This article analyzes the history of tobacco industry funding for the AIDS response – a largely ignored aspect of private donor involvement. Primary documents from the Legacy Tobacco Documents Library and AIDS organizations are analyzed, alongside existing literature on the tobacco control and AIDS responses. Research on the tactics of transnational tobacco companies has documented how they have used various charitable causes to subvert tobacco control efforts and influence public health policy. This raises questions, which this paper seeks to answer, about if donations by tobacco companies to AIDS organizations have been used for similar means, and if so how AIDS organizations have responded to tobacco industry overtures. Two examples illustrate how tobacco companies initially tried to use the AIDS response to counter tobacco control measures: (1) During the 1990s, Philip Morris, one of the largest corporate donors of the AIDS response in the USA, used its connections with AIDS organizations to create competition for health resources, improve its reputation, and market tobacco products to the LGBT community; (2) In both Latin America and sub-Saharan Africa, Philip Morris and British American Tobacco championed the AIDS response in order to delegitimize efforts to develop the World Health Organization’s Framework Convention on Tobacco Control. However, from the late 1990s onwards, AIDS organizations began to refuse tobacco funding and partnerships – though these policies have been not comprehensive, as many tobacco companies still fund programs in sub-Saharan Africa. The article concludes that tobacco companies aimed to exploit competition between health issues, and use the high-profile AIDS response to improve their reputation and market access. However, AIDS organizations, adhering to broader health goals and drawing on extensive resources and networks, were able to shut the tobacco industry out of much of the response, though pockets of influence still exist. This demonstrates the importance of co-operation and policy convergence across health sectors and suggests that tobacco control advocates, and other charitable sectors that receive funding from the tobacco industry, may be able to draw lessons from the experiences of AIDS organizations. Research on private donor involvement in the HIV/AIDS response to date has largely focused on the role of pharmaceutical companies , ‘on the protection of public health policies with respect to tobacco control from commercial and other vested interests of the tobacco industry’ WHO . The FCTFollowing a brief description of the research methodology, the article provides two examples of tobacco industry involvement in the AIDS response: PM’s support for the early AIDS response in the United States (US), and PM’s and BAT’s efforts to delegitimize the World Health Organization’s (WHO) FCTC process by arguing that AIDS was a more important global health concern than tobacco control. It then documents the measures AIDS organizations took to restrict tobacco industry interference, and the limitations on these efforts, particularly in sub-Saharan Africa. It concludes with policy implications for the AIDS response specifically, and global health governance more generally.2.The research began with a review of secondary literature to identify key processes, actors, events, and initiatives in the relationship between organizations involved in the AIDS response and tobacco industry. The Legacy Tobacco Documents Library, a collection of around 14 million industry documents that were released following US litigation, was then searched using keywords related to the global AIDS response including terms such as ‘National AIDS Fund’ and ‘Global Business Coalition’. The methodology and limitations of analyzing internal tobacco industry documents have been discussed elsewhere , and searching well-known global AIDS organizations’ , and Funders’ Concerned about AIDS) sites with the term ‘tobacco’ as well as the names of the largest tobacco corporations. In some cases, more specific online searches were conducted to verify reference to specific events or partnerships. Two authors reviewed all materials independently to ensure that relevant data were identified, interpreted appropriately and properly contextualized.et al. There were a number of limitations to the research. Many AIDS organizations have not kept historic policy documents or put them online. For example, records from some organizations, such as the Global Business Coalition on Health (GBC) and National AIDS Fund, from the 1990s (when both worked closely with the tobacco industry) were not available. The restriction to online searches meant that results were limited to what documents were made public. Furthermore, it is demonstrated that the TTCs destroyed many documents that should have been made available on the Legacy database , in 1990, when it found out that the TTC made donations to Senator Jessie Helms’ campaigns for re-election McGovern , as wellFor the Philip Morris family of companies, the AIDS epidemic of the 1980s hit close to home. Because of our support of the arts, we became aware of the devastating loss to the arts community wrought by the spread of this disease. PM , 2Over the next decade, PM became one of the largest corporate donors to the National AIDS Fund. The fund had been created in 1988 to generate resources and provide support for ASOs and received donations from a variety of sponsors, including the Ford Foundation and Elton John Foundation NAF . One of PM also explained support for AIDS nutrition programs, such as Positive Helpings, as an extension of Kraft’s (a food corporation that was at the time a subsidiary of PM) programs, which prioritized nutrition-related charitable giving.An additional option would be to identify other groups who feel they are not receiving adequate federal or state funding for their programs. Examples could include, public literacy programs, AIDS research, programs to help the physically challenged, etc … .This effort may lead to opportunities to encourage groups to divert federal or state funds away from tobacco control to other vital public programs during the appropriations process. Slavit , 9However, PM also hoped to use its relationships with ASOs to foster competition for resources that were being directed toward tobacco control. As evidence mounted regarding the harms of smoking and second-hand smoke, the US government increased funding to civil society organizations to conduct anti-smoking youth education and tobacco control advocacy. PM used the networks it had established through its AIDS programs to contest this support. In 1996, the company initiated a strategy entitled ‘Fair Play’, which aimed to limit the effectiveness of tobacco control advocates. Fair Play documents reveal a proposal to mobilize social activists, including those fighting AIDS, to compete with tobacco control groups for government funds. As suggested by Joshua Slavitt, PM Issues Manager:PM planned to achieve this through ‘work with Corporate Contributions and other internal departments to identify individuals or organizations that can act as “emissaries” to reach out to these groups’ Slavit . By lettPM also needed allies to help it counter negative publicity and improve its corporate image. The Commissioner of the Food and Drug Administration testified before US Congress in 1994 that, despite TTC assertions to the contrary, nicotine was indeed a highly addictive drug in tobacco. Lawsuits against the industry, mounted by several US states and whistleblowers, led to the release of internal industry documents revealing how TTCs had attempted to obscure evidence about the harms and addictive elements of smoking. Scholarly analysis and media reporting based on the revelations from these documents severely damaged the reputation of the tobacco companies Ashley . By 2000We’re proud of our relationship with Philip Morris U.S.A. and Virginia Slims, and we support their belief in freedom of choice. Philip Morris, through its diverse food, beer and tobacco companies, has been very supportive of organizations that are leading the fight against AIDS. They were among the first corporations to assist in AIDS research, AIDS prevention/education and direct care for people with AIDS. The Virginia Slims brand has established a history of its own, through various promotions, that have benefited AIDS research over the years. Let’s remember, AIDS does not discriminate and neither should we. PM , 1PM used AIDS fundraising events to improve its public image. For example, its largest AIDS fundraiser was the Virginia Slims Legends Tours – a series of tennis tournaments and all-female musical performances. The tour raised approximately US$ 4 million for the National AIDS Funds between 1995 and 1998 . In particular, a generalized epidemic (as opposed to epidemics contained amongst specific population groups as was the case in most high-income countries) was emerging in sub-Saharan Africa. In South Africa, HIV prevalence among pregnant women climbed from less than 5% of the population in 1990 to nearly 30% in 1997 UNAIDS . As treaMuch less attention was paid to the increasingly globalized use of tobacco products Callard . As counet al. as ‘a significant moment in globalizing tobacco control’, because it was the first meeting to concentrate on the dangers of second-hand smoke and the effects of tobacco use in LMICs , held in Buenos Aires in 1992, was the first tobacco conference held in an LMIC. The conference was described by Muggli in LMICs , 2. Confsic] AIDS increasing importance in the world and in Argentina we believe this disease to be the sole matter cable [capable] [sic] of eclipsing the [WCTOH] Conference.  in 1992, the concept of an international legal instrument for tobacco control was put forward . Established in 1984 by the tobacco industry, the ITGA was a global agricultural lobby group on tobacco farming issues , AIDS organizations began questioning the motivations behind tobacco industry funding. The Coalition of Lavender Americans on Smoking and Health was supported by the Center for Disease Control, in 1996, to organize a conference, ‘Alive With Pleasure!’, which focused on the prevention of tobacco use and alcohol problems in the LGBT communities. The Coalition met with LGBT and AIDS organizations to discuss the health impacts of smoking and suspect practices of TTCs Drabble . Their cIn response to growing awareness of smoking-related diseases within the AIDS community, as well as wider public attention to the public relations tactics of tobacco companies, many ASOs, and LGBT organizations demanded that their boards refuse tobacco-related funding. As early as 1991, a leader of Act-Up San Francisco argued in an editorial, published in the Gay Community News, that receiving funding from PM, ‘represents us taking money from walking over the bodies of those killed by cigarettes … I would characterize this as a pact with the devil’ , has until recently also been a Director of Imperial Tobacco Canada, Ltd. We are deeply disappointed by this revelation and feel this conflict is unacceptable as we work to support meaningful tobacco control programs in Africa. BMGF , 1Concurrently, a number of prominent funders developed supportive policies to limit TTC involvement in global health. The Bill and Melinda Gates Foundation, one of the largest private donors in the global health field, established a policy of not providing funding to organizations that accepted resources from or had connections with the tobacco industry BMGF . As the This case, which was widely covered in the media, propagated the message that in order to access Gates resources, organizations needed to sever all ties with the tobacco industry (see, e.g., Blackwell Our efforts for further and more widespread action on AIDS have been impeded by the difficulty of finding a suitable partner to help us. We were saddened that the head office of an international NGO prevented their regional office from partnering us in several developing countries, solely on the grounds of accepting money from a tobacco company. (Imperial Tobacco The widespread rejection of tobacco money by civil society organizations, institutions, and other private actors in the AIDS response disseminated through global networks. Imperial Tobacco reported dismay, in Overall, by the late 1990s onwards, AIDS organization in the USA and then globally turned away from tobacco funding. This was initially prompted by evidence of the harmful health effects of tobacco use on PWAs but eventually was motivated by recognition