has been found from everyday storytelling  is also Given the lack of earlier research, we did not have specific hypotheses regarding the differences between the physiological responses in the two time frames: during whole sessions and during interventions. However, we made some intriguing findings regarding the time frame of the physiological correlates of challenge that call for further work, theoretical and empirical. While in terms of heart rate, both participants respond to challenge at the session level (heart rate increases), in terms of EDA a trending effect suggests that only clients respond to challenge at the session level. However, phasically, during and immediately after the very formulations, only the therapists’ arousal—both EDA and heart rate—increased, whereas clients did not show a phasic physiological response to challenge. This is different to what was found in the earlier studies on confrontation , and mayFurthermore, the results suggest that the “professional specificity” of the therapist’s emotion regulation pertains to challenge more than to empathy: in the therapists, the regulation of empathy, unlike challenge, seems to come closer to the regularities of generic human interaction—what above was depicted as the sharing of the emotional load. In other words, the therapist’s own emotion system seems to react more “profoundly” to being empathetic than to being challenging. On the other hand, the therapists’ arousal may be connected to the effort in reflective, “controlled” type of empathy that Another effect that we did not predict but that calls for further research is the interaction effect that empathy and challenge had in predicting heart rate and facial muscle activation. In sessions where the therapist shows much empathy and challenge, along with sessions with low empathy and low challenge, heart rate was higher and positive facial muscle activity (smile) stronger than in sessions where these variables were less balanced. On the other hand, heart rate decreased over the sessions where empathy and challenge were in balance. This suggests that the close proximity of empathy and challenge in therapeutic work found in earlier interaction research  is refleThis study has demonstrated the effort and sophistication in the psychotherapists’ professional work with their clients’ emotion. The study confirms that the therapists responses to their clients have immediate effects in both the therapist’s and the clients’ emotion system. Recognizing the physiological load and its successful regulation in standards sessions contributes to understanding of the psychotherapist’s professional role, and highlights the importance of the therapist’s self-care.A limitation of the current study is the small amount of data and that some of the effects were found only as trending. Our physiological data shows that the client’s autonomic activation is constantly relatively high during the sessions, and this can amount to a roof-effect that prevents activation related to subtle differences from showing in the statistics. Data from more dyads would be helpful in solving this problem. Another limitation of this study considers the “fair to good” inter-coder reliability for challenge, and the “fair” inter-rater reliability for empathy. In our view, the common disagreement between coders and raters reflects the nature of the data , and that the coding and rating schemes allowed context-sensitive interpretation. We acknowledge that more coders and raters would probably improve the reliability. Another methodological aspect in the coding is that our coding scheme is applicable especially in the type of psychodynamic therapy that our data represents. To study challenge in other types of therapies, a coding scheme sensitive to that context would be needed. Finding a best solution considering both good reliability and context-sensitivity in the coding of interactional phenomena remains as a challenge for further research.AP, LV, NR, MS, and PH: conception and design of the study. LV, PH, MK, AP, and NR: acquisition, analysis, and interpretation of data. LV, PH, and AP: drafting the manuscript. AP, LV, NR, MS, PH, and MK: revising the manuscript, final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sei-1 is a potential oncogene that plays an important role in promoting genomic instability. Double minute chromosomes (DMs) are hallmarks of gene amplification and contribute to tumorigenesis. Defects in the DNA double-strand break (DSB) repairing pathways can lead to gene amplification. To date, the mechanisms governing the formation of DMs induced by Sei-1 are not fully understood. We established DMs induced by Sei-1 in the NIH-3T3 cell line. RNA-sequencing was used to identify key characteristics of differentially expressed genes. Metaphase spreads were used to calculate DM numbers. Immunofluorescence was employed to detect γH2AX foci. Western blot and Akt pathway inhibition experiments were performed to reveal the role of the PI3K/Akt/BRCA1-Abraxas pathway in Sei-1-induced DMs. Luciferase reporter assay was employed to explore the regulatory mechanisms between Sei-1 and BRCA1. DM formation was associated with a deficiency in DSB repair. Based on this finding, activation of the PI3K/Akt/BRCA1-Abraxas pathway was found to increase the DM population with passage in vivo, and inhibition resulted in a reduction of DMs. Apart from this, it was shown for the first time that Sei-1 could directly regulate the expression of BRCA1. Our results suggest that the PI3K/Akt/BRCA1-Abraxas pathway is responsible for the formation of DMs induced by Sei-1. As an identified oncogene, Sei-1 is located in a frequent amplification region, 19q13.1, in ovarian cancer3 and is associated with mechanisms that lead to chromosomal instability, such as increasing the number of chromosomes, and double minute chromosomes (DMs) and micronuclei formation5. In addition, Sei-1 also inhibits apoptosis by stabilizing the X-linked inhibitor of apoptosis protein6 and by promoting metastasis through AKT or ILK modulation in HER2/neu-suppressed or expressing cancer cells7. However, few reports about the mechanisms that govern the formation of DMs and Sei-1 have been published so far, and as a result, further exploration in this field is needed.Sei-1  is a member of the TRIP-Br family, which acts as a nuclear factor that regulates the cell cycle by interacting with cyclin-dependent kinase 4 (CDK4) and E2F/DP-19. DMs can be detected in many tumors, such as acute myeloid leukemia10, ovarian carcinoma12, glioblastoma multiforme14, and colonic carcinoma16. Moreover, genes located on DMs can play crucial roles in the development of cancer, such as DHFR amplification in methotrexate resistance17 and MYC promotion of proliferation and invasion18. Therefore, the study of the DM formation is important for exploring the mechanisms underlying the development of tumors.DMs are paired, circular, acentric, and self-replicating DNA and are the cytogenetic hallmark of extrachromosomal gene amplification20. When a DSB occurs, it can initiate two main repair mechanisms, homologous recombination (HR) and classical non-homologous end joining. In addition, alternative end joining and single-strand annealing have been found to take part in DSB repair under certain conditions21. Breast cancer type 1 susceptibility protein (BRCA1) is recruited to the sites of DSBs with the help of the BRCA1-Abraxas (BRCA1-A) complex to effectively repair DNA. The BRCA1-A complex is composed of BRCA1 and Abraxas  as well as other components, including Rap80, BRCC36, BRE, and NBA123. Abraxas possesses the domains necessary to interact with the BRCT domains of BRCA1 and other components, which reveals the role of central adaptor protein24. The BRCA1-A complex plays important roles in maintaining genomic integrity during the DNA damage response.DNA double-strand breaks (DSBs) are a form of DNA damage that can lead to genomic instability or apoptosis if not appropriately repaired4. Moreover, DMs can appear during the passages in vivo in this model, which has aroused interest in exploring how these DMs are produced during this process. Although we previously reported the role of the met pathway in the promotion of the formation of DMs induced by Sei-1 in NIH-3T3 murine fibroblasts5, deep-seated and unknown mechanisms are also involved. Therefore, we selected cells from different passages in vivo with high and low DM populations for RNA-sequencing (RNA-seq) to explain the process governing the formation of DMs.Previous studies have shown that Sei-1 not only transforms the NIH-3T3 passage in vivo model to possess characteristics of cancerous formations but it also increases the incidence of genomic instability5. Consistent with previous results, the Sei-1-transfected group appeared to generate xenografts while the vector group failed. The primary cell culture from the original generation in vivo was named CPX1, and the sixth generation in vivo passage was named CPX6. The mean number of DMs for Vec, the control group transfected with null-vector-NIH-3T3 cells, and CPX1 and CPX6 was measured as ~0, 10, and 50, respectively  showed that the number of γH2AX foci increased significantly as the population of DMs increased  were analyzed and shown in supplementary figure 5. All of these suggested that DEGs associated with changes of DNA structures and PI3K/Akt pathway