This research aims to understand the influence of bodily practices, especially gymnastics, in the construction of representations of a healthy body conveyed in a Brazilian women’s magazine in the 1940s and 1950s. We use records from the Jornal das Moças magazine for the analysis based on the theoretical and methodological assumptions of cultural history. The results show that gymnastics for women was linked to body maintenance and used as a tool for establishing a body standard, thus disciplining and shapingthe construction of women’s health concepts, determined by the aesthetic bias of that period: a slim body as an ideal standard of beauty and health. This study aims to understand the influence of bodily practices, especially gymnastics, on the construction of representations of a healthy body conveyed in a Brazilian women’s magazine in the 1940s and 1950s. The records from this magazine are analyzed in view of the theories of the body and methodological assumptions of cultural history. According to Chartier [Bodily practices have approached the meanings of human movement actions beyond those that are thought of in their biological sense of adaptation. Aiming to advance in another direction and considering the current discussions about the complexity to conceptualize them ,3,4, theCurrently, the greater sharing of the space for the production of the discourse that guides the role of each individual in society, previously monopolized by the old-school, military and religious pedagogical forms, provides a course for changes, expanding the customs and institutions that lead to social rules of behavior. According to Vigarello  and BaumAppearance, behavior and silhouette are increasingly incorporated into personalities and personal particularities, so there is a more intimate relationship of existence from what I am to what my body is. Within this process, the changes of being a woman in contemporary times are involved with the changes that cross her body, as Vigarello  observedThe conceptions of women’s bodies and their representations within bodily practices can be presented as a set of elements that expose within them the influences of cultural formation, in which social concepts are enabled to build standards of fundamental structures of organization of life. To think about health in a context of transformation enables us to understand how it can reflect the intentions of a social situation and its ways of production. On the other hand, it also brings evidence of variations when considering power relations that stratify and hierarchize certain groups, discriminating them within the same space based on gender, race, economic conditions, religion and other aspects.Throughout the structure of the social system of the 20th century, in the West, health was observed preponderantly through the bias of positivist rationality, thus denouncing incorrect techniques for the use of the body, composed essentially of biological matter in which the muscles were its driving machine . At the At the same time, in Brazil, political projects on women’s education via body control were formulated, which had the support of the media press. During the period called Estado Novo (1937–1945), the Brazilian national project ruled by President Getúlio Vargas had women as an essential and central element for the betterment of the Brazilian people.The increase in the working mass in precarious conditions of hygiene, health and housing, resulting from industrialization and urbanization, made women’s health a source of concern in several countries due to the accelerated growth of the world population. However, the emphasis remained on reproductive aspects. The woman that the state considered deserving attention was the one who was in the fertile period .It is possible to find in the Brazilian press (newspapers and magazines) signs of the construction of an ideology of that time, including aspects related to modern civility and a new standard of aesthetics . In the Among the bodily practices assigned to women, gymnastics stands out compared to others. We can observe a representation of gymnastics different from those known today. Some of these representations are linked to the concept of body practices of the period, modernly called “gymnastics”, which, when directed to women, had specific objectives and constructions. That is, in that period, it was common to call “gymnastics” the body practices and/or physical exercises that were done daily, such as outdoor walks and morning stretching practices, among others . AnotherDuring the 19th and 20th centuries, gymnastics developed in the West as an integral element of the modernization project, thus indicating a reorganization of the interpretation of reality. In this social dynamic, the conceptions of the human body were reconfigured by a distinct understanding of the meaning of relationships in the world . In viewThere is a diversification in the way content is analyzed based on the discourses in the documents that were gathered for this research, as well as the variety of format in which the information contained in them was recorded. Establishing perspectives that direct to a methodology is fundamental in order to focus on the reasons and the results consistent with the theoretical framework ). For CeTherefore, the analyses of the sources accessed for this historical research were submitted to the process of identifying whether there was correspondence between the information; that is, the narrated facts communicate with extratextual aspects of the period in order to situate the events in the midst of the interrelationships with the social phenomena that are formed in the modern conjuncture .One of the main sources used by historians to identify traces of the past are periodicals, specifically newspapers and magazines, according to Barros . For theThe reports of a single newspaper are not enough to explain the complex network that underlies the emergence of bodily practices linked to the health of the female body, but as the theory of cultural history demonstrates, they can constitute a historical source presenting evidence of a particular interpretation of the phenomenon in question . The focJornal das Moças, a magazine published every two weeks in Rio de Janeiro that circulated in Brazil from 1914 to 1965, according to its founders, was designed to: “Cultivate, by illustrating, and at the same time delight in the charming spirit of the Brazilian women, to whom this magazine is dedicated, will be its, if not sole scope, at least its most lively and ardent concern” . Five yeginástica (gymnastics), we obtained 52 occurrences of this bodily practice within the chosen period. After this procedure of collecting the articles, in order to extract the information from the referred issues of the magazine, they were organized and stored as files in folders divided by themes and periods in common. The files were named by author, title of the report, date, issue of publication and page number, among other complementary information important for the elaboration of the source reference.The archive with all the issues of Jornal das Moças is available in the Digital Newspaper Library of the National Library of Brazil. However, only the issues published between 1945 and 1950 were consulted, since this wasthe magazine’s best-selling period, that is, with the widest audience reach, thus establishing the time period of the research. Using the search system available in the Digital Newspaper Library of the National Library of Brazil, with the term Of this total (52), we found that 29 occurrences were linked to health, both implicitly and explicitly. When it was implicit, the word “gymnastics” was not registered literally in the text, but it was in between the lines of the narrative that it referred to the health theme. In the explicit form, the terms “gymnastics” and “health” were registered directly in the same sentence in the text. After this step, we considered that six reports met the criteria established for the selection of information: the report had to contain aspects related to the use of some body technique in order to give meaning to human movement.With this, the heuristic construction began, using two procedures approached by comparative history. Veyne  would usFor Flick  (p. 295)According to Bauman , modernibiopower with “the administration of bodies through the calculating management of life”. These movements would promote new breakdowns of the different spheres of human social life, thus causing the accentuation of more demanding and precise rules of body appearance, making body fat, for example, also a reflection of personality and even associated with ways of ordering one’s thoughts [Observing political life under the light of the articulation of modern states, this power was characterized by the consolidation of the promotion of social welfare through investment in life, and no longer through death as presented by the traditional sovereignty regime, forming what Foucault  (p. 131)thoughts .Among the devices that bring society closer to a new world of modernities and new social ruptures, in this research we focus on women’s magazines. Such magazines began to be published at the end of the 19th century with the intention of catering to the minds of readers’ new perceptions of the world and, consequently, new representations of health of women’s bodies. It is worth recalling the statement by Barbosa et al.  (p. 24):In the mid-1940s, the entire world was undergoing a radical transformation, including the reorganization of the center of economic power among nations with the end of the Second World War. The United States of America and the Soviet Union would form a bipolar order of command over government systems, through which structures would be set up to shape advantages that could favor their models of life management. It was during this period that the United Nations (UN), United Nations Children’s Fund (UNICEF), Food and Agriculture Organization of the United Nations (FAO/UN), United Nations Educational, Scientific and Cultural Organization (UNESCO), International Labor Organization (ILO) and World Health Organization (WHO) were created, which would serve as a legal, political and ideological instrument for an internationalism necessary for the interest of free-investment capitalism, enforcing a certain type of planning for postwar life .3 Meses de Ginástica em Quatro Páginas (Three Months of Gymnastics in Four Pages) [Distinct cultures are propagated among countries through processes of transference and reception of body standards. An example of this cultural dynamic was evidenced in the article from the Jornal das Moças, whose title was r Pages) , which sr Pages) . In anexr Pages) .Moreover, in the excerpt mentioned above, we can identify a relationship between the meanings proposed for physical culture within the manifestation of the concept of culture built within the European context during the 18th and 19th centuries. In that period, the term was configured as a representation linked to human development, which soughtalignment with the aspect of civilization from a historical reconstruction, which denoted shades of the advance of humanity to greater freedom from the bonds of irrationality . In the In the Jornal das Moças magazine, the expression “physical culture” appears in the texts since the 1930s and maintains its incidence until the beginning of 1960, referring to the idea of the mind–body dichotomy, indicating the importance of understanding that in order to achieve proper mental development, one must exercise the body. Thus, exercise through gymnastics would be a way to improve the power of psychic control over movement, which, when performed correctly, would generate alignment with the expected aesthetic standard; the correct posture of the body is perceived by the symmetry of its parts. Furthermore, in the case of women, exercise through gymnastics should express a greater control over the body (understanding that the psyche is part of that body) and not the demonstration of some ability.Physical culture is related to a specific knowledge of how to control the body by directing its movements to the designs of modern society, and gymnastics is one of the composing elements. These notions cross over into the conceptions of bodily practices aimed at women, presenting themselves as a tool for maintaining beauty, which can be conquered by willpower, rigor and discipline . Among tHowever, as part of the culture, the construction of images of what it is to “be