The continuation of teacher preparation activities after a 3-year Physics Teacher Education Coalition (PhysTEC) grant is used as a case study to examine multi-faceted aspects of sustainable change in higher education. Since teacher preparation is outside typical physics departmental activities, success is highly dependent on finding a department and institution who values this cause. Throughout the history of providing grants, PhysTEC has identified ten components of successful sites that they consider during the selection process. In this paper, we retrospectively analyze characteristics of six comprehensive PhysTEC sites, to see how department histories, values, and activities affect long-term sustainability as sites moved from grant funding to matched institutional funding and beyond.The most important components required to sustain these programs were (1) institutional commitment—both financial support as well as intellectual and cultural support for potential teachers—(2) champion, a respected change agent at the university who ensures program success through advocacy and support, and (3) activities that enhance not only the production of teachers but also the undergraduate education activities of the department. Of the six PhysTEC sites, three sites were able to institutionalize the majority of PhysTEC activities into departmental routine. These three sites have departmental leadership and administrators who valued and invested in physics teacher preparation. At these sites, PhysTEC symbiotically supported typical departmental activities including increasing majors, improving courses, and involving undergraduates to support teaching. Two sites were sustaining activities at the time of study but attitudes toward teaching as a profession were mixed so continued sustainability is precarious and reliant on external funding. One site discontinued the majority of PhysTEC activities because of a lack of alignment with a different physics teacher initiative on campus.Because physics teacher preparation is not often prioritized as a part of undergraduate departmental activities, success emerges when departmental and institutional value systems align with this goal. PhysTEC funding is not enough to create this culture; it must exist prior to funding. Sustaining PhysTEC activities is easier when they are seen as enhancing the undergraduate experience as a whole. The PhysTEC grant helped bring physics teacher preparation to the forefront, and a well-respected champion in a leadership position can help set this tone and advance departmental activities accordingly. This study has implications for sustaining reforms of typically undervalued activities in higher education or secondary teacher preparation programs in any discipline. Every year, the Department of Education and National Science Foundation (NSF) spend millions of dollars on projects to improve STEM education. While these initiatives aim to create positive and lasting change, the continuation of benefits can be threatened after funding has ceased. Changing departmental demands, competing priorities for faculty members, and administrative and staff turnover can threaten reforms, especially when knowledge and expertise are lost along the way Coburn . EffortsIn past studies, we found that hard-to-reverse structural changes, such as replacing lecture halls with studio classrooms like SCALE-UP, are one way to counter this trend  as a case study because it helps make the role of value systems in change efforts explicit, because creating physics teachers is not a common priority in physics departments. Since 2001, PhysTEC has been using NSF funding to help universities develop and transform physics teacher programs according to national models. They have modified their application and funding policies to explicitly focus on sustainability  physics instruction in high school, few high school students are inspired to pursue further study of physics. This limits the potential for physics majors, putting physics degree programs at risk of elimination due to low enrollment. The jobs of physics faculty, and the resources allocated to the department, are affected by students’ experiences in secondary school. Maintaining the status quo has negative implications for physics as a discipline and the United States’ ability to maintain scientific and technological competitiveness  maintenance of a program’s intended benefits , (2) institutionalization of the program within the department, and (3) capacity building in the local setting . Capacity building is any activity that builds durable resources and enables the organization to continue the delivery of the change after external funding ends. This can involve training of internal staff, identification of alternative resources, and building internal assets  to campus to lead introductory courses and coordinate on-going field-based experiences , and collaboration with education departments. This study intends to shed insight on which of these pre-conditions might be more important than others in creating sustainable outcomes.Under the grant, comprehensive PhysTEC sites are encouraged to adopt additional components including recruiting pre-service physics teachers, offering early teaching experiences, advising and mentoring teacher candidates, and connecting graduates to a network of local teachers. Learning assistants, talented undergraduates who help support student learning in undergraduate courses, are often used as a way to provide early teaching experiences, while enhancing the quality of undergraduate teaching . Often these students participate in supplemental courses in physics teaching methods, where they learn pedagogical content knowledge.PhysTEC has changed their list of components over the years to prioritize activities that do not typically exist at universities. For example, they used to include “course transformation” because that was linked to demonstrating high-quality teaching practices that could attract more majors, which expanded the number of potential teachers. Now, the listed components have more direct impacts on producing physics teachers. While each site adopts key components as a part of the grant, sites are encouraged to implement them according to their local context, taking advantage of institution-specific resources and expertise  committed upfront to sustain their programs for three or more years beyond the PhysTEC award period. Expectations for sustainability were much less clear at the beginning of the project, which included several sites in the first sustainability paper  at each institution, conducting a total of eleven interviews. The second person we talked to was often a champion in education . At Towson, we talked to two science educators in the physics department. We had not originally planned to interview TIRs because we expected them to execute and implement the vision developed by champions, faculty members, and administrators. However, in the case of two sites (BU and VTech), the TIRs ended up staying more than the year recommended by PhysTEC and took a leadership role in ensuring the continued existence of their position as well as supporting other PhysTEC efforts. In these cases, when the TIR became an active advocate and creator of change, we felt their perspectives were relevant to our research questions.The hour-long telephone interviews highlighted key factors that our interviewees perceived as important. Interviewees were asked open-ended questions about the context , implementation , and transfer . The interviewer used follow-up questions to obtain specific details about the interviewee’s experiences. The common interview protocol was adjusted to the interviewee’s role, e.g., slightly different for teachers in residence (TIR) compared to the department heads. Interviews were transcribed for analysis.Interviews were supplemented and cross checked with a variety of documentation most of which was submitted to PhysTEC throughout the grant period, which involved the perspectives of external and internal stakeholders. While the interviews are short, this documentation provides incredibly valuable information about details of how the grant unfolded over time. Documentation included annual, midyear, and data reports, as well as notes from external site visits conducted by the APS PhysTEC staff. The documentation and interviews were triangulated to explore the history, evolution, stresses, opportunities, motivations, and constraints on physics teacher preparation at each site.Individual interviews were transcribed, reviewed, and summarized with structured note-taking. Interviews at the same institution were compared and crosschecked with PhysTEC documentation  and university websites to ensure consistency. Both researchers separately summarized the interviews and documented components of the change process and aspects related to sustainability. We compared our notes, resolved any discrepancies, and typically combined responses in a more complete summary. Finally, interviews were compared across institutions to highlight thematic findings.The PhysTEC team and featured sites checked these findings for accuracy. Furthermore, the documentation submitted to PhysTEC over the grant period was validated by its members before being viewed by us. We sent the draft of this manuscript to each site for further verification.In “Boston University (BU) is a private, urban, R1 institution and was the first PhysTEC site in New England. The principal investigators (PIs) on the PhysTEC grant were a Master Lecturer in Physics and a Clinical Associate Professor in Education. Prior to the grant, the PIs worked together to provide professional development to teachers at local schools when the district switched to a Physics First model where physics is the first high school science course. Because of this project with in-service teachers, the PIs started attending PhysTEC meetings, where they met their future TIR and where they were inspired to apply for the grant and start an LA program.The TIR has been a critical part of BU’s PhysTEC efforts, actively recruiting students and career changers through listservs for physics teaching. He started the Boston University Physics Teachers Network that meets on campus five times annually with high participation (200 teachers on the mailing list) in the local community. The TIR has been involved on additional projects to secure external funding, including creating an online and in-person AP physics course for local schools and Noyce scholarship work.BU’s PhysTEC efforts included expanding their LA program, including using LAs to support studio teaching in first year courses. After their PhysTEC funding ended, BU’s LA program has continued to flourish, with funding from the institution to support over 140 undergraduates throughout the institution per year. The LA program is essentially institutionalized, but the undergraduates view it more as a way to prepare them to teach during graduate school, instead of inspiring them to teach high school. The TIR helped the department navigate the transition to studio teaching, and now that is also institutionalized.One of BU’s PhysTEC aspirations was to establish a teaching track for undergraduates (in addition to the existing pathway of a Masters of Arts in Teaching), which passed the faculty vote in physics but was ultimately voted down by the School of Education. The PI in physics identifies the failed pathway as his biggest regret, but does not believe he could have done anything to change the outcome.While the TIR believes faculty in the physics department are supportive