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Bidirectional links between HIV and intimate partner violence in pregnancy: implications for prevention of mother-to-child transmission | <sec id="st1"><title>Introduction</title><p>Prevention of mother-to-child transmission (PMTCT) has the potential to eliminate new HIV infections among infants. Yet in many parts of sub-Saharan Africa, PMTCT coverage remains low, leading to unacceptably high rates of morbidity among mothers and new infections among infants. Intimate partner violence (IPV) may be a structural driver of poor PMTCT uptake, but has received little attention in the literature to date.</p></sec><sec id="st2"><title>Methods</title><p>We conducted qualitative research in three Johannesburg antenatal clinics to understand the links between IPV and HIV-related health of pregnant women. We held focus group discussions with pregnant women (<italic>n</italic>=13) alongside qualitative interviews with health care providers (<italic>n</italic>=10), district health managers (<italic>n</italic>=10) and pregnant abused women (<italic>n</italic>=5). Data were analysed in Nvivo10 using a team-based approach to thematic coding.</p></sec><sec id="st3"><title>Findings</title><p>We found qualitative evidence of strong bidirectional links between IPV and HIV among pregnant women. HIV diagnosis during pregnancy, and subsequent partner disclosure, were noted as a common trigger of IPV. Disclosure leads to violence because it causes relationship conflict, usually related to perceived infidelity and the notion that women are “bringing” the disease into the relationship. IPV worsened HIV-related health through poor PMTCT adherence, since taking medication or accessing health services might unintentionally alert male partners of the women's HIV status. IPV also impacted on HIV-related health via mental health, as women described feeling depressed and anxious due to the violence. IPV led to secondary HIV risk as women experienced forced sex, often with little power to negotiate condom use. Pregnant women described staying silent about condom negotiation in order to stay physically safe during pregnancy.</p></sec><sec id="st4"><title>Conclusions</title><p>IPV is a crucial issue in the lives of pregnant women and has bidirectional links with HIV-related health. IPV may worsen access to PMTCT and secondary prevention behaviours, thereby posing a risk of secondary transmission. IPV should be urgently addressed in antenatal care settings to improve uptake of PMTCT and ensure that goals of maternal and child health are met in sub-Saharan African settings.</p></sec> | <contrib contrib-type="author"><name><surname>Hatcher</surname><given-names>Abigail M</given-names></name><xref ref-type="corresp" rid="cor1">§</xref><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Woollett</surname><given-names>Nataly</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Pallitto</surname><given-names>Christina C</given-names></name><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Mokoatle</surname><given-names>Keneuoe</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Stöckl</surname><given-names>Heidi</given-names></name><xref ref-type="aff" rid="AF0004">4</xref></contrib><contrib contrib-type="author"><name><surname>MacPhail</surname><given-names>Catherine</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0005">5</xref></contrib><contrib contrib-type="author"><name><surname>Delany-Moretlwe</surname><given-names>Sinead</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>García-Moreno</surname><given-names>Claudia</given-names></name><xref ref-type="aff" rid="AF0003">3</xref></contrib> | Journal of the International AIDS Society | <sec sec-type="intro" id="S0001"><title>Introduction</title><p>Prevention of mother-to-child transmission (PMTCT) has reduced new infant HIV infections from an estimated 32% in the absence of treatment [<xref rid="CIT0001" ref-type="bibr">1</xref>, <xref rid="CIT0002" ref-type="bibr">2</xref>], to as low as 1% [<xref rid="CIT0003" ref-type="bibr">3</xref>, <xref rid="CIT0004" ref-type="bibr">4</xref>]. However, major gaps in achieving PMTCT coverage remain. In 21 priority African countries, PMTCT coverage is estimated to be 65% [<xref rid="CIT0005" ref-type="bibr">5</xref>]. A recent meta-analysis in low- and middle-income settings suggests that while 75% of pregnant women adhere to antiretroviral therapy (ART) during pregnancy, only 53% maintain adequate adherence levels in the postpartum period [<xref rid="CIT0006" ref-type="bibr">6</xref>]. Ensuring PMTCT adherence is crucial, particularly as countries increasingly move towards “Option B+,” a policy that offers immediate, lifelong treatment for pregnant women living with HIV [<xref rid="CIT0007" ref-type="bibr">7</xref>].</p><p>Many structural drivers influence PMTCT uptake and adherence. The literature has noted that structural factors such as stigma [<xref rid="CIT0008" ref-type="bibr">8</xref>–<xref rid="CIT0013" ref-type="bibr">13</xref>], poverty [<xref rid="CIT0011" ref-type="bibr">11</xref>] and transport costs [<xref rid="CIT0010" ref-type="bibr">10</xref>] inhibit women's ability to adhere to PMTCT. Another key structural factor shaping access and adherence to PMTCT may be intimate partner violence (IPV). Fear and experience of IPV influence pregnant women's decisions to take up HIV services [<xref rid="CIT0014" ref-type="bibr">14</xref>, <xref rid="CIT0015" ref-type="bibr">15</xref>], and anticipated violence is associated with declines in HIV testing among pregnant women [<xref rid="CIT0012" ref-type="bibr">12</xref>, <xref rid="CIT0016" ref-type="bibr">16</xref>–<xref rid="CIT0022" ref-type="bibr">22</xref>]. A history of physical or sexual violence decreases the likelihood of HIV-positive women using ART when medically eligible [<xref rid="CIT0023" ref-type="bibr">23</xref>, <xref rid="CIT0024" ref-type="bibr">24</xref>], and those who experience abuse are more likely to miss clinic visits and delay linkage to care [<xref rid="CIT0025" ref-type="bibr">25</xref>].</p><p>Little research to date has explored the association between IPV and PMTCT. In one qualitative study in South Africa, IPV was described as a common barrier to ART adherence in pregnancy [<xref rid="CIT0011" ref-type="bibr">11</xref>]. Healthy intimate partner relationships improve PMTCT uptake: male involvement in antenatal care predicted better adherence to nevirapine in one South African study [<xref rid="CIT0026" ref-type="bibr">26</xref>]; male antenatal attendance halved the risk of MTCT in a Kenyan study, an association that persisted after controlling for maternal viral loads [<xref rid="CIT0027" ref-type="bibr">27</xref>].</p><p>Using qualitative research methodology, we explored IPV as a potential structural driver of HIV-related health among pregnant women. This research aimed to contribute to literature suggesting that structural drivers shape the health and well-being of those already living with HIV, and may pose barriers to uptake of proven prevention strategies.</p></sec><sec sec-type="methods" id="S0002"><title>Methods</title><p>We conducted qualitative research to explore the links between IPV and HIV-related health among pregnant women and service providers in Johannesburg, South Africa. This research was a portion of a larger formative study, intended to help our team design an intervention to address IPV in pregnancy. In this setting, an estimated 29% of pregnant women are HIV positive [<xref rid="CIT0028" ref-type="bibr">28</xref>] and between 25 and 35% experience physical or sexual IPV in the 12 months leading up to pregnancy [<xref rid="CIT0029" ref-type="bibr">29</xref>–<xref rid="CIT0032" ref-type="bibr">32</xref>].</p><sec id="S0002-S20001"><title>Conceptual framework</title><p>To explore the relationship between IPV and HIV-related health of pregnant women, we used an adapted socio-ecological conceptual framework (<xref ref-type="fig" rid="F0001">Figure 1</xref>), which posits that broader structural factors and relationship characteristics influence a woman's HIV-related health [<xref rid="CIT0033" ref-type="bibr">33</xref>]. This type of approach has been embraced by social scientists, who note that broader social and societal factors shape how women are able to adhere to ART [<xref rid="CIT0034" ref-type="bibr">34</xref>] and the extent to which they experience IPV [<xref rid="CIT0035" ref-type="bibr">35</xref>]. A socio-ecological framework highlights that the structural context influences the conditions and health outcomes of both IPV and HIV.</p><fig id="F0001" position="float"><label>Figure 1</label><caption><p>Conceptual framework.</p></caption><graphic xlink:href="JIAS-17-19233-g001"/></fig></sec><sec id="S0002-S20002"><title>Data collection</title><p>We conducted an exploratory qualitative study using in-depth interviews (IDIs) and focus group discussions (FGDs) with a wide range of stakeholders with the potential to take part in, deliver, or scale-up an intervention for violence in pregnancy. Participants included pregnant women, pregnant women experiencing IPV, health workers, non-governmental organizations, community leaders and policy makers (<xref ref-type="table" rid="T0001">Table 1</xref>).</p><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Data collection methods</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Participant group</th><th align="center" rowspan="1" colspan="1">Group size</th><th align="center" rowspan="1" colspan="1">Method</th><th align="center" rowspan="1" colspan="1">Sampling</th><th align="center" rowspan="1" colspan="1">Example participants</th><th align="center" rowspan="1" colspan="1">Topics</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Pregnant women at ANC</td><td align="center" rowspan="1" colspan="1"><italic>n</italic>=13 women in 4 FGDs</td><td align="left" rowspan="1" colspan="1">Focus group discussions</td><td align="left" rowspan="1" colspan="1">Convenience</td><td align="center" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">Social and structural drivers of IPV; types of IPV in pregnancy; patterns of help seeking and available community resources for violence and HIV; barriers to disclosing IPV; receptivity to an antenatal intervention</td></tr><tr><td align="left" rowspan="1" colspan="1">Pregnant abused women</td><td align="center" rowspan="1" colspan="1"><italic>n</italic>=5</td><td align="left" rowspan="1" colspan="1">Semi-structured interviews</td><td align="left" rowspan="1" colspan="1">Convenience</td><td align="center" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">Existing needs and concerns of abused women; patterns of help seeking and available community resources for violence; links between IPV and HIV; receptivity to an antenatal intervention</td></tr><tr><td align="left" rowspan="1" colspan="1">Policy makers</td><td align="center" rowspan="1" colspan="1"><italic>n</italic>=10</td><td align="left" rowspan="1" colspan="1">Semi-structured interviews</td><td align="left" rowspan="1" colspan="1">Purposive</td><td align="left" rowspan="1" colspan="1">Department of Health managers, academic experts</td><td align="left" rowspan="1" colspan="1">Types of IPV in pregnancy; current health sector response to IPV; potential integration with HIV activities, including PMTCT</td></tr><tr><td align="left" rowspan="1" colspan="1">Health providers</td><td align="center" rowspan="1" colspan="1"><italic>n</italic>=8</td><td align="left" rowspan="1" colspan="1">Semi-structured interviews</td><td align="left" rowspan="1" colspan="1">Purposive</td><td align="left" rowspan="1" colspan="1">Doctors, nurses, lay counsellors in antenatal clinics</td><td align="left" rowspan="1" colspan="1">Types of IPV in pregnancy; knowledge and practice responding to IPV; receptivity of health workers to antenatal intervention; existing capacity in clinic</td></tr><tr><td align="left" rowspan="1" colspan="1">Non-governmental organizations</td><td align="center" rowspan="1" colspan="1">
<italic>n</italic>=6</td><td align="left" rowspan="1" colspan="1">Semi-structured interviews</td><td align="left" rowspan="1" colspan="1">Purposive</td><td align="left" rowspan="1" colspan="1">Shelters, police, counselling services</td><td align="left" rowspan="1" colspan="1">Psycho-social, legal and other needs of abused women; referral options for women living with IPV</td></tr><tr><td align="left" rowspan="1" colspan="1">Community leaders</td><td align="center" rowspan="1" colspan="1">
<italic>n</italic>=4</td><td align="left" rowspan="1" colspan="1">Semi-structured interviews</td><td align="left" rowspan="1" colspan="1">Convenience</td><td align="left" rowspan="1" colspan="1">Pastors, neighbourhood representatives, traditional healer</td><td align="left" rowspan="1" colspan="1">Community factors that support or prevent women from seeking IPV assistance during pregnancy</td></tr></tbody></table></table-wrap><p><italic>Pregnant women seeking antenatal care</italic> from two antenatal clinics were recruited for four FGDs (a total of <italic>n</italic>=13 women participated). Women were given group information about the study while they waited in queue for a clinic appointment. All FGDs were conducted in a private room in the clinic, led by trained moderators who were the same sex as participants and fluent in multiple local languages (Sotho, Zulu, Tswana). Semi-structured discussion guides explored several topics (<xref ref-type="table" rid="T0001">Table 1</xref>). Discussions were audio-recorded after obtaining participants’ permission and signing an informed consent form. The discussion groups lasted for about 1 hour and 30 minutes, and women were reimbursed R50 (US $6). Because of the nature of focus groups, additional confidentiality measures were implemented: during the informed consent process, researchers explained that questions about women's individual experiences of violence or HIV would not be asked, but rather the discussion would address the issue as observed in the community.</p><p>
<italic>Pregnant women who were experiencing IPV</italic> were identified during the FGDs. Trained researchers explained that those women who had personal experience of IPV and were interested in participating in IDIs could approach the research team outside of the information giving session and privately indicate their interest in taking part in an interview. The interviews (<italic>n</italic>=5) took place in a private room at the clinics while the pregnant women were still waiting to be seen by clinic staff. As shown in <xref ref-type="table" rid="T0001">Table 1</xref>, the topics explored through structured interview guides were more focused on IPV-related help seeking and the relationship between IPV and HIV. On average, these interviews lasted about 60 minutes.</p><p>In depth interviews with <italic>Other Key Stakeholders</italic> were led by the research team and covered similar topics. This group comprised policy makers (<italic>n</italic>=10), health workers (<italic>n</italic>=8), non-governmental organizations (<italic>n</italic>=6) and community-based organizations (<italic>n</italic>=4). Stakeholder interviews focused on service provision and asked questions about available resources for women experiencing IPV. Some anecdotes of cases were shared, but this was not the main rationale for these interviews.</p><p>Several steps were taken to ensure confidentiality and provide additional support for participants during the research. In keeping with ethical considerations of researching IPV in pregnancy, all researches were conducted based on the World Health Organization's guidance on ethical and safety considerations in researching violence against women [<xref rid="CIT0036" ref-type="bibr">36</xref>]. Study staff were trained to describe research as the “social barriers” to use health services in the community, so as to reduce any undue risk associated with participating in a violence-related study. All participants were offered an information sheet containing contact information of local resources (counselling, legal advice and health care). Given the high prevalence of IPV in South Africa, it was likely that participants in categories other than “pregnant and experiencing IPV” category had experienced or witnessed IPV. If any individual demonstrated a need for additional assistance, that individual was offered an opportunity to speak to someone about his or her experience of IPV, and given referrals to organizations that could assist him or her. However, no participants asked for this referral during the course of the formative research.</p><p>All participation in this formative research was sought on the basis of informed consent and good clinical practice guidelines. Ethics approval was obtained by the World Health Organization (WHO A65780) and University of the Witwatersrand (HREC M110832).</p></sec><sec id="S0002-S20003"><title>Data analysis</title><p>The interview and FGD data were transcribed verbatim in the language in which they were conducted and, as necessary, translated from the local language (Sotho, Zulu, Tswana) into English by professional translators. To ensure accurate translation, each transcript was reviewed by a researcher, and queries were resolved through discussions among the researchers via phone or email. All identifying information about the participant or clinic setting was removed and transcripts were saved by a file name with no personal details.</p><p>Data were managed in QSR Nvivo 10, a qualitative analysis software package, following a two-day qualitative management and analysis training of the research team. Members of the research team collaboratively built an analytical framework of broad codes by creating a “start list” of possible themes and building upon the research questions. Each broad code, or wide thematic basket of ideas [<xref rid="CIT0037" ref-type="bibr">37</xref>], was applied to each transcript by two researchers using NVivo. The research team then held a series of meetings to collectively develop “fine codes” using an inductive approach – deriving meaning from the data itself rather than imposing pre-formed ideas [<xref rid="CIT0038" ref-type="bibr">38</xref>]. Fine codes were developed by printing out a full set of excerpts (from each database) related to each code and identifying sub-themes emerging from the data.</p></sec></sec><sec sec-type="results" id="S0003"><title>Results</title><p>We found qualitative evidence of strong bidirectional links between IPV and HIV among pregnant women. Here, we present a conceptual framework (<xref ref-type="fig" rid="F0002">Figure 2</xref>) for understanding the ways in which IPV is related to HIV-related health of pregnant women.</p><fig id="F0002" position="float"><label>Figure 2</label><caption><p>IPV and HIV-related health among pregnant women.</p></caption><graphic xlink:href="JIAS-17-19233-g002"/></fig><sec id="S0003-S20001"><title>Pathway 1: HIV diagnosis leads to IPV via partner disclosure</title><p>HIV diagnosis during pregnancy was noted to be a trigger of IPV. One pregnant woman described how severe violence began following disclosure of her HIV-positive status during pregnancy:<disp-quote><p>He started telling me things, hurting me emotionally, telling me that I'm a fool, and stupid, I'm an idiot. And then he strangled me, That's when it started … Maybe it's pregnancy, I don't know. I told him that I am HIV positive, so I don't know if that's what made him to do all these things. – Pregnant abused woman 1</p></disp-quote>
</p><p>HIV may lead to violence because it causes relationship conflict during the disclosure process. Usually, the conflict is related to perceived infidelity and blaming women for “bringing” the disease into the relationship:<disp-quote><p>Yes, if you're HIV positive, you start blaming each other. Because maybe the husband will be saying the wife brought it. So sometime, there's a connection [between HIV and violence] because you end up blaming each other. – Pregnant woman, FGD 3</p></disp-quote>
</p><p>Because HIV testing is coupled with antenatal care, women often learn of their HIV status in a clinic during access to health care during pregnancy. Within this health care context, women bear the brunt of disclosure to partners, who tend to use women's status as a “proxy” for their own.</p><p>In addition to physical violence, pregnant women described experiencing emotional abuse and abandonment following disclosure of HIV to a partner:<disp-quote><p>I have a sister, she was pregnant, … then she came to be tested. When she tested she found out she is positive, and when she told her boyfriend everything turned around. And there was violence at home. He started coming late and when she started asking for things for her and the baby, he started to react badly up until he ended up leaving her. – Pregnant woman, FGD 2</p></disp-quote>
</p><p>Within a context where women fear violence, blame and abandonment, it is perhaps not surprising that many pregnant women chose not to disclose their HIV status to partners. Several pregnant women spoke about the fear of partner disclosure when women live in violent relationships:<disp-quote><p>Women who are in this abusive relationship, they do get HIV and they are scared what their partner will say. – Pregnant abused woman 1</p></disp-quote>
</p><p>Health workers talked about how women in violent relationships would be hesitant to disclose their status to partners:<disp-quote><p>When you counsel them … after they have tested positive and when you have to issue the treatment she'll be saying, ‘I am not going to disclose. I mustn't take this, I must hide it’. Then you find out is it a problem for her to disclose because there's some emotional abuse or physical abuse from the partner. – Female Health Worker 4</p></disp-quote>
</p><p>Thus, fear of partner disclosure may be an early warning sign that pregnant women are in violent or unsupportive relationships and require additional assistance during antenatal care.</p></sec><sec id="S0003-S20002"><title>Pathway 2: IPV worsens HIV-related health via non-adherence</title><p>For women in violent relationships, adherence to PMTCT services was challenging, since taking medication or accessing health services might unintentionally alert male partners of their HIV status.</p><p>Health workers noted that non-adherence also served as warning sign that HIV-positive patients were in a violent relationship:<disp-quote><p>
It was the very same patient that you had told she was HIV positive that was scared to go and disclose to their partner. It is the very same patient that will default on their medication because their partner does not know that they are taking the medication. – Policy Maker 9</p></disp-quote>
</p><p>In the antenatal clinic, an HIV diagnosis in the context of living with violence may cause patients to default on clinic visits:<disp-quote><p>But in your normal facility it is a bit difficult to avoid losing patients. I think we do. Especially the mere fact that you say to a patient, “you are HIV positive.” And this is a patient who is facing domestic violence! Some will just disappear. – Policy maker 3</p></disp-quote>
</p><p>Thus, the fear of being identified by a male partner as being HIV positive may preclude women from returning to clinic services that are essential for their health. While no participants mentioned this directly, it is important to note that non-adherence to PMTCT regimens greatly increases the risk that pregnant or breastfeeding women will transmit HIV to the infant.</p></sec><sec id="S0003-S20003"><title>Pathway 3: IPV worsens HIV-related health via mental health</title><p>Declines in mental health were noted in women experiencing IPV in pregnancy. In response to persistent violent relationships, women described internalizing the abuse and assuming that they had done something wrong to deserve it:<disp-quote><p>He used to beat her while she was pregnant. She just accepted it, and sometime she'd blame herself. Saying maybe I'm the one who's wrong that's why he's beating me. – Pregnant woman, FGD 1</p></disp-quote>
