Evidence-based clinical practice guidelines (CPGs) are relevant sources for generating quality indicators (QIs). The objective of this study was to compare guideline-based QIs of German and international CPGs and their underlying methodological approaches.n = 35) that report QIs, which were identified in a preceding study. Additionally, we searched the websites of the particular CPG providers for separate documents with regard to QIs. We included evidence-based CPGs which report QIs. Reported QIs, the underlying guideline recommendations, and information on methods of development were extracted. The selection and extraction of CPGs were conducted by one reviewer and checked by another. For each matched pair of CPGs, we assessed whether the suggested QIs matched or were not directly comparable.We conducted systematic searches in the guideline databases of G-I-N  and NGC  between February and June 2017 to identify international CPGs matching the topics of German evidence-based CPGs . We found 27 QI pairs with QIs “not different or slightly different”, corresponding to 13% (27 of 212) of the QIs in German CPGs and 16% (27 of 166) in international CPGs. Only two QI pairs were judged to be “different/inconsistent”. For 183 of 212 (86%) QIs from German CPGs and 137 of 166 (83%) QIs from international CPGs, no direct comparison could be made. An explicit link to one or more guideline recommendations was found for 136 of 152 (89%) QIs from German CPGs and 82 of 166 (49%) QIs from international CPGs. Some information on methods for the development of QIs existed for 12 of 18 (67%) German CPGs and 8 of 25 (32%) international CPGs.The majority of QIs in German and international CPGs were not comparable. Various reasons for this are conceivable. More transparent reporting of the underlying methods for generating guideline-based QIs is needed.The online version of this article (10.1186/s13012-019-0918-y) contains supplementary material, which is available to authorized users. Quality measurement and improvement play an important role in the provision of healthcare. For this purpose, quality indicators (QIs) can be used. There is no clear-cut definition of a QI. According to Lawrence and Frede, a QI is a “measurable element of practice performance for which there is evidence or consensus that it can be used to assess the quality, and hence change in the quality, of care provided” . The JoiAs evidence-based clinical practice guidelines (CPGs) are designed to reflect current best practice, they are relevant sources for generating QIs , 9. The In Germany, the AWMF  provides a methodological framework for the development of CPGs by the scientific medical societies. The guideline classification scheme of the AWMF differentiates between S1-, S2k-, S2e-, and S3-CPGs depending on the methodological approach . Thus, SAlthough a working group of the Guidelines International Network (G-I-N) recently proposed a set of reporting standards for guideline-based performance measures , there iThis study was part of the project “Systematic analysis of the translation of guideline recommendations into quality indicators and development of an evidence- and consensus-based standard”, supported by the German Research Association (DFG). Our analysis provided information for another part of the research project, a qualitative study which consisted of structured interviews with developers, methodologists, and users of international guidelines . The intention of both studies was to add information to existing research on methods for the guideline-based development of QIs , 17. TheThe objective of this study was to compare guideline-based QIs of the 35 previously identified German S3-CPGs, as well as their underlying methodological approaches, with those of international CPGs on related topics.The study was aligned with the PRISMA guidelines , althougQIs are reported.The CPG is an evidence-based CPG.The topic and recommendations are comparable with those of at least one of the 35 previously identified German S3-CPGs , they had to be linked explicitly with the particular CPG.An example for such a separate document containing guideline-based QIs is a document from the website of the National Institute for Health and Care Excellence (NICE): “NICE menu of general practice and clinical commissioning group indicators” . The menAre based on a systematic literature searchAre clearly identifiable and assigned with a grade of recommendation (GoR) and/or a level of evidence (LoE)Are linked to the references of the underlying evidence.Evidence-based CPGs were defined in this analysis as CPGs whose recommendationsWe conducted systematic searches in the guideline databases of G-I-N and NGC  between February and June 2017 to identify international CPGs matching the topics of the previously identified German S3-CPGs which report QIs. The search strategies included keywords related to the clinical topics, both as full terms and with appropriate truncations, connected with Boolean operators. For six of the CPGs from the CPG program oncology and for all German S3-CPGs on diabetes, we conducted one combined search each ; for the remaining German S3-CPGs, separate searches were performed Label of the quality measure, e.g. QI, quality criteria and performance measureCategorization of QI into structure, process, or outcome indicators according to the definition of Donabedian   × 7 (items) × 2 (appraisers)) .In case reviewers had been involved in the development of an included CPG, they did not participate in their quality assessment.Data synthesis involved a descriptive analysis and a tabular comparison of the QIs of the international and German S3-CPGs for each clinical topic and, where applicable, for each underlying recommendation. We collected the number of CPGs that provided information on the QI development group, methods of QI development, as well as the rationale and intended purpose of QIs. On the basis of reported QIs, we collected the number of QIs for which quality objectives and measurement properties were reported as well as the number of QIs that were explicitly or implicitly based on guideline recommendations.For each matched pair of CPGs, we compared the suggested QIs and assessed whether the QIs matched or not.Our definition of QI-matching was that both QIs on the same clinical topic either agreed or disagreed in content and definitions regarding a specific clinical issue, e.g. a specific intervention or diagnostic procedure and either addressed or did not address the same population. Then, we assigned QIs either to the category “not different/slightly different” or “different/inconsistent”. QIs were considered not to match whenever no direct comparison could be made because the QIs differed fundamentally in contents and definitions. Thus, the QIs either addressed different specific issues within a clinical topic, or were reported in only one of the matched CPGs, even though both CPGs addressed that particular clinical topic. For example, the topic “screening” was addressed by both CPGs of a matched pair, but only one had defined QIs for that topic. Those QIs were extracted under the category “QI only defined in the international or the German S3-CPG”, respectively. For each of the categories described above, we collected the number of QIs or QI pairs. The assignment of the QIs to the categories was conducted by one reviewer and checked by another. Disagreements were resolved through discussion and consensus. Furthermore, the methods described for QI development were presented as a narrative summary.The searches in the CPG databases identified 4889 records. We found seven additional potential eligible international CPGs by crosschecking the reference lists of included CPGs. After the initial screening of the titles, 289 full texts were reviewed, out of which 264 were excluded  for the domain “Methodological Rigor of Development” for the German S3-CPGs and 62% (standard deviation 12.7) for the international CPGs. For the individually rated items and resulting scores for each CPG, see Additional file Overall, the German S3-CPGs and international CPGs contained 152 and 166 QIs on related topics, respectively. The median number of QIs per CPG was 8 (range 0–37) in the German S3-CPGs and 4.5 (range 1–15) in the international CPGs. With regard to the 30 CPG pairs, we compared 212 QIs from German S3-CPGs to 166 QIs from international CPGs . The QIs in 85% of German S3-CPGs (129 of 152) and 84% of international CPGs (139 of 166) were presented as ratios or proportions (defining numerator and denominator or quoting percentages).In 17% (3 of 18) of German S3-CPGs and 28% (7 of 25) of international CPGs, a categorization of QIs into structure, process, or outcome indicators was made by the CPG authors themselves. According to our own assignment, we found mainly process indicators: 123 of 152 (81%) in the German S3-CPGs and 133 of 166 (80%) in the international CPGs. However, for 12 of 64 (19%) QIs, we disagreed with the categorisation made by the authors of the international CPGs and therefore changed the category. For all nine QIs that were categorised by the authors of the German S3-CPGs, we agreed with the assignment.The intended purpose of the QIs was reported in 13 of 18 (72%) German S3-CPGs and in 21 of 25 (84%) international CPGs. The rationale for the QIs was stated in only one of 18 (6%) German S3-CPGs and in one of 25 (4%) international CPGs.An explicit link to one or more guideline recommendations was found for 136 of 152 (89%) and 82 of 166 (49%) QIs from 15 German S3 and 12 international CPGs, respectively.Among these, 77% (104 of 136) of QIs from German S3-CPGs and 93% (76 of 82) from international CPGs were based on strong recommendations. Of these strong recommendations, 43% (45 of 104) in the German S3-CPGs were consensus-based. This means they were based on the expert opinion of the CPG group, given that none or insufficient evidence exists for generating an evidence-based recommendation . No recommendation in international CPGs was explicitly stated to be consensus-based, but all were evidence-based. However, the quality of the underlying evidence in five international CPGs from KCE and ICSI is mostly designated as “low” or “moderate”. The underlying evidence of the strong recommendations in the seven included CPGs by NICE was mostly not clearly stated. For one of 152 (0.7%) QIs in the German S3-CPGs and 23 of 166 (14%) in the international CPGs, we found an implicit connection, as we identified one or more corresponding recommendation(s) in the particular CPG.Quality objectives were stated for 39 of 152 (26%) QIs in the German S3-CPGs and for 39 of 166 (23%) QIs in the international CPGs. Properties were not reported for any QI measurement.An overview