feminine” appears in different spaces and times under different forms, strategies and discourses. According to Pinsky ,33, modeThe concern with a healthy appearance was surrounded by having a youthful appearance, which could be maintained, acquired and corrected through gymnastics, also with the promise of a rejuvenating body, as can be seen in this publication on gymnastics for the face: “Five minutes a day of this highly beneficial gymnastics, in every way, completes this wonderful new treatment, which makes the face fresher, younger, giving it hardness. and normalizing the secretions of the skin, which gives the features and muscles the necessary elasticity to perform more graceful gestures” . In thisHistory identifies that the traditional requirement of beauty refers to the description of a slender, perfect body, and new artifices can be used to correct its flaws . In lighSometimes, the representations of health and beauty appear as synonyms in the reports, which leads women to a total dissatisfaction with their bodies. Yoga, for example, is represented by the magazine as a practice that leads to “conservation of health and beauty” , (p. 46)Throughout the 19th century, Western medicine sought its organization in the face of modernity through the precepts of the paradigms of a positivist science, in which knowledge about the body emerged from clinicopathological anatomy. Thus, the human body came to be constituted as if by right of biological matter, thus being a space of origin and distribution of disease . From thTherefore, it is noticeable that there is a growing concern for women’s thinness as a health component, but also as an aesthetic element revealed by the fashion magazines that address getting fat as one of the greatest fears of women. The female body has been required to occupy public spaces by being endowed with mobility through a slim silhouette, which took into account thin, muscular, fat-free limbs with tapered lines . This isBodily practices have become the main tool for achieving an ideal healthy body for women in Brazil, with the encouragement of the press. In light of this, in the 1940s and 1950s paradigms shifted about women’s bodies. Performing bodily practices was a requirement for those considered modern high-class women entering life , a behavIn an article entitled “Código da Linha” (Code of the Line) , Jornal In this scenario, the applied concept of “female gymnastics” comes from the medicalhygienist perspective. In this sense, gymnastics has as its guiding principle, in its conception, the strengthening of women’s bodies, because it was considered that strengthened women would have male children endowed with strength, who would be responsible for building and protecting the country . HoweverHowever, this discourse on body standards, present in Jornal das Moças, socially builds the idea that being overweight means assuming the failure of evolution, thus pushing women away from the idea of being in progress in life, prevented from experiencing the world due to having a silhouette that goes against the dominant culture and thus making them strangers even to themselves . In thisSince its beginning, the 20th century has been marked by many discussions about a possible universal concept of health. Influenced by the organizational policies of nations that sought in the greater intervention of the state a way to systematize control over the bodies of the population, in the idea of construction and preparation of knowledge aimed at the insertion of individuals in the new dynamism of modern life, the political scenario of the 1940s experienced the effervescence of these ideas. This is because at the end of this decade, the World Health Organization (1948) was established, which would define health as “the state of complete physical, mental and social well-being and not merely the absence of disease”, thus raising its scope in the social structure to a level not yet understood by many nations. This concept certainly promoted a reorganization of the discipline over the bodies, by placing health in an even more vigilant, or even unattainable level, in which bodily practices are the living manifestation of the transformations.The representation itself of a healthy female body that the bodily practices were intended to create would not be historical if it were not noticed as an action of deviation of meaning, which only becomes possible when articulated to a social place and scientific operation related to cultural models or contemporary theorists. Therefore, the writing that articulates the facts of the past is given by the presence in the present of problems that lead to question the reasons for which the reality is being juxtaposed.The evidence found in Jornal das Moças suggests that gymnastics in the analyzed period had different representations, but when aimed at women, it was linked to the maintenance of bodies and beauty. In addition, it was used as a tool to achieve a body standard, aiming to regulate, standardize and discipline women’s behaviors. Therefore, we consider that gymnastics played an important role in the construction of women’s health concepts in Brazil in the 1940s and 1950s.Every narrative comes from a context, seeks bases in ideas and elements that pursue specific views and perspectives of a time, builds symbolic connections that are convenient as a way of interpreting reality and seeks meaning from the previous belief that the past was exactly what is presented in the present, because no human discourse arises out of nowhere and is always surrounded by intentions that circulate subjectivities. For Foucault , the disIn light of the above, it is possible to understand how bodily practices, in particular gymnastics, created representations of healthy bodies in the magazine Jornal das Moças through the aesthetic bias of that period: a slim and beautiful body. Conceptions of healthy bodies were built through the narratives of Jornal das Moças in the country. After all, when the women’s press was established in Brazilian society, it became an important vehicle of communication and injunctions that disciplined and educated women both culturally and socially.A limitation of this study could be the ability to develop the search for the particularities of the phenomenon studied within the Brazilian context, because for this it would be important to select other sources that could present the modifications and preferences that were formed through adialogue with the local culture. This is a gap that this research leaves, but that does not disqualify the contribution made by this study to research on women, gymnastics, health and body practices, since it is in line with the rigor of historical research that, according to Veyne , takes pAmong the gender issues that we can address in future studies is the debate that, when analyzing the representations of the Jornal das Moças magazine, we are talking about a reality of hegemonically white, literate upper-class Brazilian women. This is because the target audience of the magazine was these women. The phenomena that permeate the issues of black and lower middle-class women, for example, are different, and may be presented differently in reports, constituting an interesting aspect for analysis and a deepened investigation.The inductions in the disseminated texts proposed an understanding about the achievement of the ideal body, and ways in which women could reach this “model,” placing discipline, the will to achieve this goal and the responsibility of maintaining beauty and health as key elements. We can conclude that the notions of body formed from the characteristics linked to gymnastics in the cultural-historical perspective of the period weremixed and wentthrough a diversity of scenarios, in which both the rhetoricand narratives acted directly on women’s bodies and helped to construct gender stereotypes.


Improved breastfeeding practices have the potential to save the lives of over 823,000 children under 5 years old globally every year. The Baby-Friendly Hospital Initiative (BFHI) is a global campaign by the World Health Organization and the United Nations Children’s Fund, which promotes best practice to support breastfeeding in maternity services. The Baby-Friendly Community Initiative (BFCI) grew out of step 10, with a focus on community-based implementation. The aim of this scoping review is to map and examine the evidence relating to the implementation of BFHI and BFCI globally.This scoping review was conducted according to the Joanna Briggs Institute methodology for scoping reviews. Inclusion criteria followed the Population, Concepts, Contexts approach. All articles were screened by two reviewers, using Covidence software. Data were charted according to: country, study design, setting, study population, BFHI steps, study aim and objectives, description of intervention, summary of results, barriers and enablers to implementation, evidence gaps, and recommendations. Qualitative and quantitative descriptive analyses were undertaken.A total of 278 articles were included in the review. Patterns identified were: i) national policy and health systems: effective and visible national leadership is needed, demonstrated with legislation, funding and policy; ii) hospital policy is crucial, especially in becoming breastfeeding friendly and neonatal care settings iii) implementation of specific steps; iv) the BFCI is implemented in only a few countries and government resources are needed to scale it; v) health worker breastfeeding knowledge and training needs strengthening to ensure long term changes in practice; vi) educational programmes for pregnant and postpartum women are essential for sustained exclusive breastfeeding. Evidence gaps include study design issues and need to improve the quality of breastfeeding data and to perform prevalence and longitudinal studies.At a national level, political support for BFHI implementation supports expansion of Baby-Friendly Hospitals. Ongoing quality assurance is essential, as is systematic (re)assessment of BFHI designated hospitals. Baby Friendly Hospitals should provide breastfeeding support that favours long-term healthcare relationships across the perinatal period. These results can help to support and further enable the effective implementation of BFHI and BFCI globally.The online version contains supplementary material available at 10.1186/s13006-023-00556-2. Globally, improved breastfeeding practices have the potential to save the lives of over 823,000 children under 5 years old every year . ExclusiGlobal Strategy for Infant and Young Child Feeding is to ensure that every maternity facility practices the BFHI’s ‘Ten Steps to Successful Breastfeeding’. Hospitals or maternity facilities can be designated “Baby-Friendly” if they pass an external examination that verifies that they comply with the Ten Steps to Successful Breastfeeding and with the ‘International Code of Marketing of Breastmilk Substitutes’ and subsequent relevant World Health Assembly resolutions (the Code). Table The Baby-Friendly Hospital Initiative (BFHI), launched by WHO and United Nations Children’s Fund (UNICEF) in 1991, has been implemented globally in over 150 countries and is a pillar of the WHO/UNICEF Global Strategy for Infant and Young Child Feeding . One of th step of the Ten Steps to Successful Breastfeeding and of the BFHI overall [th step and associated separate initiatives are often critical to support breastfeeding mothers beyond the initial days of giving birth. While almost all countries in the world have implemented the BFHI at some point in time [The Baby-Friendly Community Initiative (BFCI) is an extension of the BHFI’s 10 overall . Its foc in time , it appe in time  and High in time  and the  in time .There have been a number of attempts to review the literature on the BFHI –12. MostTo provide an overview of interventions and/or approaches to implement the BFHI/BFCITo identify barriers and enablers to implementation of the BFHI/BFCITo identify knowledge gaps in relation to research on the BFHI/BFCIThis scoping review asks the question: what is known about the implementation of the BFHI and the BFCI globally? The aim is to map and examine the evidence relating to the implementation of BFHI and BFCI globally. Review objectives include:. [Scoping reviews map the range of evidence on a particular topic, identify gaps in the knowledge base, clarify concepts, and document research that informs and addresses practice . This sc.  and prog. , 17. Acc. .A pilot search of the literature and