of his efforts, getting widespread participation in secondary outreach efforts is difficult. BU continues to struggle to recruit undergraduates who fail to see teaching as a prestigious and lucrative career, considering the high tuition for their degree. The co-PI in education developed a two-credit course modeled after UTeach to give undergraduates an early teaching experience but enrollments were low.When we first interviewed representatives from BU, the continuity of the TIR, who led the majority of PhysTEC efforts, was contingent on finding an external funding source beyond the Noyce scholarship and digital learning grant related to developing the online AP physics course. Since then, the faculty champion worked with the TIR to secure NSF funding to support his continued employment. The PIs have asked for departmental funds to sustain on-campus networking events with their extensive physics teacher network.maintaining teacher production but only moderately institutionalized because funding to keep the TIR, who leads recruitment efforts, is dependent on external funding. The undergraduates at the university and many faculty within the department do not view teaching as a prestigious career, the department is not particularly eager to embrace secondary teacher preparation as part of its mission.Before PhysTEC, BU only graduated one physics teacher per year. At one point during the grant, they were producing more than five per year. Now, BU graduates 3–4 teachers per year. We characterize BU as California State University Long Beach is a public, urban, masters’ institution with an excellent reputation for teacher education historically. California State University Long Beach (CSULB) produces about 400 elementary school teachers a year and has more applicants than they can accept. The PI in physics became interested in the project because she was disappointed with the quality of her son’s science education and thought it might help remedy the low number of physics majors (around ten per year). She joined forces with the Chair of Science Education and an undergraduate advisor who was a popular instructor and advocate for instructional technology to apply for PhysTEC funding.Because the Chair in Science Education had extensive experience with educational grants, she recommended limiting PhysTEC activities to high impact, sustainable activities with low ongoing costs. Because of this, almost all activities were sustained. The Chair of Science Education, who had the most experience with teacher preparation projects explains,We picked… things that I think had the biggest impact for our local teacher community and for our prospective teachers and that opportunity to get the networking… For our future teachers can be in the same room with the experienced teachers on a somewhat regular basis, they’re starting to build their network. They’ve got classrooms they can go to and they have to do early fieldwork experiences. Now they know five or six or ten physics teachers in the area.Specifically, they started a biannual open house—attended by the Provost, monthly demonstration-sharing lunches with local teachers and created a monthly newsletter in collaboration with the Southern California American Association of Physics Teachers. Initially, it took time and effort to seek out secondary teachers and create interest in these activities. However, once the networks were established, the logistics for Open House events were arranged, the format of the demonstration sharing lunches were determined, and it required minimal resources to continue them, costs that were easily picked up through some Sumner Foundation funding once the grant ended.Since recruiting a qualified full-time TIR was difficult, they chose to have a part-time TIR and did not use the full comprehensive funding of $100,000/year. CSULB was the only site in this sample that was awarded the grant before PhysTEC introduced the matched institutional funding requirement. The part-time TIR started two new courses: a LA pedagogy course and a physics pedagogical content knowledge course. While PhysTEC usually recommends full-time TIRs, CSULB chose to accept less grant money for a part-time position, which also made it easier to recruit teachers. The champion from CSULB also believed PhysTEC award helped distinguish physics in the faculty of science and helped secure support from administration. For example, when the grant ended, the position continued with Math Science Teacher Initiative funds from the Chancellor’s office.sustain all the major components with slight modifications. Specifically, they reduced the frequency of the Open House and newsletter. Looking back, the Physics Chair and PhysTEC champion reflects that their highly focused application made it possible to accomplish activities and continue to do those things after PhysTEC ended. For CSULB, setting limited, high-impact but realistic targets resulted in extremely high sustainability.CSULB was able to The hardest part of maintaining PhysTEC efforts is funding for 20 LAs salaries per semester. With encouragement from PhysTEC, CSULB took data to show that students connect strongly with LAs, pass rates went up, and attitudes toward science improved. This data has helped secure university funding for now, but the program may be at risk for being scaled down.Overall, the PhysTEC champion at CSULB—who was Chair until a year ago—believes the department supports all main components and believes it “wouldn’t come down easily” especially because it has led to an increased number of physics majors.CSULB has been able to maintain the programmatic efforts started during their PhysTEC funding period, graduating around five physics teachers per year, significantly more than before the grant. They sustain their part-time TIR through teaching responsibilities with the LA and physics pedagogical content knowledge courses. We characterize them as institutionalizing changes and capacity building with PhysTEC through on going recruiting, outreach to local schools, and continued employment of the TIR through LA and physics pedagogy courses.Middle Tennessee State University (MidTenn) is a public, suburban institution with moderate research activity. The economic recession and impending budget cuts threatened the existence of MidTenn’s physics department. Introductory courses had high drop, failure, and withdrawal (DFW) rates (around 55%), physics majors were few, and they had not graduated a physics teacher in over 20 years. Within the physics department, there was an obvious need and urgency for change  who could fulfill that role. MidTenn’s PhysTEC efforts involved course reform, adding a departmental concentration in physics teaching, marketing materials, LA program, and a program where high school students visit the campus.The TIR was deemed a failure because the UTeach responsibilities occupied most of their time and their location on the UTeach part of campus meant they were not a physical presence for physics students. The full teaching degree requires a whole year of student teaching before you can qualify for licensure, which makes it a lengthy process for students. Many students jump into teaching and take classes at night, leading to an underestimate of the number of graduated physics teachers.Higher administration continues to be supportive especially since the reformed classes have contributed to physics having the lowest drop, failure, and withdrawal rates within the faculty of science. The president of the university does not know much about the drop, failure, and withdrawal rates but noticed the PhysTEC site visits and acknowledged physics as a “program of distinction for undergraduates”.MidTenn maintained many of these activities including the course transformation and LA program with university funds. The marketing aspect was maintained because once the materials and strategy were developed, the resources remained applicable. The PhysTEC PI is currently the department chair. He spends time inviting high school teachers to campus, in the hopes they will recommend the program to their students. While he believes most PhysTEC activities would continue, he does not know who else in the department would spend time and energy on recruiting.maintaining teacher production (2–3 per year), institutionalization of LA program and course reforms, and moderate capacity building since PhysTEC activities have continued through departmental and UTeach activities.We characterized MidTenn as Towson University is a public, suburban, masters’ institution. The Chancellor of the University of Maryland wanted to triple the numbers of science teachers. One of the PIs was one of the first science educators embedded within the physics department, hired 11 years of secondary education experience. This educator partnered with an elementary science education colleague in the department who had started an LA program years prior. The department chair had been active in the AAPT, taught using innovative strategies, and supported PhysTEC efforts as a way to increase physics majors. Their PhysTEC efforts included hiring a TIR, marketing materials, and increasing student recruitment.While it was difficult to recruit a TIR, they eventually found a veteran teacher who stayed for 2 years, was respected by and connected to local teachers, and extensively recruited potentially secondary education majors. The TIR was respected within the physics department so physics faculty started sending students to talk to him about a future in physics teaching. The TIR offered professional development around Physics with Inquiry, a pedagogical innovation, and started a newsletter with local teachers.While they were successful in increasing the number of physics graduates prepared to teach, PhysTEC efforts have been challenging after grant funding ended. The university adopted UTeach Brainard  as a topWhen PhysTEC ended, supposedly physics would have access to a Master Teacher as part of the sustainability plan. However, UTeach at Towson lacked a Master Teacher with a physics emphasis, so all the recruiting and marketing activities were essentially discontinued.Their LA program, which existed prior to the PhysTEC funding, was sustained through funding from the Dean. However, without a TIR to talk to LAs about high school teaching, the interviewees comment that the LAs do not tend to become secondary education majors.not maintaining production of physics teachers because numbers have dropped to pre-PhysTEC numbers. We do not consider PhysTEC activities institutionalized, since without a TIR, LAs are not associating teaching experiences with teaching high school. The TIRs play an important role as a physics teacher ambassador, a positive role model for teaching who can explicitly encourage students to pursue it as a future career  used the PhysTEC grant to fund a TIR, who focused a large fraction of their efforts on the LA program. The TIR recruited and hired LAs, teaches a science pedagogy course for LAs, and successfully helped get course credits from the LA pedagogy course to count toward the Master’s degree in teaching. Data was useful to advocate to the dean to support the LA program. UMN also developed a new program “DirecTrack to Teaching” which allows undergraduates to start taking courses to lead to certification.While students enjoy being LAs, it can be difficult to find professors who want LAs in their courses and many LAs are not interested in high school physics teaching.Additionally, faculty attitudes toward teaching are variable and there is a perception that “students do not attend UMN to become