of the QIs is presented in Fig. Twelve of the 30 CPG pairs comprised 27 QI pairs that were “not different or slightly different”. This corresponds to 13% (27 of 212) of the QIs in German S3-CPGs and 16% (27 of 166) in international CPGs. Only two QI pairs were judged to be “different/inconsistent”. For the majority of Qis, no direct comparison could be made, i.e. those QIs were found only in either the international or the German S3-CPGs Table .Table 3CInformation on how QIs were developed was provided in 12 of 18 (67%) German S3-CPGs and eight of 25 (32%) international CPGs. Nine of the German S3-CPGs and one of the international CPGs  searchedRegarding the underlying evidence for QIs in the German S3-CPGs of NDMG and GGPO, it is stated that QIs should be derived from strong recommendations. This methodological approach was implemented in 11 of the 18 (61%) German S3-CPGs. None of the CPGs of the scientific medical societies gave information on underlying evidence. Among the international CPGs, eight of the 25 (32%) CPGs originating from KCE and NICE provided information on which recommendation or grade of recommendation should be considered. For the KCE-CPG, it was explicitly stated that only strong recommendations were considered for the derivation of QIs. The NICE-CPGs required proposed QIs to be linked by evidence to improved outcomes. For the remaining 17 international CPGs, no information was given.None of the QIs from German S3-CPGs were reported to be piloted or evaluated, whereas eight international CPGs included a report on pilot testing during the development of QIs. Those international CPGs originated from only two CPG providers (KCE and NICE).An overview on methodological aspects is presented in Table Information on the composition of the QI development group was given in 14 of 18 (78%) German S3-CPGs and in 12 of 25 (48%) international CPGs. In the international CPGs, this information originated from three CPG providers . In four German S3-CPGs and 13 international CPGs, no information on the QI development group was given.Clinicians, methodologists, and representatives of cancer registries were involved in the development of QIs of the KCE-CPG. According to the process guide of NICE, the QI development groups were multidisciplinary . However, there was no information on the actual composition of QI development groups for each individual included NICE-CPG. In one SIGN-CPG, it is stated that the QIs were defined by the CPG group.Among the German S3-CPGs, all CPGs of the NDMG and GGPO and three CPGs developed by scientific medical societies gave information on the QI development group. For seven of the included German S3-CPGs, the QI development group comprised clinicians of different medical specialties, methodologists, and patient representatives, and in another seven German S3-CPGs, a participation of patient representatives was not reported.An overview on the information on QIs, methods of development, and composition of QI development groups is given in an additional file for each included CPG  reported information on the methods used to develop QIs. However, information was missing in these cases as well, such as reporting of the selection and extraction of CPG recommendations and their translation into QIs. Among the German S3-CPGs, all CPGs of the NDMG and the GGPO provided information on methods, whereas almost none of the CPGs of the medical societies contained methodological information. The quality appraisal score for the domain “Methodological Rigor of Development” ranged from 50 to 83% and from 48 to 83% in the German S3-CPGs and international CPGs, respectively. High scores were not inevitably related to better description of the methods of developing the QIs or better reporting of QIs. Although it is assumed that the degree of credibility of QIs is associated with the methodological quality of CPGs, the evidence for this is lacking so far.Various reasons are conceivable that would explain that QIs of different CPGs on the same clinical topic often did not cover the same quality aspect of care. One factor could be the different methodological approaches, e.g. to defining selection criteria for recommendations, to appraising the relevance of a QI for health care improvement, or to assessing feasibility of measurement. However, because it was rarely reported how QIs were generated , we were unable to analyse this point in further detail. Therefore, for a better understanding of how guideline-based QIs are generated, a better reporting of the underlying processes is necessary. A proposal for reporting standards for guideline-based performance measures has been developed by a working group of G-I-N .Furthermore, although we compared only QIs on clinical topics that were addressed in both CPGs of a CPG pair, several recommendations of the German S3-CPGs and the related international CPGs varied to some extent in content and definitions. Most of the recommendations reported in international and German S3-CPGs were not inconsistent but had a different focus or depth of detail. For example, the German S3-CPG “Type 2 diabetes training” recommended to offer a structured education program, whereas the international CPG conducted by ICSI on “Diagnosis and Management of Type 2 Diabetes Mellitus in Adults” comprised a specific recommendation of nutrition therapy. Nutrition therapy was also considered in the particular German S3-CPG within the explanatory text. However, no specific recommendation on nutrition was made. Further, there were other cases where both CPGs of a CPG pair comprised comparable recommendations, but only in one of the CPGs, a QI was derived from the recommendation(s).Also, different definitions of QIs may result from an inconsistent composition of the QI development groups with methodologists, relevant health care professionals, stakeholders, and patients. A study about the consistency of QI selection for cardiovascular risk management across different consensus methods and panels found, in part, considerable variation, but could not explain the underlying factors . FurtherThe analyses of the two inconsistent QI pairs found that the underlying recommendations are also inconsistent, even though the link between QI and recommendation in the international CPG is only implicit. For example, the SIGN-CPG on ovarian cancer recommends that first-line chemotherapy should include a platinum agent either in combination or as a single agent , whereasStudies on the transferability of non-guideline-based QIs between the USA and the UK and between the USA and the Netherlands found that about 56% and 67%, respectively, were “exactly or nearly equivalent” or “(nearly) identical” , 71. AccOur analysis found that over 40% of the strong recommendations in the German S3-CPGs are based exclusively on the expert opinion of the CPG group. Furthermore, the quality of the underlying evidence of many strong recommendations in the international CPGs was designated as “low” or “moderate”. This appears to contradict the methodological requirement that QIs should be based on scientific evidence, where possible , 8. HoweEspecially in the international CPGs, information on the composition and responsibilities of the QI development group is lacking. Further understanding of the interaction between the QI and CPG development groups are needed, if they work independently. In this context, cooperation and mutual feedback between these stakeholders are reasonable. For example, the QI development group might need further background information regarding recommendations, or the results of the QI development could lead to a revision of recommendations.None of the QIs from German S3-CPGs were piloted or evaluated. However, this step ought to be seen as an essential element in the process of developing QIs , 76. To The strength of our analyses is the systematic methodological approach which followed a pre-defined protocol. However, although we conducted systematic literature searches in the two main guideline databases, we may have missed CPGs not included in the databases. A further limitation of our analysis is that we probably missed information on methodological issues from further CPG providers, as we only included CPGs that matched substantively with a German S3-CPG. Furthermore, potential limitations arise from the fact that both the selection of CPGs and data extraction were performed by only one reviewer and checked by another. This pragmatic approach was chosen because of the large number of hits obtained by the diverse searches, as well as the low level of complexity regarding inclusion criteria in our study. Moreover, the data extraction is in agreement with a recent methodological guide on systematic reviews of CPGs .We did not analyse the aspect of evidence underlying the QIs in the German S3- and international CPGs in depth, as we found various systems rating the quality of evidence and grading the strength of recommendations in the CPGs.Finally, the interpretability of our results might be limited as we compared the QIs on clinical topics that were addressed in both CPGs of a CPG pair directly, rather than at the recommendation level. As noted above, although the CPG pairs addressed the same clinical topics, the recommendations varied to some extent and, in some cases, resulted in QIs that were not comparable. However, it should be noted that only about half of the QIs reported in international CPGs were based explicitly on guideline recommendations. The underlying approaches for generating such QIs were not reported in sufficient detail.The majority of QIs in German and international CPGs were not comparable. Various reasons for this are conceivable, such as methodological issues or contextual differences between countries. However, no clear reason could be deduced from the available data. Detailed information on the methodological approaches of generating QIs is lacking. More transparent reporting of the underlying methods for generating guideline-based QIs is recommended.Additional file 1:German S3-CPGs which report QIs. (DOCX 27 kb)Additional file 2:Search strategies. (DOCX 17 kb)Additional file 3:Excluded articles. (DOCX 55 kb)Additional file 4:Quality appraisal of included CPGs. (DOCX 35 kb)Additional file 5:Topic-related QIs in international CPGs and German S3-CPGs and if applicable corresponding recommendations. (DOCX 238 kb)Additional file 6:Comparison of topic-related QIs in international CPGs and German S3-CPGs. (DOCX 37 kb)Additional file 7:Table a. Data-Extraction of German S3-CPGs - general and methodical aspects and Table b. Data-Extraction of international CPGs - general and methodical aspects. (DOCX 60 kb)


Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3 However, over half of the patients transplanted with human islets returned to the insulin-dependent, diabetic state within 5 years4. The causes for this late islet graft loss are still controversial. Previous reports encompass a higher rate of islet apoptosis due to endoplasmic reticulum (ER) stress6, hypoxia8 in end-portal venules within the liver, and recurrent autoimmunity9. In addition, there were evidences of metabolic deterioration due to lipid accumulated around the islets (lipotoxicity)11 and toxicity of immunosuppressive drugs13, which could all result in graft loss. Last but not least, insufficient immunosuppression could also be an important cause of chronic islet loss especially due to antibody-mediated rejection processes14. However, none of the above could clearly and single-handedly explain the exact causes of chronic islet graft loss.The Edmonton protocol was introduced in 200015. This unique opportunity allowed us to examine how the porcine islets are lost in the late phase of islet xenotransplantation. Here, we selected two monkeys with the same immunosuppressive regimen to analyze the cause of late graft loss in islet xenotransplantation: one (R051) had stable graft function for the entire follow-up periods and the other (R080) lost graft function around 160 days post-transplantation (DPT). Peripheral blood RNA-seq and subsequent bioinformatics analyses using Time-series RNA-seq analysis package (TRAP)16 hinted on the possibility of immune rejection in R080. Further in silico analyses focused on the interactions of graft loss period-related activated pathways (GLPAPs) proposed that lymphocytes- or platelet-mediated rejection might have been the cause of late graft loss.Recently, we reported consistent long-term (≥6 months) porcine islet graft survivals in five independent monkeys15). However, vital signs and routine laboratory examinations including complete blood cell count (CBC), liver function test (LFT), C-reactive protein (CRP), kidney function test (blood urine nitrogen/creatinine), electrolyte panel , lipase and amylase had shown no abnormal findings in both of the monkeys (data not shown). Also, monitoring of peripheral blood lymphocyte subsets by flow cytometry and ELISPOT  exhibited relatively early hyperglycemia around DPT160, suggesting a graft failure. Intravenous glucose tolerance test (IVGTT) had shown that the porcine islet graft loss in R080 was in progress between DPT120 and 180  select up-regulated pathways with p-value under 0.05 from the results yielded by TRAP comparing t3 and t2 in R080, ii) select up-regulated pathways with p-value under 0.05 from the results yielded by comparing R080 and R051 at t3 and then take the pathways belonging to the intersection of those sets  profile was established. Then, a protein-protein interaction (PPI) network was mapped with the seed genes and the global effects were measured using network propagation. After the genes were ranked under each hypothesis, Pearson’s correlation analysis was performed between the DEG profile ranks and the ranks calculated by the network propagation. This was to test which of the five hypotheses represented by the seed genes produced the gene expression profile similar to the DEG profile we measured. In other words, we tried to see how much the participation of genes in the actual biological process that had induced the graft loss coincided with the perturbation in the expression of genes in the given condition. Furthermore, we performed random simulations for 1000 times and calculated empirical p-values to test the significance of the coefficient as shown in Methods. The result is shown in Table To reconfirm our findings, we sought to computationally test each hypothesis which could explain the islet graft loss. We selected five hypotheses that are known to cause the late graft loss31. Although the engraftment of islets has been successful short-term, it relatively lacked long-term durability, resulting in late-phase graft failure in some islet transplant recipients2. Likewise, we and others have found that the porcine islet grafts were also lost in the transplant recipient monkeys within 6~30 months after transplantation in pig-to-NHP islet xenotransplantation32. Luckily, we were able to experience consistent, prolonged graft survival in rhesus monkeys15, and thus this retrospective analysis was performed in hopes to unearth the cause of late graft loss in pig-to-NHP islet xenotransplantation. To our knowledge, there have not been studies that were focused on the cause of late graft loss nor the mechanism of immune rejection in islet transplantation models of higher mammals.Pancreatic islet transplantation is currently one of the best treatment options for end-stage type 1 diabetes patientsBecause we were not able to find any definitive explanation of islet loss with routine laboratory tests including biochemical and immunological assays in peripheral blood from R080, we used RNA-seq technology to quantify the amount of transcript in the samples obtained from whole blood taken at various time points after transplantation. Then, we carried out pathway analysis and pathway interaction network analysis based on TRAP and PINTnet, respectively. By performing pathway analyses, we found 59 activated pathways and named those GLPAPs Table . Further29. There also have been reports indicative of platelet’s contribution in the chronic rejection of transplanted organs33. T cells have been mentioned very little regarding the chronic rejection of transplanted organs, but our results might have discovered that T cells may partake significantly in the chronic rejection of transplanted islets, at least in pig-to-NHP settings.Computationally, the above findings were reinforced by hypothesis testing. We can measure the influence of some nodes of interests to other nodes on a network using the network propagation. Likewise, we can measure the influence of genes on a biological network or a gene regulatory network. If we map genes on a curated biological network, select some genes as seed genes and run network propagation, we can measure the influence of the seed genes to other genes and rank the genes by the influence they received. Top 100 genes, for example, are the most influenced 100 genes. In our study, network propagation and subsequent correlation analysis revealed that chronic rejection-related genes had been the most related to the late graft loss Table . It is nIt would be of dire importance to show that the RNA-seq bioinformatics data truly represented the immunobiology of the recipient which experienced late graft loss. We retrieved the archives of liver biopsy materials of R080, and performed IHC that could prove the pertinence of the three GLPAPs  to the rejection. Accordingly, we could observe the infiltration of T cells to the graft site as well as antibody and fibrinogen accumulation Fig. . Since t34, and it was reported by Xie et al. that microbiota alteration could result in acute rejection of rat liver allografts35. Because intestinal blood is drained to the liver via portal vein, we hypothesized that intestinal infection might have activated strong innate immunity and this in turn triggered adaptive immune response to the graft through heterologous immunity. In line with this notion, the immunologically hostile effects of infection on the transplanted allografts were reported36 and infection even might break down established tolerance to the graft in murine heart transplantation models37.It is intriguing that only R080 experience immune system activation compared with R051 despite the usage of the same immunosuppressive regimen. To find out putative reason(s), we carefully reviewed pre-clinical symptoms, signs and laboratory data. Interestingly, we noticed that R080 had experienced two times of severe giardiasis-induced diarrhea around DPT90 and DPT120. This finding indicated that intestinal infections preceded the sampling for the RNA-seq about one month. There is increasing evidence that microbiota products could activate the innate immune system of the liverThere are some limitations in our work. First, we only had one monkey which experienced relative early graft loss within the chronic phase after transplantation. Thus, our study cannot give a definite conclusion, but rather an intriguing insight to our field. We are planning to scale up our study using more animals to validate our bioinformatic analysis method. Second, although we suggested that three pathways might have been involved in immune rejection in the chronic phase, we only found the presence of the effectors in each pathway and could not present their actual involvement. Third, because rhesus pathways in KEGG were relatively insufficient, we were not able to analyze our data in high-resolution maps. For example, even though we were interested in CD40L or IL-6 signaling pathways in our setting, we were not able to analyze them because KEGG did not support those pathways. Lastly, we were not able to pinpoint single candidate molecule or a set of molecules which could be critically responsible for graft rejection. Our next works will focus on these questions.Macaca mulatta), 3–4 years of age were used in our study. All animal experiment procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health . The experiments were approved by Seoul National University (SNU) Institutional Animal Care and Use Committee (IACUC no. 15-0297-S1A0). Islet donor pigs, the Seoul National University (SNU) miniature pigs were bred as in designated pathogen-free (DPF) grade38.Rhesus monkeys  was administered subcutaneously 2~3 hrs before islet infusion with dose of 5 mg/kg. 106 to 107ex-vivo expanded regulatory T cells were adoptively transferred after ATG depletion.Induction immunosuppression included a regimen with anti-human CD154 monoclonal antibody, sirolimus , and anti-thymocyte globulin . Sirolimus was administered daily to achieve stable trough levels between 3 and 8 ng/ml. CVF  was administered on day -1 of the transplant to prevent complement activation. TNF-α neutralizing monoclonal antibody, adalimumab . The resulting spots were counted on a computer-assisted ELISpot Reader System .ELISPOT analysis was performed by the method previously describedFlow cytometry analyses of peripheral blood leukocytes were performed using the following monoclonal antibodies: CD3-FITC , CD4-APC-Cy7 , CD8-PE-Cy7 , FoxP3-PE , HLA-DR-PerCP-eFluor710 , CD20-PE , CD14-Alexa488 , CD16-APC . Absolute counts of leukocytes were measured with 123count eBeadsTM (Thermo Fisher Scientific). FACSCanto II flow cytometer  and FACSDiva software (BD Biosciences) were used for analyses.TM-Blood kit (Thermo Fisher Scientific) as recommended by the manufacturer. 