The transcriptional repressor Blimp-1 originally cloned as a silencer of type I interferon (IFN)-β gene expression controls cell fate decisions in multiple tissue contexts. Conditional inactivation in the mammary gland was recently shown to disrupt epithelial cell architecture. Here we report that Blimp-1 regulates expression of viral defense, IFN signaling and MHC class I pathways, and directly targets the transcriptional activator Stat1. Blimp-1 functional loss in 3D cultures of mammary epithelial cells (MECs) results in accumulation of dsRNA and expression of type III IFN-λ. Cultures treated with IFN lambda similarly display defective lumen formation. These results demonstrate that type III IFN-λ profoundly influences the behavior of MECs and identify Blimp-1 as a critical regulator of IFN signaling cascades. By virtue of its ability to recruit chromatin modifiers such as HDAC1/2, LSD-1, and G9a, Blimp-1 governs epigenetic reprogramming required for germ cell specification in the early mouse embryo6. Blimp-1 null embryos die at around embryonic day 10.5 (E10.5) due to defective placental morphogenesis7. Loss of Blimp-1 disrupts specification of the spiral artery-associated trophoblast giant cell lineage (SpA-TGC’s) the crucially important cell subpopulation that invades and remodels maternal blood vessels9. Lineage tracing experiments in combination with expression profiling and single cell RNA-Seq analysis have defined the cell type specific transcriptional signature governing these specialized functional activities9.The zinc finger transcriptional repressor Blimp-1 (PRDM1) originally identified as a post-inductive silencer of interferon beta (IFN-β) gene expression in virally infected cells12. Recent studies have further characterized Blimp-1-dependent gene expression changes and chromatin remodeling at its transcriptional targets associated with plasma cell maturation13. Similarly Blimp-1 regulates cell fate choices made during differentiation of CD4+ T cell subsets, and controls the balance of cytolytic effectors vs the generation of memory CD8+ T cells18.In contrast in B and T lymphocytes, Blimp-1 function is not required at early stages during lineage commitment. Rather in B cells Blimp-1 directly silences expression of key transcription factors such as c-Myc, Pax5, and CIITA that maintain B cell identity to dramatically shift the developmental programme towards plasma cell terminal differentiation19. Blimp-1 function is required to prevent premature activation of the adult enterocyte biochemical signature19. ChIP-Seq analysis of E18.5 small intestine demonstrate Blimp-1 preferentially binds to promoter regions upstream of genes associated with metabolism20 and interestingly revealed a subset of highly conserved target sites, including the promoters of IFN-inducible components of the MHC class I antigen processing machinery such as Psmb8, Psmb10, Tapbp, and Erap1, that are also recognized by interferon regulatory factor (IRF) -1 a positive regulator of the MHC class I peptide loading pathway21. Thus Blimp-1 occupancy directly antagonizes IRF-1 to prevent premature activation of the MHC class I pathway in fetal enterocytes and maintain tolerance in the neonatal intestine in the first few weeks after birth during colonization of the intestinal tract by commensal microorganisms20.Recent experiments have shown that Blimp-1 governs postnatal reprogramming of intestinal enterocytes22. Recent experiments demonstrate that Blimp-1 expression within a rare subset of luminal progenitors is up-regulated in response to pregnancy hormones and conditional inactivation results in defective mammary gland morphogenesis23. Strikingly Blimp-1 functional loss disrupts epithelial architecture and lumen formation both in vivo and in three-dimensional (3D) primary cell cultures23.Survival at these early stages depends on the highly specialized mammary glands that produce milk and enable the mother to feed her newborn offspring. The mammary epithelium contains two structurally and functionally distinct cell subpopulations: the outer myoepithelial/basal cells and the inner luminal cell population. Considerable progress has been directed towards understanding cell fate decisions during ductal morphogenesis, lumen formation, and alveologenesis. The functional contributions made by distinct mammary stem cell subpopulations, including both unipotent and bipotent progenitors have been extensively described in lineage tracing experimentsin vivo, we exploited the inducible ROSA26:CreERT2 allele to activate Cre-mediated Blimp-1 deletion via tamoxifen treatment of 3D mammary epithelial cell (MEC) cultures. As expected Blimp-1 deficient (cKO) MEC cultures display defective lumen formation and fail to establish apical-basal polarity. Surprisingly however functional annotation analysis of up-regulated genes identified highest enrichment scores for categories associated with innate immunity and IFN signaling pathways. This gene list substantially overlaps with those recently described as conserved Blimp-1 targets via ChIP-Seq analysis, including key components of the MHC class I peptide-loading pathway20. Additionally IFN-stimulated genes and pathway regulators including Usp18, Oligoadenylate synthetase (OAS) family members, and the key transcriptional activator Stat1, were identified here as direct Blimp-1 targets. Finally the present experiments demonstrate that Blimp-1 loss of function results in up-regulated expression of double stranded (ds)RNA and type III IFN lambda (IFN-λ). Moreover type III IFN-λ treatment of wild type MEC cultures causes defective lumen formation and maturation. These results demonstrate for the first time that the zinc finger transcriptional repressor Blimp-1 silences type III IFN expression in mammary epithelial cells and establish its actions as a global regulator of the IFN signaling cascade.To further investigate the underlying causes of these tissue disturbances, here we performed transcriptional profiling experiments. To avoid studying possible contributions made by strongly Blimp-1-positive endothelial cells within this highly complex vascularized tissue To learn more about Blimp-1 functional contributions during mammary gland morphogenesis, we used the Illumina array platform to compare transcripts in wild type versus Blimp-1 cKO MEC cultures harvested at Day 3 (D3) and Day 4 (D4), when tissue disturbances become readily visible. The greatest differences in gene expression profiles were detectable at Day 4. The relatively few down-regulated genes (n = 138) in D4 Blimp-1 cKO MECs showed no significant enrichment for any GO annotated pathways or biological processes and were not investigated further  small intestine25. Results above demonstrate increased Stat1 expression readily detectable at Day 3 the earliest time point examined  18.5 small intestine is severely hypomorphic elsewhere20 and FACS sorting is problematic due to relatively weak eGFP fluorescence intensity even in plasma cells. For these reasons, we elected to perform qPCR validation of Blimp-1 occupancy at candidate target sites using transiently transfected CommaDβ cells, an established cell line that shares many functional and behavioral characteristics with bona fide mammary stem cells26, in combination with our expression construct encoding the C-terminal eGFP-tagged Blimp-1 fusion protein and the proven GFP mAb27. Results shown in Supplementary Figure Blimp-1 expression in the mammary gland is restricted to a rare subset of luminal progenitors, representing less than 1% of the total luminal cell population recovered from pregnant females29. Our transcriptional profiling experiments shown above reveal that Blimp-1 conditional loss causes up-regulated expression of several dsRNA binding proteins including the DEXD/H box helicases Ddx60, Dhx58 (LGP2), and RIG-1 (Ddx58)  Fig. . However33. Considerable evidence demonstrates that type III IFN-λ plays a crucial protective role at mucosal barrier surfaces lining the respiratory, gastro-intestinal, and reproductive tracts33. Blimp-1 was previously shown to silence IFN-λ1 expression in human lung epithelial cells34. However in mice IFN-λ1 is a pseudogene. To test whether Blimp-1 regulates type III IFN-λ expression in mouse mammary epithelial cells, here we performed RT-PCR experiments using primers designed to detect IFN-λ2/3 transcripts. As shown in Fig. Recent experiments demonstrate that type III IFN-λ via its unique receptor complex selectively activates anti-viral defense pathways in epithelial cells21. Irf-3 and Irf-7 have been shown to induce Type I and Type III IFN expression in virally infected cells35. Interestingly we found here that Irf-1, 2, 3, and 6 are constitutively expressed by mammary epithelial cells whereas Blimp-1 inactivation leads to increased expression of Irf-7 and Irf-9  that recognize viral RNA trigger membrane bound signaling proteins such as MAVS that in turn activate assembly of transcriptional complexes driving IFN-stimulated gene expression. In contrast to these disturbances downstream of viral entry and replication, the striking morphological changes described here associated with up-regulated IFN-λ expression are simply caused by Blimp-1 inactivation. Interestingly Blimp-1 cKO also resulted in accumulation of dsRNA recognized by the J2 monoclonal antibody. Thus, activation of IFN signaling responses could simply be due to the presence of dsRNA.Recent studies implicate a functional relationship between epithelial cell polarity and IFN-λ expression from peroxisomesThe J2 mAb recognizes dsRNA greater than 40 bp in length but its fine specificity remains ill defined. An extensive literature has described diverse host mechanisms responsible for controlling expression of endogenous retroviral sequences scattered across the genome. It is tempting to speculate that as for many other zinc finger proteins, Blimp-1 may directly silence ERV transcription in a sequence specific manner. However mapping ChIP seq peaks to repetitive elements requires the application of sophisticated bioinformatics strategies. An important long term goal is to characterize dsRNAs that accumulate in the absence of Blimp-1 and test this attractive model.23. We now understand in the absence of Blimp-1 that type III IFN-λ secretion by rare luminal progenitors acting in a paracrine fashion has the ability to influence the behavior of neighboring cells. Our results demonstrate for the first time that type III IFN-λ not only activates anti-viral responses in mucosal epithelial cells, but also in mammary epithelial cells. While the role played by type III IFNs in innate immune protection at mucosal barrier surfaces has been extensively investigated, relatively little is known about viral infections in the mammary epithelium or how this impacts on neonatal health. It will be important to learn more about protective and potentially negative effects mediated by type III IFN-λ.The present results help to resolve the paradoxical finding previously described in our recent studies, namely how could Blimp-1 inactivation in rare progenitor cell population cause such dramatic widespread phenotypic changes throughout the entire epithelial cell populationPrdm1BEH/+ and Prdm1CA/CA mice23 were crossed with the ROSA26:CreERT2 line47 to generate Prdm1BEH/CA;ROSA26:CreERT2 females. Mice were genotyped by PCR as described in the original reports. All animal experiments were performed in accordance with Home Office regulations and were approved by the University of Oxford Local Ethical Committee.Female C57BL/6 mice (8–10 weeks of age) were used as the wild type strain. For inducible Blimp-1 gene deletion in mammary epithelial cell (MEC) cultures, 26. Primary MECs were collected from 15.5- and 16.5-day pregnant mice and cultured as described48. Cells (2 × 104) were seeded onto growth factor reduced Matrigel  to form acini and cultured in growth media  supplemented with 2% growth factor reduced Matrigel. Cultures were fed every 48 hrs and grown for 3–6 days. Cre-mediated deletion of Blimp-1 in primary MEC cultures (hereinafter referred to as Blimp-1 cKO) was achieved by collecting MECs from Prdm1BEH/CA;ROSA26:CreERT2 mice and treating with 1/1000 dilution of 4-Hydroxytamoxifen (4-OHT) dissolved in ethanol  for 24 hrs. As controls, MECs from WT mice were similarly treated with 4-OHT. In some experiments, MitoTracker Red CMXRos (Invitrogen) dissolved in MEC growth media  was added at day 2 of culture for 45 min at 37 °C. Recombinant mouse IFN-β , IFNγ  or IFN-λ2  was added on day 0 of culture.CommaDβ cells  were maintained in DMEM/F12 medium supplemented with B27 (GIBCO), 20 ng/mL epidermal growth factor , 20 ng/mL basic fibroblast growth factor  and 10 μg/mL insulin (Sigma-Aldrich) as previously describedFor immunofluorescence staining, acini were fixed with 4% PFA in PBS and permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were blocked with 10% normal goat serum/2% BSA in PBS for 2 hrs at RT, and incubated with Blimp-1, Stat1, pStat1 (Tyr701), Stat2, or dsRNA (J2) antibodies  with on column DNase treatment according manufacturer’s protocol. Microarray transcriptional profiling experiments were performed using triplicate RNA samples of each genotype at both Day 3 and 4 as previously described using Illumina Mouse WG-6 v2 Expression BeadChips8. Differential probe expression was determined following rank-invariant normalization using the Illumina custom error model option of Gene Expression Analysis Module V1.6.0 of GenomeStudio V2009 (Illumina) with Benjamini and Hochberg false discovery rate. Probes with significant different expression  were analyzed by WebGestalt using default parameters . qPCR analysis was performed on 5 RNA samples for each genotype at day 3 and 4. First-strand cDNA was reverse transcribed from RNA (1 μg) using Superscript III  according to manufacturer’s protocol and qPCR performed using QuantiTech SYBR Green master mix  on a Rotor-Gene Q (Qiagen). Relative gene expression was calculated using the ΔΔCt method in comparison with Hprt as the reference. Reverse transcription-PCR (RT-PCR) was performed using the OneStep RT-PCR kit (Qiagen) according to manufacturer’s protocol. Primer sequences are provided in Supplementary Table Total RNA from five independent MEC 3D cultures each from 20 and Stat124 genomic binding sites. ChIP-peaks were functionally annotated with GREAT version 3.0.049, using the basal extension rule linking peaks to the nearest transcription start site (TSS) ± 100 kb.ChIP-Seq datasets corresponding to NCBI GEO accession numbers GSE66069, GSE60204 and GSE33913 were used to identify Blimp-127 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. GFP positive cells, sorted by FACS at 24–48 hours post transfection were cross-linked with 1% formaldehyde in growth media for 20 min at RT and subsequently processed for ChIP using 10 μg of mouse anti-GFP IgG2a , as described previously50. Non-transfected CommDβ cells were used as negative controls. Eluted DNA samples were recovered using ChIP DNA Clean and Concentrator column kit (Zymo Research) and qPCR analysis of quadruplicate ChIP and input samples performed using QuantiTech SYBR Green master mix  on a Rotor-Gene Q (Qiagen). Primers were designed to amplify 100–200 bp regions central to Blimp-1 binding sites identified in ChIP-Seq dataset GSE66069. Selected genes included c-Myc, Prdm1, Stat1 . P values ≤0.05 were considered significant.Statistical analyses were performed using two-tailed unpaired The microarray data has been deposited in NCBI GEO with the accession number GSE100747.Dataset 1Supplementary information


Improving child nutritional status is an important step towards achieving the Sustainable Development Goals 2 and 3 in developing countries. Most child nutrition interventions in these countries remain variably effective because the strategies often target the child's mother/caregiver and give limited attention to other household members. Quantitative studies have identified individual level factors, such as mother and child attributes, influencing child nutritional outcomes.We used a qualitative approach to explore the influence of household members on child feeding, in particular, the roles of grandmothers and fathers, in two Nairobi informal settlements. Using in‐depth interviews, we collected data from mothers of under‐five children, grandmothers, and fathers from the same households.Our findings illustrate that poverty is a root cause of poor nutrition. We found that mothers are not the sole decision makers within the household regarding the feeding of their children, as grandmothers appear to play key roles. Even in urban informal settlements, three‐generation households exist and must be taken into account. Fathers, however, are described as providers of food and are rarely involved in decision making around child feeding. Lastly, we illustrate that promotion of exclusive breastfeeding for 6 months, as recommended by the World Health Organization, is hard to achieve in this community.These findings call for a more holistic and inclusive approach for tackling suboptimal feeding in these communities by addressing poverty, targeting both mothers and grandmothers in child nutrition strategies, and promoting environments that support improved feeding practices such as home‐based support for breastfeeding and other baby‐friendly initiatives. Grandmothers have central roles in child nutrition as they are key advisors to younger mothers in addition to being child caregivers themselves. They are important targets for child health interventions and should not be undermined in strategies aiming to reduce child malnutrition in urban informal settings.Child health interventions in urban informal settings not only should focus on increasing mothers' knowledge on child nutrition but also should include promotion of optimal child‐feeding practices and promotion of interventions that support mothers to combine breastfeeding and their other occupations.Reinforcement of interventions towards sustainable poverty reduction is fundamental in dealing with child undernutrition.1Nutrition is one of the most important factors that impact on children's development and is linked to short‐ and long‐term cognitive development and survival . The NUHDSS collects birth, death, and migration data every 4 months, and the Maternal and Child Health study collected child anthropometric measurements. From these data, households with stunted and not stunted under‐five children were identified. Community health workers in the study area helped identify the selected households based on their NUHDSS unique identifier. In total, 30 in‐depth interviews were conducted in the two sites: 12 with mothers of stunted children, eight with mothers of nonstunted children, six with grandmothers, and four with fathers, as shown in Table 2.3The interviews were audio‐recorded then transcribed before translation into English. Using a deductive coding approach, the analysis was done using Nvivo 10 and guided by the conceptual framework on child health described in Figure 2.4The study was approved by the African Medical and Research Foundation Ethics and Scientific Review Committee in Kenya and the Human Research Ethics Committee  at the University of Witwatersrand in South Africa. All interviews were conducted in private, and written informed consent was sought from all participants.3Table The results below are structured around the three thematic areas explored during the study: knowledge on child stunting, views and practices on child feeding, and decision‐making mechanisms on child feeding.3.1Mother nonstunted child, ViwandaniThe child looks small and yet he is older. You will notice from the body, the body does not match the number of years, and if you have such a child, he is sickly and will not want to play with other children. Mother stunted child, ViwandaniThe child is older but the body frame is small. My youngest is really small. His age mates are much taller …. Respondents were asked to describe symptoms they associated with stunting and its perceived causes. The results show a broad knowledge on child stunting and its symptoms. Regardless of the child's stunting status, there was quasiunanimity among respondents in the two slums that stunted children were usually too small for their age, as two mothers noted.Grandmother, KorogochoSuch child can have swollen legs, pot belly, swollen cheeks; you might think he is fat which is not being fat. You are able to see he is not in good health, which is malnutrition. His hair turns red. You will get the child has sores all over the body. The same statement was echoed by grandmothers from the two slums who associated child stunting symptoms with an imbalance between the age and the height of a child. However, one respondent did not differentiate between stunting and signs of acute malnutrition:Mother stunted child, ViwandaniThe main problem was lack of food because at times in the morning when she wakes up, there was nothing to eat and she could only get strong tea in the morning, at lunch time maybe you cook white rice with nothing else. ugali and cabbage. The child will then sleep and when morning comes there is not even a cup of tea, the child will then stay hungry till evening again. Grandmother, KorogochoAnd this is because this child has been hungry all day and even when the mother comes in the evening she will bring When the causes of stunting were explored, respondents unanimously identified poverty and food insecurity and unbalanced diets as the major reasons. In particular, most of the respondents reported their limited access to food, especially food that is appropriate for feeding young children.Poor child health outcome including stunting was seen as a consequence of deprivation and the poor quality of life in the slums, parents' own poor health status, and parents' lack of interaction with their children. For instance, a caregiver's HIV status was related to child stunting in a situation where the affected parent or caregiver essentially prioritizes his or her own health rather than the feeding and health care of the child.Father, KorogochoI could tell you … the problem was the food given to the child by the mother. Mostly we, men, are out hustling for the most part of the day, so it is the responsibility of the mother to look after the child. Exploring respondents' views on individual responsibilities regarding child stunting, some fathers were reportedly not fulfilling their role to provide for their families' needs. However, from their own point of view, fathers did not appear to perceive themselves to have a direct responsibility when their child is stunted; the mother or caregiver was identified as the person primarily responsible.We noted that no reference was made to common practices that may be driven by cultural or religious norms and standards in relation to child feeding. Finally, we also noted that caregivers of stunted children were not always aware that their children were stunted.3.2Mother nonstunted child, KorogochoI used to breastfeed her, but after one month I had to do my KCSE (Kenya Certificate of Secondary Education). Then I started giving her Nan milk (infant formula). ugali and that is how I have been bringing him up. Mother stunted child, ViwandaniI felt that if I am going to sit here breastfeeding this child, I will suffer even more. So after one week I introduce other food like milk and porridge and any other food that I have found. Even if it is Respondents' views and actual practices on child feeding were explored from two angles in this study. We first asked specific questions on exclusive breastfeeding in the first 6 months, and then the introduction of complementary feeding was explored. The results show that most of the respondents in the two slums, regardless of the child's stunting status, recognized the importance of exclusive breastfeeding for a newborn but found it hard to sustain up to 6 months. In many cases, there were attempts to exclusively breastfeed in the first 2 or 3 months of child's birth, but for different reasons, mothers opted to stop exclusive breastfeeding before their child reached 6 months. Among the reasons, mothers found it impossible to combine their occupation , with exclusive breastfeeding of their child. The findings were observed among both mothers of stunted children and those of nonstunted children.Mother nonstunted child, ViwandaniWhen I just realized that I was pregnant (five months after birth), I stopped breastfeeding. Mother stunted child, ViwandaniI stopped breastfeeding because I was taking family planning contraceptives. I thought the contraceptives were making him sick. However, misinformation about health effects of contraception, often promoted in the postnatal period, was also found.Grandmother, KorogochoExclusive breastfeeding is good because it is vital in development in the child's brain. Then the milk of the mother helps prevent diseases. The mother's milk is always ready, it needs neither boiling nor cooking, and it has no budget. Grandmother, ViwandaniI think it is important because I noticed it with this baby, when he breastfed for six months exclusively, I have not seen this child disturbing at all. Being