could play essential roles in the process of DM formation. Therefore, many DEGs that were involved in DSB repair . The qPCR was used to verify the accuracy of the RNA-seq data. All genes listed in the table were downregulated and only BRCA1, Rad51, and Chek1 reached the same degree of significance as the original data , and phospho-Akt (Thr308), while there was no change in total Akt, and a significant reduction in BRCA1  and phospho-Akt (Thr308) in addition to significant decreases in total Akt. BRCA1 and Abraxas showed dramatic increases in expression after 10 µM drug treatment for 48 h 8 h Fig. . PI3K wa8 h Fig. . Collect38. In the present study, we aimed to sift through the DEGs related to DM formation, and in doing so identified BRCA1 as an essential gene. This was done using RNA-seq, which differs from the previous study we performed5. In addition, we found that most genes associated with the DSB repair pathway were downregulated, which indicated that a defect existed in repairing DSBs. Gene amplification increases when the factors involved in the occurrence of DSBs, such as IR, DNA synthesis inhibitors, and restriction endonucleases, are added. Defects in either HR-relevant genes such as ATM39, Rad51D40, and Rad5441 or in NHEJ interrelated DNA-PKcs42 can enhance the predisposition of gene amplification mechanisms that include homogeneous staining regions and DMs. Consequently, these phenomena hint that Sei-1 may act as a factor that promotes DSBs and that there is a relationship between the production of DSBs and DMs in the Sei-1-transformed NIH-3T3 model.RNA-seq, which is a deep-sequencing technology, is used to map and quantify transcriptomes and has revealed new transcriptomic insights into gene and exon boundaries, transcript complexity, novel transcription mechanisms, and DEG identification43, but its relationship with the generation of DMs is still unknown. We found that the activation of PI3K/Akt promoted the appearance of DMs in a PTEN-independent manner. Likewise, not only can loss of BRCA1 lead to aneuploidy44 but it also contributes to activating the PI3K/Akt signaling pathway[30]. The BRCA1-A complex is important for genomic stability. However, the detailed mechanisms underlying DMs formation for BRCA1 or the BRCA1-A complex deficiency are not completely known. Our results uniquely support the claim that activating the PI3K/Akt pathway induced loss of function of the BRCA1-A complex to cause the production of DMs under the action of Sei-1 in NIH-3T3 cells, and Sei-1 could also target the promoter and regulate the expression of BRCA1  and authenticated by means of the STR (short tandem repeat) test . The detailed procedures of cell culture, cell passage, and transfection were previously reportedThe following antibodies were used for the experiments: anti-BRCA1 and PTEN antibodies ; anti-Rad51, Chk1, and Abraxas antibodies ; anti-PI3K, pan-Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), and anti-γH2AX antibodies ; IRDye 800DX-conjugated affinity-purified anti-mouse IgG and IRDye 700DX-conjugated affinity-purified anti-rabbit IgG ; and anti-mouse IgG/HRP and anti-rabbit IgG/HRP secondary antibodies . The MK2206 2HCl inhibitor  was also used.Cells in metaphase reached 70–80% confluence and were treated with Colchicine  for 1.5 h. The cells were trypsinized and then washed with phosphate-buffered saline (PBS) and centrifuged. The cell precipitate was re-suspended with 0.075 M KCl and incubated in a 37 °C water bath for 13 min. After undergoing fixation three times, the cell suspension was dropped on an ice-cold clean slide. The drops spread evenly and were stained with Gimsa . The pictures were then photographed using an Olympus BX41 microscope  equipped with a JVC TKC75U color video camera .48.Cell precipitates were treated with Trizol  to extract the RNA. cDNA samples were obtained using the Transcriptor First Strand cDNA Synthesis Kit . Semi-quantitative and quantitative real-time qPCR were performed as previously reportedg at 4 °C for 30 min to extract the total protein. The protein samples were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a PVDF membrane. After blocking with 5% blocking buffer, the membranes were incubated with the appropriate antibodies and then scanned using the Odyssey Imaging system  or the Fluorchem R system . ImageJ software was used for band analysis.Cells were lysed with RIPA buffer and centrifuged at 12,000 × The cells covered 80–90% of the glass coverslips. The cells were washed three times with PBS and fixed in 4% paraformaldehyde . Then, the cells were permeabilized with 0.1% Triton X-100 and blocked in 4% bovine serum albumin. Images were obtained using a DM-RXA2 fluorescence microscope (Leica) after incubation with γH2AX antibody , a fluorescence-conjugated secondary antibody, and DAPI  stain .5. The cells were sent to the Novogene Company  for RNA-seq and analysis. We then independently screened, analyzed the data by R , and validated the DEGs.Primary cells were cultured from mouse xenografts as previously describedThe construction of Sei-1 vectors (EX-A3731-Lv103) and pGL3-BRCA1-promoter luciferase reporter vectors were completed by GeneCopoeia company  and Aizhe Biological Technology Co., Ltd , respectively. The 293T cells were plated in 24-well plates at 50,000/well. The co-transfection of Sei-1 and luciferase reporter vectors were accomplished according to the instruction of HilyMax reagents . The detection of luciferase activity was based on the instruction of Luc-Pair Duo-Luciferase Assay Kits 2.0  and completed by microplate reader .t test and one-way analysis of variance with the Student–Newman–Keuls test were employed for data analysis of DM counts, relative mRNA expression, luciferase reporter assay, and western blots. All data are presented as the mean ± S.D or mean ± S.E.M. Significant differences were defined as P < 0.05.Two-tailed Student’s Figure legends of supplementary materialsSupplementary figure 1Supplementary figure 2


The VACNTs were grown underneath the graphene and lifted up the graphene. The graphene, with its edges anchored on the Al2O3, provided a constrained boundary condition for the VACNTs and hence affected the growth height and mechanical strength of the VACNTs. We prepared three groups of samples: VACNTs without graphene, VACNTs with graphene transferred once (1-Gr/VACNTs), and VACNTs with graphene transferred twice (2-Gr/VACNTs). A nano-indentation system was used to measure the reduced compressive modulus (Er) and hardness (H). The Er and H of Gr/VACNTs increased with the number of transfers of the anchored graphene. The 2-Gr/VACNTs had the largest Er and H, 23.8 MPa and 912 KPa, which are 6.6 times and 5.2 times those of VACNTs without the anchored graphene, respectively. In this work, we have demonstrated a simple method to increase the mechanical properties and suppress the height of VACNTs with the anchored graphene and number of transfers.We fabricated a 3D sandwich hybrid material composed of graphene and vertically aligned carbon nanotube forests (VACNTs) using chemical vapor deposition. The graphene was first synthesized on Cu foil. Then it was transferred to a substrate which had a pre-deposited catalyst Fe film and a buffer film of Al PaulD method . However2 of 30 sccm and argon (Ar) of 600 sccm. The Cu foil was annealed for 30 min at that temperature to remove oxides of Cu in H2 and Ar atmosphere. The mixed reaction gas of CH4, H2, and Ar was admitted into the CVD at flow rates of 10, 30, and 600 sccm for 5 min to grow the graphene sheet. Afterward, the CH4 flow was stopped and the sample was rapidly cooled to room temperature. Next, the as-grown graphene sheet was transferred onto the target substrate with a clean-lifting transfer technique utilizing electrostatic force [2O3 and a 1.9 nm catalyst Fe film. It was defined the deposited zone by the tape as shadow mask pattern and released it after the deposition of buffer layer and catalyst film. In addition, the attached graphene on PET was transferred onto one side of the pre-patterned Fe film for comparison of the mechanical properties with or without graphene, as shown in 2 flow of 400 sccm and C2H4 gas was supplied as the carbon source at a flow rate of 40 sccm for 60 min at 750 °C. The growth processes were conducted under 1 atm. The mechanical properties of the 3D hybrid structure of substrate/VACNTs/graphene were experimentally measured with a nanoindentation instrument.In this work, a 3D hybrid structure with the VACNTs sandwiched between the substrate and graphene was constructed. The graphene was synthesized with a three-temperature-zone CVD (TTZ-CVD) process. The as-grown grapheme was first transferred to the substrate, after which the VACNTs were synthesized by CVD method to form the hybrid structure of substrate/VACNTs/graphene. As-cleaned Cu foil (99.8% purity) was first placed on a quartz boat and inserted into a TTZ-CVD furnace, where it was heated to 1000 °C at 30 min under Hic force . The tra−1) and 2D band (~2700 cm−1) of the graphene [2D/IG ~0.67, and the full width at half-maximum of the 2D band peak was 63.1 cm−1. The results indicated few or more layers of graphene [graphene . The peagraphene ,20. A scgraphene , which rFrom the VACNTs growth process, To further examine the constraining effect of the multi-layer graphene on the mechanical properties, the graphene sheets were transferred once and twice onto different areas of the substrate and VACNTs were grown, as shown in the inset of In this work, a 3D hybrid nanostructure was fabricated with graphene and VACNTs. The mechanical properties of the Gr/VACNTs increased with the number of transfers of the anchored graphene. The height of the VACNTs was affected by the number of times the graphene was transferred. This simple method could be used to fabricate a high strength nano-material. In addition, VACNTs and graphene are the excellent performance of thermal interface materials. This may warrant new applications for the polishing  and micr