of TTCs’ use of the AIDS response to further its interest, and supported by the development of shared norms and greater resources. AIDS organizations were early recognizers of the ethical problems of partnerships with TTCs. Their stance in turn proved supportive of the FCTC process and adoption of measures limiting industry influence in health programs and policy.6.However, TTCs have not been completely shut out of the AIDS response. By 2007, Imperial Tobacco had found another AIDS organization – a French charity called Cinomade – to support in Cote d’Ivore, and continues to fund AIDS programs in sub-Saharan Africa (Imperial Tobacco TTCs have continued to be active supporters of the AIDS response in sub-Saharan Africa, despite resistance from some AIDS organizations and global policies discouraging such partnerships, for two reasons. First, the needs of AIDS initiatives in sub-Saharan Africa remain greater than available resources. Global funding for the AIDS response is projected to be $21.7 billion by the end of 2015, still short of the $22 to $24 billion required to fund programs in LMICs UNAIDS . As a reTTCs continue to use AIDS projects to improve their image and reputation in sub-Saharan Africa and have even been celebrated for their contribution to the African AIDS response. In 2007, BAT sponsored an African–Middle East Conference on AIDS. At the conference, the Nigerian Minister of Labor praised BAT’s national workforce policy on AIDS and called on other businesses to implement similar policies John . It can The continued participation of TTCs in the sub-Saharan African AIDS response demonstrates that efforts to shut tobacco money and influence out have not been comprehensive. TTCs have redirected their resources where the AIDS response remains desperate for funding and where government tobacco control policies are weak. While TTCs claim to be supporting the AIDS response in sub-Saharan Africa because of the dire need, and out of the desire to ensure the health of their employees, their past practices of using the AIDS response to subvert tobacco control efforts and market their products raise questions about the motivations behind their current AIDS programs.Such questions are especially pertinent as rates of tobacco use are on the rise in many sub-Saharan African countries. Cigarette consumption on the continent almost doubled from just over 40 billion in 1990 to nearly 80 billion in 2010 (Blecher & Ross 7.During the early AIDS response in the USA, PM provided funding to ASOs to support marketing campaigns targeted toward LGBT communities, to enhance the company’s reputation during a time of declining public trust of TTCs, and to support arguments for the diversion of resources from tobacco control initiatives. TTCs then applied a similar strategy at the global level as the FCTC process progressed. TTCs capitalized on growing concerns about the AIDS epidemic in LMICs – markets they were also expanding into – to argue that WHO should focus on AIDS, not tobacco control. TTCs aimed to generate competition between the two health issues, for resources and policy attention, to protect their interests from stronger regulation.However, the collective decision by many AIDS organizations to refuse TTC funding, and thus the use of the AIDS epidemic to the tobacco industry’s advantage, demonstrates the capacity of health networks to restrict negative or co-opting influences through closer co-operation. ASOs used knowledge, of the harms of smoking for PWAs and the suspect practices of TTCs, to justify the termination of partnerships with TTCs. Through global networks and the creation of common policies, AIDS organizations thus pioneered practices that restricted the ability of TTCs to exert undue influence over global health policy. These actions by ASOs were supported by the availability of alternative funding sources and an agreed global governance instrument. The FCTC, while not binding on non-state actors, provided a set of shared norms which informed the actions of the AIDS community. Where the gap between resources for AIDS and health needs remains wide, and tobacco control policies are still relatively weak, however, TTCs remain actively involved in the AIDS response. In sub-Saharan Africa, notably, government ministers and TTC representatives still share the same podiums to congratulate each other on their AIDS efforts.Evidence of tobacco industry involvement in the AIDS response raises a number of policy implications for both the AIDS and tobacco control communities, as well as broader concerns regarding the strengthening of global health governance. First, it should be recognized that competition among health issues can be sown and exploited by vested interests for reasons that run contrary to public health goals. TTCs used the AIDS epidemic to try to divert resources and attention away from tobacco control. Priority setting and the allocation of scarce resources should be undertaken according to evidence-based criteria and transparent processes. Moreover, there is a need for more collaborative, rather than competitive, approaches to increase societal resources for health needs overall.Second, other public interest organizations can learn from, and build upon, the policies prohibiting funding from TTCs pioneered by AIDS organizations. TTCs continue to offer donations to a diverse range of charitable organizations concerned, for example, with the environment, domestic violence, poverty alleviation, arts, sports, and education BAT ; PM 2010Third, solidarity across health issues requires mutually supportive approaches embedded in global governance arrangements. In this case, AIDS organizations adopted Article 5.3 of the FCTC which calls for the exclusion of tobacco industry participation in policy-making. AIDS organizations were able to review their own practices around TTC funding because they had a shared normative framework to draw from. Conversely, tobacco control efforts should recognize the particular needs of PWAs. As AIDS organizations have broadened their mandates, such as the Global Business Coalition on HIV/AIDS becoming the Global Business Coalition for Health, there is an even greater need for policy coherence.Finally, to better enable AIDS organizations to choose not to accept TTC funding requires access to alternative resources. TTC funding for the AIDS response, notably in resource-scarce settings such as sub-Saharan Africa, requires replacement by other donors. Their funding could be provided with the condition of compliance with Article 5.3 to ensure that the needs of those affected by HIV/AIDS are not met at the expense of furthering the health-harming activities of TTCs. Such coordination would benefit the AIDS response, tobacco control initiatives and, most importantly, those individuals and communities worldwide affected by either major cause of ill-health and death.


Given the challenges in exploring lifelong therapy with little side effect for human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) cases, there is increasing interest in developing traditional Chinese medicine (TCM) treatments based on specific TCM syndrome. However, there are few objective and biological evidences for classification and diagnosis of HIV/AIDS TCM syndromes to date. In this study, iTRAQ-2DLC-MS/MS coupled with bioinformatics were firstly employed for comparative proteomic profiling of top popular TCM syndromes of HIV/AIDS: accumulation of heat-toxicity (AHT) and Yang deficiency of spleen and kidney (YDSK). It was found that for the two TCM syndromes, the identified differential expressed proteins (DEPs) as well as their biological function distributions and participation in signaling pathways were significantly different, providing biological evidence for the classification of HIV/AIDS TCM syndromes. Furthermore, the TCM syndrome-specific DEPs were confirmed as biomarkers based on western blot analyses, including FN1, GPX3, KRT10 for AHT and RBP4, ApoE, KNG1 for YDSK. These biomarkers also biologically linked with the specific TCM syndrome closely. Thus the clinical and biological basis for differentiation and diagnosis of HIV/AIDs TCM syndromes were provided for the first time, providing more opportunities for stable exertion and better application of TCM efficacy and superiority in HIV/AIDS treatment. By the end of 2016, ca. 36.7 million people were living with HIV and ca. 35.0 million people have died from AIDS-related illnesses worldwide3. The development of therapies for prevention and control of HIV/AIDS remains a herculean and significant task.HIV/AIDS pandemic represents the most severe global health challenge in modern history5. However, the medication involved in HAART is often limited by drug toxicity, poor treatment tolerability and drug resistance6. Traditional Chinese medicine (TCM), with more than 5000 years of clinical practice, has become one of the mainstays of complementary and alternative medication therapy7. It has been proved that TCM can reduce the side effects associated with HAART, enhance the immune functions of patients, and improve clinical symptoms and quality of life9. Furthermore, recent clinical study showed that TCM have positive implications on long-term survival of HIV/AIDS patients10, suggesting that TCM has the potential to become a functional cure for HIV/AIDS. Thus TCM is increasingly getting into the clinical application for HIV/AIDS treatments12.So far, the highly active antiretroviral therapy (HAART) is the most effective treatment for HIV/AIDS, leading to a notable decrease of mortality ratesZheng’ in Chinese, describe the process of summarizing and distinguishing comprehensive signals and symptoms of individuals from a particular stage of disease13. Central to TCM treatment, the differentiation of TCM syndromes is the key principle in guiding the prescription of TCM medications. It aims at specific TCM syndromes and guides the direction of treatment based on TCM theories. Hence accurate discrimination to TCM syndromes is the indispensable prerequisite for effective TCM treatments. However, firstly, the biological evidences for classification of TCM syndromes remain lacking. Moreover, the recognition of TCM syndromes is currently assessed by traditional inspection, auscultation, olfaction, interrogation and palpation14. Such method heavily relies on the clinical experiences of TCM practitioners, usually with certain degree of subjectivity and ambiguity from individuals, greatly hindering the stable exertion of TCM efficacy and superiority. TCM treatments of HIV/AIDS also face the problems. Hence it is urgently needed to scientifically prove the differences of TCM syndromes and provide objective diagnosis of HIV/AIDS TCM syndromes. Shao Li et al. found the metabolism-immune imbalanced network and potential biomarkers to evaluate cold and hot syndromes16, which inspired us to explore a new way to biologically understand TCM syndromes. HIV/AIDS TCM syndromes mainly include accumulation of heat-toxicity (AHT) syndrome and Yang deficiency of spleen and kidney (YDSK) syndrome, lung qi and yin deficiency syndrome, qi deficiency and blood stasis syndrome, and original qi and kidney yin deficiency syndrome19. Among them, AHT and YDSK syndromes account for the top proportions21 and thus were focused on in our investigation. Patients with AHT syndrome usually present with herpes or aphtha, skin fester or ulcer, sticky shit, and fever etc.  technology has been proposed as a powerful alternative27. A flurry of applications, e.g. detection of diagnosis biomarkers for pulmonary tuberculosis28, chronic hepatitis B29 and prenatal neural tube defects30, have been driven in past decade.Comparative serum proteomics has become a robust approach for the demonstration of overall protein levels and identification of disease biomarkers in recent31 and western blot analyses29. Differentially expressed proteins (DEPs) of AHT and YDSK patients compared with healthy people were screened out with iTRAQ-2DLC-MS/MS firstly. These DEPs were further comparably analyzed with bioinformatics assays via Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein-protein interaction (PPI) networks. Thus the biological differences between the TCM syndromes can be scientifically dissected and proved. In addition, western blot assays were carried out to measure the level of TCM syndrome-specific DEPs so that the objective diagnosis biomarkers of HIV/AIDS TCM syndromes can be confirmed.In this research, we aimed to identify the biological differences and serum biomarkers of AHT and YDSK syndromes by employing