The widely-used estimates of completeness of birth registration collected by Demographic and Health Surveys (DHS) and Multiple Indicator Cluster Surveys (MICS) and published by UNICEF primarily rely on registration status of children as reported by respondents. However, these self-reported estimates may be inaccurate when compared with completeness as assessed from nationally-reported official birth registration statistics, for several reasons, including over-reporting of registration due to concern about penalties for non-registration. This study assesses the concordance of self-reported birth registration and certification completeness with completeness calculated from civil registration and vital statistics (CRVS) systems data for 57 countries.Self-reported estimates of birth registration and certification completeness, at ages less than five years and 12–23 months, were compiled and calculated from the UNICEF birth registration database, DHS and MICS. CRVS birth registration completeness was calculated as birth registrations reported by a national authority divided by estimates of live births published in the United Nations (UN) World Population Prospects or the Global Burden of Disease (GBD) Study. Summary measures of concordance were used to compare completeness estimates.Birth registration completeness (based on ages less than five years) calculated from self-reported data is higher than that estimated from CRVS data in most of the 57 countries  higher; 43 countries according to GBD, average eight p.p. higher). For countries with CRVS completeness less than 95%, self-reported completeness was higher in 26 of 28 countries, an average 13 p.p. and median 9–10 p.p. higher. Self-reported completeness is at least 30 p.p. higher than CRVS completeness in three countries. Self-reported birth certification completeness exhibits closer concordance with CRVS completeness. Similar results are found for self-reported completeness at 12–23 months.These findings suggest that self-reported completeness figures over-estimate completeness when compared with CRVS data, especially at lower levels of completeness, partly due to over-reporting of registration by respondents. Estimates published by UNICEF should be viewed cautiously, especially given their wide usage. Complete birth registration and certification within a civil registration and vital statistics (CRVS) system provides major benefits for both individuals and societies. It ensures legal identity for individuals and provides them with citizenship and voting rights, access to social security benefits and health and education services, proof of age, and, above all, has been described as a fundamental human right –7. ComplBirth registration, however, is incomplete in many low- and middle-income countries –12. ReguThe State of the World’s Children reports , 1918, 1CRVS birth registration data includes available birth registration data reported by a national authority. Such data were available for 62 of the 119 countries that have self-reported birth registration completeness estimates; there were no CRVS data available for the other 57 countries. These CRVS data are primarily from a global database of birth registration published as part of a global assessment of the utility of birth registration data . These cBirth registration completeness according to the CRVS data was calculated as the number of registered births divided by the number of estimated live births reported in the UN World Population Prospects and also in the GBD Study , 14. In Our comparison of self-reported and CRVS estimates of birth registration completeness was conducted for all countries where both estimates of birth registration completeness data were available. Of the 62 countries with self-reported birth registration data, 57 countries also had CRVS birth registration data within the 10-year time frame defined above and so were included in our study (5 countries had CRVS data outside of the time frame) see . Of the About half (29) of the 57 study countries had birth registration completeness according to the CRVS data of at least 95%. In these countries it is very likely that differences with self-reported registration completeness will be low and, because they have virtually universal birth registration, self-reported data are less likely to be used than for countries where the CRVS data on births are less complete. We therefore separately analysed the 28 countries (22 with birth certification completeness) where birth registration completeness, according to the CRVS data, was less than 95%. These calculations were conducted with CRVS completeness calculated using both UN and GBD birth estimates. We were able to calculate self-reported registration completeness for ages 12–23 months for 39 of the 57 countries (data were not available for the other 18 countries); for 37 of these countries we could also calculate completeness based on certification.To compare CRVS and self-reported completeness we calculated the mean and median absolute difference and root squared difference in percentage points. In these summary results we included the results for the eight countries with unpublished data. In nine countries, the UNICEF birth registration database states that birth registration completeness differs from the standard definition or refers to only part of a country; for these countries we separately measured concordance with CRVS birth registration completeness .There were 57 countries with both CRVS and self-reported registration completeness estimates; using UN estimated births, the self-reported completeness was higher in 31 countries and the CRVS completeness higher in 25 countries, with the respective figures using GBD estimated births being 43 and 13  with a root mean squared difference of 6.8 percentage points (median 4.3) according to UN estimated births and 7.4 (median 4.2) according to GBD estimated births. For the 22 remaining countries with CRVS registration completeness less than 95%, self-reported certification completeness was higher in most countries  with small mean absolute differences  and a root mean squared difference of almost 10 percentage points . For all 57 countries, average CRVS birth registration completeness using the GBD birth estimates (79.9%) was lower than based on UN birth estimates (82.3%).Tables For the 39 countries where self-reported completeness could be calculated for children aged 12–23 months, self-reported completeness differed only slightly from completeness calculated for children less than five years . In factInformation routinely collected by DHS and MICS and compiled by UNICEF on the basis of self-reports about whether or not the births of surviving children have been certified or registered is widely used to inform health and social planning. Yet, our analysis suggests that self-reported birth registration data as reported by UNICEF over-estimates completeness compared with available national registration data. Excluding countries where birth registration completeness based on CRVS data is at least 95% (i.e. may be considered as complete), birth registration completeness calculated from self-reported data is higher than that suggested by CRVS data, calculated using either UN or GBD estimated births, in 26 out of 28 countries and being an average 13 percentage points higher and median of 9–10 percentage points higher. This difference is less extreme for countries where at least 95% of births are registered, since the methodological limitations of self-reported data on live children are likely to be less important in these populations. Of concern, self-reported completeness was over 30 percentage points higher than CRVS completeness in three countries . Self-reported completeness re-calculated for children aged 12–23 months is in fact marginally higher on average than when measured for children less than five years, despite it excluding births registered at least two years after occurrence. There is a smaller difference between self-reported birth certification completeness and CRVS birth registration completeness, with the mean absolute difference being less than one percentage point and root mean squared difference nine percentage points.State of the World’s Children reports, largely based on self-reported data. that 73% of children aged less than five years have had their birth registered, the actual level of birth registration completeness, where births are registered within one year of the birth, is likely to be significantly lower .