scoping exercise was undertaken by our research team to examine empirical studies that have focused on the implementation of the BFHI in Africa . During A three-step search strategy, as documented in the JBI manual was followed. Step one was a limited search for peer-reviewed, published papers on the PubMed and CINAHL databases. An academic research librarian was consulted and an analysis of the words contained in the titles, abstracts and index terms generated a list of keywords. Search terms were then piloted to assess the appropriateness of databases and keywords. The second step was conducted with the librarian which involved refining the search terms. The third step was to examine the references of key articles that were identified for full text review that met the inclusion criteria. Draft inclusion and exclusion criteria were tested on a sample of 15 articles to check the criteria’s suitability. The following databases were selected in consultation with the academic librarian: PubMed, Embase, Web of Science, Global Health and CINAHL. The timeframe for the search was from when the first article was published in a given database, which was 1993, to September 2022.Inclusion criteria were guided by the Population, Concepts, Contexts approach , as showAll research designs were included: qualitative, quantitative and mixed method studies. Quantitative studies included both experimental  and observational  study designs. Qualitative studies included designs such as grounded theory, ethnography, phenomenology, action research and qualitative descriptive design. In addition, all types of reviews of empirical research were included. Grey literature was not included, due to the large numbers of results that were obtained. A full list of search terms is detailed in Additional file Inclusion criteria: Articles that:describe the implementation of the BFHI and/or BFCIevaluate the BFHI (any of the 10 steps) and/or the BFCIfocus on experiences of accessing/delivering supports and services through the BFHI and/or BFCIfocus on breastfeeding outcomes as a result of the BFHI and/or BFCIfocus on any country or group of countriesare in the peer reviewed literatureempirical studiesAll types of literature reviews Exclusion criteria: Articles that:focus on other breastfeeding initiatives, supports/interventions in the hospital and/or community other than the BFHI/BFCIthe site is a baby friendly hospital but the study aim/objectives are not focused on the implementation of the BFHI/BFCIare published in a language other than Englishcommentaries, opinion pieces, editorials, evaluations, theses and book chapters and conference proceedingsThe screening process consisted of two phases: i) title and abstract screening; ii) full-text screening. In stage i) all titles and abstracts were screened by two reviewers in pairs . Screening was undertaken in Covidence and duplicates were removed. Where there was disagreement between reviewers as to whether an article should be included or excluded, a third reviewer arbitrated. At full text screening stage, the same process was undertaken. The original search was undertaken in 2020, an updated search was undertaken in 2022, and the overall results are shown in Fig. A data charting form was developed, piloted by all members of the team on five articles, amended and applied to all the included articles, according to the JBI framework , 17. DatIt was originally planned to use the PAGER  methodological framework  to analyFindings from the review will be prepared for stakeholders who have expertise in relation to the BFHI and the BFCI. These will include researchers, practitioners and policy makers at the global level and at WHO regional levels.n = 210) of studies focused on the BFHI overall/all steps, nine on the BFCI with 25 focusing on becoming BFHI/pre-BFHI (see Table n = 266), with five focused at the national level. In terms of study design, 46 were qualitative, 139 quantitative, six mixed methods, 28 reviews, and 39 intervention studies (see Table n = 144), with 60 studies focused on health professionals of various kinds , the creation of a breastfeeding-friendly environment and the removal of formula advertising from the hospital and the neonatal intensive care unit (NICU) (step 6), and the creation of a support system after the mother’s discharge (step 10) . The oveStudies that related specifically to infant formula marketing at hospital level were from Canada , the UK Overall, successful BFHI implementation was associated with higher rates of initiation and continuation of breastfeeding across studies , 76–78. Studies often measured which of the Ten Steps were fulfilled and concluded that the more Baby-Friendly hospital practices mothers met, the better the breastfeeding outcomes. This was found in Malawi  and HongSome studies highlighted the importance of specific steps. Step 1 was found to be an important factor for exclusive breastfeeding duration in Turkey . Staff sStep 4, specifically skin-to-skin contact, was the focus of several studies, such as in Brazil  and the Hospital lactation policies, high rates of surgical deliveries and nurses having limited education in breastfeeding initiation best practices, were noted as barriers to best practices related to step 5 in breastfeeding initiation, in Colorado State, USA . Step 6 A longer length of stay in hospital was seen as important to breastfeeding in Japan . This duWhere studies measured the support for the 10 steps, there was considerable variation, for example, least support (28%) for step 1 and greatest support (93%) for step 3. Inconsistencies in implementation of the other steps were common in a study in the USA . There wThere was limited evidence about the Baby Friendly Community Initiative (BFCI), with just nine studies in total, focusing on Kenya, Italy and Turkey. In Italy, counselling or education being provided concurrently in various settings was seen to be most effective . A natioMany different sorts of educational and training interventions were covered in the research. These were focused on improving both knowledge of breastfeeding, increasing support for the BFHI and improving attitudes towards breastfeeding , 108 acrKnowledge of the BFHI varied across professional groups. The least understood steps in medical and nursing students were steps 1, 3, 8, and 10  in IndiaTime pressures, out of date practices and a lack of commitment to BFHI by experienced midwives was found to have a major impact on newly graduated midwives seeking to develop their breastfeeding support skills, in an Australia-based study . In a USEnablers: Many studies emphasised the need to monitor ongoing learning. Some studies of interventions focused on single professions, for example nurses in Singapore [ingapore , where tingapore  accordiningapore . A studyingapore  highlighingapore , where iingapore . Continuingapore . Howeveringapore . The neeingapore . Gavine ingapore  highlighingapore . Lay breingapore .Barriers: Overall, a lack of health professionals’ education was found to be a barrier to BFHI implementation across many studies. Inadequate training of health staff and a high volume of patients was a barrier in Pakistan [Pakistan . A studyPakistan  highlighPakistan  and NigePakistan . Health Pakistan . ChanginPakistan , and simPakistan  and the Pakistan . WhenevePakistan . A studyPakistan . Poor coPakistan .enablers for BFHI implementation in the USA [Key  the USA  were fou the USA . Both me the USA  and Cana the USA . It was  the USA . A netwo the USA . The spe the USA , Taiwan  the USA  and in t the USA . Giving  the USA . Lactati the USA . The inv the USA .barriers such as the medicalisation of childbirth and inter-professional struggles were highlighted as hindering inter-professional teamwork and collaboration and, therefore, the implementation of BFHI and its integration into practice in Austria [Various  Austria .interventions for pregnant and lactating women were highlighted. Some interventions were Step specific, such as breastfeeding education in the prenatal setting, step 3 [Many educational , step 3  and a tr, step 3 . Mothers, step 3 . Objecti, step 3 . Confide, step 3 . Women w, step 3 . A breas, step 3 . Other eproviding sufficient information for mothers and the public about the BFHI, the benefits of breastfeeding, disadvantages of not breastfeeding, and benefits of going to accredited facilities  .mothers who gave colostrum as the first food had more frequently taken lactation counselling support than mothers who gave prelacteal foods (Turkey) .a client-focused practice development approach was found to be effective in Australia .viewing short videos increased breastfeeding knowledge, particularly about hand expression, and increased confidence in both skills (UK and China) .greatest improvements in breastfeeding were seen when counselling or education were provided concurrently in various settings .Barriers: A study in Cyprus found that a large proportion of pregnant mothers received limited information and/or education on the benefits and ways to achieve exclusive breastfeeding [tfeeding . Administfeeding . Culturatfeeding . In Lebatfeeding .Cultural beliefs of mothers, their family members and others were seen as important across studies, as a wider context for BFHI implementation. A study in Iran  found thMany studies identified gaps in the existing evidence. Patterns in study design and data collection that arose were as follows:Study design issues: The importance of conducting studies with a control group and the need to carry out more experimental studies was highlighted multiple times [le times , 145–147le times . Ducharmle times  highlighle times –151. Stale times . Mäkelä le times  also emple times . Shing ele times  highlighle times .Data issues: There was a stated need to improve the quality and validity of the collation of breastfeeding data  and to perform prevalence studies. Limited size of samples was noted several times [al times , 155. Nual times , 157 sugal times , 158–160al times  noted poWithin many studies, recommendations for practice and policy were highlighted, across different levels and settings. At a national level, political enforcement of, and support for, BFHI implementation can assist in expanding the designation process of Baby-Friendly Hospitals , 76, 162Advocacy for additional government resources is needed to support scale-up of BFCI , alongsiOngoing quality assurance is crucial, as is systematic (re)assessment of BFHI designated hospitals , 169–174At the hospital level, there were many recommendations highlighted. Hospital administrators should establish and monitor breastfeeding policies , 179. UpHospitals with Baby-Friendly status should consider models of breastfeeding support that favour long-term healthcare relationships across the perinatal period. There is a need for a continuous healthcare model , includiIn this scoping review, we sought to identify and map what is known about the implementation of the BFHI and the BFCI globally. We have included evidence from a wider range of sources than before, across all settings. A limitation of the review is a lack of critical appraisal of included studies, which may have resulted in studies of low quality being included. Studies that were published in a language other than English were excluded. Evidence from over 48 countries globally, and gathered from many different stakeholders—from women, health professionals and policy makers—is presented and organised here, to highlight six key patterns associated with implementation of the initiatives. These patterns mapped, to some extent, on to the Ten Steps for Successful Breastfeeding themselves, and range from national health system level to community level interventions between women and health professionals and others supporting breastfeeding. The BFHI has been revitalised in many countries. It seems that the potential of the BFCI has not been realised in settings beyond the initial countries in which it was implemented. Evidence gaps highlighted the need for having longer term follow-up outcome data, and having experimental designs where appropriate.These results can help to support and further enable the effective implementation of BFHI and BFCI globally. Researchers can build on this evidence base to plan and carry out higher quality studies to advance understanding and improve future implementation of the BFHI and BFCI.Additional file 1. Search terms.Additional file 2. Overview of studies.Additional file 3. Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist.