iTRAQ-2DLC-MS/MS coupled with bioinformaticsTo gain biological insights into the differences of TCM syndromes and further discover serum protein biomarkers, 45 participants were obtained for each TCM syndrome, including 15 AHT patients, 15 YDSK patients and 15 healthy volunteers as the control. The clinical baseline data  or less than 0.8-fold change (down-regulated) were screened out as the DEPs , staphylococcus aureus infection (hsa05150), tuberculosis (hsa05152), viral myocarditis (hsa05416), PI3K-Akt signaling pathway (hsa04151) and phospholipase D signaling pathway (hsa04072) , and signed informed consent was obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations.AIDS Treatment Guidelines (2011 version)’ Then, AHT and YDSK patients were diagnosed according to ‘Traditional Chinese Medicine Treatment of AIDS Clinical Technology Program (2004 Edition)’. Patients were filtered when they satisfied one of the following criteria: pregnant women or nursing mothers; with malignant tumors; with unconsciousness, dementia or mental disease; family members disagree; with primary immunodeficiency or other secondary immunodeficiency caused by hormone chemotherapy; with hematological disorders; with central nervous system diseases or severe organ diseases that were not caused by HIV/AIDS.Both western and Chinese medicine criteria were used to divide HIV/AIDS patients into two groups. First, participants were satisfied with diagnostic criteria of western medicine derived from ‘A total of 87 HIV/AIDS patients (aged 21 to 61 years) were obtained from Chengdu infectious disease hospital, where is the demonstration area for prevention and control of national infectious disease in Liangshan prefecture. These patients were filtered with clinical evaluation as described above. In addition, 35 randomly chosen healthy volunteers, aged from 24 to 54 years were also included  according to the manufacturer’s instructions. The prepared proteins were stored at −80 °C for subsequent analysis. Protein concentration was determined using the Bradford protein assay .200 μg) were digested with Trypsin Gold  with the ratio of protein:trypsin = 30:1 at 37 °C for 16 h. Digested peptides were dried by vacuum centrifugation, reconstituted in 0.5 M TEAB and processed in preparation for 8-plex iTRAQ . The peptides were labeled with isobaric tags and incubated for 2 h at room temperature. The labeled peptide mixtures were then pooled, dried by vacuum centrifugation and separated by strong cation exchange (SCX) chromatography on a 20AD high-performance liquid chromatography (HPLC) system .Proteins from 15 patients in the same group were pooled for iTRAQ labelling. Pooled protein samples (100 μm). The samples were loaded with buffer B  at 400 nL/min. MS data acquisition was performed with a Q-Exactive .The fractions were resuspended in buffer A  and centrifuged at 20000 g for 10 min. The supernatant was loaded onto a C18 trap column (2 cm) on a LC-20AD nanoHPLC  and eluted onto an analytical C18 column . The MASCOT version 2.3.0 search engine was used for all searches. All searches were performed against the human protein sequence database (NCBInr_2014_human). For protein identification, the following criteria were used: trypsin digestion allowing a maximum of one missed cleavages; Carbamidomethyl (C) was set as fixed modifications. Oxidation (M), Gln → Pyro-Glu , iTRAQ 8 plex (Y), iTRAQ 8 plex (N-term) were selected as variable modifications. The peptide mass tolerance and MS/MS tolerance were set to ±15 ppm and 20 mmu, respectively.http://www.interactivenn.net/).The quantification was processed using Proteome Discoverer version 1.3 with the following settings: the fold change threshold for up-/down-regulation was set at 20; only peptides unique for a given protein or protein group and with the 99% confidence were selected for quantitation; the number of the minimum unique peptides was set at one; experimental bias was corrected by normalization to the median in each sample. Venn diagram of up- and down-regulated proteins was analyzed by InteractiVenn (http://www.ebi.ac.uk/GOA/). Proteins were classified by GO annotation based on three categories: biological process, cellular compartment and molecular function. KEGG database were used to annotate pathways. First, we annotate proteins using KEGG online service tools KAAS. Then we map the annotation result on the KEGG database using KEGG online service tools KEGG mapper. PPI were analyzed for identified acetylated proteins using Cytoscape software. PPI network obtained from STRING database, a weighted interaction database with physical and functional interactions33. We fetched all interactions that had a confidence score ≥0.7 (high confidence).GO annotation proteome was derived from the UniProt-GOA Database (www. μL) from 15 AHT-HIV/AIDS patients, 15 YDSK-HIV/AIDS patients and 15 healthy individuals were treated with gel-loading buffer for SDS-PAGE electrophoresis at 120 V for 1 h. The samples were then transferred to a PVDF membrane (Amersham Biosciences) at 15 V for 20 min. The membranes were blocked with 5% bovine serum albumin at 37 °C for 2 h and then incubated with primary antibodies  at 4 °C overnight, followed by incubation with alkaline phosphatase-conjugated secondary antibodies (1:1000) at 37 °C for 1 h in darkness. The bands were visualized with enhanced chemiluminescence  and the band intensities were quantified with ImageJ . Albumin was used as reference to calculate the relative intensity of each protein. The calculation of mean ± standard deviation and Students’ t tests were performed with GraphPad Prism 6.0 software.The prepared protein samples (10 Supplementary Information


The diagnosis of Immunoglobulin G4 (IgG4)-related disease (IgG4-RD), in general, depends on serum IgG4 concentrations and histopathological findings; therefore, diagnosis of IgG4-RD in cardiovascular organs/tissues is often difficult owing to the risk of tissue sampling.Prevalence of IgG4-positive lymphoplasmacytic infiltration in 103 consecutive cardiovascular surgical samples from 98 patients with various cardiovascular diseases was analyzed immunohistochemically.n = 8; thoracic, n = 9); aortic dissection (n = 20); aortic stenosis (n = 24), aortic regurgitation (n = 10), and mitral stenosis/regurgitation (n = 17). In total, 10 (9.7%) of the 103 specimens showed IgG4-positive cell infiltration with various intensities; five of these were aortic valve specimens from aortic stenosis, and IgG4-positive cell infiltration was present at >10 /HPF in three of them. In one aortic wall sample from an abdominal aortic aneurysm, various histopathological features of IgG4-RD, such as IgG4-positive cell infiltration, obliterating phlebitis, and storiform fibrosis, were observed.The diagnoses of the enrolled patients included aortic aneurysm (abdominal, IgG4-positive cell infiltration was observed in 9.7% of the surgical cardiovascular specimens, mainly in the aortic valve from aortic stenosis and in the aortic wall from aortic aneurysm. Whether IgG4-positive cell infiltration has pathophysiological importance in the development or progression of cardiovascular diseases should be investigated in future studies. Immunoglobulin G4 (IgG4)-related disease (IgG4-RD), first documented by Hamano et al. in 2001 . At presSeveral previous reports have demonstrated IgG4-RD in various-size arteries and pericardium –13. MostIn this study, histological analysis was carried out on 103 consecutive histologic specimens obtained from 98 patients who underwent cardiovascular surgery at Department of Thoracic and Cardiovascular Surgery, Osaka Medical College between January 2014 and December 2014. None of the enrolled patients had been diagnosed with or was suspected to have IgG4-RD at the time of surgery. Due to the retrospective design of the current study, serum IgG4 concentrations of the enrolled patients at the time of surgery were not available. Histological evaluation was carried out on formalin-fixed and paraffin embedded specimens. Immunostaining was performed with mouse monoclonal antibody against IgG4 . IgG staining was performed with anti-IgG antibody . For some specimens, owing to the high background of IgG staining, anti-human CD138 antibody  was used to stain plasma cells. In specimens where IgG4-positive cell infiltration was observed, we counted three ×40 fields with the highest number of IgG4-positive plasma cells, and then calculated the average number of IgG4-positive plasma cells within these fields. For the purpose of calculating the IgG4-to-IgG, or IgG4-CD138, ratio, the same three fields were counted [n = 8) or thoracic (n = 9), was diagnosed for 17 patients. There were no patients who were diagnosed with inflammatory aortic aneurysm. From patients with either mitral stenosis (n = 4) or mitral regurgitation (n = 7), 11 mitral valve samples and 6 myocyte samples were obtained. Five tumors were analyzed, comprising atrial myxoma (n = 3), thymoma (n = 1), and pulmonary artery sarcoma (n = 1).In total, 103 histological samples from 98 patients were analyzed. About one fifth of the patients had a history of malignancy, and nine patients were undergoing chronic hemodialysis Table . Aortic P = 0.062 by Fisher’s exact test).Of the 103 histological samples, IgG4-positive cell infiltration was demonstrated immunohistochemically in 10 samples (10 patients): five in aortic valve samples, and five in aortic wall specimens (Table P = 0.047 by Fisher’s exact test). IgG4-positive cell infiltration was not observed in any of the mitral valve samples (n = 11), myocardium samples (n = 6), cardiac tumors (n = 3), pericardium samples (n = 3), mediastinal thymoma (n = 1), or pulmonary artery sarcoma (n = 1).Three of the 10 patients with a histological sample showing IgG4-positive cell infiltration were undergoing chronic hemodialysis; this prevalence of hemodialysis (30%) was significantly higher than that among patients without IgG4-positive cell infiltration  are demonstrated in Fig. According to clinical records, during the median follow-up period of 464 days , neither occurrence of IgG4-RD nor dysfunction of the prosthetic valve  was noted in any of the five patients with positive IgG4-positive cell infiltration in their aortic valve sample.n = 2), aortic dissection (n = 2), and thoracic aortic aneurysm   Table . The preThe sample of case 1, who was diagnosed with AAA, showed not only IgG4-positive cell infiltration, but also other pathological features that are characteristic of IgG4-RD, such as adventitial thickening, lymphoid follicle formation, eosinophil infiltration, perineural infiltration, storiform fibrosis, and obliterating phlebitis Fig. . Based oAccording to clinical records, during the median follow-up of 367 days , occurrence of IgG4-RD in tissues other than cardiovascular tissues was not noted in any of the five patients with positive IgG4-positive cell infiltration in the aortic wall samples. In addition, in case 1, neither clinically significant systemic inflammation nor graft problems occurred during the follow up period.In the current study, whether, and if so to what extent, IgG4-positive cell infiltration was present in 103 consecutive surgical samples from 98 patients undergoing cardiovascular surgery. In total, IgG4-positive cell infiltration was observed in 10 histological samples. IgG4-positive cell infiltration was most frequently observed in aortic wall of the AAA  followed by the aortic valve of aortic stenosis , and the aortic wall of dissecting aneurysm . On the other hand, IgG4-positive cell infiltration was not observed among the remaining 93 histological specimens. These findings indicate that IgG4-positive cell infiltration may occur in certain limited cardiovascular disease. In one patient with AAA (case 1), in addition to IgG4-positive cell infiltration, other histopathologic features characteristic of IgG4-RD listed in the International Consensus Diagnostic Criteria (ICDC)  were obsThere are several previous studies examining the prevalence of IgG4-RD or IgG4-positive lymphoplasmacytic infiltration in cardiovascular histological specimens. Kasashima et al. reported that 13 (5.2%) of 252 surgically-treated AAA cases  and 5  cases of 24 aortic stenosis and 5 cases of aortic aneurysm or aortic dissection. These findings collectively indicate that IgG4-positive cell infiltration is not a rare finding in cardiovascular diseases, especially in aortic stenosis, aortic aneurysm, and aortic dissection. The pathophysiological importance of IgG4-positive infiltration in these disorders should be investigated in further studies.


