Community Health Workers (CHWs) play an instrumental role in promoting socio-behavioural change at the community level, which results in changed indicators of community health. While outcomes are mostly reviewed for achieving program objectives, it is pertinent to understand the process of program implementation mainly from the perception of participating CHWs.A qualitative study to understand the perception of Sakhi’s (CHWs) regarding the outcomes of their participation in Home-Based Neonatal Care (HBNC) Program implemented by a non-governmental organization (NGO). Data consisted of 3 FGDs and 20 in-depth unstructured interviews with participating Sakhis.Sakhis perceived their ability to take decisions at critical phases of the program as an important factor influencing their performance. The opportunity to participate as a Sakhi in the health programme initiated a process of change at the personal level. The changes perceived by Sakhis were enhancement in knowledge, skills and capabilities of Sakhis. The combination of improved skills, knowledge and attitude had culminated in the process of experiencing self-empowerment for the participating Sakhis. Their ability to positively influence the individuals and community with their initiatives to improve women and child health and save lives in critical situations facilitated development of a new identity and improved societal status in their communities. Changed power-relations at the family and community level promoted the involvement of Sakhis in the broader development agenda. Sakhis’ ability to strategize goals, evaluate their own abilities, their willingness to upgrade knowledge and take others along in bringing social change, was an evident movement towards self-development.An opportunity for local women to participate in development programs creates potential for self-development as a cascading effect in addition to the accomplishment of planned program objective. The use of community health workers (CHWs) has for long been one of the important strategies to address the issue of healthcare outreach. India has a rich history of engaging CHW in health prevention and promotion programmes. A large national CHW scheme was established in the late 1970s that aimed to provide one CHW for every 1000 people in order to provide adequate health care to rural people and to educate them in matters of preventive and promotive health care , and theBeing firmly rooted in communities, the CHW play an instrumental role in promoting socio-behavioural change at the community level, which is expected to result in improvements in individual and community’s health indicators. While several studies –8 have sStarted in 2005, a HBNC program was implemented for over a decade in 160 villages of three blocks in Chandrapur District, Maharashtra by Ambuja Cement Foundation, the corporate social responsibility (CSR) arm of Ambuja Cements Ltd. In this program women from villages who wanted to become CHW were selected and trained to provide services in communities on the lines of the well-known HBNC model proposed by Bang et al.  The SakhThe umbrella term “community health worker” (CHW) embraces a variety of community health aides selected, trained and working in the communities from which they come. The CHW’s roles and activities are tailored to meet the unique needs of their communities. It also depends upon whether they work in the healthcare or social services sector . AlthougSelf-development and empowerment are inherently interrelated. In the context of health and social development, the concept of “empowerment” has received much more attention. Empowerment, both as a process and a goal, has taken considerable prominence within community organisation theories, especially as operationalized by NGOs since 1970s . It has The data presented in this paper are from an exploratory qualitative study that was conducted to understand the challenges faced and mitigation strategies used by Sakhis. The study was conducted adopting a grounded theory approach and data acquired was from 3 focus group discussions and 20 in-depth interviews with Sakhis who met the qualifying criterion of having at least 2 years of experience. A more detailed description of the study’s setting, conceptual framework, process of data analysis, and findings related to challenges and mitigation strategies are reported in another paper .An unexpected recurrent theme emerged while analysing data. While narrating challenges and mitigation strategies many Sakhis had expressed about how they had undergone positive changes at a personal level. This theme was beyond the scope of conceptual framework, and therefore the authors revisited the data to develop a nuanced understanding of the Sakhi’s perception of the effect of her work on herself. Each interview was independently coded by at least two authors and the validity of coding was ensured through joint review of coded sections by three authors . This helped to minimize bias of the authors and ascertained that findings were firmly rooted in experiences shared by the Sakhis. After engaging in several deep reflective discussions on the emerging theme, the authors concurred that the pattern reflected “self-empowerment” that had resulted from the process of working as a CHW.Through the analysis of the data we reviewed the perception of Sakhis regarding the critical phases of their process of engagement and its outcome in terms of empowerment and self-development.There were certain common characteristics observed among the local women willing to participate as Sakhis. Many of them were in the 20–35 year age-group, married, possessed minimal literacy levels, and in most cases their work was restricted to the household chores, though few were involved in farm labour to support the family livelihood.“I got married at the age of 15 and had a baby when I was 18. I was not allowed to talk to anybody. I hated to work in the farm and hence was annoyed with it but did not have any choice. It was then this opportunity for training came up. My in-laws and my husband opposed, but I argued with them to join this work. I left my six-month old baby to be taken care by my sister-in-law for five days during the first training. I suffered a lot because I missed my baby but didn’t quit the training”.In this phase the decision to participate was considered to be the most critical by the local women and the process of empowerment originated at this stage. Their decision was influenced by their family, chiefly by their spouses and in-laws. The Sakhis shared about the nature of support they experienced in joining the program. While some Sakhis experienced enthusiasm and support from their spouse and the immediate family, others did not. Despite lack of support many decided to become a Sakhi. The experiences shared by Sakhis showed a distinctive feature of women who had opted to become a Sakhi – a very strong desire to participate in activities beyond the realm of their household work and familial responsibilities. Though their motivation differed in degree, in most cases women regarded this as an opportunity to perform. High motivation and positive attitude influenced the ability of Sakhis to take a decision about participation. A Sakhi recalled,For many Sakhis it was the first opportunity to independently express and assert their views. Once the Sakhis had asserted and decided to become a CHW, the decision itself helped Sakhis to further build their confidence to initiate work for the community’s health and development. For few Sakhis, an additional factor that helped move toward independent decision making was their selection by the community to become a CHW for their village. In certain cases however local women experienced dissuasion from the community. Their intention and interest in taking on the work as Sakhi were questioned and were mistrusted for their capability and eligibility to become a Sakhi.ACF field supervisors also recommended some potential local women based on the previous interaction with the community. Sakhis perceived recommendation of their name as recognition of their efforts as well as their potential, which encouraged them to participate. A sense of self-esteem emerging out of recognition of their potential by the community enhanced the level of confidence of the women turned Sakhis. Initial fear was replaced with confidence to take upon challenges with the process of engagement. The Sakhis shared that gaining ability to respond to challenges was an elevating experience.During the training we were prepared for this phase. We were told that the patience to take the community with you will count first followed by your ability to provide needed health care services”.“The action phase began with the orientation training followed by field outreach. Each Sakhi developed a customised outreach plan and communication strategy for her village as part of the training. This helped Sakhis to have an improved acceptability for their health messages. Sakhis, being local and always available, got an opportunity to demonstrate their knowledge and skills. Their constant communication and follow-up helped in spreading health awareness especially in case of pregnancy. Sakhis experienced non-acceptance from the community during the initial period, however they continued their efforts to reach individuals and families with health care inputs and support especially during the critical conditions. In this regard, explaining how they were able to deal with difficulties, a Sakhi shared,Sakhis were able to adapt the style of communication through different phases of interaction with the community. They mentioned to have experienced improvement in communication skills, develop a poised attitude and ability to think creatively in a given context. The strong commitment and persistence among the Sakhis helped initiate the process of changing beliefs and practices among the community members. The process of influencing beliefs and attitudes of community also contributed to Sakhi’s improved confidence to act further.