unable to breastfeed exclusively until 6 months was common in the two slums with no difference between mothers of stunted children and those of nonstunted children. Grandmothers, however, were mostly favourable towards exclusive breastfeeding, even though some of them remained unclear on the duration. In many cases, they advised young mothers to comply with the practice and offered their support to take care of babies when mothers were away. However, they seemed not to be cognizant of the complexity of caring for an exclusively breastfed child without having the needed breast milk within easy reach.Mother nonstunted child, ViwandaniWhen I gave birth, after six months I started giving the baby water, fruits, and continued like that, as I introduced other feeds slowly by slowly until the baby was one‐year‐old and able to feed by himself. The findings also reveal that there is no standard or informed practice in the study area when breastfeeding is interrupted. Mothers and caregivers usually give the child the types of food that are available depending on their economic capability; a mother of a nonstunted child reported that she would not prepare something different, the child would just eat what is prepared for everybody, because there is no money to prepare weaning foods. In few cases, mothers tried to provide a more structured diet for their newborn.ugali followed by some boiled milk. After all these efforts, I would find his health is improving, and he is growing normally. Mother stunted child, KorogochoI changed it … so I would give him fruits, milk, bananas, all to boost his appetite. I would make him a mixture with a red fruit and give him. After that he would eat some plain Stunted children usually need an appropriate diet in order to improve their nutritional status. During the interviews, mothers, who recognized that their children were stunted, were asked if they thought it was necessary to feed their child differently to help them recover. Affirmative answers were noted from mothers in the two slums. The moment when the child is seen as not growing well and taken frequently to the hospital for care was usually the moment when mothers of stunted children realized the need to take care of their child differently, including changing feeding practices to avoid fatal consequences.However, this finding may not necessarily reflect usual responses to stunting in the slums as a number of mothers provided the food that was available to them as determined by their economic situation. Overall, economic circumstances were more important in determining child‐feeding practices.Another result is that child‐feeding practices in the slums did not differ by background characteristics, particularly by site or ethnic group. For instance, difficultly to exclusively breastfeed children for 6 months was found among all mothers regardless of their ethnic group.3.3Mother stunted child, ViwandaniWhen I introduced cow's milk and porridge at six months, my mother is the one who advised me to do that. Mother nonstunted child, KorogochoMy mother used to advise me on the importance of breastfeeding the child for long. Our interest focused on how decisions on child feeding are taken in the study areas and who the main persons involved are. This information could be the basis of targeted strategies to address child suboptimal feeding in the slums. The findings show that mothers are not the only decision makers on the feeding of their child. Beside health providers, grandmothers were commonly reported to have a key role in that process. Most of the mothers recognized and valued advices from grandmothers, and this was the case for mothers of children who were stunted and not stunted.Grandmother, KorogochoIt is good for me to sit down with them (young mothers) and teach them how they should feed these children and the types of food that the children should be given, how they should bring up the kids and the importance of taking the children for clinics. Grandmothers considered it normal to advise young mothers on how to take care of their babies including child feeding. Most grandmothers proudly assumed that role and justified it by their intention to ensure good health and a better future for their grandchildren. The role of grandmothers in child nutrition in this community is central as it goes beyond the basic advisory tasks. They are also child caregivers and mentors for young mothers as one reported:Grandmother, KorogochoThese days, you can see how they (young mothers) behave … If you talk to them, they would say ‘what this old woman can teach me?’ I would not be surprised if their children are fed on French fries at night. However, some grandmothers did not always think their advice was valued. Indeed, their comments implied generational shifts in norms and suggested intergenerational conflict between old and young mothers in this community, as a grandmother reportedLooking at the roles of fathers in child feeding, the results show that most of the fathers interviewed indicate that their role is limited to that of providing food, and they have developed strategies to cope with their expected duties. Their role did not extend to feeding children.4This qualitative study aimed to explore the influence of household members on child feeding, in particular, the roles of grandmothers and fathers.The study shows broad knowledge on child stunting among mothers and grandmothers. Although respondents mostly described stunting as an imbalance between the age and the height of a child, some did not differentiate stunting from acute malnutrition. Other studies from the same setting also found broad knowledge on child stunting among mothers  increasing mothers' knowledge on child nutrition including removing the misconceptions around breastfeeding and pregnancy or use of contraceptives; (b) prioritizing interventions that support mothers to combine breastfeeding and other occupations, through human milk banking, and other baby‐friendly initiatives; (c) promoting optimal child‐feeding practices, in particular, when exclusive breastfeeding ends; and (d) reinforcing interventions for more sustainable poverty reduction programmes in these settings.


The idea of screening patients for low health literacy has had a polarizing effect in the health literacy community. One side feels that universal precautions and screening for low health literacy are mutually exclusive; meaning, if you screen patients for low health literacy, you are not adhering to the universal precautions approach. Others may be concerned about the feasibility of fully scaling universal precautions across a clinical enterprise and favor a more targeted approach that identifies patients with risk factors for low health literacy so that interventions that rely on limited resources can be allocated where there is greatest potential for benefit. Given recent changes in health care delivery models, we propose that the time has come to consider a hybrid approach that employs both the foundation of universal precautions for all patients, as well as identification of those for whom universal precautions alone may not suffice, due to extreme needs. When this combined approach is used, the limitations of each approach are mitigated by the other. This integrated approach is well aligned with recent innovation in the health care landscape and should be considered by researchers, providers, and policymakers.Health literacy universal precautions were first operationalized in 2010 to address the complex demands faced by patients in health systems in the United States . The appAlthough health literacy universal precautions have been broadly supported by researchers, policymakers, and promoters of health literacy, recent publications have revealed barriers to their adoption in health systems. Indeed, some of the patient-level universal precautions  may be challenging to integrate routinely into busy clinical environments, whereas others  are resource-intensive. Weiss  posits tResearchers have developed efficient methods of screening patients for low health literacy using validated questions that assess patients' self-reported understanding of medical information and forms . The quePatient-level health literacy data make it possible for clinical staff who have completed training in clear health communication to provide additional assistance to patients with low health literacy levels. At a population level, these screening data are also useful for examining the association of health literacy with processes and outcomes of care ; for detThroughout the last decade, concerns have arisen regarding the risk of stigmatizing and embarrassing patients through health literacy screening . In addiOther studies have shown that patients feel that it is useful for their providers to know about their health literacy challenges , and whe“Health literacy 2.0,” a hybrid approach to addressing health literacy in health systems, integrates universal precautions with targeted assistance for patients with lower health literacy levels. This approach supports striving for clear communication with all patients but recognizes that resources are limited and gaps in implementation of universal precautions are prevalent. Thus, the approach also involves screening to identify patients at greatest risk, so that resources can be directed to support their care.System-wide screening and documentation in EHRs produces data that can be used for targeted interventions, population health opportunities, and point-of-care best practices that are data-driven, rather than universal or result from profiling. Further, screening patients for low health literacy is in alignment with risk-based models of medical care and models of precision medicine that are prevalent in the current health care landscape and include attention to social determinants of health .“Health literacy 2.0” should acknowledge the limitations of past and current attempts to address health literacy on large scales, learn from research and evidence, and embrace the landscape of innovation that is the context of our current health systems. The hybrid approach of universal precautions and screening for health literacy holds promise in this future. As health care shifts toward more tailored approaches to care, additional evidence is needed about how to best identify and deliver personalized care to patients with risk factors for poor outcomes related to health literacy.Historically, conceptual and analytical models of health literacy reveal causal pathways that position health literacy as an influencer of health behaviors that determine health outcomes . Most he(Table 1).A notable health literacy innovation that has influenced how care is and should be delivered in the U.S. is the Ten Attributes of Health Literate Care Organizations model . This moOne of the most influential innovations in the health care landscape in the U.S. is the adoption of the Institute for Healthcare Improvement's Triple Aim , which fhttps://innovation.cms.gov/initiatives/ACO/), can use health literacy “risk” data to identify patients for whom an evidence-based intervention and/or best practices will likely lead to improvement of specific health outcomes. As health systems personalize medicine and medical care, patients' individual capacity to understand information and choices becomes essential. Health literacy screening in patient EHRs can provide data that can also be used in population health strategies, quality and satisfaction efforts, and a host of medical informatics initiatives. There are vast opportunities for health systems researchers and administrators to test and disseminate novel ways of integrating health literacy in this new and evolving landscape.Other health care delivery model innovations have created a space for health literacy. Health systems, especially those that are a part of an Accountable Care Organization  are applied as a safety net for all patients, and screening is used to identify patients at greater risk, we can better serve both population and individual patient needs. Screening patients for low health literacy requires a modest amount of staff training to normalize the questions and ask them respectfully, as well as time to collect data . HoweverAlthough integrating these two approaches may provide opportunity to better serve patients and health systems, the need for evidence-based interventions is significant. As researchers and practitioners respond to the call to innovate, we must ensure that we continue to develop, test, and disseminate new research on interventions that are effective in this new context.