P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70,  Human non‐small‐cell lung cancer cell line A549 was purchased from American Type Culture Collections (ATCC). Human normal mesothelial cell line LP‐9 was purchased from the Cell Culture Core Facility at Harvard University . The 5 mesothelioma cell lines and A549 cells were maintained in RPMI‐1640 medium supplemented with 10% heat‐inactivated foetal bovine serum (FBS) and penicillin (100 IU/mL). LP‐9 was maintained in M199 supplemented with 15% heat‐inactivated FBS, 10 ng/mL EGF, 0.4 μg/mL hydrocortisone and penicillin (100 IU/mL). All cell lines were incubated at 37°C in a humid incubator with 5% CO2.2Fresh primary MPM and adjacent normal pleural tissues were obtained from 70 patients with MPM whose primary tumours were surgically resected. There are few exception normal pleural tissues used as normal control from non‐tumour patients on the same array. Primary human MPM samples and normal pleural samples were fixed in formalin and embedded in paraffin in 4‐μm tissue microarray sections. All human tissue samples were obtained and analysed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714–22 942–01).Rabbit anti‐YAP antibody  and rabbit anti‐PD‐L1 antibody  were used for immunohistochemistry (IHC) staining. The tissue microarray slides were deparaffinized and rehydrated and then immersed in boiled 10 mmol/L sodium citrate buffer (pH 6.0) for 10 minutes. After cooling down, slides were incubated in 3% hydrogen peroxide for 10 minutes and washed in PBS 3 times for 5 minutes. Slides were incubated with 10% normal goat serum in PBS for 30 minutes. After being washed in PBS 3 times, the slides were incubated overnight at 4°C with rabbit anti‐YAP antibody  and rabbit anti‐PD‐L1 antibody . The next day, the slides were incubated with biotin‐labelled secondary antibodies and streptavidin‐peroxidase (1:30) for 20 minutes each. Slides were stained for 5 minutes with 0.05% 3,3′‐diaminobenzidine tetrahydrochloride freshly prepared in 0.05 mol/L Tris‐HCl buffer (pH 7.6) containing 0.024% hydrogen peroxidase. The slides finally were counterstained with haematoxylin, dehydrated and mounted in Diatex.The following scoring system was used: −, no stain; +, weak staining (≥10% and <30% stained cellularity considered as positive); ++, moderate staining (≥30% and <50% stained cellularity considered as positive); +++, strong staining (50% or above stained cellularity considered as positive). All scoring was carried out under a low power objective lens (10X) with a Zeiss Axioskop 2 microscope . Images were taken under 10× or 40× objective lens.2.3The SMARTpool siRNA targeting YAP (YAP siRNA‐1) and a non‐targeting siRNA used as control were purchased from Thermo Scientific Dharmacon . Another YAP siRNA (AM16708) targeting the 3′UTR end of the YAP gene (YAP siRNA‐2) was purchased from Life Technologies . The YAP plasmid DNA (pcDNA4/HisMaxB‐YAP) used to overexpress the YAP gene in the cells was purchased from Addgene .The mesothelioma cells were plated in 6‐well plates (for Western blot or PCR) or 24‐well plates (for reporter assay) 24 hours before transfection. Cells were transfected with 100 nmol/L of siRNA using Lipofectamine RNAiMAX . For the YAP rescue procedure with YAP forced overexpression, cells cultured in 6‐well plates were transfected with 4 μg/well of YAP plasmid DNA, and those in 24‐well plates were transfected with 0.8 μg/well. After transfection for 48 hours, cells were harvested for further analysis.2.4The total amount of protein for each sample was 20 μg. The samples were run on 4%‐20% gradient SDS‐polyacrylamide gels  and then were transferred to Immobilon‐P nitrocellulose membranes . The membranes were probed with rabbit anti‐YAP , rabbit anti‐phospho Yap Ser127 , rabbit anti‐PD‐L1 antibody  and mouse anti‐GAPDH  in 4°C overnight after being blocked with 5% non‐fat milk. The membranes were then incubated with species‐specific conjugated secondary antibodies (GE Dharmacon) at room temperature for 1 hour. An ECL blotting analysis system  was used for detecting protein expression.2.5Total RNA was extracted from cells using the High Pure RNA Isolation Kit . RNA was transcribed to the cDNA using the iScript cDNA Synthesis Kit (Bio‐Rad), and the cDNA was used as the template for real‐time PCR. Primers and probe sequences used to detect YAP, CD274, AREG, CTGF and endogenous control gene b‐glucuronidase (GUSB) gene expression were purchased from Life Technologies . Real‐time PCR detection was carried out using TaqMan Technology on an Applied Biosystems 7000 sequence detection system (Applied Biosystems). Relative Quantification Software (Applied Biosystems) was used for mRNA expression analysis.2.6The 8× GTIIC‐luciferase plasmid (Addgene) and Renilla luciferase pRL‐TK plasmid  were cotransfected into cell lines using the transfection reagent Lipofectamine 2000 (Invitrogen). After 48 hours, cells were lysed and the lysate was transferred into a 96‐well plate for analysis using the Dual‐Luciferase Reporter Assay Kit (Promega). Luminescent signalling was measured on a GloMax‐96 Microplate Luminometer (Promega) according to the manufacturer's instructions.2.7Fragmented chromatin from H2052 cells was incubated with anti‐IgG (negative control), anti‐YAP and anti‐POL‐II (positive control). Recruited DNA was subjected to PCR using the primers for distal enhancer regions of PD‐L1, and PCR products were electrophoresed in agarose gel. The ChIP assay was conducted using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore Corporation). Polyclonal antibodies for YAP  and control rabbit antibody for IgG  were used for ChIP. Two‐pair primers were used for RT‐PCR to amplify the PD‐L1 gene. One pair consisted of 5′‐TCGGTCTGTGAAGGACTGC‐3′ and 5′‐ACCGTTGAGGAATGGATGAA‐3′, resulting in a product size of 203 bp, and the other consisted of 5′‐CCACCACCATTATCTAATTCCA‐3′and 5′‐AAGGAGCCAGACACAAAAGG‐3′, resulting in a product size of 210 bp.2.8Mesothelioma cell lines 211H, H2452, H290 and H2052 were cultured in a 96‐well plate. The cell count placed in each well was 3300. Cells were harvested at the time‐point of 0, 24, 48 and 72 hours. Cells were lysed, and CellTiter‐Glo Luminescent Cell Viability Assay reagent (Promega) was added to generate luminescent signalling. Luminescent signalling was detected using the GloMax‐96 Microplate Luminometer. Proportional cell proliferation was analysed with GraphPad Prism 5.0 software .2.9P value from a 2‐tailed test was <.05.Data are presented as mean ± standard deviation (SD) from 3 independent experiments. All statistical analyses were performed with GraphPad Prism . One‐way ANOVA followed by Scheffe multiple comparisons were used to compare the differences among multiple groups. A significant difference was considered when the 33.1P < .001)   . In 5 mesothelioma cell lines , the p‐YAP S127)/YAP protein expression ratio was low   Figure C.These results suggest that PD‐L1 expression is regulated by YAP expression in H2052, 211H, H2452 and A549 cells.3.4To investigate whether YAP regulates PD‐L1 at the transcriptional level in MPM cells, we examined the PD‐L1 promoter region and found 2 putative TEAD‐binding sites (CATTCC), which are 7911 and 7941 bp upstream of the PD‐L1 transcription start site . However, these patient samples were initially studied 10‐20 years ago, and there was not enough tissue left to evaluate the further molecular status between YAP and PD‐L1. Although PD‐1/PD‐L1 inhibitors can be a new treatment option for unresectable MPM with high PD‐L1 expression, the efficacy of PD‐1/PD‐L1 inhibitors is under investigation in clinical trials currently.Currently, tumour PD‐L1 expression is used as a predictive biomarker for immunotherapy targeting on the PD1/PD‐L1 axis.The regulation of PD‐L1 in tumours is not well understood. Two recent studies reported that the EGFR pathway is involved in the regulation of PD‐L1 expression in EGFR‐mutant human non‐small‐cell lung cancer (NSCLC) cells, whereby inhibition of EGFR signalling decreased PD‐L1 expression in some EGFR‐mutant NSCLC cells, suggesting that tumour PD‐L1 expression may be regulated by cancer‐driven mutation.In our MPM tumour microarray samples, some tumours with weak nucleus YAP staining show PD‐L1 expression . YAP is one of the factors that regulate PD‐L1 expression, and other factors may be involved in regulating PD‐L1 expression in various cancers.To our knowledge, our study is the first to show that inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further investigation of how YAP regulates tumour PD‐L1 expression in various cancers is warranted. In the future, treatments that target YAP may be an alternative to immunotherapy targeting on PD‐1/PD‐L1.All authors have no conflict of interest.PC. Hsu and L. You conceived and designed the study. PC. Hsu, J. Miao, Z. Huang, Z. Xu, J. You, and YL. Yang contributed to the development of methodology. PC. Hsu, J. Miao, WQ. Zhang and D.M. Jablons acquired the data  CW Wang analysed and interpreted the data . PC. Hsu, J. Miao, CT.Yang and L.You wrote, reviewed and revised the manuscript. PC. Hsu, J. Miao, WQ. Chang, Z. Xu and L. You contributed to administrative, technical or material support (organizing data). L. You supervised the study. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