</p><p>Although IPV is associated with common mental health disorders in pregnancy, few patients or providers recognized these as having clinical implications. Most health providers equated mental health to severe cases of psychopathology and said they rarely encountered mental health disorders. For example, one health worker only considered mental health in relation to bipolar depression and pharmacologically treated patients:<disp-quote><p>Mental health, yes, I remember we've had three that we already on treatment, and will tell you, I have a bipolar patient. – Health worker 8</p></disp-quote>
</p><p>We found that health workers often fail to notice the mental health dynamics of IPV in pregnancy, choosing instead to focus on physical health sequelae of pregnancy. For example, one health provider was asked about stress related to IPV, but responded only in terms of how stress impacts hypertension while ignoring the relevant impact on a woman's mental health:<disp-quote><p>Stress is one of the predisposing factors to the development of hypertension. So it is still there, this stress, but as a predisposing factor. Sometimes because of the pregnancy itself, you can develop hypertension of pregnancy. – Health worker 6</p></disp-quote>
</p><p>This tendency towards equating mental health with severe illness may be related to the lack of capacity within South Africa's public health system. As one policy maker explained, in overlooking mental health issues, current health systems make it unlikely that patients will receive the crucial support that they require:<disp-quote><p>No one has time for mental health because there are so many other crises in the health system that need to be addressed, that are much more manifested. So that means that things like depressive disorder or mental health disorders, they're not addressed – including partner depression, mental health and abuse and all of that. And people are not really encouraged to go and get support that they require. – Policy Maker 9</p></disp-quote>
</p><p>The notion of overlooking mental health is illustrated in an interview with one pregnant, abused woman when she described severe physical violence as leading to a state of being “a little depressed”:<disp-quote><p>Interviewer (I): Are you enjoying your pregnancy so far?</p><p>Participant (P): Being honest, a little depressed but I'm enjoying it.</p><p>I: Ok, so the depression is from what, if I may ask?</p><p>P: From the father of the baby. We are having problems.</p><p>I: What did he do, if you don't mind telling me.</p><p>P: He strangled me and then he let his cousin beat me up. – Pregnant abused woman 1</p></disp-quote>
</p><p>Not everyone in our sample ignored the impact of mental health on a woman's health and wellbeing. For example, poor mental health had concomitant effects on physical health for one HIV-positive participant, who described “going low” emotionally because of violence, and thereafter feeling worse physically:<disp-quote><p>I'm HIV positive and I'm in this domestic violence. And if you are HIV positive and then you have a partner who is abusing you emotionally … or physically hits you, people can't talk. Maybe you can go low, maybe you can go sick. – Pregnant abused woman 3</p></disp-quote>
</p></sec><sec id="S0003-S20004"><title>Pathway 4: IPV leads to secondary HIV risk via lack of relationship control</title><p>IPV led to secondary HIV risk when women were in relationships with forced sex or without power to negotiate condom use.<disp-quote><p>When we are in relationships where our partner is abusive, sometimes we can't even negotiate things like using the condom. Let's say, for instance, you know that your partner is the kind of person that has other girlfriends, but because he uses power over you, you can't negotiate those things. – Pregnant woman, FGD 4</p></disp-quote>
</p><p>Male partners used their control over the relationship to dictate the terms and timing of sexual activity. In one instance, a FGD revealed a story about a newly diagnosed HIV-positive woman whose partner insisted that she have sex without condoms:<disp-quote><p>There's a friend of mine that was tested alone and she had a lot of problems. The man said, I'm not HIV positive, so I'm not going to test. So the man forced her to sleep with him without a condom. And that man said ‘No! Why? We've been sleeping without a condom, but because today you went to the clinic, you're telling me we've to use a condom?’ – Pregnant woman, FGD 1</p></disp-quote>
</p><p>Pregnant women described balancing risks to physical safety (absence of physical harm to themselves or foetus) with health risks (of onwards HIV transmission to partners). They described making compromises between protecting themselves and the foetus and protecting themselves and partners from sexually transmitted infections:<disp-quote><p>If you are not compromising at all and you start saying “let's use condom,” he'll start having questions. Some things are better left unsaid, just for the safety part of it. – Pregnant woman, FGD 2</p></disp-quote>
</p><p>Many preferred staying silent on condom negotiation, in order to stay physically safe during pregnancy.</p></sec></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>We found that IPV and HIV-related health were connected concerns in the lives of pregnant women in Johannesburg. IPV and HIV seemed to have distinct pathways linking them to one another within the context of pregnancy. The initial HIV disclosure could serve as a trigger for violence in pregnancy. IPV, in turn, worsened HIV-related health through key pathways of lack of adherence and poor mental health. Finally, the experience of IPV led to secondary transmission risk behaviours – both in terms of vertical transmission due to PMTCT non-adherence or secondary transmission due to risky sex.</p><p>According to our participants, IPV shapes HIV-related health outcomes among pregnant women primarily because it leads to non-adherence. While the effect of IPV on adherence has been confirmed in small studies in the United States [<xref rid="CIT0039" ref-type="bibr">39</xref>–<xref rid="CIT0043" ref-type="bibr">43</xref>], this association is yet to be explored among pregnant women. Pregnant and postpartum women are a crucial population within which to understand IPV and adherence, since non-adherence leads not only to morbidity and mortality of the woman but also to risk of onwards HIV transmission to her infant [<xref rid="CIT0003" ref-type="bibr">3</xref>, <xref rid="CIT0004" ref-type="bibr">4</xref>]. Antenatal care provides a crucial moment to enable adherence, since a pregnant woman accesses the health system routinely and this is when many are first diagnosed with HIV.</p><p>Poor adherence among pregnant women may relate to challenges around partner disclosure [<xref rid="CIT0044" ref-type="bibr">44</xref>]. In a recent systematic review of PMTCT, partner disclosure was associated with poor PMTCT uptake in a majority of both quantitative (6 of 9) and qualitative (17 of 24) studies [<xref rid="CIT0045" ref-type="bibr">45</xref>]. We found that partner disclosure following HIV diagnosis in pregnancy led to enacted or feared violence. This aligns with extant literature, which suggests that fear of new or continued IPV may lead women to avoid disclosure of their status to male partners [<xref rid="CIT0011" ref-type="bibr">11</xref>]. In one Nigerian study among HIV-positive pregnant women, the prevalence of IPV was 17% before HIV testing and increased to 63% after testing for HIV and disclosing status [<xref rid="CIT0046" ref-type="bibr">46</xref>]. A Zimbabwean study showed that the risk of IPV in pregnancy was greatest among those women testing positive for HIV in antenatal care [<xref rid="CIT0047" ref-type="bibr">47</xref>]. Non-disclosure among pregnant women is a health risk in its own right, since it poses a risk for sexual transmission of HIV if the male partner is still HIV negative [<xref rid="CIT0048" ref-type="bibr">48</xref>–<xref rid="CIT0051" ref-type="bibr">51</xref>] and may have an impact on the implementation of PMTCT [<xref rid="CIT0052" ref-type="bibr">52</xref>].</p><p>A related but distinct pathway linking IPV to PMTCT uptake may be mental health. A growing body of literature shows that IPV leads to depression and anxiety among pregnant women [<xref rid="CIT0029" ref-type="bibr">29</xref>, <xref rid="CIT0053" ref-type="bibr">53</xref>, <xref rid="CIT0054" ref-type="bibr">54</xref>], yet this link has been largely unexplored in sub-Saharan Africa in HIV-positive populations. Our findings reflect those of a qualitative study in Zambia, in which IPV, mental health and HIV are closely related in the experience of women [<xref rid="CIT0055" ref-type="bibr">55</xref>]. Such interrelated “syndemic” issues [<xref rid="CIT0056" ref-type="bibr">56</xref>] should be explored in future sub-Saharan African studies.</p><p>Existing research shows poor mental health has significant impact on ART adherence [<xref rid="CIT0057" ref-type="bibr">57</xref>–<xref rid="CIT0060" ref-type="bibr">60</xref>]
and among pregnant women depressive symptoms are associated with HIV disease progression and mortality [<xref rid="CIT0061" ref-type="bibr">61</xref>]. It is possible that IPV is one condition exacerbating the relationship between mental health and HIV outcomes. Indeed, one new study suggests that the link between mental health and ART adherence may be partly driven by partner conflict [<xref rid="CIT0062" ref-type="bibr">62</xref>]. Despite high rates of common mental health disorders in antenatal care [<xref rid="CIT0063" ref-type="bibr">63</xref>], little screening or treatment exists in South Africa [<xref rid="CIT0064" ref-type="bibr">64</xref>]. Mental health will be crucial to address among HIV-positive pregnant women because of its strong relationship to IPV and its association with the uptake of PMTCT regimens [<xref rid="CIT0065" ref-type="bibr">65</xref>].</p><p>Finally, IPV may worsen secondary prevention behaviours in pregnancy. Non-adherence to PMTCT regimens greatly increases the risk that pregnant or breastfeeding women will transmit HIV to the infant [<xref rid="CIT0066" ref-type="bibr">66</xref>], potentially in drug-resistant form [<xref rid="CIT0067" ref-type="bibr">67</xref>]. High viral loads related to non-adherence also increase the likelihood of secondary transmission to partners, particularly in the context of unsafe sex. Our research reflects existing knowledge by suggesting that IPV inhibits women's ability to negotiate condoms [<xref rid="CIT0068" ref-type="bibr">68</xref>]. These findings explore such dynamics within the context of pregnancy, thereby suggesting a dual risk of mother-to-child infection and secondary transmission risk to a partner.</p><p>Our findings echo calls for addressing IPV in pregnancy [<xref rid="CIT0069" ref-type="bibr">69</xref>]. Scholars note that antenatal care provides an important “window of opportunity” for women who are regularly accessing the health system [<xref rid="CIT0070" ref-type="bibr">70</xref>]. Although universal screening is not recommended in settings with limited referral options and overstretched providers [<xref rid="CIT0071" ref-type="bibr">71</xref>], some type of IPV assessment, provider training and targeted response may be appropriate for South African antenatal care. Indeed, a comprehensive health response to IPV will likely require either screening or case-finding – both methods that may be acceptable in South African clinics [<xref rid="CIT0072" ref-type="bibr">72</xref>, <xref rid="CIT0073" ref-type="bibr">73</xref>].</p><sec id="S0004-S20001"><title>Limitations</title><p>The findings of this formative research should be examined in light of several limitations. Firstly, this study is exploratory in nature, resulting in small sample sizes of each participant group. While analysis suggested that we began to reach saturation through FGDs with pregnant women, the IDIs with pregnant women experiencing IPV were not sufficient to reach thematic saturation [<xref rid="CIT0074" ref-type="bibr">74</xref>]. Secondly, the socio-ecological perspective was brought to the data analysis process after data collection. Ideally, this conceptual approach would have informed the entire data collection process, rather than simply guiding the final interpretation of findings. However, since this was a preliminary, exploratory study, it was designed to explore several intersecting issues and we applied the conceptual framework during data analysis. Finally, some of the findings may be applicable for any woman experiencing IPV, and do not necessarily highlight the specific context of pregnancy. Further research should explore the perinatal time-period in detail to determine whether the link between IPV and HIV is somehow distinct during this life stage.</p></sec></sec><sec sec-type="conclusions" id="S0005"><title>Conclusions</title><p>IPV in pregnancy leads to declines in the physical and mental health of pregnant women. Our findings underscore the negative effects of IPV as a health issue in its own right and as a barrier to PMTCT. The connection between IPV and HIV medication adherence among pregnant women has yet to be explored quantitatively in sub-Saharan Africa. In future studies, it would be ideal to find systematic methods for recruiting more robust numbers of pregnant women who experience IPV and who are living with HIV. In the parent study [<xref rid="CIT0075" ref-type="bibr">75</xref>], we anticipate that by training health providers to ask about IPV confidentially and skilfully, it may be increasingly possible to reach this crucial population.</p><p>Beyond its marked impact on physical and mental health of women, IPV in pregnancy may have important implications for Option B+, as current cost-effectiveness models assume that women are willing and able to achieve 100% adherence [<xref rid="CIT0076" ref-type="bibr">76</xref>]. If Option B+ is to be adopted more broadly, the effect of IPV on adherence and mental health should be carefully considered. Addressing the inter-related issues of violence and HIV will be crucial to ensure that goals of maternal and child health are met in the sub-Saharan African region.</p></sec> |
Service, training, mentorship: first report of an innovative education-support program to revitalize primary care social service in Chiapas, Mexico | <sec id="st1"><title>Background</title><p>The Mexican mandatory year of social service following medical school, or pasantía, is designed to provide a safety net for the underserved. However, social service physicians (pasantes) are typically unpracticed, unsupervised, and unsupported. Significant demotivation, absenteeism, and underperformance typically plague the social service year.</p></sec><sec id="st2"><title>Objective</title><p>Compañeros en Salud (CES) aimed to create an education-support package to turn the pasantía into a transformative learning experience.</p></sec><sec id="st3"><title>Design</title><p>CES recruited pasantes to complete their pasantía in CES-supported Ministry of Health clinics in rural Chiapas. The program aims to: 1) train pasantes to more effectively deliver primary care, 2) expose pasantes to central concepts of global health and social medicine, and 3) foster career development of pasantes. Program components include supportive supervision, on-site mentorship, clinical information resources, monthly interactive seminars, and improved clinic function. We report quantitative and qualitative pasante survey data collected from February 2012 to August 2013 to discuss strengths and weaknesses of this program and its implications for the pasante workforce in Mexico.</p></sec><sec id="st4"><title>Results</title><p>Pasantes reported that their medical knowledge, and clinical and leadership skills all improved during the CES education-support program. Most pasantes felt the program had an overall positive effect on their career goals and plans, although their self-report of preparedness for the Mexican residency entrance exam (ENARM) decreased during the social service year. One hundred percent reported they were satisfied with the CES-supported pasantía experience and wished to help the poor and underserved in their careers.</p></sec><sec id="st5"><title>Conclusions</title><p>Education-support programs similar to the CES program may encourage graduating medical students to complete their social service in underserved areas, improve the quality of care provided by pasantes, and address many of the known shortcomings of the pasantía. Additional efforts should focus on developing a strategy to expand this education-support model so that more pasantes throughout Mexico can experience a transformative, career-building, social service year.</p></sec> | <contrib contrib-type="author"><name><surname>Van Wieren</surname><given-names>Andrew</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Palazuelos</surname><given-names>Lindsay</given-names></name><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Elliott</surname><given-names>Patrick F.</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Arrieta</surname><given-names>Jafet</given-names></name><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Flores</surname><given-names>Hugo</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Palazuelos</surname><given-names>Daniel</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib> | Global Health Action | <p>Ever since the President of Mexico and the Dean of the Universidad Nacional Autónoma de México established an agreement in 1937, graduating Mexican medical students have been required to complete a year of primary care social service (called pasantía) before obtaining their full medical license (<xref rid="CIT0001" ref-type="bibr">1</xref>, <xref rid="CIT0002" ref-type="bibr">2</xref>). Distributing social service physicians (pasantes) throughout Mexico is intended to provide a safety net for the underserved. In reality, however, pasantes are typically unsupervised and unpracticed (meaning that they are both inexperienced and have not undergone enough mentored training to adequately practice without further oversight). In addition, they are often distracted by pending residency entrance exams, and regularly try to secure placements in more comfortable urban environments (<xref rid="CIT0003" ref-type="bibr">3</xref>, <xref rid="CIT0004" ref-type="bibr">4</xref>). When they are assigned rural sites, lack of support, absenteeism, and underperformance often define their experience <xref rid="CIT0004" ref-type="bibr">4</xref>–<xref rid="CIT0006" ref-type="bibr">6</xref>).</p><p>Compañeros en Salud (CES), the Mexican branch of Partners in Health (PIH), is a non-governmental organization (NGO) whose mission is to build a model of excellence in rural primary care in partnership with the Ministry of Health (MOH). The founders of CES have each worked in the Sierra Madre Mountains of the state of Chiapas – one of the poorest areas in Mexico – for 5–10 years, and found that access to primary care was limited by physician shortages (<xref rid="CIT0005" ref-type="bibr">5</xref>). Although Mexico's health system reform in 2003–2004 expanded public health insurance coverage to the poorest Mexicans, its full promise has yet to be realized, as well-trained and logistically supported health care professionals are often not available to provide high-quality care in the rural areas where many of the poorest Mexicans live
<xref rid="CIT0007" ref-type="bibr">7</xref>–<xref rid="CIT0009" ref-type="bibr">9</xref>)
. In an effort to expand effective access to care in the Sierra Madre region, CES began to recruit pasantes to work in MOH clinics that previously lacked a physician.</p><p>CES recognized that working with social service physicians also provided an opportunity to address known shortcomings of the pasantía through creating a transformative educational experience. The CES approach to transformative education was inspired by a call to action from the Lancet Commission for Health Professions Education asking educators to not just inform students by imparting knowledge and skills or form them through teaching professional values, but rather transform students through developing leadership attributes and enlightening them as agents of change (<xref rid="CIT0010" ref-type="bibr">10</xref>). In this transformative spirit, CES staff and US physician collaborators created an innovative education-support program that aims to: 1) train pasantes to effectively deliver primary care, 2) expose pasantes to central concepts of global health and social medicine, and 3) foster continuing medical education and career development of pasantes. This paper describes the components, evaluation, and insights of the first 2 years of the CES education-support program.</p><sec sec-type="methods" id="S0002"><title>Methods</title><sec id="S0002-S20001"><title>Pasante recruitment</title><p>CES staff (LP, JA, HF, and DP) worked with faculty and a student group at the Monterrey Institute of Technology Medical School (ITESM) – and subsequently with several other medical schools – to identify graduating medical students interested in completing their pasantía in Chiapas. Initial connections between CES and the ITESM were established through several of the CES staff being alumni or having previous affiliations with ITESM. CES staff have subsequently connected with global health-oriented student groups from medical schools throughout Mexico at national and international conferences, which has facilitated recruitment of pasantes from medical schools in many different areas of Mexico, including Nuevo León, Mexico City, Querétaro, Durango, Veracruz, Tamaulipas, Chihuahua, and Puebla. During recruitment, CES sought candidates with the following characteristics: academic excellence, interest in global health and social medicine, perceived ability to adapt to an impoverished rural community, and expressed desire to provide primary care to marginalized patients. To ensure that income is not a barrier to participation, CES offers pasantes a matching stipend to that provided by the government for a total combined stipend of about US$380 monthly (<xref rid="CIT0004" ref-type="bibr">4</xref>).</p></sec><sec id="S0002-S20002"><title>Site selection</title><p>CES staff (LP, HF, and DP) identified communities in the Sierra Madre region of Chiapas with public clinics that lacked a full-time physician. Local and state MOH authorities granted CES permission to recruit pasantes to these clinics and provide a package of training and logistical support. CES staff then met with community leaders to introduce the organization, offer a pasante to the local clinic, and request collaboration (including willingness to help pasantes find room and board). Two communities chose to participate in February 2012, and an additional four joined in August 2012. CES aims to expand to a total of 10 clinics by the end of 2015.