teachers”, as one interviewee stated. This attitude is shared by some faculty and administrators at the institution, including the director of undergraduate studies who had some reservations about recommending high school teaching as a career.The challenges were succinctly described in one of the final reports: “We are philosophically committed to continuing support of the teachers prepared in our program. However, our resources are currently stretched very thin, both in terms of funding and time.” The PI was writing grants in collaboration with other departments in hopes of getting funding to sustain the LA program and the TIR position.When we interviewed the PI, the TIR position had been maintained with department funds by paying them to support the LA program. However, funding was extremely uncertain and it remains unclear whether efforts would continue without the PI. The PI is passionate about science education and outreach, but the departmental culture does not widely embrace these as a primary part of its mission.Thus, we characterized this as maintaining production of teachers and unstable institutionalization because PhysTEC activities and the TIR employment is contingent on continuing to find funding sources.Virginia Polytechnic Institute and State University (VTech) is a public, rural, land grant, R1 institution. At VTech, several senior faculty members in leadership positions cared about high school teaching and it aligned with institutional priorities. Around the time of application, the VTech president signed a letter to former President Obama alongside other land grant universities promising to address the nationwide shortage of STEM teachers. The Dean of College of Science, and a former chair of the physics department, started the Physics Outreach Team in the mid-1990s. This team continued as an established part of departmental activities, creating a precedent of valuing outreach. The Department Chair applied for PhysTEC funding with a program leader for Science Education. When this Department Chair left the university to pursue a leadership position elsewhere during the grant, they passed PhysTEC responsibilities to another senior physics faculty member. The new PI in physics had a long friendship with the colleague in education who had fostered his interest in teacher preparation so there was a strong collaboration between the physics and education departments. Although their PhysTEC team lost a leading member, there was no significant harm done because of the collective leadership.Even before PhysTEC, the collaboration between departments helped support students transition from undergraduate physics to graduate teacher licensure program by funding a Masters in Teaching through a Teaching Assistantship. There were two champions within the physics department so efforts continued seamlessly even when the principal applicant moved to another institution.The PIs on the grant credit much of their success to the TIR, who was a VTech alumna who had taught for a few years prior to returning to VTech as the TIR. Before PhysTEC funding ended, she became a full-time instructor in the department in introductory and outreach courses. Between outreach and LA opportunities, students are exposed to a wide variety of early teaching experiences available to any physics student. The TIR continues to be a highly visible resource for any students contemplating a career in high school physics teaching. The university switched to studio teaching, and the TIR supported the transition. Administrative support simplified financial transitions and secured the TIR’s continued employment as a departmental instructor.Since VTech has a rural location, it is difficult for teachers to visit the university for Teacher Advisory Group meetings. Now that the TIR has more instructional duties, they have less time to visit local schools. It was also slightly difficult to recruit LAs because of busy schedules.maintaining teacher production because it continues to meet/exceed the rate produced during the grant. VTech has institutionalized PhysTEC efforts and built capacity by internalizing costs, hiring the TIR as a full-time instructor, and supporting potential teachers through Teaching Assistantships as they earn their Master’s in teaching.We categorized VTech as In Table The following section highlights key themes regarding how sites did (or did not) create fertile soil for sustainable change. As mentioned by King , fertileOur study reinforced Scherr et al.’s  conclusiSites can strategically leverage this affect by consciously tying “new” activities to essential departmental tasks. For example, both VTech and BU started Studio Physics and an LA program around the same time as PhysTEC. For both institutions, the introductory course transformation helped increase student engagement and interest in physics in general—cutting failure rates and increasing the number of majors. Attracting more students into physics creates a bigger pool of potential teachers; however, the number of teachers does not automatically increase linearly with the number of majors. For example, undergraduates may enjoy serving as LAs but without explicit encouragement, they may not associate that with a career in high school teaching.sufficient to increase the number of potential teachers, it can be leveraged for sustainability purposes. Delivering high-quality undergraduate courses falls directly within a department’s mission, unlike producing secondary teachers. If the TIR’s expertise can be utilized to lead these efforts, it can cause an increased reliance on their expertise. Specifically, the TIR at VTech had a big role in starting studio and the LA program. She recruited undergraduates through multiple channels and taught courses in physics, physics pedagogy, first year experience, and physics outreach (designed to improve communication skills regardless of career path). She advised the Society of Physics students and hosted pizza parties where undergraduates can learn about outreach opportunities. She had a highly visible role in the department, impacted essential departmental activities, and VTech made her a full time instructor before matched funding ended.While introductory course transformation is not In contrast, the contributions of the BU TIR could be considered “auxiliary” to typical departmental duties. He did provide advice for starting studio and provided mentoring for the LAs, but he spent most of his time recruiting career changers into the Masters of Teaching program and developing a network of over 200 local teachers. While these accomplishments are impressive, a department head at a research institution may not find recruiting future teachers vital to university operation. The department did not offer to make his role permanent. When PhysTEC funding ran out, the continuation of his position was dependent on finding external funding.Both VTech and BU were successful in producing over five physics teachers a year, but because the VTech TIR’s activities were tightly tied to essential, undergraduate activities and eliminated the potential obstacle of her being auxiliary rather than essential, it was easier for VTech to justify integrating her as a full-time instructor in the physics department.A strategic initiative that enhances the established organizational culture has a high probability of success. When the change conflicts with the organizational culture, culture can diminish the power of the change initiative  to highlight physics teacher preparation as a valued departmental activity. Not coincidentally, the three most successful sites  had PhysTEC PIs that served as department chairs at some point during the grant. As departmental leadership, PIs have the influence and funding to help change departmental norms to value physics teacher preparation. For example, the current Associate Chair of Physics at VTech states,None of the sites that struggled with sustainability had a PhysTEC champion in a leadership position, and the champion might have lacked credibility due to disciplinary research. At BU, the champion in physics was a Master Lecturer. At Towson, the co-PIs were science educators, one of whom was a new employee. At UMN, the champion was research faculty in Astrophysics but did not have a leadership role. Seymour (2001) shows that the research credentials of the champion are more effective in persuading other faculty about the worth of a teaching innovation than either the data that supports its efficacy or video that demonstrates its merits or shows that students like it.Successful sites had clear communication to ensure a unified vision , or as a panacea, but in the ability to implement them selectively and adaptively in order to best match the culture and environment of the organization (p. 316).Throughout the paper, we included insights from a variety of change literature under the belief that the field needs to consider reforms from multiple frameworks that were developed in diverse contexts. As Sidorko  points oThe case studies and change model presented here are included so reformers increase their awareness of possible ways to cultivate fertile ground for sustainable change from the start.While our data was acquired over time and we have tried to ensure that our data reflects multiple perspectives, these case studies reflect a small number of people and events. While we spoke to multiple people at each institution, the perspectives reflected here are still limited.A central claim of this paper is that sustainable change requires a climate that aligns with the reform. Successful sites realized PhysTEC could be a solution to institutional needs, such as producing more majors, increasing the quality of undergraduate education, and providing more career options for graduates. This climate includes both values and culture. Values are explicit or implicit expressions of the “desirable” that influence individuals’ means and ends of action . Reform activities that simultaneously benefit undergraduate education and physics teacher preparation are easier to sustain, partially because of alignment around a unified vision that encompasses the reform.The PhysTEC grant provided resources to sites that were primed to flourish. The grant brought financial resources, prestige, and support to bring physics teacher preparation to the foreground. The selection criteria funds sites already have some ingredients for success: a departmental champion, support of the physics department head, a collaboration with the School of Education, and a commitment from higher administration in the form of matched funding. PhysTEC supports sites in defining their vision, providing advice and connecting sites in a supportive community.A common vision and collaboration is important for widespread buy-in. The weakest point for sustainability of these programs seems to be attracting new champions to the cause, so a “team effort” to reform is advantageous  does not appear to be a significant improvement over what was reported by Scherr et al. . In ScheAlthough this study examined the PhysTEC reform, we believe results may apply to sustaining other reforms that hope to advance undervalued activities at universities. A key message is change agents should focus on seeding reform efforts at sites where further growth and propagation is likely. The change must align with the existing departmental and institutional culture and a well-respected champion can help further advance the change. Activities should be undertaken strategically to support existing departmental activities as well as furthering the reform effort.