This ensues a new methodological challenge for computational biology which consists in objectively extracting from these data the We deployed the fast multi-scale community mining algorithm based on spectral graph wavelets to characterise the networks of intra-chromosomal interactions in human cell lines. We observed that there exist structural domains of all sizes up to chromosome length and demonstrated that the set of structural communities forms a hierarchy of chromosome segments. Hence, at all scales, chromosome folding predominantly involves interactions between neighbouring sites rather than the formation of links between distant loci.Multi-scale structural decomposition of human chromosomes provides an original framework to question structural organisation and its relationship to functional regulation across the scales. By construction the proposed methodology is independent of the precise assembly of the reference genome and is thus directly applicable to genomes whose assembly is not fully determined.The online version of this article (doi:10.1186/s12859-017-1616-x) contains supplementary material, which is available to authorized users. At smomosomes . Howeveromosomes , 9, our omosomes –18. Hi-Comosomes , 20 is ∼omosomes . Mean inomosomes . Changesomosomes . Neverthomosomes . Principomosomes . Projectomosomes  omosomes –29. In oomosomes , 26–34. omosomes , 27,  and foetal lung fibroblast cell line IMR90 Hi-C data for which TADs are available , allowinMyelogenous leukemia cell line K562 and lymphoblastoid cell line GM06990 Hi-C data  for the Cervical cancer cell line HeLaS3 Hi-C data  where thHere we used Hi-C data obtained in different human cell lines: masked positions.Hi-C intra-chromosomal co-localisation frequency matrices for non-overlapping 100 kb loci correspond to the downloaded matrices that were down-sampled to 100 kb when necessary or were constructed from the alignment files  can be quantified as the correlation , the matrix of correlations between wavelets at scale s is approximated by the correlation between η (≪n) wavelet transforms of random Gaussian functions on the graph ). Sorting the (d has a match in We say that a domain b has a match in bi.e ± 1 pixel away. associated lengths (see below), we consider them in groups We say that a border As discussed in Domain length is an intuitive quantity to order a set of domains. In the same manner, we associated a length with each border of the genomic domains used in this work. TAD borders can be shared by at most 2 consecutive TADs, so we associated them with the length of the shortest TAD they border. At a fixed scale of analysis, a border of the novel interval-communities  delimits two consecutive interval-communities, so  we associated it with the minimum length of the two bordering communities. However these borders also present a strong pattern of conservation from one scale to another . We systematically applied the wavelet-based multi-scale community detection method to all the connected interaction networks scanning 100 scales logarithmically distributed in the range of available scales  reducing them to their main interval. This allowed us to adopt a simple representation of the structural-communities obtained across scales  where the interval-communities reduce to singletons in IMR90 . We also filtered out the communities that more than double in size when reintegrating the masked regions of the genome, e.g. interval-communities spanning the centromers. This leads to 386 (resp. 537) non trivial interval-communities in IMR90 (resp. H1 ES) for the chromosome 12. When applied to the 6 Hi-C datasets considered (Methods), the methodology presented for human chromosome 12 in H1 ES and IMR90 resulted in few thousands interval-communities per dataset . The original study  showed tλ=50  organised in fully connected interval-communities with no specific organisation at scales larger than the community size: the matrix is built as a series of 40 pairs of domains of size 20 nodes and 30 nodes with internal domain interaction set to 60, with the two first (resp. second) sub-diagonals set to 80 (resp. 70) to assure connectivity and with an additive Poisson noise over all interaction pairs of mean value vs distance curves for different interval-community length categories show that on average there are more interactions within the communities than between communities, regardless of the cell line and the community length: the interaction ratio systematically increases to some maximal value at distances ∼ 1–2 Mb, from a maximal value ∼ 1.6 in GM06990 and K562, to ∼ 2.2 in H1 ES and ∼ 3 in IMR90. Over larger distances, the ratio remains rather constant in GM06990 and K562 and decreases to ∼ 1.5 in H1 ES and IMR90  between TADs and interval-communities is slightly higher in H1 ES as compared to IMR90 for all TAD lengths  and a coarser  resolution  Fig. . The prethe TADs  are recomaster replication initiation zones that present similar insulating properties as the ones observed for TADs and interval-communities borders  resolution has provided some refined partitioning of the human genome by TADs of mean size ∼180 kb , much closophila . Interesll types , 20, 59.


Therefore, we generated mice that lack CaBP2 or CaBP1/caldendrin (–/–Cabp2 and –/–Cabp1) to test whether these CaBPs are essential for normal retinal function. By immunohistochemistry, the overall morphology of –/–Cabp1 and –/–Cabp2 retinas and the number of synaptic ribbons appear normal; transmission electron microscopy shows normal tethered ribbon synapses and synaptic vesicles as in wild-type retinas. However, whole-cell patch clamp recordings showed that light responses of retinal ganglion cells of –/–Cabp2 and Cabp1–/– mice differ in amplitude and kinetics from those of wild-type mice. We conclude that CaBP1/caldendrin and CaBP2 are not required for normal gross retinal and synapse morphology but are necessary for the proper transmission of light responses through the retina; like other CaBPs, CaBP1/caldendrin and CaBP2 likely act by modulating presynaptic Ca2+-dependent signaling mechanisms.Calcium-binding proteins (CaBPs) form a subfamily of calmodulin-like proteins that were cloned from the retina. CaBP4 and CaBP5 have been shown to be important for normal visual function. Although CaBP1/caldendrin and CaBP2 have been shown to modulate various targets  Ca2+-binding proteins (CaBPs), including the calmodulin-like CaBPs, exert Ca2+-dependent effects on specific target proteins—e.g., ion channels. To determine whether CaBP1/caldendrin and CaBP2 are important for normal retinal function, we used CaBP1/caldendrin- and CaBP2-deficient mice. Although these proteins are not required for retinal development and maintenance, CaBP1/caldendrin and CaBP2 are important for normal transfer of light signals through the retina.Electrical signals generated by the photoreceptors in response to incident light are processed by diverse retinal neurons before being sent to the brain. Ca2+-binding proteins with high homology to calmodulin  and long (L-) forms of these proteins . A thirdby CaBP2 .–/–Cabp1) and newly generated CaBP2 KO (–/–Cabp2) mice to investigate the importance of CaBP1/CD and CaBP2 for normal visual function. We find that both CaBP1/CD and CaBP2 regulate ganglion cell light responses and have different effects on their contrast sensitivity.Despite the identification of a large variety of interacting partners, the contributions of CaBP1/CD and CaBP2 to the transmission of light responses through the retina is unknown. In this study, we used CaBP1/CD knockout , Alexa Fluor 488 goat anti-rabbit , Alexa Fluor 555 goat anti-rat , Alexa Fluor 568 goat anti-mouse IgG2a , Alexa Fluor 488 goat anti-mouse IgG1 , anti-calretinin , anti-synaptotagmin 2 , anti-Ctbp2/ribeye , anti-NK3R , anti–green fluorescent protein , anti-calsenilin , and anti-CaBP1, anti-CaBP2, and anti-CaBP5 .m glycine buffer, pH 2.5, and dialyzed overnight against PBS.Anti-CaBP1/CD and anti-CaBP2 polyclonal antibodies were raised in rats by subcutaneous immunization with purified S-CaBP1 or S-CaBP2 recombinant proteins mixed with Freund’s adjuvant . For affinity purification of rat anti-CaBP1/CD or anti-CaBP2 antibodies, purified CaBP1 or CaBP2 was coupled to cyanogen bromide–activated Sepharose  according to the manufacturer’s protocol. After loading of a 10-fold dilution of the sera in PBS, the columns were washed with 20 volumes of PBS. The bound antibodies were eluted with 0.1 Cabp2 gene was amplified first from C57Bl/6 genomic DNA by PCR with primer FH939 (5′-GTCGACTAAGTAGCTGAGACCAGAAGAGATCGAAG-3′) that was extended with a SalI restriction site and includes a stop codon in all three open reading frames and primer FH940 (5′-GGTACCAGGAGGGCTCAGTTGCTCACATTA-3′) that was extended with a KpnI site. After sequencing of this 2.1-kb fragment, it