“I can’t tell you how happy I feel to see the saved child grow and it is also the same with the family. I built relations with those families. The families have also shared with their child about their gratitude for the efforts we had made to save her”.The experiences shared by Sakhis indicated that the first experience of handling a critical case played a significant role in boosting their confidence. It was evident in the data that technical knowledge and timely decision making ability among the Sakhis got sharper with each critical case handled by them. The experience of handling critical child birth cases was perceived to be a significant differentiator by Sakhis. They placed highest value on handling critical cases that led to saving human life, considering it to be a noble task. Further, Sakhis mentioned it to be a gratifying experience that also elevated their status in the community. The ability to respond to critical cases, ability and courage to take timely decisions and recognition for this ability from their own community and other stakeholders developed an immense sense of self-esteem among the Sakhis. The aspects of saving lives of babies also provided Sakhis with huge satisfaction. As a Sakhi mentioned,“This is our reward. Money doesn’t matter to us but it is the respect that we get in the community which becomes important”.Another Sakhi stated,panchayat initially for health issues and expanded their reach to discussing other development schemes by government, being representative on various committees or to support social action campaign on issues such as alcoholism and tobacco use. Sakhis also helped women to get their ration card or access loan from a bank. These added roles further demanded mobility for a Sakhi. It was evident from the following example that the enhanced mobility and interface of Sakhis with the external world stimulated their aspirations to grow further and influenced choices made by them. A Sakhi-turned-master-trainer mentioned mobility adding to her confidence in being independent.“Now I can travel very far for work, I don’t feel afraid even if I am alone. Feel like my home wherever I go”.The Sakhis’ experiences revealed that their performance of roles was also shaped by their ability to influence social customs and practices. Acknowledgment by community members and health providers encouraged Sakhis to take initiative and also go beyond their predetermined role in the project and contribute in other aspects of community health and village development issues. Beginning with women and child care, they moved on to working on addressing issue of child marriage, sanitation, nutrition, decisions regarding contraception and quality of health care provided by public health system. Sakhis believed their effort helped in improving awareness among the community about relationship of health issues with social customs such as early age of marriage. Sakhis engaged with “I feel my village should always remain good and healthy, and zero mortality is my aim”.Another Sakhi discussed the value she found in interacting with experts and external contacts. Interestingly, these aspirations are not limited to herself or her family. A Sakhi mentioned,Based on the experiences shared by Sakhis about the process of their participation and self –growth through this process, an empowerment framework seems to be emerging. The process appeared to have begun with personal changes at an intrinsic level, which over the period of time expanded to the Sakhis’s standing in the society as an empowered woman involved in managing affairs of the community. The components of this process are described below sequentially, however, they are likely to have occurred parallel to each other with substantial overlaps.“I might have remained just like that, uneducated and unhappy about not utilising my potential, if I had not been part of this program”. Sakhis appeared to have learned to overcome their fears by confronting the fear and continuing with their work. As expressed by a Sakhi, “I feel when we do something, initially we are scared but later the fear just withers”. Over time, Sakhis were able to replace their initial fears with larger dreams for their own community’s development.Sakhis experienced an emergence of a new identity for themselves which is clearly highlighted from an example. As a Sakhis mentioned, “I have two children and both are studying. One is pursuing BSc and other one is in 11th. When it came to paying their fees I used my earned money and took a decision to pay it off. My dream today is to ensure that my children are well educated”.Sakhis earned honorarium for their work. The earning, though not very substantial, provided Sakhis with respect and confidence which was highlighted in the example shared by a Sakhi.It was evident in most cases that Sakhi’s earning became an additional support for the family and in most cases it was utilised for domestic needs and education of their children. Sakhis shared that, with access to finance, their involvement in household financial decisions improved. The finding corroborates with another study conducted by ACF which suggested the access to finance through self-earning regardless of the magnitude develops a sense of confidence among the women working as veterinary health workers .“The decision benefited me a lot and my husband too turned supportive seeing the work I did”. Another Sakhi shared, “Now our family also doesn’t disallow us. In fact they say they are very proud of our work and earning money is inconsequential”. In other words, as Sakhis started acquiring financial independence, they started getting a say in their family affairs and their role as Sakhi was accepted.The journey of Sakhis began with influencing family members for their consent to participate in the program. Non-cooperation and lack of support at family level in certain cases were replaced with acceptance and trust. A Sakhi who faced resistance initially mentioned, “Yes, I am respected today. If a meeting is held, they invite me in it. Whenever anew committee is formed, they always offer a post to me”.Sakhis started being included in consultations related to other development agenda at the village level. The communities started seeking Sakhis to represent their interests by being on village level development committees such as education or sanitation committees. This elevated their status in the society. As a Sakhi expressed, “After being a Sakhi, I got habituated to being occupied. I am eager to work more in whatever way it may be”.The major learning as perceived by Sakhis in this case has specifically been in terms of improved knowledge, skills and attitudes. These aspects appeared to be interdependent and having a mutually fostering effect. The earned knowledge and learnt skills promoted positive attitude in the Sakhis, which in turn helped them take calculated risks and informed decisions while addressing difficult situations. The experience of successfully addressing difficult situations further accentuated the Sakhis’ self-image, increased their self-esteem, through vicarious learning and also strengthened their knowledge and skills. This ongoing process appeared to have triggered the process of empowerment in which the Sakhis’ growth was not limited to the personal level, but also at the societal level. Some Sakhis grew to become ASHAs or their supervisors and master trainers, others pursued education at secondary level and some also started working as CHW on other development projects. In other words, the Sakhis developed aspirations to perform effectively in future and not be without any constructive work. A Sakhi put this very simply and clearly – Sakhis, the key actors in the program, experienced a significant transformation in terms of improved ability in decision making which helped them to evolve their own identity. Sakhis place significant importance to creation of their identity. The elevated status for Sakhis in their community and an emergence of leadership reflected in their involvement in domain areas apart from health signifies empowerment.Women, an imperative segment of the society, have often been denied a fair role in the process of development, especially in underprivileged areas. The program played an instrumental role in encouraging local women to participate and contribute in working for their communities’ health and development. Further, it paved a path for gender integration in development at a local level by providing opportunities for women to enhance and use their knowledge and skills for the development of their own community.


EGFR基因突变状况的不一致性。表皮生长因子受体（epidermal growth factor receptor, EGFR）突变是晚期非小细胞肺癌（non-small cell lung cancer, NSCLC）患者获益于酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI）治疗的预测因子，本研究旨在探讨NSCLC原发灶与相应转移灶之间EGFR基因突变状况。应用TaqMan RT-PCR的方法检测35例病理确诊为NSCLC患者原发灶和相应转移灶的EGFR基因突变型，余下6例为EGFR野生型。35例转移灶中18例为EGFR基因突变型，17例为EGFR野生型。35对配对标本中，11对（31.43%）标本出现原发灶EGFR基因突变，而转移灶为EGFR基因野生型，18对原发灶及转移灶均为EGFR基因突变型，且突变具体位点相同，6对原发灶及转移灶均为EGFR基因野生型。NSCLC原发灶与转移灶的EGFR基因表达不一致率为31.43%（11/35, P=0.008）。原发肺癌病灶中有29例为EGFR基因表达存在不一致性。NSCLC原发灶与转移灶的 Louis, MO, USA）上进行扩增；⑦应用SDS 2.0软件对扩增曲线进行分析并判断EGFR基因突变阴、阳性。实验大致过程如下：①两位病理科医生分别在显微镜下核对石蜡组织的H E染色玻片，选取肿瘤组织>80%的石蜡块；②切取每片10 μm厚的蜡片4张；③使用TIANamp基因组DNA提取试剂盒（TIANGEN BIOTECH, 北京)从切片中提取出原发肺癌组织和转移灶的基因组DNA；④进行DNA提取物的验证：甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase, GAPDH）管家基因（300 bp）PCR扩增，1.3Fisher’s检验。P < 0.05为有统计学差异。统计分析采用SPSS 16.0统计学软件。计数资料分析采用22.135对NSCLC病例的配对标本中，原发灶均是已经完整切除的肺部病灶，35例配对的转移灶中，29例是纵隔淋巴结转移灶（9枚隆突下淋巴结，8枚主动脉下淋巴结，8枚下气管旁淋巴结，4枚上气管旁淋巴结），4例是脑转移病灶，2例是锁骨上淋巴结转移灶。所有病例在获得肿瘤标本前未曾接受过EGFR靶向治疗。病例的一般情况数据如下：中位年龄为57岁（38岁-72岁）；女性14例（40%），男性21例（60%）；病理学类型为：鳞癌1例（2.9%），腺鳞癌3例（8.6%），腺癌31例（88. 5%）；肿瘤分期：Ⅲa期24例（68.6%），Ⅲb期3例（8.6%），Ⅳ期8例（22.8%）；吸烟史：不吸烟者16例（45.8%），既往吸烟者13例（37.1%），现时吸烟者6例（17.1%）。2.2EGFR基因突变RT-PCR扩增曲线见EGFR基因突变型，余下6例为EGFR野生型。29例原发肺癌病灶EGFR突变类型分别为第19号外显子突变15例、第21号外显子突变14例。其中第19号外显子突变中均为E746-A750缺失；第21号外显子突变中13例为第858位密码子碱基替换突变，1例为第861位密码子碱基替换突变。EGFR基因突变型，17例为EGFR野生型。18例EGFR基因突变型转移灶的具体突变类型为：第19号外显子突变11例、第21号外显子突变7例。其中第19号外显子突变中均为E746-A750缺失；第21号外显子突变中均为第858位密码子碱基替换突变（35例转移灶中18例（18/35, 51.43%）为2.3EGFR基因野生型的病例，其相应的转移灶分别为：3例脑转移病灶、2例锁骨上淋巴结转移灶和1例上气管旁淋巴结转移灶，这6例转移灶的EGFR基因均为野生型，与原发病灶一致。