Taxus cuspidata is well known worldwide for its ability to produce Taxol, one of the top-selling natural anticancer drugs. However, current Taxol production cannot match the increasing needs of the market, and novel strategies should be considered to increase the supply of Taxol. Since the biosynthetic mechanism of Taxol remains largely unknown, elucidating this pathway in detail will be very helpful in exploring alternative methods for Taxol production.Taxus cuspidata transcriptomes with next-generation sequencing (NGS) and third-generation sequencing (TGS) platforms. After correction with Illumina reads and removal of redundant reads, more than 180,000 nonredundant transcripts were generated from the raw Iso-Seq data. Using Cogent software and an alignment-based method, we identified a total of 139 cytochrome P450s (CYP450s), 31 BAHD acyltransferases (ACTs) and 1940 transcription factors (TFs). Based on phylogenetic and coexpression analysis, we identified 9 CYP450s and 7 BAHD ACTs as potential lead candidates for Taxol biosynthesis and 6 TFs that are possibly involved in the regulation of this process. Using coexpression analysis of genes known to be involved in Taxol biosynthesis, we elucidated the stem biosynthetic pathway. In addition, we analyzed the expression patterns of 12 characterized genes in the Taxol pathway and speculated that the isoprene precursors for Taxol biosynthesis were mainly synthesized via the MEP pathway. In addition, we found and confirmed that the alternative splicing patterns of some genes varied in different tissues, which may be an important tissue-specific method of posttranscriptional regulation.Here, we sequenced Taxus spp. using molecular breeding or plant management strategies and synthesizing Taxol in microorganisms using synthetic biological technology.A strategy was developed to generate corrected full-length or nearly full-length transcripts without assembly to ensure sequence accuracy, thus greatly improving the reliability of coexpression and phylogenetic analysis and greatly facilitating gene cloning and characterization. This strategy was successfully utilized to elucidate the Taxol biosynthetic pathway, which will greatly contribute to the goals of improving the Taxol content in The online version of this article (10.1186/s12870-019-1809-8) contains supplementary material, which is available to authorized users. Taxus cuspidata, an evergreen woody plant from the Taxaceae family that is native to Northeast China, Korea, Japan and the extreme southeast of Russia [T. cuspidata is well known worldwide for its ability to produce the antitumor metabolite Taxol, a complex tetracyclic diterpenoid that is mainly produced by plants from the Taxus genus [Taxus species and artificial semisynthesis from the extracted intermediates baccatin III or 10-deacetylbaccatin III (DAB) [Taxus spp. and the high costs of purifying Taxol and its intermediates [Taxus cell culture, metabolic engineering and synthetic biology methods.f Russia , has beef Russia , 2. Howeus genus . Since Tus genus , 5. The us genus . Furtherus genus . At presII (DAB) , 8. Unfomediates . TherefoTaxus spp., which are responsible for hydroxylation at the C-2, C-5, C-7, C-10, and C-13 positions [Elucidating the biosynthetic pathway of Taxol in detail is essential in exploring alternative methods for Taxol production. In plants, all terpenoids arise from the common precursors dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP), which are typically synthesized by either the mevalonic acid (MVA) pathway in the cytoplasm or the methylerythritol phosphate (MEP) pathway in the plastid , 10. Forositions . All of ositions , 12. HowT. chinensis to be involved in transcriptional activation of the DBAT gene. Lenka et al. [T. cuspidata play negative roles in Taxol biosynthesis. However, in some cases, TFs from the same families were shown to potentially play opposite roles in regulating Taxol biosynthesis. Zhang et al. [T. chinensis. The functional diversity of TFs has led to difficulty in understanding the regulatory mechanism of Taxol biosynthesis.Understanding the regulatory mechanism of Taxol biosynthesis is a necessary prerequisite for improving the Taxol content in intact plants, tissues and cell cultures using biotechnology. Most transcription factors (TFs) are very important for regulating plant growth and development as well as for the biosynthesis of secondary metabolites , 15. A na et al.  demonstrg et al.  reportedTaxus spp.Alternative splicing (AS) can produce multiple transcript isoforms from a single pre-mRNA via variable splice site selection . In planZea mays [Sorghum bicolor [Arabidopsis thaliana [Transcriptome analysis-based next-generation sequencing (NGS) technology is a powerful and economical way to obtain genetic information on a large scale and has been widely used to uncover genes involved in the biosynthesis of secondary metabolites , 25. AltZea mays , Sorghum bicolor , Arabidothaliana , and strthaliana .T. cuspidata genome is currently available, inhibiting elucidation of the Taxol biosynthetic pathway. Here, we developed a practical strategy to mine candidate genes involved in Taxol biosynthesis based on an Iso-Seq analysis of the T. cuspidata transcriptome. The candidate sequences and in-house pipelines produced from this study provide valuable resources for the elucidation of Taxol biosynthesis and will be beneficial for future studies on the production of Taxol or its precursors with synthetic biology technology.Due to its large genome size, little information about the T. cuspidata grown in Institute of Medicinal Plant Development served as the source of plant material in this study. Roots, stems and leaves were collected in three duplicates. After collection, all samples were immediately frozen in liquid nitrogen and stored at − 80 °C prior to RNA extraction. Total RNA was extracted using an RNAprep Plant Kit  and quantified by Qubit . The RNA integrity was evaluated on an Agilent 2100 Bioanalyzer .Total RNA from different tissues was mixed at equal ratios. Poly(A) RNA was isolated from total RNA using Dynal oligo(dT)25 beads  and used for construction of the Iso-Seq library. The first cDNA strand was synthesized from purified polyA RNAs using the Clontech SMARTer PCR cDNA Synthesis Kit . After PCR optimization, large-scale PCR was performed to synthesize the second cDNA strand without size selection. Equimolar mixed libraries of unfiltered fragments and > 4 kb fragments were prepared with the SMRTbell Template Prep Kit 1.0. Sequencing was performed on a PacBio Sequel platform. A total of four SMRT cells were utilized in this study.The raw data were processed using SMRTlink 4.0 software. Circular consistency sequences (CCSs) were generated from subread sequences by mutual correction and then classified into full-length or non-full-length reads by examining whether the 5′ primer, 3′ primer, or polyA tail was present. Full-length reads were corrected by isoform-level clustering (ICE) to obtain clustered consensus sequences, and then final arrow polishing was performed with non-full-length reads to obtain polished consensus sequences. Finally, the high-quality consensus transcripts of multiple libraries were merged, and redundant reads were removed based on CD-HIT-EST (−c 0.99) to obtain nonredundant transcripts . The CodA total of twelve RNA samples from the roots, stems and leaves were used in four duplicates to construct the transcriptome sequencing library. The transcriptome library was pair-end sequenced on the Illumina HiSeq™ 2000 platform. Clean reads were used for error correction to obtain the polished consensus sequences as described above. For comparison of Iso-Seq and RNA-Seq data, Illumina data from the same samples were assembled with Trinity and SOAP to produce unigenes. Coding sequences (CDSs) from RNA-Seq unigenes predicted using Swiss-Prot and NCBI Non-redundant Protein (Nr) data and the CDSs from unigenes assembled by Cogent using ANGEL software  were comhttp://www.kegg.jp/) [https://www.uniprot.org/uniprot/) [https://pfam.xfam.org) [ftp://ftp.ncbi.nih.gov/pub/COG/KOG/) [https://www.ncbi.nlm.nih.gov/protein/) [https://www.ncbi.nlm.nih.gov/nucleotide/) and Gene Ontology (GO)(http://www.geneontology.org/) [https://CRAN.R-project.org/package=pheatmap).Gene functions were annotated using the following databases: Kyoto Encyclopedia of Genes and Genomes (KEGG) (egg.jp/) , Swiss-Pniprot/) , Pfam (hfam.org) , euKaryoOG/KOG/) , NCBI Norotein/) , NCBI Nogy.org/) . The expgy.org/)  with IllSeveral tools have been used to evaluate the coding potential of unigenes, such as Coding Potential Calculator (CPC) , Coding-T. cuspidata. The classification of TcuCYP450 proteins was based on reference sequences from a P450 database established by Nelson. BAHD ACTs were identified by searching for the key word “PF02458” in the Pfam database. Unigenes were submitted to the iTAK online program (version 1.7.0b) [Arabidopsis thaliana and Salvia miltiorrhiza used for TF comparative analysis were derived from NCBI.Based on functional annotations from the Swiss-Prot and Pfam databases, the unigenes of CYP450s were identified in  1.7.0b)  for idenThe CYP450, WRKY, bHLH and ERF phylogenetic trees were constructed using the neighbor-joining (NJ) method with the “Poisson correction” and “pairwise deletion of gaps” functions in MEGA6 software [–△△Ct method.RNA samples were isolated from the roots, stems and leaves in three biological replicates. Reverse transcription was performed using the GoScript™ Reverse Transcription System kit . For each sample, reverse transcription was performed using 2 μg of total RNA and 200 U M-MLV Transcriptase (Promega) in a 40 μl volume. The reaction was carried out at 25 °C for 5 min, 42 °C for 60 min and 70 °C for 15 min. A qPCR analysis was then conducted in triplicate using SYBR Premix Ex Taq  and a 7500 Real-time PCR system (ABI). The reaction mixture (20 μL) contained 10 μL of 2 × SYBR Premix Ex Taq, 0.5 μL of each forward and reverse primer, and 1 μL of template cDNA. PCR amplification was performed under the following conditions: 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 15 s; and 95 °C for 10 s. The primers used in this study are listed in Additional file T. cuspidata plants were pooled together and reverse transcribed into cDNA. To minimize bias that favors sequencing shorter transcripts, unfiltered and > 4 kb cDNA fragments were equally mixed and used to construct sequencing libraries. Using the PacBio Sequel platform, a total of 5,678,524 subreads with an average length of 2047 bp were generated. According to the bioinformatics procedure shown in Figs. T. cuspidata transcriptome.To identify as many transcripts as possible, equal amounts of total RNA from the roots, stems and leaves of To compare the integrality of transcripts from Iso-Seq and RNA-Seq, RNA-Seq data from the same samples were assembled with Trinity and SOAP, producing 63,872 and 53,781 unigenes, respectively. As shown in Fig. To capture the most informative and complete annotation information, we used a basic local alignment search tool (BLAST) to annotate all the unigenes based on