In this study, three chemistry research themes closely associated with the Nobel Prize are bibliometrically analyzed—Ribozyme, Ozone and Fullerene—as well as a research theme in chemistry not associated with the Nobel Prize (a Nobel snub theme): Brunauer–Emmett–Teller equation. We analyze, based on an algorithmically constructed publication-level classification system, the evolution of the four themes with respect to publication volume and international collaboration, using two datasets, one of them a subset of highly cited publications, for each considered time period. The focus of the study is on international collaboration, where co-occurrence of country names in publications is used as a proxy for international collaboration. For all four themes, especially for Brunauer–Emmett–Teller equation, the publication volumes increase considerably from the earliest period to the later periods. The international collaboration rate shows an increasing trend for each theme. For Ozone, Fullerene and Brunauer–Emmett–Teller equation, the international collaboration rate tend to be higher for the highly cited publications compared to full datasets. With regard to the evolution of number of countries per international publication and per highly cited international publication, a vast majority of the distributions are positively skewed, with a large share of publications with two countries. With respect to the last four periods of the study, the concentration to two countries per publication is more pronounced for the Brunauer–Emmett–Teller equation theme compared to the three Nobel Prize themes. The scientific Nobel Prizes receive much attention, not only within the scientific community, and might be regarded as the ultimate accolades in science Merton . A numbeAs stated by Bruckner et al. , citatioIn this study, three chemistry research themes closely associated with the Nobel Prize are bibliometrically analyzed—Ribozyme, Ozone and Fullerene—as well as a research theme in chemistry not associated with the Nobel Prize but a Nobel snub theme: Brunauer–Emmett–Teller equation (BET equation). The purpose of the study is analyze the evolution of the four themes with respect publication volume and international collaboration. In particular, we aim at comparing the three Nobel Prize themes with the BET equation theme with respect to potential evolution differences. For instance, one might ask if the evolution is driven by the Nobel Prize or not. To shed light on such questions, it is important to include at least one  non-Noble Prize theme in the study.In order to fulfill the purpose of the study, an automatically constructed, hierarchical publication-level classification system and two datasets are used. The focus of the study is on international collaboration, where co-occurrence of country names in publications is used as a proxy for international collaboration. We believe it is useful study international collaboration in view of its potential important influences on the evolution of scientific research. Indeed, international collaboration, considered as a common and functional characteristic in the production and development of research, has been increasingly analyzed, varying from the analysis of its patterns to designing policies for supporting research .Ozone: The work in atmospheric chemistry, particularly concerning the formation and decomposition of ozone (1995).Fullerene: The discovery of fullerenes (1996).BET equation, named after its three inventors : The equation results from BET theory, which aims to explain the physical adsorption of gas molecules on a solid surface.The official web site of the Nobel Prize was explored in order to select three chemistry themes associated with the prize.Article or Review, with the largest number of shared references (with the seed publication) were recorded.The Nobel laureate lectures corresponding to the three themes (a)–(c) appear as publications in Web of Science, and these publications were retrieved and used as seed publications. For BET equation, we obviously had to take another approach. We did a topic searchArticle or Review have been algorithmically grouped into classes based on direct citation links between them. The classification consists of four hierarchical levels: level 4 with 22 broad classes, level 3 with 673 classes, level 2 with 3935 classes, and level 1 with 35,192 classes. For each class in the system, content labels have been obtained automatically. The terms used as content labels are keywords, journal names, names of Web of Science journal categories, and titles of review articles. For each class, the three most relevant terms, where relevance is based on the tf-idf scheme  at level 1 to which the three related publications, for a given seed publication, belong was identified. However, the number of publications of the identified classes turned out to be too small for a meaningful analysis. Therefore, for a given seed publication, the class at level 2 of the three related publications was identified.Note that (a), (b), (c) and (d) in this list corresponds to (a), (b), (c) and (d), respectively, in the first list of this section. In the rest of this work, we denote the class associated with (a) by Ribozyme_c, with (b) by Ozone_c, with (c) by Fullerene_c and by (d) by BET_c. Further, we refer to the four classes by the term “research fields”.For the analysis of the evolution over time, with respect to publication volume and international collaboration, of the four research fields, the publication period of the study, 1980–2015, was divided into the following 3-year periods: T1 = 1980–1982, T2 = 1983–1985, T3 = 1986–1988, T4 = 1989–1991, T5 = 1992–1994, T6 = 1995–1997, T7 = 1998–2000, T8 = 2001–2003, T9 = 2004–2006, T10 = 2007–2009, T11 = 2010–2012, and T12 = 2013–2015.S, and the subset of S of its highly cited publications. We define a highly cited publication as a publication belonging to the 20% most frequently cited publications in S.For each research field and each time period, we considered two datasets: the set of publications in the research field published during the time period, say P: Number of publications.international publications, defined as publications co-authored by two or more countries.IP: Number of Ph: Number of highly cited publications.IPh: Number of international publications among the highly cited publications.IPR: Percentage of IP relative to P.IPRh: Percentage of IPh relative to Ph.ΔIPR(Tn): Degree of alteration of IPR at Tn relative to T(n − 1) (ΔIPR(Tn) = | IPR(Tn) − IPR(T(n − 1))|, 1 < n ≤ 12).ΔIPRh(Tn): Degree of alteration of IPRh at Tn relative to T(n − 1) (ΔIPRh(Tn) = | IPRh(Tn) − IPRh(T(n − 1))|, 1 < n ≤ 12).The indicators used in the study can be divided into two categories: indicators based on publication volume, and indicators based on country (name) volume within publications. The following indicators are based on publication volume:9.CIP: Number of countries per international publication.10.CIPh: Number of countries per highly cited international publication.We collectively refer to the indicators 5 and 6 in the list above as “International collaboration rate”. The alteration indicators 7 and 8, inspired by Sangwal , facilitAdmittedly, the selection of the four themes, especially the three Nobel Prize themes, is quite arbitrary. However, as indicated above, Bibmet has 1980 as the earliest publication year. In view of this, we decided to select Nobel Prize themes that were awarded the prize no earlier than approximately 1990. Obviously, for a theme to be selected it should not have been awarded the prize too late. Therefore, it was decided to select themes that were awarded the prize no later than approximately 2005. A further condition was that for each of the four seed publications, its three related publications should belong to the same class at level 2 of the Bibmet publication-level classification system (cf. footnote 7). If a theme does not satisfy this condition, the union of two or three classes can be used. However, such a union might involve a lot of publications that are not connected, or weakly connected, to the theme. Expressed in another way, the precision of the union might be low. Several initially considered chemistry Nobel Prize themes did not satisfy the condition.Table The number of publications in the Ribozyme_c field increases strictly from T1 to T11, from 68 publications in T1 to 1419 in T11  increases strictly, since these constitute 20% of the number of publications for each period). This yields that the publication volume is about 21 times higher in T11 compared to T1. In the Ozone_c field, we observe a strict increase in publication volume from T1 to T6, from 930 to 2000 publications. The peak occurs in the period T10 . From T6 onwards, the number of publications is relatively stable. In the Fullerene_c field, publication volumes are relatively small up to the period T4. A steep increase occurs from T4 to T6, which is the period of the volume peak . From T6, the publication volume strictly decreases. Ozone_c has much larger publication volumes in initial periods than Ribozyme_c and Fullerene_c, and Ozone_c and Fullerene_c have total publications volumes more than twice the corresponding volume of Ribozyme_c  from the period T3 onwards are positively skewed, with a large share of publications with two countries. However, this share, which generally is above 80%, is 74%  in T12 for both sets. In this period, we observe small shares of publications with five or six countries . With respect to Ozone_c, for both publication sets, all distributions are positively skewed, with a large share of publications with two countries. In the last four periods, however, the distributions are less concentrated, and publications with more than six countries can be observed. However, the absolute frequencies of such publications are small .According to Cech , the conOur results show that the international collaboration rate tends to be higher for the highly cited publications regarding the research fields Ozone_c, Fullerene_c and BET_c, but not regarding the field Ribozyme_c. The latter was quite unexpected in view of earlier research on the relationship between international collaboration and citation rates (e.g., Aksnes We have to point out four limitations of our study. First, to obtain a deeper understanding of the evolution of the themes of the study, we believe it is proper to go further back in time than to the year 1980. However, we were not able to do that, since the earliest publication year of Bibmet is 1980. Second, the number of publications is low in the earlier periods for the research fields Ribozyme_c, Fullerene_c and BET_c. The results that concern these periods and fields should therefore be interpreted with caution. Third, the three algorithmically constructed fields used in the study cannot be assumed to be perfect representations of the three corresponding research themes. Some publications that clearly belong to the Ribozyme theme, for instance, might be missing in the field Ribozyme_c, whereas some publications that are only weakly connected to the theme might be present in Ribozyme_c. Forth, we are not chemists, which makes it difficult for us to put forward causes for the observed differences. Our understanding of these are, then, quite restricted. The use of subject experts as result interpreters is an interesting possibility in this respect, and we might realize this possibility in future, and similar, studies.For future research, we would like to further explore how the algorithmically constructed publication-level classification system employed in this study can be used for the analysis and mapping of research themes. For a given theme, an alternative starting point might be a comprehensive review article with several cited references. The classes of the system to which the cited references, covered by Web of Science, belong can be identified for each hierarchical level of the system. By this, publication classes strongly connected to the theme might be obtained.