6例NSCLC原发灶为EGFR基因突变型的病例中，只有18例EGFR基因突变，而其余11例转移灶为EGFR基因野生型。18例EGFR突变型的转移灶中，11例为第19号外显子突变，病灶分别为：3例下气管旁淋巴结转移灶、3例隆突下淋巴结转移灶、3例主动脉下淋巴结转移灶及2例上气管旁淋巴结转移灶，第19号外显子突变均为E746-A750缺失；另外7例为第21号外显子突变，其相应转移灶分别为：2例下气管旁淋巴结转移灶、2例隆突下淋巴结转移灶、2例主动脉下淋巴结转移灶及1例脑转移灶，第21号外显子突变均为第858位密码子碱基替换突变。转移灶的EGFR基因突变位点与原发病灶的突变位点一致。29例原发灶P=0.008）原发灶EGFR基因突变阳性而转移灶为EGFR基因野生型的患者其转移灶分别为：4例隆突下淋巴结转移灶、3例下气管旁淋巴结转移灶、3例主动脉下淋巴结转移灶。其原发灶EGFR基因突变类型为：第19号外显子突变4例（均为E746-A750缺失）、第21号外显子突变7例（6例为第858位密码子碱基替换突变，1例为第861位密码子碱基替换突变），其相应转移灶均为EGFR基因野生型，与原发灶EGFR基因表达不一致。11例（11/35, 31.43%, P=0.008）标本出现原发灶EGFR基因突变，而转移灶为EGFR基因野生型，18对原发灶及转移灶均为EGFR基因突变型，且突变具体位点相同，6对原发灶及转移灶均为EGFR基因野生型。研究结果显示转移灶与原发灶的EGFR基因表达存在不一致性，其不一致率为31.43%（11/35, P=0.008）。35例原发NSCLC病灶与相应转移灶配对标本中，11例（31.43%, 3EGFR突变者对TKI治疗的反应率及PFS均较EGFR野生型者明显升高。检测EGFR基因突变来筛选获益于TKI治疗的患者已经推广使用。目前转移病灶的EGFR基因状况是否与原发病灶保持一致尚不明确。当晚期NSCLC患者在决定是否使用EGFR TKI治疗时，进行EGFR基因突变状况检测，是否原发病灶与转移病灶同时检测更合适，也无法定论。在晚期NSCLC患者中EGFR基因突变状况的一致性，共检测了35对配对的原发肺癌病灶与相应转移灶的EGFR基因。研究结果显示转移灶与原发灶的EGFR基因表达存在不一致性。入组病例中有11例（31.43%）出现原发灶EGFR基因突变，而其对应的转移灶为EGFR基因野生型。这一发现与最近几项同类研究的结果相似，Park和Gow等研究结果显示在NSCLC原发灶与相应转移灶之间EGFR基因表达的不一致率分别为11.9%（12/101，直接测序法），16.8%（17/101，异源双链分析法）、27%（18/67，TaqMan RT-PCR法) 、33%（16/45，免疫组化）、28%（7/25，直接测序法）。各个实验检测EGFR基因突变的方法不同及病例入组标准的差异可能是造成不一致率存在差异的原因。本研究比较了NSCLC原发病灶与相应转移病灶之间EGFR基因突变型，表达一致。另一个研究对比8例肺癌患者原发灶与转移灶的基因表达，EGFR基因、KRAS、P53在原发灶与转移灶之间都保持了一致性。然而这些研究的入组病例数都很少。然而，某些研究却发现相反的结果。Matsumoto在研究中有8例肺腺癌脑转移灶患者可找到相应的肺部原发病灶，其中6例脑转移灶和肺部原发病灶均为EGFR基因突变型晚期NSCLC患者中有明显疗效，能有效延长PFS、提高生活质量-16。TKI治疗是否是晚期肺癌患者合适的治疗，根据EGFR突变类型来选择是关键，然而EGFR基因在原发灶与转移灶之间存在着不一致性，所以如果对肺癌原发灶及转移灶同时进行EGFR基因检测，可能对患者的治疗有更好的指导意义。EGFR TKI治疗在总之，本实验发现在NSCLC原发病灶与转移灶之间的EGFR基因状况的不一致率为31.43%。这种原发灶与转移灶EGFR基因状况的不一致性可能对晚期NSCLC患者选择EGFR TKI治疗有一定程度的影响，同时也是将来相关临床试验设计时需考虑到的因素之一，尚有待大样本量的研究来进一步验证其临床意义。


Online health community (OHC) moderators help facilitate conversations and provide information to members. However, the necessity of the moderator in helping members achieve goals by providing the support they need remains unclear, with some prior research suggesting that moderation is unnecessary or even harmful for close-knit OHCs. Similarly, members’ perceptions of moderator roles are underexplored. Starting January of 2013, WebMD moderators stopped working for WebMD communities. This event provided an opportunity for us to study the perceived role of moderators in OHCs.We examine the OHC members’ perception on OHC moderators by studying their reactions toward the departure of moderators in their communities. We also analyzed the relative posting activity on OHCs before and after the departure of moderators from the communities among all members and those who discussed moderators’ departures.We applied a mixed-methods approach to study the posts of all 55 moderated WebMD communities by querying the terms relating to discussions surrounding moderators’ disappearance from the WebMD community. We performed open and axial coding and affinity diagramming to thematically analyze patients’ reactions to the disappeared moderators. The number of posts and poster groups (members and moderators) were analyzed over time to understand posting patterns around moderators’ departure.Of 821 posts retrieved under 95 threads, a total of 166 open codes were generated. The codes were then grouped into 2 main themes with 6 total subthemes. First, patients attempted to understand why moderators had left and what could be done to fill the void left by the missing moderators. During these discussions, the posts revealed that patients believed that moderators played critical roles in the communities by making the communities vibrant and healthy, finding solutions, and giving medical information. Some patients felt personally attached with moderators, expressing they would cease their community participation. On the other hand, patients also indicated that moderators were not useful or sometimes even harmful for peer interactions. The overall communities’ posting activity, which was already in decline, showed no significant difference before and after the moderators’ departure. In fact, the overall posting activities of the communities were declining well before the moderators’ departure. These declining posting activities might be the reason why WebMD removed the moderators.Compassionate moderators who provide medical expertise, control destructive member posts, and help answer questions can provide important support for patient engagement in OHCs. Moderators are in general received positively by community members and do not appear to interfere with peer interactions. Members are well aware of the possibility of misinformation spreading in OHCs. Further investigation into the attitudes of less vocal community members should be conducted. In-person patient support groups, often organized by hospitals and clinical moderators, are a well-established mechanism to encourage peer-patient interaction, help patients improve self-efficacy, and educate patients about self-care management -4. As WeTo successfully administer OHCs, we need to understand the critical role that moderators have in OHCs. A study revealed effective moderation styles for various negative online behaviors  . AlthougUnlike in face-to-face support groups, where moderators are often clinicians known to participants, moderators in OHCs do not have an immediate connection and the trust of members. Moderators are also unable to provide medical consultation. Furthermore, one prior study found that moderators urging patients to talk to their health care providers instead of consulting the community was highly associated with decreased peer-patient interaction . Health WebMD , an onliHealth professional moderators at WebMD were those with clinical backgrounds, providing clinical expertise to patient members by answering members’ questions and participating in conversations. Nevertheless, the HPMs would follow the same guidelines given to STMs, where HPMs would not be allowed to give personalized medical consultation. Health professional moderators could clarify clinical concepts and knowledge that would be useful for self-management, or direct ways to discuss with the members’ own health care providers.WebMD, however, made a decision to let go of the STMs starting January of 2013. Members were not notified of this change in staffing prior to its occurrence, but quickly picked up on the absence of moderators. The departure of STMs triggered patient members to talk about their experiences with moderators, including HPMs, as part of the communities. The sudden departure of moderators from WebMD provided critical information about how patients perceived the benefits and disadvantages of having moderators in their communities when faced with loss of moderation.While prior research has clearly demonstrated the benefit of moderators in OHCs, there is a limited understanding of the community member’s perceptions of moderators in their participation with OHCs. In this paper, we will investigate: (1) member’s perceptions of moderators in OHCs, and (2) the summary statistics analysis on OHC retention and moderators’ removal to understand both qualitative and quantitative aspects of moderators’ roles and influences in OHCs.We first walk through the data collection process, including community selection, followed by the descriptions on our mixed-methods analysis.Our institutional review board (IRB) determined this study to be unregulated because our data were publicly available and did not involve human subjects. WebMD has a total of 55 moderated communities. In January of 2015, we wrote an automated crawling program, which downloaded all 55 communities and saved the data to a local database software, MySQL (Oracle) . The craa) moderators leaving or (b) members’ perceived moderators’ roles, 2 coders manually went through each post to determine the relevance  and (7) The communities to investigate using our qualitative and quantitative analysis procedures were finalized. The result of this process is delineated in To best select active communities that can lend us more data regarding members’ discussions on moderators’ roles at WebMD, the following process was adopted: (1) From the total of 55 WebMD moderated communities, the top 20 communities which had the most posts in total after excluding nonhuman health-related community  were selected. (2) Posts made before November 2012 and those made by moderators were excluded. (3) A regular expression query with the keywords related to moderators was conducted: “moderator[s],” “mod[s],” “[moderator names of that community].” The query was conducted on both the title and the message body of each post; (4) All posts under the threads that contained the query result posts were then collected. (5) The communities that came with zero query results when (3) was performed were excluded. (6) To further filter whether the thread was about  of the data collection, 2 coders worked together with the first 5 communities on determining the criteria for relevance, asking about the posts that were related to discussion about moderators. If the post contained any dialogue about WebMD moderators—either STMs or HPMs, the post was flagged as relevant. The resulting criteria for relevance were as follows: First, the post should be about moderators, not other synonym that did not mean community moderators . Second, even though the post does not explicitly mention moderators’ removal from WebMD, if it contains any opinion about the moderators, it is included. The 2 coders divided the rest of the communities to code for relevance.We analyzed the whole thread in which related posts were its part. One main reason for excluding the thread was that when the whole thread of a post ended without someone intervening to inform the poster that the moderator had left, in case a moderator left without the poster’s knowledge.For the posts determined as relevant, a mixed-methods approach was adopted to understand the phenomenon using thematic analysis in addition to the quantitative methods employed. The thematic analysis  was usedWith the resulting codes, an affinity diagram exercise was performed  See Mul to conduIn