UCH-L1）是近期研究较多的一类癌基因，在多种恶性肿瘤如食管癌、肺癌、乳腺癌等中均有异常表达和活性增强。本研究旨在探讨脂质体转染UCH-L1 siRNA对人肺腺癌H157细胞增殖、凋亡的影响。泛素羧基末端水解酶-1（ubiquitin C terminal hydrolase-L1, UCH-L1基因的siRNA片段，通过脂质体转染到H157细胞内，显微镜下观察其形态学变化；流式细胞术检测各组细胞周期变化和凋亡指数；RT-PCR检测UCH-L1 mRNA的表达；Western blot检测UCH-L1蛋白表达。人工合成抑制转染siRNA后的H157细胞，细胞增殖受到显著抑制，细胞出现明显凋亡，UCH-L1 mRNA表达显著下降，UCH-L1蛋白水平显著下降。UCH-L1基因可以显著抑制H157细胞增殖，促进调亡。UCH-L1可能成为肺癌基因治疗的一个新靶点。应用RNA干扰靶向抑制


King Vision and McGrath MAC video laryngoscopes (VLs) are increasingly used. The purpose of this study was to evaluate the performance of nasotracheal intubation in patients with predicted difficult intubations using non-channeled King Vision VL, McGrath MAC VL or Macintosh laryngoscope by experienced intubators.Ninety nine ASA I or II adult patients, scheduled for oral maxillofacial surgeries with El-Ganzouri risk index 1–7 were enrolled. Patients were randomly allocated to intubate with one of three laryngoscopes . The intubators were experienced with more than 100 successful nasotracheal intubations using each device. The primary outcome was intubation time. The secondary outcomes included first success rate, time required for viewing the glottis, Cormack-Lehane grade of glottis view, the number of assist maneuvers, hemodynamic responses, the subjective evaluating of sensations of performances and associated complications.p < 0.001). Both King Vision and McGrath groups had a 100% first attempt success rate, significantly higher than Macintosh group . The laryngoscopy time was comparable between King Vision and McGrath group (16.7 ± 5.5 s vs. 15.6 ± 6.3 s) and was shorter than Macintosh group  also. Compared with Macintosh laryngoscope, Glottis view was obviously improved when exposed with either non-channeled King Vision or McGrath MAC VL (p < 0.001), and assist maneuvers required were reduced (p < 0.001). The maximum fluctuations of MAP were significantly attenuated in VL groups . Most device insertions were graded as excellent in McGrath group, followed by Macintosh and King Vision group (p = 0.0014). The tube advancements were easier in VLs compared with the Macintosh laryngoscope (p < 0.001). Sore throat was found more frequent in Macintosh group compared with King Vision group (p < 0.05).The intubation time of King Vision and McGrath group was comparable (37.6 ± 7.3 s vs. 35.4 ± 8.8 s) and both were shorter than Macintosh group  contains supplementary material, which is available to authorized users. The video laryngoscope (VL) has been well established as an approach in airway management for patients with difficult direct laryngoscopy –5. HowevThe success of a VL assisted intubation depends on multiple factors, such as blade design ; quality of the image on the monitor, as well as the experience of the intubator , 10. RecA recent systematic review comparing VL versus DL for NTI showed that VL is particularly beneficial for patients with difficult airways . Howeverclinicaltrials.gov (NCT03126344). Written consents to participate were obtained from all participants after enrollment. Our study was adhered to the applicable Consolidated Standards of Reporting Trials (CONSORT) guidelines.This trial was approved by IRB (2017–308-T228) from Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and registered at 2)] were excluded from the study.Consecutive patients, between 18 and 60 years old with American Society of Anesthesiologists (ASA) classification of I or II, and requiring NTI for elective oral and maxillofacial surgery, were screened in the Preoperative Evaluation Unit of our institute. Prediction of difficult intubation is graded by El-Ganzouri multivariate risk index (EGRI) based on seven parameters  , 18  was used as control.Patients were asked which nostril was clearer. If both sides were equal and the surgeon had no objection, the right nostril was chosen . Patient2 (pulse oximetry), and end expiratory carbon dioxide. A Bispectral (BIS) index sensor was attached to the patient’s forehead in conjunction with the BIS Monitor. Cannulation of right radial artery was performed under local anesthesia for invasive blood pressure monitoring.No premedication was administered. Lactated Ringer’s solution infusion was started intravenously to deal with the fluid loss from the overnight fast after entering the operating theatre. A standard preparation was then performed, including heart rate (HR), lead II ECG, SpOAll patients were preoxygenated by a facemask in the position of neutral. Prior to anesthesia induction, the nasal mucosa was well prepared with 1% tetracaine hydrochloride jelly for 2 min and five drops of ephedrine hydrochloride nitrofurazone (containing approximately 2 mg ephedrine) in all patients. Baseline hemodynamic data were recorded by an investigator after a stabilization period of 10 min.The nasotracheal tube used was reinforced endotracheal tube  and was well lubricated with 1% tetracaine hydrochloride jelly. Dosing of induction medications was given at the discretion of the attending anesthesiologists. Induction agents included midazolam (0.02 mg/kg), propofol (1.5~2 mg/kg) and fentanyl (2 μg/kg). Upon loss of consciousness and jaw relaxation, manual ventilation was tested. If manual ventilation was available, cissatracurium besilate (0.15 mg/kg) was administrated and post induction values were recorded 3 min after induction. Unsuccessful manual ventilation led to study exclusion.The anesthesiologist tried to intubate when the Train of Four (TOF) count reached zero and BIS value decreased to 50. NTI was performed in a standard manner. First, a preformed ETT was inserted into the nostril and advanced to the posterior nasopharyngeal wall. Second, a laryngoscope blade was introduced into the mouth to expose the glottis. If it’s necessary, the BURP maneuver  on the thyroid cartilage was attempted to obtain good glottis visibility . And ult2 waves were appeared on the monitor. Since the time required for SpO2 to decrease during apnea was about 150 s ) was used to describe the parametric data. The number (percentage) was used to describe nonparametric data. Statistical analyses were performed with Prism 5.0 for Windows . Binary data for three groups were analyzed using chi-square test and each two groups were compared with chi-square segmentation method or Fisher’s exact test as appropriate. One-way analysis of variance (ANOVA) with post-hoc Bonferroni’s Multiple Comparison test was used to analyze parameter data for changes within groups. The Kruskal-Wallis ANOVA with post-hoc Dunn’s test was used to analyze ordinal data. A In total, 99 patients were enrolled in this study between June 2017 and January 2018 and both were significantly shorter than Macintosh group  in King Vision and McGrath groups were comparable and both were significantly attenuated compared with Macintosh group . Quality of view on display did not differ between King Vision and McGrath groups. The ease of tube advancement was comparable between King Vision and McGrath groups, and both were much better than Macintosh group (p < 0.001). There were no cases of desaturation and dental injury during NTIs. Sore throat was found more frequent in Macintosh group compared with King Vision group (p < 0.01). Occurrence of bleeding and hoarseness seemed more frequent in Macintosh group, but failed to show significance. These symptoms were minor and ceased spontaneously without intervention.Results of the subjective sensations between devices were listed in Table Although many studies about indirect laryngoscopes were carried out, only two randomized controlled trial, to our knowledge, have compared the VLs (Airtraq and C MAC respectively) with Macintosh laryngoscope in patients showing predictors of difficult nasal intubation , 17. KinOur main result was that the time to successful NTI with both VLs was significantly faster than with Macintosh DL. The intubation time mainly comprises two parts: time to view the vocal cords and time required for tube passage through glottis. Firstly, we confirmed both non-channeled King Vision and McGrath MAC VL significantly shortened the laryngoscopy time in predicted difficult patients, which was certainly a reason for a shortened intubation time. The result showed less association between predictors of difficult intubations and glottis exposure using non-channeled King Vision or McGrath MAC VL than using Macintosh DL. Secondly, laryngoscopy with non-channeled King Vision or McGrath MAC caused less anterior elevation of the larynx than invasive direct laryngoscopy because airway axes alignment was not needed. This might provide a more direct route from nasopharynx to glottis and therefore ease advancing the tube into the trachea. In current study, we confirmed it was easier to advance the ETT through the glottis, accordingly less frequency of assist maneuvers was required in VL groups. Thirdly, when doing oral intubation with VLs, we often bend the styletted tracheal tube to a greater degree (‘hockey stick’ like) to follow the curvature of the video blade, which always hinder stylet removal and increase intubation time , 25. HowThe predictors of difficult airways used in our study are reliable . To compBoth non-channeled King Vision and McGrath MAC VLs improved the Cormack-Lehane grade significantly which was the main superiority of VLs. St Mont et al. demonstrated first attempt success rate of NTI by Airtraq was 94% in predicted difficult airway . HazarikTo