In particular, AT can be quantified accurately in live zebrafish using fluorescent lipophilic dyes. Although this methodology offers considerable promise, the comprehensive identification and classification of zebrafish ATs has not been performed. Here, we use fluorescent lipophilic dyes and in vivo imaging systematically to identify, classify and quantify the zebrafish AT pool. We identify 34 regionally distinct zebrafish ATs, including five visceral ATs and 22 subcutaneous ATs. For each of these ATs, we describe detailed morphological characteristics to aid their identification in future studies. Furthermore, we quantify the areas for each AT and construct regression models to allow prediction of expected AT size and variation across a range of developmental stages. Finally, we demonstrate the utility of this resource for identifying effects of strain variation and high-fat diet on AT growth. Altogether, this resource provides foundational information on the identity, dynamics and expected quantities of zebrafish ATs for use as a reference for future studies.The zebrafish model system offers significant utility for  Summary: A standardized nomenclature and classification system for zebrafish adipose tissues and regression models to predict expected adipose size during the course of zebrafish development. The chronic consumption of hypercaloric diets in modern societies causes excessive accumulation of lipid within adipose tissue (AT), AT dysfunction and increased risk for cardiovascular disease and diabetes . A centrAT is a vertebrate innovation and forms during development . In bothin vivo imaging systematically to identify, classify and quantify zebrafish ATs. We identify 34 distinct ATs and classify them according to anatomical location. Furthermore, we quantify the area of each AT over a range of postembryonic zebrafish sizes and construct regression models to predict expected AT sizes. These data provide vital information on the expected complement and size of zebrafish ATs. Finally, we apply this classification and quantification system to identify effects of strain variation and diet on zebrafish AT. Altogether, the new approach established in this resource provides the framework for large-scale analysis of AT distribution dynamics and supports the expanded use of zebrafish as a model for AT research.In this resource article, we use FLDs and whole-animal P=0.486), suggesting that these cohorts were comparable and could be analysed together. To help relate AT development to established postembryonic stages, we mapped EKW SL to standardized SL (SSL)  wild-type fish derived from 37 independent clutches A. These SL (SSL)  and usedSL (SSL) .Fig. 1.Figs S5-S22). For classification purposes, we adapted a system previously used for human ATs , abdominal visceral adipose tissue (AVAT) and caudal fin ray subcutaneous adipose tissue (CFRSAT)], then dissected each AT and quantified triacylglyceride content by fluorimetric assay . In addition, we also applied models to predict consolidated AT categories (i.e. VAT and SAT) (Table S1). SL explained the majority of variance for the 43 adiposity traits tested  (Table S1). Models for the single adiposity trait for which SL was not an accurate predictor  could not be improved by use of different, or multiple, predictor variables . Intriguingly, however, age had the greatest ability to predict UHD size (Table S3). For all ventrally located ATs, including UHD, other hyoid apparatus (HYD) ATs [basihyoid (BHD) and ceratohyal (CHD)] and subcutaneous abdominal adipose tissue (ASAT), SL had a reduced predictive ability (mean R2=0.51), suggesting that measurement of these depots from a lateral view was prone to experimental error (Table S1). However, overall these statistical models allow the prediction of expected AT levels in wild-type animals of different sizes. Charts depicting the growth dynamics for each AT are included in Figs S5-S22, and the raw measurement data are included in Table S4.We next systematically quantified all identified zebrafish ATs within our EKW cohort. For each of the 34 ATs, we constructed regression models to predict AT size across a broad range of postembryonic EKW fish  and have utility for defining stages of zebrafish AT development. We interspersed adiposity ‘milestones’ with existing postembryonic ‘milestones’ to provide comprehensive coverage across postembryonic development . IAT was largely composed of VAT, with NVAT never contributing >12% of IAT . However, SAT composition was more dynamic and evenly distributed between the three main subdivisions . PVAT and AVAT were the primary components of IAT, whereas subcutaneous lateral adipose tissue (LSAT) was the largest SAT . Analysis of body fat percentage  showed a marked slowing, coincident with the appearance of SAT . Although not striking by eye, pelvic fin ray (PR) appearance stage ; however, transitions between postembryonic stages and appearance of ATs were not significantly different between control and high-fat-fed fish , suggesting that regionally distinct ATs have different responses to high-fat diet.To evaluate the utility of the AT classification system further, we next sought to increase adiposity experimentally using 5% chicken egg yolk (CEY) as a high-fat diet supplement . We expofed fish ,C. Furthfed fish D. High-ffed fish ,F. Furthfed fish G, suggesTo facilitate identification of zebrafish ATs, included below are detailed descriptions on the individual morphologies and growth patterns of each AT.Fig. S5A). We confirmed PVAT localization to the exocrine pancreas using the ptf1a:eGFP transgenic line . The mechanism by which the pancreas promotes AT development is unclear; however, pancreatic acinar cells can transdifferentiate into adipocytes, suggesting the potential for a shared lineage . This morphological change was consistent with similar changes occurring to the exocrine pancreas, and PVAT shape closely corresponded to exocrine pancreas shape . It is likely that the morphological changes in PVAT and the exocrine pancreas occur simultaneously with gut looping, leading to the posterior pancreatic ‘bulb’ filling a gap created by the looped intestine. As PVAT was associated with the exocrine pancreas, it was asymmetrically localized to the right flank of larvae . However, from ∼SP:10 mm SL, small deposits of PVAT appeared on the left flank and exhibited distinct morphology consistent with a multi-lobed pancreas interspersed between a looped intestinal tract  . The relationship between SL and PVAT area was nonlinear , and the rate of PVAT growth noticeably plateaued from ∼10 mm SL. Of note, AT was found immediately basal to the intestinal submucosa . In humans, omental AT juxtaposed to the intestine is often used for metabolic studies . In previous studies, PVAT was called pancreatic WAT (peroxisome proliferator-activated receptor γ (pparγ), fatty acid-binding protein 11a (fabp11a), adiponectin (adipoqb) and adipsin  (Pancreatic VAT (PVAT) was the first zebrafish AT to appear and was detected immediately ventral to the swim bladder  . Owing t studies ; howeveratic WAT , and adi d; cfd) .Fig. S5A). AVAT was the second zebrafish AT to appear at ∼AC:5.5 mm SL and was often the largest AT . Although they become tightly pressed together in larger fish, a clear gap was visible between AVAT and PVAT until ∼10 mm SL when viewed laterally . In larger fish, when a gap was not evident, a distinguishing line dividing these ATs was often observed  . Taking these points into account, AVAT appeared entirely symmetrical which, together with its central location, might play a role in buoyancy and movements associated with swimming. AVAT first appeared at the posterior extent of the anterior SB and, subsequently, formed between the anterior and posterior SB lobes . Occasionally, a small AVAT cluster was located at the anterior end of the anterior SB, but as AVAT expanded, all three clusters merged to form a final AVAT with a consistent and distinctive morphology . Histology revealed that AVAT connected to the lateral extremes of the kidney . Running bilaterally within the AVAT of adult males were testes, which themselves contain adipocytes; however, this distinct depot could not be discerned by whole-animal stereomicroscopy . SL was a highly accurate predictor of AVAT area . We refer to this AT as abdominal VAT (AVAT) (ig. S6C) . Analysi and cfd .Fig. S7A and Fig. S7G,H). Histology revealed that RVAT could be located in an entirely subcutaneous location ; however, at different anterior-posterior levels, RVAT expanded between body wall musculature  and was found contiguous with the kidney . Therefore, surprisingly, RVAT bridges both subcutaneous and visceral locations. In mammals, renal AT can be either perirenal  or pararenal  . Therefore, by morphology alone, our data suggest that RVAT is analogous to mammalian perirenal AT. As previously noted, a small portion of AVAT, at its dorsalmost extremity, was located in a perirenal position . Similar to PVAT and AVAT, SL accurately predicted RVAT area  was a far smaller and simpler AT than either PVAT or AVAT. RVAT appeared immediately posterior to the operculum at ∼RVAT:7.9 mm SL ( fascia) . By hist fabp11a .Fig. S9); a location that is likely to correspond to pericardial AT in humans. Zebrafish CVAT was best viewed and quantified ventrally, and consisted of two deposits that grew to merge . CVAT was first detected at the anterior heart (aCVAT) at approximately ventral opercular (∼vOPC):9.7 mm SL where the bilateral cleithrum meets ventrally . A second CVAT deposit at the posterior extent of the heart (pCVAT) appeared from ∼pCVAT:12.5 mm SL . aCVAT expanded posteriorly and connected with pCVAT to form a single depot with a distinct bulbous morphology when viewed ventrally . By histology, it was evident CVAT was contained within the wall of the parietal pericardium ; however, even in adults the body wall musculature did not fully close around CVAT, suggesting that CVAT could also be classified as subcutaneous . CVAT was also in close proximity to ASAT . ASAT ran along