this section, we first report the community selection and the summary statistics of posting activities. We then further examine qualitative aspects of how members perceived having moderators, providing us with insights for how moderators should be implemented in OHCs. The follow-up posting activity analysis of the members who participated in the discussions was then reported.A total of 14 communities were found to have included moderator-related keywords after having filtered out nonhuman health-related communities and relatively less active communities.In this section, the results of the overall posting activities, identification of related posts for further qualitative analysis, and the posting activity analysis of the members who participated in the discussions about moderators leaving WebMD communities have been reported.For the 14 resulting WebMD communities for us to analyze, 6-15 STMs and 1-7 HPMs have been moderating each community. It was found that these communities had, on average, 650 members per STM (ranging from 260 members per moderator on the Breast Cancer community to 1526 members per moderator on the Sex and Relationships community). On average, STMs started 255 threads and posted 1115 replies . On average, HPMs started 36 threads and posted 690 replies (from 0 to 119 thread starting posts and from 0 to 3348 replies). Further detailed breakdown of the activities are delineated in the Multimeda As shown in The last date of the STMs’ posts ranged between November 17 and December 20, 2012. Members’ posts related to moderators’ departure started as early as November 17, 2012 (the last posting date of the Sex and Relationships community) and persisted till December 20, 2012. There was a one-time post, an identical one, made by one STM in March to a few of the communities, asking members to share their experiences around insurance to a WebMD email address. Other than this particular post, no other posts have been made by STMs after December 20, 2012. One STM came back to the Fibromyalgia community with a regular member profile, updating about her current situation of finding a new job and health issues she is dealing with.In December 2012 (when STMs last posted) and January 2013, the discussion about moderator leaving WebMD peaked. Since then, members continued to talk about how moderators left, sharing opinions and solutions around STMs’ departure.The thematic analysis of 821 posts resulted in generating 166 codes. From these codes, 6 subthemes grouped into 2 main themes from conversations about moderators during the period from December 2012 to February 2014 see  were ideThe process by which the members figured out the news that the STMs have left WebMD was consistent across the 11 communities and is as follows: members first noticed moderators were gone; next, the groups shared the news individuals received from WebMD that the moderators had been removed from the forums; and finally, members pondered why the moderators had been removed, shared members’ initial reaction to the decision, and discussed how the community would continue to function in their absence .In figuring out moderators’ absence, members (1) noticed that moderators no longer participated, and (2) together they attempted to understand reasons for why moderators left. These discussions led to (3) discussions about solutions for not having moderators in their communities.WebMD community members’ posts revealed that they had not received any public notification about STMs’ departure from the communities. Instead, members slowly started realizing in mid-January 2013 that their moderators were not posting on their communities any more. This finding demonstrates that moderators’ roles were salient enough that their absence was quickly detected by members. Members started noticing that moderators were no longer posting, and they were seeking reasons for the absence of moderators’ posts:It seems that there are no more moderators on the community boards, and nobody is posting. Is anyone still here?LupusMembers who participated in multiple communities were able to publicize the idea that something suspicious was happening at the WebMD level, not just in one community:I am active in four WebMD communities. No mods posting in any of them. Maybe they were given time off for the holidays? DiabetesTo answer these questions, a number of members emailed the website directly and shared the replies from WebMD that WebMD had removed STMs from communities in December 2012 see . MembersThese communities no longer have moderators. The members are expected to inform others of the rules and policies. I am simply informing you of the facts.Pain managementMembers voiced their frustration about not being told about the change in the communities in advance:NO GOODBYE? Just over. The top of the site still says moderator mediated. No one has said anything to us? FibromyalgiaAs members realized moderators no longer participated in their communities, they attempted to figure out why moderators left.Members reacted strongly against moderators being removed from the communities and attempted to understand the rationale behind the decision. Members from the community suspected that budget cuts were the primary reasons for removal of the moderators. Members shared concerns about the moderators’ employment status, demonstrating the close relationships many had forged with the moderators. One member from a community shared evidence that WebMD had a budget cut, of which laying off moderators was a part. The following quote exemplifies the strong reaction to WebMD’s decision:Why??? Why would they deliberately sabotage a great site like Web.M.D.??? For what purpose??? Where are the BIG CHIEFS ??? What have they to say for themselves???Sex and Relationshipsthis is part of the changes which take place every day....businesses finding ways to cut or reduce costs....I am sure the moderators were paid for being here as are the other professional people who were here as well.FibromyalgiaIn one community, a member posted a press release on WebMD’s budget cut, which they believed provided a reason for the layoffs:For all those who have made inquiries about our missing leaders, apparently this is the answer: Forbes news release of December, 11, 2012 at 9:29AM(Breast Cancer)WebMD Cutting Staff 14%--250 Jobs. In Cost Cutting Program WebMD this morning disclosed plans to cut 250 jobs about 14% of the company’s staff as part of a plan to reduce annual operating expenses by about $45 million. The company said the plan is designed to “streamline its operations, reduce costs, and better focus its resources on increasing user engagement, improving customer satisfaction, and driving innovation.” As members accepted that moderators were no longer with them and attempted to understand why such a decision was made, the discussion moved on to how they, as WebMD community members, should proceed in maintaining their communities.Members discussed ways to independently manage their communities—to “stand united”  and make sure members regulate and facilitate conversations themselves. Some members predicted lack of moderators would produce problems, and advocated for strong mechanisms for self-regulation; others believed that communities could thrive without moderators, so long as they could provide a strong support system for each other.A few members stated that they no longer wanted to participate in the communities, as they were upset by WebMD’s decision to remove the moderators and bothered by increased spammers and trolls. One participant in the Pain Management community urged members to “ignore the trolls” and not “give [the trolls] any power to mess up” the community.To sustain the communities, members needed to stay and support each other. For instance, members discussed “staying strong for each other and the new ones that come [to WebMD] for help” and urged that they “do [their] best to help [new comers] in any way [they] know how” (Breast Cancer). In response to some members stating they would leave, other members asked them to stay:I’m sorry to see you go [David]. There are too few of us who have remained here over all the changes during the years (I’ve been around about 15 or more). Please reconsider. You help so many people. Anyway, hope you stay well and will contribute as you can to other groups.DiabetesI understand your needing a break, but you will be missed. Your insight and experience are a valuable asset to the other members of this community.Pain managementMembers also discussed that peer knowledge would not be enough and moderators were essential in communities requiring immediate answers to medical problems. Members discussed concrete actions they could take to fill the void left by the departure of the moderators, such as facilitating conversations by posting questions to elicit responses from “the wonderful people here” (Breast Cancer). After focused conversations about solutions to moderators being gone, members continued to encourage community members to monitor spam and advertisements. A member also shared one tactic of controlling malicious posts: “to not reply.” (Diabetes) we can report it and see what happens and then FLAG IT AS SPAM by the FMILY here so no one bothers to read it! Members began to practice self-moderation on their communities. They would tell other members not to share medical advice and provided alternative ways to get help at low cost (Pain Management), thus mirroring the prior activities of paid moderators. Members also put self-moderation into practice by reporting people; this task was not favorably received and caused one member to note that she or he “really miss(es) the moderators” (Pain Management). Furthermore, members shared websites that members should be careful of visiting. Members encouraged those who stated they would leave the community to stay, in an effort to sustain the community.While making sense of moderators being gone, patients shared their past experiences of having moderators as part of their communities. Throughout this process, even though HPMs still stayed on WebMD, members