avoid missing any hemodynamic response, invasive blood pressure was used in current study. In addition, the maximum fluctuation of MAP was chosen to reflect the hemodynamic change. This could partially explain why our data were different from previous study . During Most participants felt McGrath blade insertions were easiest. That was because of its slim design as we discussed before. McGrath MAC was lightest and more portable than the others. The monitor could be adjusted to an optimized angle for intubation. Although it was claimed that the King Vision VL could provide a better vision condition, we did not see the difference. Quality of view on display did not differ between King Vision and McGrath VLs. Reducing the usage of assist maneuvers, fewer demanding of the physical workload and lower anterior pressure exerted on the soft structures could be linked to reduced sore throat and hoarseness occurrences in both VL groups. The King Vision group had the fewest cases of sore throat. We guessed the length and angle of the King Vision non-channeled blade might be more beneficial to exposure of glottis compared with Macintosh like blade, and accordingly less workload was required. However, oral surgical procedures might confound these results since they tend to cause similar symptoms, and further study is required.Some limitations of our study should be considered carefully. First, neither the intubator nor the independent observer could be blinded from the groups. However, we have minimized adverse effects by defining robust outcome measures. Second, if we compared both Cormark-Lehane and POGO scores, our results should be more convincing. Third, our results might be biased by the variable experience of the intubator with different laryngoscope. It is believed that intubation with VL require a complex hand-eye-coordination competencies which grow with a learning curve . On the In summary, we observed that NTIs with non-channeled King Vision and McGrath VLs in the setting of predicted difficult intubations resulted in shorter intubation time, higher first success rate, better qualities of glottis view, attenuated hemodynamic responses, and fewer incidences of side effect compared with Macintosh DL. These data provided evidence that NTI using King Vision and McGrath were comparable, and both devices were superior to Macintosh DL in managing the difficult intubations.Additional file 1:El-Ganzouri risk index. (DOC 44 kb)


Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder. Major depressive disorder (MDD)Ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1) is an enzyme that hydrolyzes small C-terminal adducts of ubiquitin and generates ubiquitin monomer . UCH-L1 17In the present study, using 2D-PAGE-based proteomic analysis, we identified sixty-three stress-responsive proteins in the hippocampus of chronic restraint stress mouse model and found the most significant changes in UCH-L1. We focused on oxidative modifications of Cys of UCH-L1 and suggest that oxidative regulation of UCH-L1 plays a key role in neuroprotection in major depressive disorder. We further demonstrated that the overexpression of wild-type (WT) UCH-L1 in HT-22 hippocampal cells afforded protective effects against oxidative stress, whereas its Cys to Ser mutants abolished this effect and Cys to Asp mutants behaved like WT. These results reveal a novel neuroprotective function of UCH-L1 via stress-induced Cys oxidative modifications.Chronic restraint stress mouse model was produced as described previously . Six-weeOn the last day of stress treatment, the animals were anesthetized with intraperitoneal injections of Zoletil 50  and Rompun  and transcardially perfused with chilled heparin/PBS solution  using a 20 ml syringe to remove red blood cells in the tissue. Whole mouse brains were separated, and the hippocampi were dissected using a needle of 1 ml syringe under microscopic view. Tissue samples were stored in a −80 °C deep freezer until use for proteomic analysis.3VO4, trichostatin A, sodium butyrate, PMSF, acetone, urea, iodoacetamide, bromphenol blue, ACN, formic acid, methanol, ethanol, xylene, and bis-acrylamide from Sigma-Aldrich  unless otherwise mentioned.Antibodies used in this study were purchased from following manufacturers: anti-UCH-L1 , anti-DPYL2 , anti-ARPC5 , anti-PRDX2 , anti-Myc  and anti-tubulin . Anti-HDHD2 antibody was custom-made from AbClon . Dulbecco's Modified Eagle Medium (DMEM), F-12 medium, Opti-MEM, FBS, trypsin and Pen Strep for HT-22 cell culture were purchased from Gibco Life Technologies , Eagle's Minimum Essential Medium (EMEM) was from WELGENE , EDTA, DTT, and Tris from Duchefa , SDS from Amresco , and all other chemicals used in proteomic analysis including HEPES, NaCl, KCl, Triton X-100, glycerol, protease inhibitor mixture, Nam HEPES, pH 7.4, 50 mm NaCl, 40 mm KCl, 1% (w/v) Triton X-100, 10% (w/v) glycerol, 0.5% (v/v) protease inhibitor mixture, 2.5 mm Na3VO4, 5 mm trichostatin A, 5 mm sodium butyrate, 1 mm PMSF, 1 mm EDTA). The lysates were centrifuged at 20,000 × g for 30 min at 4 °C. The supernatants were collected and centrifuged again under the same condition. Protein concentration of each sample was determined using BCA assay kit . Proteins were precipitated by adding acetone containing 1% DTT to cell lysate, vortexing the tube and incubating samples at −20 °C for 1 h. After centrifugation at 10,000 × g for 5 min at 4 °C, the pellets were collected and dissolved in tissue lysis buffer by incubating at room temperature for 2 h with vortexing at 30 min intervals. Proteins (150 μg) of each sample were loaded on a rehydrated strip gels , 18 cm, pH 4–7). 2D-PAGE was performed as previously described  SDS, 30% (w/v) glycerol) added with 65 mm DTT and the second equilibration buffer supplemented with 2.5% (w/v) iodoacetamide and bromphenol blue. SDS-PAGE was performed using PROTEAN II xi 2-D cell apparatus (BIO-RAD) and all gels were simultaneously stained by silver staining method in the same tray.To compare the protein expression profiles of control and stressed mice and to study specifically post-translationally modified proteins, we employed 2D-PAGE for protein separation. Whole hippocampal protein extracts from each sample were obtained by grinding the tissue with liquid nitrogen and lysing it with lysis buffer  were considered statistically significant and were targeted for protein identification.For image analysis, gel images were obtained using Image Scanner III . Progenesis SameSpots  auto-processing 2D-PAGE gel analysis software was used for gel image alignment, normalization, spot detection, quantification of spot intensities and statistical analysis. Spots showing at least 1.3-fold difference in three replicates of each group  as previously described with a few modifications . ModificMus musculus (house mouse) and Homo sapiens (human) for overexpressed UCH-L1 samples . Trypsin was the only protease used to generate peptides. Maximum number of 1 missed cleavage was permitted. No fixed modifications were considered. As variable modifications, acetylation and formylation of Lys, N-terminal pyroglutamylation of Gln and Glu, oxidation of Met, carbamidomethylation of Cys, acrylamide adduct propionamide of Cys and phosphorylation of Ser or Thr were considered for identification of 2D-PAGE protein spots. For PTM analysis, we employed SEMSA  PTM searching algorithm  were considered significant. Ions score was calculated as −10 x Log(P) (P: the probability that the observed match is a random event) and automatically provided as Mascot result. Regarding the protein identification result, the best match with the highest score among identified results was used as the identification result for the spot. All spectra were manually verified to sort out meaningful results. For PTM search results, modified peptides with minimum total mascot score 30 was considered good enough for PTM analysis, but if the same peptide with the same modification was found in other spots, the result was included even if the score was lower than 30 for comparison. PTM results were verified not just by score threshold, but by checking each spectrum carefully. Other PTMs with low scores were presented in supplemental Table S1 for ambiguous PTMs. In the case of peptides that could not be annotated accurately, they were classified as ambiguous PTMs  even if the scores were high.For further processing of raw mass spectrometry data, peaklist files (.pkl) were generated using Protein-Lynx Global Server (PLGS) 2.3 data processing software (Waters Co.). Peaklists were searched against protein sequence databases NCBInr  and SwissProt  using a global search engine Mascot . Taxonomy filter for 2D-PAGE samples was ed SEMSA  methodollgorithm . For Cyswww.pantherdb.org)  .5 cells were lysed in 75 μl of SDS gel sample buffer. The amount of overexpressed UCH-L1 and tubulin was detected using anti-Myc and anti-tubulin antibodies. Anti-UCH-L1 antibody was used to detect endogenous UCH-L1.Hippocampal proteins from control and stressed mice were separated by mini 2D-PAGE and transferred to hydrophobic PDVF membrane (Millipore). Distributions of UCH-L1, DPYL2, HDHD2, ARPC5, and PRDX2 spots were verified by Western blotting using each antibody. Amersham Biosciences ECL Prime Western blotting detection reagent  and LAS-3000 imaging system  was used to gain chemiluminescent signals and Multi Gauge V3.0 (Fujifilm) was used for image analysis. For 1D Western blot analysis performed in HT-22 cell experiment, 5 × 102O2/Methanol; 3:200, v/v) for 15 min. Following epitope retrieval and immunostaining were conducted using anti-UCH-L1 antibody (Millipore #Ab1761-I) and Bethyl immunohistochemistry accessory kit  according to the manufacturers' instructions. In brief, brain sections were incubated in 96 °C with retrieval buffer for 20 min to retrieve epitopes. After treating with blocking solution for 15 min at room temperature, brain sections were incubated overnight at 4 °C with anti-UCH-L1 (1:100) or antibody diluent solution (for negative control). Anti-rabbit secondary antibody and 3,3′-diaminobenzidine (DAB) staining for 1 min were used to detect UCH-L1. Hematoxylin counterstaining for 2 min was used to stain cell nuclei. Slides were then treated with IHC-Bluing solution, dehydrated in ethanol, cleared in xylene and covered with coverslip using permanent mounting media . Image analysis was performed using Axio Scope.A1 microscope  and AxioVision software version 4.9. (Carl Zeiss).Paraffin-embedded coronal sections of mouse brains were kindly provided by Professor Kyunglim Lee (Ewha Womans University). Brain sections were deparaffinized by incubating slides in Xylene, three times for 10 min each, and washing with 100% ethanol three times for 5 min each. Endogenous peroxidase quenching was performed with quenching solution . Cells were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin . SH-SY5Y cells were purchased from ATCC. Cells were maintained in 1:1 mixture of Eagle's Minimum Essential Medium (EMEM) and F-12 medium supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. All cells were incubated in a 37 °C incubator containing 5% COHuman UCH-L1 clone inserted into pcDNA3.1/myc-His(-) A vector  was obtained as previously described . Cys to m. Cell medium was changed 6 h after transfection to reduce cell toxicity. Following WST-1 cell viability assay was performed 72 h post-transfection.For transient overexpression of Myc-UCH-L1 and its Cys mutants, Lipofectamine 2000 (Invitrogen) reagent was used to transfect HT-22 cells in accordance with the manufacturer's protocol. Empty pcDNA3.1/myc-His(-) A vector was used as control plasmid and transfected to HT-22 cells in parallel with the plasmids carrying UCH-L1 DNA constructs. In brief, cells were plated on 60 mm culture plate 24 h before transfection and treated with 3 μg of plasmid and 7.5 μl of Lipofectamine 2000 reagent diluted in Opti-MEM. Cells were incubated for another 24 h before used for further analysis. For silencing UCH-L1 gene in SH-SY5Y cells, UCH-L1 small interfering RNA (siRNA) and control siRNA were obtained from Bioneer . Cells were transfected with Lipofectamine RNAiMAX reagent (Invitrogen) following the manufacturer's protocol at a final siRNA concentration of 100 n5 cells/60-mm plate and grown for 24 h. Control plasmid pcDNA3.1/myc-His(-) A vector, and plasmids carrying wild-type (WT) and Cys mutants  of UCH-L1 were delivered into the cells respectively using Lipofectamine 2000 transfection reagent. At 24 h post-transfection, cells were washed twice with 1 ml Hanks' Balanced Salt Solution (HBSS) to remove serum. Cells were treated with 0 or 0.3 mm of H2O2 in HBSS for 2 h . Real-time cell growth was measured using xCELLigence RTCA instrument . Briefly, 50 μl of DMEM-CM/well in 96-well RTCA E-plate was used to read the blank. After 2 h of H2O2 treatment, H2O2 was removed by washing cells sequentially with 1 ml HBSS and 1 ml PBS. Cells were trypsinized and 1 × 104 cells in 100 μl DMEM-CM were added into the well containing 50 μl DMEM-CM. Cell index was monitored at 15 min intervals for 72 h. All samples were tested in triplicate.HT-22 cells were seeded at a density of 5 × 105 cells/60 mm plate) were plated and transfected with control and UCH-L1 siRNA at 24 h after seeding. At 48 h after transfection, cells were transferred to 96-well plate (1 × 105 cells/well) and grown for 24 h. At 72 h after transfection, cells were treated with various concentrations of H2O2  for 2 h at 37 °C in a 5% CO2 incubator as previously described. After 2 h, HBSS containing H2O2 was replaced with 50 μl culture medium and 5 μl WST-1 reagent  per each well was added. Cells were incubated for 1 h in the incubator  and absorbances at 450 nm and 690 nm were measured using SpectraMax 190 Microplate Reader  and SoftMax Pro 5.4.1 software (Molecular Devices). OD450–690 was used as cell viability index and data were normalized to viability of cells treated with 0 mm H2O2. UCH-L1 gene silencing was confirmed by Western blot analysis.SH-SY5Y cells  and stressed mice that were chronically restrained for 9 days (n = 3) or 14 days (n = 3). Two biological replicates were performed. 2D-PAGE was performed twice  in one animal experiment with varying amounts of proteins loaded and remaining samples were used for further verifications. Because chronic restraint stress causes extreme pain to the mouse, we used a minimal number of animals to receive IACUC approval. We chose three animals for each group because that is the minimum number needed for statistical analysis. Twelve is also the maximum number that can be used to minimize errors in the handling of 2D-PAGE procedure and the silver staining procedure which is performed in a single tray. Based on our previous study  analysis was applied for samples satisfying normality and Kruskal-Wallis test (supported by pairwise comparisons using the Dunn-Bonferroni approach post-hoc test) was used for samples not satisfying the normality. Statistical analyses for 2D-PAGE spots were performed using Progenesis SameSpots  and other statistical analyses were performed using IBM SPSS Statistics .In this study, we used freshly isolated hippocampus respectively from unstressed controls . We found, in triplicated samples, that the expression of sixty-three proteins changed by more than 1.3-fold in the stressed mouse; 21 proteins decreased  and those of 42 increased  following exposure to stress .Recently we reported that C57BL/6 mice treated with restraint daily 2 h for 14 days exhibited depressive-like behaviors in the tail suspension test and forced swim test, and these behavioral changes lasted for more than 3 months . In lineFig. 1A) and protein interaction network analysis  with additional clustering methods , we found that proteins functioning in synaptic plasticity, neurite outgrowth and neuronal morphology (20.6%) were the most enriched, followed by proteins related to protein metabolic process (15.9%), protein aggregation, neurodegenerative diseases (7.9%), neural differentiation (7.9%), intracellular protein transport, vesicle mediated transport (7.9%), antiapoptotic role (6.3%), reactive oxygen species generation (6.3%), calcium-dependent regulation of neural plasticity (6.3%), carbohydrate metabolic processes (6.3%), haloacid dehalogenase (HAD)-type phosphatases (4.8%), extracellular antioxidant defenses (3.2%), generation of precursor metabolites and energy (3.2%), neuronal migration, cell motility (1.6%), and vitamin metabolic processes (1.6%). As shown in Fig. 2B, these identified proteins could be classified with respect to three major functions; neural plasticity, metabolic process, and protein aggregation, which is novel resource for molecular understanding of depression.Each of 63 protein spots was subjected for protein identification by peptide sequencing using nanoUPLC-ESI-q-TOF tandem mass spectrometry . Upon fuFig. 2http://pantherdb.org/) revealed that these proteins are related to cytoskeletal protein and hydrolase.We further classified the identified proteins into three groups on the basis of the kinetics of expression changes as shown in heat map . The firThe second group includes proteins that were up-regulated by cumulative restraint stress, and they were then divided into three subgroups (Type I to III). A group of 18 proteins including PDXK and DLDH (Type I) peaked on day 9 and slightly decreased thereafter. Therefore, we classified these proteins as “early response to stress” proteins. Another group of 8 proteins including VATB2 and DPYL2 (Type II) was classified as “late response” proteins, which showed gradual increases up to day 14. The third group including PGP and NCALD (Type III) was called the 'on and off' response proteins because the kinetic changes of these proteins showed an increase on day 9, but a sharp decrease on day 14. As shown in protein class analysis data, proteins in this group mainly consisted of cytoskeletal protein (Type I) and hydrolase (Type II and III).The last group contains ten proteins downregulated by stress, including KCRB and 1433T. They were at minimum expressions on day 9 or day 14. Most of these proteins are chaperone and membrane traffic proteins, implying that chronic restraint stress decreases chaperone activity. These results suggest that the amount and expression kinetics of the stress-regulated proteins were dynamically regulated by the length of stress treatment.To validate the proteins identified by proteomic analysis, hippocampal tissue lysates from control and stressed mice were examined by Western blot analysis on 2D-PAGE using specific antibodies . Mini-geA). Western blot analysis indicated that the spots of DPYL2 (B), HDHD2 (C), and ARPC5 (D) also moved to the acidic direction in chronically stressed samples. Using 