This article discusses the pioneering role of the ILO not in terms of its contribution to labour law, but in terms of its epistemic relevance: it was the first international organization which cut through the classic borderline between national law and international law. In order to do so, the article sketches pre-ILO legal doctrine, and discusses the creation and particular structure of the ILO at some length: why even create an organization to address labour issues, instead of concluding a convention? This is followed by outlining just how relevant the role of the ILO has been. Contrary to popular opinion, the role of law, both in domestic societies and in international affairs, is not first and foremost about constraining action. Law is not about telling people how to behave, and inflicting punishment when they behave differently—not solely, at any rate. To think this, as many do, is to view criminal law as the template for law generally. Instead, much of the law, both in domestic societies and in international affairs, follows a different template, and is about facilitating action.What is more, in addition to (or in the process of) facilitating action, law also helps to structure the way we think about things. We cannot begin to think of the state without invoking the criteria for statehood; we cannot seriously discuss agreement without bearing legal concepts of treaty or contract in mind; and we cannot characterize the military presence of state A in state B without some term from the legal vocabulary, and it matters a great deal which exact term we employ, for calling something an “invasion” or an “attack” evokes different associations than labelling the same act an “intervention”.4Given that rules, norms and decisions invariably have distributive effects, they are typically employed as weapons and arms in struggles for power and hegemony between people  with diverging political agendas. Those weapons may have long fuses, and their effects may only manifest themselves over time, but this makes them only more effective, for the most effective form of power is the power to influence how people think about things.6With this in mind, the creation of the ILO can be seen as a crucial step in the development of public international law, and its singular relevance resides in having sensitized international law to addressing the situation of individuals, whether as employers or as workers. The relevance of the ILO is not just that it helped create and enforce labour rights, although it did and does that too. But part of its relevance also resides in something else, on a deeper level so to speak; this has little to do with labour rights per se, but rather more with opening up international law, with making visible that international law is not just about inter-state relations. The ILO is probably the first international organization—the first manifestation of international law—to take individuals and companies seriously, thus paving the way for the involvement of international law in more recent years with individuals, something we now almost take for granted. Human rights involve the individual, as do international criminal law, EU law, the law on investment protection, et cetera. It is impossible to prove (and silly even to try) that none of this would have happened without the ILO. But what can be demonstrated is that the ILO marked a significant step in the creation of the individual as an entity of relevance to international law.7In what follows, I will substantiate that particular claim, demonstrating first that the international legal vocabulary prior to the ILO’s creation did not facilitate thinking about individual rights under international law, in thrall as it was to the idea that international law only operated between states and would only affect states, in their capacity as states. Thereafter, I will discuss the creation of the ILO, indicating just how creating the ILO marked a seismic shift. This is followed by a discussion as to how and why the international legal vocabulary—the landscape—changed with the establishment of the ILO.Traditionally, international law was always nominally concerned with relations between states. International law, in a collated textbook definition from the late nineteenth century, was the law made by states, to regulate relations between states, and for the benefit of those states. Oppenheim, e.g., in the second edition of his classic treatise published in 1912, defined international law as “the body of customary and convention rules which are considered legally binding by civilized States in their intercourse with each other”.res inter alios acta), with the only relationships envisaged being those between the organization and its member states, but never with the outside world.13The epistemic priority of the state also extended to international organizations. These were always derivative creatures, deriving their existence and powers from the states that founded them.And when international law even deigned to think about individuals, it was only in relation to the state, only as state representatives. This applied formally with respect to protection of diplomats or the conclusion of treaties. It applied more artificially with the protection of property abroad: injuring the individual was seen as injuring the state, and entitling the state  to take action.15All this suggested that international law and domestic law would never need to be in touch with one another: international law stayed on the inter-state level, and the rest was the concern of domestic law.But founded on the logic that international affairs are by definition merely inter-state affairs,This was never a fully accurate or convincing picture, but it worked until the 1920s, and generated an understandable popularity. It entailed that domestic parliaments, which had fought hard and long to acquire a say over domestic legislation, could not be outflanked or overruled by governments entering into international commitments. Over international commitments, after all, typically those same parliaments had no influence. If dualism thus respected concerns about local democracy , a side-effect was the re-affirmation of the role of the state and a re-affirmation of the strict separation between international and domestic law.Triepel himself pointed out that his theory was empirically-based; it was built on the finding that there actually were no contacts between the international and the domestic legal order. These things are always in the eye of the beholder , the Universal Postal Union (UPU), International Bureau of Weights and Measures, the Union of International Transport by Rail, the International Sugar Union, the International Institute for Agriculture: all late-nineteenth century and early twentieth century creations were thought of as creations of states, affecting those very states (the member states) in their very capacity as states. And in the case where they were not thought of in state-centric terms, as creatures of states, then they were not considered part of international law. The Red Cross  in the late 1920s.28But in 1919, when the ILO was created in Versailles, this was still something for the future.Versailles saw the creation of the League of Nations, the clearly still highly state-centric creature to guarantee collective security.The obvious answer—or the beginning of an answer—is that the ILO marks a response to the October revolution of 1917, and reading contemporary papers and books, there is a clear sense of urgency on this point. Part of the idea behind the ILO was to set it up as an answer to the red threat, to communism. As David Morse, long-time Director-General of the ILO, much later put it in admirably bureaucratic and anodyne style, “there was general recognition that the ferment and instability which characterized the world of labor and industry in 1918 and 1919, particularly in Europe, called for immediate and constructive action.”31But still, a set of intellectual problems emerged. The drafters realized all too well that economic circumstances differ from country to country, from state to state. Thus, there is a quasi-natural competitive obstacle that needs to be overcome. What made things more difficult still was the realization that protecting labour comes at the expense of capital, and that the costs and benefits might not be evenly distributed. Some industries would be harder hit than others; for some industries, protecting workers would come at bigger costs than for other industries, not because those others would have been doing so earlier, but because they would be less dependent on night work, or would be less involved with dangerous materials. A third problem that emerged revolved around colonialism: some of the bigger states benefitted from cheap labour being available in their colonies. In fact, as one of the founding fathers, Britain’s George Barnes, openly confessed in relation to the imperial issue: “To be quite candid, our motives were not altogether humanitarian.”32Instead, while the communist threat was perceived as very real, it had to be met in such a way as not to distort global competition. The same George Barnes notes, in his work on the ILO written a few years after its creation, that the “need had arisen for levelling out industrial competition between the nations by raising the conditions of labour in the lower-paid countries”,34This proved quite a riddle. Capitalism requires competition, after all, and one of the more obvious arenas for industrial competition is in the sphere of labour, both by keeping wages low and not spending much on decent working conditions. Yet allowing for the race to the bottom to occur was thought to play in the hands of communism, putting the capitalist world economy at risk. It seemed a veritable catch-22: either allow for unhampered competition and invite communism to take over, or limit competition as far as labour issues are concerned and in that way implicitly accommodate communism as well. Clearly, this left a delicate balancing act: infusing just enough worker protection into the system so as to save the system: too much would make the capitalist economy collapse, and too little would have pretty much the same result. The logic was well-put by a contemporary observer, Leonard Woolf: “If it is in the interest of every State to regulate the conditions of employment within its territory, but it is prevented from doing so unless all the other States do likewise”, so Woolf wrote, “then clearly the solution ought to be found in unification of the Labour laws of the different countries through international agreements.”35One possible way—hypothetically at any rate—to solve the problem was to leave it entirely to the market, and open the borders for unmitigated migration. If the capitalist logic would work, after all, then people would move to the place where there would be work and a decent wage. This, however, was never realistic. As John Hobson observed at the time, Asia may be a “rich reservoir” of labour, but “the difficulty of procuring the general assent of civilized nations to “an open door” for Asiatic labour would, of course, be insuperable”One thing that became reasonably clear was that a single convention on worker’s rights was unlikely to do the trick. What was needed instead was a careful and continued balancing of the interests of workers, capital and states, and this, in turned, required permanent management, not a one-off arrangement in the form of a treaty; for a single treaty could never be comprehensive enough to cover all industries, cover all kinds of situations that might arise, and accommodate all conflicting interests.38tout court, it can be said with considerable hindsight, was due to the combination of organizational form and tripartite structure—even if the precise limits of this organizational form remained subject to debate, and resulted in the Permanent Court of International Justice being asked several questions.42The ILO’s success in performing the balancing act of navigating between unfettered labour competition possibly leading to communism, and