discussed their perceptions about health professionals as moderators. Contrasting opinions emerged about HPMs. Patients had conflicting views of whether moderators gave pragmatic help. Patients expressed that moderators were critical in ensuring the community did not include hostile messages, spams, and misleading information. Patients reported they felt personally attached with moderators. On the other hand, some expressed moderators were not useful and that peer support was more critical.Of the members who commented on the utility of moderators, most mentioned moderators to be helpful. Although STMs were the ones who left the communities, members discussed experiences around HPMs as well, allowing us to understand members’ experiences around not just STMs but also HPMs:I notice very few of the boards have doctors posting to them anymore. They were one of the reasons why I joined WebMD.Sex and Relationships. ” Similarly, a member stated, “While the support from people who are “suffering” from the same health problems is the most important thing on these blogs, expert advice [was] invaluable to [them] all. Members from the Diabetes community also felt that “the presence of mods experts in their fields to answer questions” was helpful. Members from Breast Cancer also noted the importance of moderators who are “ experts” on the topic: and that they “had always awaited a promised ‘expert’ for this board” (Breast Cancer).Many members did not draw a clear distinction between HPMs and STMs until some members clarified that there is a difference between STMs and HPMs. This perception was best illustrated by the sentiment that medical expertise was a unique resource that moderators could provide the communities. For instance, members in Sex and Relationships discussed how they missed doctors on the forum and expressed “personal experience doesn’t always match having a medical opinion” and that although members “have tons of personal experience, [they] sure as heck don’t have the required learning Although the STMs did not have the medical expertise, members appreciated the unique help provided by these STMs. These moderators helped keep the communities interested by posting articles, news, and information about WebMD. Members felt confident that moderators would find solutions to problems that arose on the forum. Members believed moderators ensured communities kept their “focus and no bickering.” Members did not “feel as safe posting here”  as they did when the presence of moderators was obvious. Being monitored by moderators made some members feel safe to participate in the community. Members declared their intent to leave, saying that they “joined because it was not like all the other social networking sites" (Diabetes). They “did not feel comfortable here now” because “mods used to tend to these issues [monitoring spams] in a timely fashion and keep an eye out” (Breast Cancer). Apart from a policing role, moderators helped maintain communities by picking up conversation when it slowed down. Members felt people were not posting anymore because the moderators were gone.Members from all communities saw an effect of not having moderators—spams lasting too long on the boards before being deleted, and that “New members are posting with rather radical ideas and trying to convince others that they are right” (Diabetes). The community being a “public, world-wide site with the communities open to anyone” (Diabetes), not having moderation made many members no longer feel safe posting.Members not only considered moderators to be helpful, but developed personal ties with them. They demonstrated these ties by voicing concerns about them losing their jobs. Several posters commented that moderators were like family members; for example, one wrote, “It is like losing a family member [...] To you all we will miss you” (Breast Cancer) and another similarly posted “we really loved having you on this board. We felt we were part of a large family” (Breast Cancer). In the Fibromyalgia community, members coordinated sending a card for one of the moderators. In the Depression community, a member started a thread to thank one of the moderators and wish her well. Some posters tried to petition WebMD to bring back the moderators, on the Sex and Relationships community, a thread called “BRING BACK OUR MODERATORS!!!” was posted with 17 replies in which the members vented on why moderators had to leave: “Complete madness to fire them.”Several members even threatened to leave the communities after learning about STMs leaving WebMD. One member wrote how she had been on WebMD for over 10 years and said:It was and has been my life-‐line and support for info from very good ladies. But Excuse ME-‐-‐-‐You are NOT the only Medical Information site on the Internet. I do believe you have just Stepped in IT [Breast Cancer].Some members wondered where they could find their former community members on other websites. One member suggested to start a discussion titled ”Refugees from that other site“ for the members to meet up and continue to support one another (Breast Cancer). Another suggested Facebook site where members can find the other members. Several Breast Cancer community members discovered that former members had posted about a discussion thread called “Friends from WebMDs Support Board” at another breast cancer forum.While most patients who commented about moderators believed they were helpful and important members of their communities, some patients discussed how they did not find moderators’ help to be useful. These members considered peer-support as the main benefit to participation in OHCs, and felt that moderators could deter the dynamic exchange of ideas among patients and peers.For example, a member stated that HPMs did not weigh in with medical expertise when needed. Certain moderators’ questions were thought to be unhelpful for the community, such as “What turns you on?” in the case of Sex and Relationships community, which seemed to minimize the seriousness of the medical focus of the community. Other examples of ineffective moderation included inappropriate language spreading out even in the presence of moderators.Members who saw moderators’ roles as providing medical expertise did not find HPMs to be helpful, as they cannot “treat, diagnose, or exam the people” (Sex and Relationships). In addition, members discussed how the “one size fit all” advice coming from the HPMs was not helpful; rather, peers’ “one-on-one, spiritually rewarding” support would be more beneficial for the community (Diabetes).A few members considered others’ shared personal experience as more critical than moderators’ help. In an exchange between 2 members on the Sex and Relationships community, they shared their belief that their community did not seem to need HPMs’ involvement:Member 1: It’s got to a point where we do not need a doctor, we’re too good Member 2: I think you are right? We really are better than any doctor, any day, Right???A similar sentiment was found in the Fibromyalgia community:It’s the people that make the community and the support we gain from each other that keep us coming back.Some members believed that the absence of moderators might facilitate increased sharing among peers. Members believed that self-efficacy was essential and that moderators could not help with disease management. This thought was especially apparent on the Diabetes community. Patients felt that the communities did a good job of self-regulating and making sure that no one was claiming to share medical advice; rather, they were sharing anecdotal experiences. As one poster commented:I think there are a lot of very smart people who post on the various boards and I have never seen anyone claim to be an expert. We post what we know through personal experiences or something that has worked for us [Sex and Relationships].Although members believed that moderators may not improve the chemistry of communities, they did acknowledge that the website was losing long-term membership with the absence of moderators. Several members of the Diabetes community mentioned that it is up to the patients themselves to manage their disease, and that moderators cannot help. Similar sentiments were echoed on the Sex and Relationships community, noting that peer’s expertise was important.. Many believe that patients gravitate to these communities because they are free from professional governance [OHCs were primarily developed to connect patients to others with similar disease processes in an attempt to provide emotional and informational support -28. Whilvernance ,31. By iThis analysis of the departure of STMs from WebMD communities offers several insights into the role that moderators play on OHCs. Longstanding community members quickly detected moderators’ absence. The departure spurred conversation and debate on the communities about the utility of moderators. This rapid detection of the departure of moderators appeared to be indicative of the important role moderators played on many of the communities, such that the void was noted quickly. This void was validated by our quantitative analysis demonstrating that many of the moderators were active on communities. The close personal ties that many community members felt with moderators was demonstrated by the fact that many members worried about the employment status of the moderators, and some further petitioned WebMD to bring the moderators back.Although many participants appeared to be especially appreciative of the pragmatic help that moderators had provided to their communities, such as monitoring for trolls and stimulating conversation when it had slowed, many of these roles were quickly assumed by community members. However, members did not enjoy these tasks, which often made them comment on how they missed the moderators. Community members also worked to create a sense of camaraderie and encourage their peers to continue to post, thus filling in the void left by the moderators. These tasks appeared to be less bothersome to posters. Such altruistic behavior is often observed in closely knit online