[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This paper address a very important topic about the difference between self-reported birth registration and registers. I believe this paper makes an important contribution to the literature on this topic.Reviewer #2: A good, interesting, and timely paper that does what it says on the tin.A couple of pointers/concerns that the authors may wish to take into account in any revision.1) I understand the need to take on UNICEF for its role in presenting such aggregated data; however, the real target of the paper is on the data that UNICEF use in aggregating the data (MICS / DHS). I would rather the focus were on those; with UNICEF being a prime example of how those data are used and prone to misinterpretation.2) In comparing completeness with UNWPP and GBD, not enough is made of how dependent the assessment of completeness is on the relaibiltiy of those series. Yes, Lebanon is picked out as the extreme; but it points to a more fundamental issue, about those underlying data. In many instances the resulting estimates of completeness differ by more than ten percentage points. Should the authors not be drawing some kind of conclusion about the relative utility of the two series, where the GBD seems to produce somewhat lower estimates of births (and hence higher completeness) than does the WPP? (Table 1 could be better formatted to make it clearer which series is which (the heads of the sub-sections get lost in the welter of data).3) Around lines 113-118. Another important consideration is that in much of the global South, registration may also occur when (and be delayed until) a child needs to go to school, which may require proof of birth. However, such a delay obviously attenuates the utility of the registration data, since any child dying in the interim is not covered.Minor: Line 501. Months. Not years.Reviewer #3: The objective of this paper is to assess the reliability of self-reported estimates of birth registration completeness obtained from surveys. It compares self-reported estimates with estimates computed using birth registrations reported by a national authority and estimates of the number of live births (UN and GBD).This paper is interesting and relevant. It is well written and clearly preented. Overall, I found the results plausible, but I am not (yet) entirely convinced that self-reported estimates overestimate birth registration completeness.I would encourage you to better discuss the impact of the denominator on estimates of completeness calculated from CRVS systems. In some countries, the numbers of live births are estimated using the number of births reported in CRVS. So, the denominator and the numerator are not independent. It will probably be found in places with high level of completeness, so it should not alter your results, but I think it is worth mentioning. In other countries, numbers of live births are based mainly on fertility estimates and estimates of population size by age groups, which may not be very reliable. In case numbers of births are overestimated, this could lead to lower estimates of completeness. Again, it may not substantially influence your results, but you could discuss this possibility.I also think you should discuss the differences you find in CRVS registration completeness between UN and GBD (Table 2). In some countries, differences are huge (e.g. Lebanon), and often are non-negligible . This indicates that the estimates of the number of births is far from perfect.You could also provide analyses without the outliers. With GBD estimates, Paraguay and Lebanon are clear outliers, and Paraguay is also an outlier with UN data. Actually, if you remove these two countries, differences remain, but are smaller. Some other countries with big differences are also very small, and it may be worth mentioning it.I did not understand why countries in Table 3 are not presented in the same way as in Table 2.I also think you could report results at several points in time for the countries. If we find strong variations over time in either source, this would suggest there are some issues with the date. Moreover, since monitoring progress in birth registration is mentioned as important topic in the introduction, your results would be all the more relevant.More literature would be useful to understand in more detail why survey data may lead to overestimating completeness. Research by Hertrich and Rollet in Mali (in French) worked on self-reported estimates in census data, and found that these were overestimated because of a wrong understanding of the rules by a few interviewers. This may be relevant to your paper.Other commentsCould you mention the countries where estimated births are greater than registerd births? (line 197)**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  27 Apr 2021PONE-D-21-03554How reliable are self-reported estimates of birth registration completeness? Comparison with vital statistics systemsResponses to reviewers’ commentsWe thank the editor for the opportunity to revise this manuscript. Please see our responses below.When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfWe have revised the title page and file names to required format.2. In the Methods section please provide further clarification how results from right countries with unpublished data was collected [line 224].We have now added that the data for the eight countries with unpublished data “were made available to the authors through established collaborations”. We felt it most appropriate to add this statement in Lines 193-94. Reviewer #1: This paper address a very important topic about the difference between self-reported birth registration and registers. I believe this paper makes an important contribution to the literature on this topic.We thank the reviewer for their positive review of the manuscript.Reviewer #2: A good, interesting, and timely paper that does what it says on the tin.A couple of pointers/concerns that the authors may wish to take into account in any revision.1) I understand the need to take on UNICEF for its role in presenting such aggregated data; however, the real target of the paper is on the data that UNICEF use in aggregating the data (MICS / DHS). I would rather the focus were on those; with UNICEF being a prime example of how those data are used and prone to misinterpretation.The reviewer makes a good point about making the distinction between the data collectors and the disseminators of the data (UNICEF). We now refer to the data as being self-reported estimates of completeness of birth registration collected by DHS and MICS and published by UNICEF. This change has been made in several places: Line 22-23, 90, 127, 133, 153, 214, 346, 366, 430, 445-462) In comparing completeness with UNWPP and GBD, not enough is made of how dependent the assessment of completeness is on the relaibiltiy of those series. Yes, Lebanon is picked out as the extreme; but it points to a more fundamental issue, about those underlying data. In many instances the resulting estimates of completeness differ by more than ten percentage points. Should the authors not be drawing some kind of conclusion about the relative utility of the two series, where the GBD seems to produce somewhat lower estimates of births (and hence higher completeness) than does the WPP? (Table 1 could be better formatted to make it clearer which series is which (the heads of the sub-sections get lost in the welter of data).The reviewer makes a good point about the lower estimates of births (and therefore birth registration completeness) made by the GBD compared with the UN WPP. We have chosen to present completeness estimated using both UN WPP and GBD birth estimates because they are both prominent sources of country birth estimates. We do not however make conclusions about the relative utility of the two estimates because, firstly, it is not the primary aim of the manuscript  but also that the two estimation processes utilize different approaches for estimating fertility from vital registration data (often incomplete), complete birth histories and summary birth histories. In the limitations (lines 416-423) we have pointed out that “There is some uncertainty regarding the accuracy of the completeness estimates calculated using the GBD and UN estimated births because the two sets of estimates are derived from different analytical approaches that result in higher completeness where GBD estimated births are used when compared with UN estimated births (because GBD commonly estimates lower births). Additionally, their estimates of total births are derived from age-specific fertility rates applied to age-specific population data of women of reproductive age that is also subject to uncertainty. As a result, there can be significant differences in estimated completeness between the two sources, as in Lebanon, however