500 μl of peripheral blood from the recipient monkeys was used for each sample. Eluted total RNA was stored in −80 °C. Next, RNA sequencing was performed. Purified total RNA was sequenced by Theragen Etex (Korea).Total RNA from peripheral blood of rhesus monkey was extracted using the Ambion Ribopure15. Briefly, liver biopsy samples from the recipient monkeys were fixed in 10% neutral buffered formalin, and embedded in paraffin. Paraffin-embedded tissues were 4-μm sectioned using a microtome. Each de-paraffinized and hydrated section was incubated for 30 min with primary antibody cocktails for insulin , CD3 , CD4 (Santa cruz Biotechnologies), CD8, CD20, and CD68 , and then washed four times in TBST. After staining procedure, all of the stained slides were dried at 60 °C, and mounted with aqueous mounting medium (Thermo Fisher Scientific). For immunofluorescence staining, anti-fibrinogen antibody and anti-insulin antibody (both from Abcam) were used as primary antibodies and, Alexa 488-conjugated anti-rabbit IgG and Alexa 647-conjugated anti-mouse IgG were used as secondary antibodies, respectively. FITC-conjugated anti-monkey IgG  was used to detect antibody involvement at the graft site. The stained sample was observed by Carl Zeiss Axio Imager A1 microscope and images were taken with a micrograph with AxioVision software .Biopsy and immunohistochemical staining was conducted as described previously16 and pathway interaction network20 were performed as described previously. Network propagation was performed as follows: The genes related to the five hypotheses were collected as seed genes by the literature search and domain knowledge, and each hypothesis was represented by a set of genes. Next, DEG profile was established by measuring the expression change of each gene with calculating the log2 fold change between R080 and R051 at time point 3 and ranking the genes. At that time, we removed the genes of which the expression value was smaller than 1 in either R080 or R051 to prevent extremely high or low fold change yielded by the comparison between small numbers. Then, we mapped the seed genes on a PPI network. The number of nodes and edges in the network are 6,780 and 117,963 respectively. The number of seed genes are ten, nine, eight, ten, and nine for ER stress, islet exhaustion, lipotoxicity, chronic graft rejection, and toxicity of immunosuppressant, respectively. After that, the global effect of the seed genes was measured using network propagation and the genes in the PPI network were ranked for each hypothesis. Then, Pearson’s correlation coefficients were calculated between the ranking in the DEG profile and the ranking by the network propagation for each hypothesis. One thousand random simulations were run and empirical p-values were calculated to test the significance of the coefficient, as shown in the equation below:i indicates each hypothesis and it ranges from 1 to 5. pi indicates the empirical p-value of i-th hypothesis. N is the number of random simulation and it is 1,000. j indicates the j-th random simulation. cij is the coefficient of j-th random simulation of i-th hypothesis. i-th hypothesis.TRAPSupplementary Information


Thermogravimetry–Fourier transform infrared spectroscopy (TG-FTIR) analysis showed that the main pyrolysis products included water, carbon dioxide, and methane.In this study, X-ray diffraction (XRD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) methods were used to study the structure, the thermal degradation kinetics, and the thermogram of sweet potato starch, respectively. The thermal decomposition kinetics of sweet potato starch was examined within different heating rates in a nitrogen atmosphere. Different models of kinetic analysis were used to calculate the activation energies using the thermogravimetric data of the thermal degradation process. The activation energies got from Kissinger, Flynn–Wall–Ozawa, and Šatava–Šesták models were 173.85, 174.87, and 174.34 kJ·mol Starch is one of the most widely investigated biopolymers made from rice, potato, and maize. It is an unusual material with a more complicated structure than that of synthetic polymers. This polymer is a mixture of amylopectin, highly branched polysaccharides with α-1,4 backbone chains linked by α-1,6 bonds, and amylose, a dominant linear structure of α-1,4-linked glucose units [Starch decomposes when heated, and the degradation of starch is a complicated process. In practical applications, it may be enough to consider the basic characteristics of the thermal decomposition process using some simple mechanisms. From the point of view of science and industry, thermal degradation and stability is a very important problem. The heat treatment of starch has been widely used in the food industry and in starch-based materials ,3. Thermal analysis is a group of techniques for the rapid assessment of thermal stability, decomposition parameters, adsorbed water content, crystal water content, and the thermal degradation dynamics . ThermogThere have been some reports about the degradation of raw or processed starches from many different sources using different thermal analysis models ,10. DewaThe main purpose of this study was to investigate the thermal behavior of sweet potato starch using TG and DSC. In this study, three methods were used: XRD to study its structure, TG to investigate the thermal degradation processing, and DSC to survey the thermogram of sweet potato starch. The thermal degradation data were used to analyze the thermal degradation kinetics with different isoconversional models like Kissinger, Šatava–Šesták, and Flynn–Wall–Ozawa models, which were used to evaluate the activation energy of sweet potato starch and its activation energy of thermal degradation. The decomposition activation energies obtained in this study can help to evaluate the thermal stability of sweet potato starch used in the food and chemical processing industry.The sweet potato starches used in this study were purchased from Guiyang Jincheng Co., Ltd. , and the moisture content is less than 1%. The dried samples were crushed and sieved using a sieve of 0.15 mm in order to get uniform particles for TG/DSC measurements. The samples were kept in airtight packages.θ range from 5° to 72° with a step size or sampling interval of 0.02°. From the XRD patterns, the interplanar spacings (d) were calculated by Bragg’s equation. In order to get the crystallization fraction of sweet potato starch, the software MDI Jade 5.0 was used.The X-ray diffraction was measured using a diffractometer  instrument, which was operated at 40 kV and 40 mA, and 2−1. The starting temperature was 35 °C. The rates of heating were 5, 10, 15, and 20 °C·min−1, respectively. Each sample was about 5 mg.A thermal analyzer  was used for obtaining TG and DSC curves. The thermal analyses were carried out in alumina crucibles under dry a argon atmosphere with a flow rate of 10 mL·min2 from room temperature to 600 °C with a heating rate of 20 °C·min−1.Thermogravimetry–Fourier transform infrared spectroscopy (TG-FTIR) analysis was obtained by TG 209F3/Tensor 27 , heating under Nc) was obtained using the following formula:c and Ha are the intensities of the crystalline and amorphous parts, respectively.The crystallinity index is an important parameter, which has an important effect on the utilization of materials. X-ray diffraction can provide a qualitative or semi-quantitative assessment of the crystallinity index. The value of the crystallinity index  .dα/dt =The following expression is obtained in conjunction with Equations (1) and (2):The following equality was obtained in conjunction with Equations (3) and (4):Equation (5) is a general non-isothermal kinetic function.m and Tm are the fastest conversion ratio and the corresponding temperature, respectively and (dα/dt)m and n(1 − αmax)n−1 were approximately 1. The Ea value was obtained from the slope of a line of ln(β/(Tm2))vs.(1000/Tm).The intercept of the line was expressed with the following formula:The Kissinger technique was a differential approach for studying the thermal decomposition process. The activation energy Ea was calculated with the following formula based on Kissinger .Y[ln =m).The Ea value was obtained from the slope of the line of lgβ vs.  of sweet potato starch is 0.56. Since the crystallinity depends mainly on the crystallization of amylopectin, the Xc value of sweet potato starch is positively related to the content of amylopectin.The crystallinity index  vs. 1000/Tm, where slopes give −E/R, and the correlation coefficient (R2) is 0.9930. Thus, the Kissinger model was appropriately applied in sweet potato starch. The Kissinger model was used to roughly calculate the activation energy of the thermal degradation using the peak value of the thermogravimetric curves. The activation energy and pre-exponential factor got from the line were 173.85 kJ·mol−1 and 27.85 min−1, respectively. In general, the activation energy value was in the maximum weight loss rate or in the largest heat absorption [The Kissinger model was used to analyze the thermogravimetric curves of sweet potato starch. sorption . m, were obtained. m, where slopes give −E/R. The linear correlation coefficient was 0.9981, and the activation energy was 174.87 kJ·mol−1. Thus, the Flynn–Wall–Ozawa model can also be applied to sweet potato starch.The Flynn–Wall–Ozawa model was also employed to process the thermogravimetric curves of sweet potato starch and calculate the corresponding dynamics parameters. From m, where slopes give −0.4567E/R. The fitting curves were approximately parallel, and the mean correlation coefficient (R2) of the fitted curves was −0.9935. Activation energy is usually regarded as the energy barrier to control the bond breaking or bond reapportioning step [−1.The Šatava–Šesták model was also used to process the thermogravimetric curves of sweet potato starch. The mechanism function G(α) = −ln(1 − α) was ultimately chosen after comparison with the others. ing step . The actThe activation energies obtained by the three models are similar, and the Student’s t-test analysis showed that the three values belong to this same population. The high consistency of the values confirmed the reliability of the calculation and also confirmed the predictive ability of dynamics theory . TherefoThe DSC curve was used to study the thermal transformation occurring in the process of heating under an inert atmosphere. The typical DSC curve of sweet potato starch in a range of 40–580 °C is presented in −1. Mainly water vapor was found, which was characterized by peaks at 3736 cm−1. The band at about 2150 cm−1 indicated the existence of carbon monoxide. The narrow band at 3014 cm−1 was the significant peak of methane. Some other gases were released at the same time, which led to more difficultly in finding the peak of gases with small quantity. The speculative chemical pathway for the conversion of sweet potato starch is indicated in The three-dimensional (3D) FTIR diagram of gaseous products from the pyrolysis of sweet potato starch is shown in −1 based on Kissinger, Flynn–Wall–Ozawa, and Šatava–Šesták models, respectively. The activation energy values allow the development of a simplified approach to generally understand the thermal behavior of sweet potato starch in relation to food processing. The gaseous products from the pyrolysis of sweet potato starch were water, carbon dioxide, carbon monoxide, methane, and so on. The relationship between activation energy and conversion rate and the mechanism of degradation of sweet potato starch will be discussed in the future.X-ray diffraction, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) methods were applied for studying the main characteristics of sweet potato starch. The crystallinity index of sweet potato starch is 0.56. Thermogravimetric curves were used to study the thermal degradation kinetics of sweet potato starch by a non-isothermal process. The activation energy values of sweet potato starch were 173.85, 174.87, and 174.34 kJ·mol