communities, which is one of the strongest sources of maintaining online participation ,33.Several posters commented they did not feel as safe participating after moderators had left their communities, even as members tried to step in and report inappropriate posting activity. This finding suggests that even on vibrant communities, the role of the professional moderators may be unique and irreplaceable. This role of professional moderators, however, should be distinguished from general moderators or informal leaders in online community literature ,35. The Although the general sentiment of community members toward moderators was positive, this sentiment was not universal. The departure of the moderators spurred debate on some communities, which felt that the utility of the moderators might vary according to the disease process the community dealt with. Members were insightful in their discussions about the tension between having moderators versus not having moderators. Having moderators can potentially impede conversation but limit the exchange of erroneous information. The lack of moderators or self-moderated communities can have strong community dynamics but requires strong commitment by the members to self-moderate bothersome, unmoderated exchanges by visitors and new comers with either ill intent or lack of knowledge around the community norms and common ground. Furthermore, not having moderators and committed members would lead to reduced access to health professional knowledge or validated information.A few members threatened to, and eventually left their communities, after removal of the moderators. Some spoke about attempting to find people who had left for other websites. The remaining members believed that their communities were less active. However, this analysis has revealed that the overall posting activity of the WebMD health communities was declining throughout the study period, so it is difficult to infer that the decline in posting activity was a result of the moderators’ departure. Rather, the decline of member participation was followed by STMs’ and HPMs’ increased posting activity. Moderators’ increased activities showed WebMD’s effort to increase participation, which was evidenced by a study that reported qualitative analysis on the post content of WebMD’s moderators . The resThe participation decline observed in WebMD is a canonical retention problem in social media. Thus, user migration has been widely studied to understand how to retain and attract users in social media platforms -42. MainAnother reason for decline in member participation might be due to the members not seeing health benefits as Su et al have discussed in their literature review of eHealth Systems . HoweverWe also found the lack of distinction community members drew between HPMs and STMs. Although WebMD only dismissed STMs, members did not initially pick up on the fact that HPMs were still active members of their communities. Similarly, even on communities where there were HPMs participating , some members were frustrated because they did not realize that there were HPMs (and such moderators were desired). Online health information consumers are keen on finding indicators for the source of information -46. ThusOur study has implications for future OHC development. By exploring the impact of the removal of STMs from several well-established communities and the discussions ensued among participants, an insight was gained into what aspects of moderators’ presence and posting activity are most appreciated by members and helpful:OHCs need explicit, systematic policing activities put into place to help members feel safe and agree on continued participation. This was a universal sentiment expressed on the communities; members appreciated this role that moderators had previously played and did not enjoy assuming this responsibility. Given the disproportionate number of verbal members who volunteer to take on the moderating role, strong dependence on these members will threaten the sustainability of the communities.The importance of HPMs may vary according to health topics of the communities. It is difficult to characterize which communities benefited most from moderators from our study, given the more or less similar participation pattern across all communities. However, members’ conversations revealed the needs are different among different communities, where communities such as diabetes or heart disease might need immediate help from health professionals. Further work should clarify and identify what characteristics of illness experiences drive members to seek health professionals’ knowledge online rather than peers’ social support.The background of moderators  should be explicit. Some WebMD community members demonstrated a poor understanding of the distinction between HPMs and STMs, where some thought HPMs have left the communities. Even in communities with an HPM, posters noted that they had been promised such an expert on the community and yet had still not encountered him. Explicit signatures should be available for every post and profile, clarifying if the moderator has undergone any professional health training, or is an STM. If an STM, whether the STM has any professional knowledge on health should be presented explicitly.Major community changes, such as removing moderators, should be communicated to members with advanced notice. Much of the discussion about moderators centered around communities attempting to understand what had happened to them after they noticed their absence. Members’ frustration about the decision to remove moderators, and the lack of notification, was apparent in their posts. Members felt betrayed, and as many of them had developed close bonds with moderators, were upset about not being able to say their goodbyes to these members of their communities.Our data source was limited to observed, publicly available posts. Thus, our analysis is limited to those who are verbal and have expressed opinions. Other rich metadata sources can be used to further analyze deeper insights about OHC activities, such as user logs, page views, and click streams . The resThe analysis of STMs’ departure from WebMD communities provided a critical insight into the utility of moderators in OHCs. By analyzing the reactions of community members in 11 different illness communities to moderators’ departure, it was found that members perceive that moderators play an important role in OHCs, stimulating discussion and making them feel safe. However, this quantitative analysis of the posting activities shows that moderators’ efforts were not enough to increase the already decreasing member participation. Our work disputes a widely held belief about patients’ blindly accepting other patients’ shared information in OHCs and preferring nongovernance. Although moderators’ efforts did not play a large-scale influence on member participation, their presence was effective and favored by the core members. The discussion on member attrition and attraction in OHCs contributes to the call for analyzing attrition as a core research in eHealth systems . This st


The incorporation of pseudoallylic strain by N-methylation at the solvent exposed loop in proteins leads to a stark increase in their thermodynamic stability that can be tuned by altering the amino acid composition. N-methylation of the i+1–i+2 amide bond at the reverse turn results in well-folded and stable β-sheets in aqueous solution at room temperature. The developed superactive turn inducing motif is tolerant to a wide variety of functional groups present on coded amino acids making the designed turn fully compatible with bioactive loops in proteins. We demonstrate that the steric restraint and the functional groups at the reverse turn act in synergy to modulate the folding of re-engineered β-sheets. Introduction of the turn motifs onto a three-stranded β-sheet protein, Pin 1 WW domain, resulted in various analogs showing a cooperative two-state transition with thermal stability (TM) ranging from 62 °C to 82 °C. Despite modulating the stability of Pin 1 variants by ∼2.8 kcal mol–1 (ΔΔGf), the native fold in all the protein variants was found to be unperturbed. This structural stability is brought about by conformational preorganization at the engineered reverse turn that results in strong intramolecular hydrogen bonds along the three dimensional structure of the protein. Thus, this simple loop engineering strategy via two amino acid substitution provides us a “toolkit” to modulate the stability of β-sheet containing peptides and proteins in aqueous solution that will greatly expand the scope of de novo protein and foldamer design.Reverse turns are solvent-exposed motifs in proteins that are crucial in nucleating β-sheets and drive the protein folding. The solvent-exposed nature makes reverse turns more amenable to chemical modifications than α-helices or β-sheets towards modulating the stability of re-engineered proteins. Here, we utilize van der Waals repulsive forces in tuning the steric restraint at the reverse turn. The steric restraint induced upon  Tight packing in the hydrophobic core of a protein by optimizing the van der Waals interaction R-nipecotic acid-S-nipecotic acid,via synthetic and semi-synthetic methods.Free peptide bond rotation is a major driving force for the unfolding of a protein.i+1–i+2 amide bond at the turn by N-methylation.N-methylation was found to introduce a favorable pseudoallylic strain between various atoms of the i+1 and i+2 residues in the β-turn (CNMe/i–1)(Cβ and i)(CNMe/i) strictly favors a trans peptide conformation, whereas when present between two homochiral residues (l-/l- or d-/d-), there is an equal probability for the N-methylated peptide bond to adopt a cis or a trans conformation , the introduction of pA1,3 strain increases the Ktrans/cis in the heterochiral system (L3) and significantly decreases the Ktrans/cis in the homochiral system (L2). The reduced Ktrans/cis in the homochiral system is a result of the unfavorable 1,3-diaxial strain in the trans conformation, which is absent in the heterochiral