2D-western, several populations of each protein were identified in addition to those found in silver stained gels . The stress blot of ARPC5  was obtained by high exposure to show the moved spot (acidic spot) more clearly, so the spot in the control position (basic spot) seemed to have a higher intensity than in control blot (upper panel). This difference was confirmed by the intensities of standard spots. Overall, these spot shifts examined by Western blots were consistent with the results seen on 2D-PAGE (B). Acidic protein spot shift to lower pI values could happen when Cys oxidation to sulfinic and sulfonic acid, phosphorylation at Ser, Thr and Tyr, or acetylation at Lys occurs. Employing MS analysis of UCH-L1, we concluded that the Cys oxidations were the major response induced by chronic stress , as the result on silver stained spots on 2D-PAGE (B). Because oxidized PRDX2 is readily removed , HDHD2 C, and ARnd ARPC5 D also mo 2D-PAGE B. Acidicc stress . These rc stress . Western 2D-PAGE B. Becaus removed , these s removed . These rAsupplemental Fig. S2). GAPDH was used as loading control because ACTB and ACTG increased almost 3-fold in the CRS mouse . Increased PDXK may cause change in vitamin B6 level which is associated with various enzymatic pathways, however, these changes were not dramatic as the UCH-L1 showed.Because TPIS, PGAM1, and PDXK identified in RS mouse . Only PDA) led us to identify at least four major UCH-L1 spots, which were re-numbered and presented in Fig. 5A. Spots 1  and 2 decreased on day 9 of stress treatment, whereas spots 3 and 4  increased. On day 14 of stress treatment, these changes were partially reversed, whereas the total intensity of the four spots remained unchanged.Extensive analysis of UCH-L1 spot shifts A led us i  (B), which is a major cause of spot shift. Dioxidation at Cys152, presented in supplemental Table S1, was classified as ambiguous PTM because the accurate spectra for that modification could not be annotated, but because Cys152 is the only Cys in that peptide (136NEAIQAAHDSVAQEGQCR153) we included the modification for the calculation in this case. Spot 1 from stressed sample had no identified cysteine modifications, probably because of the low abundance of spot 1, although we analyzed the PTMs of several pooled spots (stress spot 1). Other PTMs of UCH-L1 are summarized in  +64 Da)  occurred +64 Da) B, which supplemental Table S1). Generally, phosphorylation was equally well detected in all spots . Acetylations at Lys71 and Lys123 were not found in spot 1 . In the hippocampus, UCH-L1 (right panel) was expressed in pyramidal cells of the CA1  and CA3  and in granule cells in the dentate gyrus . Negative controls are in left panels. UCH-L1 appeared to be also expressed in microglia and astrocytes.To determine in which type of cells in hippocampus UCH-L1 exerts its activity, we examined the distribution of UCH-L1 in mouse hippocampal tissue . ImmunohFig. 6m H2O2 for 2 h, and real-time cell growth patterns were monitored employing xCELLigence RTCA apparatus . This protective effect was abolished in HT-22 cells overexpressing C90S, C152S, and C90/152S mutants. HT-22 cells overexpressing C152S and C90/152S mutants were more sensitive to oxidative stress than control cells, indicating that Cys152 plays a key role in mitigating oxidative stress. We reported previously . Because endogenous level of UCH-L1 in HT-22 cells is low compared with the overexpressed UCH-L1 , these protective effects on cell viability are mainly caused by overexpression of UCH-L1. These results suggest that redox sensitive Cys residues in UCH-L1 protect the hippocampal cells from oxidative damage.Because the kinetics of oxidation of UCH-L1 at Cys90 and Cys152 following chronic restraint stress are different , we invepparatus . As showeviously , that Cym of H2O2 for 1 h at 37 °C and cellular proteins were subjected to 2D-PAGE for MS analysis . Stoichiometry changes to oxidized spots of overexpressed UCH-L1 in HT-22 cells were lower than those of tissue ones . These low abundant oxidized spots can be identified as UCH-L1 with MS/MS, however, were not enough for PTM analysis. In our experience, PTMs of a protein are well identified with higher stoichiometry in tissues than in cultured cells, and PTMs of endogenous proteins are much better identified than transiently overexpressed protein in cells. supplemental Fig. S5 shows overall protein profiles of HT-22 cells , indicating the very low expression of endogenous UCH-L1 in this cell line.To investigate UCH-L1 Cys modifications in HT-22 cells as well as in the tissue, HT-22 cells overexpressing Myc-tagged wild-type UCH-L1 were treated with 0 or 1 mA). Intriguingly, these Cys to Asp mutants showed protective effect comparable to that of wild-type UCH-L1 in response to oxidative stress (0.3 mm H2O2) (B). Cells overexpressing C90/152D mutant also showed some increase in cell viability when treated with 0.3 mm H2O2, but the difference was not statistically significant. Overexpression of each UCH-L1 mutant was verified by Western blot analysis (C). This study demonstrated that the protective effect of wild-type UCH-L1 was not from massive overexpression of protein containing reactive thiols. Cys to Asp mutants of UCH-L1 seemed to mimic Cys oxidized wild-type UCH-L1, which suggests that oxidized UCH-L1 itself is exerting the neuroprotective function.To further investigate whether the protective function of UCH-L1 is related to the loss of its enzyme function caused by oxidative modifications of its Cys residues, we examined the protective effects of various Cys to Asp mutants  of UCH-L1, which mimic oxidized Cys residues. We determined the cell viabilities of HT-22 cells overexpressing WT and C90D, C152D and C90/152D of UCH-L1 subjected to oxidative stress, employing xCELLigence RTCA. Overexpression of Cys to Asp mutants of UCH-L1 had no effect on cell viability without treatment of oxidative stress A. Intrigmm H2O2) B. Cells analysis C. This sm H2O2 . We previously reported that UCH-L1 is a highly soluble protein and that the N-terminal truncated form of UCH-L1, which serves as an ROS scavenger in cells, is relatively easily aggregated and degraded . Because the amounts of insoluble fractions were small, they were concentrated 5-fold for comparison with soluble fractions, to obtain UCH-L1 bands for further densitometry assays . The significant difference in protein solubility caused by H2O2 was only found in the insoluble fraction of WT UCH-L1  and C152D . Specifically, Cys to Ser mutants tended to show patterns of increasing insoluble fraction after H2O2 treatment, whereas Cys to Asp mutants tended to show no changes or decreasing patterns, although these differences were not statistically significant. Wild-type UCH-L1 is readily oxidized by H2O2, consistent with the results in To investigate whether oxidized form of UCH-L1 is causing its precipitation, we fractionated HT-22 cells overexpressing Cys mutants (both Cys to Ser and Cys to Asp mutants) treated with 0 or 0.3 mdegraded . For thi , HT-22 cells are appropriate model system to examine the gain-of-function of UCH-L1. To investigate the loss-of-function of UCH-L1, we employed SH-SY5Y human neuroblastoma cells expressing high endogenous UCH-L1. SH-SY5Y cells were transfected with the specific siRNA to knock down UCH-L1 or control siRNA for 72 h, and cell viabilities were assessed employing WST-1 cell viability assay in response to various concentrations of H2O2  for 2 h at 37 °C (A). SH-SY5Y cells knocking down UCH-L1 showed significant decrease in cell viability when treated with oxidative stress of 0.25 mm H2O2, but not cells treated with 0 or 0.1 mm mild oxidative stress. Knockdown of UCH-L1 was verified by Western blot analysis (B). The above finding is consistent with the gain-of-function results of studies in which UCH-L1 overexpression and its oxidized form protects cells from oxidative damage . These proteins were classified into three groups by their function, respectively in synaptic plasticity, metabolic processes and protein aggregation. Of these 63 proteins, UCH-L1 , DPYL2 (dihydropyrimidinase-related protein 2 (CRMP2)), HDHD2 , ARPC5 (actin-related protein 2/3 complex subunit 5) and PRDX2 (peroxiredoxin-2) showed significant spot shifts on 2D-PAGE because of post-translational modifications (PTMs) caused by chronic stress. We demonstrated that site-specific oxidative modifications at Cys90 and Cys152 of UCH-L1 had occurred after chronic restraint stress. Overexpression of UCH-L1 in HT-22 hippocampal cells protected these cells from H2+ signaling and regulation of synaptic plasticity and memory function , DPYL2 (dihydropyrimidinase-related protein 2 (CRMP2)) and CNPY2 (protein canopy homolog 2) regulate neurite outgrowth cooperatively –36. DPYL3745function .l-lactate dehydrogenase B chain) and TPIS (triosephosphate isomerase) function in carbohydrate metabolic process. NDUS3  is associated with energy metabolism; DLDH , classified in ROS generation group, is also involved in brain energy metabolism . IF5A1 (eukaryotic translation initiation factor 5A-1) and UBE2K are involved in cellular stress response , and haloacid dehalogenase (HAD)-type phosphatases. In this group, CH60 , OTUB1 (ubiquitin thioesterase OTUB1) and UBE2K (ubiquitin-conjugating enzyme E2 K), increased after chronic stress, are involved in protein metabolic process and facilitating the correct folding of protein (CH60) and ubiquitin proteasome system (OTUB1 and UBE2K). PGAM1 (phosphoglycerate mutase 1), VATB2 , LDHB , SYUA , SEPT5, UBE2K, TPIS, MK01 (mitogen-activated protein kinase 1), GNAO (guanine nucleotide-binding protein G(o) subunit alpha), 1433T (14-3-3 protein theta), 1433E (14-3-3 protein epsilon) and KCRB are associated with the onset of neurodegenerative diseases such as AD and PD , 57–63. 