sequence similarity searches against public databases, including the Swiss-Prot, KEGG, GO, KOG, NCBI Nr, and NCBI Nt databases. In addition, annotation was performed with hmmScan based on a domain similarity search against the Pfam database. In total, 42,920 unigenes were successfully matched to known sequences or domains in at least one of the seven databases, and 10,388 unigenes were annotated in all the databases    Fig. a. The laGs) Fig. b. In othPAM gene, involved in side chain synthesis, exhibited expression patterns different from those of TS, as PAM was expressed at the lowest level in the root, followed by the stem, and exhibited the highest expression in the leaf. Among ten genes encoding enzymes modifying the Taxol skeleton, six had expression patterns similar to those of TS. In the left four genes, TAT showed high expression in all three tissues, while T2αH and T7βH showed moderate expression in all tissues; T13αH exhibited an expression trend opposite that of TS, showing the lowest expression in the root and the highest expression in the leaf. According to our coexpression analysis, we hypothesized that the enzymes involved in the stem biosynthetic pathway are TS, T5αH, TAT, T10βH, TBT, DBAT, BAPT and DBTNPT. In addition, several genes in the first part of the biosynthetic pathway were expressed at substantially higher levels than those in the final steps. In particular, the gene encoding the last enzyme, DBTNPT, was expressed at the lowest level in all plant tissues, which is one of the reasons underlying the extremely low Taxol content in T. cuspidata. Therefore, we hypothesized that dramatically improving DBTNPT expression will substantially contribute to Taxol production. We also investigated the coexpression of Taxol-specific genes with several upstream genes and found that GGPPS and two genes from the MEP pathway, DXS and DXR, were consistently coexpressed with most of the downstream genes, such as TS, T5αH, T10βH, TBT, DBAT, BAPT and DBTNPT, while HMGS and HMGR from the MVA pathway had expression patterns different from those downstream genes, suggesting that the isoprene precursors for Taxol biosynthesis may primarily derive from the MEP pathway, which is consistent with previous reports [As shown in Fig.  reports .AS is a regulated process that increases the diversity of an organism’s transcriptome and proteome, mediating plant biological processes ranging from plant development to stress responses , 22. In Six unigenes were used to validate the authenticity of the AS events using the RT-PCR method, including two genes encoding CYP450s, one encoding a TF and three encoding splicing factors  and 3 subfamilies .Plant CYP450s are heme-containing enzymes that play roles in a wide variety of both primary and secondary metabolism reactions –51. To dTaxus CYP450s via analysis of high-throughput RNA sequencing data from G. biloba and found that G. biloba suspension cells exhibit taxoid 9α-hydroxylation activity. This CYP450 belongs to the CYP716B subfamily, suggesting that C9 hydroxylases in T. cuspidata may belong to the CYP716B subfamily , which catalyzes taxa-4(20),11(12)-diene-5α-ol into taxa-4(20),11(12)-diene-5α-yl acetate in the Taxol pathway. However, we did not find the other four characterized BAHD ACTs in the T. cuspidata transcriptome. According to the real-time results, the four enzymes were expressed at substantially lower levels in T. cuspidata than TcuACT31  had the same expression patterns as TAT and TBT, as their expression quantities in root tissue were similar to that in leaf tissue but significantly higher than that in stem tissue  exhibited expression trends similar to those of BAPT, DBAT and DBTNBT, as their levels in roots were significantly higher than those in stems and leaves. We inferred that these seven BAHD ACTs are possible candidates for the Taxol pathway.All BAHD ACTs from S. miltiorrhiza (1948 TFs), and to that of the model plant A. thaliana (2357 TFs). As shown in Fig. T. cuspidata, an obviously higher number than those in the angiosperm A. thaliana and S. miltiorrhiza. Plant C2H2 zinc finger proteins are mainly involved in the growth and development of plants at various stages and the regulation of gene expression under environmental stress, including extreme temperatures, salinity, drought, oxidative stress and excessive light [T. cuspidata genomes during evolution, resulting in the rapid expansion of these genes, and they may have special functions in T. cuspidata. Seven TFs have been identified to be involved in Taxol pathway regulation, including one WRKY, three bHLHs, two ERFs and one AP2 TF [We herein identified 1940 unigenes representing putative TFs distributed across 61 families and including bZIPs, bHLHs, WRKYs, and MYBs. The number of TFs is comparable to that of another diterpenoid-producing plant, liana 235 TFs. As ve light . The phee AP2 TF –19.Fig. T. chinensis is the sole WRKY TF that regulates Taxol biosynthesis. TcWRKY1 was reportedly able to specifically bind to W-box elements (TTGAC(C/T)) within the DBAT promoter and activate DBAT expression [T. cuspidata transcriptome, although six TcuWRKYs were divided into the same subgroup, Group IIa, with TcWRKY1  were then subjected to further analysis. An alignment-based method was used to cluster and remove redundant reads to generate unigenes. For example, with this pipeline, 813 nonredundant transcripts annotated as CYP450 were clustered into 139 CYP450 unigenes, while with Cogent, among all CYP450 nonredundant transcripts, only 595 transcripts were constructed into the 136 UniTransModels, and 218 CYP450 transcripts were left. According to the manually curated CYP450 unigenes, two types of mistakes were evident in the Cogent output. One is that transcripts from one CYP450 were constructed into different UniTransModels, and the other is that transcripts from two CYP450s were constructed into one UniTransModel. Our in-house pipeline was more accurate than Cogent, partly because manual evaluation was used, and was thus utilized for the subsequent gene family analysis. The candidate genes related to Taxol biosynthesis were screened from these gene families by analyzing phylogenetic relationships, conserved motifs and expression profiles. This strategy avoids the mistakes introduced by short-read assembly in NGS and can produce assembly-free, highly accurate full-length and near full-length unigenes, which not only substantially contribute to specific gene cloning and characterization by molecular biology methods but also significantly improve the accuracy of gene annotation and gene expression quantification. With this strategy, we successfully identified 9 CYP450s and 7 BAHD ACTs as lead candidates for Taxol biosynthesis.T. cuspidata transcriptome, which is comparable to the number of TFs in another diterpenoid-producing plant, S. miltiorrhiza, and the number of TFs in the model plant A. thaliana. However, it is interesting that significant expansion of the C2H2 family was observed in T. cuspidata, suggesting that C2H2 TFs may play important roles in the survival of T. cuspidata. Three categories of TFs are reportedly involved in the Taxol pathway, including WRKY, bHLH and ERF/AP2 TFs. Using a strategy similar to that used for the discovery of enzymes involved in Taxol biosynthesis, 14 TFs were identified as candidates for the regulation of Taxol biosynthesis, including six WRKYs, one bHLH and seven ERFs.Long-read transcriptome sequencing also provided a substantial amount of genetic information for transcriptional and posttranscriptional regulation analyses, such as TFs, AS and lncRNAs. TFs that regulate transcriptional initiation by binding to cis-regulatory elements in promoters or enhancers are key players in regulating the biosynthesis of secondary metabolites . In geneT. cuspidata [AS is one of the most important posttranscriptional regulations and can affect gene expression by multiple regulatory mechanisms, and we herein observed that TFs easily underwent AS events in uspidata . In totauspidata , 67. Recuspidata . The rolLncRNAs are another important group of regulators that play vital roles in plant stress responses , 69. LncIn summary, compared to NGS, the TGS transcriptome provides substantially longer and more accurate sequence resources for gene discovery and AS analysis. However, for the Iso-Seq analysis of species without a reference genome assembly, the currently used software Cogent does not effectively cluster transcripts into unigenes or identify AS events. Here, we developed an in-house pipeline to analyze gene families and their AS events to screen candidate genes related to Taxol biosynthesis and regulation. This pipeline can plausibly be used for unigene generation and AS analysis at the gene family level, as it was successfully used to identify candidate genes related to the Taxol biosynthesis pathway. However, to analyze AS at the whole-transcriptome level, the effectiveness of Cogent needs to be improved or a more effective, novel software program needs to be developed to better analyze TGS transcriptome data without a reference genome.T. cuspidata transcriptome sequencing with PacBio SMRT technology. With this strategy, we identified 9 CYP450s and 7 BAHD ACTs as the lead candidate genes in the Taxol biosynthetic pathway and 6 TF genes that may regulate this pathway. We also investigated the coexpression of known genes in Taxol biosynthesis and elucidated the stem biosynthetic pathway based on the rule that genes in the same pathway are coexpressed. A coexpression analysis also suggested that the isoprene precursors for Taxol biosynthesis are mainly synthesized via the MEP pathway. In addition, we found and confirmed the existence of tissue-specific AS events, which represent a possible posttranscriptional mechanism in the regulation of Taxol biosynthesis. Our study provides not only a valuable resource for investigating novel genes in Taxol biosynthesis but also a practical procedure for screening candidate genes involved in secondary metabolite biosynthesis and analyzing AS events in organisms without reference genomes based on long-read transcriptome sequencing.We developed an in-house pipeline to search for candidate genes involved in Taxol biosynthesis based on Additional file 1:Table S1. Proteins derived from GenBank used for the phylogenetic analysis. (XLSX 13 kb)Additional file 2:Table S2. Primers used in this study. (XLSX 20 kb)Additional file 3:Table S3. RNA-Seq data of identified genes and candidates for Taxol biosynthesis in T. cuspidata. (XLSX 46 kb)Additional file 4:Figure S1. Venn diagram of unigene numbers of Iso-Seq from the KEGG, Swiss-Prot, Pfam, NT, NR, and KOG databases for T. cuspidata. (TIF 1151 kb)Additional file 5:Figure S2. Identification of lncRNAs. (TIF 394 kb)Additional file 6:Figure S3. GO annotation of unigenes. (TIF 6225 kb)Additional file 7:Figure S4. Unigene functional classification by KEGG. The abscissa indicates the number of genes annotated to the pathway, and the ordinate indicates the subcategories. The pathway is divided into five categories in this analysis, including Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism, and Organismal Systems. (TIF 1344 kb)Additional file 8:Figure S5. Functional classification of unigenes exhibiting increased expression in roots by KEGG analysis. (TIF 294 kb)Additional file 9:Figure S6. Phylogenetic tree of 139 CYP450 proteins in T. cuspidata. The image shows an NJ tree made using CLUSTAL Omega . The tree was drawn with Figtree v1.4.4 and labeled in GIMP2.8.2. (PNG 664 kb)Additional file 10:Figure S7. Phylogenetic tree of WRKY domains among T. cuspidata and A. thaliana. Filled green diamonds represent unigenes in T. cuspidata, and the filled red circle indicates the identified gene in Taxus. (TIF 2580 kb)Additional file 11:Figure S8. Phylogenetic analyses of bHLH domains in T. cuspidata and A. thaliana. Filled green diamonds represent unigenes in T. cuspidata, and the filled red circle indicates the identified gene in Taxus. (TIF 1382 kb)Additional file 12:Figure S9. Phylogenetic analyses of ERF domains in T. cuspidata and A. thaliana. Filled green diamonds represent unigenes in T. cuspidata, and the filled red circle indicates the identified gene in Taxus. (TIF 2809 kb)