system  alone does not provide sufficient conformational restriction to favor the trans conformation; however, in combination with the pA1,2 strain (L3) it increases the Ktrans/cis. These results clearly indicate that the differential behavior of an N-methylated peptide bond depends on the neighboring substituents with the highest propensity to adopt a trans conformation when the flanking residues are heterochiral.This finding encouraged us to evaluate the role of pseudoallylic strain in governing the peptides  using NMystem ESI. FurtherN-methylation of the peptide bond, we analyzed the Ramachandran plot of N-methylated linear tetrapeptides with varying pseudoallylic strain. We observed that irrespective of the chirality of N-methylated residue, N-methylation restrains Ψ of the preceding residue , due to van der Waals repulsion, provides added stability to the β-sheet.Initially, we synthesized four molecules with varying pseudoallylic strain in the reverse turn to analyze the critical role of tability . We acquand 2, 3 , we obse > 4 > 1  which isments ESI. This ordom coil .20 We al peptide .21 The bi+1 and i+2 residues of the reverse turn in the β-sheet peptide with diverse functional groups, but fixed N-methylation at the i+1–i+2 amide bond.Encouraged by this finding, we set out to systematically scan the 3 that showed the maximum foldedness. Since the pseudoallylic strain pA1,3 is critical in folding the peptide, keeping the i+1 substitution constant, we initially sought to substitute the i+2 residue with various l-amino acid side chains and assess their influence on the β-sheet stability using ∑CSD  between i+2 N-methyl and i+2 Cγ  leads to destabilization of 3b and 3a. In contrast, in 3e the β-branch demonstrates almost a two-fold increase in the ∑CSD resulting from the restricted χ1 rotation due to a probable intramolecular hydrogen bond between Thr–OH and its C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbdri0Nd" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O , we obtained a more than two-fold increase in the ∑CSD compared to that of 3b. Another notable observation was the increase in the foldedness of the β-sheet by incorporating positive charges, e.g., in 3l and 3n at the termini of an unbranched amino acid side chain 3f. These results show how modulation of local steric interactions and charges at a single site in the β-turn can alter the overall foldedness of the β-sheet.Our primary library was based on ing ∑CSD .21 The cing ∑CSD  indicateg>O ESI. Moreovei+2 substituted linear peptides, we estimated the folded population of selected β-sheets and their free energy of folding (ΔGfold) in water at 25 °C . The presence of a hydrophobic β-branch (3a and 3b) is detrimental to the folding of the peptide, whereas a –CH3 to –OH substitution dramatically alters the folded population . Thus, we observe that a combination of pA1,2 and polar functional groups enhances the stability of the β-sheet by –1.0 kcal mol–1 (ΔΔGf between 3n and 2).Next, towards the thermodynamic analysis of at 25 °C  utilizind-amino acid substitutions at the i+1 site with N-methylated alanine at the i+2 site. To our satisfaction, we observed the strong tolerance of various functional groups at the reverse turn (i+2 site (3b), by virtue of the unfavorable pA1,2 strain destabilizes the β-sheet, whereas when present at the i+1 site, the favorable pA1,3 strain enhances the stability of the β-sheet (4g) which is comparable to the stability of the ring constrained reverse turn motif pG (–1 destabilization (ΔΔGf between 3 and 3b) or stabilization (ΔΔGf between 3 and 4g) when present at the i+2 or i+1 site, respectively . To our amazement, the introduction of the i+1 isopropyl group into 3n resulted in an additional 0.58 kcal mol–1 stabilization (ΔΔGf between 5 and 3n) yielding a super stable monomeric β-sheet 5. This result suggests that the i+1 and i+2 substituents act synergistically to enhance the foldedness of the engineered β-sheet. Additionally, although almost all functional groups are tolerated at the reverse turn, certain amino acids, leucine, lysine, glutamine, and arginine at the i+2 site and d-aspartate, d-threonine, d-lysine, d-tyrosine, d-phenylalanine and d-valine at the i+1 site, display high propensity to stabilize the β-sheet  < –2.3 ppb K–1 (2) < –1.6 ppb K–1 (3) < –1.0 ppb K–1 (3n) ≅ –1.0 ppb K–1 (4g) < –0.6 ppb K–1 (5)). Thus, it is tempting to speculate that the increased conformational restriction in the β-turn possibly results in an enhanced strength of the intramolecular hydrogen bond stabilizing the β-turn.Thus, this conformational restriction results in a well-formed β-turn with a strong hydrogen bond between Lys8Hin.Gfold  corrobor3n and 5 typical of β-sheet rich proteinsi+2 site in the reverse turn. The observed right-handed twist was also indicated by the presence of strong NOEs between Lys9Hβ/γ and Tyr2Hδ in the ROESY spectrum3n and 5 as opposed to 4g , also shows the involvement of the guanidinium group of arginine at the i+2 site in side chain-backbone hydrogen bonding.Another remarkable feature was the stark right-handed twist observed in to 4g ESI.28 Althoi+1 and i+2 side-chain functional groups which imposes a favorable steric constraint on the β-turn. Such preorganization of the main chain torsion angles by side-chain substitution confers extraordinary conformational stability to proteins and peptides as was observed in collagen triple helices by substituting proline with 4-fluoroproline and 4-methylproline.32The enhanced thermodynamic stability of the monomeric β-hairpins in water is fundamentally attributed to the conformational preorganization of the β-turn attained by different et al. that the introduction of a type II′ β-turn affords the fastest folding of the WW domain.This encouraged us to utilize the designed turn motifs with myriad side-chain functional groups to engineer the conformational stability of proteins. Since antiparallel β-sheets are quite prevalent in proteins and have found great utility towards the development of non-antibody based- and hyperstable peptide scaffolds,TM and free energy of folding (ΔGf), respectively. Unlike the model 12-mer peptide, the incorporation of pA1,3 strain at the reverse turn in 6 was sufficient to impart reasonable thermodynamic stability (TM of 68.7 ± 0.2 °C) to the Pin 1 protein due to multiple hydrophobic interactions along the β-stands in the entire protein. The TM values of all the Pin 1 variants studied were independent of the protein concentration over a range of 5–50 μM indicating their monomeric nature  in 7 (i+2 site (8) by virtue of the unfavorable pA1,2 strain destabilizes the Pin 1 by ∼0.9 kcal mol–1 (ΔΔGf). However, when incorporated at the i+1 site (9), it stabilizes the protein by ∼1.2 kcal mol–1 (ΔΔGf), further strengthening the critical role of steric interaction at the reverse turn in governing the thermodynamic stability of proteins. Nevertheless, the most exciting observation was the synergism between i+1 and i+2 residues at the reverse turn in enhancing the thermodynamic stability of the Pin 1 protein as observed in 10  and 11  with the incorporation of lysine and arginine at the i+2 site, respectively, which corroborated our findings in the 12-mer model peptide.All the Pin 1 variants were chemically synthesized, which showed the characteristic maximum at 227 nm in CD spectra indicating the presence of a folded protein ESI. Their tature ESI. FurtherGf) in 7 . The int8 and 11 (ΔTM of ∼20 °C) corresponding to about 2.8 kcal mol–1 (ΔΔGf) results from significant perturbation in the secondary structure of the re-engineered Pin 1. To identify the structural alterations in the solution conformation of Pin 1 variants, we acquired the 1H, TOCSY, and NOESY spectra of each Pin 1 variant (40–60 μM) at 15 °C. On comparing the backbone Hα CSD of the re-engineered Pin 1 variants with the wild-type apo-Pin 1 WW domainNG)1H resonances  were observed in Arg11 show strong hydrogen bonding as compared to 8 to the amino acids of the flanking N-term and C-term strands indicating higher stability of the intramolecularly hydrogen bonded region in 11.Overall, we observed more positive amide temperature coefficient values in 1 than 8 ; howeverN-methylation of a peptide bond differentially modulates the population of cis/trans isomers about the tertiary amide bond. This property of an N-methylated peptide bond along with its heavily restricted conformational space allowed us to probe the strength of pseudoallylic strain pA1,2 and pA1,3 in engineering reverse turns with different functional groups present on coded amino acids. Utilizing the combination of pseudoallylic strain and the functional groups on coded amino acids, we could impart remarkable conformational stability to isolated β-sheets and proteins in aqueous solution at room temperature. This enhanced conformational stability is presumably achieved by preorganization of the β-turn by the formation of stable hydrogen bonds at the reverse turn in the transition state.i+1 and i+2 sites of a β-turn, which induces favorable steric constraints upon the main chain. Thus, by modifying the side-chain substituents at the turn, we could successfully tune the stability of β-sheets. Interestingly, a single amino acid substitution at the reverse turn also contributes towards modulating the right-handed twist in isolated β-sheets.In summary, we demonstrate that the non-covalent constraint induced by Gf of ∼2.8 kcal mol–1 between the least and the most stable Pin 1 analogs). Hence, with the remarkable advancement in tools to generate semi-synthetic proteins45Since reverse turns are solvent exposed, and the pseudoallylic strain provided a basic conformational stability to the reverse turn, it allowed us to probe the effect of various amino acid substitution on the stability of a mini-protein, Pin 1 WW domain. We show that re-engineering the non-repetitive motif like β-turns with non-natural amino acids can have a large contribution to protein stability (ΔΔThere are no conflicts of interest to declare.Supplementary informationClick here for additional data file.