adopting communism This dovetailed nicely with a second invention: tripartism. During World War I, the major industrialized states had all seen fit to mobilize labour and capital for the war effort, and in Britain in particular this was welcomed as an experiment well worth repeating. Britain made an effort to transplant the model to the nascent ILO, also because it realized that if it were alone among the major powers to continue to practice tripartism, it might suffer a competitive problem. Cox puts it well: “As the leading trading nation, Britain might have been disadvantaged in world markets if a peacetime prolongation of tripartism were to have the effect of raising labor costs. Hence the concern of British officials to internationalize the experiment.”43One unexpected implication of the establishment of tripartism is that it cemented a place for non-state interests in the work of an international organization. It became clear that the interests of all stakeholders could not be reduced to those of the member states. This had been the traditional idea: what is good for the state, is good for everyone within the state, and things can be kept on the inter-state level. But with the ILO now, it was clearly understood that whatever the ILO would decide, adopt and promulgate, would affect workers and capital—not just the state and its competitive position. Thus, tripartism set in motion an accidental revolution, by incorporating other than direct state interests in the institutional structure of an international organization, and therewith acknowledging that the work of this organization did not just affect member states in their mutual relations, but could potentially affect every worker in every member state, and every employer in every member state.accidental in that the inspiration behind tripartism had been to secure Britain’s competitive position, rather than any grand design about popular consultation or great philosophy of the quod omnes tangit variety.revolution because it opened the door to changing conceptions of international law. The establishment of the ILO slowly created the possibility for thinking of international law as directly affecting the real lives, the real interests, the real blood and real guts, of real people. If until the creation of the ILO international law could still with some sense be said to apply to inter-state relations only (if only because everyone seemed to agree that this was the case), once the ILO was created this was no longer possible: the toothpaste had been squeezed out of the tube; and once the bell tolls, its sound can no longer be unheard.The revolution was It is generally acknowledged that the ILO’s tripartite structure was, at the time, unique—and by and large it still is, at least in the sense in which the formal constitution of an international organization formally involves representatives of social actors other than government representatives, as the ILO does with insisting that states representations include representatives from government, labour and capital.45But if the ILO’s structure is still unique, the past century has developed several variations on the same theme. In some organizations, it is possible for states to be represented by specialists: meteorologists in the case of the World Meteorological Organization; police officers in the case of Interpol  are directly represented. The European Forest Institute has two categories of membership: state membership, and membership of research institutions (it started out as an association of research institutes), requiring an intricate institutional balance when it comes to decision-making. Some organizations participate in joint ventures with private sector actors: these are particularly prevalent in the global health domain, where an important role is played by the Bill and Melinda Gates Foundation.51And then there are organizations where societal interests are represented in all sorts of advisory organs or through consultative status: think of the EU’s Committee of the Regions, or the hundreds of actors having consultative status with the UN General Assembly or the UN Economic and Social Council. Member states might be happy to include domestic actors in their national missions, whether senators or parliamentarians from the opposition or more straightforward interest representatives. And where consequential decisions are taken, lobbying is never far away. This applies not only to private interests, but also to civil society actors: it is a public secret that the Assembly of States Parties to the International Criminal CourtThe ILO was pioneering in its tripartite structure, ensuring the representation of social interests in its standard-setting work. But it was also pioneering in a different sense: it was the first organization explicitly devoted to improving the plight of individuals, regardless of the then prevailing template according to which international organizations would only affect member state interests. At any rate, that was always an impossible conceit: it may be the case that the telegraphic pipelines regulated by the ITU were mostly publicly owned, but the senders and recipients of telegraph messages were, most often, private individuals and private companies.55In a sense then, by focusing on protection of workers, the ILO made explicit what was already implicit with other organizations: that the ultimate addressee and stakeholder would be the individual, whether as worker or as industrialist, with member states mostly involved as conduit. The member states make the rules together and have to implement them in one way or another, but it would be insufficient to say that the regime only affected those member states, and not any one residing within them. With the ILO this was, no doubt, the result of turning vice into virtue: the focus on the individual was occasioned by the distrust of other states. The risk of facilitating “free riding” was simply too big to organize worker protection in any other way than through the combination of continuous law-making while respecting sovereignty, and to do so through a permanent entity rather through a single convention or small group of related conventions.It was only once the ILO had sensitized international law to the possibility of piercing through the mystifying veil of the state, that the international community could come to think of protecting human rights. And even then it took a while still, with direct protection of individual human rights hesitantlyVan Gend & Loos and Costa v ENEL, both decided early in the EU’s existence.This would be further developed by the EU, that wonderful and occasionally somewhat tragic experiment in governance and authority beyond the state.auctor intellectualis, Jean Monnet, worked for a while close by the ILO as the Deputy Decretary-General of the League of Nations, and may have had a look at how the ILO was set up and how it worked in practice.62This is so not only because its main raison d’état. That is an insight that is slowly gaining acceptance, but credit where credit is due: possibly the first venue where this was made visible was the ILO, partly because it incorporated social interests through its tripartite structure, and partly because it may well have been the first venue  which recognized that individuals could be addressed under international law. While there is some ground to suggest that the bilateral treaties of the nineteenth century aided in bringing slavery to an end, these treaties were still the result of paternalist impulses. What made the ILO different was that to the extent that paternalist thought was involved, it was counterbalanced by the self-interests of employers. The ILO took the form, eventually, of states making law to protect individuals, but beneath this surface layer, the ILO’s output is the outcome of serious social struggle between labour and capital—governance beyond the state, rather than governance between states.But the main reason why the EU would have looked differently without the ILO experience is the circumstance highlighted above: the ILO was the first to clear the state-centric cobwebs from international organization, and the first to open up international law to recognition and embrace of interests other than those presumed to be of states. One might argue, of course, that states have few interests of their own, other than the circular concept of the It may well be the case that, as far as the concrete standard-setting and effectiveness thereof is concerned, the ILO may have become somewhat marginalized over the course of its first century.64But even so, the world would look differently, and most likely considerably worse, without the ILO. Its main contribution has not just been in concrete standard-setting, but perhaps even more so, as this paper has argued, in opening up the closed universe of inter-state international law, therewith paving the way for later developments,


Many children receiving chemotherapy struggle with therapy-induced side effects. To date, there has been no literature investigating the needs, knowledge, or implementation of osteopathic manipulative treatments (OMT) as a supportive care option in pediatric oncology. We hypothesized that pediatric oncology clinicians, caregivers, and patients have (a) limited knowledge of OMT and (b) dissatisfaction with current supportive care options and (c) would be interested in having OMT available during chemotherapy, once educated.Participants included three cohorts: (1) children aged ≥ 9 years, diagnosed with cancer and actively receiving chemotherapy; (2) their caregivers; and (3) oncology clinicians at Nationwide Children’s Hospital. Participants completed 1:1 semi-structured interviews, which were audio-recorded, transcribed, and analyzed for thematic content regarding their perception of supportive care measures and views on OMT. Quantitative data was summarized descriptively.A total of 60 participants completed the interview. Participants demonstrated limited awareness of osteopathic medicine; no participant had more than “some” knowledge of OMT. After education about OMT using a brief video, all clinicians, caregivers, and 95% of patients were receptive to OMT as a supportive care option. Major themes included the following: (a) patients have uncontrolled chemotherapy side effects, (b) improved supportive care options are desired, and (c) osteopathic medicine is a favorable supportive care adjunct.Pediatric oncology clinicians, caregivers, and patients reported a need for better management of chemotherapy-associated side effects and an interest in utilizing OMT. These findings support further investigation into the safety, feasibility, and efficacy of implementing OMT in the pediatric oncology clinical setting.The online version of this article (10.1007/s00520-020-05612-9) contains supplementary material, which is available to authorized users. Survival rates for childhood cancer continue to rise, yet the side effects and long-term sequelae of anticancer chemotherapy remain an area of concern for clinicians, caregivers, and patients. Chemotherapy is the primary treatment for many types of pediatric cancers, and advancements continue to be made in the discovery of novel, more targeted, chemotherapy agents. Common therapy side effects can include pain, nausea, fatigue, constipation, and psychosocial struggles , 2. AddiOsteopathic medicine is a distinct form of medical practice, defined by the National Center for Complementary and Integrative Health (NCCIH) as a “mind and body” complementary method that offers the added benefit of a hands-on approach to diagnoses and treatment of many medical conditions . Doctor Osteopathic manipulative treatment (OMT) is one of the specialized therapeutic modalities used by DO physicians and incorporates techniques such as gentle stretching and manipulation to target specific muscle and nerve groups . StudiesFollowing institutional review board approval, we recruited eligible clinicians and families from December 2018 to February 2019 at Nationwide Children’s Hospital. Inclusion criteria included (1) patients