</p></sec><sec id="S0002-S20003"><title>Program development and components</title><p>The education-support program strives to transform the entire pasantía into a learning experience. The program harnesses both clinic and classroom time with five fundamental components: 1) monthly multi-day supportive supervision visits by CES staff, 2) biannual multi-week mentorship by medical resident and attending physician volunteers, 3) access to clinical information resources such as locally tailored evidence-based treatment algorithms and UpToDate©, 4) a monthly 2–3 day interactive seminar utilizing adult learning best practices, and 5) improved functioning of the clinics where pasantes work. The CES approach to both patient care and pasante mentorship utilizes a style called ‘accompaniment’, which emphasizes solidarity and collective problem solving.</p><sec><title>Supportive supervision</title><p>CES hires a select group of graduated pasantes and external applicants as supervisors (in a ratio of approximately one supervisor per three to four pasantes) to provide holistic support for the next generation of pasantes. CES uses a model of supportive supervision informed by the World Health Organization (WHO) concept: supervision is helping to make things work, rather than checking to see what is wrong (<xref rid="CIT0011" ref-type="bibr">11</xref>). Supervisors visit each site for about 3 days per month and review a portfolio of functions with the pasante, including troubleshooting difficult cases, clinic management, facilitating referrals, and negotiating community relations. The pasante and supervisor jointly identify priority areas for improvement and make plans for how to address them. The system encourages performance by recognizing and praising when standards are met, and using a supportive rather than primarily punitive approach when they are not.</p></sec><sec><title>Clinical mentorship</title><p>Throughout the year and with intensified focus when pasantes first arrive in February and August, volunteer residents from the USA and Mexico of different specialties (e.g. Family Medicine, Internal Medicine, Pediatrics, Obstetrics/Gynecology, Psychiatry, and Neurology) pair with pasantes to provide intensive bedside teaching. Clinical mentors do not directly provide care, but rather precept and reinforce core skills, talking through each patient's diagnosis, counseling, and treatment with the pasante.</p></sec><sec><title>Clinical information resources</title><p>In an effort to promote high-quality and standardized care among pasantes, CES developed a series of clinical algorithms to guide pasante decision-making for common primary care problems (e.g. acute cough, dysuria, hypertension, diabetes mellitus, etc.). CES algorithms respect official MOH norms, incorporate likelihood ratios for history and physical exam findings, and use principles from care delivery value chains to encourage pasantes to provide evidence-based and locally tailored care <xref rid="CIT0012" ref-type="bibr">12</xref>–<xref rid="CIT0014" ref-type="bibr">14</xref>). CES also provides pasantes with free access to UpToDate©.</p></sec><sec><title>Interactive seminar</title><p>CES designed a monthly course utilizing adult learning best practices such as building on existing knowledge, using a ‘teach-back’ method, and project and case-based exercises. ITESM accredited the seminar as a 12-month certificate course in Global Health and Social Medicine. The curriculum has undergone two rounds of formal evaluation in an effort to improve its contents and delivery. Because new pasantes arrive to work with CES every 6 months, fundamental components of the curriculum are delivered every 6 months, allowing pasantes in the second half of their year to take on a ‘teach-back’ role, whereas other components of the curriculum are delivered once every 12 months.</p><p>The course content focuses on three main areas: 1) clinical medicine taught through problem-based learning cases, case presentations done in ‘morning report’ style, and use of ultrasound and other diagnostic tests in the primary care setting; 2) global health and social medicine delivered via case-based health systems analysis, socioeconomic determinants of health, policy, and cultural competency and humility; and 3) quality care delivery (e.g. clinical leadership skills, team-based care, quality improvement projects, morbidity and mortality review, feedback sessions, and humanistic curriculum). CES staff, volunteer residents, guest speakers, graduated pasante supervisors and, in many cases, pasantes in the latter half of their pasantía all teach different sessions of the course, with all sessions supervised by CES leadership and often using a team-teaching strategy. The 2–3 day monthly seminar also provides pasantes an opportunity to build community and support each other throughout the challenging social service year. As part of the course, pasantes also receive complementary EXARMED© study materials for the Mexican residency entrance exam (ENARM).</p></sec><sec><title>Improved function</title><p>CES pasantes leave their assigned communities for approximately 7–8 pre-planned consecutive days each month, 3–4 days of which are spent between the 2–3 day educational seminar at the CES office, 1 day of turning in paperwork at the jurisdiction offices, and 1–2 days of travel time. The remaining 3–4 days are assigned as vacation. This model of concentrating days away from the community purposely differs from the traditional model of pasantes having 1–2 days off a week, because the long trips required to exit and then re-enter isolated rural communities tend to diminish actual time off and promote absenteeism (<xref rid="CIT0004" ref-type="bibr">4</xref>, <xref rid="CIT0005" ref-type="bibr">5</xref>). CES-supported clinics also receive supplemental supplies to ensure there are no stock-outs of essential medicines, diagnostic tests, and basic infection control supplies.</p></sec></sec><sec id="S0002-S20004"><title>Motivating and retaining pasantes</title><p>Adequately supporting pasantes during their social service year requires many complex and interdependent investments. In a systematic review of factors that help motivate and retain health workers in underserved areas, Willis-Shattuck found that seven critical strategies combat ‘brain drain’ and promote ‘brain gain’ (<xref rid="CIT0015" ref-type="bibr">15</xref>). <xref ref-type="table" rid="T0001">Table 1</xref> provides examples of how CES has learned to address these seven areas programmatically. Furthermore, because new pasantes coming from different educational backgrounds have varied levels of preparedness for both the clinical and psychosocial demands of the social service year – similar to new interns starting residency programs – CES strives to quickly identify pasantes who require additional support to catch up to their peers and then provides supplemental supportive supervision and clinical mentorship during the first month of the pasantía.</p><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Seven key motivating factors (<xref rid="CIT0015" ref-type="bibr">15</xref>) and corresponding CES strategies to attract and retain pasantes</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Motivating factor</th><th align="center" rowspan="1" colspan="1">CES strategies</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Financial rewards</td><td align="left" rowspan="1" colspan="1">-Match pasante stipend, increasing the total from approximately ~US$190 to ~US$380 monthly</td></tr><tr><td align="left" rowspan="1" colspan="1">Career development</td><td align="left" rowspan="1" colspan="1">-Provide opportunities for pasantes to work in management roles within CES after completing the pasantía<break/>-Provide opportunities for pasantes to visit other Partners in Health sites</td></tr><tr><td align="left" rowspan="1" colspan="1">Continuing education</td><td align="left" rowspan="1" colspan="1">-Monthly CES course<break/>-Supervisor and resident accompaniment in clinic<break/>-Clinical information resources such as clinical algorithms and access to UpToDate©</td></tr><tr><td align="left" rowspan="1" colspan="1">Work environment</td><td align="left" rowspan="1" colspan="1">-Providing electronic medical record and computers for clinic notes and for automatic generation of government paperwork that previously took hours to complete by hand</td></tr><tr><td align="left" rowspan="1" colspan="1">Resource availability</td><td align="left" rowspan="1" colspan="1">-Working with Mexican government to prevent ‘stock-outs’ of essential medications<break/>-Supplementing medication stocks<break/>-Helping pasantes refer complex patients</td></tr><tr><td align="left" rowspan="1" colspan="1">Workplace management</td><td align="left" rowspan="1" colspan="1">-Teaching pasantes how to work as part of and lead a clinical team<break/>-Regular visits by supervisors and leadership to help build teamwork among clinic staff<break/>-Advising pasantes on how to manage patient flow and limit wait times</td></tr><tr><td align="left" rowspan="1" colspan="1">Recognition/appreciation</td><td align="left" rowspan="1" colspan="1">-Providing a certificate through the ITESM for the Global Health and Social Medicine course<break/>-Supporting pasantes to integrate with the communities they serve</td></tr></tbody></table></table-wrap></sec><sec id="S0002-S20005"><title>Survey instrument</title><p>The authors collected data about the education-support program and its influence on pasantes using pre-, mid-, and post-intervention surveys that ranged in length from 18 to 23 questions. The surveys incorporated both Likert scale and open-ended questions, and explored the following areas: pasantes’ self-reported medical knowledge (overall medical knowledge, and specialized knowledge in Internal Medicine, Pediatrics, Obstetrics/Gynecology, and Emergency Medicine); clinical leadership skills and understanding of the Mexican health care system; preparedness for the ENARM; residency and career plans (planning on applying for residency, type of medicine planning to pursue, desire to work with the poor or underserved); best and worst experiences as a pasante; best aspects and recommended changes for the education-support program; and degree of satisfaction with the CES program. Content of the three surveys was similar to facilitate measuring changes throughout the year. The survey instruments were written by AVW, LP, and DP in English and then translated into Spanish by a bilingual native Spanish-speaking CES volunteer.</p></sec><sec id="S0002-S20006"><title>Study design, data collection, and analysis</title><p>The present study was conceived as a case study of pasantes completing their social service requirement as part of the CES program. The data reported were collected from February 2012 through August 2013. The data reflect completed pre-, mid-, and post-intervention questionnaires for six pasantes, representing the first two classes of pasantes to complete the CES education-support program. Data are not reported for two pasantes who did not complete the study, one who left the CES-affiliated pasantía due to a desire to be closer to family, and one who had incomplete data. To put the current size of the CES program in context, 263 of 932 (28.2%) public primary care clinics in Chiapas are staffed exclusively by pasantes (<xref rid="CIT0004" ref-type="bibr">4</xref>). Surveys were distributed to pasantes during a designated time at the seminars and pasantes were given as much time as needed to complete them. Pasantes wrote a code that only they would understand on their three surveys in order to concurrently track data and preserve anonymity. AVW, LP, and DP performed data analysis, which included conducting descriptive statistics on quantitative data, and identifying predominant themes and supporting quotations from qualitative data. This study was reviewed by the Partners Healthcare Institutional Review Board (Brigham and Women′s Hospital) in Boston, MA, and was given IRB exemption.</p></sec><sec id="S0002-S20007"><title>Evaluation of the program</title><p>CES staff evaluates the CES education-support program on both an informal continuous basis and also at designated formal intervals at the end of each pasante cycle (every 6 months in January and July). Pasantes evaluate the program through the aforementioned pre-, mid-, and post-intervention questionnaires and through informal feedback obtained by staff and volunteers throughout the social service year. CES staff members verbally evaluate the program during regularly scheduled monthly meetings with CES leadership. CES volunteers provide either verbal or written evaluations of the program at the conclusion of their volunteer period. CES then uses feedback from pasantes, staff members, and volunteers to implement new changes to both the educational and support aspects of the curriculum at the beginning of each new pasante cycle (every 6 months in February and August). CES implements changes to the education-support program using a Plan-Do-Study-Act model, in which feedback is reviewed by CES leadership to form plans for change, which are then implemented, subsequently studied in the next semester's review process, and then the change plans are either adopted, adapted, or abandoned.</p></sec></sec><sec sec-type="results" id="S0003"><title>Results</title><p>The six pasantes who completed the study ranged in age from 22 to 26 years, included three men and three women, had all completed required coursework at an accredited medical school in Mexico making them eligible to complete their social service requirement, and all had completed the pasantía as part of the CES education-support program in Chiapas.</p><sec id="S0003-S20001"><title>Knowledge and skills</title><p>Overall, pasantes reported that their medical knowledge and clinical skills improved while completing the education-support program during their social service year in Chiapas. <xref ref-type="fig" rid="F0001">Figure 1</xref> demonstrates how pasante self-report of general clinical knowledge and preparedness to practice Internal Medicine, Pediatrics, Obstetrics/Gynecology, and Emergency Medicine changed throughout the course of the education-support program. When asked to reflect on changes in knowledge and skills during the pasantía, one pasante remarked, ‘I have gained greater medical knowledge because of the accompaniment I received from residents, the feedback I received from patients, and from being able to longitudinally observe the patients I treated in my community’. Another observed, ‘My abilities to create differential diagnoses and treat patients are constantly improving because of the feedback and accompaniment I have received from the CES staff, residents, and volunteers’.</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Percent of pasantes reporting good or very good general clinical knowledge and preparedness to practice several medical specialties before, halfway through, and at the end of the CES education-support program.</p></caption><graphic xlink:href="GHA-7-25139-g001"/></fig><p>Pasantes reported that clinical leadership skills and understanding of the Mexican health care system also improved throughout the course of the education-support program. The percentage of pasantes who reported feeling well or very well prepared to lead a primary care clinic increased from 50% at the beginning of the program to 83% by the end of the program. Whereas 67% of pasantes reported understanding the organization or hierarchy of the Mexican health care system well or very well at the beginning of the program, 100% of pasantes felt that way by the end of the program.</p><p>Throughout the course of the education-support program, pasante report of perceived preparedness for the Mexican residency entrance exam (ENARM) decreased and, by the end of the year, most pasantes had decided not to take the ENARM in the year following their pasantía. Although 100% of pasantes believed the pasantía would have a positive or very positive effect on their performance on ENARM at the beginning of the program, by the end of the year 83% of pasantes had decided against taking the ENARM in the year following their pasantía.</p><p>Notably, pasantes reported learning more than just rote clinical knowledge. Pasantes also recalled developing more nuanced clinical skills – such as the ability to use a referral network for more complex cases – and beginning to appreciate and address the social determinants of health. One pasante reflected, ‘I found that I was actually able to accurately diagnose and treat the majority of my patients; for the more complicated cases in which I did not know what to do, I fortunately had the support of the entire CES team to first figure out the diagnosis and then implement the best treatment plan’. Another explained, ‘I am beginning to understand how to break the cycle of poverty and disease by treating diseases that were previously incurable in the Sierra, diseases that affected whole generations and society as a whole’.</p></sec><sec id="S0003-S20002"><title>Career goals and plans</title><p>Pasante career goals and plans changed in several ways throughout the course of the education-support program. When asked if they were considering a career in primary care at the beginning of the year, 33% responded ‘probably no’, 33% ‘undecided’, and 33% ‘probably yes’. By the end of program, 67% of pasantes reported they were either definitely or probably not considering a career in primary care, but 33% reported they definitely were considering a career in primary care and no pasantes remained undecided. The percentage of pasantes who reported a desire to work primarily with poor and underserved populations during their career increased from 83 to 100% between the beginning and end of the program. By the end of the year, 83% of pasantes felt the social service year had a positive or very positive effect on their career goals and plans. One pasante detailed this transition as follows: ‘The trajectory of my life has taken a significant turn after working with CES. I would like to work with different communities, especially the poor communities where CES works’. Another pasante reflected, ‘Before I arrived to work with CES, my life plan was set. Now I'm not sure what I want to do. The only thing I'm sure of is that, whatever happens, I want to continue collaborating with CES in the short- and long-term’.</p></sec><sec id="S0003-S20003"><title>Satisfaction with pasantía</title><p>When asked at the end of the year, 100% of pasantes reported they were satisfied with their experience as a pasante and that they were glad they had done their social service year in CES-supported government clinics in Chiapas. When asked why they were satisfied with the pasantía, one pasante wrote, ‘I grew personally and professionally’, whereas another pasante explained ‘[the year] provided me a totally new perspective regarding professional opportunities that I had never imagined’. At the end of the year, 67% of pasantes reported that they had achieved their goals for the pasantía. Among the pasantes who reported achieving their goals, cited reasons why included: ‘I learned how to practice medicine and feel confident as a doctor’, and ‘I completed all of my goals and even others I formed during the year’. One of the pasantes who denied having achieved his or her goals for the year explained: ‘I gained many clinical skills and helped many patients. However, I did not study for ENARM as much as I had hoped’.</p></sec><sec id="S0003-S20004"><title>Satisfaction with education-support program</title><p>
<xref ref-type="table" rid="T0002">Table 2</xref> shows highlights of what pasantes felt were the best and worst aspects of both the overall pasantía and the CES education-support program. Regarding the overall pasantía, pasantes appeared to appreciate the following: gaining clinical knowledge and confidence, developing relationships with their patients over time, seeing their patients improve, having resident and supervisor accompaniment, attending the monthly global health seminar, working on quality improvement projects, and exploring a new area of Mexico. In critiquing the overall pasantía, pasantes cited the following: running out of available treatment options, patients not following up as requested, social isolation in the communities, frustration and fatigue throughout the course of the year, limited collaboration with nurses in the clinics, and growing pains of CES as a new organization. When asked to describe the best aspects of the CES education-support program, pasantes praised: the program's global health and social medicine focus, its case-based approach, the program being new and unique in Mexico, CES always striving to improve the program, resident and supervisor accompaniment, the ITESM certificate awarded at the conclusion of the program, and the program being tailored to the local reality of Chiapas. Asked to identify the worst aspects of the CES education-support program, pasantes criticized: the seminar's classes being too long and dense, the curriculum being poorly defined, the teaching approach being too informal, too many different instructors teaching the courses, too much focus on quality improvement, and limited distribution of written resources.</p><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Pasante report of best and worst aspects of the overall pasantía, and best and worst aspects of the CES education-support program</p></caption><table frame="hsides" rules="groups"><tbody><tr><td align="left" rowspan="1" colspan="1">Best aspects of overall pasantía:</td><td align="left" rowspan="1" colspan="1">Worst aspects of overall pasantía:</td></tr><tr><td align="left" rowspan="1" colspan="1"> Gaining clinical knowledge and confidence<break/> Seeing patients improve<break/> Long-term relationships with patients<break/> Resident and supervisor accompaniment<break/> Traveling to new area of Mexico<break/> Monthly global health seminar<break/> Working on quality improvement projects</td><td align="left" rowspan="1" colspan="1"> Running out of available treatment options<break/> When patients did not follow up as requested<break/> Being alone in the community<break/> Frustration and fatigue<break/> Growing pains of CES as a new organization<break/> Limited collaboration with nurses</td></tr><tr><td align="left" rowspan="1" colspan="1">Best aspects of education-support program:<break/> Global health and social medicine focus<break/> Case-based approach<break/> Program is new and unique in Mexico<break/> CES is always working to improve program<break/> Resident and supervisor accompaniment<break/> Certificate awarded at end of course<break/> Locally oriented to Chiapas</td><td align="left" rowspan="1" colspan="1">Worst aspects of education-support program:<break/> Classes are too long<break/> Classes are too dense<break/> Curriculum is not defined well enough<break/> Too informal<break/> Too many different people teaching classes<break/> Too much focus on quality improvement<break/> Limited distribution of written resources</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr></tbody></table></table-wrap></sec></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>Building on changes introduced by the Mexican health system reform of 2003–2004, the Mexican health care system has recently been celebrated for providing universal health coverage to all Mexicans (<xref rid="CIT0007" ref-type="bibr">7</xref>, <xref rid="CIT0008" ref-type="bibr">8</xref>). Despite this high level of enrollment, gaps in effective coverage persist (<xref rid="CIT0016" ref-type="bibr">16</xref>). Truly achieving equitable access to high-quality care within Mexico will require an ongoing commitment to ensuring primary care providers arrive at the most underserved regions of Mexico and are provided logistical and training support so that they can perform at a high level (<xref rid="CIT0017" ref-type="bibr">17</xref>).