the ventral midline from the anus to the heart  and was likely to join or closely abut CVAT. Unlike other VATs, the rate of CVAT growth did not decrease in larger fish, suggesting that CVAT undergoes uninhibited expansion throughout the fish from our cohort .In humans, cardiac VAT (CVAT) around the heart is found in two locations; epicardial  and pericardial  . These dIAT is also classified as ‘non-visceral’ in humans . In zebrFig. S11A-G). We term these stripes according to their position along the dorsal-ventral axis; dorsal (dPOS), central (cPOS) and ventral (vPOS). cPOS was the largest POS and was initially detected in posterior locations at ∼cPOS:9.1 mm SL before spreading anteriorly . dPOS was detected at ∼SP:10 mm SL, again in a posterior location before extending anteriorly . vPOS was detected from approximately dorsal fin ray subcutaneous adipose tissue (∼DFRSAT):11.5 mm SL, and appeared as a small streak originating in posterior regions before terminating at the anal fin . All POS ATs appeared internal and segmented, suggesting that they are localized to reiterated structures . As such, cluster number and cluster length were useful metrics of POS development . By histology it was possible to discern that dPOS was located immediately dorsal to the neural tube  and was found in a large cluster at its anteriormost point, which we term the dPOS cluster . cPOS was localized around the outside of the notochord , and vPOS was present associated with ventral hemivertebrae . Quantification of FLD+ POS ATs appearance was variable , most probably because of experimental error attributable to its internal location. cPOS and dPOS were shown to express fabp11a by Paraosseal AT (POS) was composed of multiple deposits associated with the zebrafish skeleton. When labelled by FLDs and viewed laterally, POS constituted three stripes that ran along the flank of zebrafish . We term these IM ATs according to their position running dorso-ventrally at the midline of the tail; dorsal (dIM), central (cIM) and ventral (vIM) . These deposits were usually very small and consisted of individual adipocytes when viewed on transverse section . In situ hybridization by fabp11a.AT associated with skeletal muscle can be in intramuscular (within a muscle), perimuscular (outside muscle) or intermuscular (between muscles) locations . PerhapsCSAT was the first SAT to appear (at ∼DR:6.6 mm SL) and comprised a diverse and intricate collection of SATs that, for simplicity, we have subdivided into ATs located at the operculum (OPC), eye  and hyoid apparatus (HYD).Fig. S14). HYD ATs were named mandible WAT by Fig. S14A-D′; S4). However, the appearance of UHD was inconsistent and its area poorly estimated by SL and other measures of zebrafish size . fabp11a.Three distinct AT clusters were deposited in close proximity to the hyoid apparatus. These ATs were best viewed and quantified from ventral, and were classified according to their proximity to the closest hyoid structure: basihyoid (BHD), ceratohyal (CHD) and urohyoid (UHD) cartilages  . We term these dorsal opercular (dOPC) and ventral opercular (vOPC) ATs. Both dOPC and vOPC were fairly loose collections of adipocytes, but formed in very consistent anatomical positions . SL accurately predicted OPC area . OPC were termed cranium WAT by Two loose LD clusters were evident in dorsal and ventral regions of the operculum . Posterior ocular AT (pOCU) and ventral ocular AT (vOCU) were closely positioned  and often merged in larger animals . Anterior ocular AT (aOCU) was sometimes observed in two clusters; immediately proximal to the eye and further dorsally . OCU were termed cranium WAT by Four distinct AT clusters formed around the eye from ∼PB:7.7 mm SL . LSAT grew to become the largest zebrafish SAT . LSAT first appeared at ∼LSAT:8.2 mm SL in an anterior location at the level of somite #1 . LSAT expansion occurred primarily along its anterior-posterior axis . However, expansion also occurred along its dorsal-ventral axis to produce a long, thin depot . At the horizontal myoseptum, clusters of LSAT appeared constrained within somite boundaries . By both histology and transgenic reporter methods, LSAT was partly localized to the wedge of slow-type skeletal muscle present at the horizontal myoseptum . However, LSAT expanded ventrally and also juxtaposed fast-type fibres . SL was an accurate predictor of LSAT area ; however, LSAT length  and width  could also be used to quantify LSAT development.LSAT was located peripherally and ran laterally along the trunk at the horizontal myoseptum . DSAT was categorized into anterior (aDSAT) and posterior (pDSAT) compartments intercepted by the dorsal fin . pDSAT was first detected at ∼vOPC:9.5 mm SL in a posterior location close to the caudal fin, and expanded anteriorly along its anterior-posterior axis . aDSAT appeared at ∼SP:10 mm SL and initially consisted of two clusters; one located anteriorly towards the head  and another posteriorly at the base of the dorsal fin . These distinct aDSATs expanded and eventually connected to form a single aDSAT (not shown). Iridescent iridophores were especially prominent at aDSAT .Extensive SAT was present in long, thin streaks in the dorsalmost trunk (DSAT) . VSAT appeared at ∼vOPC:9.8 mm SL . As with DSAT, VSAT was closely positioned with pigment cells; however, melanophores appeared most prominent around VSAT .In a similar manner to DSAT, SAT was also present in a ventral stripe, termed VSAT, between the anal and caudal fins . We term this deposit ASAT. ASAT was initially located in a cluster close to the ventral region of PVAT  and could be hard to distinguish from PVAT when viewed laterally . ASAT expanded within a restricted ventral region along the entire anterior-posterior extent of the zebrafish belly . ASAT did not fully fulfill the definition requirements of SAT, as it was not positioned between muscle and skin but, like CVAT, appeared as a fatty island surrounded by body wall musculature . ASAT connected with CVAT at its anterior extremity, suggesting that CVAT and ASAT are conjoining ATs and could potentially form a single large SAT . At the position of the pelvic fin girdle, ASAT narrowed and there appeared to be an imperfect ‘join’ between two ASAT parts . ASAT was called subcutaneous WAT by From ∼SA:10.5 mm SL, SAT was located at the abdomen in a streak along the ventral belly . PELSAT appeared as discrete clusters immediately anterior to the base of the pelvic girdle  and grew to form symmetrical ‘wedges’ anterior to the pelvic girdle . When viewed laterally, PELSAT could be mistaken for ASAT or even PVAT . In larger fish, ∼CFRSAT:11 mm SL, PELSAT formed loose adipocyte-LDs along its edges . PELSAT was shown to express fabp11a by Bilaterally symmetrical ATs appeared at the base of the pelvic fins at ∼cPOS:9.1 mm SL. We term these PELSAT . AFRSAT appeared at ∼vOPC:9.7 mm SL with only one or a few clusters initially evident . In larger animals, the number of AFRSAT clusters increased, and clusters were evident between each fin ray . AFRSAT area was highly correlated with SL . Analysis of the number of clusters relative to SL revealed that additional AFRSAT clusters were added between fin rays during a short developmental time frame from ∼SP:10 mm SL .Discrete LD clusters were evident at the base of anal fin rays, which we term anal fin ray SAT (AFRSAT) . Dorsal fin ray SAT, termed DFRSAT, appeared at ∼DFRSAT:11.5 mm SL and expanded by both adding new clusters and growth of existing clusters .In a similar fashion to AFRSAT, adipocyte-LDs were clustered between the rays of the dorsal fin . We term this collection of AT the anal fin cluster SAT (AFCSAT) . AFCSAT appeared at ∼SP:10 mm SL and initially formed a large cluster at the anterior of the anal fin and immediately posterior to the anus . A second cluster appeared and extended into horizontal stripes in both anterior and posterior directions . Histology revealed that these horizontal stripes were in a subcutaneous location  and deposited at the base of the anal fin musculature . At later stages, these AFCSAT stripes also connected with sparse LDs forming ventral extensions of LSAT .Positioned dorsally and anterior to AFRSAT was a complex collection of subcutaneous LDs that formed in a highly stereotypical fashion with consistent and distinctive morphology . Unlike AFRSAT and DFRSAT, CFRSAT was not deposited in discrete clusters between fin rays, but instead formed a continuous mass . CFRSAT extended the full extent of the dorsal-ventral axis of the caudal fin, and connected to VSAT and pDSAT . We defined the extent of CFRSAT at these extremes by its striated appearance relative to VSAT and pDSAT . CFRSAT was shown to express fabp11a by AT at the base of the caudal fin (CFRSAT) appeared at ∼CFRSAT:10.8 mm SL . When viewed laterally, aPECSAT and pPECSAT could be mistaken for CVAT . However, when viewed from ventral, aPECSAT was clearly observed close to aCVAT and positioned at the pectoral fin base from ∼pPECSAT:11.8 mm SL . From ∼pCVAT:12.8 mm SL, pPECSAT was observed in more distal regions of the pectoral fin . These PECSAT depots expanded but retained consistent morphology . Loose LD clusters, collectively termed lPECSAT, were sometimes evident at more lateral regions of the pectoral fin from ∼DFRSAT:11.5 mm SL and formed large clusters , posterior (pPECSAT) and ‘loose’ (lPECSAT)  to identify 34 regionally distinct zebrafish ATs; (2) to describe detailed morphological characteristics for each of these ATs; (3) to identify ‘milestones’ useful for delineating postembryonic stages relevant to AT development in zebrafish; (4) to develop standardized nomenclature for zebrafish ATs; and (5) to construct statistical models that predict expected AT size and variation. Furthermore, we use this methodology to compare adiposity dynamics across zebrafish strains and analyse regional adiposity levels after exposure to a high-fat diet. Altogether, this study generates a reference resource useful for future studies investigating adiposity dynamics in zebrafish.Of particular note, the