in most countries these are small.”We have split Table 1 into Tables 1 (UN estimated births) and 2 (GBD estimated births), to make the data presentation clearer.3) Around lines 113-118. Another important consideration is that in much of the global South, registration may also occur when (and be delayed until) a child needs to go to school, which may require proof of birth. However, such a delay obviously attenuates the utility of the registration data, since any child dying in the interim is not covered.The author makes a good point here. We have now added a statement that an example of a delay in birth registration may occur because the birth is only registered when the child is about to commence school (Lines 113-114). We also state that the impact of child mortality on the completeness of birth registration data is more likely where registration is delayed (Lines 122-123).Minor: Line 501. Months. Not years.We have corrected this error.Reviewer #3: The objective of this paper is to assess the reliability of self-reported estimates of birth registration completeness obtained from surveys. It compares self-reported estimates with estimates computed using birth registrations reported by a national authority and estimates of the number of live births (UN and GBD).This paper is interesting and relevant. It is well written and clearly preented. Overall, I found the results plausible, but I am not (yet) entirely convinced that self-reported estimates overestimate birth registration completeness.We thank the reviewer for their valuable comments on the manuscript.I would encourage you to better discuss the impact of the denominator on estimates of completeness calculated from CRVS systems. In some countries, the numbers of live births are estimated using the number of births reported in CRVS. So, the denominator and the numerator are not independent. It will probably be found in places with high level of completeness, so it should not alter your results, but I think it is worth mentioning.The reviewer makes a valid point. We have now added (in Lines 202-204) that “The GBD and UN do use birth registration as a source of fertility estimates where such data are complete, which may create dependence between the numerator and denominator; we therefore filter our analyses to countries with completeness less than 95% (see below).” “In countries with incomplete birth registration, both the UN World Population Prospects and GBD estimate live births predominantly from census and survey data using demographic and statistical models.” In other countries, numbers of live births are based mainly on fertility estimates and estimates of population size by age groups, which may not be very reliable. In case numbers of births are overestimated, this could lead to lower estimates of completeness. Again, it may not substantially influence your results, but you could discuss this possibility.The reviewer makes a good point, similar to Reviewer #2. In Lines 416-423 we have now added the following sentences about the limitations of GBD and UN birth estimates: “There is some uncertainty regarding their accuracy because their two sets of estimates are derived from different analytical approaches that result in higher completeness where GBD estimated births are used when compared with UN estimated births (because GBD commonly estimates lower births). Additionally, their estimates of total births are derived from age-specific fertility rates applied to age-specific population data of women of reproductive age that is also subject to uncertainty. As a result, there can be significant differences in estimated completeness between the two sources, as in Lebanon, however in most countries these are small.”I also think you should discuss the differences you find in CRVS registration completeness between UN and GBD (Table 2). In some countries, differences are huge (e.g. Lebanon), and often are non-negligible . This indicates that the estimates of the number of births is far from perfect.This issue relates to our response to the previous comment – we recognize and state that there is uncertainty in the UN and GBD birth estimates (and therefore birth registration completeness estimates), hence we have presented both sets of estimates. As noted, this can result in large differences (as in Lebanon) but smaller differences elsewhere. The new text in Lines 416-423 addresses this issue.You could also provide analyses without the outliers. With GBD estimates, Paraguay and Lebanon are clear outliers, and Paraguay is also an outlier with UN data. Actually, if you remove these two countries, differences remain, but are smaller. Some other countries with big differences are also very small, and it may be worth mentioning it.We have already identified three countries with at least 30 percentage points difference between self-reported completeness and birth registration completeness (both UN and GBD) – Paraguay, Solomon Islands and Rwanda  – two of these countries have populations of at least 7 million. The calculation of the median difference in completeness overcomes the potential distortion of results due to these outliers, but differences still remain: in countries with completeness less than 95%, the median difference is 9-10 percentage points and the average difference is 14 percentage points.I did not understand why countries in Table 3 are not presented in the same way as in Table 2.The data for the eight countries in Table 3 (now Table 4) were unpublished data and were made available to the authors through established collaborations with colleagues in these countries. Given their unpublished nature, we decided to only report absolute differences with self-reported completeness. We added this statement in Lines 193-94.I also think you could report results at several points in time for the countries. If we find strong variations over time in either source, this would suggest there are some issues with the date. Moreover, since monitoring progress in birth registration is mentioned as important topic in the introduction, your results would be all the more relevant.The reviewer makes a good point. However, for many countries there is only one data point of self-reported completeness. As more data become available, this suggested approach could be the subject of a future study.More literature would be useful to understand in more detail why survey data may lead to overestimating completeness. Research by Hertrich and Rollet in Mali (in French) worked on self-reported estimates in census data, and found that these were overestimated because of a wrong understanding of the rules by a few interviewers. This may be relevant to your paper.We thank the reviewer for making us aware of this research. We have now included this as a possible reason for over-estimation of self-reported completeness as well as a reference to this research (Lines 100-102).Other commentsCould you mention the countries where estimated births are greater than registerd births? (line 197)The countries where registered births exceed either GBD or UN estimated births is commonly due to the stochastic variation of national registered birth numbers when compared to the smoothed trends used in estimating national births.The countries where the registered births exceed GBD estimated births are: • Armenia• Cuba• Egypt• Panama• Suriname• TunisiaThe countries where the registered births exceed UN estimated births are: • Argentina• Armenia• Barbados• Cuba• Egypt• Mongolia• Montenegro• Suriname• Thailand• Tunisia• Ukraine 11 May 2021How reliable are self-reported estimates of birth registration completeness? Comparison with vital statistics systemsPONE-D-21-03554R1Dear Dr. Adair,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Bernardo Lanza Queiroz, Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 31 May 2021PONE-D-21-03554R1 How reliable are self-reported estimates of birth registration completeness? Comparison with vital statistics systems Dear Dr. Adair:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Bernardo Lanza Queiroz Academic EditorPLOS ONE


Apolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma triglyceride levels and its glycosylation may have an effect on the clearance of triglyceride-rich lipoproteins by directing these particles to different metabolic pathways. Large-scale sample cohort studies are required to fully elucidate the role of apo-CIII glycosylation in lipid metabolism and associated cardiovascular disease. In this study, we revisited a high-throughput workflow for the analysis of intact apo-CIII by ultrahigh-resolution MALDI FT-ICR MS. The workflow includes a chemical oxidation step to reduce methionine oxidation heterogeneity and spectrum complexity. Sinapinic acid matrix was used to minimize the loss of sialic acids upon MALDI. MassyTools software was used to standardize and automate MS data processing and quality control. This method was applied on 771 plasma samples from individuals without diabetes allowing for an evaluation of the expression levels of apo-CIII glycoforms against a panel of lipid biomarkers demonstrating the validity of the method. Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced by apo-CIII sialylation. Interestingly, the association of apo-CIII glycoforms with triglyceride levels was found to be largely independent of body mass index. Due to its precision and throughput, the new workflow will allow studying the role of apo-CIII in the regulation of lipid metabolism in various disease settings. Lipid metabolism is regulated by complex biological mechanisms in which apolipoproteins – proteins embedded in lipoprotein particles – modulate the transport and availability of blood lipids . Apolipo0a) and three O-glycosylated variants with a core 1 (T-antigen) glycan structure, which is either non-sialylated (apo-CIII0c), monosialylated (apo-CIII1) or disialylated (apo-CIII2) (2/apo-CIII1 ratio and triglyceride levels has been confirmed by two independent studies (Apo-CIII exists in four major proteoforms: one non-glycosylated form (apo-CIIIo-CIII2) . Low-abuo-CIII2) . It has o-CIII2) . For exa studies . It has  studies  and that studies  and the  studies . Since tin vivo (We have developed a high-throughput method based on magnetic-bead extraction and matrix-assisted laser desorption/ionization (MALDI) and ultrahigh-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) for the analysis of serum apo-CIII proteoforms . Apo-CIIin vivo , sample in vivo . The preIn the current study, we have applied a modified workflow employing a previously established MALDI FT-ICR MS method preceded by a chemical oxidation step for complete oxidation of apo-CIII methionine residues. This results in highly reproducible high-throughput measurements for relative quantification of apo-CIII proteoforms in a large number of plasma samples varying in protein oxidation levels. Furthermore, we have adopted sinapinic acid (SPA) as a MALDI matrix to minimize the loss of sialic acid induced by MALDI. The high-throughput quantitation software, MassyTools , was herBlood plasma samples from a group of individuals without diabetes of the DiaGene Study were used. The DiaGene Study is a case-control study comprising 1886 type-2 diabetes patients and 854 controls without diabetes, from the areas of Eindhoven and Veldhoven, in the Netherlands. The study is described in detail elsewhere . For theClinical information and blood samples were obtained at baseline, as described previously . TriglycMagnetic beads (Dynabeads RPC-18) were purchased from Invitrogen Dynal AS, Oslo, Norway. VisuCon-F plasma standard from Affinity Biologicals, Ancaster, Canada. Hydrogen peroxide 30%, ethanol and acetone were purchased from Merck, Darmstadt, Germany. Acetonitrile (ACN) was from Biosolve Chimie SARL, France. Trifluoroacetic acid (TFA) was purchased from Thermo Fisher Scientific, Tewksbury, MA. Sinapinic acid (SPA) and α-Cyano-4-hydroxycinnamic acid (HCCA) from Sigma-Aldrich. Ultrapure milliQ water (18 MΩ cm at 25°C) was used throughout.2O2/0.5% TFA in water) was added to each sample. The plate was sealed with a pierce foil seal  and incubated for 1 h at 37°C. Subsequently, the plate was cooled at 4°C for 30 min and centrifuged briefly at 800 × g. The pierce foil was removed and the plate was transferred onto a liquid handling robot  where solid-phase extraction (SPE) was carried out as follows: the RP-C18 beads were activated by three washes using acetonitrile (ACN) and trifluoroacetic acid (TFA) solution in water (first wash using 50% ACN/0.1% TFA followed by two washes with 0.1% TFA). Next, plasma samples were transferred to the activated beads and incubated for 10 min at room temperature. The incubation was followed by three washes: one wash using 15% ACN and two washes with 0.1% TFA. Proteins were eluted by adding 15 µL of 50% ACN/0.1% TFA in water and incubating for 5 min at room temperature. For MALDI spotting, 2 μL of sample eluates were mixed with either 16 μL of sinapinic acid solution (1.3 g/L in 2:1 v/v ethanol/acetone) or 15 μL alpha-cyano-4-hydroxycinnamic acid solution (1.4 g/L in 2:1 v/v ethanol/acetone). 1.5 μL of each sample mix was spotted in duplicate onto a MALDI AnchorChip target plate  and allowed to air-dry before MALDI MS analysis.Plasma standards (VisuCon-F) were randomized over cohort sample plates. 10 µL of human blood plasma was transferred from the cohort sample plates into 96-well skirted PCR plates . 15 µL of an oxidizing solution  each using 524,288 data points. The analyzer parameters were set as previously reported  equipped with a Smartbeam II™ laser system (355 nm wavelength) and a ParaCell detector. All spectra were acquired in the reported . Brieflyin vivo  although this latter form requires harsher oxidizing conditions (Apo-CIII contains two methionine residues which can be oxidized to form methionine sulfoxide (MetO) and methionine sulfone  are preferentially cleared by faster-acting low-density lipoprotein (LDL) receptor (LDLR) and LDL receptor-related protein 1 (LRP1), whereas apo-CIII2 directs VLDLs to syndecan 1 (SDC1) receptors that are characterized by a slower but larger capacity metabolism of TRLs. The same study also showed that a 13 weeks antisense oligonucleotide treatment for apo-CIII, which, as expected, reduced plasma TG levels, also altered relative abundances of these two glycoforms leading to an increase of apo-CIII2 and a decrease of apo-CIII1. The increase of the apo-CIII2/apo-CIII1 ratio in a response to the antisense oligonucleotide therapy was explained by a differing capacity and clearance speed of the hepatic TRL receptors. In support of this, we observed in our cohort study of individuals without diabetes a positive association of the relative abundance of apo-CIII1 glycoform with TG levels, and a negative association for apo-CIII2 (So far, the inhibitory effect of apo-CIII on LPL has been linked to total apo-CIII concentration, but not to the relative proportion of apo-CIII glycoforms . Recent  in mice . In detapo-CIII2 ; Table 3Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced, by apo-CIII glycosylation, specifically sialylation. However, other aspects have to be considered. For instance, defective LDLR/LRP1-driven metabolic pathways might lead to decreased clearance of TGs. Expression and stability of LDLR and LRP1 in the liver might be affected by naturally occurring genetic variants . MoreoveExpression levels of apo-CIII were not investigated in this study. The differences in apo-CIII glycosylation profiles observed between individuals may be caused by varying expression levels of apo-CIII  or apo-C2/apo-CIII1 ratio is associated with improved triglyceride levels (Apo-CIII is a novel potential drug target in the management of cardiovascular disease driven by multiple studies demonstrating that plasma levels of apo-CIII are predictive of coronary heart disease and the risk of disease-related events . Previoue levels . In humae levels , therefoHere, we present a workflow for high-throughput MALDI FT-ICR MS analysis of apo-CIII glycosylation in human plasma samples varying in protein oxidation levels. The controlled oxidation of apo-CIII methionine residues, the use of sinapinic acid as a MALDI matrix, and the use of MassyTools software for semi-automated, standardized spectra processing have been implemented to achieve highly repeatable measurements of intact apo-CIII proteoforms. The new analytical workflow allowed us to overcome the problem of the high spectral heterogeneity produced by methionine oxidation thus allowing the robust screening of a large cohort of plasma samples for the relative quantitation of apo-CIII proteoforms. Importantly, the evaluation of MS spectra-derived quality parameters was implemented to minimize biases and ensure accuracy of collected data.The cohort analysis confirmed that the level of apo-CIII sialylation is strongly associated with lipid biomarkers, especially with triglyceride levels. The relation between relative abundances of apo-CIII glycoforms and cardiovascular disease development should be further explored. More insight into the role of apo-CIII glycosylation in disease pathophysiology could provide new drug targets. Also, understanding of the mechanisms of existing drugs might increase by considering apo-CIII glycosylation. The methods presented, will enable such large-scale studies.