</p><p>In Mexico, despite the fact that pasantes are still technically considered students until they complete their social service year, as many as one-third of public primary care clinics are staffed exclusively by pasantes and nearly three-quarters of pasantes are assigned to rural health centers (<xref rid="CIT0004" ref-type="bibr">4</xref>). This represents a large work force addressing rural health needs, but there is a growing concern that, similar to interns beginning residency, Mexican pasantes would provide higher quality and safer care if they were supervised, supported, and continued to receive education during the social service year (<xref rid="CIT0004" ref-type="bibr">4</xref>, <xref rid="CIT0005" ref-type="bibr">5</xref>). Innovative approaches to supporting and training pasantes during their social service year are sorely needed to help turn Mexico's universal enrollment into true universal effective coverage.</p><p>Given the limited number of studies evaluating strategies to address human resources concerns and strengthen primary health care systems in low- and middle-income countries (<xref rid="CIT0018" ref-type="bibr">18</xref>, <xref rid="CIT0019" ref-type="bibr">19</xref>), this program and its evaluation offer important insights to those interested in strengthening the primary health care system in Mexico and wherever social service providers are dispatched. Our results suggest that the CES education-support program has several important strengths and insights.</p><p>The results of this study show that the CES education-support program is a transformative experience for participants. The Lancet Commission for Health Professions Education has called for efforts at medical education reform to be guided by two main principles: transformative (rather than only informative or formative) learning, and interdependent and interdisciplinary education (<xref rid="CIT0010" ref-type="bibr">10</xref>). Involvement in the CES program fundamentally changed the way the pasantes considered their future; all experienced an increased commitment to work with the poor and underserved in the future. In addition, they reported greater clinical leadership skills and a more nuanced understanding of the Mexican health care system. The Lancet Commission for Health Professions Education has highlighted several programs that model transformative education, such as the Public Health Foundation of India which uses a similar public-private partnership as the CES model to transform public health professionals during a 12-month program that couples field work with classroom learning (<xref rid="CIT0010" ref-type="bibr">10</xref>). CES aims to draw from both internal program evaluation and lessons learned from others in an effort to constantly improve the CES program and make it as transformative as possible for pasantes. Although the MOH did provide pasantes with some basic supervision and training before development of the CES program, the public-private partnership between the MOH and CES has resulted in the development of a program that now teaches pasantes leadership skills and how to potentially be agents of change within the Mexican health care system.</p><p>The findings also highlight the importance of classroom training that goes beyond technical proficiencies to integrate systems-level concepts of global health, social medicine, and quality improvement. The Global Health and Social Medicine course appears to instill a sense of mission within pasantes, thus invigorating the care they provide to underserved patients. Interest in global health is rising to unprecedented levels among medical trainees, and seems to represent a revitalization of the sense of mission and altruism that is core to the medical profession (<xref rid="CIT0020" ref-type="bibr">20</xref>, <xref rid="CIT0021" ref-type="bibr">21</xref>). Inviting residents and attending physicians from Mexico, the USA, and elsewhere to offer clinical ‘accompaniment’ of pasantes represents a powerful way in which interest in global health can be harnessed to improve training for not only Mexican providers but also the visiting physicians themselves (<xref rid="CIT0010" ref-type="bibr">10</xref>, <xref rid="CIT0019" ref-type="bibr">19</xref>). Efforts to create effective and sustainable approaches to pasante training and mentoring will benefit from new partnerships that have not been previously considered, including partnerships between the public sector, private NGOs, and academic institutions.</p><p>Although critically evaluating this type of education-support program will inform future efforts to support and educate pasantes, we must also consider how public policy changes could influence pasante behaviors and promote effective universal access (<xref rid="CIT0015" ref-type="bibr">15</xref>, <xref rid="CIT0019" ref-type="bibr">19</xref>). Indeed, some limitations of the pasantía model may be better addressed through public policy changes than educational programs alone. For example, entrance to residency programs in Mexico is determined largely by the ENARM test, typically taken after the social service year. Despite providing complementary study preparation materials for ENARM, pasantes’ self-reported preparedness for the ENARM exam decreased during the social service year and most pasantes had decided to either postpone or not take ENARM by the end of the pasantía. We suspect this phenomenon is the result of clinical responsibilities precluding pasantes from having sufficient study time. Perhaps a more realistic and transformative approach is that used within the Chilean social service system. In Chile, rather than having to worry about how social service time will negatively affect performance on residency entrance exams, pasantes who complete their social service in rural undeserved areas are awarded additional points on the medical residency entrance exam (<xref rid="CIT0004" ref-type="bibr">4</xref>).</p><p>Another example of how public policy changes could improve the social service year is by securing support within the most recent Mexican health reform to expand education-support programs for pasantes within the public sector. Although the CES program has created a unique educational environment within six government-run clinics, the state of Chiapas alone has 263 primary care clinics that are staffed exclusively by pasantes (<xref rid="CIT0004" ref-type="bibr">4</xref>). The CES program relies upon private donations and is run by a NGO, and is thus likely not sustainable at a large scale. We believe that the question is not whether there is enough money to better train and support the providers who care for the most underserved Mexicans, but rather whether sufficient political will exists to align funds for this purpose. As the Lancet Commission for Health Professions Education points out, despite the labor-intensive nature of medicine, only 2% of global health expenditures are spent on training – a percentage that is even smaller in low-income countries (<xref rid="CIT0010" ref-type="bibr">10</xref>). Furthermore, in his critical appraisal of the pasantía in Mexico, Nigenda proposes that, with relatively modest financial investment, the Mexican government could feasibly hire licensed and fully trained physicians to work alongside pasantes, guiding and training them during their social service year (<xref rid="CIT0004" ref-type="bibr">4</xref>). In fact, some Mexican districts already provide pasantes with limited intermittent training and supervision. However, our data support the deployment of more comprehensive training and support packages that convert the social service year into a transformative experience.</p><sec id="S0004-S20001"><title>Study limitations</title><p>Our study results have several important limitations. First, because we did not incorporate a comparison group of pasantes not participating in the education-support program into the study, the results could simply show the effect of completing a social service year rather than that of the education-support program. Furthermore, because many of the survey questions were retrospective about experiences during the preceding months, the results are subject to recall bias. Finally, because our sample size of pasantes is small (<italic>N</italic>=6), our quantitative results should be interpreted as suggesting possible trends rather than establishing statistically significant patterns.</p></sec><sec id="S0004-S20002"><title>Opportunities for future work</title><p>Ultimately, if we are to engage in true health systems strengthening, we will need to develop a strategy to fully transition the CES education-support program to the public sector (<xref rid="CIT0022" ref-type="bibr">22</xref>). Large-scale expansion of this type of education-support program with public funding, however, should only be undertaken after more rigorous studies have demonstrated a clear benefit of the education-support program by directly comparing it to the social service year alone. Notably, recruitment of pasantes for the program and study was likely facilitated by affiliations with ITESM, PIH, and Harvard Medical School – all well-known and respected private institutions – and, thus, efforts to expand this type of program within the public sector would need to consider how those organizational affiliations could either be maintained at larger scale or explore other types of recruitment incentives. However, there is an expanding body of literature indicating that a large percentage of medical students throughout the world are interested in global health suggesting that, if the pasantía in Mexico (and elsewhere in Latin America) could be appropriately re-branded as a global health effort, enthusiasm for the social service requirement might surge at the national and international level (<xref rid="CIT0020" ref-type="bibr">20</xref>). Finally, subsequent studies of pasante education and support should attempt to move from pasante perceptions of outcomes, such as clinical knowledge and skills, to more robust measures such as test scores, and peer or patient evaluation of pasantes.</p></sec></sec><sec sec-type="conclusions" id="S0005"><title>Conclusions</title><p>Guaranteeing universal access to high-quality health care for the poor in Mexico requires not only financial and organizational reform, but also achieving an equitable distribution of well-trained and operationally supported health care providers. In Mexico and much of Latin America, the poor depend largely upon health care services provided by pasantes who are often inadequately prepared to practice independently. Efforts that focus on training and mentoring during the social service year represent a unique opportunity to improve the care that the poor and vulnerable receive, and build a cadre of providers motivated to serve them. Based on our data, education-support programs that combine on-site accompaniment of pasantes, access to clinical information resources, and regular seminars appear to be an effective way to recruit pasantes to underserved areas, increase their clinical knowledge and leadership skills, and address many of the known shortcomings of the social-service year. Additional efforts could focus on fully transitioning this type of education-support program to the public sector and developing an expansion model to reach pasantes throughout Mexico and Latin America.</p></sec><sec id="S0006"><title>Disclosures</title><p>AVW received funding for his travel, living expenses while in Chiapas, and limited supplies through a Martin Solomon Primary Care Scholarship through Brigham and Women′s Hospital Department of Internal Medicine. CES is funded primarily by donations received through Partners in Health.</p></sec> |
The effect of immunonutrition (glutamine, alanine) on fracture healing | <sec id="st1"><title>Background</title><p>There have been various studies related to fracture healing. Glutamine is an amino acid with an important role in many cell and organ functions. This study aimed to make a clinical, radiological, and histopathological evaluation of the effects of glutamine on fracture healing.</p></sec><sec id="st2"><title>Methods</title><p>Twenty rabbits were randomly allocated into two groups of control and immunonutrition. A fracture of the fibula was made to the right hind leg. All rabbits received standard food and water. From post-operative first day for 30 days, the study group received an additional 2 ml/kg/day 20% <sc>L</sc>-alanine <sc>L</sc>-glutamine solution via a gastric catheter, and the control group received 2 ml/kg/day isotonic via gastric catheter. At the end of 30 days, the rabbits were sacrificed and the fractures were examined clinically, radiologically, and histopathologically in respect to the degree of union.</p></sec><sec id="st3"><title>Results</title><p>Radiological evaluation of the control group determined a mean score of 2.5 according to the orthopaedists and 2.65 according to the radiologists. In the clinical evaluation, the mean score was 1.875 for the control group and 2.0 for the study group. Histopathological evaluation determined a mean score of 8.5 for the control group and 9.0 for the study group.</p></sec><sec id="st4"><title>Conclusion</title><p>One month after orally administered glutamine–alanine, positive effects were observed on fracture healing radiologically, clinically, and histopathologically, although no statistically significant difference was determined.</p></sec> | <contrib contrib-type="author"><name><surname>Küçükalp</surname><given-names>Abdullah</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Durak</surname><given-names>Kemal</given-names></name><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Bayyurt</surname><given-names>Sarp</given-names></name><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Sönmez</surname><given-names>Gürsel</given-names></name><xref ref-type="aff" rid="AF0004">4</xref></contrib><contrib contrib-type="author"><name><surname>Bilgen</surname><given-names>Muhammed S.</given-names></name><xref ref-type="aff" rid="AF0002">2</xref></contrib> | Food & Nutrition Research | <p>The classical definition of a fracture is the breaking of the entirety of a bone from internal or external forces. The bone is healed by reconstruction of osseous tissue without any scarring (<xref rid="CIT0001" ref-type="bibr">1</xref>). Fracture healing starts immediately after the injury and continues until the broken ends unite with the new bone tissue (<xref rid="CIT0002" ref-type="bibr">2</xref>).</p><p>Studies have been made on many factors to accelerate fracture healing. Glutamine is an amino acid with an important role in several chemical reactions of many cell and organ functions (<xref rid="CIT0003" ref-type="bibr">3</xref>). Among these are the energy source of cells such as fibroblasts, epithelial cells, enterocytes, lymphocytes, and macrophages. In addition, it is the most important precursor substance regulating protein synthesis in the nucleic acid biosynthesis of cells. By protecting tissues from free radical damage, and as a major antioxidant, glutamine shows positive effects by promoting glutathione synthesis (<xref rid="CIT0004" ref-type="bibr">4</xref>–<xref rid="CIT0006" ref-type="bibr">6</xref>). Several studies in literature have shown positive healing effects of glutamine on wound healing and on patients requiring critical care (<xref rid="CIT0007" ref-type="bibr">7</xref>, <xref rid="CIT0008" ref-type="bibr">8</xref>). It is thought that this study can make a positive contribution to fracture healing by considering the accelerating effect of glutamine on wound healing.</p><sec sec-type="materials|methods" id="S0002"><title>Materials and methods</title><p>This study was approved by Uludag University Animal Experiments Local Ethics Committee and was carried out on 20 New Zealand white rabbits, aged 3 months, weighing between 2.5 and 3.0 kg, obtained from the Experimental Animal Breeding and Research Centre with the permission of the Deanery of Uludag University Medical Faculty.</p><sec id="S0002-S20001"><title>
Study plan – surgical technique</title><p>The rabbits were randomly allocated into two groups of 10 as the immunonutrition group and the control group. Anaesthesia of 5 mg/kg xylazine and 35 mg/kg ketamine was administered intramuscularly to both groups. The rabbits were then laid down with the right hind leg uppermost. After disinfection of the fibular area with 10% free alcohol containing 10 g povidone iodine, a 1–2 cm skin incision was made approximately 1 cm to the posterior of the fibula. By subcutaneous dissection, continuing to slide the skin over the fibula, the fibula shaft was reached. An oblique fracture was created using tissue scissors on the fibula corpus (<xref ref-type="fig" rid="F0001">Figs. 1</xref> and <xref ref-type="fig" rid="F0002">2</xref>).</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Surgical fracture of the exposed rabbit fibula.</p></caption><graphic xlink:href="FNR-58-24998-g001"/></fig><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>Short oblique surgical fracture.</p></caption><graphic xlink:href="FNR-58-24998-g002"/></fig><p>After washing the surgical site with saline, the skin was closed. Povidone iodine was applied with a dressing, then transparent film dressing spray, leaving the wound uncovered. From the post-operative first day for 30 days, the immunonutrition group were given standard rabbit food and water with the addition of 2 ml/kg/day 20% L-alanine and L-glutamine solution via a gastric catheter. The control group received 2 ml/kg/day isotonic via gastric catheter in addition to standard rabbit food and water. All the rabbits were sacrificed at the end of 30 days. By disarticulating the lower extremity where the fracture had been made, from the hip joint, the surrounding soft tissue was removed without any damage to the callus tissue. The fractures were examined clinically, radiologically, and histopathologically in respect to the degree of union.</p><p>Anterior–posterior and lateral radiographs were taken of the lower extremities removed from the rabbits. These radiographs were evaluated by five specialist doctors from the orthopaedics and traumatology department and five separate specialist doctors from the radiology department using the Lane and Sandhu (<xref rid="CIT0009" ref-type="bibr">9</xref>) radiological scoring system to give a numerical value. In this system, the radiological findings are scored 0–4, where 0=no healing, 1=callus formation, 2=the start of bone union, 3=the fracture line is starting to disappear, and 4=full union. The fibula union was subjectively evaluated by examination in two planes as described by Dimar et al. (<xref rid="CIT0010" ref-type="bibr">10</xref>). This scoring system grades the amount and quality of bone callus tissue through physical movement. A score of 0 indicates movement in both planes and therefore no union, a score of 1 indicates movement in one plane with moderate union and with a score of 4, there is no movement and full union.</p><p>After the clinical and radiological evaluations, tissue from the prepared samples from the fracture union site were fixed in 10% concentration formalin and stored for 3 days. The electrolytic method was used for decalcification and accordingly the tissues were kept for 2 weeks in a decalcification solution, and changed every 2 days (<xref rid="CIT0011" ref-type="bibr">11</xref>). The tissues were washed for 4 h in running water, then for 1 h in each of the following strengths of alcohol; 70, 80, 90, 96, and 100%, kept overnight in xylol, then for 2 h in liquid paraffin before being fixed in paraffin blocks. The lamina prepared from 5-micron thickness sections cut by rotary microtome were stained with haematoxylin and eosin and examined by light microscope. Callus tissue was scored histopathologically using the system recommended by Huo et al. (<xref rid="CIT0012" ref-type="bibr">12</xref>).</p><p>Statistical analysis was made by SPSS 13.0 packet program. Descriptive statistics were given as median, minimum, and maximum values. The Mann–Whitney <italic>U-</italic>test was used for comparison of the two independent groups and the Wilcoxon test for comparison of dependent groups. Spearman correlation analysis was used in the examination of the relationships between variables. A significance level of <italic>p</italic><0.05 was accepted.</p></sec></sec><sec sec-type="results" id="S0003"><title>Results</title><p>In the first week of the study, two rabbits in the immunonutrition group aspirated the dipeptiven solution given by gastric catheter. One of the rabbits died within minutes of aspiration and the other on the following day. In the control group, in the first week, one rabbit trapped its right lower extremity in the cage while it was being cleaned and sustained a femoral fracture. A pelvipedal plaster was applied but the rabbit died 3 days later. In the second week, one rabbit in the control group was found dead in its cage of unknown causes. The study continued with eight rabbits in the immunonutrition group and eight rabbits in the control group. Throughout the study no surgical site infection developed in any of the rabbits. Apart from the rabbit that sustained a femoral fracture, no functional problems were observed in any rabbit other than the created fibula fracture.</p><sec id="S0003-S20001"><title>Radiological findings</title><p>The radiological evaluation results determined the control group points from the orthopaedic and traumatology specialists to be mean 2.5 (minimum 0.4–maximum 3.6), and from the radiology specialists, mean 2.65 (minimum 0.6–maximum 3.8) (<xref ref-type="fig" rid="F0003">Figs. 3</xref> and <xref ref-type="fig" rid="F0004">4</xref>).</p><fig id="F0003" position="float"><label>Fig. 3</label><caption><p>Control group anterior–posterior radiograph.</p></caption><graphic xlink:href="FNR-58-24998-g003"/></fig><fig id="F0004" position="float"><label>Fig. 4</label><caption><p>Immunonutrition group anterior–posterior radiograph.</p></caption><graphic xlink:href="FNR-58-24998-g004"/></fig><p>The immunonutrition group points from the orthopaedic and traumatology specialists were determined to be mean 3.42 (minimum 3.0–maximum 3.8), and from the radiology specialists, mean 3.35 (minimum 2.4–maximum 4.0) (<italic>p</italic>=0.13 orthopaedists, <italic>p</italic>=0.279 radiologists, <xref ref-type="fig" rid="F0005">Fig. 5</xref>).</p><fig id="F0005" position="float"><label>Fig. 5</label><caption><p>Mean points distribution given by Orthopaedics and Radiology physicians.</p></caption><graphic xlink:href="FNR-58-24998-g005"/></fig></sec><sec id="S0003-S20002"><title>Clinical examination results</title><p>An orthopaedic doctor who was not involved in the experimental study made a subjective clinical examination of the relevant disarticulated extremities after removal from the soft tissue. In the clinical evaluation, the control group mean points were 1.875 (minimum 1.0–maximum 2.0), and the immunonutrition group mean points were 2.0 (minimum 2.0–maximum 2.0) (<italic>p</italic>=0.721, <xref ref-type="fig" rid="F0006">Fig. 6</xref>).</p><fig id="F0006" position="float"><label>Fig. 6</label><caption><p>Clinical points distribution of callus tissue.</p></caption><graphic xlink:href="FNR-58-24998-g006"/></fig></sec><sec id="S0003-S20003"><title>Histopathological results</title><p>Following the clinical and radiological examinations, the preparations were histopathologically evaluated according to the criteria described by Huo et al. (<xref rid="CIT0012" ref-type="bibr">12</xref>). In this evaluation, the control group mean points were 8.5 (minimum 8.0–maximum 9.0), and the immunonutrition group mean points were 9.0 (minimum 9.0–maximum 9.0) (Figs.