Cajanus cajan and one of its wild relatives, Cajanus platycarpus. Illumina sequencing revealed 0.11 million transcripts in both the species with an annotation of 0.09 million (82%) transcripts using BLASTX. Comparative transcriptome analyses on the whole, divulged cues about the wild relative being vigilant and agile. Gene ontology and Mapman analysis depicted higher number of transcripts in the wild relative pertaining to signaling, transcription factors and stress responsive genes. Further, networking between the differentially expressed MapMan bins demonstrated conspicuous interactions between different bins through 535 nodes (512 Genes and 23 Pathways) and 1857 edges. The authenticity of RNA-seq analysis was confirmed by qRT-PCR. The information emanating from this study can provide valuable information and resource for future translational research including genome editing to alleviate varied stresses. Further, this learning can be a platform for in-depth investigations to decipher molecular mechanisms for mitigation of various stresses in the wild relative.Pigeonpea is a major source of dietary protein to the vegetarian population of the Indian sub-continent. Crop improvement to mitigate biotic and abiotic stresses for realization of its potential yield and bridging yield gap is the need of the hour. Availability of limited genomic resources in the cultivated germplasm, however, is a serious bottleneck towards successful molecular breeding for the development of superior genotypes in pigeonpea. In view of this, improvement of pigeonpea can be attempted through transgenesis or by exploiting genetic resources from its wild relatives. Pigeonpea wild relatives are known to be bestowed with agronomic traits of importance; discovery and deployment of genes from them can provide a lucrative option for crop improvement. Understanding molecular signatures of wild relatives would not only provide information about the mechanism behind desired traits but also enable us to extrapolate the information to cultivated pigeonpea. The present study deals with the characterization of leaf transcriptomes of  Escalating global food demand and the potential impact of climate change have created an increased need for effectual crop improvement programmes. Management of resilience to an array of biotic and abiotic stresses and improvement of productivity through appropriate utilization of available genetic resources requires attention –3. ConsiCajanus cajan (L.) Millspaugh], also known as red gram, is the sixth most important grain legume crop grown in the semi-arid tropics of Asia, Africa and the Caribbean [Pigeonpea [aribbean ; India baribbean  due to aCajanus cajanifolius Maesen in peninsular India [Cajanus is composed of 34 species [C. cajan is the only cultivated member, while the wild relatives are assigned to secondary or tertiary gene pools [Wild relatives, the ancestors of crop plants have been exposed to natural challenges compared to their cultivated counterparts. Continuous exposure to severe climatic conditions has helped them evolve at the genetic level, making them not only more genetically diverse but also more resilient towards the adverse impacts of climate change and incidence of pests and diseases . It is kar India . The gen species , amongstne pools . Studiesne pools –13.Cajanus platycarpus from the tertiary gene pool is one of the wild relatives of C. cajan found growing along the hedges with slender and climbing plant type. The leaves and pods are extremely pubescent and produce rectangular dark brown seeds on maturity. C. platycarpus has the same chromosome number as that of cultivated pigeonpea (2n = 22) [C. platycarpus. In view of this, the major aim of the study has been the characterization of baseline transcriptomes of the cultivated and wild pigeonpea as it can provide fascinating insights on the basic differences in their molecular signatures. Such comparative baseline transcriptomes have been developed in numerous important crops as a step towards broadening the genetic base and better molecular understanding [2n = 22)  and is b2n = 22) –18. Seve2n = 22) , 19, 20.2n = 22) , 21. It 2n = 22)  as human2n = 22) . This st2n = 22) , 25 and standing –28.C. cajan vis a vis C. platycarpus.Corroboratory efforts have been made in various crops for extrapolation of the information obtained through molecular characterization of crop wild relatives for revelation of traits endowed to them both under stressed and non-stressed conditions , 29–31. C. cajan  procured from UAS, GKVK, Bangalore, India and C. platycarpus  procured from ICRISAT, Hyderabad, India were used in the present study. Seeds of both the species were sown in plastic pots (14 inch diameter and 60 inch height) and maintained under greenhouse conditions. In order to obtain enough plant material for RNA isolation, at least two plants were maintained per pot. Fully expanded and healthy leaves from 3rd or 4th positions were collected from 45 days old plants. Samples were collected from six different plants separately and made into two individual pools; two such pooled samples were considered as replicates. The samples were frozen in liquid nitrogen and stored at -80°C until use.Two species of pigeonpea, C. cajan and C. platycarpus leaf samples using Spectrum Plant Total RNA kit (Sigma) following manufacturer’s instructions. RNA samples (5μg) were later treated with DNase to remove the residual genomic DNA and integrity was checked on 1% formaldehyde agarose gel. Total RNA quality control was performed using Agilent 2100 Bioanalyzer  and samples with an RNA integrity number (RIN) of 8.0 were used for mRNA purification. mRNA was purified from 1 μg of intact total RNA using oligodT beads . The purified mRNA was fragmented at an elevated temperature (90 0C) in the presence of divalent cations and reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen Life Technologies) by priming with random hexamers. Second strand cDNA was synthesized in the presence of DNA polymerase I and RNaseH. The cDNA was further cleaned using Agencourt Ampure XP SPRI beads (Beckman Coulter) and Illumina adapters were ligated after end repair and addition of an ‘A’ base followed by SPRI clean-up. The resultant cDNA library was amplified using PCR for enrichment of adapter ligated fragments, quantified using a Nanodrop spectrophotometer (Thermo Scientific) and validated for quality with a Bioanalyzer (Agilent Technologies). The cDNA library was sequenced using Illumina Hi-Seq 2500 platform with 100 bp read length obtained in paired end module. Paired end FASTQ files were subjected to standard quality control with Phred Score >20 using NGSQC Tool Kit [Total RNA was extracted from Tool Kit  to obtaide novo transcriptome assembly. For this study, we chose de novo bruijn graph-based Trinity Assembler [All the HQ filtered paired end libraries were subjected to ssembler  with crissembler . Clusterhttp://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Protein). BLAST hits with e-value cutoff ≤ 1e-14 and query coverage of >80% were considered as annotated homologous proteins and AWK script was used for filtering reciprocal best hits. BLAST hits were processed to retrieve associated Gene Ontology (GO) terms describing biological processes, molecular functions, and cellular components. Expression levels of all the transcripts in the individual libraries in replicates were assessed by mapping high quality (HQ) filtered reads using BOWTIE2 [Annotation of the unique transcripts (>200 bp) was performed using BLASTX homology search against NCBI non-redundant (nr) protein database  followed by GO categorization using another online server, Wego [Transcripts annotated in both the er, Wego . For Maper, Wego  analysisDifferential gene expression analysis of the expressed transcripts was performed using DESeq  softwareCajanus spp and pathway clusters.Enriched biological categories along with differentially expressed genes were used as input for Bridge Island Software  for identifying key edges that connect genes and biological categories. Statistical scores from differential expression and biological analyses were used as attributes to visualize the network. Output of Bridge Island Software was used as input to CytoScape V 2.8 . The nodIF4α gene in each sample was used for normalization. RT PCR conditions were set as: initial denaturation at 95°C for 5 min, followed by 40 cycles each of 95°C for 10 sec, 60°C for 15s and 72°C for 15s. qRT-PCR was performed in two independent biological replicates with three technical replicates along with no template control. For analysis, C. platycarpus was considered as the test and C. cajan as control. The data was first normalized by subtracting internal reference gene from test and control samples and fold change was calculated [About 2 μg total RNA was used for cDNA synthesis by Superscript Vilo cDNA synthesis kit (Invitrogen). The diluted cDNA was used as a template in qRT PCR and amplified with gene specific primers  transcripts were annotated  transcripts with <10 Read count out of the total 114781 transcripts. We also observed 13203 (11.5%) transcripts to be C. platycarpus-specific and 11402 (10%) to be C. cajan-specific  were seen to be expressed in more numbers compared to other TFs. Comparatively, WRKY transcription factors showed higher expression in C. platycarpus whereas more number of MYB TFs displayed higher expression in C. cajan. However, highest expression level of both the transcription factors was seen in C. platycarpus. In contrast to MYB, TFs belonging to homeobox-leucine zipper were found to be expressing more in C. cajan  were seen to be abundant. Among the transcripts pertaining to calcium mediated signaling, calcium-transporting ATPase, calcium-dependent protein kinase, calcium-binding protein, calmodulin-like protein, and calmodulin-binding protein/transcription activators were displayed in higher levels in C. platycarpus. Nevertheless, calcineurin B-like protein and CBL-interacting serine/threonine-protein kinases were predominantly expressed in C. cajan (S3 