The transport properties of few-layer graphene are the directly result of a peculiar band structure near the Dirac point. Here, for epitaxial graphene grown on SiC, we determine the effect of charge transfer from the SiC substrate on the local density of states (LDOS) of trilayer graphene using scaning tunneling microscopy/spectroscopy and angle resolved photoemission spectroscopy (ARPES). Different spectra are observed and are attributed to the existence of two stable polytypes of trilayer: Bernal (ABA) and rhomboedreal (ABC) staking. Their electronic properties strongly depend on the charge transfer from the substrate. We show that the LDOS of ABC stacking shows an additional peak located above the Dirac point in comparison with the LDOS of ABA stacking. The observed LDOS features, reflecting the underlying symmetry of the two polytypes, were reproduced by explicit calculations within density functional theory (DFT) including the charge transfer from the substrate. These findings demonstrate the pronounced effect of stacking order and charge transfer on the electronic structure of trilayer or few layer graphene. Our approach represents a significant step toward understand the electronic properties of graphene layer under electrical field. The peculiar band structure features of massive Dirac fermions in multilayer graphene are driving intense activity in fundamental research, as well as for applications in next generation optoelectronic devicesπ and π* bands near the Fermi level in monolayer graphene are modified in FLG, showing strong dependence on the stacking sequence as well as the number of layer4P6/mmm), ABA stacking , and ABC stacking 8In few-layer graphene (FLG), the interlayer coupling introduces perturbations of the low-energy band dispersions. Consequently, the linear Among the different methods to obtain few layer graphene, SiC graphitization is a viable method for a large-scale graphene production11Here, large-area uniform trilayer graphene layers were successfully synthesized on off-axis 4H-SiC(0001). The morphology of the trilayer graphene consisting of large and flat domain was confirmed by LEEM. The band structure and the charge transfer from the substrate to graphene layer were studied using ARPES. The electronic properties and the stacking order trilayer graphene are studied by STM/STS and density functional theory (DFT). Our studies indicate that trilayer epitaxial graphene exhibits two structure with Bernal  and rhom−1. The peak at 1522 cm−1 is considered to be an overtone of the L-point optical phonon from the SiC. The Raman signals from the graphene show prominent characteristic peaks at 1595 cm−1 (G) and 2705 cm−1 (2D), which gives evidence of carbon sp2 reorganization. The G peak value of 1595 cm−1 indicates n type doping. Also the blue-shifted position of the 2D peak at 2705 cm−1 could be the result of a compressive strain of the graphene layer during the post-growth cooling down procedure or charge transfer doping from the substratefwhm) of about 75 cm−1. This broadening can be attributed both to a defect scattering and to the formation of two or more graphene layers. The intensity ratio of the defect-induced D band to the graphite G band (ID/IG) has been widely used to estimate the density of defects in grapheneLa)laser is the Raman excitation wavelength (λlaser = 532nm) and the measured ratio μm. The regions with two different graphene thickness can be distinguished by differences in the reflected intensity25The trilayer graphene used in this study was obtained by annealing 4° off-axis 4H-SiC(0001) at 1550 °C in 800 mbar argon for 10 min. We used micro-Raman spectroscopy to probe the quality and electronic properties of the sample. p spectrum. The spectrum was fitted by sums of Voigt curves . The Lorentzian width (fwhm) was fixed at 45 meVp peaks are made up of spin-orbit split doublets  substrate while the component at 100.9 eV is attributed to the presence of Si clusters formed when Si-C bonds are broken during graphitization301s spectrum showed three components at 283.4, 284.5, and 285.3 eV in binding energy. The sharp C 1s peak, labeled G, located at 284.5 eV in binding energy, indicates the presence of sp2 hybridized C–C bonds and is the signature of the graphene layers. Due to its metallic nature this component was fitted using a Doniach-Sunjic line shape with an asymmetry factor α of 0.1 and a fwhm of 0.5 eV. The two other peaks labelled SiC (283.4 eV) and IL (285.3 eV) are attributed to the SiC bulk and to the interfacial layer respectively. Due to the non-metallic nature of these components, the peaks are symmetric (asymmetric factor α = 0 eV). From the C 1 s spectrum, the thickness of the graphene layers can be calculated from the ratio between the intensities of the G and SiC components. This ratio corresponded to an exponential decay of roughly 3 ML of carbon coverage supposing a continuous and homogenous coverage of the surfaceThe chemical bonding environment of the sample has been probed using X-ray Photoelectron Spectroscopy (XPS) experiments. High-resolution spectra of Si 2p and C 1 s recorded under surface sensitive conditions  are shown in F and ED  surface. The surface is formed by self-ordered periodic structures consisting of pairs of a (0001) basal plane terrace and a (1–10n) nanofacet with a characteristic periodicity of ∼600 nm, as shown in  zone A,  of Fig.  zone B, . The dif zone B, . From th−3 electrons per unit cellD in the 150–200 meV range. z orbitals of the uppermost graphene layer. That this is a reasonable approximation follows from the fact that the STM signal is proportional to the LDOS integrated between the Fermi energy and the corresponding bias voltagez orbitals and since the pz orbitals of the uppermost layer of the multilayer sample have the largest extend into the vacuum region they will contribute most to the signal. We also verified this for the ABC trilayer by calculating the numerical derivative of the STM signal as obtained with the Tersoff-Hamann approximationdI/dV versus bias (V) spectra and the calculated LDOS shown in e.g in the case of ABCA stacking, the variation in charge transfer induces different intensity ratio between the peak above the Dirac point and the secondary peaks at 200–300 meV.In order to understand how the tunneling spectra depend on the stacking of the layer we compare the STS results with the local density of states (LDOS) as calculated by DFT. This comparison reported in α or β atoms of the outermost layers. The DFT studies taking into account the doping of graphene due to the interaction with the SiC underlayer are able to reproduce the observed STS results which are different from the case of undoped rhombohedral stacked graphene. The present analysis is only possible because the STM/STS are very sensitive to the stacking, and because of the very high energy of the STM/STS experiment. Note that the total energy difference between ABA and ABC stacked trilayer graphene is very small (0.18 meV/atom)As compared to the Bernal stacking, the rhombohedral stacked few layer graphene exhibits band dispersions near the Dirac point. Specifically, the low-energy bands near the Dirac level (flat band) in ABC-stacked few layers graphene has a surface states with its wave functions distributed on either In summary, we identify the three bands due to interlayer interactions and two stacking sequences of the trilayer graphene using ARPES and STM/STS measurements. We find that the stacking and the charge transfer change significantly the LDOS of the few layer graphene, which illustrate the unique electronic properties of graphene layers. The STS and DFT spectra show a clear evidence for flat bands near Dirac energy in trilayer graphene on SiC. The flat bands, which contribute to a pronounced peak in the tunneling density of states, arise from ABC stacking. The morphology of the trilayer graphene consisting of large and flat domain was confirmed by LEEM. The lateral domain size is only limited by the step edges pre-existing in the substrate. STS showed the presence of epitaxial trilayer graphene with a two- stacking domain arrangement of carbon atoms.n-type 4H-SiC(0001) substrates that were etched in hydrogen at 1550 °C and annealed at 1525 °C in argon partial pressure of P = 800 mbar. The samples were then cooled down to room temperature and transferred ex-situ from the growth chamber to undergo STM/STS, XPS/ARPES measurementsThe few layers graphene were synthesized on off axis T = 4.2 K, the I(V) characteristics were acquired while the feedback loop was inactive, the differential conductivity dI/dV , proportional to the LDOS, was measured directly by using a lock-in technique. For this purpose a small AC modulation voltage Vmod was added to V  and the signal dI detected by the lock-in amplifier was used to determine the differential conductivity dI /dVmod.STM/STS measurements were carried out using an Omicron ultra-high vacuum low temperature scanning tunnelling microscope (UHV-LT-STM)XPS/ARPES measurements were conducted at the TEMPO beamline of the SOLEIL synchrotron 2 electron-momentum mesh with an 1 mRy Methfessel-Paxton first-order spreading2 electron-momentum grid. In all figures of the paper, the DFT band energy was rescaled by a 1.18 multiplicative factor to correct for the DFT underestimation of the Fermi velocityThe electronic structure calculations were performed using the QUANTUM-ESPRESSO codeHow to cite this article: Pierucci, D. et al. Atomic and electronic structure of trilayer graphene/SiC(0001): Evidence of Strong Dependence on Stacking Sequence and charge transfer. Sci. Rep.6, 33487; doi: 10.1038/srep33487 (2016).