<xref ref-type="fig" rid="F0007">7</xref>–<xref ref-type="fig" rid="F0011">11</xref>
).</p><fig id="F0007" position="float"><label>Fig. 7</label><caption><p>Histopathological points distribution.</p></caption><graphic xlink:href="FNR-58-24998-g007"/></fig><fig id="F0008" position="float"><label>Fig. 8</label><caption><p>Control group stage 8 recovery: Image shows immature bone (IB) and little cartilage (C) tissue (haemotoxylin and eosin staining ×100 magnification).</p></caption><graphic xlink:href="FNR-58-24998-g008"/></fig><fig id="F0009" position="float"><label>Fig. 9</label><caption><p>Control group stage 8 recovery: Image shows immature bone (IB) and little cartilage (C) tissue (haemotoxylin and eosin staining ×200 magnification).</p></caption><graphic xlink:href="FNR-58-24998-g009"/></fig><fig id="F0010" position="float"><label>Fig. 10</label><caption><p>Immunonutrition group stage 9 recovery: section from fracture line (FL) showing complete immature bone tissue (IB) (haemotoxylin and eosin staining×40 magnification).</p></caption><graphic xlink:href="FNR-58-24998-g010"/></fig><fig id="F0011" position="float"><label>Fig. 11</label><caption><p>Immunonutrition group stage 9 recovery: complete immature bone tissue (IB) (haemotoxylin and eosin staining ×100 magnification).</p></caption><graphic xlink:href="FNR-58-24998-g011"/></fig></sec></sec><sec id="S0004"><title>Discussion and conclusion</title><p>In recent years several scientific studies have focussed on glutamine as a non-essential amino acid as it has been determined to have significant physiological roles. Glutamine facilitates the process of rapid cell reproduction as it is a leading source of both energy and biosynthesis (<xref rid="CIT0013" ref-type="bibr">13</xref>, <xref rid="CIT0014" ref-type="bibr">14</xref>). Particularly in critical cases such as multiple injuries, sepsis, or burns, glutamine is used as an essential amino acid as it undertakes the precursor task for nucleic acids, nucleotides, amino sugars, and proteins (<xref rid="CIT0003" ref-type="bibr">3</xref>). Under catabolic conditions, glutamine takes on the aspects of an amino acid (<xref rid="CIT0003" ref-type="bibr">3</xref>, <xref rid="CIT0015" ref-type="bibr">15</xref>). When muscle tissue is exposed to excessive stress, the most important of the amino acids which provide a repository of some amino acids, is glutamine (<xref rid="CIT0016" ref-type="bibr">16</xref>). A fall in the level of glutamine is associated with poor clinical results, as starting with a decreased amount in the process of muscle destruction, morbidity and mortality rates may increase. Parenteral or enteral administration of glutamine to critical patients leads to decreased complications associated with sepsis and better functional results (<xref rid="CIT0017" ref-type="bibr">17</xref>, <xref rid="CIT0018" ref-type="bibr">18</xref>). Glutamine has the essential properties of an amino acid in respect to affecting lymphocyte proliferation in the most appropriate way by neutrophil and macrophage functions <xref rid="CIT0019" ref-type="bibr">19</xref>–<xref rid="CIT0022" ref-type="bibr">22</xref>). By eliminating a lack of glutamine, it is possible to restore nitrogen balance and improve immunosuppression (<xref rid="CIT0017" ref-type="bibr">17</xref>).</p><p>To the best of our knowledge there are no studies on the effect of orally administered glutamine on fracture healing. In this study, taking various beneficial effects into consideration, it was thought that glutamine could make a positive contribution to fracture healing, and in this context, the similarity of wound healing to fracture healing was the guide.</p><p>As a single indeterminate structure, glutamine quickly becomes denatured in water. The dipeptide structure formed when glutamine is combined with alanine may remain stable in aqueous solutions. L-amino acids are more easily absorbed than d-amino acids (<xref rid="CIT0016" ref-type="bibr">16</xref>). Therefore, in this study an l-alanine l-glutamine solution was used to achieve absorption with active transport of the glutamine and to prevent rapid denaturing.</p><p>In an experimental study of 50 rabbits with fibula fracture and femoral condyle defects, and orally nourished with essential amino acids containing lactose, Fini et al. (<xref rid="CIT0023" ref-type="bibr">23</xref>) reported a positive effect on fracture healing. Although the current study investigated the effect on rabbit fibula fracture of glutamine as a different essential amino acid, both studies showed a similarity in respect to the chosen animal model and the choice of amino acid given. In the fracture model created in the rabbits, cartilage islands started to form on the sixth day of fracture healing, on the 12th day the centre was filled with cartilage tissue, and in the third and fourth week bone tissue replaced the cartilage showing complete union. Thus, in the current study, the experiment was terminated in the fourth week as a sufficient time for fracture healing in rabbits.</p><p>Although various techniques are used for the radiological evaluation of fracture healing, most of these have been shown to be inadequate as they are not objective and vary from person to person. In some studies, the bridging status between the fractured ends has been evaluated from direct radiographs (<xref rid="CIT0009" ref-type="bibr">9</xref>, <xref rid="CIT0024" ref-type="bibr">24</xref>). In the current study, the widely used Lane and Sandhu (<xref rid="CIT0009" ref-type="bibr">9</xref>) scoring system was used in the radiological evaluation by more than one orthopaedic, traumatology, and radiology specialist (<xref rid="CIT0025" ref-type="bibr">25</xref>–<xref rid="CIT0028" ref-type="bibr">28</xref>). We are of the opinion that this provides a more objective evaluation.</p><p>In clinical application, bone union is monitored with both physical examinations and radiological tests. Similarly, in the current study, the presence of bone union was investigated by forced movements to the fractured fibula in two planes, and radiology provided an additional evaluation. Dimar et al. (<xref rid="CIT0010" ref-type="bibr">10</xref>) emphasised clinical examination in two planes for stability evaluation and the approach in the current study supports this.</p><p>Some histopathological evaluations related to fracture healing have given qualitative results rather than numerical values (<xref rid="CIT0029" ref-type="bibr">29</xref>, <xref rid="CIT0030" ref-type="bibr">30</xref>). Allen et al. (<xref rid="CIT0031" ref-type="bibr">31</xref>–<xref rid="CIT0033" ref-type="bibr">33</xref>) reported a 5-stage histopathological evaluation. However, Huo et al. (<xref rid="CIT0012" ref-type="bibr">12</xref>) recommended a more comprehensive method, which was used in the current study. By examination of 10 stages in this system, more detailed and accurate results are obtained (<xref rid="CIT0028" ref-type="bibr">28</xref>, <xref rid="CIT0034" ref-type="bibr">34</xref>, <xref rid="CIT0035" ref-type="bibr">35</xref>).</p><p>Physiological and pathological events in living organisms occur as a result of substances which may have harmful effects. The most important of these are free oxygen radicals which are balanced by the body's antioxidant system (<xref rid="CIT0036" ref-type="bibr">36</xref>). The effects of antioxidant molecules on fracture healing have been examined with particular focus on some vitamins and essential amino acids (<xref rid="CIT0004" ref-type="bibr">4</xref>, <xref rid="CIT0031" ref-type="bibr">31</xref>, <xref rid="CIT0033" ref-type="bibr">33</xref>, <xref rid="CIT0037" ref-type="bibr">37</xref>–<xref rid="CIT0039" ref-type="bibr">39</xref>). As part of the antioxidant system, glutamine being a precursor of the glutathione-peroxidase enzyme, also serves as an antioxidant. Studies by Durak et al. (<xref rid="CIT0040" ref-type="bibr">40</xref>) and Sarisözen et al. (<xref rid="CIT0031" ref-type="bibr">31</xref>) emphasised that the effect of antioxidants in reducing free oxygen radicals found in fracture haematoma may have a positive effect on fracture healing. Therefore, in the current study it was thought that as well as other effects, glutamine as an antioxidant could positively influence fracture healing by reducing free oxygen radicals found in the fracture area, but indicators related to this were not examined.</p><p>In a study on rabbits by Sinha and Goal (<xref rid="CIT0041" ref-type="bibr">41</xref>), where the amino acids lysine and arginine were administered orally, increased callus vascularisation and mineralisation was reported and the healing period was accelerated to 2 weeks. In an experimental study by Kdolsky et al. (<xref rid="CIT0042" ref-type="bibr">42</xref>) a femoral diaphysis defect was created in guinea pigs and intramedullary fixation was applied to the femur with a k-wire. The findings were evaluated histologically, mechanically, and radiologically and it was concluded that the animals which had received L-Arginine had better fracture healing and mechanical stability. Comparing the current study to that one, the amino acid used was glutamine, the fibula was used rather than the femur, and there was no mechanical evaluation. Therefore, although the idea of the effect of amino acids on fracture healing is similar, from a technical aspect there is no similarity to the current study.</p><p>In the histological evaluation of the current study, all the rabbits that had received glutamine were seen to be at stage 9 full recovery, whereas half of the control group were at stage 8 recovery with cartilage and immature bone together and the others were at stage 9 recovery. This finding justifies the view that glutamine has a positive effect on fracture healing. However, no statistically significant difference was determined between the two groups. Similarly, in the radiological evaluation, the immunonutrition group was determined as mean 3.42 points from a total of 5 by the orthopaedic and traumatology specialists and mean 3.35 points by the radiology specialists whereas the control group points were low at mean 2.5 and 2.65 respectively. Despite the difference between them, there was no statistically significant difference. This is thought to be because of the low number of animals used in this study. It is also thought that histological evaluation at an earlier stage of healing may yield significant results.</p><p>In conclusion, despite the beneficial effects of glutamine on the speed of wound healing and reducing morbidity and mortality rates in sepsis, and the positive effect observed on bone healing in the immunonutrition group clinically, radiologically, and histopathologically, no statistically significant difference was determined compared to the control group in this study. Additional light will be shed on this subject in literature by subsequent research considering different parameters.</p></sec> |
Foot-and-mouth disease virus leader proteinase: Structural insights into the mechanism of intermolecular cleavage | Could not extract abstract | <contrib contrib-type="author" id="au0005"><name><surname>Steinberger</surname><given-names>Jutta</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au0010"><name><surname>Grishkovskaya</surname><given-names>Irina</given-names></name><xref rid="aff0010" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au0015"><name><surname>Cencic</surname><given-names>Regina</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au0020"><name><surname>Juliano</surname><given-names>Luiz</given-names></name><xref rid="aff0015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au0025"><name><surname>Juliano</surname><given-names>Maria A.</given-names></name><xref rid="aff0015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au0030"><name><surname>Skern</surname><given-names>Tim</given-names></name><email>timothy.skern@meduniwien.ac.at</email><xref rid="aff0005" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib><aff id="aff0005"><label>a</label>Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria</aff><aff id="aff0010"><label>b</label>Max F. Perutz Laboratories, University of Vienna, Department of Structural and Computational Biology, Campus Vienna Biocenter 5, A-1030 Vienna, Austria</aff><aff id="aff0015"><label>c</label>Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil</aff> | Virology | <sec id="s0005"><title>Introduction</title><p id="p0030">Virally encoded proteinases play essential roles not only in the processing of the viral proteins but also in cleavage of host cell proteins in order to manipulate cellular processes to the advantage of the virus. One of the first such reactions to be documented was the modification of cellular translation factors during picornaviral replication leading to the shut-off of protein synthesis from capped cellular mRNA (<xref rid="bib13" ref-type="bibr">Etchison et al., 1982</xref>, <xref rid="bib27" ref-type="bibr">Leibowitz and Penman, 1971</xref>). This reaction was subsequently shown to be performed by the 2A proteinase (2A<sup>pro</sup>) in enteroviruses (<xref rid="bib24" ref-type="bibr">Kräusslich et al., 1987</xref>), a chymotrypsin-like cysteine proteinase (<xref rid="bib35" ref-type="bibr">Petersen et al., 1999</xref>), whereas in aphthoviruses, the proteolysis is performed by the leader proteinase (L<sup>pro</sup>, illustrated in <xref rid="f0005" ref-type="fig">Fig. 1</xref>) (<xref rid="bib11" ref-type="bibr">Devaney et al., 1988</xref>), a papain-like cysteine proteinase (<xref rid="bib20" ref-type="bibr">Guarn<bold>é</bold> et al., 1998</xref>). The targets of both proteinases are the two homologues of the host protein eukaryotic initiation factor (eIF) 4G (<xref rid="bib15" ref-type="bibr">Gingras et al., 1999</xref>). Cleavage of the eIF4G homologues prevents recruitment of capped mRNAs to the ribosome (<xref rid="bib26" ref-type="bibr">Lamphear et al., 1995</xref>) whereas viral RNA can still be translated under these conditions as it initiates via an internal ribosome entry segment (IRES) (<xref rid="bib29" ref-type="bibr">Martinez-Salas and Ryan, 2010</xref>). In addition, L<sup>pro</sup> has been shown to be involved in impairing the host innate immune defence by influencing NF-κB activation and to have deubiquitinase activity (<xref rid="bib8" ref-type="bibr">de Los Santos et al., 2007</xref>, <xref rid="bib9" ref-type="bibr">de los Santos et al., 2009</xref>, <xref rid="bib40" ref-type="bibr">Skern and Steinberger, 2014</xref>).<fig id="f0005"><label>Fig. 1</label><caption><p>Schematic drawing of FMDV L<sup>pro</sup> self-processing and eIF4G cleavage. (A) The FMDV RNA genome is shown as a black line, the single open reading frame as a box with the names of the mature proteins and the position of the IRES. L<sup>pro</sup>, being expressed either as Lab<sup>pro</sup> or Lb<sup>pro</sup>, is indicated in red. (B) Synthesis of the polyprotein from the FMDV genome showing that Lb<sup>pro</sup> can either be freed by an intramolecular or intermolecular reaction. sLb<sup>pro</sup> (shown in orange) is generated by self-processing at the C-terminus of Lb<sup>pro</sup>. (C) The effect of eIF4G cleavage by Lb<sup>pro</sup> or sLb<sup>pro</sup>. The cellular mRNA is shown as a black line with the cap structure as a filled circle. Lb<sup>pro</sup> and sLb<sup>pro</sup> are shown in red and orange, respectively. The 40S ribosomal subunit, the polyA-binding protein (PABP), eIF4G, eIF4E, eIF4A and eIF3 are shown in different shades of grey. Following cleavage of eIF4G by Lb<sup>pro</sup> or sLb<sup>pro</sup>, the capped mRNA is no longer connected to the 40S subunit and cannot be translated. In contrast, the viral RNA can bind to the C-terminal fragment of eIF4G and thus to the 40S subunit via eIF3.</p></caption><graphic xlink:href="gr1"/></fig></p><p id="p0035">Given these involvements in such different reactions as intramolecular and intermolecular self-processing, eIF4G cleavage and deubiquitination, it is not surprising that L<sup>pro</sup> has unusual specificity determinants. These are well illustrated by the sequences of the three L<sup>pro</sup> cleavage sites that have been determined directly by protein sequencing: KVQRKLK⁎GAGQSS for both intra- and intermolecular cleavage on the viral polyprotein between the C-terminus of L<sup>pro</sup> and VP4 (<xref rid="bib43" ref-type="bibr">Strebel and Beck, 1986</xref>), PSFANLG⁎RTTLST on eIF4GI (<xref rid="bib23" ref-type="bibr">Kirchweger et al., 1994</xref>) and VPLLNVG⁎SRRSQP on eIF4GII (<xref rid="bib17" ref-type="bibr">Gradi et al., 2004</xref>). Studies on L<sup>pro</sup> intramolecular self-processing and cleavage of peptide substrates have revealed that L<sup>pro</sup> can cleave before or after basic residues provided that the other amino acid before or after the scissile bond is glycine (<xref rid="bib16" ref-type="bibr">Glaser et al., 2001</xref>, <xref rid="bib33" ref-type="bibr">Nogueira Santos et al., 2012</xref>, <xref rid="bib38" ref-type="bibr">Santos et al., 2009</xref>). However, a peptide that contained basic residues before and after the scissile bond was refractory to cleavage and was subsequently shown to be an inhibitor in the micromolar range (<xref rid="bib38" ref-type="bibr">Santos et al., 2009</xref>). This information was then used to develop a nanomolar epoxide inhibitor based on E64, termed E64-R-P-NH<sub>2</sub> (<xref rid="bib38" ref-type="bibr">Santos et al., 2009</xref>); the structure and inhibitor parameters are shown in <xref rid="f0010" ref-type="fig">Fig. 2</xref>, together with those of the other inhibitors used or referred to in this work. The slow formation of the tight enzyme–inhibitor complex indicates that inhibition follows slow-binding kinetics (<xref rid="bib38" ref-type="bibr">Santos et al., 2009</xref>, <xref rid="bib49" ref-type="bibr">Zhou et al., 1998</xref>).<fig id="f0010"><label>Fig. 2</label><caption><p>Chemical structures of inhibitors referred to in this work. The structures and kinetic parameters of the inhibitor E64-R-P-NH<sub>2</sub> (<xref rid="bib33" ref-type="bibr">Nogueira Santos et al., 2012</xref>) crystallised with sLb<sup>pro</sup> are shown together with those of the inhibitors NS-134 (<xref rid="bib42" ref-type="bibr">Stern et al., 2004</xref>) and CA074 (<xref rid="bib48" ref-type="bibr">Yamamoto et al., 1997</xref>) whose structures were determined in complex with cathepsin B. The correspondence of side-chains in the inhibitors to substrate side-chains is shown using the nomenclature of <xref rid="bib39" ref-type="bibr">Schechter and Berger, (19670</xref>.</p></caption><graphic xlink:href="gr2"/></fig></p><p id="p0040">The structural basis for this unusual specificity has not been elucidated, as the present structures determined by X-ray crystallography and NMR (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>, <xref rid="bib20" ref-type="bibr">Guarné et al., 1998</xref>, <xref rid="bib19" ref-type="bibr">Guarné et al., 2000</xref>, <xref rid="bib41" ref-type="bibr">Steinberger et al., 2013</xref>) only provide information on the S binding region but not on the S′ binding region of L<sup>pro</sup>. The nomenclature for sites (S) on the enzyme binding to residues of substrate (P) is that of <xref rid="bib39" ref-type="bibr">Schechter and Berger (1967)</xref>; prime site residues are those C-terminal to the scissile bond. Indeed, information on the nature of the S′ region from related papain-like proteinases is also sparse (<xref rid="bib46" ref-type="bibr">Turk et al., 2012</xref>), with structural information only being available for cathepsin B (<xref rid="bib42" ref-type="bibr">Stern et al., 2004</xref>, <xref rid="bib45" ref-type="bibr">Turk et al., 1995</xref>, <xref rid="bib48" ref-type="bibr">Yamamoto et al., 1997</xref>) determined with inhibitors similar to E64-R-P-NH<sub>2</sub> (<xref rid="f0010" ref-type="fig">Fig. 2</xref>). However, cathepsin B is also unusual in being an exopeptidase, with an occluding loop that prevents access beyond the S2′ site, that is the site on the enzyme interacting with the P2′ residue of the substrate (<xref rid="bib42" ref-type="bibr">Stern et al., 2004</xref>). Thus, any information on the S′ binding region of FMDV L<sup>pro</sup> will shed light on the nature of this region in papain-like cysteine proteinases generally.