was cloned into the targeting vector SalI and KpnI opened between the neomycin phosphotransferase gene and herpes simplex virus thymidine kinase gene. The long arm of ∼5.2 kb covering the promoter region of the Cabp2 gene upstream the ATG was amplified in two fragments. The upstream fragment of 2 kb was amplified by PCR with primer FH937 (5′-GCGGCCGCTCGTGGTTTCAGGTGCTCTACACA-3′) that was extended with a NotI site and primer FH947 (5′-TAAGGTCTTAGAGGGTCTGACAGG-3′) that covers a SpeI restriction site. A 3.2-kb fragment upstream of the CaBP2 initiation codon was amplified by PCR with primer FH938 (5′-ACCCAGGTTTCTGGCCTTATGTCT-3′) that also covers the SpeI restriction site and FH948 (5′-TACCGACTGACTCATGCCTAGGTT-3′) that hybridizes a few bases downstream of the CaBP2 initiation codon. All fragments were cloned in the pCRII-TOPO vector and sequenced. A tdTomato vector  was modified by mutagenesis using QuikChange Lightning Multi Site-Directed Mutagenesis  to introduce a NheI site after the SV40 polyadenylation site with primer FH1043 (5′-GTATCTTAAGGCGTAGCTAGCAAGCTTTAATATTTTGTTAAAATTCGC-3′) and delete the internal NcoI site in tdTomato with primer FH1044 (5′-CGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCC-3′). tdTomato was then fused to the CaBP2 promoter as a fragment NcoI-NheI and transferred together in the targeting vector as NotI-BglII and BglII-NheI fragments.All animal procedures were performed in accordance with the University of Washington Animal Care and Use Committee’s regulations. A ∼2.1-kb fragment covering part of exon 2 to part of intron 5 of neo cassette and FH1139 (5′-TACACAGGCTCACCGAGACATCAT-3′) hybridizing approximately 163 bp downstream of the 3′ end of the short arm in the Cabp2 gene and amplifying a fragment of ∼2.3 kb. A control PCR for the wild-type (WT) gene was made with primers FH1139 and FH1140 (5′-ACCAGGCATGGAGTTGGGTATGAA-3′) hybridizing in intron 2A, 480 bp upstream of the 5′ end of the short arm of the Cabp2 gene and amplifying a fragment of ∼2.75 kb. Targeted disruption of the Cabp2 gene was then confirmed by Southern blot analysis. Ten micrograms of genomic DNA was digested with MfeI and hybridized with a 0.6-kb 5′-end probe located 100 bp upstream of the 5′ end of the long arm (Cabp2 gene is targeted.The KpnI linearized targeting vector was electroporated into B6/BLU embryonic stem cells. Recombinant clones were selected on medium containing G418. Transfected embryonic stem (ES) cell clones were first screened through PCR analysis. To screen for homologous recombination, we used primers FH 1064 (5′-GGGTCGTTTGTTCGGATCCTCTAGAGTC-3′) located in the long arm . This pragouti offspring were genotyped by PCR to verify germline transmission. Confirmation of Cabp2 gene targeting was first performed with primers FH1139, FH1140, and FH1064 as indicated above. For routine genotyping of the offspring, the WT allele was identified with primers FH1214  and FH1218  that generate a PCR product of 0.4 kb. The targeted Cabp2 allele was identified with primers FH1218 and FH1384  that produces a PCR product of ∼1.07 kb.One targeted ES clone was injected into C57BL/6J blastocysts. One 80% male chimera was crossed with C57BL/6J mice, and Total RNAs were isolated from mouse retina using RNeasy kit . Total RNA (1 μg) was subjected to first-strand cDNA synthesis using Superscript III reverse transcriptase and oligo(dT) in a volume of 20 μl according to the manufacturer’s protocol (Invitrogen). Short and long CaBP1 were amplified with common forward FH705 (5′-CACCATGGGCAACTGCGTCAAGTCG-3′) and reverse FH706 (5′-TCAGCGAGACATCATCCGGACAAAC-3′) primers. Caldendrin was amplified with primers FH1089 (5′-ACACCAATCATATCTGCCGTCTCC-3′) and FH1093 (5′-GCGATGGGGAGGAACGGGGGCT-3′). Short and long CaBP2 were amplified with common forward K122 (5′-TCCGGGCCTGGCATGGTTC-3′) and reverse FH1410 (5′-CCGAACAAATTCTTCAAAGTCAACC-3′). Amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward (5′-GAAGGGCTAATGACCACAGTCCAT-3′) and GAPDH reverse (5′-TAGCCATATTCGTTGTCGTACCAGG-3′) was used as a positive control. The PCR conditions were as follows: 94°C for 2 min, 35 cycles of 94°C for 15 s, 64°C for 30 s, and 72°C for 1 min, then 72°C for 7 min.–/–Cabp1, –/–Cabp2, –/–Cabp1/–/–Cabp2, or WT mice of either sex were fixed in 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4 (PB), for 1 h. After fixation, tissues were immersed in a graded sucrose series to 20% sucrose in 0.1 m PB and embedded in 33% optimum cutting temperature compound  diluted with 20% sucrose in PB before being frozen. Eye tissues were cut in 12-µm sections. To block nonspecific labeling, retinal sections were incubated with 3% normal goat serum in PBST buffer  for 20 min at room temperature. Sections were then incubated overnight at 4˚C with purified rat anti-CaBP1/CD 1:50 dilution or rat anti-CaBP2 1:20 dilution. Alexa Fluor 555–conjugated goat anti-rat IgG and Hoechst stain (1:2000) were reacted with the sections for 1 h at room temperature. The sections were rinsed in PBST and mounted with Prolong antifade reagent (Invitrogen). As indicated, some sections were incubated overnight at 4°C with a mix of rat or rabbit anti-CaBP1/CD or anti-CaBP2 antibodies and rabbit anti-NK3R at 1:500 dilution, mouse anti-Syt2 at 1:200, mouse anti-calretinin at 1:1000, chicken anti-GFP at 1:1000, mouse anti-calsenilin at 1:1000, or rabbit anti-CaBP5 at 1:200. A mix of Alexa Fluor 555–conjugated goat anti-rat IgG (1:400) and Alexa 488–conjugated goat anti-rabbit IgG (1:400), Alexa Fluor 488–conjugated goat anti-mouse IgG (1:400), or Alexa Fluor 488–conjugated goat anti-chicken IgG (1:400) was then reacted with the sections for 1 h at room temperature.Eyecups from 6-week-old to 3-month-old For the analysis of whole-mount retinas, the mouse retinas were fixed for 1 h in 4% paraformaldehyde in PB before dissection, incubated with 5% normal goat serum in PBST buffer overnight at 4°C, and incubated overnight at 4°C with a mix of anti-Syt2 (1:200) or anti-NK3R (1:500) and anti-Ctbp2 (1:1000). After three washes for 15 min in PBST, a mix of Alexa Fluor 568 goat anti-mouse IgG2a (γ2a) and Alexa Fluor 488 goat anti-mouse IgG1 (γ1) or a mix of Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit was reacted with the sections overnight at 4°C. After three washes in PBST, the retinal whole mounts were mounted with the photoreceptor side down in Prolong antifade reagent and analyzed under a confocal microscope . Immunofluorescent images were obtained with 63×/1.4, 40×/1.1, or 40×/1.3 NA objectives (Zeiss).–/–Cabp1, and –/–Cabp2 retinas were acquired using a 63×/1.4 objective at a voxel size of 0.028 × 0.028 × 0.359 µm (x-y-z). The sublamina containing the axon terminals of cone OFF bipolar type 1 and type 2 was identified by colabeling with anti-NK3R and anti-Ctbp2. The axon terminals of cone ON bipolar type 6 were visualized by colabeling with anti-syt2 and Ctbp2. Ctbp2-labeled synaptic ribbons were counted in images of six randomly chosen 25-μm2 areas across the retina of four mice for each phenotype. The statistical analysis was done using the two-tailed Student’s t-test. Data are expressed as mean ± SE.For quantification of the Ctbp2-labeled synaptic ribbons, confocal images of en face views in whole mounts of WT, –/–Cabp1, and –/–Cabp2 mice of either sex were fixed in 4% glutaraldehyde in 0.1 m sodium cacodylate buffer at 4°C. After four washes in 0.13 m PB, eyecups were dissected and postfixed in 1% buffered osmium tetroxide for 2 h on ice. After another four washes in PB, the retinas were en bloc stained in 1% uranyl acetate overnight at 4°C, dehydrated through a graded series of ethanol and two incubations in propylene oxide, and infiltrated in a 1:1 mixture of propylene oxide:epon araldite and two incubations in epon araldite, before polymerization at 60°C. Seventy-nanometer sections were cut on a Leica UC7 ultra microtome and stained with Reynolds lead citrate. The sections were viewed and images recorded on a JEOL 1230 electron microscope . Images of bipolar cell terminals were analyzed in the outer part of the inner plexiform layer within 2–5 μm from the inner nuclear layer where type 1 and type 2 cone OFF bipolar cell axon terminals are located. Images of photoreceptor synapses with bipolar cells were taken in the outer plexiform layer.Eye cups from WT, –/–Cabp1, or –/–Cabp2 mice (2/5% CO2) Ames medium  at ∼32–34°C. Isolated retinas were flattened onto poly-l-lysine slides : 105 Cs methanesulfonate, 10 tetraethylammonium chloride, 20 HEPES, 10 EGTA, 2 QX-314, 5 Mg-ATP, 0.5 Tris-GTP . Alexa 594 dye (100-200 microM) was included in the intracellular solution to image the ganglion cells post recording as shown in t-test; *p < 0.05, **p < 0.01, ***p < 0.001; error bars indicate SD.Experiments were conducted on whole-mount retinal preparations taken from dark-adapted WT, –/– mice . Isolate–/–Cabp2 mice, we designed a targeting vector to replace exon 1 and exon 2a of the Cabp2 gene with tdTomato and a PGK-neo cassette as shown in Cabp2 allele was further verified using Southern blot -PCR using retinal RNA of –/–Cabp1 mice , and OFF sustained (OFF S). Extensive work on these alpha-like ganglion cells has identified type-specific response properties generated from distinct circuit mechanisms . Recordi–/–Cabp2 retinas ; this suggests that alterations in the transmission of the light signal likely occur downstream of the bipolar cell