EKW fish used in this study were derived from two highly related stocks, but fed different diets and raised in distinct facilities. Considering this environmental heterogeneity, it was surprising that variance in AT size was so small, suggesting that genotype exerts a strong influence on adiposity traits in zebrafish. This conclusion is supported by strong heritability estimates for human adiposity traits, including body fat distribution and early-onset obesity . CompariPrevious studies have established that ATs in zebrafish and mammals share many homologous molecular markers . HoweverAll zebrafish experiments conformed to the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, using protocols approved by the Institutional Animal Care and Use Committee of University of North Carolina-Chapel Hill and Duke University. Wild-type Ekkwill (EKW) strain zebrafish were used in this study. EKW fish were raised at either University of North Carolina-Chapel Hill (UNC-EKW) or Duke University (Duke-EKW) under a 14 h light/10 h dark cycle. WIK fish were raised at UNC.Artemia franciscana , and larvae were gradually weaned onto an Artemia-only diet over the next few days. From 30 dpf, the Artemia diet was supplemented with a 5:2:1:1:1 mixture of TetraMin flakes, AquaTox flakes, spirulina, freeze-dried Artemia (Aquatic Eco-Systems) and Cyclopeeze (Argent Labs).UNC-EKW or WIK embryos were collected from adult pairs and ∼50 embryos raised in 100-mm-diameter Petri dishes containing 30 ml of system water at 28.5°C. At 5 dpf, embryos were transferred to static 2 litre tanks at a density of 10 larvae/l. At 10 dpf, tanks were moved onto a slow-drip water supply, and at 14 dpf the rate of water supply was increased to a steady flow. From 5 dpf, UNC-EKW larvae were fed twice daily a 3:2 powdered mixture of Active Spheres Golden Pearls  and spirulina powder . At 10 dpf, the diet was changed to a 3:2 powdered mixture of Rotifer Size I Golden Pearls and spirulina-fed twice daily. From 14 dpf, powdered diets were supplemented with live Artemia twice daily supplemented with a twice daily feed of Gemma Micro 75. From 28 dpf, the Micro 75 diet was replaced with Micro 150. Postembryonic zebrafish were exposed to 5% chicken egg yolk (CEY) as described by Tg(ptf1a:eGFP)jh1Tg and Tg(smyhc1:eGFP)i104 transgenic strains (referred to as ptf1a:eGFP and smyhc1:eGFP) were maintained on EKW backgrounds  with slow dripping water at a density of 20 larvae/l. From 5 to 14 dpf, larvae were fed Zeigler Larval AP100 (Zeigler Bros. Inc.) twice daily, supplemented with Gemma Micro 75 (Skretting) powder twice daily. From ∼14 dpf, larvae were fed kgrounds . The ‘cokgrounds . The staBODIPY 505/515 , Nile Red (Sigma Aldrich) and HCS LipidTOX Red  were used to label neutral lipid. LipidTOX dyes were preferred for both confocal analysis and when using fluorescent transgenic lines, as fluorescence emission from LipidTOX is restricted to single channels. BODIPY and Nile Red are brighter dyes and were preferred when using stereomicroscopy to image whole-animal adiposity. Staining was conducted as described by Fig. S23).All image analysis was conducted in FIJI/ImageJ (v1.50e) . Two copThe area of three ATs  were measured in 24 zebrafish following the protocol above. The zebrafish were then individually housed in glass beakers overnight to allow the Nile Red stain to wash out. The following morning, ATs were dissected and triacylglyceride quantified using the fluorimetric triacylglyceride assay kit (Biovision) and a Synergy HT plate reader (BioTek).Fixation, embedding, sectioning and Masson's trichrome staining of juvenile and adult EKW zebrafish was conducted as described by P>0.5 was used to determine when an AT was most likely to be present. Linear ordinary least squares (OLS) regression was used to test the ability of SL to predict AT area. To select the most accurate model, transformations to linearize nonlinear relationships and addition of power functions were tested. Full and reduced models were compared with a partial F statistic. Where error variances were unequal, observations were weighted with the absolute value of each residual. The best model for each AT is included in Table S1 (EKW) and S2 (WIK). As the models contain power functions, care must be taken in extrapolating beyond the range of the data or at data extremes.To determine the SL at which specific ATs appear, we performed logistic regression as per


Hybrid materials with superior structural and functional properties can be obtained by incorporating nanofillers into polymer matrices. Polyhedral oligomeric silsesquioxane (POSS) nanoparticles have attracted much attention recently due to their nanometer size, the ease of which these particles can be incorporated into polymeric materials and the unique capability to reinforce polymers. We review here the state of POSS-containing polymer nanocomposites. We discuss the influence of the incorporation of POSS into polymer matrices via chemical cross-linking or physical blending on the structure of nanocomposites, as affected by surface functional groups, and the POSS concentration. Polymers are widely used in industry due to their light weight and ductility. However, they have a lower modulus and strength when compared to metals and ceramics , which m1.5, where R may be a hydrogen atom or an organic functional group, e.g., alkyl, alkylene, acrylate, hydroxyl or epoxide unit  (PCL-b-P4VP)-POSS blends revealed that increasing the amount of POSS in the PCL-b-P4VP diblock caused a change in the morphology due to increased hydrogen-bonding interactions [The miscibility and self-assembly of poly[, amount of POSS, crystallinity, morphology, The influence of POSS surface functionality on mechanical properties  has been studied by incorporating POSS with three different surface functional groups  into polypropylene (PP) via melt-blending . AdditioIncorporation of trisilanolisobutyl-POSS was also found to enhance mechanical properties of polylactide (PLA), as evidenced by improved ductility, storage modulus and tensile strengths . This prThe amount of POSS present can play a very important role in determining the mechanical properties of POSS-polymer composites. POSS-containing methycrylate composites could exhibit superior or inferior mechanical properties , dependi<10 wt.%) was found to improve significantly the mechanical properties of the polymer . Conversely, a large amount of MA-POSS (≥10 wt.%) led to less desirable mechanical properties. By reinforcing poly(methyl methacrylate) with a small amount of MA-POSS, Gao et al. [Methacrylate-based polymers have been extensively used as dental implant materials for several years . Dental o et al.  effectivo et al. .The morphology of POSS-containing polymeric composites played an important role in determining the mechanical properties of the composite. For example, the incorporation of POSS into PdMPO/PA6 polymer blends was found to influence the mechanical properties of the blend by changing its morphology from droplet to co-continuous . POSS-coPOSS-containing polyethylene (PE) displayed superior mechanical properties than neat PE . The mecApplications of polymeric materials are limited in extreme conditions because of the poor thermal stability of polymers. Incorporation of POSS in polymer matrices can improve the thermal stability of polymers because of the rigid structures of the POSS cage. The thermal stability of POSS-containing composites depends on POSS-polymer surface interactions, the amount of POSS and cross-linking. The role of POSS-polymer surface interactions on the thermal stability of the POSS-containing POM nanocomposites was investigated by dispersing apib-POSS, ge-POSS and PEG-POSS . Incorpoet al. [POSS-containing epoxy nanocomposites exhibited improved thermal stabilities . The suret al.  reportedet al. .et al. [The amount of POSS present in the POSS-polymer composites also influenced the thermal stability. Zheng et al.  studied et al. . At a 5%et al. [Huang et al.  studied et al. ,92 synthet al. . The resIncorporation of POSS in polymeric matrices can alter the mobility of polymers adjacent to particles. Therefore, the incorporation of POSS particles can alter the glass transition temperature Tg of the POSS-containing composite material, which depends on several factors, such as particle-polymer surface characteristics, amount of POSS, and crystallinity of polymer.POSS-polymer surface interactions were found to influence the Tg. The effect of three different surface functional POSS groups, isobutyl (iBu), cyclopentyl (Cp) and cyclehexyl (Cy), on the linear viscoelastic behavior of polystyrene revealed that the Tg was strongly dependent on surface functional groups of POSS particles . Figure et al. [To understand the effect of the amount of POSS incorporation on the Tg, Turan et al.  conducteet al. .Thermal studies carried out on the p(MA-POSS) system, whose morphology was described in 8M8H), Maitra and Wunder [n = 2) was attached to Q8M8H, resulting in reduced chain mobility compared to the linear material. A more dramatic effect was observed for PEO (n = 4), where the material turned completely amorphous from its original crystalline state, indicating four PEO repeat units were insufficient to induce crystallization at the surface. Allyl−PEO (n = 4) is completely crystalline, and no Tg was observed even after rapid quenching. After attachment to silsesquioxane to form Q8M8PEO(n=4), melting was entirely suppressed, and the resulting amorphous liquid showed a Tg value quite close to the Tg obtained for n = 2. Since Q8M8H did not exhibit a Tg before sublimation or decomposition, the authors argued that the observed Tgs resulted from the PEO segments. The attachment of PEO to the inorganic Q8M8 surface was said to restrict the mobility of the chains and suppress their crystallization. The PEO oligomers crystallized with an increasing side chain length. It is worth noting that Q8M8n=2)PEO ( and Q8M8n=4)PEO octasilsesquioxane (Qd Wunder  found thications .versus frequency for pristine polystyrene (PS) and two PS-POSS composites at the same temperature [versus frequency for pristine polystyrene and two PS-POSS nanocomposites at the same temperature. By adding 38 wt.