currently receiving chemotherapy, ≥ 9 years of age; (2) caregivers of an oncology patient receiving chemotherapy, 0–21 years of age; and (3) allopathic or osteopathic physicians or advanced nurse practitioners in the Division of Oncology at Nationwide Children’s Hospital. We excluded respondents unable to comprehend written English and participants unable to complete the study due to medical or learning disabilities. Total participants approached included 24 oncology clinicians, 21 pediatric oncology patients, and 20 caregivers of pediatric oncology patients. Of those approached, 20 clinicians, 20 caregivers, and 20 patients (including 6 patient/caregiver dyads) completed a semi-structured, qualitative interview in clinic.A single trained investigator (J.B.) provided participants a brief education on OMT which included a script detailing osteopathic medicine followed by a 1-min video demonstrating OMT on a child . Participants then completed 1:1 semi-structured qualitative interviews with open-ended questions and self-reported quantitative questions in REDCap. Participants were asked about the following: their experience with chemotherapy-induced side effects, how they have managed these side effects, how they felt OMT could potentially be utilized in the oncology setting, and any hesitancies about incorporating OMT into oncology care. Furthermore, clinicians and caregivers were asked how and when they felt OMT could be best introduced to their patient or child, respectively. Following the qualitative interview portion, participants were asked whether they would want to have OMT available as a supportive care option.All quantitative responses were stored securely in a REDCap database. Interviews were audio-recorded and stored using an encrypted Apple iPad. All interviews were conducted by the same trained investigator (J.B.) to ensure consistency. Participants were compensated $10 for their time. Basic participant demographic information, such as age and gender, was collected and entered into a de-identified database.Digital recordings were transcribed verbatim by an external service. Using an iterative process, two investigators on the research team  independently analyzed and coded interview transcriptions using constant comparison method . AnalysiA total of 60 participants completed the interviews. Interviews lasted between 1.3 and 12.0 min (mean = 8.4 min). Twenty oncology clinicians participated , including 15 attending physicians and 5 nurse practitioners with a median of 7 years of clinical practice . Twenty caregivers of pediatric oncology patients  with a median age of 40 years (range 27–71) and 20 pediatric oncology patients  with a median age of 17 years (range 10–28) completed the study. Leukemia (33%) and sarcoma (29%) were the most common cancer diagnoses, with a median of 15 years of age at diagnosis (range 9–21). Demographic characteristics are summarized in Table n = 20) of clinicians had heard of osteopathic medicine, only 50% (n = 20) of caregivers and 0% (n = 20) of patients had heard of osteopathic medicine. Responses on their knowledge varied from 70% of clinicians knowing “some” to 30% knowing “very little” or “none at all.” In contrast, 85% of caregivers and 100% of patients knew “very little” or “none at all” about osteopathic medicine. Neither clinician, caregiver, nor patient knew “a lot.” On quantitative assessment, after receiving the video and script education, 100% of clinicians, 100% of caregivers, and 95% of patients responded “yes” when asked if they were interested in having osteopathic medicine available as an adjunct supportive care treatment option  pediatric oncology patients have uncontrolled chemotherapy side effects, (b) desire for better supportive care options, and (c) osteopathic medicine is a favorable supportive care option Table .Table3MaParticipants were interviewed regarding their experiences with chemotherapy and disease side effects. All three groups reported specific instances where chemotherapy side effects were difficult to manage. Most commonly, participants experienced poor symptom management particularly for nausea, vomiting, constipation, neuropathy, and pain. Vincristine was a major identified source of uncontrolled side effects, including constipation and neuropathy. Some clinicians further expressed that vincristine side effects often lead to unwanted dose reductions. Clinicians, caregivers, and patients expressed frustration with currently available treatments including insufficiency of multiple pharmacologic interventions and a lack of alternative available options Table . One cliAll three cohorts expressed a need for better supportive care options to help control side effects. Participants expressed concern regarding a lack of available alternative supportive care therapies. Clinicians were often approached by patients and caregivers who specifically desired non-pharmacologic interventions to help with side effects and often felt clinician responses were inadequate. One clinician stated, “I’ve definitely had parents ask about more, or instead of medicine, is there something else we can try. I haven’t been able to give them a great answer.” Patients viewed taking pills as a burden and expressed a desire for non-pharmacologic interventions, with multiple patients having similar views to a 12-year-old patient who stated “It’s just I wish I wasn’t on the pills. I wish there was a different way, because I hate taking pills.”Following brief description and education about OMT, participants discussed various ways OMT may be utilized for chemotherapy side effects. Clinicians and caregivers expressed a desire to consider OMT when patients experience side effects that are not well-managed with pharmacologic-based therapies. They perceived OMT as an intervention that could potentially benefit nausea, vomiting, constipation, neuropathy, or pain. Clinicians, caregivers, and patients viewed OMT as an attractive therapy with the potential to be utilized at home and decrease as-needed medications. Some caregivers described the benefits of hands-on techniques, specifically the benefit of physical touch for healing.While no major barriers were identified, participants elicited several smaller barriers before integrating OMT into their current supportive care plans. After education about OMT, clinicians uniformly stated that they had minimal to no hesitation of recommending or referring their patients for OMT, yet they sought further education, as to when best to refer and how to introduce OMT to families. Similarly, caregivers also stated they would like printed or video educational materials describing OMT techniques. One clinician and one caregiver expressed concern about the availability of an osteopathic physician to perform treatments.Participants were asked about their perceptions of the osteopathic techniques, specifically if they had any fears or hesitations. After watching the scripted video, caregivers and patients did not report any fears or hesitations. One patient stated, “Nothing scares me about OMT, I would love it.” Two patients expressed not wanting to be touched when they were ill, but one stated that if OMT were helpful, they would be willing to tolerate the physical touch. Other clinician’s comments centered on the need for additional research on osteopathic medicine in pediatric oncology patients to demonstrate efficacy and the feasibility of integration into clinic.Clinicians and caregivers agreed that introduction to osteopathic medicine should occur early in the chemotherapy course. Several clinicians and caregivers stated that osteopathic medicine should be one of the first supportive care options discussed with families when chemotherapy side effects are explained during the initial consent process. Other clinicians and caregivers had some concerns about families being overwhelmed at the time of diagnosis and suggested opting for an introduction shortly after diagnosis. Regardless, most believed that having OMT available and offered within the first few weeks of therapy, before side effects arise, would be optimal. Many caregivers expressed at the end of interviews a desire to start OMT immediately, with one mother of a child with leukemia stating, “I’d try it right now if it were an option.”Using a mixed methods approach, we assessed current knowledge and perceptions of pediatric oncology clinicians, caregivers, and patients, regarding pediatric oncology supportive care and osteopathic medicine. In addition to confirming the universal desire for improvement in supportive care options, this study verified the relative lack of knowledge of OMT or its potential roles in supportive care. Most oncology clinicians had “some” to “very little” knowledge of osteopathic medicine, while caregivers had “very little” to “none at all,” and zero patients had knowledge of osteopathic medicine.Current supportive care measures alone are frequently insufficient in treating pediatric chemotherapy side effects –22. PartFamiliarity with osteopathic medicine was low in all participants, indicating a significant knowledge gap. Our findings demonstrated that pediatric clinicians had less knowledge of osteopathic medicine compared to publications from adult-based literature, where most physicians felt “extremely knowledgeable.” This could be due to few osteopathic physicians at the institution practicing osteopathic medicine. Additionally, this discrepancy may be the result of a general lack of OMT practiced by osteopathic physicians at large pediatric institutions compared to rural adult hospitals .Current literature does not include a role for OMT as a supportive care option, as it has not been studied in the childhood cancer population. Our study begins to address this gap by demonstrating minimal to no hesitation for OMT integration in all three participant groups. After education, participants were receptive to OMT, perceived it to be a low-risk, non-pharmacologic intervention and expressed minimal hesitations to implementation. All oncology clinicians and caregivers were interested in having osteopathic medicine available to patients, and all but one patient were interested. This acceptance is necessary in order to move from pilot to efficacy studies, as this study acknowledges a yet unmet opportunity for further osteopathic research to establish its safety, feasibility, and efficacy within pediatric oncology.While there were no major barriers or hesitations identified, participants recognized several minor concerns about integrating OMT into their current supportive care plans, although most of these concerns would be alleviated through OMT education and a logistical plan for billing and workflow. Depending on the institution and resources available, any DO that practices OMT is able to treat pediatric cancer patients. Recognizing that the feasibility of osteopathic medicine in pediatric patients has not been well studied, several institutions have dedicated osteopathic clinics or osteopathic clinicians that are skilled in performing simple osteopathic techniques, feasibly during a patient clinic visit. Billing for OMT is commonly coverage by insurance providers and for oncology patients can be bundled with their outpatient clinic visit or hospitalization . The maiDespite promising findings, the authors acknowledge limitations of the study. This is a small sample, single institutional study which may not be representative of the wider pediatric oncology population. Nonetheless, we obtained perspectives from clinicians with a wide range of clinical experience and areas of oncology expertise, as well as patients and families that represented a variety of cancer diagnoses, ages, and duration of cancer treatment. Another limitation was that the lead investigator was an osteopathic physician which could lead to implicit bias from participants. To best negate this, all quantitative responses were blinded from the interviewer, and an iterative script was used for qualitative questioning. As with most qualitative research, there is the potential for observation bias, which was mitigated by having two researchers that independently code the data. Although there was not participant checking, rigor in coding was ensured by two independent coders with high interrater reliability. Utilizing a mixed methods approach to interview three different populations, including the clinicians who dictate and offer supportive care measures, as well as the patients and families who receive that care, is a strength of our study.This study investigates current knowledge and perspectives of pediatric oncology supportive care management and investigates the views of implementing OMT as an adjunctive treatment option. We identified a need from clinicians, patients, and their caregivers for better supportive care options and, after education, the desire to have osteopathic medicine available as an adjunct treatment. These findings support the need for future, scientifically rigorous clinical trials investigating the feasibility, safety, and efficacy of OMT as a non-pharmacologic adjunctive supportive care therapy option for childhood cancer patients.ESM 1(PDF 81 kb)ESM 2(MP4 3821 kb)ESM 3(MP4 6305 kb)ESM 4(MP4 4283 kb)