57This protein profile study demonstrates that CRS induces many proteome-level changes in proteins functioning in synaptic plasticity, metabolic processes and ROS generation which coordinate to influence the physiological conditions of stressed mice. PTM analysis reveals that Cys90 and Cys152 of UCH-L1 in the hippocampus are oxidized with different sensitivities. Cell survival studies showed that overexpression of UCH-L1 WT or Cys to Asp mutants  protects cells from oxidative stress, whereas the Cys to Ser mutants  do not. This was not because of the solubility changes of UCH-L1. These results suggest that oxidative modifications of Cys90 (active site of hydrolase) and 152 residues of UCH-L1 confers a protective function in response to oxidative stress and the effect is independent of the hydrolase activity of UCH-L1. C90/152S and C152S seem to act as dominant negatives because of their deteriorating effect on cell viability in response to oxidative stress, indicating that Cys152 particularly play a key role in neuroprotective effect.UCH-L1, as a deubiquitinating enzyme, which degrades misfolded proteins and recycles ubiquitin molecules under stress condition appears to play a key role in changes in protein profiles and neuroprotection in MDD. However, previous several studies demonstrate the function of UCH-L1 independently of its hydrolase activity , 67, 68,et al.  into AD model mice improved their contextual memory . Cultureet al. , which sIn summary, this study demonstrated that UCH-L1 exerts a neuroprotective effect against oxidative stress, in the hippocampus of animals exposed to chronic stress, and that Cys oxidative modifications of UCH-L1 occur from chronic stress. The study also identified the post-translational molecular changes that occur in the brain of animals exposed to chronic stress. This is the first study that highlighted the role of UCH-L1 in the brain under chronic stress as an MDD model. These results might help to understand biochemical changes in the brain of depression patients and have the potential to lead to new therapeutic approaches.http://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer) (http://msviewer.ucsf.edu/prospector/cgi-bin/mssearch.cgi?search_name=msviewer&report_title=MS-Viewer&rows_per_page=100&search_key=sqp0rufe0d&.The mass spectrometry proteomics data for protein identification  are uplosviewer)  repositosupplemental Table S1) have been deposited to the ProteomeXchange (Raw data for UCH-L1 PTM analyses (eXchange  ConsortieXchange  partner


Breast cancer (BC) is the most common cause of cancer among women, with a high incidence rate occurrence every year worldwide despite advances in its management. BC is characterized by a spectrum of subtypes which respond differently to treatments due to their biological features, representing the main issue in the control of this type of malignancy. Androgen receptor (AR) is emerging as a target to investigate among hormone receptors, since it seems to play a role at various stages of development of specific BC subsets. For this reason, in recent years AR has become very important in the clinical practice, although its role remains controversial. A number of studies have proposed a correlation between microRNAs (miRNAs), a class of gene expression modulators, and AR in prostate cancer (PC), but there are still few evidences about the relationship between miRNAs and AR in BC. The purpose of this review is to present a state of the art scenario with consideration to the most recent discoveries about miRNAs involved in the AR associated pathogenesis of BC, in order to provide new insights into the role of miRNAs as key drivers in the modulation of AR, and possible actors in the development and progression of BC. Moreover, we consider findings about involvement of AR signaling in all stages of BC, highlighting its association with different subsets of breast carcinomas and with pre- and postmenopausal state of patients. Breast cancer (BC) is the most common cause of cancer among women and in 2018 in the United States 266,120 new cases and 40,920 deaths have been estimated . DespiteAndrogen receptor is a member of the family of steroid nuclear receptors which mediates the biological effects of androgens. It is well established that AR is considered an oncogenic driver at all stages of PC, but its role in BC remains controversial. In fact, expression of AR splice variants (ARVs) has been elucidated in PC, but only recently the presence of multiple known and novel ARV transcripts has been demonstrated in a panel of BC cell lines and human tissues: AR-V1, -V3, -V4, -V7, and -V9 . In partIn the last years several studies have focused on the role of AR in ER-α positive (ER+) BCs, since there would seem to be a correlation between its expression grade and some clinical advantages. In fact high levels of AR are associated to reduced lymph node involvement, better DFS, RFS, and OS, response to endocrine therapies and chemotherapy, lower tumor grade, Ki67 expression, smaller tumor size and less necrosis, suggesting for AR a possible role as a tumor-suppressor in malignant breast epithelial cells . Rangel in vitro using BC cell lines whose growth was promoted by AR expression. Robinson et al. demonstrated that in the absence of ER-α more than a half of AR binding events showed an analogous pattern to that of ER-α in ER+ cells, promoting the expression of ER target genes, and suggesting a role of AR as a ER-α mimic  subtype had a distinct transcriptional landscape independent of HER2-amplificated (HER2A) that reflected and confirmed how AR signaling can replace ER-driven tumorigenesis . In fact, it is estimated that up to 90% of primary BC and up to 75% of metastatic lesions expressed AR (in situ (DCIS)  , althougu (DCIS) , Schrijv with ET . This ne with ET . Usually with ET , and E2  with ET . Whereas with ET . Severaln levels . How horn levels .MicroRNAs are the most explored non-coding RNAs, and give rise to a large family of short (19–24 nucleotides) single-strand RNAs which take part in a variety of biological processes, such as cell proliferation, death, differentiation, and stress response . They opIQWD1 (IQ motif and WD repeats-1) and miR-363: under low androgens levels IQWD1 was downregulated by miR-363, but this negative modulation did not occurred after DHT administration  is known to promote EMT, thereby progression, invasion and metastasis, and is found abnormally overexpressed. Yang et al. identified an AR negatively induced long non-coding RNA (lncRNA) ARNILA that correlated to poor PFS, tightly connected to AR and able to sequester miR-204, in turn facilitating the expression of its target Sox4. Particularly in AR+ carcinomas, ARNILA is suppressed by the action of DHT and AR, resulting in the decreased adsorption of miR-204 thus favoring Sox4 expression inhibition. On the other hand, in AR- tumors, the action of ARNILA leads to Sox4 expression by increasing the sequestration of miR-204, leading to the induction of EMT and metastatic propagation (A number of studies have proposed a correlation between miRNAs and AR in PC , but thestration . Interesoteasome . In AR+/oteasome . Similaroteasome . Resultsoteasome . Moreovec target . Anotherc target . In TNBCpagation . All togpagation . Again ipagation  (Al-Othmpagation . In anotpagation . Again, pagation . Interespagation . This hypagation .Many studies support the idea that BC is a heterogeneous pathology and this consideration is mainly motivated by the existence of different subtypes classified on the basis of hormone receptor expression. About 70–80% of BC express considerable level of ER and are estrogen-dependent , but, asSince the findings that miRNAs play a role in carcinogenesis and are deregulated in several types of tumor, they have obtained a lot of interest about their potential use as therapeutic agents in chemotherapy. Furthermore, the supposing of the existence of a specific uptake can help the purpose to customize miRNAs as therapeutics alone or, more likely, in combination with today’s anti-cancer therapies. Surely more stress should be placed on understanding the balance between AR and ER in relation to the different subtypes, which gives rise to main questions regarding a different response to endocrine therapies in BC. For instance, the interesting positive feedback mechanism identified by Lyu et al. between AR and miR-30a could be a starting point for further studies about the role of miRNAs as a therapeutic predictive markers, besides the identification of other miRs that are able to target AR, or molecules involved in the AR pathway, can certainly help to find more answers about this interaction . Also, nEB wrote the first draft of the manuscript. FF contributed to the writing of the manuscript. FF and EB drew figures and tables. Both authors critically reviewed the manuscript and approved the final version of the manuscript.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.