</p><p id="p0045">Understanding of the mechanism of L<sup>pro</sup> is complicated by the presence of different forms of the protein in the infected cell (<xref rid="bib37" ref-type="bibr">Sangar et al., 1987</xref>, <xref rid="bib36" ref-type="bibr">Sangar et al., 1988</xref>). Two isoforms, Lab<sup>pro</sup> and Lb<sup>pro</sup> (<xref rid="f0005" ref-type="fig">Fig. 1</xref>), arise from the presence of two in-frame AUG codons for the initiation of protein synthesis on the viral RNA (<xref rid="bib37" ref-type="bibr">Sangar et al., 1987</xref>). Consequently, the Lab<sup>pro</sup> possesses an additional 28 amino acids at the N-terminus than Lb<sup>pro</sup>. <xref rid="bib4" ref-type="bibr">Cao et al. (1995)</xref> demonstrated in cell culture that Lb<sup>pro</sup> was essential whereas Lab<sup>pro</sup> was not; nevertheless, there may still be as yet unknown roles for Lab<sup>pro</sup> during infection in the host organism. In addition, a shortened form of Lb<sup>pro</sup> (sLb<sup>pro</sup>) lacking 6 or 7 amino acids at the C-terminus has long been known (<xref rid="bib36" ref-type="bibr">Sangar et al., 1988</xref>). The truncation arises through Lb<sup>pro</sup> self-cleavage (<xref rid="bib36" ref-type="bibr">Sangar et al., 1988</xref>) and can be observed in vitro when Lb<sup>pro</sup> expressed in rabbit reticulocyte lysates (RRLs) is incubated for longer time periods (e.g. 1 h)(<xref rid="bib30" ref-type="bibr">Mayer et al., 2008</xref>). A separate function for sLb<sup>pro</sup> has not been identified; however, one report suggested that Lb<sup>pro</sup> and sLb<sup>pro</sup> may differ in their cleavage efficiencies in intermolecular cleavage of the polyprotein substrate (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>).</p><p id="p0050">One clear difference between Lb<sup>pro</sup> and sLb<sup>pro</sup> is the ability of Lb<sup>pro</sup> to form homodimers through interactions of the C-terminal extension (CTE) of one monomer and the substrate binding site of the neighbouring one and vice versa. sLb<sup>pro</sup> cannot form homodimers in this way because it lacks the six most C-terminal residues. The Lb<sup>pro</sup> homodimer has been observed by X-ray crystallography and NMR (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>, <xref rid="bib20" ref-type="bibr">Guarné et al., 1998</xref>, <xref rid="bib19" ref-type="bibr">Guarné et al., 2000</xref>, <xref rid="bib41" ref-type="bibr">Steinberger et al., 2013</xref>) with the K<sub>D</sub> being estimated from NMR analyses to be in the millimolar range (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>). Therefore, formation of the homodimer at concentrations of Lb<sup>pro</sup> achieved when it is synthesised in the infected cell seems unlikely unless there is a high local concentration. In contrast, both Lb<sup>pro</sup> and sLb<sup>pro</sup> use an exosite featuring residues Tyr183 to Leu188 as well as Cys 133 to recognise binding sites located on the eIF4G homologues, located in both cases 20 to 30 amino acids from the cleavage site (<xref rid="bib14" ref-type="bibr">Foeger et al., 2005</xref>). How this binding favours Lb<sup>pro</sup> or sLb<sup>pro</sup> cleavage of the eIF4G homologues is not known.</p><p id="p0055">To investigate further the properties of sLb<sup>pro</sup>, we set out to determine the structure of sLb<sup>pro</sup> complexed with the inhibitor E64-R-P-NH<sub>2</sub> and to define differences in the cleavage of intermolecular polyprotein substrates by sLb<sup>pro</sup> and Lb<sup>pro</sup>.</p></sec><sec id="s0010"><title>Materials and methods</title><sec id="s0015"><title>Materials</title><p id="p0060">The bacterial expression plasmid pET-11d sLb<sup>pro</sup> (FMDV residues 29–195) was created by site-directed PCR mutagenesis of pET-11d sLb<sup>pro</sup> C51A, described earlier (<xref rid="bib20" ref-type="bibr">Guarné et al., 1998</xref>, <xref rid="bib23" ref-type="bibr">Kirchweger et al., 1994</xref>), to restore the catalytic cysteine.</p><p id="p0065">The plasmids that were used as templates for in vitro transcription pCITE-1d Lb<sup>pro</sup> (residues 29–201 of Lb<sup>pro</sup>), pCITE-1d sLb<sup>pro</sup> (residues 29–195 of Lb<sup>pro</sup>) and pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2 (residues 29–201 of Lb<sup>pro</sup>, all 85 residues of VP4 and 78 residues of VP2) have been described (<xref rid="bib16" ref-type="bibr">Glaser et al., 2001</xref>). The constructs pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2 containing the mutations at position P1 and P1′ of the Lb<sup>pro</sup>-VP4 cleavage site (VQRKLG⁎RAGQ, VQRKLK⁎RAGQ, VQRKLG⁎AAGQ) were created by site-directed PCR mutagenesis of pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2. The construct pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2 containing the eIF4GI sequence SFANLG⁎RTTL at the Lb<sup>pro</sup>-VP4 cleavage site, termed pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2 SFANLG⁎RTTL (FMDV residues 29–195 of Lb<sup>pro</sup>, residues 669–678 of eIF4GI, residue 5–85 of VP4 and 78 residues of VP2), has been described (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>). The construct pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2 containing residues 599–678 of eIF4GI, termed pCITE-1d Lb<sup>pro</sup> C51A eIF4GI<sub>599-668</sub> VP4/VP2 SFANLG⁎RTTL, (FMDV residues 29–195 of Lb<sup>pro</sup>, residues 599–678 of eIF4GI, residue 5–85 of VP4 and 78 residues of VP2) was created by PCR amplification of residues 599–678 of eIF4GI using the plasmid pKS eIF4GI 400–739 as template and cloning of this fragment into pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2 via the restriction sites <italic>Bpu10</italic>I and <italic>Sac</italic>I.</p><p id="p0070">The inhibitor E64-R-P-NH<sub>2</sub> was prepared as described (<xref rid="bib33" ref-type="bibr">Nogueira Santos et al., 2012</xref>).</p></sec><sec id="s0020"><title>Protein expression and purification</title><p id="p0075">Protein expression and purification were performed as described by <xref rid="bib41" ref-type="bibr">Steinberger et al. (2013)</xref> with the following modifications. Proteins were expressed from the construct pET-11d sLb<sup>pro</sup> transformed into BL21(DE3)LysE bacteria. To avoid degradation of the active protease, all purification steps were carried at a maximum of 10 °C.</p></sec><sec id="s0025"><title>Preparation of the sLb<sup>pro</sup>-E64-R-P-NH<sub>2</sub> complex</title><p id="p0080">Purified sLb<sup>pro</sup> was incubated with a fivefold molar excess of E64-R-P-NH<sub>2</sub> over night at 4 °C to allow complex formation. Subsequently, the complex was dialysed against a buffer containing 50 mM NaCl, 10 mM Tris HCl pH 8, 1 mM TCEP, 5% glycerol to remove excess inhibitor. The concentration was adjusted to 18 mg/ml and centrifuged at 18,000<italic>g</italic> for 10 min at 4 °C to remove precipitated protein.</p></sec><sec id="s0030"><title>Crystallisation, data collection, structure determination and refinement</title><p id="p0085">Crystals of the sLb<sup>pro</sup>-E64-R-P-NH<sub>2</sub> complex were initially obtained in the Wizard I and II screen crystallisation screen (Emerald Bio), using the sitting-drop vapour diffusion technique and a nanodrop-dispensing robot (Phoenix RE; Rigaku Europe, Kent, United Kingdom), and optimised to 0.1 M sodium acetate pH 4.8, 0.9 M NaH<sub>2</sub>PO<sub>4</sub> and 1.2 M K<sub>2</sub>HPO<sub>4</sub> using the hanging drop vapour diffusion technique at 22 °C and seeding technique. The seed stock was produced by a “seed-bead” kit from Hampton Research (<xref rid="bib28" ref-type="bibr">Luft and DeTitta, 1999</xref>). The crystals were flash-frozen in liquid nitrogen in a reservoir solution supplemented with 25% glycerol prior to data collection.</p><p id="p0090">Diffraction data sets were collected at the ESRF Synchrotron (Grenoble) at beamline ID14-1 at 100 K using a wavelength of 0.93 Å to 1.6 Å resolution, processed using the XDS package (<xref rid="bib22" ref-type="bibr">Kabsch<bold>,</bold> 2010</xref>), converted to mtz format using POINTLESS and scaled with SCALA (<xref rid="bib47" ref-type="bibr">Winn et al., 2011</xref>).</p><p id="p0095">The crystal structure was solved by difference Fourier techniques using the protein atomic coordinates of the inactive mutant of sLb<sup>pro</sup> from the Protein Data Bank (accession code 1QMY). Model building and refinement steps were performed with REFMAC and COOT. The structure was refined using the programs REFMAC (<xref rid="bib31" ref-type="bibr">Murshudov et al., 1997</xref>) and Phenix Refine (<xref rid="bib1" ref-type="bibr">Adams et al., 2010</xref>) and model building was done with the program Coot (<xref rid="bib12" ref-type="bibr">Emsley and Cowtan, 2004</xref>). Data collection and refinement statistics are shown in <xref rid="t0005" ref-type="table">Table 1</xref>. Stereo-chemistry and structure quality were checked using the MolProbity web server (<xref rid="bib7" ref-type="bibr">Davis et al., 2007</xref>).<table-wrap position="float" id="t0005"><label>Table 1</label><caption><p>X-ray parameters and refinement statistics.</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="2" valign="middle"><bold>Data collection</bold></th></tr></thead><tbody><tr><td valign="middle">Source</td><td valign="middle">ID14-1, ESRF</td></tr><tr><td valign="middle">Wavelength (Å)</td><td valign="middle">0.93</td></tr><tr><td valign="middle">Resolution (Å)</td><td valign="middle">45.35–1.6 (1.69–1.6)<xref rid="tbl1fna" ref-type="table-fn">a</xref></td></tr><tr><td valign="middle">Space group</td><td valign="middle">P2<sub>1</sub></td></tr><tr><td rowspan="2" valign="middle">Unit cell (Å, °)</td><td valign="middle"><italic>a</italic>=45.81 <italic>b</italic>=110.68, <italic>c</italic>=56.77</td></tr><tr><td valign="middle"><italic>α</italic>=<italic>γ</italic>=90, <italic>β</italic>=98.12</td></tr><tr><td valign="middle">Molecules / a.u.</td><td valign="middle">3</td></tr><tr><td valign="middle">Unique reflections</td><td valign="middle">72906 (10232)</td></tr><tr><td valign="middle">Completeness (%)</td><td valign="middle">99.0 (95.0)</td></tr><tr><td valign="middle"><italic>R</italic><sub>merge</sub><xref rid="tbl1fnb" ref-type="table-fn">b</xref></td><td valign="middle">0.037 (0.174)</td></tr><tr><td valign="middle"><italic>R</italic><sub>meas</sub><xref rid="tbl1fnc" ref-type="table-fn">c</xref></td><td valign="middle">0.041 (0.213)</td></tr><tr><td valign="middle">Multiplicity</td><td valign="middle">4.9 (2.9)</td></tr><tr><td valign="middle">I/sig(I)</td><td valign="middle">29.9 (5.6)</td></tr><tr><td valign="middle">B<sub>Wilson</sub> (Å<sup>2</sup>)</td><td valign="middle">22.5</td></tr><tr><td colspan="2" valign="middle"><bold>Refinement</bold></td></tr><tr><td valign="middle"><italic>R</italic><sub>cryst</sub><xref rid="tbl1fnd" ref-type="table-fn">d</xref>/ R<sub>free</sub><xref rid="tbl1fne" ref-type="table-fn">e</xref></td><td valign="middle">16.9/20.1</td></tr><tr><td valign="middle">R.m.s.d. bonds (Å)</td><td valign="middle">0.011</td></tr><tr><td valign="middle">R.m.s.d. angles (°)</td><td valign="middle">1.4</td></tr><tr><td valign="middle">Ramachandran plot (%)</td><td valign="middle"/></tr><tr><td valign="middle">favored/allowed/outliers</td><td valign="middle">96.9/3.1/0</td></tr></tbody></table><table-wrap-foot><fn id="tbl1fna"><label>a</label><p id="ntp0005">Values in parentheses are for the highest resolution shell.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fnb"><label>b</label><p id="ntp0010"><inline-formula><mml:math id="M1" altimg="si0001.gif" overflow="scroll"><mml:msub><mml:mrow><mml:mi>R</mml:mi></mml:mrow><mml:mrow><mml:mi>m</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mi>g</mml:mi><mml:mi>e</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mstyle displaystyle="true"><mml:mfrac><mml:mrow><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>N</mml:mi></mml:msubsup><mml:mrow><mml:mo>|</mml:mo><mml:msub><mml:mrow><mml:mi>I</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mover accent="true"><mml:mi>I</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:mrow><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub><mml:mo>|</mml:mo></mml:mrow></mml:mrow></mml:mrow><mml:mrow><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>N</mml:mi></mml:msubsup><mml:mrow><mml:msub><mml:mrow><mml:mi>I</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub></mml:mrow></mml:mrow></mml:mrow></mml:mfrac></mml:mstyle></mml:math></inline-formula></p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fnc"><label>c</label><p id="ntp0015"><inline-formula><mml:math id="M2" altimg="si0002.gif" overflow="scroll"><mml:msub><mml:mrow><mml:mi>R</mml:mi></mml:mrow><mml:mrow><mml:mi>m</mml:mi><mml:mi>e</mml:mi><mml:mi>a</mml:mi><mml:mi>s</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mstyle displaystyle="true"><mml:mfrac><mml:mrow><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:msqrt><mml:mrow><mml:mi>N</mml:mi><mml:mo>/</mml:mo><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:mi>N</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:mrow></mml:msqrt><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>N</mml:mi></mml:msubsup><mml:mrow><mml:mo>|</mml:mo><mml:msub><mml:mrow><mml:mi>I</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mover accent="true"><mml:mi>I</mml:mi><mml:mrow><mml:mo stretchy="true">¯</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub><mml:mo>|</mml:mo></mml:mrow></mml:mrow></mml:mrow><mml:mrow><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>N</mml:mi></mml:msubsup><mml:mrow><mml:msub><mml:mrow><mml:mi>I</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub></mml:mrow></mml:mrow></mml:mrow></mml:mfrac></mml:mstyle></mml:math></inline-formula> where <inline-formula><mml:math id="M3" altimg="si0003.gif" overflow="scroll"><mml:msub><mml:mrow><mml:mover accent="true"><mml:mi>I</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:mrow><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub></mml:math></inline-formula> is the mean intensity of multiple <inline-formula><mml:math id="M4" altimg="si0004.gif" overflow="scroll"><mml:msub><mml:mrow><mml:mi>I</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mo stretchy="false">(</mml:mo><mml:mi>h</mml:mi><mml:mi>k</mml:mi><mml:mi>l</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub></mml:math></inline-formula> observations of the symmetry-related reflections, <italic>N</italic> is the redundancy</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fnd"><label>d</label><p id="ntp0020"><inline-formula><mml:math id="M5" altimg="si0005.gif" overflow="scroll"><mml:msub><mml:mrow><mml:mi>R</mml:mi></mml:mrow><mml:mrow><mml:mi>c</mml:mi><mml:mi>r</mml:mi><mml:mi>y</mml:mi><mml:mi>s</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mstyle displaystyle="true"><mml:mfrac><mml:mrow><mml:mo>∑</mml:mo><mml:mrow><mml:mo stretchy="true">|</mml:mo><mml:mrow><mml:mrow><mml:mo stretchy="true">|</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>o</mml:mi><mml:mi>b</mml:mi><mml:mi>s</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo stretchy="true">|</mml:mo></mml:mrow><mml:mo>−</mml:mo><mml:mrow><mml:mo stretchy="true">|</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>c</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo stretchy="true">|</mml:mo></mml:mrow></mml:mrow><mml:mo stretchy="true">|</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mo>∑</mml:mo><mml:mrow><mml:mo stretchy="true">|</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>o</mml:mi><mml:mi>b</mml:mi><mml:mi>s</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo stretchy="true">|</mml:mo></mml:mrow></mml:mrow></mml:mfrac></mml:mstyle></mml:math></inline-formula></p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fne"><label>e</label><p id="ntp0025"><italic>R</italic><sub>free</sub> is the cross-validation <italic>R</italic><sub>factor</sub> computed for the test set of reflections (5%) which are omitted in the refinement process.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s0035"><title>In vitro transcription and translation</title><p id="p0100">In vitro transcription reactions were performed as described (<xref rid="bib32" ref-type="bibr">Neubauer et al., 2013</xref>) with the following modifications. The plasmids were cleaved with <italic>BamH</italic>I for the expression of active proteinases (pCITE-1d Lb<sup>pro</sup> and pCITE-1d sLb<sup>pro</sup>) and <italic>Sal</italic>I for the substrates (pCITE-1d Lb<sup>pro</sup> C51A VP4/VP2 and derivatives thereof).</p><p id="p0105">In vitro translation reactions were performed as described (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>) with the following modification. To translate substrate proteins and proteinases, RNA was added to the reaction at concentrations of 14 ng/µl.</p></sec><sec id="s0040"><title>Electrophoresis and immunoblotting</title><p id="p0110">Electrophoresis and immunoblotting for protein analysis were performed as described (<xref rid="bib32" ref-type="bibr">Neubauer et al., 2013</xref>), except for the separation of translation products when SDS-PAGE gels containing 17.5% acrylamide were used (<xref rid="bib6" ref-type="bibr">Dasso and Jackson, 1989</xref>).</p></sec><sec id="s0045"><title>Structural comparisons</title><p id="p0115">Structural alignments and superimpositions were done using Coot (<xref rid="bib12" ref-type="bibr">Emsley and Cowtan, 2004</xref>, <xref rid="bib25" ref-type="bibr">Krissinel and Henrick, 2004</xref>). All drawings were created using PyMOL (<xref rid="bib10" ref-type="bibr">DeLano<bold>,</bold> 2002</xref>). The electrostatic potential of sLb<sup>pro</sup> was calculated using the Adaptive Poisson-Boltzmann Solver package (<xref rid="bib2" ref-type="bibr">Baker et al., 2001</xref>) within PyMOL.</p></sec><sec id="s0050"><title>Accession numbers</title><p id="p0120">Coordinates for the structure determined here have been deposited in the protein data bank (pdb accession code 4QBB). The PDB identifiers of the structures used for comparisons were 1QOL for Lb<sup>pro</sup>, 1GEC for glycyl endopeptidase-complex with benzyloxycarbonyl-leucine-valine-glycine-methylene, 3CH3 for SERA5 from plasmodium falciparum, 1SP4 for bovine cathepsin B-complex with NS-134, 1QDQ for bovine cathepsin B-complex with CA074.</p></sec></sec><sec id="s0055"><title>Results and discussion</title><p id="p0125">The crystal structure of the inhibitor E64-R-P-NH<sub>2</sub> bound to sLb<sup>pro</sup> has been determined. The correspondence of the side-chains in the inhibitor to substrate side-chains is illustrated in <xref rid="f0010" ref-type="fig">Fig. 2</xref>; a portion of the electron density in the final model of the inhibitor bound to the active site is shown in <xref rid="f0015" ref-type="fig">Fig. 3</xref>. Three chains, termed A, B and C were found in the asymmetric unit of the crystal lattice. Electron density was visible for sLb<sup>pro</sup> residues 29–187 of chain A, 29 to 184 of chain B and 29–185 of chain C. For the inhibitor, electron density was visible for all atoms except for those of the P3 amino-alkyl guanidinium group (referred to as Arg-m in the text and figures) and P1′ Arg. For P3 Arg-m, density was visible up to the <italic>C</italic><sub><italic>β</italic></sub> atom for chain A, for all atoms of chain B (due to favourable interactions with an Asp residue from a symmetry related molecule) and to atom <italic>N</italic><sub><italic>ε</italic></sub> for chain C. For the P1′ arginine residues, density up to the C<sub>β</sub> atom for chain A was visible whereas for chains B and C density was observed to the <italic>C</italic><sub><italic>γ</italic></sub> atom. The remaining atoms of these side-chains including the guanidinium group were modelled in <xref rid="f0020" ref-type="fig">Fig. 4</xref>, <xref rid="f0025" ref-type="fig">Fig. 5</xref>, <xref rid="f0030" ref-type="fig">Fig. 6</xref>, <xref rid="f0035" ref-type="fig">Fig. 7</xref> after the side-chain trace of <italic>C</italic><sub><italic>α</italic></sub> to <italic>C</italic><sub><italic>γ</italic></sub> in the most likely conformation. Density for the covalent bond between the active site cysteine and the inhibitor (atom C1) was very clear in all three chains. Superimposition of the structure of sLb<sup>pro</sup> bound to E64-R-P-NH<sub>2</sub> with the unbound Lb<sup>pro</sup> structure of sLb<sup>pro</sup> C51A C133S (PDB ID 1QMY, chainB) (<xref rid="bib19" ref-type="bibr">Guarné et al., 2000</xref>) gave an r.m.s.d. of 0.35 Å over 156<italic>C</italic><sub><italic>α</italic></sub> atoms superimposed. Given that the best resolution of the inhibitor was found in chain B, all structural analysis is based on this chain.