The road transportation sector is a dominant and growing energy consumer. Although investigations to quantify the road infrastructure’s impact on energy consumption have been carried out, there are currently no standard methods to measure or label the energy efficiency of road networks. Consequently, road agencies and operators are limited to restricted types of data when managing the road network. Moreover, initiatives meant to reduce energy consumption cannot be measured and quantified. This work is, therefore, motivated by the desire to provide road agencies with a road energy efficiency monitoring concept that can provide frequent measurements over large areas across all weather conditions. The proposed system is based on measurements from in-vehicle sensors. The measurements are collected onboard with an Internet-of-Things (IoT) device, then transmitted periodically before being processed, normalized, and saved in a database. The normalization procedure involves modeling the vehicle’s primary driving resistances in the driving direction. It is hypothesized that the energy remaining after normalization holds information about wind conditions, vehicle-related inefficiencies, and the physical condition of the road. The new method was first validated utilizing a limited dataset of vehicles driving at a constant speed on a short highway section. Next, the method was applied to data obtained from ten nominally identical electric cars driven over highways and urban roads. The normalized energy was compared with road roughness measurements collected by a standard road profilometer. The average measured energy consumption was 1.55 Wh per 10 m. The average normalized energy consumption was 0.13 and 0.37 Wh per 10 m for highways and urban roads, respectively. A correlation analysis showed that normalized energy consumption was positively correlated to road roughness. The average Pearson correlation coefficient was 0.88 for aggregated data and 0.32 and 0.39 for 1000-m road sections on highways and urban roads, respectively. An increase in IRI of 1 m/km resulted in a 3.4% increase in normalized energy consumption. The results show that the normalized energy holds information about the road roughness. Thus, considering the emergence of connected vehicle technologies, the method seems promising and can potentially be used as a platform for future large-scale road energy efficiency monitoring. This time series was first resampled at 50 Hz. Then, the speed data were integrated w.r.t. time to provide cumulative distance and the traction energy was calculated from Equation . Lastly,Average vehicle speed and measured traction energy from the GM cars traveling over the same road are visualized in 10 is the 10 m moving average of the IRI reported by the P79 profilometer. Moreover, It is observed from It can be seen from In order to visualize the influence of the normalization technique and validate the proposed method, the road slope versus measured and normalized energy data are plotted in For road sections where the car drives at a constant speed, the energy consumption must vary according to the longitudinal slope since all other driving resistances are constant, as verified/shown in The chart in Finally, the chart in r is 0.68–0.83. Since it is impossible to drive at an exact speed  there is some noise in the data caused by small changes in longitudinal acceleration. r is 0.93–0.96. r is 0.91–0.97.From The spikes in the data around a slope of zero in The results in quations  (see Tabquations  yields(g = 9.81 m/s2 (earth’s gravity acceleration), m, is 1706 kg, which falls within the expected range of 1500–1900 kg  indicates that the analysis results are significant.The Pearson correlation coefficient, ried out . ANOVA tFrom In order to test the hypothesis, the normalized energy data are aggregated for 15 km of road and then divided into three groups. Group no. 1 contains data from both the highway road and the urban road, group no. 2 contains data from the highway road, and group no. 3 the data from the urban road. The data are divided into five categories within each group, each representing roughness properties from very smooth  to very rough . Each category contains 20 percent of the data, i.e., from the 0–20th percentile to the 80th–100th percentile for ‘very smooth’ to ‘very rough’ (‘respectively’). Next, the linear correlation between means in each group is calculated as shown in It is observed from The ANOVA test results for group no. 1 are visualized in the box plot in p-value of zero is obtained for all three groups, indicating that differences between column means are significant. The table shows the between-category variation (‘columns’) and within-category variation (‘error’). F-statistic is the ratio of the mean squared errors. The p-value is the probability that the test statistic can take a value greater than the value of the computed test statistic. F > The results are summarized in Data from individual car passes are evaluated in windows of 100, 250, 500, 1000, and 2500 m to test the method’s reproducibility and performance for shorter road sections. The choice of window size is guided by the following: (i) standard road condition parameters are typically reported in 10 to 100-m intervals; (ii) the measured energy consumption is constant over more extended road sections compared to traditional standard road condition parameters; and (iii) to ensure overlap between time series data collected from different sources at different speeds .It is observed from p-value is 0.07–0.39 It is observed from p-values are obtained from a manufactured dataset. First, a synthetic set of normalized energy consumption data were generated for each car pass. This was done, assuming a normal distribution of data, with a mean and standard deviation equal to the corresponding data for the real car pass. Next, the manufactured dataset was utilized to produce a set of synthetic correlation coefficients. Finally, the p-values were calculated from an ANOVA test. The null hypothesis, in this case, is that the correlation coefficients in The The results of the ANOVA test for a window size of 1000 m are visualized in the box plot in p-values  device. The data were transmitted periodically before being processed, normalized, and saved in a database. The normalized energy was then linked to the road pavement roughness and visualized on a map.This is the first time such a concept has been utilized to quantify changes in vehicle energy consumption caused by pavement roughness on highways and urban roads. The approach has the advantage that it enables analysis of data across vehicle types in a real-scale setting and is, therefore, superior to other methods.The new method was first validated utilizing a limited dataset of vehicles driving on a highway road. The results from this verification effort show that energy data can be normalized and that the physical models proposed apply to the problem.Experimental data from ten nominally identical electric cars driven over 25 km of highways and urban roads were utilized to investigate the relationship between normalized energy consumption and road pavement roughness.As a first step, the normalized energy data were aggregated into three road groups/ classes. Within each group, the data were further subdivided into five road roughness categories, each containing 20% of the group data. The results showed a strong linear relationship between the normalized energy consumption and road roughness. Then, the relationship between normalized energy and road roughness for individual passes and the methods’ reproducibility were assessed. In this context, the data were analyzed in 100- to 2500-m windows. It was found that the normalized energy consumption collected from individual cars resembled the measured road roughness. The results also showed a low-to-moderate positive linear relationship for all car passes and window sizes on both highways and urban roads.Analysis of Variance (ANOVA) tests showed that the results obtained are statistically significant for both aggregated data and data from individual passes. Thus, it is concluded that the normalized energy consumption holds some information about the physical condition of the road.The method enables road network mapping of energy data. Such energy consumption maps give users/operators a quick overview of the total energy consumption on the road network and help identify critical areas of the road infrastructure. The method also enables energy data analysis in terms of physical phenomena  and source , as well as how these parameters evolve.The main findings from this study can be summarized as follows; (i) a new method for estimating road energy efficiency was proposed and successfully utilized to analyze energy data on highways and urban roads; (ii) the average normalized energy consumption is 0.13 and 0.37 Wh per 10 m for highways and urban roads (respectively); (iii) the normalized energy consumption is positively correlated to surface roughness—the average correlation coefficient is 0.88 for aggregated data and 0.32 and 0.39 for 1000-m road sections on highways and urban roads (respectively), and (iv) an increase in IRI of 1 m/km results in a 3.4% increase in normalized energy consumption.Considering the emergence of connected vehicle technologies, the method seems promising and can potentially be used as a platform for future large-scale road energy efficiency monitoring. The normalized energy consumption is a new pavement condition indicator that supports decision-making and may contribute to improved pavement management. Additionally, the normalized energy can be applied to initiatives to create labeling systems for road infrastructure similar to those used in consumer sectors. In this context, it is envisioned that the proposed concept will be combined with other important factors affecting vehicle energy consumption, such as road pavement type, road classification, road geometry, traffic information, and weather conditions. Hence, this issue is of critical importance and requires further research.