Data).With respect to various transcripts belonging to signaling and G- proteins, a total of 6076 transcripts were expressed in both the species. Among them, 977 and 701 transcripts were seen to be specific to tycarpus . It was C. cajan ; S3 DataC. platycarpus along with proline-rich and LysM domain RLKs. Further, probable/putative receptor like kinases and threonine-protein kinases were specifically up-regulated in C. platycarpus whereas, G-type lectin-RLKs (GsRLKs) showed higher level of expression in C. cajan (Leucine-rich repeat receptor-like protein kinases (LLR-RLKs) were seen to be abundantly expressed in both the species compared to other RLKs. Distinctively, LLR-RLKs were predominantly expressed in C. cajan ; S3 DataC. platycarpus. However, L-type lectin-domain containing receptor kinase was equally expressed in both the species. Different isoforms of protein phosphatases were also found to be up-regulated in both the species.Protein kinases and phosphatases are important regulators of proteins in biological systems. Based on differential gene expression analysis, mitogen-activated protein kinase, casein kinase, receptor protein kinase TMK1, wall-associated receptor kinase, and putative receptor protein kinase ZmPK1 were seen to be specifically up regulated in The study illustrated that 48 transcripts that belonged to G-proteins were differentially expressed in both species, out of which, 27 transcripts were found to be up-regulated in the wild relative. Particularly, the up-regulated transcripts included, extra-large guanine nucleotide-binding protein 1-like, EVI5-like protein isoform X4, ras-related protein Rab11A-like and rop guanine nucleotide exchange factor 5-like isoform X1. In the domesticated pigeonpea, transcripts belonging to 22B isoform X1, a member of TBC1 domain family proteins and GTP-binding protein SAR1A were seen to be up-regulated ; S3 DataC. platycarpus and C. cajan specific transcripts being 160 and 169 respectively. However, no major difference was seen in the types of transcripts between both the species  and 1857 edges . It was tycarpus . The secTwenty genes  homology domain in plants, an intracellular domain common among the identified plant R-proteins [The quality of leaf transcriptomes of both the species was found to be reliable with respect to parameters like N50, number and length of transcripts. Based on KEGG analysis, proteins . Similarproteins , plant Tproteins –56.In-depth analysis of the transcriptome would be definitely fascinating for a better perception of the species under study. Towards this, MapMan, an advanced bioinformatics tool for comprehensive interpretation of transcriptome data and visualization of functions of associated genes was used. This analysis allowed us to explore gene categories from the large data sets to get meaningful information. Through MapMan, it was evident that significant variation between the two species was conspicuous with respect to genes related to transcription factors (TFs), signaling, secondary metabolites, and stress response. Exploration of specific bins was attempted in order to decipher major similarities as well as differences between the two systems.C. platycarpus expressed more number of WRKY transcripts when compared to its cultivated counterpart depicting its role in regulation of various abiotic and biotic responses [In general, TFs are seen to be involved in various plant processes like growth, development and stress signaling , 57–59. esponses , 61. Simesponses  indicatiC. platycarpus as a large number of varied kinases, especially receptor like kinases and those involved in calcium-mediated signaling [Further support for this assumption was established based on the analysis of genes involved in cellular signal transduction. Response of plants to various environmental and developmental signals is pertinent for successful growth and reproduction , 64. Proignaling  were seeignaling , 67.Cajanus platycarpus. Tocopheral plays a crucial role in wax accumulation in plant leaves. It is a known fact about C. platycarpus that it portrays more pubescence, increased hardening of leaves (sclerophyly) by cuticular wax accumulation, cell wall thickening and lignifications. These traits are expected to prevent plants from insect attack by making them non-preferable, unpalatable and undigestable [Secondary metabolism produces a large number of specialized molecules that are required for the plant to survive in its environment and essential for communicating with other organisms in a mutualistic  or antagonistic (eg. to combat herbivores and pathogens) manner. Under baseline or non-stress conditions, it is expected that mutualistic metabolites or those required for normal physiological processes are expressed , 69. Thigestable , 70, 71.C. platycarpus. This variation presented in the study repeatedly depicted intrinsic differences between the two species at transcriptome level thus reconfirming earlier evidences in other categories like TFs and signaling.Furthermore, another interesting feature observed was the variation in the transcripts pertaining to biotic and abiotic stress. Though, the study did not involve imposition of stress, still majority of stress-related gene transcripts were seen to be up-regulated in C. platycarpus were found to be interacting in the developed network.Perfect corroboration was evident from interactions between the differentially expressed genes of specific bins derived from MapMan analysis. The inherent variation in the kind and specific function of transcripts between the two species was clear when it was observed that distinct clusters densely packed with transcripts dominated by Therefore, considering different aspects of the study, clear disparity was seen in the transcriptome profiles of the two pigeonpea species, with the wild relative demonstrating skewed expression of transcripts pertaining to signaling, transcription factors and certain biotic stress related genes. However, dynamics of the transcriptome under specific stress conditions will provide intriguing insights and reasoning for the variety of desirable agronomic traits persisting in the wild relative. This learning can be a platform for further investigations with respect to the wild relative in deciphering the hidden molecular mechanisms towards mitigation of various biotic/abiotic stresses.S1 FileTable A. List of primers used in the study; Table B. QC statistics; Table C. Assembly statistics.(DOCX)Click here for additional data file.S1 DataTable A. Complete list of transcripts with annotation; Table B. Differential gene expression analysis of C. platycarpus vs C. cajan (given in separate tabs).(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.


Avian influenza A (H5N6) virus poses a great threat to the human health since it is capable to cross the species barrier and infect humans. Although human infections are believed to largely originate from poultry contaminations, the transmissibility is unclear and only limited information was available on poultry environment contaminations, especially in Fujian Province.Chi-square test or Fisher’s exact probability test was used to compare the AIV and the viral subtype positive rates among samples from different Surveillance cities, surveillance sites, sample types, and seasons. Phylogenetic tree analysis and molecular analysis were conducted to track the viral transmission route of the human infection and to map out the evolutions of H5N6 in Fujian.A total of 4901 environmental samples were collected and tested for Avian Influenza Virus (AIV) from six cities in Fujian Province through the Fujian Influenza Surveillance System from 2013 to 2017. Two patient-related samples were taken from Fujian’s first confirmed H5N6 human case and his backyard chicken feces in 2017. p < 0.05) in the positive rates in samples from different cities, sample sites, sample types and seasons. The viruses from the patient and his backyard chicken feces shared high homologies (99.9–100%) in all the eight gene segments. Phylogenetic trees also showed that these two H5N6 viruses were closely related to each other, and were classified into the same genetic clade 2.3.4.4 with another six H5N6 isolates from the environmental samples. The patient’s H5N6 virus carried genes from H6N6, H5N8 and H5N6 viruses originated from different areas. The R294K or N294S substitution was not detected in the neuraminidase (NA). The S31 N substitution in the matrix2 (M2) gene was detected but only in one strain from the environmental samples.The overall positive rate of the H5 subtype AIVs was 4.24% (208/4903). There were distinctive differences  contains supplementary material, which is available to authorized users. China’s first H5N6 strain was isolated in March 2014 from a domestic duck in Guangdong Province .Wild migratory birds and waterfowls are often considered as natural hosts of AIVs. Through their migration, the viruses are transmitted globally , 5. The Epidemiological investigations have confirmed that most cases of H5N6 human infections had contact with infected poultry –16. LiveTherefore, in order to reduce the risk of human H5N6 infection and the impacts of viral circulation in poultry, and also to trace the origin of the human H5N6 virus, we collected and analyzed the samples from poultry environments and the human H5N6 virus in Fujian Province, China.chi-square test or Fisher’s exact probability test.Data for this study were collected through the Fujian Influenza Surveillance System, which has been collecting at least 40 environmental samples monthly from locations such as LPMs, poultry farms, households and slaughter factories in Fuzhou, Xiamen, Quanzhou, Sanming, Nanping and Zhangzhou cities since 2013. Sample types includes surface wipe samples of poultry cage and slaughter or placing board, poultry fecal samples, poultry cleaning sewage samples and poultry drinking water samples. Samples were kept under 4 °C to 8 °C and sent to FJCDC within 24 h for viral detections. Surveillance data from 2013 to 2017 were retrieved and analyzed using SPSS 20.0. The differences of positive detection rates of avian influenza A and the H5 subtype in different network labs, surveillance sites, sample types, and seasons were tested by The two case-related samples were injected into 9-to-10-day-old specific pathogen-free (SPF) embryonated chicken eggs and inoculated at 37 °C for 72 h in a biosafety level 3 laboratory. Hemagglutination assay and hemagglutination inhibition assay of the allantonic fluids were sequentially used for subtype identifications.Viral RNA was amplified using Qiagen One-Step RT-PCR Kit  with 32 pairs of primers . Full genetic sequences of all six Fujian H5N6 strains from 2016 were retrieved from the China Influenza Virus Genetic Sequence Database and the GISAID EpiFlu Database  using the ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) following the manufacturer’s instructions. The sequencing primers were M13F (5′–TGTAAAACGACGGCCAGT–3′) and M13R (5′–CAGGAAACAGCTATG ACC–3′). Sequences were assembled using the SeqMan program of the Lasergene Package . The genetic sequences of both strains were submitted