<fig id="f0015"><label>Fig. 3</label><caption><p>Stereo view of the arrangement of the inhibitor E64-R-P-NH<sub>2</sub> and the substrate binding site of sLb<sup>pro</sup>. 2F<sub>0</sub>–F<sub>c</sub> maps contoured at 1 σ are shown as grey mesh for the inhibitor and the sLb<sup>pro</sup> residues Asp49, Cys51, Glu96 and Glu147. The inhibitor is shown as green sticks. Residues of sLb<sup>pro</sup> interfacing with the inhibitor are shown as grey sticks. Oxygen, nitrogen and sulphur atoms are coloured red, blue and yellow, respectively. Due to the lack of electron density, no structure is shown for the P1‘ Arg residue of E64-R-P-NH<sub>2</sub> from the Cδ atom onwards.</p></caption><graphic xlink:href="gr3"/></fig><fig id="f0020"><label>Fig. 4</label><caption><p>Comparison of the binding of E64-R-P-NH<sub>2</sub> and P1-P3 of the CTE. (A) The inhibitor (green sticks) is shown in the substrate binding site of sLb<sup>pro</sup>. Side-chains of the inhibitor are labelled. In <xref rid="f0020" ref-type="fig">Fig. 4</xref>, <xref rid="f0025" ref-type="fig">Fig. 5</xref>, <xref rid="f0030" ref-type="fig">Fig. 6</xref>, <xref rid="f0035" ref-type="fig">Fig. 7</xref>, the atoms of the P1′ Arg residue from C<sub>δ</sub> onwards are modelled based on the most favourable conformation. Residues of the active site (Cys51, His148, Asp163) as well as the three acidic residues discussed in the text are shown as sticks. (B) As in A, with the P1-P3 residues (in yellow and labelled) of the CTE superimposed for comparison.</p></caption><graphic xlink:href="gr4"/></fig><fig id="f0025"><label>Fig. 5</label><caption><p>Electrostatic interactions involved in sLb<sup>pro</sup> interaction with E64-R-P-NH<sub>2</sub> and the P1-P3 residues of the CTE. The electrostatic potential of sLb<sup>pro</sup> was calculated using the Adaptive Poisson–Boltzmann Solver package (<xref rid="bib2" ref-type="bibr">Baker et al., 2001</xref>) within PyMOL (<xref rid="bib10" ref-type="bibr">DeLano<bold>,</bold> 2002</xref>). The surface is coloured according to the electrostatic potential ranging from −5 (red) to +5 (blue) kT/e. (A) The inhibitor E64-R-P-NH<sub>2</sub> is shown as green sticks, (B) residues P1-P3 of the CTE as yellow sticks. The representations on the right are rotated 90° on the <italic>x</italic>-axis relative to those on the left.</p></caption><graphic xlink:href="gr5"/></fig><fig id="f0030"><label>Fig. 6</label><caption><p>Comparison of arrangement of negatively charged residues in the substrate binding sites of sLb<sup>pro</sup>, glycyl endopeptidase and SERA5. (A) sLb<sup>pro</sup> bound to E64-R-P-NH<sub>2</sub> (green sticks). (B) Substrate binding site of SERA5. (C) Glycyl endopeptidase bound to the inhibitor benzyloxycarbonyl-leucine-valine-glycine-methylene (yellow sticks). (D) Stereo view of the superimposition of the three structures in A–C. Residues referred to in the text are shown as sticks.</p></caption><graphic xlink:href="gr6"/></fig><fig id="f0035"><label>Fig. 7</label><caption><p>Comparison of the S1′ and S2′ binding sites of FMDV sLb<sup>pro</sup> and cathepsin B. (A and B) E64-R-P-NH<sub>2</sub> (green sticks) bound in the substrate binding site of Lb<sup>pro</sup> with the inhibitors NS-134 (A, blue sticks) and CA074 (B, magenta sticks) superimposed. (C and D) NS-134 (C) and CA074 (D) bound to the substrate binding site of cathepsin B with E64-R-P-NH<sub>2</sub> superimposed. The colour coding is as in A and B. The structure of Trp221 for which there is no equivalent in Lb<sup>pro</sup> is shown as sticks as are residues referred to in the text.</p></caption><graphic xlink:href="gr7"/></fig></p><p id="p0130">To determine the binding of E64-R-P-NH<sub>2</sub> to sLb<sup>pro</sup>, we first compared its arrangement in the substrate binding site of sLb<sup>pro</sup> to that of the last three residues of the CTE observed in the crystal structure of Lb<sup>pro</sup> (<xref rid="bib20" ref-type="bibr">Guarn<bold>é</bold> et al., 1998</xref>). <xref rid="f0020" ref-type="fig">Fig. 4</xref> shows that the positions of the P3 Arg-m side-chain of the inhibitor and the P3 Lys side-chain of the CTE occupy similar positions in the two structures. The <italic>C</italic><sub><italic>γ</italic></sub> atom of the P1′ Arg residue of the inhibitor lies between the side-chains of Asp49 (distance from <italic>C</italic><sub><italic>γ</italic></sub> to carboxy group of Asp49 is 4.2 Å) and Glu147 (distance from <italic>C</italic><sub><italic>γ</italic></sub> to <italic>C</italic><sub><italic>β</italic></sub> of Glu147 is 4.5 Å). Given the uncertainty in the position of the guanidinium group (as mentioned earlier, the remaining atoms were modelled as no density was observed), a closer localisation is not possible. Nevertheless, the superimposition in <xref rid="f0020" ref-type="fig">Fig. 4</xref>B shows that the P1 Lys of the CTE lies almost equidistant between Asp49, Glu96 and Glu147. The disorder of the P1′ Arg in the structure of the inhibitor presented here indicates that the side-chain is flexible; in contrast, in the previously published structure of Lb<sup>pro</sup> C51A, good density was observed to the P1 Lys residue in the substrate binding site of Lb<sup>pro</sup> (<xref rid="bib20" ref-type="bibr">Guarn<bold>é</bold> et al., 1998</xref>). Given that the polypeptide chain is fully extended in both the CTE and E64-R-P-NH<sub>2</sub> bound structures, this explains how a peptide containing Lys and Arg at P1 and P1′ can be refractory to cleavage (<xref rid="bib33" ref-type="bibr">Nogueira Santos et al., 2012</xref>). If the Lys at P1 points away from the globular domain, an Arg side-chain at P1′ would have to point towards it. Thus, on oligopeptide substrates at least, the enzyme can only accommodate a basic residue at one of the positions, presumably because it requires a glycine with its greater freedom of rotation at the other. However, the data do not answer the question why a peptide containing Lys and Arg at P1 and P1′ can inhibit Lb<sup>pro</sup> (<xref rid="bib33" ref-type="bibr">Nogueira Santos et al., 2012</xref>). This implies that the inhibitor may bind in a mode that has not yet been observed that moves the scissile bond out of the active site. However, additional structural information will be required to elucidate the nature of the binding of this peptide.</p><p id="p0135">Overall, comparison of the binding of the E64-R-P-NH<sub>2</sub> and the CTE residues (<xref rid="f0025" ref-type="fig">Fig. 5</xref>) show that the P1/P1′ binding area is a deep cleft surrounded by the acidic residues Asp49, Glu96 and Glu147. We set out to determine whether other papain-like cysteine proteinases have been identified that have a similar arrangement of three acidic residues in the vicinity of the S1/S1′ binding sites. Berti and Storer (<xref rid="bib3" ref-type="bibr">Berti and Storer, 1995</xref>) compared the sequences of 48 representative papain-like cysteine proteinases. Only one, SERA5 (Serine repeat antigen 5, termed PfalI in (<xref rid="bib3" ref-type="bibr">Berti and Storer, 1995</xref>)) from <italic>P. falciparum</italic> showed acidic amino acids at the equivalent positions to those in sLb<sup>pro</sup>; these are Asp594, Glu638 and Asp761 which are equivalent to Asp49, Glu96 and Glu147 of sLb<sup>pro</sup> ((<xref rid="bib21" ref-type="bibr">Hodder et al., 2009</xref>); <xref rid="f0030" ref-type="fig">Fig. 6</xref>A and B). However, little is known about the biochemistry of this protein; indeed, proteolytic activity has not been shown. Furthermore, the putative active site residue is serine, not cysteine. In addition, the authors suggested that Asp594 (equivalent to Asp49) of SERA5 is too near to the substrate binding site to allow substrate to bind.</p><p id="p0140">A second enzyme, glycyl endopeptidase (ppiv in <xref rid="bib3" ref-type="bibr">Berti and Storer, (1995)</xref>), also possesses two acidic residues, Glu23 and Asp158, equivalent to Asp49 and Glu147. The third residue (Asn64, equivalent to Glu96 in sLb<sup>pro</sup>) is however not acidic and is followed by Arg65. As can be seen in <xref rid="f0030" ref-type="fig">Fig. 6</xref>C, the presence of Glu23 and Arg65 preclude the entry of any substrates with amino acids larger than glycine at P1, thus conferring the specificity referred to in the name glycyl endopeptidase.</p><p id="p0145">It should be noted that only these three papain-like enzymes have an amino acid other than glycine at the position equivalent to Gly23 in papain (equivalent to Asp 49 in sLb<sup>pro</sup>). Superimposition of the three structures (<xref rid="f0030" ref-type="fig">Fig. 6</xref>D) shows that Asp49 in sLb<sup>pro</sup> is further away from the substrate binding site than Glu23 or Asp594 in glycyl endopeptidase (<xref rid="bib34" ref-type="bibr">O’Hara et al., 1995</xref>) and SERA5 (<xref rid="bib21" ref-type="bibr">Hodder et al., 2009</xref>). This is due to the presence of only four residues in sLb<sup>pro</sup> lying between the oxyanion hole defining residue (Asn46) and the active site Cys51. In all other papain-like cysteine proteinases, five residues are present between the oxyanion-hole residue Gln19 and the active site nucleophile Cys25. Interestingly, Glu23 of glycyl endopeptidase is closer to the substrate binding site than Asp594 in SERA5, suggesting that the substrate binding site of SERA5 may be more open than previously thought. In contrast, Glu96 does not superimpose well with Glu638, with the <italic>C</italic><sub><italic>α</italic></sub> lying 3.7 Å apart (<xref rid="f0030" ref-type="fig">Fig. 6</xref>D). Finally, as an important control for the accuracy of the structural superimposition, we note that the <italic>C</italic><sub><italic>α</italic></sub> of the catalytic histidines (H148, H762 and H159) superimpose well (<xref rid="f0030" ref-type="fig">Fig. 6</xref>D).</p><sec id="s0060"><title>Comparisons with inhibitor complexes from cathepsin B</title><p id="p0150">Information on the structural details of the S1′ and S2′ binding sites in papain-like cysteine proteinases is limited, especially for the S2′ site (<xref rid="bib44" ref-type="bibr">Turk et al., 1998</xref>, <xref rid="bib46" ref-type="bibr">Turk et al., 2012</xref>). Indeed, structures of compounds with residues bound in the S2′ position are only available for cathepsin B complexed with the inhibitors CA030, CA074 and NS-134 (<xref rid="f0010" ref-type="fig">Fig. 2</xref>; (<xref rid="bib42" ref-type="bibr">Stern et al., 2004</xref>; <xref rid="bib45" ref-type="bibr">Turk et al., 1995</xref>; <xref rid="bib48" ref-type="bibr">Yamamoto et al., 1997</xref>)). To compare the binding of the inhibitors CA074 and NS-134 to cathepsin B (CA030 differs only in the length and chemical bond of the N-terminal aliphatic moiety (<xref rid="bib45" ref-type="bibr">Turk et al., 1995</xref>)) and that of E64-R-P-NH<sub>2</sub> to sLb<sup>pro</sup>, the structures of cathepsin B complexes with CA074 and NS-134 were superimposed on that of sLb<sup>pro</sup> using the SSM tool of Coot (<xref rid="bib25" ref-type="bibr">Krissinel and Henrick, 2004</xref>). The r.m.s.d. values were 2.44 Å for sLb<sup>pro</sup> superimposed on cathepsin B complexed to CA074 (1QDQ) and 2.31 Å for sLb<sup>pro</sup> superimposed on cathepsin B complexed to NS-134 (1SP4). <xref rid="f0035" ref-type="fig">Fig. 7</xref>A and B show the positions of the inhibitors NS-134 and CA-074 relative to E64-R-P-NH<sub>2</sub> on sLb<sup>pro</sup>; <xref rid="f0035" ref-type="fig">Fig. 7</xref>C and D show the relationships on the structure of cathepsin B.</p><p id="p0155">The P1′ residues of CA074 and NS-134 are Ile and Leu, respectively, in contrast to the Arg found in E64-R-P-NH<sub>2</sub>. Despite the difference in chemical composition, however, the side-chains of these residues superimpose well and occupy the same relative space. The specificity of cathepsin B for these large hydrophobic residues is derived from the presence of a hydrophobic pocket made up of residues Phe174, Val176, Phe180, Leu181, M196 and Trp221 (<xref rid="f0035" ref-type="fig">Figs. 7</xref>C and D). In contrast, in sLb<sup>pro</sup>, there is no equivalent loop to those in cathepsin B bearing residues Phe174 to Leu181 and Met196 or Trp221. This region is thus open and, as mentioned before, the presence of Glu147 and Asp49 enable sLb<sup>pro</sup> to accept well the arginine residue.</p><p id="p0160">At the P2′ position, all three inhibitors have a proline residue. It is clear that the positions of the proline residues from CA074 and NS-134 on the one hand and sLb<sup>pro</sup> on the other are different. In sLb<sup>pro</sup>, the proline P2′ residue of E64-R-P-NH<sub>2</sub> lies closer to Asp163, the third member of the catalytic triad. Two factors appear to be responsible for this. The first is the absence of a residue equivalent to Trp221 in cathepsin B (Trp177 in papain) that pushes the proline residue away from Asn219 (equivalent to Asp163 in sLb<sup>pro</sup>). Second, albeit only in chain A, the sLb<sup>pro</sup> residue Asp164 forms a hydrogen bond (2.7 Å) to the terminal nitrogen on the proline residue whereas in cathepsin B, the terminal carboxyl group of the proline is co-ordinated by His110 and His111 in the occluding loop, a structure unique to cathepsin B that is responsible for its exopeptidase activity. It is clear from the structure that there is no binding pocket for the P2′ residue in sLb<sup>pro</sup>.</p></sec><sec id="s0065"><title>Lb<sup>pro</sup> and sLb<sup>pro</sup> differ in cleavage efficiencies on intramolecular substrates</title><p id="p0165">The structural analysis illustrates how sLb<sup>pro</sup> can bind to an inhibitor bearing Leu, Gly and Arg at the P2, P1 and P1′ sites, respectively, the very residues found at the eIF4GII cleavage site. Nevertheless, a peptide corresponding to the eIF4GI peptide (SFANLG⁎RTTL) was a poor substrate for both Lb<sup>pro</sup> and sLb<sup>pro</sup> ((<xref rid="bib38" ref-type="bibr">Santos et al., 2009</xref>); unpublished data). However, the eIF4GI cleavage site when introduced into the background of the polyprotein substrate was efficiently cleaved by Lb<sup>pro</sup> but was still refractory to cleavage by sLb<sup>pro</sup> (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>). Examination of the state of the endogenous eIF4GI in RRLs used for the experiments showed that it was cleaved by both Lb<sup>pro</sup> and sLb<sup>pro</sup> (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>).</p><p id="p0170">To understand these observations and illuminate differences in cleavage efficiencies between Lb<sup>pro</sup> and sLb<sup>pro</sup>, we decided to investigate further the cleavage of intermolecular polyprotein substrates using the system described by <xref rid="bib5" ref-type="bibr">Cencic et al. (2007)</xref>. Here, a fragment of the FMDV polyprotein encoding an inactive form of Lb<sup>pro</sup>, VP4 and part of VP2 (termed Lb<sup>pro</sup> C51A VP4/VP2) is labelled with <sup>35</sup>S methionine by translation in RRLs (<xref rid="bib5" ref-type="bibr">Cencic et al., 2007</xref>). Subsequently, cold methionine is added and an mRNA encoding an active, mature Lb<sup>pro</sup> or sLb<sup>pro</sup> is added. The enzyme was synthesised from an RNA molecule rather than adding purified recombinant proteinase for two reasons. First, preparations of purified active Lb<sup>pro</sup> always contain some sLb<sup>pro</sup> that arises from self-processing, even when all purification steps are done at 4 °C (<xref rid="bib23" ref-type="bibr">Kirchweger et al., 1994</xref>). Second, the translation of the RNA followed by Lb<sup>pro</sup> cleavage of the eIF4G isoforms in the RRLs resembles more closely the in vivo situation during an FMDV infection. The labelled substrate and products are separated by SDS-PAGE and detected by fluorography. Although it is very difficult to vary either the enzyme or substrate concentrations in this assay, it still provides qualitative information on differences in the rates of reaction between different forms of L<sup>pro</sup>.</p><p id="p0175">A typical experiment is shown in <xref rid="f0040" ref-type="fig">Fig. 8</xref>A, with Lb<sup>pro</sup> cleaving the wild-type sequence between 15 and 30 min. The Lb<sup>pro</sup> moiety has four methionine residues compared to only two in the VP4/VP2 part, providing a partial explanation for the lower intensity of the latter band (<xref rid="bib16" ref-type="bibr">Glaser et al., 2001</xref>). In addition, we have evidence that the VP4/VP2 part is degraded in the RRLs (data not shown), with degradation being enhanced when a residue other than the wild-type glycine is present at the N-terminus of VP4 (see <xref rid="f0040" ref-type="fig">Fig. 8</xref>B–E). We first investigated the effect of substituting residues at the P1 and P1′ positions with residues (underlined in <xref rid="f0040" ref-type="fig">Fig. 8</xref>B–D), several of which had been shown to be detrimental to peptide cleavage (<xref rid="bib19" ref-type="bibr">Guarné et al., 2000</xref>, <xref rid="bib33" ref-type="bibr">Nogueira Santos et al., 2012</xref>, <xref rid="bib38" ref-type="bibr">Santos et al., 2009</xref>). However, none of the modifications affected the cleavage efficiency of Lb<sup>pro</sup> (<xref rid="t0010" ref-type="table">Table 2</xref>). These results show that there are clear differences between the activity of Lb<sup>pro</sup> on peptides and polyprotein substrates, indicating that the conformation of the substrate may be different in the background of the polyprotein. In addition, as previously observed, replacement of the P5-P4′ residues of the polyprotein cleavage sequence with those of the eIF4GI site (<xref rid="f0040" ref-type="fig">Fig. 8</xref>E) also did not affect the efficiency of Lb<sup>pro</sup> cleavage.<fig id="f0040"><label>Fig. 8</label><caption><p>Effects of P1 or P1′ site mutations on the intermolecular cleavage efficiency of Lb<sup>pro</sup>. Intermolecular processing of the precursor Lb<sup>pro</sup> C51A VP4/VP2 (A) and variants thereof (B–E) by Lb<sup>pro</sup>. The cleavage sequence present in the background of the polyprotein is shown in grey boxes. Differences from the wild-type sequence of the precursor are underlined. RRLs were programmed with RNA (14 ng/µl) coding for the polyprotein Lb<sup>pro</sup> C51A VP4/VP2. Translation was performed for 20 min at 30 °C in the presence of [<sup>35</sup>S]-Met and terminated by the addition of unlabelled Met for 10 min. RNA (14 ng/µl) coding for Lb<sup>pro</sup> was added and translation was continued at 30 °C. The reaction was terminated by placing the samples on ice and the addition of Laemmli sample buffer containing excess unlabelled Met and Cys. 10 µl aliquots were analysed by 17.5% SDS-PAGE gels, followed by fluorography. Uncleaved precursor Lb<sup>pro</sup> C51A VP4/VP2 and cleavage products Lb<sup>pro</sup> C51A and VP4/VP2 are indicated. The asterisk (*) indicates an aberrant cleavage product. Negative controls devoid of any RNA (-sub, -prot) or comprising only RNA encoding the precursor (+sub, -prot) are shown on the right of each gel. Protein standards are shown on the left. Each cleavage reaction was performed twice; a representative autoradiogram for each is shown.</p></caption><graphic xlink:href="gr8"/></fig><table-wrap position="float" id="t0010"><label>Table 2</label><caption><p>Summary of the mutational analysis of the intermolecular cleavage efficiency of Lb<sup>pro</sup> and sLb<sup>pro</sup>. Data are taken from <xref rid="f0040" ref-type="fig">Fig. 8</xref>, <xref rid="f0045" ref-type="fig">Fig. 9</xref> and <xref rid="bib16" ref-type="bibr">Glaser et al., 2001</xref> for cleavage of eIF4GI by Lb<sup>pro</sup>. The experiments were performed twice.