This work examined the effect of ultrasound and microwave treatments, separate and in combination, on the physicochemical and functional properties of chestnut starch. The results revealed that the ultrasonic-microwave (UM) and microwave-ultrasonic (MU) dually modified samples exhibited more severe surface damage, weaker birefringence, and lower relative crystallinity and gelatinization enthalpy than the native and single-treated starches. The UM samples showed the highest oil absorption capacity, and the MU samples showed the highest water absorption capacity and the best freeze-thaw stability (five cycles) among all samples. The swelling power, peak, trough, final, and breakdown viscosities, and pasting temperature all decreased regardless of single or dual modification. This study provides a reference for potential industrial applications of ultrasound and microwave treatments for the modification of chestnut starch. The annn starch ,4,5,6. Ln starch . Starch The development of improved strategies for the physical modification of starch has attracted considerable interests from researchers and consumers particularly due to the safe, non-polluting, simple, and eco-friendly of mechanical processes . UltrasoSeveral studies have examined the individual effects of ultrasound or microwave treatments on starch, such as potato starch, waxy corn starch, and lotus seed starch, but very limited studies have focused on chestnut starch. In addition, the dual physical modification, also referred to as the synergistic effect of physical modification, is very promising because it can improve the properties of starch without involving the use of chemical reagents. The literature included dual modification by annealing and ultra-sonication treatments on potato starch , and heaCastanea mollissima) fruits “Dabanhong” were obtained from Kuancheng, Hebei Province of China. Chestnut starch was isolated by alkaline solution according to the method of our previous report [Chinese chestnut . The suspension was treated using an ultrasonic breaker  Co., Ltd., Shanghai, China) with a power of 500 W and a frequency of 20 kHz for 60 min  [Native chestnut starch (2 g) was suspended in distilled water  . The sonFor microwave modification, the chestnut starch (2 g) was dispersed in 20 mL distilled water, and the suspension was treated in a microwave oven  at 2450 MHz and 30 W for 90 s, as described in our previous report . The treThe obtained UC as mentioned in Similarly, the MC as described in The morphology of the starch samples was observed by scanning electron microscopy (SEM) . A small amount of each sample was evenly coated on dual-sided tape and subjected to gold plating. The accelerating voltage was 10.0 kV .2O, v/v) to make a 1% starch suspension. A drop of the starch suspension was observed by polarizing light microscope (PLM) , and images were taken at 25× magnification [The starch sample was mixed with a 1:1 glycerin solution  at 40 kV and 40 mA. The scanning area was 5° to 45°, with a scanning rate of 2° /min, and a step size of 0.02° as a function of 2θ. The relative crystallinity of the chestnut starch was calculated by equation :Crystallw/w) and pressed in a mold to obtain tablets, and KBr was calibrated as a blank. The infrared spectra were obtained (32 scans) with a scan range of 4000 to 400 cm−1 at a resolution of 4 cm−1 [The FTIR spectra of starches were recorded using a FTIR spectrometer . The chestnut starch sample was thoroughly ground with dry KBr powder , peak (pT), and conclusion (cT) temperatures, and gelatinization enthalpy (ΔH) were obtained.The thermal properties of starches were studied using the method of Yang et al.  on a Q20w/w) starch slurry with a total weight of 28 g was placed in a TecMaster Rapid Viscosity Analyzer (RVA)  to obtain a viscosity curve. To do this, the starch slurry was held at 50 °C for 1 min, heated from 50 to 95 °C at 12 °C/min, and held at 95 °C for 2.5 min, then cooled to 50 °C at the same rate and finally maintained at 50 °C for 2 min. Stirring was performed at 960 rpm for the first 10 s and then maintained at 160 rpm during operation. The measured parameters were peak, trough, final, breakdown and setback viscosities, and pasting temperature.The pasting properties of the samples were analyzed following the method of Joshi et al.  with somW) starch sample was mixed with 10 mL of distilled water and heated in a water bath at 55, 65, 75, 85, or 95 °C for 30 min, cooled to room temperature, and then centrifuged at 12,000× g for 15 min. The supernatant was dried in an oven at 105 °C until reaching constant weight (A), and the weight of the swollen starch in the centrifuge tube was expressed as P. SP was calculated according to the following equations [A 0.2 g  and M2 is the mass of the starch precipitate after centrifugation (g).The freeze-thaw stability of starch can be measured by syneresis. Lower syneresis indicates better freeze-thaw stability. The 5% starch suspension was heated in a 95 °C water bath for 30 min with constant stirring. The paste was transferred to a pre-weighed centrifuge tube to determine the weight of the paste. The samples were subjected to alternating freezing and thawing cycles  for 5 days, followed by centrifugation at 8000× equation .SyneresW0) of starch powder was dispersed in 5 mL deionized water (for WAC) or canola oil (for OAC) in a centrifuge tube and weighed (W1). The mixture was vortexed for 30 min and then centrifuged at 4500× g for 10 min at room temperature. The supernatant was decanted, and the precipitate was weighed (W2). The WAC or OAC of starch was then calculated by the following equation.The WAC and OAC were analyzed as described in Nawaz et al.  with somp < 0.05). All analyses were carried out in triplicate. Analysis of the variance (ANOVA) of the experimental data was performed by SPSS 23.0 software  with a significance level of 95% (The microstructure of the native and modified chestnut starch samples was observed by SEM. The native chestnut starch (NC) exhibited a variety of shapes, ranging from round and oval, to the horn and irregular shapes , and theThe crystal structure of the starch granules was observed by PLM, and the polarized micrographs of chestnut starches are shown in The crystallinity of starch reflects its long-range molecular order. Analysis of NC revealed a typical C-type crystal with strong characteristic peaks at 5.6°, 15°, 17°, and 23° (2θ) A, which −1 indicates the short-range order of the inner granules of the starch molecule [−1 (O-H stretching region), 3000–2800 cm−1 (C–H stretching regions), 1500–800 cm−1 (the fingerprint region), and below 800 cm−1 [−1 of the MC increased by 0.1249, but the ratios of the other modified samples decreased  and pT (62.2 °C), while UMC had the highest oT (61.8 °C) and pT (64.3 °C). The cT of all modified samples was reduced compared to NC (68.0 °C). oT and cT are related to the melting temperatures of the weakest crystallites and high-perfection crystallites in the starch granules, respectively [oT might reflect the partial melting of the weakest crystallite of the particles becoming more stable and structural changes within the particles affected by microwave heating [cT was attributed to the formation of unstable crystallites by the modification treatment [The thermal properties of chestnut starches modified by ultrasonic and microwave treatments are shown in ectively . The inc heating . Previou heating . The decreatment . The difreatment .r = 0.824, p < 0.01). The decrease in ΔH demonstrated the destruction of the double helices structure under ultrasonic or microwave treatment [The ΔH of NC was 2.91 J/g and consistent with the results of Guo et al. (0.65–5.52 J/g) [reatment , which wreatment , and micreatment .r = 0.818, p < 0.01) and ΔH . Ultrasound and microwave treatment could disrupt the crystallinity structure of starch granules, rupture the macromolecular chains, and reduce the rigidity and integrity of the granules, thus reducing the peak viscosity [The pasting properties of native, ultrasonic-, and microwave-modified chestnut starches determined by RVA are shown in iscosity . The breiscosity . The breiscosity . A similiscosity , quinoa iscosity . The setiscosity . Analysiiscosity . The setr = 0.716, p < 0.01) and peak viscosity . The structures of dual-modified starches (UMC and MUC) at a high temperature were more seriously damaged than those subjected to a single treatment (UC and MC), which restricted the starch swelling due to the fewer starch crystallites, resulting in a lower SP.NC showed the highest SP (31.83 g/g) at 95 °C . Comparep < 0.05) after the single (UC and MC) and dual treatments (UMC and MUC). The freeze-thawed starch gel formed a spongy structure, and the treatment might improve the stability of the gel by delaying the formation of this spongy network [The freeze-thaw stability reflects the degree of syneresis after the thawing of frozen foods, where the lower the syneresis rate, the better the freeze-thaw stability. The syneresis of chestnut starches increased with the number of freeze-thaw cycles . After 5 network . Additio network . Consequp < 0.05) after ultrasonic and microwave treatment. The highest WAC was observed in MUC (2.37 g/g), followed by UMC (2.14 g/g). Ultrasonic and microwave treatments caused degradation of starch to dextrin, maltose, and glucose. These monosaccharides had a higher affinity for water than starch, thus increasing the water absorption capacity of starch [r = –0.939, p < 0.01) and ΔH value . Alimi and Workneh [The WAC of chestnut starches ranged from 1.05 to 2.37 g/g . The WACf starch . In addi Workneh  also fouThe OAC of the modified chestnut starches was higher than that of NC (1.23 g/g), with UMC exhibiting the highest value 1.69 g/g). An increase in OAC might be due to the destruction of the starch structure by ultrasonic and microwave treatments, resulting in more hydrophobic portions of the amylose–lipid complex being exposed . Similar g/g. An After single ultrasonic or microwave treatment and dual-modification on chestnut starch, UMC and MUC showed lower SP, relative crystallinity, and ΔH than UC and MC, due to the synergistic effect of ultrasonic and microwave treatment, and the layered structure and double helix structure of starch was more severely disrupted. This was confirmed by the observation of more wrinkles and grooves on the particle surfaces and fewer Maltese crosses of dually modified starch. The pasting properties of all modified starches decreased, whereas the freeze-thaw stability was better than that of the native chestnut starch, suggesting its potential application in frozen foods. In addition, the highest WAC was observed in MUC, and the UMC showed the largest OAC. The results of this study can provide theoretical support for the dual physical modification of starch, and provide a new reference for the industrial production of starch-based products that require specific properties, e.g., starches with high water absorption can be used as thickeners.