Due to the essential role of cyclin D1 in regulating transition from G1 to S phase in cell cycle, aberrant cyclin D1 expression is a major oncogenic event in many types of cancers. In particular, the dysregulation of ubiquitination-dependent degradation of cyclin D1 contributes to not only the pathogenesis of malignancies but also the refractory to cancer treatment regiments with CDK4/6 inhibitors. Here we show that in colorectal and gastric cancer patients, MG53 is downregulated in more than 80% of tumors compared to the normal gastrointestinal tissues from the same patient, and the reduced MG53 expression is correlated with increased cyclin D1 abundance and inferior survival. Mechanistically, MG53 catalyzes the K48-linked ubiquitination and subsequent degradation of cyclin D1. Thus, increased expression of MG53 leads to cell cycle arrest at G1, and thereby markedly suppresses cancer cell proliferation in vitro as well as tumor growth in mice with xenograft tumors or AOM/DSS induced-colorectal cancer. Consistently, MG53 deficiency results in accumulation of cyclin D1 protein and accelerates cancer cell growth both in culture and in animal models. These findings define MG53 as a tumor suppressor via facilitating cyclin D1 degradation, highlighting the therapeutic potential of targeting MG53 in treating cancers with dysregulated cyclin D1 turnover. The expression of cyclin D1 is induced by mitogenic stimulations. Then cyclin D1 enters nucleus and couples with CDK4/6 to inactivate retinoblastoma tumor suppressor (Rb) by phosphorylation, which enables the transcription of E2F-dependent genes. When cells enter S phase, cyclin D1 is phosphorylated and transported back to cytosol where it is ubiquitinated followed by degradation via proteasome. In addition, various stress conditions, such as radiation, DNA damage, drug treatment, or starvation, may also induce cyclin D1 degradation and subsequent cell cycle exit.13Cyclin D1 plays multiple roles in tumorigenesis, including enabling cell cycle progression, promoting cell migration, facilitating DNA damage repair, and driving chromosome instability.16 Thus, innate insensitivity or acquired resistance to CDK4/6is constitutes a major hindrance in the clinical application of these drugs. To date, two large E3 ligase complexes, S-phase kinase-associated protein 1-Cullin 1-F-box complex (SCF) and anaphase-promoting complex/cyclosome (APC/C), have been reported to mediate the degradation-related polyubiquitination of cyclin D1.1 SCF E3 ubiquitin ligase complexes contain different substrate-interacting proteins. Among them, SCFFBX4/αB crystallin is a well-validated E3 ligase complex of cyclin D1. The mRNA levels of αB crystalline and FBX4 are downregulated in prostate, thyroid, and breast adenocarcinomas, as well as lymphomas.11 In addition, mutations of FBX4 were found in esophageal tumors.17 SCFFBXW8 is identified to facilitate cyclin D1 ubiquitination in HCT116 and SW480 colon cancer cells and T98G glioblastoma cells, although there is no direct evidence linking SCFFBXW8 to tumorigenesis in vivo.18 β-transducin repeat-containing protein (β-TrCP1) is another adaptor that interacts with cyclin D1 in response to the treatment of the peroxisome proliferator-activated receptor γ agonist STG28.19 Intriguingly, β-TrCP1 induces cyclin D1 degradation when prostate cancer cell LNCaP are challenged with glucose starvation, while other E3 ligases are not seem to be involved under this condition. Another E3 ubiquitin ligase complex APC/C mediates irradiation-induced degradation of cyclin D1 via its subunit Cdc27/APC3.21 It is noteworthy that the expression of Cdc27/APC3 is repressed in many types of cancer cells, but whether Cdc27/APC3 dysregulation promotes tumorigenesis via cyclin D1 is not reported. It is not uncommon that a protein has several E3 ligases, but the functions of the E3 ligases of cyclin D1 might be cancer-type and context dependent. Furthermore, it has been shown that the ablation or knockdown of the components of either SCF or APC/C E3 ligase complex does not reduce cyclin D1 abundance in vivo or in vitro.22 Therefore, it is important to identify other mechanisms contribute to the dysregulation of cyclin D1 protein turnover in the context of different cancer types.Emerging evidence has pinpointed a pivotal role of dysfunctional E3 ubiquitin ligase in the cancer-associated cyclin D1 accumulation. Moreover, recent studies have implicated dysfunction in ubiquitination-dependent degradation of cyclin D1 as a key mechanism underlying insensitivity towards CDK4/6 inhibitors (CDK4/6is) in treating certain types of cancers.24 On the other hand, MG53 facilitates membrane repair26 and activates survival signaling pathways,28 and thereby playing a protective role against acute damage of multiple organs.31 An unbiased screening has identified MG53 as one of the 4 essential regulators that limits the proliferation and metastasis of non-small cell lung cancer,32 however how MG53 functions as tumor suppressor is unclear. To delineate the underlying mechanism, we examined the function of MG53 in different cancer cell lines and searched for the cell cycle regulators that interacted with MG53 by proteomic analysis. We further validated our findings using MG53 transgenic and knockout mice, as well as samples from patients with colorectal and gastric cancers.MG53  is an E3 ligase of insulin receptor and insulin receptor substrate 1. Thus, increases in MG53 expression contribute to metabolic disorders by impairing insulin signaling and glucose metabolism.Specifically, we have demonstrated that the expression of MG53 is overtly decreased in more than 80% of the tumors examined relative to the normal tissues from the same patient. The downregulation of MG53 is strongly correlated with reduced overall survival of patients with colorectal or gastric cancers. Mechanistically, MG53 functions as an E3 ligase of cyclin D1, thereby suppressing tumor growth in mouse models with xenograft tumors as well as carcinogen-induced colorectal cancer. In contrast, depletion of MG53 has the opposite effects of accelerating cancer cell proliferation and exacerbating tumorigenesis in mice. Furthermore, adenoviral delivery of MG53 expression vector into xenograft tumors significantly retards tumor growth. These results reveal the mechanism of action of MG53 in suppressing tumor growth and demonstrate the potential of targeting MG53 in cancer therapy.To get mechanistic insights in the function of MG53 in cancer, we performed Kaplan–Meier survival analysis of two cohorts of colorectal cancer. Results showed that patients defined as having “high expression” of MG53 had significantly prolonged survival Fig. . Remarka33To reveal the mechanism underlying the tumor-suppressive function of MG53, we firstly examined MG53 expression in several cancer cell lines, and found that colorectal cancer cell HCT116 had endogenous MG53 expression. Next, we overexpressed MG53 in HCT116 and found that upregulation of MG53 markedly attenuated  assay revealed a strong interaction between MG53 and cyclin D1 in HEK293 cells when both proteins were overexpressed  abolished the ubiquitination by MG53, substantiating that MG53 is an E3 ligase of cyclin D1  treatment using MG53 overexpressing (MG53-TG) or deficient (MG53-KO) mice  via catalyzing its ubiquitination in hepatocellular carcinoma cells.46 In this study, we have shown that MG53 represses tumor growth via destabilizing cyclin D1. Thus, there was a negative correlation of the amount of MG53 and cyclin D1 in cultured cancer cells and tumors derived from animal models as well as patients with gastric or colorectal cancers. Moreover, MG53 induces cell cycle arrest at G1, and cyclin D1 is the key G1/S regulator that interacts directly with MG53. Most importantly, supplement of cyclin D1 can restore the cell proliferation attenuated by MG53 overexpression. Although we cannot fully exclude other potential mechanisms, our results strongly suggest that the anti-cancer effect of MG53 is largely mediated by facilitating cyclin D1’s ubiquitination and subsequent degradation in multiple cancer cell types that depend on cyclin D1 for cell proliferation. Therefore, understanding the detailed mechanism(s) of action of MG53 in each particular cancer type is fundamental for implementation of precision medicine in cancer treatment.Several studies have demonstrated that MG53 is a tumor suppressor,26 which has inspired the application of recombinant MG53 protein (rhMG53) in treating acute injury of a variety of organs.49 In a recent study using mouse xenograft model of colorectal cancer cell SW620/AD300, injection of rhMG53 inhibits tumor growth and displays synergistic antitumor effect with doxorubicin.42 We showed significant therapeutic effects of adenoviral delivery of MG53 in delaying HCT116 tumor progression in vivo. Moreover, as the dysfunction of cyclin D1 E3 ligases is involved in desensitizing of cancer cells towards CDK4/6is in clinical practice,16 we utilized MG53 in combination with palbociclib and successfully enhanced the sensitivity of cancer cells to palbociclib and further repressed tumor growth. These proof-of-concept tests demonstrate the possible application scenarios of MG53 in cancer therapy.MG53 has originally been reported to play an important role in membrane repair,In summary, we have shown that MG53 is a tumor suppressor that targets cyclin D1 for ubiquitination-dependent degradation. Upregulation of MG53 is sufficient to suppress tumor growth. Most importantly, increased expression of MG53 not only inhibits tumor growth in animal models, but also is associated with markedly improved survival probability of cancer patients. These findings highlight the therapeutic potential of MG53 in treating cancers with high cyclin D1 abundance and improving the efficacy of CDK4/6is.Patient samples were obtained from Beijing Cancer Hospital and Institute, Beijing, China; and Wuhan Union Hospital, Hubei, China . The animals were maintained in the AAALAC-accredited Laboratory Animal Center at Peking University, Beijing, China. Male mice were randomly assigned to experiment groups for treatments. MG53 overexpressing transgenic mice (MG53-TG) and MG53 knockout mice (MG53-KO) were generated as described previously.6 HCT116 cells expressing either MG53-GFP or MG53-specific shRNA (shMG53) into one axilla, and cells expressing GFP or GFP-specific shRNA (shGFP) were transplanted into the axilla on the other side of the same animal as a control. Once palpable tumors were established, a caliper was used to measure their sizes every 2 days. The formula (A × B2)/2 was used to calculate the volume of a tumor, where A and B were the tumor length and width, respectively. After the indicated time course, mice were imaged. The xenograft tumors were dissected and weighed. For tests of palbociclib treatments, tumors were monitored till the average size of xenograft MG53-overexpressing tumors reached 100 mm3, at which point mice were randomly assigned to treatment group of either vehicle or palbociclib (40 mg/kg) via oral gavage for 7 consecutive days. For evaluating therapeutic potential of MG53, 3 × 106 HCT116 cells were xenoplanted into male 6-week-old BALB/c nude mice. Once the average tumor size was around 200 mm3, the mice were randomized into two groups and injected with either adenovirus expressing β-gal or MYC-tagged MG53. The adenovirus was injected very slowly into the tumor at two different sites once every 2 days . Mice were sacrificed after they received five doses of adenovirus.For the xenograft mouse model, male BALB/c nude mice at 6 weeks of age were injected with 3 × 102, 1.2 mM CaCl2, 25 mM NaHCO3, 2.4 mM K2HPO4, 0.4 mM KH2PO4, and 2% PMSF, pH 7.4). Colons were slit open longitudinally and the number of tumors were counted. The snap-frozen small intestine, colon, and tumors were stored at −80 °C for further analysis.For AOM/DSS-induced colorectal carcinogenesis, mice were injected with AOM intraperitoneally . 