</p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th colspan="5"><bold>50% cleavage (min)</bold><hr/></th><th/></tr><tr><th/><th colspan="5"><bold>pCITE Lb</bold><sup><bold>pro</bold></sup><bold>C51A VP4/VP2</bold><hr/></th><th><bold>endogenous eIF4GI</bold><hr/></th></tr><tr><th/><th><bold>VQRKLK<sup>⁎</sup>GAGQ</bold></th><th><bold>VQRKL<underline>G</underline><sup>⁎</sup><underline>R</underline>AGQ</bold></th><th><bold>VQRKLK<sup>⁎</sup><underline>R</underline>AGQ</bold></th><th><bold>VQRKL<underline>G</underline><sup>⁎</sup>AAGQ</bold></th><th><bold><underline>SFAN</underline>L<underline>G</underline><sup>⁎</sup><underline>RTTL</underline> (eIF4GI)</bold></th><th><bold><underline>SFAN</underline>L<underline>G</underline><sup>⁎</sup><underline>RTTL</underline></bold></th></tr></thead><tbody><tr><td><bold>Lb</bold><sup><bold>pro</bold></sup></td><td align="center"><bold>15–30</bold></td><td align="center"><bold>0</bold>–<bold>15</bold></td><td align="center"><bold>15</bold>–<bold>30</bold></td><td align="center"><bold>0</bold>–<bold>15</bold></td><td align="center"><bold>15</bold>–<bold>30</bold></td><td align="center"><bold>0</bold>–<bold>15</bold></td></tr><tr><td><bold>sLb</bold><sup><bold>pro</bold></sup></td><td align="center"><bold>30</bold></td><td align="center"><bold>30</bold></td><td align="center"><bold>45</bold>–<bold>90</bold></td><td align="center"><bold>90</bold></td><td align="center"><bold>No cleavage</bold></td><td align="center"><bold>0</bold>–<bold>15</bold></td></tr></tbody></table></table-wrap></p><p id="p0180">We next investigated the ability of sLb<sup>pro</sup> to cleave the same five substrates. In all cases, sLb<sup>pro</sup> cleavage was delayed compared to the Lb<sup>pro</sup> cleavage. 50% cleavage of the wild-type substrate and a substrate bearing P1 Gly and P1′ Arg occurred at 30 min (<xref rid="f0045" ref-type="fig">Fig. 9</xref>A and B) compared to 15–30 min and 0–15 min, respectively, with Lb<sup>pro</sup> (<xref rid="t0005" ref-type="table">Table 1</xref>). 50% cleavage of the substrate bearing two basic amino acids at the scissile bond was observed between 45–90 min whereas the substrate lacking basic amino acids at the cleavage site only showed 50% cleavage after 90 min. Finally, as was shown previously by <xref rid="bib5" ref-type="bibr">Cencic et al. (2007)</xref>, the substrate with the eIF4GI cleavage site was not cleaved at all, even after 120 min of incubation. Thus, the cleavage efficiencies of sLb<sup>pro</sup> on modified polyprotein substrates are similar to those on analogously modified oligopeptides, in contrast to those of Lb<sup>pro</sup>. As a control, we examined the state of the endogenous eIF4GI present in RRLs by performing a Western blot of the cleavage reaction using an anti-eIF4GI antiserum (<xref rid="f0045" ref-type="fig">Fig. 9</xref>F). Endogenous eIF4GI was cleaved within 15 min after the start of incubation, an efficiency comparable to that previously observed with Lb<sup>pro</sup> and sLb<sup>pro</sup> (<xref rid="bib16" ref-type="bibr">Glaser et al., 2001</xref>). This cleavage efficiency on endogenous eIF4GI was also observed in all other reactions in <xref rid="f0045" ref-type="fig">Fig. 9</xref> (data not shown).<fig id="f0045"><label>Fig. 9</label><caption><p>Effects of P1 or P1′ site mutations on the intermolecular cleavage efficiency of sLb<sup>pro</sup>. The intermolecular processing of the precursor Lb<sup>pro</sup> C51A VP4/VP2 (A) and variants thereof (B–E) by sLb<sup>pro</sup>. The cleavage sequence present in the background of the polyprotein is shown in grey boxes. Differences from the wild-type sequence of the precursor are underlined. The translation and analysis was done as shown in <xref rid="f0040" ref-type="fig">Fig. 8</xref>. (F) The intermolecular cleavage of endogenous eIF4GI present in the RRLs from panel E. 10 µl aliquots were analysed on a 6% Dasso & Jackson SDS-PAGE (<xref rid="bib6" ref-type="bibr">Dasso and Jackson, 1989</xref>), followed by immunoblotting with an antiserum detecting the N-terminal part of eIF4GI. Uncleaved eIF4GI and cleavage products (cp<sub>N</sub>) are indicated. Negative controls devoid of any RNA (−sub, −prot) or comprising only RNA encoding the precursor (+sub, −prot) are shown. Protein standards are shown on the left. Each cleavage reaction was performed twice; a representative autoradiogram for each is shown.</p></caption><graphic xlink:href="gr9"/></fig></p><p id="p0185">How can we explain the differences observed above between Lb<sup>pro</sup> and sLb<sup>pro</sup> in their behaviour towards oligopeptide and polyprotein substrates (<xref rid="t0010" ref-type="table">Table 2</xref>)? Why do the introduced mutations only affect sLb<sup>pro</sup> cleavage and why is sLb<sup>pro</sup> not capable of recognising the eIF4GI cleavage site in the background of the polyprotein? The difference in the cleavage efficiencies between Lb<sup>pro</sup> and sLb<sup>pro</sup> on polyprotein protein substrates can be explained by the ability of Lb<sup>pro</sup> to bind to the cleavage site on the substrate with its canonical substrate binding site and through its own CTE to the “substrate binding site” of the substrate as shown in <xref rid="f0050" ref-type="fig">Fig. 10</xref>A and B. In contrast, sLb<sup>pro</sup> can only make one of these interactions, as it lacks an intact CTE (<xref rid="f0050" ref-type="fig">Fig. 10</xref>C and D).<fig id="f0050"><label>Fig. 10</label><caption><p>Model of the intermolecular cleavage of polyprotein substrates by Lb<sup>pro</sup> and sLb<sup>pro</sup>. (A and C) Cleavage of wild-type Lb<sup>pro</sup> C51A VP4/VP2 by Lb<sup>pro</sup> and sLb<sup>pro</sup>, respectively. (B and D) Cleavage of Lb<sup>pro</sup> C51A VP4/VP2 containing the eIF4GI cleavage sequence by Lb<sup>pro</sup> and sLb<sup>pro</sup>, respectively. E. Cleavage of Lb<sup>pro</sup> C51A eIF4GI<sub>599-668</sub> VP4/VP2 SFANLG*RTTL by sLb<sup>pro</sup>. Lb<sup>pro</sup> is in light blue, sLb<sup>pro</sup> in dark blue, VP4 in light green, VP2 in dark green and the eIF4GI<sub>599-668</sub> fragment in red.</p></caption><graphic xlink:href="gr10"/></fig></p><p id="p0190">sLb<sup>pro</sup> can efficiently cleave the eIF4GI site on the native protein present in the RRL because this involves residues Cys133 and Asp184-Leu188 of the CTE but not the last six residues of the CTE (<xref rid="bib14" ref-type="bibr">Foeger et al., 2005</xref>). Accordingly, we introduced 80 amino acids from eIF4GI containing the L<sup>pro</sup> binding and cleavage sites into the Lb<sup>pro</sup> C51A VP4/VP2 substrate (<xref rid="f0050" ref-type="fig">Fig. 10</xref>E). This modified substrate (termed Lb<sup>pro</sup> C51A eIF4GI<sub>599-668</sub> VP4/VP2 SFANLG⁎RTTL) was cleaved by sLb<sup>pro</sup> between 30 and 90 min, indicating that the availability of the two binding sites had allowed cleavage to take place (<xref rid="f0055" ref-type="fig">Fig. 11</xref>A).<fig id="f0055"><label>Fig. 11</label><caption><p>eIF4GI<sub>599-668</sub> is essential for the cleavage of the sub-optimal eIF4GI cleavage site SFANLG*RTTL by sLb<sup>pro</sup>. A-C, left panels. Intermolecular processing of Lb<sup>pro</sup> C51A eIF4GI<sub>599-668</sub> VP4/VP2 by sLb<sup>pro</sup> and sLb<sup>pro</sup> C133S Q185R E186K and Lb<sup>pro</sup> C51A VP4/VP2 by sLb<sup>pro</sup> C133S Q185R E186K. The cleavage sequence present in the background of the polyprotein is shown in grey boxes. Translation reactions and analyses of products were as in <xref rid="f0040" ref-type="fig">Fig. 8</xref>. Uncleaved precursors Lb<sup>pro</sup> C51A eIF4GI<sub>599-668</sub> VP4/VP2 and Lb<sup>pro</sup> C51A VP4/VP2 as well as cleavage products Lb<sup>pro</sup> C51A eIF4GI<sub>599-668</sub>, Lb<sup>pro</sup> C51A and VP4/VP2 are indicated. A–C, right panels. The intermolecular cleavage of endogenous eIF4GI present in the RRL. Analysis of the state of eIF4GI was as shown in <xref rid="f0045" ref-type="fig">Fig. 9</xref>. Uncleaved eIF4GI and cleavage products (cp<sub>N</sub>) are indicated. Negative controls lacking added RNA (−sub, −prot) or comprising only of RNA encoding the precursor (+sub, −prot) are shown. Protein standards are shown on the left.</p></caption><graphic xlink:href="gr11"/></fig></p><p id="p0195">As a control, we examined the ability of the variant sLb<sup>pro</sup> C133S Q185R E186K that had previously been shown to be unable to cleave endogenous eIF4GI because the variant cannot recognise its binding site on this factor (<xref rid="bib14" ref-type="bibr">Foeger et al., 2005</xref>). This variant could also not cleave the Lb<sup>pro</sup> C51A eIF4GI<sub>599-668</sub> VP4/VP2, but maintains the ability to cleave the wild-type substrate Lb<sup>pro</sup> C51A VP4/VP2 (<xref rid="f0055" ref-type="fig">Figs. 11</xref>B and C, left panels). As previously reported, sLb<sup>pro</sup> C133S Q185R E186K was however not able to cleave endogenous eIF4GI, even after 120 min of incubation (<xref rid="f0055" ref-type="fig">Figs. 11</xref>B and C, right panels).</p></sec></sec><sec id="s0070"><title>Concluding remarks</title><p id="p0200">The structural data presented here reveal that sLb<sup>pro</sup> uses three acidic residues to bind to basic residues at the P1 or P1′ positions of a substrate and that this represents a unique arrangement that is not found in cellular papain-like proteinases. Differences in the cleavage efficiency of Lb<sup>pro</sup> and sLb<sup>pro</sup> were observed on modified polyprotein substrates. The presence of sLb<sup>pro</sup> in infected cells (<xref rid="bib36" ref-type="bibr">Sangar et al., 1988</xref>) suggests that differences in the properties of Lb<sup>pro</sup> and sLb<sup>pro</sup> will be relevant to the success of viral replication. Hence, the removal of six C-terminal residues 40 Å from the active site may represent a unique mechanism to modify the properties of a proteolytic enzyme during viral replication.</p></sec> |
Transmission of allergen-specific IgG and IgE from maternal blood into breast milk visualized with microarray technology | Could not extract abstract | <contrib contrib-type="author" id="au1"><name><surname>Hochwallner</surname><given-names>Heidrun</given-names></name><degrees>PhD</degrees><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Alm</surname><given-names>Johan</given-names></name><degrees>MD, PhD</degrees><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Lupinek</surname><given-names>Christian</given-names></name><degrees>MD</degrees><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Johansson</surname><given-names>Catharina</given-names></name><degrees>PhD</degrees><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Mie</surname><given-names>Axel</given-names></name><degrees>PhD</degrees><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Scheynius</surname><given-names>Annika</given-names></name><degrees>MD, PhD</degrees><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author" id="au7"><name><surname>Valenta</surname><given-names>Rudolf</given-names></name><degrees>MD</degrees><email>rudolf.valenta@meduniwien.ac.at</email><xref rid="aff1" ref-type="aff">a</xref></contrib><aff id="aff1"><label>a</label>Division of Immunopathology, Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria</aff><aff id="aff2"><label>b</label>Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden</aff><aff id="aff3"><label>c</label>Sachs' Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden</aff><aff id="aff4"><label>d</label>Department of Medicine Solna, Translational Immunology Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden</aff> | The Journal of Allergy and Clinical Immunology | <p content-type="salutation">To the Editor:</p><p id="p0010">Data from experimental animal models have previously shown that allergen-specific IgG antibodies are transmitted from the mother to the offspring via breast milk<xref rid="bib1" ref-type="bibr">1</xref>, <xref rid="bib2" ref-type="bibr">2</xref> and have provided evidence that the transmitted allergen-specific IgG antibodies protect specifically against allergic sensitization.<xref rid="bib2" ref-type="bibr"><sup>2</sup></xref> In some studies, it has been demonstrated for humans that breast-feeding has a prophylactic effect against atopic disease but there are also reports arguing against this and the underlying mechanisms are not known.<xref rid="bib3" ref-type="bibr"><sup>3</sup></xref> There is evidence that IgG antibodies against bacterial antigens (ie, pneumococcal antigens) are transferred from the blood of mothers into their breast milk.<xref rid="bib4" ref-type="bibr"><sup>4</sup></xref> Furthermore, it was possible to detect IgA and IgG against different food antigens in human serum, saliva, colostrums, and milk samples.<xref rid="bib5" ref-type="bibr"><sup>5</sup></xref> Another study found that total IgE levels in breast milk and blood were associated but allergen-specific IgE was not analyzed.<xref rid="bib6" ref-type="bibr"><sup>6</sup></xref></p><p id="p0015">With the FP7-funded European Union research program Mechanisms of the Development of ALLergy (MeDALL; <ext-link ext-link-type="uri" xlink:href="http://medall-fp7.eu/" id="intref0010">http://medall-fp7.eu/</ext-link>), we have recently developed a microarray containing a large number of purified natural and recombinant respiratory, food, and insect allergens that allows highly sensitive measurement of allergen-specific IgE and IgG levels with minute amounts of blood.<xref rid="bib7" ref-type="bibr"><sup>7</sup></xref> A major advantage of the microarray technology is that it allows one to measure antibody reactivities toward a large panel of different allergens. Here, we investigated whether the MeDALL chip is suitable for (1) the measurement of allergen-specific IgG and IgE levels in human breast milk samples, (2) whether there is a transmission of allergen-specific antibodies from blood into breast milk, and (3) whether the reactivity profile of allergens recognized by antibodies in blood and milk is similar. For this purpose, we analyzed plasma and breast milk samples from sensitized (n = 23) and nonallergic mothers (n = 6) from the ALADDIN birth cohort.<xref rid="bib8" ref-type="bibr"><sup>8</sup></xref> None of the mothers was on allergen-specific immunotherapy. Maternal blood samples were collected in the period around delivery (−1 to +2 months), and the breast milk samples were obtained 2 months after delivery. The study was approved by the local Research Ethical Committee, and written informed consent was obtained from all families.</p><p id="p0020">The breast milk samples were centrifuged for 10 minutes at 2500<italic>g</italic> before use to remove the lipids. For comparison of IgG titers in plasma and breast milk, the plasma samples were diluted 1:50, 1:100, 1:200, and 1:400 before analysis. Microarrays were incubated with 30 μL of the plasma dilutions or undiluted breast milk samples and allergen-specific IgG and IgE antibodies were detected with fluorophore-conjugated anti-IgG and anti-IgE antibodies, respectively.<xref rid="bib7" ref-type="bibr"><sup>7</sup></xref> The fluorescence intensities were measured with a biochip scanner. Results were expressed in ISAC standardized units (Thermofisher, Uppsala, Sweden). Correlation coefficients were calculated with SPSS.</p><p id="p0025">Detailed analysis of allergen-specific IgG and IgE levels in plasma and breast milk samples indicated that allergen-specific IgG antibodies are transmitted from the blood into breast milk in a highly specific manner and that breast milk IgG mirrored the profile of IgG reactivity in the blood (see <xref rid="appsec1" ref-type="sec">Fig E1</xref> in this article's <xref rid="appsec1" ref-type="sec">Online Repository</xref> at <ext-link ext-link-type="uri" xlink:href="http://www.jacionline.org" id="intref0015">www.jacionline.org</ext-link>). A comparison of allergen-specific IgG levels measured in 4 plasma dilutions with that of undiluted breast milk samples (<xref rid="fig1" ref-type="fig">Fig 1</xref>) indicated that allergen-specific IgG levels in breast milk were approximately 200- to 400-fold lower than in plasma. Allergen-specific IgG reactivities in plasma and breast milk were significantly correlated; for the 1:200 dilution, Spearman correlation coefficient was 0.608 (<italic>P</italic> < .001) and for the 1:400 dilution, Spearman correlation coefficient was 0.604 (<italic>P</italic> < .001) (<xref rid="fig2" ref-type="fig">Fig 2</xref>). Detailed results are displayed for each allergen in the heat map (<xref rid="appsec1" ref-type="sec">Fig E1</xref>). For the vast majority of allergens, plasma- and milk-derived IgG antibody reactivities were correlated. However, in certain instances (eg, milk allergens recognized by donor 3), specific IgGs were high in plasma but did not appear in milk; in some other cases, allergen-specific IgG was detected only in milk but not in plasma (<xref rid="appsec1" ref-type="sec">Fig E1</xref>). Possible explanations for lack of allergen-specific IgG binding in milk are that certain antigens are present in milk and inhibit IgG binding and/or low affinity/avidity of IgG may prevent binding despite high titers in blood. In fact, the presence of certain respiratory and food allergens in breast milk has been recently demonstrated.<xref rid="bib9" ref-type="bibr">9</xref>, <xref rid="bib10" ref-type="bibr">10</xref> However, milk-specific IgG may appear because of local IgG production without corresponding IgG in blood.<fig id="fig1"><label>Fig 1</label><caption><p>Comparison of allergen-specific IgG levels (ISAC standardized unit [ISU]) measured in different plasma dilutions of 4 mothers with allergen-specific IgG levels in their breast milk samples.</p></caption><graphic xlink:href="gr1"/></fig><fig id="fig2"><label>Fig 2</label><caption><p>Correlation of allergen-specific IgG levels (ISAC standardized unit [ISU]) in plasma samples (<italic>left</italic>: dilution 1:200; <italic>right</italic>: dilution 1:400) from 4 mothers (<italic>the donors are labeled in different colors</italic>) with allergen-specific IgG levels in their corresponding undiluted breast milk samples.</p></caption><graphic xlink:href="gr2"/></fig></p><p id="p0030">The presence of allergen-specific IgG in breast milk may be due to specific transmission of allergen-specific IgG from the blood of mothers into their breast milk or eventually because of local production. For transmission, active transport by the IgG receptor FcRn, which in fact is expressed in the human mammary gland,<xref rid="bib11" ref-type="bibr"><sup>11</sup></xref> and/or transudation from blood into the mammary glands, as was found for allergen-specific IgG in mucosal fluids, may be considered.<xref rid="bib12" ref-type="bibr"><sup>12</sup></xref> Interestingly, for mothers with high levels of allergen-specific IgE in their plasma (donors 1, 9, 13, 15, 16, and 19), we also found that specific IgE antibodies were present in breast milk. In all but 1 case (donor 3, Phl p 5–specific IgE), the presence of allergen-specific IgE was associated with levels of IgE in the blood. The allergen-specific IgE reactivity profiles in blood and breast milk are summarized in detail in <xref rid="appsec1" ref-type="sec">Figs E1 and E2</xref> in this article's <xref rid="appsec1" ref-type="sec">Online Repository</xref> at <ext-link ext-link-type="uri" xlink:href="http://www.jacionline.org" id="intref0020">www.jacionline.org</ext-link>.</p><p id="p0035">In none of the 6 nonallergic mothers (donors 24-29), who served as negative controls, allergen-specific IgE was detected in plasma or in breast milk, demonstrating the specificity of IgE test results (<xref rid="appsec1" ref-type="sec">Fig E2</xref>).</p><p id="p0040">Our results thus demonstrate that allergen-specific IgG and IgE antibodies with similar specificity are present in blood and in breast milk. Furthermore, our study shows that the MeDALL allergen chip is suitable for the measurement of allergen-specific IgG and IgE antibodies not only in blood but also in breast milk, which opens the possibility to study the transmission of allergen-specific IgG antibodies from mothers to offsprings on the development of allergic sensitization in birth cohorts.</p> |
"Gain-of-function mutations in signal transducer and activator of transcription 1 (<italic>STAT1</it(...TRUNCATED) | Could not extract abstract | "<contrib contrib-type=\"author\" id=\"au1\"><name><surname>Frans</surname><given-names>Glynis</give(...TRUNCATED) | The Journal of Allergy and Clinical Immunology | "<p content-type=\"salutation\">To the Editor:</p><p id=\"p0010\">Heterozygous gain-of-function muta(...TRUNCATED) |
"Regulation of the calcium-sensing receptor expression by 1,25-dihydroxyvitamin D<sub>3</sub>, inter(...TRUNCATED) | Could not extract abstract | "<contrib contrib-type=\"author\" id=\"aut0005\"><name><surname>Fetahu</surname><given-names>Irfete (...TRUNCATED) | The Journal of Steroid Biochemistry and Molecular Biology | "<sec id=\"sec0005\"><label>1</label><title>Introduction</title><p id=\"par0020\">Epidemiological st(...TRUNCATED) |
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