The oxygen barrier capacity is close to zero. Particularly in the ordered magnetic montmorillonite starch composite film, the oxygen barrier ability is the best. Therefore, modified montmorillonite could serve as an excellent reinforcing agent for cassava starch films and effectively improve the oxygen barrier performance of the films.In this study, we investigated the barrier properties of a montmorillonite-reinforced biomass material, starch. Organically modified montmorillonite materials were prepared from natural montmorillonite by reacting it with dodecyl trimethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride or octadecyl trimethyl ammonium chloride under ultrasonic conditions. The composite starch films incorporated with these organically modified montmorillonite samples were characterized by scanning electron microscopy, transmission electron microscopy, infrared spectroscopy and X-ray diffraction. The results showed that the introduction of montmorillonite decreases the transmittance of the composite film by 10% in the visible region and significantly inhibits UV-light transmittance. The decomposition temperature of the composite film ranges from 200 to 500 °C, with a weight loss rate of 80%. The distance between the montmorillonite layers increases from 0.14 nm in the non-magnetized state to 1.49 nm after magnetization. The oxygen permeability of the starch film modified by organic montmorillonite (0.067 cm Starch is a naturally occurring polymer. A starch solution is typically prepared by heating starch with water. Because of its good film-forming properties, a stable starch film can be formed by casting it onto a smooth plane from a solution . The filMontmorillonite is a silicate substance with a typical lamellar structure; it has a 2:1 hierarchical structure and bond-type structures such as Al–O and Si–O chain structures in the interior. The introduction of organic modifiers can increase the distance between the montmorillonite lamella and increase the transmission path for molecules, thereby improving the barrier properties ,5. BecauIn recent years, starch-based films have been used in packaging. However, they have not found wider use in other commercial and consumer goods, owing to their poor mechanical and barrier properties . AlthougHerein, we studied the barrier properties of a montmorillonite-reinforced biomass material, starch. We prepared different organically modified montmorillonite samples (hereafter referred to as organic montmorillonite) by reacting montmorillonite with different organic modifiers (amines) at different dosages for different reaction times and used each of them as a reinforcing agent to improve the barrier properties of starch films. Furthermore, by magnetizing the organic montmorillonite using an iron chloride solution, we increased the distance between the montmorillonite layers and thus extended the passage of small gas molecules in the composite material to enhance the barrier mechanism of the composite film. The composite film can be used in the fields of packaging, agricultural moisture retention, everyday preservation and medicine and is hence of great significance.The materials used include montmorillonite , dodecyl trimethylammonium chloride , dodecyl dimethyl benzyl chloride ammonium chloride , octadecyl trimethyl ammonium chloride , anhydrous ethanol , silver nitrate , phosphotungstic acid , potassium bromide , natural tapioca starch , glycerin , ferrous chloride , ferric chloride  and sodium hydroxide .− (until no precipitate was detected with the use of a 0.2 M AgNO3 solution) and was finally dried in a blast-drying oven at 50 °C for 24 h. The dried montmorillonite was ground and passed through a 120 mesh sieve to obtain organic montmorillonite.Montmorillonite was dried in a 50 °C blast-drying oven for 24 h, then cooled in a drying dish to obtain dry montmorillonite. The dry montmorillonite (30 g) was weighed and suspended in distilled water (600 mL), then ultrasonically dispersed at room temperature (25–27 °C) for 1 h and finally magnetically stirred for 12 h (500 r/min) to obtain a montmorillonite suspension. Next, DTAC (6 g) was added to the montmorillonite suspension, stirred at room temperature for 30 min and ultrasonically dispersed for 1 h. The mixture was stirred in a water bath at 80 °C for 3 h, allowed to cool to room temperature and dispersed again for 1 h. The suspension was then centrifuged , following which the precipitate was washed with 50% aqueous ethanol to remove ClDTAC (3 g each) was added to three montmorillonite suspensions, which were then reacted under different ultrasonic conditions for 0, 1 and 2 h. Thereafter, the suspensions underwent water-bath cooling, ultrasonication, centrifugation, washing, drying, sieving and other operations to obtain samples labeled 0-1 DTAC, 1-1 DTAC and 2-1 DTAC, respectively. Further, 3, 6 and 9 g of DTAC; 6 g of DDBAC; and 6 g of STAC were separately added to five other montmorillonite suspensions, which were reacted under ultrasonic conditions for 1 h to obtain samples labeled 1-1 DTAC, 1-2 DTAC, 1-3 DTAC, 1-2 DDBAC and 1-2 STAC, respectively.3 (0.01 mol) and FeCl2 (0.005 mol) were dissolved in 100 mL of water. The iron solution was quickly poured into the Ar-filled flask and stirred, followed by the addition of a 0.2 M NaOH solution to adjust the pH of the mixed suspension to 11. This suspension was stirred for 1 h at 80 °C in a water bath, after which the stirring was stopped and the suspension was ultrasonicated for 30 min. The resulting product was magnetically separated out as a black precipitate, which was washed multiple times with distilled water and dried at 50 °C for 24 h in a vacuum oven to obtain magnetic organic montmorillonite.Organic montmorillonite  was added to a three-necked flask with distilled water (200 mL) and the mixture was stirred for 12 h to form a suspension. This suspension was then dispersed ultrasonically for 30 min. Thereafter, the device was vented with argon to exhaust the air in the flask. Subsequently, FeClThe organic montmorillonite (0.5 g) was dispersed in water (50 mL), sonicated for 1 h and magnetically stirred for 12 h to form a suspension. Subsequently, tapioca starch (5 g), glycerol (1.5 g) and water (100 mL) were added to the organic montmorillonite suspension and the resulting mixture was stirred under heating in a water bath at 80 °C for 30 min. The heating was then stopped while stirring was continued at room temperature [A three-necked flask was purged with Ar gas to create an inert atmosphere in the flask. The modified montmorillonite was weighed out at 0.25–1 g  and added into the three-necked flask containing 50 mL of distilled water, then sonicated for 1 h and mechanically stirred for 12 h to form a magnetic organic montmorillonite suspension. Subsequently, tapioca starch (5 g), glycerin (1.5 g) and water (100 mL) were added to the montmorillonite suspension and the resulting mixture was mixed well. This mixture was then heated to 80 °C and mechanically stirred for 30 min, followed by stirring while cooling to room temperature. Additional ultrasonication for 30 min yielded a magnetic starch/montmorillonite film-forming suspension. Further, 48 g of the film-forming suspension was taken in a 12 cm × 12 cm square plastic Petri dish. To obtain a disordered material, the film-forming suspension was dried in a drying box at 50 °C for 10 h, while an ordered material was obtained by drying the film-forming suspension naturally in air under parallel magnetic induction lines produced by a strong magnet for 24 h, to obtain magnetic organic montmorillonite/starch films ,14.The apparent morphology and size of the organic montmorillonite and magnetic organic montmorillonite were characterized by transmission electron microscopy  at an acceleration voltage of 80 kV. Prior to TEM, a droplet of the suspension of organic montmorillonite was loaded onto a copper mesh and dried.The microscopic morphology of the organic montmorillonite and organic montmorillonite/starch composite films were characterized by scanning electron microscopy  at the test voltage of 10 kV. The sample used for observation was placed on the conductive adhesive of the electron microscope loading table and then treated with a gold spray.The moisture permeability of the magnetic organic montmorillonite/starch composite films and organic montmorillonite/starch composite films with different contents  of organic montmorillonite was determined according to the GB/T1037-1988 standard using a water vapor transmittance tester .The prepared film-forming liquid (60 g) was placed in a circular plastic Petri dish of diameter 15 cm. The ordered and disordered magnetic starch films were dried by the same method to test their oxygen permeation performance.The tensile strength and elongation at break of the magnetic montmorillonite/starch composite films and montmorillonite/starch composite films with different amounts of montmorillonite were measured and analyzed using an electronic universal material testing machine . The ASTM-D882-02(2002) test method was used, with samples 100 mm × 10 mm in size, an initial fixture distance of 50 mm and stretching speed of 1 mm/s.–1 and the resolution was 4 cm−1.The magnetized and non-magnetized organic montmorillonite powders were ground to 200 mesh (a grain for filtering) and characterized by Fourier-transform infrared  spectroscopy. The scanning wavelength range was 400–4000 cmd is the montmorillonite spacing in nanometers, θ is the diffraction angle of the characteristic crystal plane in degrees and λ is the X-ray wavelength in nanometers.The magnetized and non-magnetized organic montmorillonite powders dried at 50 °C for 2 h were characterized by X-ray diffraction (XRD) . The scanning range was 2θ = 3–10° and the scanning speed was 5°/min. The spacing in montmorillonite was calculated according to the following formula :(1)2dsin2 gas flow of 40 mL/min at the heating rate of 10 °C/min in the temperature range of 30–60 °C.The thermal weight loss of the montmorillonite samples prepared under different ultrasonication durations using different modifiers at different dosages was analyzed by synchronous thermogravimetric analysis , to analyze the thermal stability of each sample. TGA was conducted under a NThe hydrophobicity of the montmorillonite/starch composite films was studied with the Kruss contact angle measurement . The montmorillonite/starch film was cut into a slender specimen (25 mm × 20 mm) and the specimen was tightly attached to a glass slide. The slide containing the sample was placed on the carrier table of the measuring instrument and the water droplet size was set to 3 μL.3∙24 h) to 518.1933 g/(m3∙24 h). The water absorption capability of the hydroxyl groups in the magnetic montmorillonite allows the montmorillonite to act as a barrier to water vapor in the composite film, because of the change in its Fe–O bond. However, the composite films with both, non-magnetized and magnetized organic montmorillonite, have a relatively small range of moisture permeability, because starch itself is