7 days after AOM injection, 2.5% DSS  was administered via drinking water for 7 consecutive days, then replaced with normal drinking water for another 14 days. At the end of 10 weeks after repeating this treatment scheme for 3 times, the mice were sacrificed. Colons were collected, and feces were washed off with Ringer’s solution  was from Raybiotech. Palbociclib (#S1116) was from Selleck Chemicals. Cycloheximide  was from APExBio.Unless specifically indicated, all the chemicals used were from Sigma-Aldrich. The antibodies used in this study are listed in Supplementary Table The expression vector of HA-Ubiquitin was from Addgene (RRID: Addgene_18712). The full-length MG53 cDNA was amplified from a human ORFeome v8.1 Entry library by PCR and the expression vector was generated using pcDNA4/TO/myc-HisB Expression Vector . Cyclin D-FLAG expression vectors were also constructed by inserting PCR product of the human ORFeome library into the vector c-FLAG pcDNA3 (RRID: Addgene_20011). The constructs expressing MG53 D-RING and truncated cyclin D1 were generated from their corresponding wild type full-length expression vectors. All mutant and truncation expression vectors were generated using a Q5 Site-Directed Mutagenesis Kit  as described by the manual.All the cells, including HEK293 , HepG2 , H460 , H1975 , A549 , H1299 , AGS , Hs746T , SW480 , and HCT116  cells, were from ATCC. HEK293, HCT116, HepG2, and Hs746T cells were cultured in Dulbecco’s modified Eagle’s medium . H1975, A549, H1299, and AGS cells were maintained in RPMI1640 , all of which were supplied with 10% fetal bovine serum . SW480 cells were cultured in Leibovitz’s L-15 medium  with 15% fetal bovine serum. Cell number was determined by Cellmeter Auto T4 (Nexcelom). When cells reached 90% confluency, gene transfer was performed by adenoviral or lentiviral infection (Suzhou GenePharma), or plasmid transfection using Lipofectamine 3000TM . The sequences of the MG53-specific shRNAs are as follows:shMG53#1: 5′-GACTGAGTTCCTCATGAAATA-3′,shMG53#2: 5′-GGGTTGAAGCTTAGGTCTCCT-3′.4 per well and infected with the indicated adenoviruses. Cell proliferation was assessed with MTT assay . Specifically, cells were incubated with MTT at 37 °C for 4 h. The medium was then replaced with dimethyl sulfoxide and cells were solubilized. MTT cleavage was quantified using a spectrophotometer to measure absorption at 490 nm with absorption at 630 nm subtracted as background. CellTiter-Glo® Luminescent Cell Viability Assay  was utilized to assess cell viability. For monitoring cell proliferation at real-time, 4 × 104 cells in a well of 24-well plate were subjected to the measurement by an impedance-based real-time instrument system .Cells were seeded in 24-well plates at 4 × 10Cells were synchronized using transient serum starvation and infected with the indicated adenovirus. Twenty-four hours after infections, cells were fixed at 4 °C overnight in 75% ethanol, then washed in PBS before stained in 1 mL PI staining solution  containing RNase A  in the dark at room temperature for 30 min. For each sample, around 10,000 PI-stained cells were evaluated with a FACScan flow cytometer (Becton Dickinson).23 The relative mRNA level was determined by normalizing to the level of 18S rRNA. Each real-time PCR  experiment was performed in triplicate, and the data were presented as the average of at least three independent experiments. Primers for real-time PCR are:Total RNA was extracted for reverse transcription and real-time PCR reactions.18S forward: 5′-GTAACCCGTTGAACCCCATT-3′;18S reverse: 5′-CCATCCAATCGGTAGTAGCG-3′.Human cyclin D1 forward: 5′-TGCATGTTCGTGGCCTCTAA-3′;Human cyclin D1 reverse: 5′-GAACTTCACATCTGTGGCAC-3′.After transfection with the indicated plasmids or infection with the indicated adenovirus, cells were stained with indicated antibodies, and the immunofluorescent images were examined using a confocal microscope (A1RSi+).Tissue or cell lysate was homogenized in lysis buffer  and lysed on ice for 10 min. The supernatant was collected after 10 min centrifugation at 13,000 rpm and used for western blotting and co-immunoprecipitation. All original and uncropped images of western blots were provided in the Cells were infected with adenovirus expressing c-terminus MYC-tagged MG53. Upon harvest, cell lysate was prepared by centrifugation at 13,000 rpm for 10 min. MG53 was immunoprecipitated with anti-MYC antibody or IgG on protein A Sepharose 4 Fast Flow . The non-specific bindings were removed with ice-cold lysis buffer and then loading buffer (BIO-RAD #1610737) was added to elute immunoprecipitated protein complex. The eluent was resolved by SDS–polyacrylamide gel electrophoresis (BIO-RAD #1610183) and stained with Coomassie blue. The specific bands were cut out for mass spectrometry analysis, and the IgG control lane was used to subtract background hit in the MS.m/z, 120,000 resolution) is followed by 3 s data-dependent MS/MS scans in an Ion Routing Multipole at 30% normalized collision energy (HCD). The MS/MS spectra from each LC-MS/MS run were identified using Proteome Discovery searching algorithm (version 1.4) to search against the selected database.For LC-MS/MS analysis, peptides were separated using a Thermo-Dionex Ultimate 3000 HPLC system with a 120 min gradient elution at a flow rate of 0.300 μL/min. The analytical column was homemade by packing C-18 resin  in a fused silica capillary column . Mobile phase A and B are 0.1% formic acid, and 100% acetonitrile and 0.1% formic acid, respectively. Xcalibur3.0 software was used to control and process data obtained from an Orbitrap Fusion mass spectrometer operating in a data-dependent acquisition mode. A single Orbitrap scan across full mass spectrum  and lysed on ice for 10 min. The ubiquitinated proteins were immunoprecipitated with indicated antibodies from the cell lysate and resolved on SDS-PAGE for western blotting. The in vitro ubiquitination assay of cyclin D1 was carried out using an ubiquitination kit from Enzo Life Science (cat. no. BML-UW9920-0001) and UBE2H was used in ubiquitination reactions.Cells were incubated with MG132  for 12 h and then harvested in ice-cold PBS. Then cells were re-suspended in RIPA buffer  following manufacturer’s protocol.R2: Genomics Analysis and Visualization Platform (http://r2platform.com). Patients were ranked based on the expression levels of MG53 in their tumors obtained from the TCGA RNAseq and GEO databases. Kaplan–Meier survival analyses and log-rank significance tests were used to compare the survival outcome.Data was analyzed in t test or paired t test. P < 0.05 was considered as statistically significant. Reproducibility and statistics were described in detail in the figure legends.Statistical analyses were performed using GraphPad Prism 8. Data were presented as mean ± s.e.m. The statistical significance of differences between groups were examined using two-tailed unpaired Supplementary MaterialsSupplementary Table S1


Filamentous fungi possess an array of secreted enzymes to depolymerize the structural polysaccharide components of plant biomass. Sugar transporters play an essential role in nutrient uptake and sensing of extracellular signal molecules to inhibit or trigger the induction of lignocellulolytic enzymes. However, the identities and functions of transceptors associated with the induction of hemicellulase genes remain elusive.l-arabinose transporter MtLat-1 is associated with repression of hemicellulase gene expression in the filamentous fungus Myceliophthora thermophila. The absence of Mtlat-1 caused a decrease in l-arabinose uptake and consumption rates. However, mycelium growth, protein production, and hemicellulolytic activities were markedly increased in a ΔMtlat-1 mutant compared with the wild-type (WT) when grown on arabinan. Comparative transcriptomic analysis showed a different expression profile in the ΔMtlat-1 strain from that in the WT in response to arabinan, and demonstrated that MtLat-1 was involved in the repression of the main hemicellulase-encoding genes. A point mutation that abolished the l-arabinose transport activity of MtLat-1 did not impact the repression of hemicellulase gene expression when the mutant protein was expressed in the ΔMtlat-1 strain. Thus, the involvement of MtLat-1 in the expression of hemicellulase genes is independent of its transport activity. The data suggested that MtLat-1 is a transceptor that senses and transduces the molecular signal, resulting in downstream repression of hemicellulolytic gene expression. MtAra-1 protein directly regulated the expression of Mtlat-1 by binding to its promoter region. Transcriptomic profiling indicated that the transcription factor MtAra-1 also plays an important role in expression of arabinanolytic enzyme genes and l-arabinose catabolism.In this study, we reveal that the M. thermophila MtLat-1 functions as a transceptor that is involved in l-arabinose transport and signal transduction associated with suppression of the expression of hemicellulolytic enzyme-encoding genes. The data presented in this study add to the models of the regulation of hemicellulases in filamentous fungi.The online version contains supplementary material available at 10.1186/s13068-023-02305-3. Trichoderma reesei, Myceliophthora thermophila, and Penicillium oxalicum, have been developed as the platforms to produce cellulolytic enzymes for industrial applications [Non-edible plant biomass is recognized as a potential sustainable source for production of biofuels and commodity chemicals in biorefinery processes . Howeverications –5.Neurospora crassa, cellodextrin transporters CDT-1 and CDT-2, and cellobionic acid transporter CLP1 are involved in the induction of expression and secretion of cellulases. The function of CDT-1 and CDT-2 on signal transduction for the activation of cellulolytic gene expression is not dependent on the transporting activities [T. reesei, a dual cellobiose/glucose transporter Stp1 is involved in the carbon catabolite repression response (CCR) and repressed induction of cellulase and hemicellulase genes [N. crassa [Saccharomyces cerevisiae Snf3 and Rgt2 [N. crassa RCO3 [In fungi, lignocellulolytic enzyme-encoding genes are regulated by the complex signaling networks. Sugar transporters play an essential role in substrate uptake and sensing extracellular signal molecules to inhibit or trigger enzyme induction –8. The ttivities . In T. rse genes . Similar. crassa . Some prand Rgt2 , and N. ssa RCO3 , 13. Snfssa RCO3 . HoweverN. crassa, Aspergillus nidulans, and P. oxalicum [T. reesei and Aspergillus niger [N. crassa, M. thermophila, and Fusarium graminearum, XlnR homologs regulate xylanase expression and d-xylose utilization [Fusarium oxysporum, a strain carrying a deletion of xlnR lacked transcriptional activation of structural xylanase genes and exhibited dramatically reduced extracellular xylanase activity [N. crassa and Aspergillus spp. [N. crassa [Magnaporthe oryzae [T. reesei [A. niger [Fungal cellulase and hemicellulase gene expression is tightly regulated at the transcriptional level upon appropriate nutrient sensing . Severaloxalicum –20. Tranus niger –23, Xyr1lization , 24, 25.activity . CreA/CRlus spp. , 28. In . crassa , Magnapoe oryzae  and T. r. reesei , while aA. niger .Myceliophthora thermophila is a thermophilic fungus known for its capability to efficiently secrete a complete set of thermostable carbohydrate-active enzymes [l-arabinose transporter MtLat-1 and demonstrated that MtLat-1 exhibits specific transport activity for l-arabinose [M. thermophila. On the basis of comparative transcriptomic analysis and the assays of a non-transporting MtLat-1 mutant, we found that MtLat-1 functions as a transceptor, which is involved in l-arabinose transportation, sensing, and downstream signaling cascades in M. thermophila. In addition, we also demonstrated that Mtlat-1 expression is directly regulated by transcription factor MtAra-1, which was involved in l-arabinose release and metabolism. enzymes , 32. Rec enzymes  and as a enzymes , 33–35.  