Contraceptive prevalence in Pakistan has plateaued near 34 percent for over a decade, suggesting that fertility levels are likely to stay high unless effective interventions are designed. We evaluate the Family Advancement for Life and Health 2007–2012 (FALAH), a family planning project implemented in 31 districts of Pakistan. Deviating from previous programs, FALAH emphasized birth spacing—as opposed to limiting family size—as the primary purpose of contraceptive use. We use Pakistan Demographic and Health Survey to evaluate FALAH's impact on continuous and binary measures of birth intervals. To estimate the causal effects of the project, we compare the outcomes for multiple children born to the same mother before and after the project. We find that FALAH increased interbirth intervals by 2.4 months on average and reduced the proportion of short birth intervals by approximately 7.1 percentage points. This finding suggests that birth spacing as a goal of contraceptive use may resonate better with Pakistani couples than limiting family size. The project's effects were more pronounced for women with high education, in rural areas, and in the middle of the wealth distribution. The couIn 2007, a project called Family Advancement for Life and Health (FALAH) was initiated which deviated from the usual practice of promoting small family size as the goal of contraceptive use and repositioned family planning as a health intervention aimed at improving the health of mothers and children through adequate birth spacing . Our study thus complements the existing literature on FALAH. Second, we evaluate a large project from a setting where contraceptive use is low, birth intervals are narrow, and fertility has been difficult to reduce, despite decades of effort from the government and development agencies. Pakistan's experience from FALAH may inform policy design in other similar settings around the world, thus contributing to the Sustainable Development Goals on improving the health and well‐being of women and children.To preview the results, we find that FALAH increased birth spacing by 2.4 months, which translates to an approximately 9.3 percent increase in birth intervals from the average. As such, the proportion of birth intervals deemed short by WHO fell by approximately 7.1 percentage points. These findings, which are robust to a range of checks, suggest that birth spacing as a goal of contraceptive use may resonate better with Pakistani couples than limiting family size. The project had heterogeneous effects based on mother's education, area of residence, and household wealth, with the effects more pronounced for those with high education, in rural areas, and in the middle of the wealth distribution.The rest of the paper is organized in the following manner. In the second section, we provide a brief overview of the FALAH project. We discuss our data and the empirical strategy in the third and fourth sections, respectively. In the fifth section, we present our findings, including results from various robustness checks. We conclude in the sixth section with a discussion of the key limitations, policy implications, and areas for future research based on our findings.The FALAH project was launched in 2007 with the objective of improving the well‐being of families through increased demand and utilization of birth spacing and quality family planning services Mahmood . The proLike many family planning programs around the world, FALAH's full package of interventions included behavior change communication and measures to strengthen the health system along with community mobilization activities , women were told about the health benefits of birth spacing and how HTSP was consistent with Islamic beliefs. Project monitoring documents show that the beneficiaries of these training acknowledged that the new messages about birth spacing were new to them and that they appreciated the focus away from limiting family size . There To estimate the causal effect of the project on these outcomes, we compare the outcomes for multiple children born to the same mother before and after the project, using mother‐fixed effects. We estimate the coefficients in the following equation:ijtY is the outcome for a child i, born to mother j, in year t. As mentioned above, we use interbirth interval both as a continuous variable and as a dichotomous variable. ijtD is a binary variable that measures exposure to the FALAH project and varies by births even for the same mother. ρ is the expected change in birth interval due to exposure to FALAH. ijtX represents characteristics of the child that can vary over time between multiple children born to the same mother. λj are the mother‐fixed effects intended to capture confounding due to time‐invariant characteristics of the mother. εijt is the usual error term. We cluster the standard errors at the district level to allow arbitrary correlation between observations within a district.In this equation, ijt warrants further discussion. As mentioned above, implementation of FALAH varied across districts in terms of roll‐in and duration  as exposed, even if the project was not implemented for the entire period. We do so because the educational component of FALAH may have continued to influence persons in the project district even after its cessation. However, we also conduct a separate analysis by categorizing the districts as fully implemented and partially implemented.As an illustration, take three children born to the same mother in Lasbella district, where the project started in June 2007  framework. While health policy researchers have used this approach widely . We start by controlling for the birth order, then added death of a child in the family (binary), proportion of sons among the older children born to the mother, sex of the preceding birth, and mother's age at the time of index birth. All columns include mother‐fixed effects.In Table For interpretation, we focus on the results from the fully specified regression (Model 6). Based on that regression, exposure to the FALAH project resulted in an increase in interbirth intervals by 2.4 months after controlling for the time‐variant factors. Substantively, this is a large effect, which translates to an increase in birth intervals of approximately 9.3 percent from the mean (=100 × 2.45/26.25).r = 0.63).The coefficients on the covariates are in the expected direction except in the case of the regression that includes both birth order and mother's age. For example, the death of a child in the family is negatively associated with birth interval; on average, the occurrence of the death of a child in the family reduces the birth interval before a successive birth by 2.4 months. We find no association between the proportions of sons a mother already has and the birth interval before a subsequent birth. Having a son is also not associated with an increase in the next birth interval. In the regression that does not include mother's age (Model 5), birth order is positively associated with birth interval. However, this relationship is reversed once we control for mother's age, which is strongly and positively associated with birth interval (Model 6). While the change in the association between birth order and birth interval after we control for mother's age is surprising, it may be due to the high correlation between birth order and mother's age . After controlling for potential time‐varying confounders, we find that exposure to FALAH reduced the proportion of short intervals by 7.1 percentage points Table . Given tRecall that, in our main analysis, all districts where the project was implemented count as such, irrespective of the duration of the implementation. For a project of this nature, it is natural to expect some variation in the quality of implementation—especially when the duration of the implementation differs. To see if the magnitudes of the effects differ by exposure to this duration, we conduct a separate analysis by counting as project districts only the districts where FALAH was implemented for all five years. The nonproject districts are districts where the project was never implemented. Districts where the project was implemented for fewer than five years were eliminated from the analysis.Results from estimating equation  using thIn Table The project had the largest effect among women who had secondary or higher levels of education, followed by those who had no schooling (Panel B). This was true for both outcome measures. At the respective means, FALAH increased interbirth interval by 20.2 and 8.4 percent for women with secondary or higher levels of education and those with no education, respectively. The