more sensitive to water vapor [er vapor ,21.3/m2·d, because it is known that montmorillonite has a lamellar structure. With an increase in the distance between the montmorillonite layers, the path for organic materials  is elongated and therefore, the modified montmorillonite with increased interlayer spacing enhances the barrier properties of these films [3/m2·d) than that of the montmorillonite starch film without magnetization modification (0.097 cm3/m2·d). The oxygen barrier capacity is close to zero. The increased interlayer spacing of montmorillonite after organic modification can widen the path for molecular transmission due to the support of carbon chains. However, because the starch film particles are modified with organic montmorillonite, the formation pathway and increase in the interlayer distance are small. As a result, although the barrier performance of the composite is markedly improved, it does not reach the ideal barrier capability of approximately zero. The oxygen permeability of the ordered magnetic montmorillonite starch composite film increases with the addition amount, from 0.23 to 0.113 cm3/m2·d and the effect is stable. Owing to the high quality of the magnetic montmorillonite and its magnetic effect, a montmorillonite/starch film with layers oriented in a single direction (layer orientation) is formed with the longest possible path. Thus, the best barrier properties of montmorillonite/starch films are obtained with ordered composite films in this study. Compared with the disordered starch film, the ordered starch film can achieve a constant gas-barrier effect faster and more stably and the introduction of Fe can effectively improve the barrier properties of the montmorillonite/starch films.se films . The oxy−1 that arises from the hydroxyl vibrations in the montmorillonite and Si–O tetrahedra [−1. The vibration peak of –CH2– appears at 1033 cm−1, that of Cl− appears at 1641 cm−1, and the characteristic peak of Al–O appears at 462 cm−1 [−1 has increased markedly due to the superposition of the characteristic Fe–O peak of the incorporated magnetic material, Fe3O4 and the characteristic peak of Al–O, indicating that the magnetic material is successfully introduced. Compared with the characteristic peak of the hydroxyl groups of hydrated montmorillonite at 3438 cm−1, the intensity of the –CH2– characteristic peak at 2920 cm−1 does not change markedly, indicating that no new functional groups are introduced. These characteristic peaks indicate that organic montmorillonite does not undergo any other reaction with iron(III) oxide and that magnetic montmorillonite powder is successfully obtained.trahedra . The FTI462 cm−1 ,26 The oθ = 5.28°; the interlayer distances are different for different samples. The highest peak value, which would represent the greatest distance between the layers of montmorillonite, is calculated from the change point using Prague’s law [θ = 5.15° and an interlayer distance of 0.14 nm. After magnetization, this peak shifts to 2θ = 6.18° and the interlayer distance increases nearly ten-fold to 1.49 nm [ue’s law . Figure  1.49 nm . Magneti 1.49 nm .3/m2·d) than that of the non-magnetized montmorillonite starch film (0.097 cm3/m2·d) and the oxygen resistance capacity was close to zero. Magnetized modified montmorillonite and organic montmorillonite imparted evident oxygen-blocking ability when incorporated in starch films. In particular, the oxygen permeability of the ordered magnetic montmorillonite starch composite film increased from 0.23 to 0.113 cm3/m2·d with the increase in the additive amount in the ordered magnetic montmorillonite/starch composite films. This effect was stable and the oxygen-blocking ability reached the optimal value. However, the intercalation reaction of montmorillonite under other conditions was not clarified; the upper limit of the interlayer distance of montmorillonite was not clearly defined. We plan to explore whether the barrier properties of montmorillonite could be improved further with a further increase in the interlayer distance, in the future.This study explored the modification of montmorillonite with different ultrasonic conditions, organic modifiers and magnetization conditions. The intercalation reaction was employed as the main strategy to improve the barrier performance, while a magnetic material was used to control the paths for small molecules to pass through the material. The results reveal that due to the introduction of montmorillonite, the light transmittance of the composite film reduced by 10% at 600 nm. The maximum reduction in transmittance was observed in STAC-modified montmorillonite at 14.5%. The introduction of montmorillonite had a significant inhibitory effect on ultraviolet light, which was most apparent at 200 nm. The distance between the montmorillonite layers of the non-magnetized montmorillonite powder increased from 0.14 to 1.49 nm after magnetization. The mechanical properties of the non-magnetized organic montmorillonite/starch film increased from 5 to 6.5 MPa as the amount of montmorillonite increased; however, due to the introduction of Fe, the magnetized montmorillonite did not show improved mechanical properties. The oxygen permeability of the organic montmorillonite-modified starch film was lower (0.067 cm


In the in vitro 3-dimensional (3D) culture model, LXW7-modified collagen hydrogel significantly improved EC spreading, proliferation, and survival. In a mouse subcutaneous implantation model, LXW7-modified collagen hydrogel improved the engraftment of transplanted ECs and supported ECs to form vascular network structures. Therefore, LXW7-functionalized collagen hydrogel has shown promising potential to improve vascularization in tissue regeneration and may be used as a novel tool for EC delivery and the treatment of vascular diseases.Endothelial cell (EC) transplantation via injectable collagen hydrogel has received much attention as a potential treatment for various vascular diseases. However, the therapeutic effect of transplanted ECs is limited by their poor viability, which partially occurs as a result of cellular apoptosis triggered by the insufficient cell-extracellular matrix (ECM) engagement. Integrin binding to the ECM is crucial for cell anchorage to the surrounding matrix, cell spreading and migration, and further activation of intracellular signaling pathways. Although collagen contains several different types of integrin binding sites, it still lacks sufficient specific binding sites for ECs. Previously, using one-bead one-compound (OBOC) combinatorial technology, we identified LXW7, an integrin αvβ3 ligand, which possessed a strong binding affinity to and enhanced functionality of ECs. In this study, to improve the EC-matrix interaction, we developed an approach to molecularly conjugate LXW7 to the collagen backbone, via a collagen binding peptide SILY, in order to increase EC specific integrin binding sites on the collagen hydrogel. Results showed that in the  Endothelial cell (EC) transplantation has been widely used for the treatment of various types of vascular diseases, such as myocardial ischemia, cerebrovascular disease, and peripheral vascular disease . HoweverBiomaterial carriers are attractive tools for improving cell delivery and survival . Hydrogein vitro and promoted vascularization in a rat carotid artery bypass vascular graft model (One-bead one-compound (OBOC) combinatorial technology is an ultra-high throughput chemical library synthesis and screening method, which is suitable for integrin-based ligand discovery . Previouft model , 2020a. ft model . SILY haft model . TherefoWe used endothelial colony forming cell (ECFC) banks as described in our previous study , 2020a. 2-LXW7 and SILY-(LXW7)2  SPPS synthesis of SILY-DBCO or LXW7-DBCO, 3) DBCO-azido copper-free Click conjugation by mixing LXW7-2N3 with 2 eq. of SILY-DBCO or SILY-2N3 with 2 eq. of LXW7-DBCO, respectively. Detailed synthesis was described in 2-LXW7 or SILY-(LXW7)2. After 1 h, the wells were washed three times with PBS and blocked with 1% BSA (Thermo Fisher Scientific) for 1 h. After, the wells were rinsed three times with PBS. For the cell attachment assay, 5 × 103 ECFCs were added to the wells and incubated for 5 min at 37°C and 5% CO2. The wells were washed three times with PBS, and the adhered cells were fixed in 10% formalin (Azer Scientific) for 20 min. The wells were washed, and then nuclei were stained with 4′, 6-diamidino-2-phenylindole . After three washings with PBS, the ECFCs were imaged using a Carl Zeiss Axio Observer D1 inverted microscope. Image quantification was performed using the ImageJ software (NIH).To facilitate the LXW7 modification on 2-dimensional (2D) collagen culture surface and 3-dimensional (3D) collagen hydrogel, we synthesized (SILY)-(LXW7)2  through 2, 37°C and 5% CO2. ECFCs were seeded in 96-well plates treated with collagen, collagen and (SILY)2-LXW7, or collagen and SILY-(LXW7)2. For caspase 3 assay, the cells were cultured in ischemic-mimicking hypoxic environment for 6 h, then lysed and analyzed by using a Caspase 3 Assay Kit  according to the manufacturer’s instruction. Fluorescence (ex 380 nm/em 450 nm) was measured using a SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices). For the cell survival assay, the ECFCs were cultured for 5 d and determined using a CellTiter 96® AQueous One Solution Cell Proliferation Assay  according to the manufacturer’s instruction. The amount of soluble formazan product produced by the reduction of MTS by metabolically active cells was measured at the 490 nm absorbance using the SpectraMax i3x Multi-Mode Detection Platform.The ischemic-mimicking hypoxic environment was set up as cells cultured in EGM-2 with 1% fetal bovine serum (FBS) and low concentration growth factors  at 1% O2 for 5 days, then the number of cells was determined using MTS as described above. For the preparation and evaluation of LXW7-modified collagen hydrogel, the diluted 2 mg/mL collagen was mixed with SILY-(LXW7)2 at the different concentration of SILY-(LXW7)2/collagen (nmol/mg), such as 0, 0.005, 0.05, 0.25, 0.5, 2.5, 5, 12.5, or 25 nmol/mg. The LXW7-modified collagen (0.1 mL) was then put in 48-well plates and incubated at 37°C for 1 h, then 100 μL PBS was added into the wells. After 24 h, the PBS was collected for High-performance liquid chromatography (HPLC) analysis to quantify the amount of unbound free SILY-(LXW7)2 that was eluted to the solution. Briefly, HPLC analysis was performed on the Waters 2996 HPLC system equipped with a Waters XTerra® MS column . A linear gradient was run from 100% solution A (water/0.1% trifluoroacetic acid) to 100% solution B (acetonitrile/0.1% trifluoroacetic acid) within 20 min with a flow rate at 1.0 mL/min. The UV detection wavelength was 214 nm. The compressive modulus of the untreated collagen hydrogel and the LXW7-modified collagen hydrogel were determined using the Instron 5566 Universal Testing Machine.For optimization of collagen hydrogel concentration, the original 10 mg/mL collage type I was diluted to 1, 2, 4, and 8 mg/mL with PBS, respectively. The