% of Ph-POSS, the α-relaxation shifted by about sixty to higher frequencies, corresponding to a decrease in glass transition.Incorporation of nano-size filler-like POSS into polymeric matrices can improve the dielectric properties of the materials. The improvement in dielectric properties of polymer nanocomposites can be attributed to various factors, such as large particle-polymer interfacial area, particle-polymer nanoscopic structure, change in internal electric field (polarity) due to the presence of nanoparticles. The dielectric properties have been studied for POSS-containing polymeric nanocomposites using dielectric spectroscopy ,96,97. Fperature . ComparePOSS particles possess unique structures and superior properties that allow them to be used as fillers with polymers developed for biomedical applications, e.g., dental composites ,86, drugIn vivo degradation of polyester polyurethanes over a period of six months using cross-linked POSS as the hard crystalline segments have been evaluated by Knight et al. [t et al. . No acutt et al. . POSS-PCt et al. . Heart vt et al. .et al. [Gupta et al.  studied et al. . Chemicaet al. . For thiet al. . The PEUL-lactide)/caprolactone copolymers (P(DLLA-co-CL) covalently attached to PEG [Biodegradable drug-eluting stent coatings were recently developed using thermoplastic polyurethane (TPU) with POSS hard segments (POSS-TPU) and soft segments made of poly/POSS hybrids also supported cell growth without any toxicity recorded . The conet al.  also demPOSS-containing polymer composite materials have attracted significant interest in recent years, as they possess nanoscopic structures and functional properties that are not typically seen in conventional hybrid materials. POSS nanoparticles are 1–3 nm in size, smaller than a polymer radius of gyration, monodisperse and rigid. Incorporation of POSS nanoparticles into both thermoplastic and thermoset polymeric matrices by chemical cross-linking or physical blending methods provides excellent reinforcement. In this review, we highlight studies on POSS-containing polymer nanocomposites that have impacted the material engineering field, with an emphasis on enhancements in mechanical, thermal stability, glass transition and biomedical properties.Incorporation of POSS through chemical cross-linking into polymer can influence its structure, impart amphiphilicity, and improve mechanical properties by reinforcement. The POSS surface functional groups play a crucial role in controlling properties of POSS-containing hybrids. The incorporation of POSS nanoparticles into polymer through physical blending relies on favorable surface interactions between POSS and polymer. POSS having surface functional groups that have favorable surface interactions can disperse uniformly in the polymeric matrix. Uniform dispersion of POSS in polymeric matrices helps to improve physical properties of the nanocomposites. On the other hand, POSS having surface functional groups not compatible with the polymer matrix cannot be dispersed uniformly and lead to phase-separated systems with POSS-rich domain and polymer-rich domains. Such microphase separated systems cannot provide proper reinforcement and may even lead to a decrease in desired properties.POSS-containing polymer nanocomposites are found in diverse areas, such as thermoplastics and thermosets, packaging materials, biomedicine, flow aids, surface modifiers, flame retardants, catalyst support, coatings, membrane materials, ceramic precursors, adsorbents, and dispersants ,105,106.


Dynamics of three MET antibody constructs  and the IgG4-MET antigen complex was investigated by creating their atomic models with an integrative experimental and computational approach. In particular, we used two-dimensional (2D) Electron Microscopy (EM) images, image class averaging, homology modeling, Rapidly exploring Random Tree (RRT) structure sampling, and fitting of models to images, to find the relative orientations of antibody domains that are consistent with the EM images. We revealed that the conformational preferences of the constructs depend on the extent of the hinge flexibility. We also quantified how the MET antigen impacts on the conformational dynamics of IgG4. These observations allow to create testable hypothesis to investigate MET biology. Our protocol may also help describe structural diversity of other antigen systems at approximately 5 Å precision, as quantified by Root-Mean-Square Deviation (RMSD) among good-scoring models. Given the critical roles of the MET/HGF pathway in tumor growth and development, various groups developed MET blocking antibodies –15. Howein vitro bioassays to characterize the agonist properties of LY2875358, using HGF and agonist bivalent MET antibody 5D5 as positive controls with Phospho-AKT as the most sensitive agonist assay. Here we established that LY2875358 (IgG4 isotype) induced only a weak and transient phosphorylation of pan-AKT upon binding to MET  increases as the number of disulfide bonds decreases. A lower number of disulfide bonds resulted in more 2D class averages and a higher number of the corresponding modeled 3D domain conformations. For example, only five unique conformational states were observed for the four-hinge-disulfide-bonded IgG2, while eight and nine conformational states were found for the two-hinge-disulfide-bonded IgG1 and IgG4, respectively.The angular range of domains relative to one another , i.e., tThe EM images of the complex between the MET antigen and IgG4 allowed us to compute seven models of domain conformations. While the antigen presence did not change the average values of the domain angles, it led to a larger span of these angles and higher pairwise RMSD values  than fouIt has been shown that antibodies can adopt multiple conformational states. For example, as many as 120 different conformations were reported for the mouse IgG1 from an IPET study [Our results also indicate that antigen free IgG4 MET conformations are different from those observed in the complex with the antigen. This observation suggests the conformational states required for MET antigen recognition might be different from those accessible to the free antibody. Therefore, the complexes rather than free antibodies are more suited to study structures relevant to the biological activity. Our results demonstrate that the conformational domain variability in our models was dependent primarily on the hinge length, while the number of conformational states revealed by 2D class averages depends on the hinge disulfide patterns.It is commonly understood that negative staining may introduce artifacts, such as particle flattening . Our anaThe resolution of 2D images and the resulting class averages is lower when compared to the near atomic resolution from the state-of-the art single particle cryo-EM reconstruction –24. But Our integrative experimental and computational approach was able to provide models of four MET antibody systems with 5 Å RMSD precision. It is expected to be applicable to the determination of low-resolution 3D models of many other antibodies and for testing certain structural hypothesis at a fraction of cost needed for single-particle reconstruction. While 2D images likely reveal only a fraction of all conformational states, they can serve as a good starting point to understanding a relative structural diversity of a particular system. Therefore, lower resolution 2D imaging and resulting class averages could be complementary to 3D Cryo-EM for biological systems that have multiple conformations differing by more than 5 Å RMSD, such as in the case demonstrated here for the MET antibodies.The variable regions from LY2875358  were cloSamples of IgG1, IgG2, IgG4, and IgG4 MET antigen complex were diluted 1:500 with HBS-N pH 7.4 prior to imaging. The samples were then prepared using continuous carbon grid method. Grids were nitrocellulose supported 400-mesh copper. The samples were prepared by applying 3 μL of sample suspension to a cleaned grid, blotting away with filter paper, and immediately staining with Uranyl Formate.Caki-1 Cells were starved overnight in serum-free medium with 0.5% BSA and then treated with various doses of MET antibodies for 15 minutes. Cell lysates were analyzed for phosphorylation of pan-AKT by MSD ELISA. HGF and agonist bivalent MET antibody 5D5 were used as positive controls.2.EM experiments were performed  using anFig 7):Our integrative structure modeling proceeds through four stages , 11: (1)The individual particles were identified in the high magnification images prior to the alignment and classification. The individual particles were then selected, boxed out, and individual sub-images were combined into a stack to be processed using a reference-free classification method . IndividInitial comparative structure models of the antibody constructs were built by MOE modeling package  using anα atoms between every pair of generated models as well as the domain angles as defined in We used the Rapidly exploring Random Tree (RRT) algorithm , 35 to eThe EM2D module of the IMP program –11 was uA scoring function that can rank alternative models by their accuracy is an essential part of any structure modeling. Good scoring functions can correctly rank models over a broad range of RMSD values from the native state. We examined whether or not such “scoring funnels” existed for EM2D scoring  and deteFor every experimental 2D class average, all domain conformational models were sorted in descending order by their EM2D scores (see Stage 3) and clustered by a “leader” algorithm , 39 to fThe visual inspection, aided by PyMOL  protein The final 3D structures representing the best matches to the 2D class averages were subsequently transferred to the MOE modelling package , and minS1 FileThe file also contains two figures and three tables.(DOCX)Click here for additional data file.S2 File(ZIP)Click here for additional data file.