corresponding effects on the proportion of short intervals were 35.1 and 10.7 percent, respectively. The project had no effect on the interbirth interval or the proportion of short intervals for women with a primary or lower secondary level of education.In terms of the wealth quintiles, the project had the most pronounced effects on the interbirth interval for women in the middle of the wealth distribution Figure . Based oWe checked the robustness of our main results in a number of ways. First, using the previous rounds of the Pakistan Demographic and Health Survey data PDHS 2006–2007), we conducted regressions similar to those reported in Tables 006–2007,Recall that in the main analysis, we restrict the analysis to births that occurred within five years of the start date of FALAH. As a second robustness check, we extended this duration to six years prior to FALAH. Using this expanded analytical sample of birth histories from 2002 to 2012, we were still able to find a significant positive effect of the FALAH project on both measures of the outcome . We conducted further analysis by restricting the sample to women in the program districts, thus removing any noise from the nonswitchers. The results from these analyses, presented in Online Appendix Table In this paper, we exposed a large family planning intervention in Pakistan, called FALAH, to a rigorous evaluation technique and assessed its effect on birth spacing. Deviating from previous family planning programs in Pakistan, the FALAH project emphasized birth spacing, as opposed to limiting family size, as the primary purpose of contraceptive use. Our findings suggest that this repositioning of the goal of contraceptive use resonated well with Pakistani couples. Although some of the estimated effects may be due to other aspects of the project, such as supply‐side strengthening, given the focus on messaging, it is reasonable to conjecture that much of the estimated effect is due to the nature of the messaging. These findings are important for the context of Pakistan where fertility rates have been stubbornly high, contraceptive uptake has been low, and birth intervals have been narrow, all of which adversely affect maternal and child health.Our results agree with previous evaluations, although those evaluations have focused on different outcomes, and it is difficult to interpret their findings as causal. Using data collected during baseline and endline surveys of the married women of reproductive age, Mahmood  shows thAlthough we overcome the major limitations of previous studies, our study also has a number of limitations. The most important limitation relates to our use of mother fixed effects, which improve internal validity at the expense of generalizability , as a woman's exposure to FALAH is defined by her district of residence rather than by whether she was a direct beneficiary of the project.after FALAH was implemented in many districts.Second, due to the data limitations, we could not estimate the effect of FALAH on contraceptive use. Even though FALAH focused on birth spacing messages, these messages were about the use of contraceptives in order to achieve longer birth intervals. PDHS 2012–2013 contains information on contraceptive use between 2007 and 2012–2013 . Therefore, we only have contraceptive use data A third limitation relates to censoring. Consider a woman who was exposed to FALAH and, as a result, decided to either delay the next birth for more than five years or stop childbearing. Our analysis is set up to compare the birth intervals for births that occurred (from the same mother) pre‐ and post‐FALAH implementation. As such, it does not account for women who did not have a birth during the post‐FALAH period. This implies that our estimates may be biased downward because of censoring.Fourth, birth history data obtained through retrospective reporting are prone to multiple biases (Rustein and Rojas Finally, we are unable to account for abortion, unintentional pregnancy loss, or stillbirths in our analysis. The prevalence of abortions or pregnancy loss in a population can lead to an overestimation of the length of birth intervals (Rossi and Rouanet In addition to addressing the limitations above, future studies can fill the gap in two other important areas. Immediate among these areas is an exploration of the mechanisms behind the success of the FALAH project, perhaps through a deeper qualitative study to analyze what aspects of the project were successful in promoting birth spacing. FALAH also had a few supply‐side components, such as improving the availability of contraceptives and training providers for better management of side effects, although behavioral change communication was a defining feature of the program as discussed earlier. It is reasonable to expect that some of the effect we estimate may have come from these other components.Second, and more generally, a comparative study of messaging in family planning programs in Pakistan and other high‐fertility settings that provides broad as well as context‐specific lessons for policymakers seems critical for the design of future family planning programs. This line of research can build on the Pakistan‐based studies referenced above as well as evidence from other countries where policymakers have attempted to reframe goals of family planning interventions to fit their contexts. For instance, a qualitative study evaluating the success of the family planning program in Rwanda shows that one of the reasons for the success of the family planning program there is that the government promoted family planning program as a tool to empower individuals and families rather than as a program with a motive to curb population growth (Schwandt et al. Finally, the need for implementing large‐scale projects of this nature in a way that allows for rigorous ex post evaluation cannot be overstated. Many of the limitations of our study relate to the nonrandom roll‐in of the FALAH project. In the future, implementers should consider other approaches, such as rolling in the projects randomly rather than into more problematic areas first, so that alternative evaluation methods which allow for greater generalizability of results can be used.Online AppendicesClick here for additional data file.


GmGAMYB gene of R2R3-MYB type was induced by long-days (LDs). GmGAMYB showed higher transcriptional levels in the flowers, leaves and pods of soybean. Overexpression of GmGAMYB in transgenic soybean showed earlier flowering time and maturity in LDs and short-days (SDs). GmGAMYB interacted with GmGBP1 and might promote flowering time by up-regulating the expression of GmFULc gene in soybean. Moreover, the expression level of GmGAMYB was also induced by gibberellins (GAs) and the plant height of GmGAMYB-ox plants was significantly increased, which was caused by the enlargement of internode cell in stem. Furthermore, GmGAMYB overexpression led to increased GA sensitivity in the hypocotyl of soybean seedlings compared with WT. GmGAMYB may be a positive regulator of GA response of promoting plant height by up-regulating the expression of GmGA20ox gene in soybean. Together, our studies preliminarily showed that the partial functions of GmGAMYB in regulating flowering time and GA pathway.The flowering time and plant height of soybean are important agronomic characters, which control the adaptability and yield of soybean. R2R3 MYB transcription factor plays an important regulatory role in plant growth and development. In this study, soybean  Glycine max (L.) Merrill] is a short-day (SD) plant, and its growth and development are very sensitive to photoperiod response. SD can promote flowering, and long-day (LD) inhibit the growth of flower bud  homologs, activated the expression of floral identity gene homologs such as GmAP1, GmLFY and GmSOC1 to promote early flowering in soybean  encodes an ent-kaurene synthase (KS) and plays a key role in GA-regulated cell elongation in soybean stem internodes  is one of the most important plant hormones in determining plant height . Recent ternodes . A homol soybean . DespiteArabidopsis, GAMYB-like genes AtMYB33, AtMYB65, and AtMYB101 mediated GA signal transduction regulates petiole elongation and flowering response , a SKIP homologous gene, functioned as a positive regulator of photoperiod control of flowering time and maturity responses  and Co-lmmunoprecipitation (Co-IP). Combined with RNA-Seq analysis, the overexpression of both genes regulated the expression of GmFULc gene. Therefore, we speculated that GmGAMYB and GmGBP1 interacted to promote flowering time by upregulation of GmFULc gene expression in soybean. Moreover, RNA-seq analysis