dendrites.We investigated the light responses of alpha ganglion cells to determine the importance of CaBP1/CD and CaBP2 for proper transmission of light responses across the retina. Compared with responses of ganglion cells from WT retina, we observed a ∼50% increase in the amplitude of the excitatory postsynaptic currents of OFF T ganglion cells of –/–Cabp1 mice. The opposite changes in response range could be explained if the bipolar cells that provide input to OFF S and OFF T ganglion cells express different targets for CaBP1/CD. Alternatively, CaBP1 may differently shape the synaptic input to these two types of OFF ganglions cells.Interestingly, the two OFF alpha ganglion cell types displayed similar changes in kinetics but opposite changes in response range in The absence of CaBP2 resulted in a decrease in the response range of the ON alpha ganglion cell response. The absence of CaBP1/CD slowed response kinetics but did not change the response range of the excitatory synaptic currents of the ON alpha ganglion cells. Because CaBP1 is not found in cone ON bipolar cells, the effect of CaBP1 deficiency on ON alpha ganglion cell excitatory inputs could result from alterations in responses of amacrine cells that provide presynaptic inhibition on type 6 bipolar cells and shape the kinetics of excitatory inputs of ON alpha ganglion cells. The complex interplay between ON and OFF pathways means that other explanations are possible as well.2+ channels  and Cav3  complex. We found here that deficiency of CaBP1/CD also results in slower response kinetics than in WT animals. If CaBP1/CD were to have similar effects on synaptic vesicles priming, it could affect the kinetics of the light responses.CaBPs have also been shown to interact with other proteins playing an important role in neurotransmitter release. For example, CaBP5 interacts with Munc18-1, a component of the synaptic vesicle cycle. CaBP5 was suggested to have a role in synaptic vesicle priming by binding to a specific domain of Munc18-1 necessary for its interaction with the soluble –/–Cabp1 and –/–Cabp2 mice gives us a first insight into the important role of CaBP1/CD and CaBP2 for normal transmission of the light responses through retinal circuits. Although the effects of CaBP1/CD and CaBP2 deficiency might result from a change of modulation of voltage-gated calcium channels, other cellular processes might also be altered. Future experiments investigating the interacting partners for CaBP1/CD and CaBP2 in bipolar cells will help to explain the visual phenotypes in the CaBP KO mice and the cellular functions of these proteins in the retina.In conclusion, the phenotype of


Previous studies on hybrid dielectric-graphene metasurfaces have been used to implement induced transparency devices, while exhibiting high Q-factors based on trapped magnetic resonances. Typically, the transparency windows are single wavelength and less appropriate for polarization conversion structures. In this work, a quarter-wave plate based on a hybrid silicon-graphene metasurface with controllable birefringence is numerically designed. The phenomena of trapped magnetic mode resonance and high Q-factors are modulated by inserting graphene between silicon and silica. This results in a broader transmission wavelength in comparison to the all-dielectric structure without graphene. The birefringence tunability is based on the dimensions of silicon and the Fermi energy of graphene. Consequently, a linear-to-circular polarization conversion is achieved at a high degree of 96%, in the near-infrared. Moreover, the polarization state of the scattered light is switchable between right and left hand circular polarizations, based on an external gate biasing voltage. Unlike in plasmonic metasurfaces, these achievements demonstrate an efficient structure that is free from radiative and ohmic losses. Furthermore, the ultrathin thickness and the compactness of the structure are demonstrated as key components in realizing integrable and CMOS compatible photonic sensors. With proper dimensions of the antenna, a 90° phase difference is obtained as shown in Fig. Txx = Tyy occurs near the resonance, defining an ideal QWP condition. Additionally, by sweeping through different values of the length L2 while keeping L1 fixed (L1 = 440 nm), the resonance amplitudes associated with different electric and magnetic modes can be varied. An acceptable phase bandwidth range within ± 10° was obtained when L2 = 365 nm for RCP, and L2 = 450 nm for LCP, as shown in Fig. λ = 1.48 μm, the undoped graphene (EF = 0 eV) causes high penetration of electric fields into the silicon dipoles and a large phase difference between the x and y components of scattered light (≈150°) occurs. However, as EF approaches 0.8 eV, the in-plane properties (εx = εy) increase the surface conductivity of graphene, resulting in a reduced penetration into the silicon and a ΔΦ≈90° at λ = 1.49 μm.The graphene effect is beneficial for tuning the phase components and the phase retardation of the transmitted electric fields. Firstly, the components of the incident LP light are decomposed into the orthogonal arms of the silicon antenna. Each dipole resonance imprints a different phase pattern on the scattered light. Specifically, near the resonance, each dipole resonance shifts the phase of the incident electric field in the range [− xed L1 = 40 nm, thL1 = 450 nm, L2 = 370 nm, and W = 60 nm are shown in Fig. λ = 1.5 μm, the Stokes parameter ratio |S3/S0|≈ ± 1, where a + 1 value indicates a perfect RCP and a − 1 indicates a perfect LCP output. Here, S0 = |Ex|2+|Ey|2 and S3 = 2ExEy sinΔΦ are the Stokes parameters. The degree of transmission intensity is determined by S0, i.e., a value > 50% is acceptable. Figure The calculated Stokes parameters and polarization ellipse dimensions for the hybrid structure with Tyx and Txx are cross and co-polarization terms, respectively. Within the wavelength range λ = 1.48 μm and λ = 1.51 μm, the efficiency is ≈96% for RCP and ≈90% for LCP outputs. However, at λ = 1.52 μm, the efficiency slightly drops to ≈80% for LCP. As shown in Fig. Ex/Ey≈1 and phase shift ΔΦ≈90° are obtained in a wide range. When α = 48°, an accurate QWP condition is obtainedwhere Pi at the wavelength λ = 1.49 μm. In Fig. ΔΦ≈90°) occurs in the region where the transmittance is above 80%, as shown Fig. Additionally, the transmission phase profile defining the form birefringence was calculated as a function of the periodicities y-planes of the silicon/graphene structure was designed as shown in Fig. νF, and switches the direction of flow of electrons. Additionally, the bias voltage changes the carrier density of graphene which in turn leads to a change in its electrical conductivity and permittivity. In this configuration, the structure forms a quasi-parallel plate capacitor model with an electrostatic capacitance per unit area, C, defined as C = εsiε0/Px, where εsi is the dielectric permittivity of silicon. The Fermi energy, ns) and the electrostatic capacitance per unit area (C) scale the Fermi energy through the gate voltage, that is, ns = CVG/e. Consequently, an increment in Px decreases both the carrier concentration in graphene and the capacitance per unit area. As a result, as shown in Fig. Application of gate voltage bias across the λ = 1.5 μm, the two states of circular polarization can be encoded as two binary states, 0 and 1. The logic state 0 corresponds to the reverse voltage − 47.5V while the logic state 1 corresponds to the forward voltage 47.5V, as shown in Fig. ΔΦ≈0°, can be observed when the gate voltage is at − 25 V . This observation shows a non-linear response in the phase change at − 47.5, − 25, and 47.5 V, attributed to a variation in capacitive coupling as graphene becomes more conductive because of a change in the carrier density and gate voltage. In comparison to other wavelengths in the near-infrared, 1.5 μm shows the optimum point for switching the circular polarization states of the scattered light.At S3 illustrates the degree of circular polarization as a result of the gate voltage biasing. The − 1 and 1 limits denote the ideal polarization conversions from a linear state to LCP and RCP states, respectively. Between the wavelengths λ = 1.49 μm and λ = 1.52 μm, the degree of circular polarization approaches unity (> 90%) for both states, confirming the most appropriate operation region of the structure as a QWP.In Fig. z component of the electric field, calculated at the design wavelength λ = 1.5 μm at z = 0. The distribution shifts as the voltage is reversed from 47.5 to − 47.5 V. The change in electrical conductivity and carrier density of graphene results in a rotation of the trapped magnetic mode around the silicon structure.Figure %) in the near-infrared (λ = 1.45 to 1.54 μm). Unlike in plasmonic metasurfaces, these achievements demonstrate high efficiency devoid of radiative and ohmic losses. Additionally, the structures are compact and have an ultrathin thickness, appropriate for compatibility and integration with CMOS and photonic devices. Meanwhile, graphene is feasible and can be grown using chemical vapor deposition on the substrate while the silicon structure can be fabricated using standard lithographic methods.In summary, birefringence controllability of a hybrid silicon/graphene metasurface polarization converter has been numerically designed. Trapped magnetic modes and high Q-factors are modulated by integrating graphene and silicon. Two configurations of the hybrid structure have been shown, one with a gate voltage bias and the other without. In the voltage-biased structure, birefringence performance is shown through reversal of the gate voltage. From an incident LP light, a reverse bias voltage (- 47.5 V) produces an RCP output and a forward bias voltage (47.5 V) produces an LCP output. Hence, a dynamic switching performance is achieved. For the free-space configuration, QWP performance is shown through manipulation of the dimensions of silicon and the Fermi level of graphene. In both designs, a more stable and broader bandwidth is obtained than in structures without graphene. The designs show higher degrees of polarization conversions (>96