same number of ECFCs was loaded into the collagen hydrogel with different concentration and cultured at 37°C, 5% COin vitro and in vivo, ECFCs were transduced with the pCCLc-MNDU3-LUC-PGK-EGFP-WPRE vector as previously described  Vector Core. To track the cell fate and behavior escribed . Transdu2 and 5% CO2 for 5 days. Cell metabolic activity and proliferation was quantified every 24 h using MTS as described above. For cell survival, ECFCs were loaded in the untreated collagen hydrogel or LXW7-modified collagen hydrogel, respectively, and cultured under ischemic-mimicking hypoxic environment for 5 days. Cell metabolic activity was quantified every 24 h using MTS, as described above.For cell sprouting assay, ECFCs were loaded in the untreated collagen hydrogel, or LXW7-modified collagen hydrogel, respectively, and cultured in EGM-2 for 3 d. Images were taken at day 1 and day 3 by using the Carl Zeiss Axio Observer D1 inverted microscope. Image quantification was performed using the ImageJ software. For capillary network formation, three groups were set up: (1) ECFCs cultured in untreated collagen hydrogel, (2) ECFCs cultured in LXW7-modified collagen hydrogel, (3) ECFCs incubated with a monoclonal anti-αvβ3 integrin blocking antibody  first to block the integrin αvβ3 expressed on the ECFCs, then cultured in the LXW7-modified collagen hydrogel. Images were taken at day 5 by using the Carl Zeiss Axio Observer D1 inverted microscope. Image quantification was performed using the ImageJ software. For cell proliferation, ECFCs were loaded in the untreated collagen hydrogel or LXW7-modified collagen hydrogel, respectively, and cultured in EGM-2 at 37°C, 20% O–/–) immunodeficient mice were purchased from The Jackson Laboratory. 5 × 105 transduced ECFCs were loaded in 500 μL untreated collagen hydrogel or LXW7-modified collagen hydrogel, respectively, and injected subcutaneously to the groin at both sides of each mouse.All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, Davis. All facilities used during the study period were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). NSG (NOD/SCID/ IL2RγIn Vivo Imaging Spectrum (IVIS) system (PerkinElmer) as previously described (®2.50 (Perkin Elmer). Total intensity was measured within a defined area of the signal. Baseline intensity was determined by using the same defined area where there is no positive signal in the same animal.Cells transplanted in NSG mice were monitored via the escribed . BrieflySamples were collected, fixed with 4% paraformaldehyde for 24 h, protected by 30% sucrose dehydration for 48 h, and embedded in the O.C.T compound . Serial sections were made at the thickness of 15 μm using a Cryostat (Leica CM3050S) and collected onto microscope slides (Matsunami Glass). Tissue sections were extensively washed with PBS, blocked with 5% BSA in PBS at room temperature for 1 h, and stained with primary antibody at 4°C overnight. The dilution of primary antibody was goat anti-GFP  at 1:100. Sections were incubated with secondary antibodies diluted at 1:500 for 1 h at room temperature. The secondary antibody was donkey anti-goat (Thermo Fisher Scientific) conjugated with Alexa488. The slides were counterstained with 1:5000 dilution of DAPI for 5 min, mounted with Prolong Diamond Antifade Mountant (Invitrogen), and imaged with the Zeiss Observer Z1 microscope.t-test was used. For multiple-sample comparison, analysis of variance (ANOVA) was performed to detect whether a significant difference existed between groups with different treatments. A p-value of 0.05 or less indicates a significant difference between samples in comparison.For two-sample comparison, a student’s 2-LXW7 and SILY-(LXW7)2, that can be used conveniently to conjugate LXW7 onto the collagen backbone. The results showed that cell culture surface treated with both (SILY)2-LXW7 and SILY-(LXW7)2 significantly improved ECFC attachment compared to the untreated collagen surface 2-LXW7 treated collagen surface indicating that a higher density of LXW7 was more beneficial for ECFC attachment 2 treated collagen surfaces suppressed expression of caspase 3 in ECFCs, compared to the collagen surface under the ischemic-mimicking hypoxic environment, and only SILY-(LXW7)2 treated collagen surface significantly decreased the expression of caspase 3 in ECFCs, compared to the collagen surface (2-LXW7 or SILY-(LXW7)2. Stoichiometrically, the LXW7 binding sites provided by SILY-(LXW7)2 was four times greater compared to the LXW7 binding sites provided by (SILY)2-LXW7. Subsequently, the results showed that the SILY-(LXW7)2 treated collagen surface significantly improved ECFC survival under the ischemic-mimicking hypoxic environment 2 used to modify collagen hydrogel was up to 5 nmol/mg, and the peaks increased as the amount of SILY-(LXW7)2 increased (2 modification approach did not significantly change the stiffness of the collagen hydrogel. This was probably because only one end of the SILY-(LXW7)2 molecule carried the –SILY group that could bind to collagen, and the –(LXW7)2 end could not bind to collagen, therefore the SILY-(LXW7)2 modification approach did not induce molecular cross-linking of collagen.The effect of ECM stiffness on cell biology, signaling and response has been well-established, which has significant implications for tissue regeneration . Particul volume . The HPLncreased . These rncreased  indicatiin vitro analog of in vivo angiogenesis. Also, it is know that cell-matrix interaction could generate cellular force that can impact capillary network formation. The results of network formation showed the ECFCs cultured in LXW7-modified collagen hydrogel for 5 days formed uniform and integrated network 2-LXW7 and SILY-(LXW7)2, performed the HPLC analysis, and discussed the results. KG assisted the in vivo evaluation. CH, SH, and CZ assisted the in vitro evaluation. GS, DF, AP, and KL were involved in the results discussion. AW was responsible for conceptualization, results, discussion, and revision of the manuscript. All authors contributed to the article and approved the submitted version.DH performed the construction and the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance.The cellulolytic thermophile C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%.The rate of growth of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.This study documents inhibition of  Corn fiber represents a “Generation 1.5” biofuel feedstock, intermediate between starch and lignocellulose. Since it is already present at corn-based biofuel facilities, a corn fiber-based process can potentially be added to an existing corn ethanol plant in a ‘bolt-on’ configuration . T. T28]. TC. thermocellum growth on cellobiose. A control broth was prepared from a C. thermocellum fermentation on 4.2 g/L Avicel, which was approximately equal to the total amount of glucan consumed during the 40 g/L corn fiber fermentation. This broth controlled for fermentation products such as ethanol that are normally present in the spent broth. The C. thermocellum strain used in this experiment was LL1043, from which lactate dehydrogenase, phosphotransacetylase, and acetate kinase have been deleted. While the strain itself produces negligible amounts of acetate or lactate, some acetate was released from corn fiber.To investigate these phenomena, we first tested the effect of spent broth, collected at various times during the 40 g/L corn fiber fermentation, on C. thermocellum growth on cellobiose by spent broths from a 50 g/L switchgrass fermentation. The difference may be due to the much higher levels of hemicellulose in corn fiber relative to switchgrass or the presence of other inhibitors in corn fiber that are absent in switchgrass. At higher solids loading , the broth from switchgrass fermentations may also become inhibitory to C. thermocellum growth on cellobiose, although this remains to be shown. To study the effect of the spent broth on C. thermocellum’s hydrolytic activity, we analyzed the effect of spent broth on cellulase hydrolysis in vitro using cellulase prepared by high-molecular-weight cut-off filtration. The spent broth used for these experiments was collected after 250 h of corn fiber fermentation. The cellulase enzyme activity assay, adapted from Johnson et al. [C. thermocellum cellulase added. A titration of added cellulase shown in Fig. Figure n et al. , was basn = 2) of the control.For a quantitative measurement of the rate, the glucose release was measured at multiple sampling times  XOS (degree of polymerization of 2–6) in a mixture as well as separately; (ii) wheat arabinoxylan and arabinoxylan oligosaccharides; (iii) starch hydrolysis products such as maltose, maltodextrin, and pullulan; (iv) xyloglucan and xyloglucan oligosaccharides. All monosaccharides present in corn fiber were also tested. The full list of 28 tested compounds is shown in Additional file First, a range of commercially available carbohydrates were tested for their effect on C. thermocellum growth on cellobiose. They showed that xylobiose caused a > 50% reduction in growth rate at 20 g/L, while other XOS were slightly less inhibitory. They also showed that xylose affected growth rate at a similar level, with 50% reduction at 20 g/L. While we also observed inhibition with xylose , we did not observe inhibition by XOS at up to 20 g/L. As d-ribose was reported to be a strong inhibitor by Verbeke et al., we tested that at 20 g/L and found a 32 ± 2% reduction in growth rate. At a 10 g/L concentration of d-ribose, there was no effect. Mannose was also an interesting case, as it caused a lag phase for 2–3 days, after which the growth rate was normal.No growth inhibition was observed for the compounds tested, except xylose, mannose, and ribose. Verbeke et al.  tested mImportantly, our experiments showed no growth effects from arabinoxylan oligosaccharides and XOS, which are the commercially available compounds most closely related to the corn arabinoxylan oligomers found in spent broth. Xylose was found to be inhibitory, but since the broth only has small amounts of monomeric xylose (~ 1 g/L), it is unlikely to be the primary cause of inhibition.C. thermocellum cellulase, by measuring the OD600 over time, as described above. Figure 600 value after 24 h. To broaden the scope of this study, we decided to look at a large variety of hemicelluloses that are commonly found in lignocellulosic feedstocks for biofuel production including woods and grasses. The effect of these carbohydrates on C. thermocellum cellulases has not been studied previously. The effect of reduced enzyme loading was also tested. Cellobiose is a known inhibitor of cellulases [C. thermocellum cellulase in our study, as well. Fortunately, C. thermocellum rapidly consumes any cellobiose produced by its enzymes and we have not seen it accumulate in the broth of an actively growing culture. As shown in Fig. C. thermocellum cellulases as well, similar to what has been reported previously for fungal cellulases [C. thermocellum cellulases by the spent broth may be explained by the presence of XOS-like structures. Importantly, arabinoxylan (AX), which has not been evaluated in previous studies of cellulase inhibition to the best of our knowledge, caused less inhibition than XOS. This could be due to the higher degree of polymerization (DP) of the Wheat AX used. Beechwood and Birchwood xylan, which also had high DP, showed inhibition similar to Wheat AX (data not shown). Similarly, xyloglucan oligosaccharides, which are present in corn fiber and spent broth from C. thermocellum fermentation, caused only moderate inhibition. Galactomannan also showed a moderate inhibitory effect. No monosaccharide caused inhibition . Glucomannan, which was found to be a strong inhibitor of cellulase but had no effect on growth on cellobiose, completely inhibited growth of C. thermocellum on Avicel when present at 2 g/L, and substantially reduced the growth rate even at 0.2 g/L. This may be an important observation for fermentations of woody feedstocks like poplar and pine that are known to contain a substantial amount of glucomannan [The monosaccharides mannose, galactose, and arabinose actually led to a small increase in growth rate. As with growth on cellobiose, mannose caused a long lag phase on Avicel too, which gradually decreased after transferring the strain a few times in medium-containing mannose. The inhibitory effect of xylose on comannan .C. thermocellum can alleviate inhibition, we ran ternary cocultures with hemicellulose-utilizing thermophiles Herbinix spp. strain LL1355 and T. thermosaccharolyticum ethanologen strain LL1548. Although LL1355 can consume most of the hemicellulose without T. thermosaccharolyticum, we included T. thermosaccharolyticum to reduce formation of lactic acid, the main product of fermentation by Herbinix spp. strain LL1355.Our results indicate that soluble hemicellulose hydrolysis products inhibit cellulase activity and Avicel growth more than growth on cellobiose. To test if consuming the hemicellulose that is liberated from corn fiber by C. thermocellum by Froese et al. [C. thermocellum with Clostridium stercorarium and Thermoanaerobacter thermohydrosulfuricus were able to solubilize 58% more of the total polysaccharides in wheat straw compared to the C. thermocellum monoculture control.Figure e et al. . They shC. thermocellum by consuming the potential inhibitors, but this was not the case . Also, the spent broth from the 40 g/L corn fiber fermentation is inhibitory to the cellulase activity of C. thermocellum. This is partly explained by the buildup of hemicellulose hydrolysis products, especially arabinoxylan and XOS, in the liquid broth. However, the spent broth also inhibited growth on cellobiose, which could not be replicated with any of the model carbohydrates we tested. Therefore, there is an undiscovered inhibitory factor produced in corn fiber fermentations that requires further characterization. Xylooligomers are well-known inhibitors of cellulase enzymes which may be explained by the structural similarity between xylobiose and cellobiose. In microbial communities in nature, accumulation of hemicellulose would be unexpected, since hemicellulose-consuming organisms are typically present. It is plausible that in natural environments, there is no evolutionary pressure for C6-fermenting organisms like C. thermocellum to overcome inhibition by C5 sugars.We show here that there is a sharp decline in C. thermocellum, but the exact mechanism remains unclear. The removal of solubilized hemicellulose carbohydrates is likely to be part of the story, but the particular molecules involved are not yet defined. Enzymatic treatment of spent broths may detoxify and shed light on this topic, especially using enzymes prepared from Herbinix spp. strain LL1355. The possibility of phenolic compounds being a major inhibitor will be a worthwhile investigation. Nonetheless, inhibition by solubilized hemicellulose is likely to emerge as a significant barrier to fermentations of lignocellulosic feedstocks at high solids loading. Developing suitable coculture organisms to partner with C. thermocellum may be an important line of future research. In some cases, specialized organisms may need to be isolated, as Herbinix spp. strain LL1355 was isolated for consumption of corn fiber hemicellulose, but they will need to be engineered for high ethanol yield or their enzymatic capabilities transferred to ethanologens.The addition of coculture partners restored fermentation performance of C. thermocellum strain LL1043, also known as M1570 [T. saccharolyticum [T. thermosaccharolyticum strain LL1244, also known as ATCC 31960 and HG-8. LL1355 was a novel isolate as reported elsewhere [Fermentations were carried out with as M1570 ; the M14olyticum  and varilsewhere .C. thermocellum bioreactor fermentation on 20 g/L Avicel, harvested at the time of its peak gas production rate, and frozen in 50 mL aliquots in N2-flushed 150 mL serum bottles at − 80 °C. A 1% inoculum was used for all reactors other than one of the 10 g/L reactors which used a 5% inoculum. Samples were taken after first clearing the lines by drawing and discarding 3 mL of culture, then withdrawing a ~ 60 mL sample. While this sample was being stirred, three samples of 1 mL were taken for CipA measurements. 50 mL of sample was then measured into a Falcon centrifuge tube . The tubes were centrifuged for 15 min at 7000×g to separate solids and the liquid was pipetted out. This liquid was then used for liquid Quantitative Saccharification (QS) as described below. The solids were washed twice with DI water before being dried in a 60 °C oven till constant weight, which was noted. The residual sugars in these solids were measured by solids QS in triplicate, as described below.Destarched corn bran and growth media were prepared as previously described . The ferT. thermosaccharolyticum was grown in CTFUD media [For the coculture reactors, UD media , pH 6.7 C. thermocellum grown on CC6 media was used. A control broth was prepared from a C. thermocellum fermentation on 4.2 g/L Avicel.The spent broth collected at each sampling time point of the fermentation was centrifuged to remove solids and then filtered through a 0.22 µm syringe filter  into a sterile tube. 10 g/L MOPS as buffer (pH adjusted to 7.2), fresh CC6  media coThe source of each is given in Additional file 2SO4 was added to 1 mL of clear liquid supernatant, then put in a 4 mL shorty vial , and capped with a butyl rubber stopper. These vials were then autoclaved for 1 h on a liquid cycle, along with acidified sugar standards to control for degradation. All polysaccharides/oligosaccharides in the liquid were thus hydrolyzed and then measured on HPLC  using xylose, arabinose, and glucose HPLC standards. The separation was performed with an Aminex HPX-87H column  at 60 °C, with RI (refractive index) detection and a 5 mM sulfuric acid solution eluent at a flow rate of 0.6 mL/min. The fermentation products were measured using the same column, as described previously [The dry residual solids from a fermentation were analyzed using complete acid hydrolysis and HPLC (Quantitative Saccharification—QS) as described . The toteviously .2O, 0.1% formic acid spiked with heavy-labeled CipA peptide standards . Five microliters of each sample were autosampled onto an in-house pulled nanospray emitter (75 µm ID) packed with 30 cm C18 resin  and analyzed by 1D LC–MS/MS as previously described [The 1 mL sample taken from the reactor was washed twice to completely remove the supernatant and only retain intact cells in the pellet. Cells were lysed via bead beating in SDS lysis buffer  followed by denaturation (10 min at 95 °C), reduction, and alkylation with DTT (10 mM) and IAA (30 mM), respectively. Proteins were extracted using chloroform:methanol as previously described , dried, escribed .C. thermocellum proteome sequence was concatenated with common contaminant proteins  and reversed sequences of the target database. MS/MS data were searched against these sequences using Tide search [The e search  using the search . Retentie search . The moFCipA intensity was calculated by summing all CipA peptide intensities, excluding the labeled peptides which were used for sample normalization. Similarly, whole-cell proteome intensity was also calculated for each sample.Growth rate measurements were performed in a Powerwave XS plate reader  in an anaerobic chamber at 55 °C as previously described . The medThe Avicel growth experiments were run in 125 mL serum bottles with a final culture volume of 30 or 50 mL. The bottles with water and Avicel were sealed and purged with 20 × 45 s of alternating cycles of vacuum and nitrogen before autoclaving for 20 min on a liquid cycle. CC6 medium was used with a MOPS concentration of 10 g/L with the final pH of 6.9–7.0. The residual Avicel and pellet nitrogen was measured using the total carbon and nitrogen (TCN) analyzer, Shimadzu Scientific Instruments  as described earlier .C. thermocellum culture grown on 20 g/L Avicel for 5 days until the substrate was completely consumed. The liquid broth was centrifuged to remove cells and filtered in an anaerobic glove bag  to limit the exposure to oxygen. The filtered broth was then passed through a 50 kDa Molecular Weight Cut-off Filter (Millipore Sigma) to concentrate the enzymes four-to-fivefold. The protein concentration was measured using a standard Bradford assay as described earlier [2 solution, various enzyme concentrations, and water.The cellulase enzyme was prepared from a  earlier . The 5 mThe qualitative assays were run with 0.5–1 g/L initial Avicel. To estimate the remaining Avicel, the absorbance was measured using a portable spectrophotometer (Biochrom Colorwave WPA CO7500) at a wavelength of 600 nm. The quantitative assays were run with 6 g/L initial Avicel. The enzyme loading was 10–25 mg protein/g of Avicel. Samples were taken to determine glucose released from Avicel by performing liquid QS as described above. Liquid QS allowed for the hydrolysis of all the released glucose oligomers into monomeric glucose that was then measured by HPLC.Additional file 1: Figure S1. Timecourse of glucose release from Avicel for cellulase assay with the addition of spent broth. Figure S2. Total sugars in fermentation broth from C. thermocellum monoculture reactors on corn fiber, measured by liquid QS. Table S1. List of carbohydrates tested for effect on growth rate of C. thermocellum on cellobiose. Table S2. Sources of carbohydrates used in the study. Figure S3. Effect of different monosaccharides on C. thermocellum cellulase activity. Figure S4. Effect of adding Xylooligosaccharides to C. thermocellum Avicel fermentations in bioreactors. Figure S5. Curve fitting to measure the effect of various carbohydrates on the utilization of Avicel by C. thermocellum. Figure S6. Effect of final spent broth from a corn fiber coculture fermentation on growth of C. thermocellum on cellobiose in a plate reader.Additional file 2. Supporting data for Fig. 6. Avicel remaining over time is given for Avicel fermentations with addition of various carbohydrates, along with curve fits used to calculate maximum Avicel utilization rate.