on GmGAMYB-ox soybean plants showed that GA synthetic gene GmGA20ox was up-regulated by GmGAMYB. GmGAMYB may be a positive regulator of GA response of promoting plant height by up-regulating the expression of GmGA20ox gene in soybean. These results preliminarily proposed the partial functions of GmGAMYB in regulating flowering time and GA pathway.Soybean esponses . Recent d system . In the GmGAMYB gene. The seeds of two soybean cultivars were provided by the Northeast Agricultural University, Harbin, China.In this study, soybean “DongNong 50” was used as the wild-type (WT) control and the background plant for genetic transformation. “DongNong 42,” a photosensitive soybean variety was used to analyze the expression pattern of GmGAMYB experiments, soybean “DongNong 42” were cultured at 25°C, 250 μmol m–2sec–1 white light, LD (16/8 h light/dark) conditions (LDs). A part of seedlings was transferred to SD (8/16 h light/dark) conditions (SDs) on day 15 after emergence. When the second trifoliate leaves were expanded, samples were taken every 3 h under LDs and SDs for a total of 24 h. Samples of different tissues including roots, stems, leaves, flowers, pods and seeds of soybean plants grown under LDs and SDs were collected. To analyze the response of GmGAMYB to GA3, 15-day-old seedlings under LDs as described above were sprayed with 100 μM GA3, and trifoliate leaves were sampled at 0, 0.5, 1, 3, 6, 9, 12, and 24 h after treatment. All samples were frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted from all samples and the expression of GmGAMYB was analyzed by quantitative real-time RT-PCR (qRT-PCR).For expression pattern analysis of GmGAMYB promoter activity in different Arabidopsis tissues and the activity of GmGAMYB promoter in Arabidopsis treated with GA3, Col-0 was used as the background plant for genetic transformation. Seeds of proGmGAMYB:GUS transgenic Arabidopsis were surface sterilized with 10% hypochlorite and then planted on MS agar medium. When Arabidopsis seedlings had two true leaves, they were transplanted into 1:1 of vermiculite and turfy-soil and cultured under LDs. When Arabidopsis seedlings had four leaves, some of them were soaked in 100 μM GA3 and sampled at 0, 3, and 6 h, respectively, for staining. On the 30th day of culture, stem leaves, inflorescence, rosette leaves and roots of Arabidopsis plants were stained with X-Gluc staining solution. After 12 h at 37°C, then decolorized with 70% ethanol. After the chloroplast were removed, microscopic observation was carried out.In order to analyze 3 generation GmGAMYB-ox-1, GmGAMYB-ox-2, and WT soybean seeds were planted in plastic pots with dimensions of 30 cm high × 25 cm diameter at the top and 15 cm diameter at the bottom and cultured in a greenhouse at 25°C with 250 μmol m–2sec–1white light under LDs. The positive seedlings detected by Western blot were retained when the cotyledons fully developed. When the first trifoliate leaves were expanded, part of the seedlings were transferred to SDs under the same temperature regime. At least 15 plants of WT and two GmGAMYB-ox soybean lines were cultured under LDs and SDs, respectively. Five reproductive stages of soybean  were recorded according to the identification method of soybean growth period proposed by Fehr GATCCCA GGATCTGATTACAAGGATCATGATGGTGATTACAAGGAT CACGACATCGACTACAAGGATGACGATGACAAGCACCA TCATCACCACCATTGATCTCTAGA-3′, the sequences encoding 3F6H tag were in bold] (NotI-XbaI sites of pENTRY vector (named pENTRY-3F6H). GmGAMYB gene fragment of 1602 bp was cloned from “DongNong 42” genome using GmGAMYB-3F6H-F and GmGAMYB-3F6H-R primers  connection onto the pENTRY-3F6H vector (named 35S:GmGAMYB-3F6H-pENTRY). Recombinant plasmid 35S:GmGAMYB-3F6H-pENTRY was synthesized into pB7WG2 carrier by LR reaction (named 35S:GmGAMYB-3F6H-pB7WG2). The construct was then transferred into Agrobacterium tumefaciens (EHA105). According to the method described previously  were selected from five T3 transgenic soybean lines for further study.Firstly, the FLAG and HIS tag carrier were constructed by synthesizing the tandem repeats of 3 × FLAG and 6 × Histidine (3F6H) tags with in bold] . The syn primers . The GmGeviously , transgeGmGAMYB genome sequence of 1945 bp in front of the 5′ untranslated region (UTR) served as the promoter region of the gene. The GmGAMYB promoter sequence was amplified from the genomic DNA of “DongNong 42” using proGmGAMYB:GUS-F and proGmGAMYB:GUS-R primers (pENTR/D-TOPO (Life technologies) (named proGmGAMYB-TOPO). The recombinant plasmid was transferred to pGWB533 vector through LR reaction (named proGmGAMYB:GUS), and then the new fusion vector was introduced into Agrobacterium GV3101 for transforming into Arabidopsis thaliana (Col-0) using the floral dip method ] to extract soybean protein to detect the protein expression of GmGAMYB driven by cauliflower-mosaic virus (CaMV) 35S promoter in transgenic soybean. Each 20 μg protein sample was subjected to 10% SDS-polyacrylamide gel electrophoresis, which was separated and transferred to nitrocellulose membrane. HRP-conjugated anti-FLAG antibody  was used to detect 35S:GmGAMYB-3F6H protein. Mouse beta-actin monoclonal antibody  was used to detect actin proteins as control. Super Signal West Pico Chemiluminescent substrate kits (Thermo Fisher Scientific) and the signal was detected by chemiluminescence imaging (Amersham Imager 600).Using extraction buffer , 10 μl of Protein G-coupled magnetic beads  was used to capture anti-FLAG antibody (Sigma). After incubation at 4°C for 30 min, magnetic beads were washed three times for 5 min each time with 1 ml of Co-IP buffer without MG-132, Na3VO4, sodium fluoride, and protease inhibitor, and eluded with 2 × SDS sample buffer at 80°C for 5 min. At each sample, the immunoprecipitated proteins and 20 μl of the total extract were separated by 10% SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. Then, HRP-conjugated anti-FLAG antibody  and HRP-conjugated anti-GFP Antibody  were applied to test GmGAMYB-FLAG and GmGBP1-GFP protein, respectively.The constructed a leaves  and were® Tip Green qPCR SuperMix  according to the manufacturer’s instructions on Applied BiosystemsTM 7500 Fast Dx Real-Time PCR Instrument (ABI). The PCR cycling conditions were as follows: 94°C for 30 sec; 40 cycles of 95°C for 5 sec and 60°C for 34 sec. GmActin4 (GenBank accession number AF049106) was used as endogenous regulatory genes of soybean. The primers used in qRT-PCR analyses were shown in RNA isolation has been described previously . qRT-PCRGmGAMYB in the National Center for Biotechnology Information (NCBI) GenBank (Accession No. KC525897) was cloned from the leaves of “DongNong 42” by RT-PCR. The cDNA sequence of GmGAMYB is 2,975bp and contains 893bp 5′ UTR, 480bp 3′ UTR and 1,602 bp open reading frame, which encodes 533 amino acids with predicted molecular mass of 58.873 kDa. Multiple sequence alignment of soybean GmGAMYB and GAMYB-like proteins of Arabidopsis thaliana, Oryza sativa, Zea mays, Vitis vinifera, Manihot esculenta, Solanum lycopersicum, and Cucumis sativus showed that GmGAMYB protein contains a highly conserved R2R3 domain in the N-terminal , Spatholobus suberectus (TKY68413.1), Cajanus cajan (XP_020219565.1), Vigna unguiculata (XP_027909434.1), Vigna angularis (XP_017431637.1) and Lupinus angustifolius (XP_019449331.1) indicating that GmGAMYB proteins were relatively conservative in the evolution of leguminous plants  was constructed, and Agrobacterium-mediated transient expression of the fusion protein was transformed into tobacco leaves. Fluorescence microscope was used to observe green fluorescence in tobacco mesophyll cells. The observation results showed that the expression of 35S:GFP vector caused GFP fluorescence dispersed throughout the entire cell. In contrast, 35S: GFP-GmGAMYB fusion protein was specifically localized on the nucleus of tobacco mesophyll cells  between WT and the GmGAMYB-ox transgenic line were detected  genes are a group of downstream flowering genes that are known to play a major role in the reproductive transition. All three homologous genes GmFULc (Glyma.05G018800), GmFUL1a (Glyma.04G159300), and GmFUL2b (Glyma.17G081200) in soybean are positively regulated by GmGAMYB. GmFPF1 (Glyma.04G074800), a FLOWERING PROMOTING FACTOR 1, showed 35.32% amino acid identity with AtFPF1 (AT5G24860), which promoted flowering in Arabidopsis , GmTCP8 (Glyma.05G027400), and GmTCP12 (Glyma.06G193000). GA20ox was a gene that regulated plant height in the GA metabolic pathway, and its overexpression saved the dwarfing phenotype in rice  transcription factor family, are the best homologous matching of AtTCP14 and AtTCP15 in Arabidopsis, and