The development of humidity sensors with simple transduction principles attracts considerable interest by both scientific researchers and industrial companies. Capacitive humidity sensors, based on polyimide sensing material with different thickness and surface morphologies, are prepared. The surface morphology of the sensing layer is varied from flat to rough and then to nanostructure called nanograss by using an oxygen plasma etch process. The relative humidity (RH) sensor selectively responds to the presence of water vapor by a capacitance change. The interaction between polyimide and water molecules is studied by FTIR spectroscopy. The complete characterization of the prepared capacitive humidity sensor performance is realized using a gas mixing setup and an evaluation kit. A linear correlation is found between the measured capacitance and the RH level in the range of 5 to 85%. The morphology of the humidity sensing layer is revealed as an important parameter influencing the sensor performance. It is proved that a nanograss-like structure is the most effective for detecting RH, due to its rapid response and recovery times, which are comparable to or even better than the ones of commercial polymer-based sensors. This work demonstrates the readiness of the developed RH sensor technology for industrialization. Humidity is defined as the water vapor amount in air. It is the most abundant greenhouse gas and an important abiotic factor influencing the human life. To describe humidity in a precise way, several items are commonly used, which are absolute humidity (AH), relative humidity (RH), specific humidity (SH), and dew point . The RH Recently, the state of the art of humidity monitoring has evolved rapidly and continuously. However, the wide application range of devices being able to record humidity is behind the intensive scientific studies dedicated to this area. Their most important fields of use are indoor application, plants growth in agricultural market, and control of industrial processes, electronics, and semiconductor area, as well as medical processes ,5,6,7,8.A survey of the literature leads to a classification of humidity sensors based on their working principle as capacitive, electrical conductivity (or resistive) and optical sensors. It is obvious that, except optical sensors, a humidity sensor is defined as a combination of a transducer and a sensing material to humidity.Interdigitated transducers (IDT), also known as interdigital electrodes, have been intensively studied and used for unlimited applications due to the possibility of measuring both capacitance and resistance ,10. An I2O3, TiO2, SiO2, and spinel compounds with AB2O4 structural composition, are known to change their electrical properties, such as resistance, in the presence of humidity [Ceramic materials, like Alhumidity . The firhumidity . Each sihumidity , thus a humidity . This mehumidity . Decreas2, In2O3 [Semiconducting materials, like SnO2, In2O3 , and per2, In2O3 , are pro2, In2O3 . This le2, In2O3 .The third type of humidity-sensing material is an organic material being commonly a polymer. It is important to underline that polymers are an emerging candidate in many sensing applications due to low cost, commercial availability, and easy deposition on different transducers. Besides this, a photosensitive polymer adds advantages related to rapid polymerization, elimination of volatile organic solvents usage, and reduction of technological process steps. A polymeric layer in contact with water vapor shows a change in its conductivity or its dielectric constant ε. As water diffuses into a hydrophobic polymer layer like polyimide, poly(methyl methacrylate) (PMMA), cellulose acetate butyrate (CAB), or poly (PETT), the matrix absorbs water and only its relative permittivity ε undergoes changes . SeveralNowadays, capacitive humidity sensors dominate the market. Polymeric layers as a sensitive layer in combination with interdigitated electrodes are intensively studied . DespiteA chemical sensor is a device that transforms chemical information, i.e., the analyte concentration, into an electrically useful signal. Basically, it consists of a transducer and a sensitive layer. The chemical information is obtained either due to a chemical reaction or due to a sorption process between the analyte and the sensitive layer. In our study, we fabricated capacitive humidity sensors, which monitor any changes of in the dielectric properties of the appropriate sensing material. The transducer was a pair of planar interdigitated electrodes. The sensing material was polymer-based material called polyimide.2. Finger width, length, and pitch were set to 6 µm, 2 mm, and 12 μm, resulting in 83 and 42 fingers, respectively.A pair of interdigitated microelectrodes was produced by standard photolithography on a 620 µm thick glass substrate. For operating at elevated temperatures, quartz substrates are the best choice. After cleaning the glass substrate wafer, a thin adhesion layer of TiW (40 nm) followed by a thick gold layer (140 nm) were deposited by sputtering deposition technique. The electrodes were patterned by photolithography technique using the desired mask. The pattern included two wires around the interdigitated electrodes, one to heat the sensor and the other to measure the surrounding temperature . Each ch2 inert atmosphere. The curing temperature was 400 °C.The polyimide used for humidity sensing layers was prepared from a photosensitive solvent developable polyamide acid ester. First, the substrate surface was cleaned with a stripper in an ultrasonic bath from any contaminants and organic impurities prior to the coating process in order to ensure a good adhesion of the sensing layer to the glass and metallic electrodes surface. The polyimide material was spin coated homogeneously on top of the planar electrodes on the 6-inch glass wafer. Different layer thicknesses were obtained, ranging from 1 µm to 11 µm. The spin coating parameters (spin speed and time) varied according to the desired final film thickness and the precursor initial viscosity. After spin coating, the films underwent a soft bake for a short period of time to drive out residual solvent. Then, the photosensitive layer was exposed to an appropriate dose using a mask for pad opening. The exposure dose was chosen based on the thickest layer in case of a variation of the coating thickness on the substrate. The wet development process was realized at room temperature for an optimal time and followed by flood exposure, without a mask, applied after developing to allow a complete crosslinking reaction. The imidization reaction took place between the amino group and the ester group resulting in an imine group. After completing the photolithographic process, the film was cured in a Yes Oven under N2 large individual chips. Each chip was mounted on an adapted printed circuit board (PCB), glued, and then gold wire bonded thereto. The PCB was equipped with zero insertion force (ZIF) connectors. The coated wafer with polyimide sensing material was diced into 4.7 × 3.9 mm2 or synthetic air.A reliable gas mixing apparatus, which is a part of a gas measurement setup, is an important system for characterizing gas sensors under desired gas mixtures. The dynamic volumetric method, described in reference , was adoUp to 10 chips could be placed into a cylindrical stainless steel chamber (sensor chamber), which is connected to the gas mixing apparatus a. ConseqThe electrical properties of the sensors were monitored by impedance spectroscopy using a Solartron gain-phase frequency analyzer model 1260 A controlled by a PC, permitting automated data collection. The impedance measurements could be carried out at different temperatures (from RT to 200 °C) by heating up the chip using the integrated heater around the electrodes. The measurements were performed from 1 MHz to 10 Hz to get the optimal frequency at which the sensor capacitance change linearly as a function of RH. Within another project, it is planned to develop a miniaturized system using the AD chip. The AC Voltage excitation of the AD chip is around 38 kHz. For this reason, most of the experimental measurements are done at 40 kHz.For this study, the capacitance is recorded at 25 °C under different frequencies . The senthV: threshold voltage (1.5 V)ddV: positive supply voltage (3.3 V)REFC: reference capacitorN: number of charge transfer cycles needed to charge the sC.An evaluation kit with high speed data acquisition was designed. Additional features are low power, low cost, compact size, and network