Kangaroo mother care, an evidence based practice and a national policy for management of low birth weight newborns in Nepal, is not widely practiced. This implementation research study aimed to explore the consumer preference and acceptability of the traditional and a new ergonomic wrap on the continuation of kangaroo mother care in the facility and community following discharge.A mixed method feasibility study was done from May to October 2015. Ninety-six families of stable low birth weight newborns weighing 1800 to 2499 grams were counseled and taught to practice kangaroo mother care using both wraps. They were randomized into two groups of 48 with one group trying out the traditional wrap for the first six hours and the new wrap for the next six, and vice versa. Mothers were allowed to choose between the wraps for continuation of kangaroo mother care at the facility and post discharge. They were followed up telephonically weekly over 28 days postpartum to ascertain practice of kangaroo mother care. In-depth interviews with mothers (n = 12) and focus group discussions with health workers (n = 16) further evaluated the intervention. Descriptive statistics are presented for the quantitative part of the study.Mothers in the two groups chose the new wrap with no significant difference . Of the 96 randomized mothers, 85% chose the new wrap. During the hospital stay, six mothers dropped out and remaining 90 mothers who were discharged with the intention of continuing Kangaroo Mother Care, 78 and 12 mothers did so with the new and traditional wrap respectively. New wrap users  performed skin-to-skin contact for an extra 77.4 hours overall than traditional wrap  users from first day to 28 day postpartum. Health workers and mothers reported positive experience with the new wrap as it was easy to wear without assistance, secure and flexible to move around in kangaroo mother care position.Involvement of mothers and families with provision of ergonomic wraps showed improvement in kangaroo mother care practice during hospital stay and at home. The neonatal mortality rate at the global level fell from 36 per 1,000 live births in 1990 to 19 in 2015, but this decrease in neonatal mortality has been slower than that of post-neonatal under-five mortality: 47%, vs 58% [Kangaroo Mother Care (KMC)—defined as early, prolonged skin-to-skin (STS) contact between mothers, family members and the newborn with exclusive or near exclusive breastfeeding along with early discharge from hospital has been proposed as an alternative to incubator care for LBW infants . KMC hasKMC has been applied in different ways in different contexts, and studies show that there are several barriers to implement KMC, including the need for time, social support, medical care and family acceptance –10. SuccDespite strong evidence about improved health outcomes among LBW newborns receiving KMC, it has never been completely integrated within the health systems globally and this holds true for Nepal , 10. ThoWe implemented the study in two peripheral hospitals of Morang and Jhapa districts in the Eastern Development Region of Nepal from May to October 2015. We selected this region due to high prevalence of LBW (16%), compared to the national average (12%) . The hosintervention, inner and outer settings, individuals involved, and the process by which implementation is accomplished -we focus our efforts on the stakeholder perceptions, adoption and trialability of the intervention.The study was designed as a sequential explanatory design, with a trial for mothers of LBW newborns adopting KMC practice using different wraps, followed by collection and analysis of qualitative data on perceptions and experiences among providers and mothers. The reason for the above design was to use qualitative results to assist in explaining and interpreting the findings of the initial trial . The neeThe intervention is pictorially represented . Health We randomized mothers based on odd and even numbers in the sequence of enrollment. A coin was flipped to determine the group allotment for the first enrollee in both the facilities, which ended up as the traditional wrap first. Subsequently, all the odd numbers received traditional wrap first and even numbers received the new wrap first in both facilities. After obtaining informed consent from the mother, the health worker counseled the mother and other family members (if present) on the benefits of KMC and demonstrated the steps of correct KMC using both the wraps. Mothers and family members of the newborn practiced KMC with Preemie Natalie  using both the wraps. After the mother and other family members were comfortable and confident in performing KMC, they practiced KMC with both wraps, switching after six hours for a total of 12 hours with the practice on the first one based on the sequence of randomization. They were offered the opportunity to choose one of the wraps to continue practicing KMC at the facility and at home after discharge. The preferred wrap was provided free of cost. All mothers with LBW newborns were encouraged to stay in the health facility for three days to allow for early detection and appropriate management of early newborn sepsis, which usually manifests by the third day.The two types of wraps used as part of the study are briefly described here.The traditional cloth  for carrThe new wrap designed by Laerdal Global Health, also known as CarePlus, is an ergonomic wrap . The weapost hoc power analysis was performed to determine if the choice of wrap by mothers was associated with the sequence, in which the wraps were presented to and tried out by the mothers for six hours. For the randomized experiment, we were interested in testing the hypothesis that the sequence in which the wraps were presented to the women was not associated with the final choice of the type of wrap. A sample of 96 women (48 in each arm) would have rejected the null hypothesis with type 1 error of 0.05 and power of 80% only if the odds of the choice of wrap were at least 56% lower in one group over the other.Since the target population for this study is mothers with LBW newborns requiring KMC, the sample size was based on the feasibility of enrolling LBW newborns within the study period. A We conducted in-depth interview with subset of 12 mothers (6 per facility) who were discharged from the health facility with the intention of continuing KMC, attended four phone follow ups and postnatal care. After the quantitative study was analyzed, we randomly sampled mothers, after stratifying them into high and low KMC practice among users of both wraps. We also conducted one focus group discussion (FGD) at each hospital with 16 health workers who had been involved in the study.th day, 14th day and 21st day postnatal at home and an in-person interview during the postnatal visit around the 28th day at hospital. A qualitative researcher completed in-depth interviews with a subset of mothers of LBW newborns and FGDs with health workers involved in the study, to explore their experience with the wraps. During the phone interview, mothers were inquired about duration of STS practiced and their experience with providing KMC. No diary or paper format was used by the mother and family members to document the hours of KMC practiced. We filed and stored the completed tools and informed consent documents in a locked cabinet in the respective hospitals with access restricted to the research officers. The data files was entered and stored in password protected computers, with access limited to research personnel. The copy of the interview guide  chose the new wrap and 9 (18.7%) mothers chose the traditional wrap. Similarly, out of 48 mothers who were presented with traditional wrap first, 43 (89.6%) mothers chose the new wrap and 5 (10.4%) mothers chose the traditional wrap. The difference between the proportion choosing the different wraps did not differ significantly between the two groups (p = 0.24). There was no significant differences at baseline between mothers selecting the new wrap and traditional wrap .th phone call, of which 66 continued using the new wrap and 11 with the traditional one, and 59 mothers completed the postnatal contact on 28th day.Subsequent to the choosing of wraps for continuation of KMC, 4 mothers (5%) from the new wrap group and 2 (14%) from the traditional wrap group dropped out of the study, for various reasons, before being discharged from the hospital. Of the 90 mothers remaining in the study at discharge, one mother was discharged before 3 days, and the remaining 89 mothers were discharged after 3 days. All mothers (90) who were enrolled in the study provided exclusive breastfeeding to their newborns during hospital stay. Overall, 77(86%) mothers were followed up until the 4The mean duration of STS practiced per day from day 1 to day 28 by type of wrap selected . New wraOf the 59  mothers who came for postnatal visit, 58 (98%) reported practicing KMC at home. One mother, using traditional wrap, did not practice KMC from day 14 postpartum as the newborn suffered from buttock sores. Among 58 mothers practicing KMC, 55 (94.8%) maintained exclusive breastfeeding until 28 days postpartum, with 2 mothers from new wrap group and 1 from traditional wrap group not breast feeding exclusively.The health workers reported that their perception and understanding about KMC and its benefits before the intervention was limited to STS contact and the treatment of hypothermia. They had not counseled mothers of LBW newborns for KMC, as they