enzymes , 36, 37.rabinose . In the l-arabinose [Mtlat-1 on various carbon sources, we conducted comparative transcriptomic analysis of M. thermophila grown on plant biomass-derived monosaccharides, including d-glucose, d-xylose, and l-arabinose. When responding to l-arabinose, 26 putative sugar transporter genes  showed high expression levels (RPKM > 20), including five putative glucose transporter genes , four putative glucose and pentose transporter genes , and two putative cellodextrin transporter genes (Mycth_114107 and Mycth_43941)  of l-arabinose uptake. Therefore, we were interested in the role of the MtLat-1 in the growth of M. thermophila on plant biomass-derived monosaccharides.Our previous study revealed that sugar transporter MtLat-1 (Mycth_95427) showed a high uptake activity and specificity for rabinose . To deteMtlat-1-null strain was constructed via homologous replacement with a neomycin resistance gene (neo)-inclusive cassette using CRISPR/Cas9 system [d-glucose or the three most relevant hemicellulose side-chain sugars , the consumption rates of d-glucose, d-xylose, and d-galactose in the ΔMtlat-1 mutant were similar to those of the wild-type (WT) strain  indicated that l-arabinose uptake was reduced by approximately 15% in the ΔMtlat-1 mutant compared with that in the WT (15.4 nmol/min/mg_DCW)  l-arabinose level, but led to a growth defect at low l-arabinose level [The 9 system . The corbolished . Howeverse level .d-xylose and l-arabinose) can act as inducers of the expression of genes encoding plant biomass-degrading enzymes [l-arabinose-specific transport activity, participates in sensing and transducing an l-arabinose signal that is involved in the induction of hemicellulolytic enzymes in M. thermophila. To test this hypothesis, physiological phenotypes of the strain ΔMtlat-1 were assayed when it was grown on arabinan or xylan. Unexpectedly, the strain ΔMtlat-1 exhibited an approximately 14% increase in dry cell weight compared with the WT when grown on arabinan for 4 days  exhibited markedly up-regulated expression levels in ΔMtlat-1 compared with the WT, which is a prerequisite for efficient sugar utilization and mycelium growth. An analysis of the transcriptional profiles of Carbohydrate-Active enzymes (CAZyme) genes  indicated that genes encoding hemicellulolytic enzymes were dramatically induced in strain ΔMtlat-1 relative to the WT  and three xylanolytic enzyme genes  showed significantly increased expression levels in the ΔMtlat-1 mutant compared with the WT. In addition, the genes associated with the pentose catabolism were significantly up-regulated in ΔMtlat-1, including xylose reductase , l-arabinitol 4-dehydrogenase , xylitol dehydrogenase , l-xylulose reductase , and d-xylulose kinase , which might attributed to increased l-arabinose release resulting from the enhancement of hemicellulolytic enzyme activity in ΔMtlat-1.To further investigate the effect of MtLat-1 on hemicellulase production, comparative transcriptomic profiles of the WT and ΔS. cerevisiae strain BSW5AP with a heterogenous arabinose metabolic pathway and native l-arabinose transporter gene deleted [l-arabinose, showing that the mutation abolished the transport function of MtLat-1 , respectively. The initial l-arabinose uptake rate of strain Ptef-Mtlat-1(R136K) was lower than that of mutant Ptef-Mtlat-1  located in a cytoplasmic loop just preceding the fifth transmembrane domain of several hexose sensors, leads to the loss of substrate uptake activity, but constitutive glucose signaling , 45, 46. deleted  was usedt-1 Fig. : Fig. S5Mtlat-1 strain. When grown on arabinan, the resultant strain Ptef-Mtlat-1M showed mycelium yield, secreted protein content, and hemicellulolytic activities similar to those of the strain Ptef-Mtlat-1(R136K), revealing that the C-terminal cytosolic region of MtLat-1 was dispensable for the function of the protein in transmitting the l-arabinose signal.Previous studies have shown that C-terminal cytoplasmic sequence extension of several transceptors related to the CCR trigger or the induction of cellulase was required for their interaction with a component of the molecular signal transduction pathway , 48. To Mtlat-1, we determined transcriptomic profiles of all transcription factor genes in M. thermophila when grown on mono-saccharides  or under starvation (no carbon) conditions. As shown in Fig. Mtlat-1 was specific to the presence of l-arabinose, we considered the four genes  that were only highly induced by l-arabinose as the candidate regulators of Mtlat-1. Next, single-gene mutants of these genes were constructed and the expression level of Mtlat-1 in response to l-arabinose was detected by real-time quantitative PCR (RT-qPCR) assay. Three of the mutants showed no significant change in Mtlat-1 expression compared with the WT strain. However, the ΔMycth_2121737 strain showed dramatically decreased expression of Mtlat-1  and T. reesei (Ara1) were related to utilization of arabinan, l-arabinose, and d-galactose [To search for transcription factors essential for expression of alactose , 27. TheMtlat-1 expression, we conducted electromobility shift assays (EMSAs), involving the DNA-binding domain (DBD) of MtAra-1 and the promoter region of Mtlat-1. A glutathione S-transferase (GST)-fused MtAra-1-DBD was expressed in and purified from Escherichia coli. Two probe fragments of the promoter region of Mtlat-1  were amplified by PCR. In the EMSAs, the recombinant MtAra-1 bound the Mtlat-1 promoter regions in a typical protein concentration-dependent manner. Retardation occurred upon addition of 10 nM recombinant MtAra-1 protein  were also dramatically downregulated in ΔMtara-1 grown on l-arabinose , also catalyzes the third reaction of the oxidoreductive catabolism of d-galactose. Consequently, we also found that the ΔMtara-1 strain showed a severely decreased rate of d-galactose consumption   from N. crassa, LAT-1 from Ambrosiozyma monospora [Pichia stipitis [Penicillium chrysogenum [l-arabinose transporters, their effects on physiological phenotypes differ in various fungi when grown on l-arabinose and arabinan. In T. reesei, Lat-1 is a high-affinity symporter and shows high specificity for l-arabinose. The absence of Trire2_104072 did not result in an altered growth phenotype or different total protein secretion when grown on in high (1%) concentration of l-arabinose, lactose, or spent grain extract with a high content of arabinoxylan [N. crassa lat-1 mutant was observed on arabinan. Moreover, in N. crassa, lat-1 disruption caused a remarkably reduced l-arabinose uptake, which abolished the induction of ard-1 (gene encoding l-arabinitol-4-dehydrogenase) at low (2 μM) l-arabinose concentrations [Mtlat-1 was specifically induced by l-arabinose and its absence resulted in a decrease in l-arabinose uptake and consumption rates, but not those of d-glucose, d-xylose, or d-galactose, which is consistent with the specific l-arabinose transport activity of MtLat-1 [Mtlat-1 mutant when grown on arabinan. Comparative transcriptomic analysis revealed that deletion of Mtlat-1 enhanced the expression of genes encoding xylanolytic enzymes and arabinanolytic enzymes. These results suggested that l-arabinose transporter MtLat-1 repressed the expression of hemicellulolytic enzymes on arabinan but not on xylan. Furthermore, a point mutant of MtLat-1 in which the transport activity was abolished exhibited similar effects to the wild-type MtLat-1 on the protein secretion and hemicellulolytic activities on arabinan, clearly demonstrating that the involvement of MtLat-1 in M. thermophila hemicellulase gene expression was independent of its transport activity. These experiments have helped to separate nutrient sensing from substrate transport, and indicate that MtLat-1 protein serves as a transceptor, which might sense and transduce molecular signal of l-arabinose, one of the end products of hemicellulose degradation, to repress the induction of hemicellulase genes. The role of transceptor in the induction of genes involved in plant biomass degradation has also been demonstrated for CDT-1/2 in N. crassa and Crt1 in T. reesei [Our previous study revealed that sugar transporter MtLat-1 of rabinose . LAT-1 ionospora , and Arastipitis  and Peniysogenum . Althouginoxylan . Howevertrations . Herein, MtLat-1 . However. reesei , 52.C-terminal amino acids did not alter their transport activities, but abolished their signaling function [T. reesei, a Crt1 mutant retaining only the first five amino acids of C-terminus still had the lactose transport activity but lost the cellulase induction activity [M. thermophila, MtLat-1 has a C-terminal extension of 63 amino acids, which is markedly shorter than those of Snf3 (303 amino acids) and Rgt2 (220 amino acids), but longer than that of Crt1 (44 amino acids) [-terminus restored the growth phenotype and hemicellulolytic activities of strain ΔMtlat-1 to similar levels to those of the WT on arabinan, showing that the C-terminal cytosolic region of MtLat-1 is not required for its signaling function. Similarly, in Pichia pastoris, deletion of the C-terminal fragment (150 amino acids) of glucose sensor Gss1 slightly affected glucose catabolite repression and pexophagy, but the signaling function of the protein was maintained [T. reesei, the internalization of Crt1 was observed on cellulose, although it was also found under glucose or glycerol and may not be correlated with the induction of cellulases [The C-terminal cytoplasmic fragments of several transporters and sensors have been reported to be essential for signal transduction . Truncatfunction , 52. In o acids) , 52. Howintained . Signal intained . In T. rT. reesei, XlnR/XYR1 regulates both cellulase and hemicellulase genes, while it is a major positive activator of hemicellulase genes in N. crassa and F. graminearum [M. thermophila, Xlr1 controls the expression of xylanolytic genes and genes involved in pentose transport and catabolism, but it has a smaller impact on l-arabinose catabolism [d-galactose and l-arabinose releasing and catabolic genes. In N. crassa and T. reesei, deletion of ara-1 dramatically downregulated the expression of l-arabinose transporter Lat-1 [M. thermophila, the induced expression of Mtara-1 was observable only on l-arabinose, as also observed for Mtlat-1. Transcriptional analysis and EMSAs demonstrated that MtAra-1 directly regulates Mtlat-1 expression by binding to its promoter region. Consistent with observations in N. crassa [Transcription factors play an essential role in regulation of the expression of CAZyme genes and genes involved in sugar transport and intracellular catabolism. Several essential transcription factors associated with hemicellulose deconstruction and utilization have been studied in fungi, including CreA/CRE-1, Xyr1/XlnR, and Ara-1. CreA/CRE-1 is a major regulator of CCR, a process through which the expression of genes involved in the utilization of non-preferred carbon sources, including hemicellulose/cellulose, are inhibited , 55. In minearum , 21, 24.tabolism . In seveer Lat-1 , 42. In . crassa , 27,Mtara-1 disruption abolished the growth of M. thermophila on l-arabinose and d-galactose, but no effect on the growth of strain ΔMtara-1 was observed on d-xylose. Moreover, the ΔMtara-1 mutant exhibited a remarkable