compatibility. The used microcontroller STM32-L476RG possesses a low-power design, several Analog-to-Digital converters (ADC), and a touch sensing controller for capacitive measurements via the charge transfer method on board. The touch sensing controller consists of a number of internal switches. After loading the sensor capacitor with a constant voltage, the charge is transferred by the switches to an external reference capacitor (104 times bigger). This process is repeated N times, until a threshold voltage is reached on the reference capacitor. The capacitance of the sensor is then calculated by (1)Cs=−CAn extension circuit board was developed, which holds sensor connectors, heater driver circuit, resistance temperature detectors (RTD), and reference capacitors for the charge transfer method. Additionally, PT1000 and SHT25 were added as reference sensors and connectors for communication protocols (UART and I2C). The evaluation kit can simultaneously measure three sensors by measuring the capacitance and controlling the operating temperature by a proportional-integral-derivative (PID) algorithm. It permitted measuring the response and recovery times of the developed sensors by ensuring a short measurement interval and quick gas exchange ability. In order to use the evaluation kit together with the gas mixing apparatus, a PEEK lid was designed and screwed with a sealing ring onto the PCB, where three sensors sockets were positioned b. The sa2) were coated with a polyimide layer and examined under IR radiation at different humidity levels by a FTIR spectrometer. A background reference curve is measured with an uncoated sample from the same silicon wafer. A first measurement is performed with a polyimide coated sample under dry condition inside the FTIR interior space at nearly 0% RH  is an extremely reliable method that provides chemical bond and molecular composition present of any partially transparent material. To realize FTIR spectroscopy for our purpose, silicon test wafer substrates  is a simulation software, which applies finite element simulation to a 3D model of the structure to be studied in this work. The CAD is used to build a 3D model that consists of several layers: glass substrate, gold layer, sensitive layer , and air layer. The derived capacitance of a bare IDT by FEM simulations agree well with the one measured by the impedance analyzer. By combining simulations with different dielectric constants of the sensitive layer and the measured sensitivity of the coated IDT, the correlation between the dielectric constant of the polyimide layer and the relative humidity was determined. Therefore, at 25% RH the dielectric constant of polyimide is equal to 3.5.PI~3.5). The values of the penetration depth are obtained from COMSOL plots of the electric field. Calculation of the penetration depth is simulated to determine the maximum reasonable polyimide layer thickness. In the last years, high aspect ratio structures from nature have inspired engineering and scientists to develop new materials. The nanostructures, having superhydrophobic surface properties, is still attracting considerable attention for application in photovoltaic , biosensIn the case of polyimide material, Lee et al. proposed that etching of the layer at pressure equal to 100 mTorr for 5 min results in a nanograss structure . A pressIn the present work, the plasma etching process of polyimide films is carried out under oxygen plasma at two different pressures, 50 mTorr and 720 mTorr, using a reactive ion etching chamber from Plasmalab. Oxygen plasma is generated by a radio frequency (RF) power source. The RF power for both working pressures is fixed to 113 W. The bias voltage, which built up between oxygen source and sample chuck, is 380 V and 140 V for lower and higher etching pressure, respectively. The bias voltage reflects the kinetic energy of the etching reactants. Hence, the oxygen ions with the lower pressure hit the polyimide surface with a higher kinetic energy. The etching rate of polyimide layer is determined by measuring the layer thickness by a profilometer after an etching time of 2 min. The plasma etched samples are analyzed by SEM, as presented in Previous theoretical work established a correlation between pressure and etching rate  to explaThe realized etching process under oxygen plasma at low pressures, 50 mTorr, yields nanograss-like polyimide structures with higher etching rate . In thisA series of photosensitive polyimide layers are deposited onto IDTs. The layers present different thickness and surface morphologies. To characterize the performance of chemical sensors under development, sensors are placed into a hermitic test measurement chamber and the sensors capacitance is monitored under different humidity levels by an impedance analyzer. A commercial humidity sensor, SHT25 device with a standard calibration, is selected for reference.RH=85% and CRH=6% are the measured capacitance at high and low level of relative humidity, respectively.The sensitivity of the sensors decreases from 15 to 9.5 fF/% RH by decreasing the polyimide thickness from 4.6 to 1.5 µm with a flat morphology. As described in Although a gas measurement setup, combining the gas mixing apparatus and electrical measurements using impedance analyzer, is a standard method to characterize the performance of chemical sensors under development, the response and recovery times, typically less than 3 min, are difficult to be accurately monitored for RH sensors under test. The reason is that some minutes (5 min) are needed to ensure a complete gas exchange inside the testing chamber . To overThe response and recovery times in this study are defined as the times required for the sensor capacitance to reach 90% of the maximum measured signal, when the humidity is changed between two humidity levels (here from 6% RH to 67%). As the sensing mechanism is based on diffusion, the thinner sensing layer shows a smaller response as well as a smaller recovery time. However, the nanograss-like layer shows the best performance even in comparison to commercial humidity sensors. An optimization of the transducer design and its miniaturization will lead to a further improved sensor performance. The measured capacitance, normalized, of the polyimide-based RH sensor (for both thicknesses and morphologies) as a function of time is shown in The RH level in the test chamber is varied from dry to 65% and back to the initial level 6% after some seconds. The sensor samples with a polyimide thickness of 4.6 µm having a flat morphology and 1.5 µm rough surface and 1.5 µm nanograss-like surface present the same response and recovery times once measured in the chamber of gas mixing setup. As mentioned in 2 or synthetic air.It is worth noting that the sensor responses are uncorrelated to the used ambient air. No significant differences are detected of measuring the sensors response under carrier gases NGenerally, the adsorption and desorption of a specific analyte carried by an inert gas into a sensing material matrix is governed by either diffusion process or thermodynamically controlled process or even both processes. In case of an ideal diffusion-controlled process, the response time and the recovery time should be equal. In the thermodynamically controlled process, the adsorption is an exothermic process, whereas desorption is an endothermic process. Consequently, desorption requires higher external energy which is reflected by a higher recovery time. An adsorbed molecule on the surface of a sensing layer at certain temperature remains stable at this state. A moderate increase of the sensor temperature, in the absence of any interaction between the sensing material and the analyte, leads to desorption of the desorbed molecule from the surface and returns into its initial gas form.2 plasma etch will be subject of future studies.As confirmed from the FTIR measurements, the polyimide material presents no chemical interaction between the polymer backbone and water molecules. However, the polyimide layer with rough and nanograss-like morphologies show a large difference between their response and recovery times (72%) to humidity. This large difference between the two parameters describing the humidity sensor is probably due to structure changes into the polyimide matrix induced by the plasma process. These structure changes in the polymer induced by the O2 plasma etch parameters. The nanograss morphology exhibits the best performance among the studied morphology types. The ideal polyimide layer thickness turns out to be equal to the penetration depth of the used interdigitated transducer. The sensor signal, capacitance, follows a linear dependence with RH. Polyimide sensing layers with nanograss morphology ensure the best humidity sensor performance regarding the response and recovery times. The optimal polyimide layer thickness and morphology can be chosen as a function of the envisaged application of relative humidity sensors and the used interdigitated transducer.In summary, a systematic examination of the sensor performance of a series of polyimide layers with different thickness and morphologies  exposed to different humidity levels are measured simultaneously in the same test chambers. We have successfully demonstrated that the nanostructure of polyimide to obtain nanograss can be well controlled by adjustment of a series of O