were unaware of its benefits. After the intervention, health workers reported the realization that KMC was a standard method of care for LBW newborns which included STS contact and exclusive breastfeeding; perceived benefits included helping maintain the newborn’s body temperature, reducing infection, promoting breastfeeding, and increasing weight gain. They reported counseling mothers of LBW newborns to practice KMC. One of the FGD participants (trained nursing staff) described the change in perception of KMC as,“Before the training, we just informed the mother to keep the newborn warm, but, now, we counsel them about all aspects of KMC.” Another nursing staff providing KMC added, “We can answer the queries of visitors about KMC and confidently explain about the benefits”.Health workers appeared to have positive attitudes and experiences with the new wrap for KMC in both health facilities. The key characteristics mentioned were that the new wrap was easy to wear without assistance, provided proper support for the newborn because of the shoulder band and, hence, was comfortable to carry the newborn and move around. They also mentioned ease of breastfeeding as the mother can slacken the knot and loosen the strap of one side and feed the newborn herself without help of another people. While in the traditional wrap, there is always the need for another person to release the knot at the back for feeding, thus makes the breastfeeding cumbersome. Health workers also reported that male members found the new wrap acceptable to use on their own. According to their observation at health facility, they found that fathers were more interested and engaged in providing KMC to the newborn with the new wrap as compared to the traditional wrap. They mentioned that the traditional wrap is considered a women’s cloth and men were not willing to use it while the “decent” look of the new wrap promoted its use among male members.The health workers mentioned that traditional wrap was inconvenient, as another person was always needed to support the mother and help to keep the newborn in KMC. As the traditional wrap was thick and long it was uncomfortable for the mothers during summer due to heat and would sweat considerably. It was not easy to sleep in KMC position with the wrap because of the knot at the back.Reduced risk of infection, as there is less chance that relatives and neighbors will touch and carry the newborn while the newborn is in KMC. If the newborn was not kept in KMC, the relatives and neighbors coming to see the newborn would touch and carry the newborn thereby increasing the chances of infection.”Mothers, irrespective of the wrap they had selected, reported that KMC was beneficial for their LBW newborns and to them as parent. Mothers reported that KMC kept the newborn warm, relaxed, provided more opportunity for breastfeeding and promoted weight gain. Few mothers reported that KMC also reduced the chances of infection in the newborn, as outsiders did not carry them. One mother reported, “Some mothers expressed being satisfied and happy, as they were able to provide increased care to their newborn. Mothers also said that KMC saved money indirectly as KMC promoted the health of LBW newborn.Mothers, irrespective of the selected wrap, appeared to recommend the new wrap over the traditional one. Even those mothers choosing the traditional wrap reported that it would have been easier for them and their families to practice KMC at home if they had chosen the new wrap. They also mentioned that they would strongly recommend the new wrap to other mothers, as they do not want other mothers to experience the problems they confronted using traditional wrap. The mothers using new wrap reported the major benefit of the new wrap was that they could provide KMC alone as the wrap could be tied without anybody’s help.“Even when there’s no one at home, the new wrap is of great help since I can tie it on my own”.(New wrap user)On the contrary, mothers using traditional wrap reported that the traditional wrap was cumbersome in providing continuous KMC as they always needed extra person throughout the day to tie and untie the knot to keep the newborn in KMC, after taking out for breastfeeding, and changing diapers.I had to go through great struggle to practice KMC at home as I always needed someone to help me tie the wrap.”“New wrap is extremely convenient, not only did I practice KMC at home, but I also continued it while looking after the goats in the farm after 22 days of delivery”.The other benefit offered by the new wrap was about the security. Mothers reported that they felt secure while moving around practicing KMC with the new wrap as it was tied securely with belt. All mothers choosing new wrap performed regular household chores such as cooking, washing and drying cloths, sweeping the house etc. One mother who was a new wrap user reported, “On the contrary, none of the mothers using the traditional wrap performed household chores while practicing KMC as they were not comfortable with the security offered by the wrap especially in conditions when they had to tie the wrap themselves or when they had to seek help from untrained persons.Family member trained in the hospital for tying the traditional wrap is not always available at home, and when I seek help from other untrained relatives/neighbors, they cannot tie the wrap securely”.“Mothers using the new wrap reported that it was easy to breastfeed during KMC as she could easily slacken the strap herself for feeding. With the new wrap, it was easy to rest on the bed or chair while practicing KMC. They also reported that with the new wrap it was easier to get help from male members of the family, since the new wrap was perceived as a baby carrier by male members which led to their engagement in keeping the newborn in KMC. Husbands of new wrap users found it was easy to fasten, and considered it safe.During the hospital stay my husband preferred to carry the newborn in new wrap as he felt less embarrassed with it. However as I chose the traditional wrap, he avoided keeping the newborn in KMC at home.”“During the interview, mothers were inquired if they would have purchased the wrap if it were not provided free of cost and the amount they were willing to pay if they agreed to pay. All interviewed mothers reported that they would have purchased the wrap for KMC even if it had not been provided free of cost, as the wrap facilitated KMC which had positive effect on the health of their babies and it was comfortable to use. The price reported by mother ranged from 1 to 10 dollar for new wrap and from 5 to 12 dollars for the traditional wrap.Despite KMC being introduced in Nepal over a decade ago  and beinSeveral systematic reviews have explored the enablers and barriers of KMC –11. LackMajority of mothers included in our study were from Dalit, Janajati and Madeshi ethnic group. A study analyzing data from NDHS 2011 reported the prevalence of LBW was equal among all ethnic groups . AnotherIn our study, a majority of the mothers practiced STS contact for nearly about 24 hours during hospital stay irrespective of the selected wrap. A longitudinal study from Ghana with 202 mothers of LBW newborns reported only 18% of the continuous KMC during hospital stay , while aNo significant difference was observed in the hours of STS contact among mothers using the traditional wrap and the new wrap during hospital stay. This might be because mothers from both group received the same level of training, encouragement and support by health workers at the postnatal ward. An additional reason might be that household obligations which are identified as a major barrier for KMC did not exist in the hospital. Studies have reported that there was more time for KMC at hospital than at home due to everyday household tasks burdened on women , 22.It is essential to assure that the mothers initiating KMC in the hospitals continue it at home after discharge from the hospital to ensure optimal benefit. Most of the mothers in our study discharged from the hospital expressed an intention to continue KMC at home. This finding is consistent with the study done in Ghana where 95.5% of the mothers were willing to continue KMC at home . Almost Mothers using the new wrap appeared to report more hours of STS practiced at home compared to the traditional wrap. This could be attributed to some of the benefits provided by the characteristics of the new wrap as ease of wearing the wrap, providing security while holding the newborn and able to perform household chores. Several studies have identified that the time required for providing KMC as a major barrier for caregivers, including mothers, due to their obligations at home, and lack of support inhibits them from providing the time required to provide continuous KMC –10. MothSeveral studies have reported that practicing KMC promotes breastfeeding , 25. AllThe strength of our study is the use of a mixed methods approach to assess the feasibility of introducing a new wrap. Courtesy bias for qualitative data collection was minimized by hiring a qualitative researcher who was not part of the study team.A key limitation of the study is the small sample size in one group as compared with the other. The number of mothers choosing traditional wrap was quite low, creating an imbalance in the group numbers and further reducing the power to detect a difference in total hours of STS.Information on hours of STS contact practiced by mother and exclusive breastfeeding at home was captured by phone interviews rather than direct observations. This has the potential for both recall bias and social desirability bias. The study was not powered to evaluate the differences in the hours of STS practiced, or impact on neonatal mortality.Involvement of mothers with the provision of a new ergonomic wrap of their choice led to high acceptability, uptake and continuation of KMC practice during hospital stay and at home. This new wrap may have made a difference in KMC continuation rate at home as the new wrap was designed in a way that it securely holds the newborn, allows flexibility for performing household chores while practicing KMC and acceptable for use by male members of the family. Recently with increased attention and investment with Every Newborn Action Plan (ENAP), KMC has been highly prioritized through NENAP where government of Nepal aims to have 35% of all premature newborns receive KMC by 2035 from a present situation of 0% coverage. The involvement of mothers and families with provision of new ergonomic wrap, is very timely and can be taken up by the government of Nepal while scaling up the KMC practice.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(DOCX)Click here for additional data file.