reduction in secreted protein production and hemicellulolytic activities when grown on arabinan. Our transcriptomic data reflected the significantly downregulated expression of all arabinanolytic enzyme-encoding genes in the ΔMtara-1 mutant when it was grown on arabinan. These results demonstrated that MtAra-1 is mainly involved in regulation of the expression of arabinanolytic genes and the genes associated with l-arabinose and d-galactose catabolism in M. thermophila.l-arabinose transport and signal transduction associated with the suppression of expression of hemicellulolytic enzyme-encoding genes in M. thermophila. The absence of Mtlat-1 caused a decrease on l-arabinose uptake and consumption rates, but led to increases in mycelium growth, secreted protein production, and hemicellulolytic enzyme activities on arabinan. Furthermore, point mutation of MtLat-1 revealed that the involvement of MtLat-1 in the expression of hemicellulase genes is independent of its transport activity. Moreover, transcription factor MtAra-1 played an important role in the induction of arabinanolytic genes and l-arabinose catabolism, and directly regulated Mtlat-1 expression by binding to its promoter region.In this study, we demonstrated that MtLat-1 is involved in M. thermophila ATCC 42464 was obtained from the American Type Culture Collection (ATCC). This WT strain and its mutants were grown on Vogel’s minimal medium (VMM) with 2% (w/v) glucose at 35 °C to obtain mature conidia. Antibiotic was added when needed to screen for transformants. For media shift experiments, M. thermophila strains were precultured in 100 mL of 1 × VMM with 2% glucose for 16 h. Subsequently, the mycelia were collected, washed three times with 1 × VMM, and then transferred to 100 mL of fresh 1 × VMM with 2% l-arabinose (Sigma) or arabinan (Megazyme) for continued incubation for 2 h. For fungal growth assays, mature conidia were inoculated into 100 mL of 1 × VMM with 2% carbon source  at a concentration of 2.5 × 105 conidia/mL in 250-mL Erlenmeyer flasks. The culture was incubated at 45 °C with rotary shaking at 150 rpm.S. cerevisiae BSW5AP with heterogenous arabinose metabolic pathway [l-arabinose medium in 250-mL Erlenmeyer flasks at an initial OD600 value of 1.0. Cells were cultivated in an orbital shaker at 200 rpm at 30 °C and samples were taken at intervals. pathway , was culE. coli Mach-T1 cells were used for vector manipulation and propagation, and were cultivated in Luria–Bertani medium with 100 µg/mL ampicillin or 50 µg/mL kanamycin for plasmid selection.Mtlat-1, Mycth_95427; Mtara-1, Mycth_2121737; MtPdr-1, Mycth_53224; MtPdr-2, Mycth_46266; Mycth_2300935) were identified using the sgRNACas9 tool [M. thermophila U6 promoter sequence, the synthetic gRNA scaffold sequence, and the target DNA sequence were constructed via overlapping PCR and cloned into the pJET1.2/blunt cloning vector to generate the corresponding plasmids U6p-Mtlat-1-sgRNA, U6p-Mtara-1-sgRNA, U6p-MtPdr-1-sgRNA, U6p-MtPdr-2-sgRNA, and U6p-Mycth_2300935-sgRNA. To construct donor DNA sequences, the 5′- and 3′-flanking fragments of Mtlat-1, Mtara-1, MtPdr-1, MtPdr-2, and Mycth_2300935 were separately amplified by PCR from M. thermophila genomic DNA, fused with the selectable marker cassette PtrpC-neo from plasmid p0380-neo [The primer sequences used in this study are listed in Additional file as9 tool . Fragmen0380-neo , and cloMtlat-1 (1934 bp), was amplified from the M. thermophila genome and ligated between the BglII and BamHI sites of pAN52-PgpdA-bar [SpeI and BamHI sites, under the control of the strong constitutive tef1 (Mycth_2298136) promoter of M. thermophila, to generate the corresponding recombinant vectors. Polyethylene glycol (PEG)-mediated protoplast transformation for gene disruption or overexpression in M. thermophila was performed as described previously [To generate the complementation strain, a DNA fragment containing the upstream region (1485 bp), the downstream region (853 bp), and full-length gpdA-bar  to genergpdA-bar  at the Seviously .S. cerevisiae strain BSW5AP, the genes encoding MtLat-1, MtLat-1–GFP, MtLat-1(R136K), and MtLat-1(R136K)–GFP were separately inserted into the shuttle plasmid p426kanmx4 [S. cerevisiae transformations were carried out as described previously [For expression of transporters in 26kanmx4  with theeviously .M. thermophila, mycelia were pre-grown in VMM with 2% arabinose for 16 h at 45 °C and then the cells were observed using the Olympus BX51 fluorescence microscopy system. Recombinant S. cerevisiae BSW5AP strains expressing sugar transporters tagged with GFP were inoculated into YPD medium and grown to the exponential phase (OD600 ~ 1.0). The cells were collected, washed twice with sterile water, and then resuspended in sterile water. Next, 10 μL of culture was spotted on a cover glass for confocal microscopy. The images were processed using ImageJ software.To detect subcellular localization of MtLat-1 and its mutants in M. thermophila strains were precultured in 1 × VMM containing 2% glucose for 18 h. Then, mycelia were washed three times in 1 × Vogel’s salts without any carbon source and shifted into 0.5% arabinose medium for the additional 4 h of induction. After that, the mycelia were harvested, washed again as above, and resuspended in uptake buffer  for 20 min. The residual sugar in the supernatant was determined using HPLC with an e2695 instrument , and the fungal biomass was completely dried to determine the dry weight for data normalization.The M. thermophila strain were inoculated into 100 mL of 1 × VMM with 2% carbon source  at a concentration of 2.5 × 105 conidia/mL in 250-mL Erlenmeyer flasks and incubated at 45 °C with rotary shaking at 150 rpm. Samples were taken at the indicated time for the assays of secreted protein and enzyme activity. The total extracellular protein in culture supernatants was measured using a Bio-Rad protein assay kit. Endo-1,5-l-arabinanase, endo-1,4-xylanase, and endo-glucanase activities in the supernatants of culture of M. thermophila strains were measured using an Azo-Xylan kit (Megazyme), an endo-1,5-l-arabinanase assay kit (Megazyme), and an Azo-CM-Cellulose assay kit (Megazyme), respectively.The mature conidia of M. thermophila mycelia were shifted into 1 × VMM with 2% carbon source  for the induction of 4 h, harvested by vacuum filtration, and immediately ground to a fine powder using a pestle and mortar with liquid nitrogen for the subsequent extraction of total RNA [Mtlat-1 deletion mutant grown on arabinan, M. thermophila wild-type strain and Mtlat-1 deletion mutant were cultured in 1 × VMM with 2% arabinan (Sampled at 2 days and 4 days).otal RNA . For traRNA integrity and concentration were determined using agarose gel electrophoresis and NanoDrop spectrophotometer. Purified RNA samples, with RNA integrity number > 8.0, determined using an Agilent 2100 Bioanalyzer (Agilent Technologies), were sequenced by Novogene Corporation  using the Illumina NovaSeq 6000 platform to generate 150-bp paired-end reads.M. thermophila ATCC42446 genome sequence [2 fold-change|≥ 1, RPKM ≥ 20 (at least one sample), and DESeq P-adj value < 0.05, were considered significantly differentially expressed between two samples. The detailed data are shown in Additional file Clean reads were mapped to the sequence  using Tosequence . The cousequence . Unless M. thermophila strains were shifted into induction medium containing 2% arabinose as the carbon source and incubated for 4 h, then harvested for the subsequent extraction of total RNA using the method described previously [−ΔΔCt method with the actin-encoding gene (Mycth_2314852) as the internal control.For RT-qPCR analysis, precultured mycelia of eviously . QuantitM. thermophila cDNA and inserted between the BamHI and XhoI sites of pGEX-4T-1  to form a GST-tagged protein expression plasmid. The recombinant plasmid was introduced into E. coli BL21 (DE3) cells for protein expression and purification as previously described [The DNA sequence encoding the DBD of transcription factor MtAra-1  was amplified from escribed .Mtlat-1  were amplified from M. thermophila genomic DNA using primers shown in Additional file EMSAs were performed as described previously . BrieflyAdditional file 1. Table S1: List of PCR primers used in this study. Table S2: Profiles of RNA-Seq reads mapped to the genome of M. thermophila. Table S3: Transcriptomic profiles of 26 sugar transporters with robust expression levels (RPKM > 20) in at least one tested condition. Table S4: Genes showing significantly different transcriptional levels in strain ∆Mtlat-1 compared with the WT when grown on 1 × VMM with 2% arabinan for 4 days. Table S5: Transcriptomic profiles of genes encoding major hemicellulases from RNA-Seq data when grown on 1 × VMM with 2% arabinan. Table S6: Gene ontology (GO) analysis of up-regulated genes in strain ΔMtlat-1 compared with the WT when grown on 1 × VMM with 2% arabinan for 4 days. Table S7: Transcriptomic profiles of transcription factor genes with significantly upregulated expression levels in WT M. thermophila grown on l-arabinose, d-xylose, or d-glucose, compared with that under no carbon. Table S8: Genes showing significantly different transcriptional levels in strain ∆Mtara-1 compared with the WT strain when grown on 2% arabinan for the induction of 4 h.Additional file 2. Fig. S1: Sugar consumption by M. thermophila strains WT, ΔMtlat-1, and ΔMtara-1, when grown in 1 × VMM with 2% d-glucose (A), 2% d-xylose (B), or 2% d-galactose (C). Error bars indicate the SD from at least three biological replicates. Fig. S2: Growth phenotypes of the M. thermophilaΔMtlat-1 mutant under xylan condition. A Cell dry weight of M. thermophila strains WT and ΔMtlat-1 after growth on 2% xylan for 2 d. B Protein concentrations, C xylanase activity, and D arabinanase activity of the culture supernatants for the M. thermophila strains grown in 2% xylanase medium. Error bars indicate the SD from at least three biological replicates. Fig. S3: Growth phenotypes of the complementation strain of the ΔMtlat-1 mutant. Al-arabinose transport rates of mycelia from the complementation strain Pn-Mtlat-1. B Cell dry weight of the M. thermophila complementation strain after growth on 2% arabinan for 4 days. C Protein concentrations, D arabinanase activity, and E xylanase activity of the culture supernatants for M. thermophila grown in 2% arabinan medium. Error bars indicate the SD from at least three biological replicates. Fig. S4: Comparative transcriptomic analysis of the WT and ΔMtlat-1M. thermophila strains grown in arabinan medium for 2 d. A Total expression of genes encoding major hemicellulases from RNA-Seq data. B Transcriptional profiles of genes encoding arabinanolytic enzymes in the ΔMtlat-1 and WT strains when grown on 2% arabinan for 4 days. Fig. S5: Cell dry weight of M. thermophila strains after growth on 2% arabinan for 4 days. Fig. S6: Heatmap analysis of expression profiles for putative sugar transporter genes with statistically significant differences in transcript levels between ΔMtara-1 and the WT under l-arabinose condition. Log-transformed expression values are color-coded. Fig. S7: Protein concentrations and hemicellulase/cellulase activity of the culture supernatants for M. thermophila strain ΔMtara-1 grown in 1 × VMM with 2% xylan (A) or 2% Avicel (B) for 4 days.