their potential functions in regulating plant height  was identified by fine mapping, affecting brown seed coat/hilum phenotype in soybean  . R2R3 MY W2 gene . GmMYBJ1e stress . GmMYB73bidopsis . A speci soybean . Overexpuit size . HoweverGmGAMYB, a new MYB transcription factor, was isolated from soybean and identified. GmGAMYB was identified as a member of the GAMYB subfamily of R2R3 MYB transcription factors by amino acid sequence alignment with typical GAMYB-like proteins in several species and conservative motif analysis. According to the subcellular localization of GmGAMYB in tobacco leaf cells, the GmGAMYB-GFP fusion protein was specifically localized on the nucleus of tobacco leaf cells, which indicated that GmGAMYB is a nuclear localization protein that matches its function as a transcription factor. Soybean is a typical SD plant, which is particularly sensitive to photoperiod. Photoperiod responses lay the foundation for the adaptation of different soybean varieties and play important roles in flowering and maturation of soybean (WEREWOLF (WER), encodes an R2R3 MYB transcription factor, expressed in the epidermis of leaves and regulated flowering time through photoperiod pathway in Arabidopsis , a family of MADS-box transcription factor protein genes, is a major of downstream flowering genes, which is known to play an important role in reproductive transition. In the photoperiod-dependent flowering pathway of Arabidopsis thaliana, the Arabidopsis flowering integrator FT promotes the transition to flowering by regulating the accumulation of FUL in Arabidopsis leaves (GmFT1a inhibited the expression of GmFULa (a soybean FUL homolog) and delayed flowering  were significantly upregulated. GmGBP1 is induced by SDs and is a positive regulator of photoperiod control of flowering time  can be found below: NCBI-SRA database under the BioProject no. XY performed protein interaction and data analysis. XL and JS performed phenotypic observation and measurement. YL performed gene cloning transformation. YZ and YW performed cell morphology detection and RNA data analysis. XY, LZ, and WL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudoaneurysm formation is known to complicate arteriovenous haemodialysis access. Ultrasound guided thrombin injection is a recognised treatment option, but is not possible in pseudoaneurysms with no measurable neck. Balloon assisted techniques have been described in such cases, which transiently obstruct flow out of the pseudoaneurysm and thereby prevent non-target embolization during ultrasound guided percutaneous thrombin injection. We describe a balloon assisted technique for the treatment of a radial artery pseudoaneurysm, via retrograde access from the draining cephalic vein of an arteriovenous fistula.A 61-year-old male with a radio-cephalic fistula was found on duplex ultrasound to have a large radial artery pseudoaneurysm with no measurable neck, as well as a juxta-anastomotic cephalic vein stenosis. Endovascular treatment was selected over open surgery. Retrograde cephalic venous access was established, which allowed for concurrent treatment of both the venous stenosis and the arterial pseudoaneurysm. After balloon dilation of the juxta-anastomotic stenosis, a percutaneous transluminal angioplasty balloon catheter was advanced across the arteriovenous anastomosis and inflated across the neck of the radial artery pseudoaneurysm, to transiently obstruct blood flow. This allowed for safe injection of thrombin into the pseudoaneurysm by direct ultrasound guided sac puncture; thereby achieving thrombosis.Balloon assisted ultrasound guided thrombin injection is an endovascular treatment option that can obviate the need for open surgery in cases involving pseudoaneurysms with no measurable neck. The technique described allowed both concurrent treatment of a juxta-anastomotic venous stenosis and treatment of an arterial pseudoaneurysm from a single venous puncture. This technique avoided arterial access and its inherent complications. Pseudoaneurysm formation is a recognised complication affecting arteriovenous haemodialysis access. Cases that are not suitable for open surgical repair are often treated with minimally invasive techniques including ultrasound guided thrombin injection (UGTI) , using balloon-assisted UGTI with retrograde access from the cephalic vein draining the AVF. The procedure was undertaken on an outpatient basis, concurrently with venoplasty to treat a juxta-anastomotic cephalic vein stenosis.A 61-year-old male with a left radio-cephalic fistula presented with a low haemodialysis flow rate of 270ml/min; measured using a Transonic® flow-QC device . Duplex ultrasound (DUS) demonstrated a 24mm diameter pseudoaneurysm arising directly from the radial artery. Whilst the neck of the pseudoaneurysm was narrow, there was no measurable neck length, increasing the risk of non-target embolization with standard thrombin injection techniques , with good angiographic result.A 7 × 40mm Sterling Balloon (Boston Scientific) was then advanced over the wire and positioned across the neck of the pseudoaneurysm under ultrasound guidance. The balloon was sized to match the diameter of the radial artery, which measured 7mm on DUS. The pseudoaneurysm was punctured with a 21-guage needle under ultrasound guidance. The 7 × 40mm Balloon was then inflated, covering the pseudoaneurysm neck. 2mls of Thrombin was injected under ultrasound guidance, filling the pseudoaneurysm. The balloon was then deflated.After balloon deflation, DUS demonstrated a small amount of residual perfusion within the largely thrombosed pseudoaneurysm sac. This appeared to have resolved following ultrasound guided manual compression of the pseudoaneurysm. Follow up DUS 72 h later demonstrated a persistent 8mm area of perfusion within the pseudoaneurysm. A repeat procedure was therefore undertaken, employing the same technique as the initial procedure, requiring a further 1mls of thrombin to achieve complete thrombosis.No complications occurred during or immediately after either procedure. Both procedures were undertaken as day cases. A follow up ultrasound scan 72 h after the second procedure demonstrated complete thrombosis of the pseudoaneurysm with volume flow of 1400mls/min recorded in the brachial artery Fig. . SuccessWe present a case of a radial artery pseudoaneurysm treated with balloon assisted UGTI with retrograde venous access via an ipsilateral radio-cephalic AVF. Whilst balloon assisted techniques for pseudoaneurysm embolization have been described previously (Mary et al. Our patient presented due to low haemodialysis flow and it is likely that resultant venepuncture difficulties caused the pseudoaneurysm, which lay immediately deep to the venous needling site. The position of the pseudoaneurysm also left it susceptible to further injury from inadvertent puncture during subsequent AVF cannulation.The location of the pseudoaneurysm in the mid/upper forearm made open surgery a technically difficult, higher risk treatment option. The radial artery lies deep to pronator teres at this position and the draining cephalic vein directly overlying the pseudoaneurysm would have needed to be mobilised. The patient also wished to avoid an overnight admission due to social circumstances.Treatment with a covered stent was considered and would have been attempted had balloon-assisted thrombin injection failed. The endovascular approach via the draining vein of the AVF also allowed concomitant treatment of the juxta-anastomotic stenosis; improving flow within the fistula.Arterial pseudoaneurysms in patients with radio-cephalic AVFs have previously been treated with balloon assisted techniques via a retrograde puncture of the distal radial artery (Aytekin et al. Residual filling of the pseudoaneurysm was identified by DUS following the first attempt at Thrombin injection and a second procedure was required to achieve sustained thrombosis. DUS follow up post procedure is essential to ensure thrombosis with no re-perfusion. At the time of writing the patient continues to dialyse successfully with no further complications. Clinical surveillance continues with physical examination of the AVF and regular measurement of volume flow on dialysis.This case demonstrates the feasibility of treating AVF related arterial pseudoaneurysms with no measurable neck with balloon assisted UGTI. Venous access in these cases may be preferable as it allows for concurrent treatment of AVF stenoses and carries a low risk of puncture site complications. The technique is adaptable for different anatomy and type of AVF and offers a minimally invasive treatment option as an alternative to open surgery.