Animal Frontiers derives from a recent International Livestock Congress (ILC) presented by the International Stockmen’s Educational Foundation (ISEF) in conjunction with the Houston Livestock Show and Rodeo. The ISEF hosts industry and world leaders and scientific experts, and supports a student educational program, in part through its annual Congress, to generate discussion and exchange of global opinions on current topics of interest and concern across the livestock industry’s entire supply chain. The most recent ILC focused on “Science-based strategies for meat in the diet and new perspectives on global trade.” This topic arose from the underlying concept that food security is one critical component of national security. Moreover, “nutrient security” must also be considered for adequate nutritional wellbeing of people in economically disadvantaged countries, as well as those that are more prosperous, in order to meet the challenges of adequate protein intake needs, conflicting nutritional advice, and activist agendas.Food and nutrient security in light of population growth projected to reach 9.8 billion over the next 30 yr  are becoIn this issue, David Klurfeld addresses “What is the role of meat in a healthy diet?” He describes the value of red meat as a nutrient-dense food that is a complete protein source containing all essential amino acids, iron, zinc, selenium, and B vitamins. Among animal-source foods, red meat in particular has been a target for criticism. Klurfeld describes a review process in which the World Health Organization’s International Agency for Research on Cancer ignored controlled studies and used weak epidemiological evidence in an effort to classify red meat as a carcinogen. Although questions still remain regarding dietary choices, Klurfeld notes that the most common nutrient deficiencies worldwide could be eliminated by regular consumption of a small amount of beef, emphasizing that dietary recommendations must be based on sound scientific principles.DGA) continues to provide evidence-based information about dietary practices for optimal nutrition, a National Academies’ panel has made several recommendations to improve the DGA update process. Among the recommendations is a call for increased rigor in evaluating scientific evidence (To ensure that the U.S. Dietary Guidelines for Americans (evidence . In a glThe world market is changing, and the role of the private sector leads this transformation . As descNext, two case studies of the beef industry bring an international perspective and approach to the discussion. In “Beef cattle production system capacity considerations for improved food security: A case study in Myanmar,” Herring and colleagues propose strategies for developing livestock operations and improving beef production in that country. At the other end of the spectrum, Polkinghorne describes a strategic approach to overall industry improvement—with emphasis on product quality and marketability “From commodity, to customer, to consumer: The Australian beef industry evolution.” Finally, Daigle and Ridge wrap up this issue by addressing the importance of developing the animal science workforce and suggest that “Investing in stockpeople is an investment in animal welfare and agricultural sustainability.” They describe current barriers and offer potential solutions for developing and increasing the availability of a skilled workforce.


EJNMMI Radiopharmacy and Chemistry by an international working group of experts, the EJNMMI journal family endorses the application of the guidelines in contributions to the journals.With the open letter published in Attached to the letter by the working group is a three-page summary highlighting the most relevant issues used in the notation of radiopharmaceuticals and related terms.—setting the record straight, Coenen, H.H., Gee A.D. et al., Nuclear Medicine and Biology, volume 55, v–xi (2017) (10.1016/j.nucmedbio.2017.09.004)”.The full paper with all recommendations is published in “Consensus nomenclature rules for radiopharmaceutical chemistryAll Editors-in-Chiefs of the EJNMMI journals strongly recommend all manuscripts meet these guidelines upon submission. In addition, all reviewers will be asked to be aware of the guidelines and wherever possible to check if the guidelines are followed.Editors-in-Chief,Philip ElsingaAngelika Bischof DelaloyeArturo ChitiStefaan VandenbergheFrancesco GiammarileIgnasi Carrio


The natural variation of starch phosphate content in potatoes has been previously reported. It is known that, in contrast to raw starch, commercially phosphorylated starch is more stable at high temperatures and shear rates and has higher water capacity. The genetic improvement of phosphate content in potato starch by selection or engineering would allow the production of phosphorylated starch in a natural, environmentally friendly way without chemicals. The aim of the current research is to identify genomic SNPs associated with starch phosphorylation by carrying out a genome-wide association study in potatoes.S. tuberosum L. varieties were used for phenotyping and genotyping. The phosphorus content of starch in 90 potato cultivars was measured and then statistically analysed. Principal component analysis (PCA) revealed that the third and eighth principal components appeared to be sensitive to variation in phosphorus content . PC3 showed the correlation of starch phosphorus content with allelic variations responsible for higher phosphorylation levels, found in four varieties. Similarly, PC8 indicated that hybrid 785/8–5 carried an allele associated with high phosphorus content, while the Impala and Red Scarlet varieties carried alleles for low phosphorus content. Genotyping was carried out using an Illumina 22 K SNP potato array. A total of 15,214 scorable SNPs (71.7% success rate) was revealed. GWAS mapping plots were obtained using TASSEL based on several statistical models, including general linear models (GLMs), with and without accounting for population structure, as well as MLM. A total of 17 significant SNPs was identified for phosphorus content in potato starch, 14 of which are assigned to 8 genomic regions on chromosomes 1, 4, 5, 7, 8, 10, and 11. Most of the SNPs identified belong to protein coding regions; however, their allelic variation was not associated with changes in protein structure or function.A total of 90 A total of 8 novel genomic regions possibly associated with starch phosphorylation on potato chromosomes 1, 4, 5, 7, 8, 10, and 11 was revealed. Further validation of the SNPs identified and the analysis of the surrounding genomic regions for candidate genes will allow better understanding of starch phosphorylation biochemistry. The most indicative SNPs may be useful for developing diagnostic markers to accelerate the breeding of potatoes with predetermined levels of starch phosphorylation.The online version of this article (10.1186/s12863-019-0729-9) contains supplementary material, which is available to authorized users. The potato is the third most important food crop in the world, and it has reliably held this position for centuries . The plaAnother feature of potato starch that could be improved with significant practical benefits is its phosphate content. Phosphate groups in potato starch are normally chemically bound to amylopectin molecules with approximately one phosphate group per 200–300 glucose units (0.05–0.08% P). The negatively charged phosphate groups result in better dispersion of the polysaccharide chains in water due to mutual Coulomb repulsion. In industry, phosphorylated starch is manufactured in a chemical process that results in the addition of 1–3% phosphate content by mass. Commercially phosphorylated starch is known as food additive E1410 and functions as a texturizing and stabilizing agent. In contrast to raw starch, it is chemically more stable at high temperatures and shear rates and has a higher water capacity. It has been demonstrated that the physical and chemical properties of potato starch gels is dependent on its phosphate content . Thus, gamf resulted in two contrasting effects; some plants showed higher phosphate content than the corresponding control, while others exhibited lower phosphate content, thereby generating starches with broad-scale variation in phosphate content [Lactobacillus reuteri 121 into the same lines resulted in a significant increase in starch phosphate content in the amf line, while starches from the Kardal background did not show any changes in phosphate content [There have been some successful attempts to genetically modify potatoes that produce starch with a higher phosphate content. The expression of the laforin protein in the tuber  resulted content . The int content .Arabidopsis as a model [It was shown that α-glucan, water dikinase (GWD/GWD1) and phosphoglucan, water dikinase (PWD/GWD3) enzymes play key roles in starch metabolism. Improved physical and chemical properties of starches isolated from GWD-deficient plants were described using  a model .In addition to reverse genetic approaches, the tools of forward genetics such as QTL analysis , 12 and We performed a genome-wide association study using a 22 K SNP potato array to find novel genomic regions associated with starch phosphorylation.Solanum tuberosum L.) varieties from ICG “GenAgro” collection  was grown during the period May to October 2017 in the same field in Novosibirsk region . A subset of 8 varieties was preliminary grown during the period May to October 2016 in the same field. Growing of potato plants was performed according to the standard procedure. Briefly, seed tubers of all cultivars were planted in two rows with 0.75 m spacing and 0.3 m distance between the plants on the rows. In total, 10 plants were planted in the row, so, the length of each row was 10 m. Each cultivar was planted in three replicates, and distances between the replicates’ plots were 2 m. Sowing was performed in the first decade of May and harvesting in the 3rd decade of September.The set of 90 potato  according to the standard procedure. Concentration and purity of the isolated DNA were tested by gel electrophoresis and micro spectrophotometry on Nanodrop 2000 equipment.All 90 accessions were genotyped using Illumina 22 K SNP potato array (GGP Potato V3) at the Traitgenetics GmbH . The Illumina Infinium procedure was performed according to the manufacturer’s protocol. None of the samples were failed for the analysis.Analysis of the starch samples was performed using phosphomolibdate method with spectrophotometric detection according to the standard procedure, described in GOST 7698–93 “Starch. Rules for acceptance and methods of analysis” . In brief, starch probe was decomposed in a mixture of sulfuric and nitric acids, neutralized and reacted with ammonium molibdate. Spectrophotometric detection at 825 nm allowed to determine content of phosphorus in the initial starch probe with the help of the calibration curve.The Principal component analysis (PCA) was considered with genotyping data calculated through the distance matrix. It was done using software packages STATISTICA 8 . In ordeDifferent statistical models were tested on disease resistance scores (separately for each of four isolates) with the help of TASSEL 5 package  to detecSince TASSEL have specially been developed for diploid genome analysis, we re-coded tetraploid potato genome from four-letter code to numerical, taking into account the dose of certain allele. After the re-coding, 0 was assigned to effector allele and 1 – as non-effector allele, and their intermediate forms were coded as 0.75, 0.5 and 0.25. For example, AAAA allele is reflected as 1, AAAG – as 0.75, AAGG – as 0.5, AGGG – as 0.25, and GGGG – as 0.− 6, and (ii) the false discovered rate (FDR) that was calculated for each isolate in each model. Percentage of random was < 10%.To identify significant SNPs two corrections were used: (i) the Bonferroni correction, where the significant threshold (0.05) is divided by the total number of tests, in this case, the total number of markers , giving threshold 1.8302*10Initially, the starch phosphorus content was determined for 8 varieties of potato harvested in 2016 and 2017. A strong positive correlation of this trait between 2 years (Pearson’s coefficient 0.91) was found in most of the cultivars  and revealed a set of 15, 214 (71.7%) scorable SNPs. SNPs were treated as significant only if they were discovered in > 95% of the studied genotypes. We used the chromosomal position of the SNPs  to arranPopulation structure analysis showed that for the set of 90 potato varieties, a total of 23 principal components contributed more than 1% of common genotype variance. The first three components described 31.8% of variance. Two dimensional projections of the first three principal components using PC1-PC2, PC2-PC3 and PC1-PC3  appeared to account for phosphorus content. Correlation of the starch phosphorus content with PC3 suggested that the Ladozhsky, Nevsky, Ruslan and Kuznechanka varieties carry alleles responsible for higher phosphorus content  or the less strict FDR  criteria were considered. The Bonferroni correction sets very strict criteria; overcoming it implies the greatest likelihood of SNP-trait association, but permits false negative results, omitting some trait-associated SNPs [The association-mapping plots were obtained with the TASSEL program by using the SNP-genotyping and phenotyping results for all 90 varieties. QQ- and Manhattan plots shown in Additional files ted SNPs . To counSix significant SNPs were found on chromosome 1 Table . AllelicAnother significant SNP was located about 10 Mbp from the genomic region described above Table . AllelicSBEI) and starch phosphorylation. The SBEI location is 4:71586223–71,597,347, which is approximately 13 Mbp distal from the PotVar0084444 SNP, suggesting that the phosphorus content-associated region identified on chromosome 4 in the current study is not related to SBEI.A single significant SNP was revealed on chromosome 4 Table . The SNPGWD (GWD1) gene encoding α-glucan, water dikinase (syn.: Starch-granule-bound R1 protein). Carpenter et al. [GWD gene and the phosphorus content in tuber starch. The GWD gene location is 5:9823451–9,838,970. The distance between this gene and the region found in the current study is greater than 30 Mbp.Two significant SNPs were found on chromosome 5 Table . These Sr et al.  also obsPotato chromosome 7 has never before been reported to be associated with phosphorus content in tuber starch. Here, the significant chromosomal region was found to be associated with the solcap_snp_c2_38828 marker Table . The SNPThe association between potato chromosome 8 and starch phosphorylation has not been reported previously. A significant SNP was found on chromosome 8 Table . The SNPPotato chromosome 10 has never been reported to be associated with phosphorus content in tuber starch before. Two significant SNPs were revealed in the current study , C3-phosphorylation , and phosphorylation in both positions  were uncovered . Carrena review  revealeda review . These Qmosome 9 .A genome-wide association study using a 22 K SNP potato array allowed 8 novel genomic regions on chromosomes 1, 4, 5, 7, 8, 10, and 11 associated with starch phosphorylation to be found. Some of the SNPs identified were located in non-coding genomic regions. Allelic variation in the SNPs found in protein coding regions was not related to changes in protein structure or function. Further validation of the SNPs identified and the analysis of the surrounding genomic regions for candidate genes will allow a better understanding of starch phosphorylation biochemistry. Among the genes carrying significant SNPs identified in the current study, the gene on chromosome 7 encoding a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase protein is a primary target for a reverse genetic investigation of a potential functional association with tuber starch phosphorus content.Additional file 1:Scatterplots of phosphorus content in starch of potato varieties analyzed, showing sensitivity of the third (A) and eighth (B) components to phosphorus content. The blue ellipses show varieties, which high or low phosphorous contents in starch are genetically determined. (PDF 314 kb)Additional file 2:QQ-plots (quantile-quantile plots) for the models: (1) GLM without correction for population structure; (2) GLM + Q: GLM + Q-matrix to account for population structure; (3) GLM + PCA, (4) MLM. (PDF 282 kb)Additional file 3:p-value. Chr1–12 – chromosome assignment of loci. Chr0 – unassigned loci. (PDF 787 kb)Association mapping results for phosphorous content scorings in potato starch using different models: GLM without accounting for population structure, GLM + Q (GLM + Q-matrix to account for population structure), GLM + PCA, and MLM. Dash line named “Bonferroni” corresponds the Bonferroni threshold. Dash line named “FDR” corresponds the FDR  threshold. Vertical axes show the –log (10) of marker trait association