to the Global Initiative on Sharing Avian Influenza Data (GISAID) (The DNAMAN program (version 6.0) was used for the analysis and alignment of the sequencing data. The phylogenetic trees were performed in MEGA version 6  using maximum-likelihood method based on the Tamura–Nei model with 1000 bootstrap replicates . Severalp < 0.05). The highest positive rates of the H5 subtype were observed in Sanming city (9.83%), LPMs (5.26%), and the wipe samples of poultry chopping board (7.66%). The positive rates of H5 subtype were at peak in winter and spring (Table The positive detection rates of influenza A virus and the H5 subtype were 31.92% (1565/4903) and 4.24% (208/4903), respectively. There were statistical differences in the positive rates of influenza A virus and the H5 subtype in different cities, sample sites, samples types and seasons , polymerase basic 1 (PB1), HA, matrix protein (MP) and nonstructural (NS) genes appeared to be derived from an H5N8 virus isolated from a black swan in Hubei Province, China (A/Cygnus atratus/Hubei/HF-1/2016). Their NA and nucleoprotein (NP) gene segments were in the same cluster as the H5N6 viruses that were circulating in Fujian Province during 2016  were closely related to the courtyard chicken feces strain (A/environment/fujiansanyuan/08/2017). Their PA genes were both clustered into the HThe key amino acid mutations had increased the viral mammalian transmissibility, virulence in mammals and antiviral resistance in the human strain and the other 7 environmental strains of H5N6 viruses Table .The HA oSince the H5N1 virus was first identified in Guangdong Province, China, 1996, this high pathogenic H5 subtype of AIVs has been continually detected in wild birds and domestic poultry environments . The H5NThe H5N6 viral isolates from the first human case in 2014 carried the same internal genes as the H5N1 virus identified in 2013 , which sInfluenza viruses are carried by migratory waterfowl and therefore spread globally along the avian flyways during migration . H5N6 isThe H5N6 viruses in this study were considered highly pathogenic in poultry due to the amino acids at the HA cleavage site. The affinities of the influenza virus to different sialyl-sugar structures are important determinants of rang and pathogenicity in the viral host . The α-2Our study has several strengths, including large environmental sample size, viral isolated from Fujian’s first confirmed H5N6 human case and his courtyard chicken feces. However, this study also has some limitations, for example, the signs and symptoms and mortality rates among poultry were not collected.The results of this study indicated that in Fujian Province, the clade 2.3.4.4 of the H5 subtype had become the main circulating AIVs in poultry environments. The patient with H5N6 infection was most likely to contract the virus from contaminated poultry environment. The human strain had genetic reassortment with H6N6, H5N8, and H5N6 viruses. It was still sensitive to Oseltamivir and Zanamivir. Although there has been no outbreak of human infection with H5N6 in Fujian Province, it is of great importance to continue and strengthen the surveillance of the H5Nx virus in poultry environment to monitor the spread and evolution of the virus.Additional file 1:Table S1. Composition of subtypes of AIVs positive specimens in Fujian Province, during 2013–2017. H5 + H7: Both the H5 and H7 subtype influenza virus nucleic acids were detected in the same sample and other subtypes and so on. (DOCX 18 kb)Additional file 2:Table S2. The GISAID isolate ID of H5N6 viruses. (DOCX 16 kb)Additional file 3:Phylogenetic analysis of the H5N6 viruses isolated in Fujian Province. (In red Triangle) Viral strains of human infection with avian influenza A(H5N6) virus in Fujian Province. (In green Square) Viral strains of human H5N6 viruses. (In red Circle) Viral strains of H5N6 viruses isolated from environment sample in Fujian Province. (DOC 415 kb)


However, several authors have hypothesized that this ancestor may have been the crucian carp (Carassius auratus). Previously, we generated an experimental hybrid goldfish (EG) from the interspecific hybridization of red crucian carp  × common carp . Unlike either parent, EG possessed twin caudal fins similar to those of natural goldfish . The genetic characteristics of EG, as well as the mechanisms underlying its formation, are largely unknown. Here, we identified the genetic variation in the chordin gene that was associated with the formation of the twin-tail phenotype in EG: a stop codon mutation at the 127th amino acid. Furthermore, simple sequence repeat (SSR) genotyping indicated that, among the six alleles, all of the EG alleles were also present in female parent (RCC), but alleles specific to the male parent (CC) were completely lost. At some loci, EG and NG alleles differed, showing that these morphologically similar goldfish were genetically dissimilar. Collectively, our results demonstrated that genetic variations and differentiation contributed to the changes of morphological characteristics in hybrid offspring. This analysis of genetic variation in EG sheds new light on the common ancestor of NG, as well as on the role of hybridization and artificial breeding in NG speciation.Owning to the extreme difficulty in identifying the primary generation (G Carassius auratus, NG) lineages, this conserved architecture has undergone extreme modifications due to artificial selection  in NG during ventralization in early embryonic development  belong to the same subfamily, Cyprininae. Previous phylogenic studies have shown that NG are closely related to crucian carp  × male common carp  after sequential selective breeding. A stable population of EG was successfully established by self-mating  from the hybridization of female red crucian carp , and alleles differed between EG and NG at some loci. Collectively, our results showed that certain genetic characteristics of EG, including the chordin gene mutation and the RCC-aligned SSR alleles, originated from the distant hybridization, and contributed to the observed difference in morphology. The process of EG generation, as well as the genetic characters of this hybrid, shed new light on the common ancestor of NG, as well as on the role of hybridization and artificial breeding in NG speciation.Here, we identified a stop codon mutation at the 127All RCC, CC, and EG were raised in ponds at the State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Normal University, Changsha, China. NG were purchased at a local market. All experiments were approved by the Animal Care Committee of Hunan Normal University and followed the guidelines of the Administration of Affairs Concerning Animal Experimentation of China.chordin gene between single-tail and twin-tail fish, we isolated and sequenced the 1st to 6th exons of chordin homologues from the embryonic cDNA pools of four fish: single-tail RCC and CC, and twin-tail EG and NG. After self-mating, total RNA was extracted from the gastrula-stage embryos of all four fish using Trizol . The first-strand cDNA of the chordin gene was synthesized using ReverTra Ace . The forward primer 5′-GCGTTACCCATCCAACC-3′ and the reverse primer 5′-TCTGTRTCCGCTTGTGGT-3′ were designed based on CDS of the chordin genes from Carassius auratus (AB874473.1) and Cyprinus carpio (LC092194.1), which were downloaded from GenBank. Each PCR (25 μL) contained 20 ng of cDNA template, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 μM of each primer, 1 × PCR buffer, and 1.25 U Taq polymerase . The cycling conditions were as follows: an initial denaturation at 94°C for 4 min; followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were separated and purified using 1.2% agarose gels and Gel Extraction Kits , respectively. Purified products were ligated into pMD18-T vectors and transfected into Escherichia coli DH5α. Positive clones were sequenced and further analyzed using BLAST1 and CLUSTAL W2.To compare the sequences of the Taq Buffer (Mg2+Plus), 1 μL of 2.5 mM dNTPs, 0.4 μL of each primer (5 μM), 0.4 μL of Taq DNA Polymerase (5 U/μL), and 14.8 μL of ddH2O. The PCR cycling conditions were as follows: an initial denaturation at 94°C for 3 min; followed by 35 cycles of 94°C for 30 s, a primer-specific annealing temperature for 30 s, and 72°C for 45 s; a final extension step of 72°C for 7 min. PCR products were sequenced using capillary electrophoresis on an ABI 3730XL DNA sequencer  using BigDye Terminator Cycle Sequencing kits .We randomly selected eight fish of each type  for SSR testing. Total genomic DNA was extracted from the fin tissue of each fish with a DNA extraction kit . The microsatellite regions were PCR amplified using six florescent-labeled microsatellite primers : three from the common carp  ; two froGenetic distance and genetic polymorphism indexes, including major allele frequency, numbers of genotypes, numbers of alleles, heterozygosity, and gene diversity, were calculated using Popgene32 . SSR genst to 6th exons of chordin homologues were cloned from RCC, CC, EG, and NG. All cloned sequences were identified as chordin and were submitted to GenBank . There was high nucleotide sequence similarity (> 99%) among the chordin genes from all four fish  at the 127th amino acid position of the chordin protein, while the single-tail fish had a glutamic acid codon (GAG)  .Across the four fish , each of the six amplified SSR loci (121–302 bp) had 1–8 alleles . Almost Given the uncertainty surrounding the common ancestor of NG strains, as well as the evolutionary processes underlying their emergence , the forHybridization is an important method of animal breeding because this process increases genetic variation , and becth exon of the chordin gene  includes a glutamic codon at the 127th amino-acid position, while the other allele (chordinAE127X) includes a stop codon at the same position. The allele chordinAE127X is predicted to encode a truncated protein that contributes to the formation of the twin-tail (chordin gene gave rise to a twin-tail phenotype (0) of NG, we identified the twin-tail-associated mutation at the 127th amino-acid position in the chordin gene: a stop codon (TAG) instead of a glutamic acid codon (GAG). This mutation was identical to that found in NG. In contrast, the chordin genes of RCC and CC had a wild-type glutamic acid codon at the same position  have been broadly used in studies of molecular evolution . These s1–F6, within 6 years)  . Here, oarieties . Thus, oThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Care Committee of Hunan Normal University and followed the guidelines statement of the Administration of Affairs Concerning Animal Experimentation of China. All samples were raised in natural ponds, all dissections were performed with 100 mg/L MS-222 , and all efforts were made to minimize suffering.JW and WH conceived the research, analyzed the data, and wrote the manuscript. JZ, LL, GZ, TL, CX, and MC performed the research and writing-reviewed the manuscript. SL provided substantial contributions to conception and coordination. 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.


Croatian Medical Journal. The authors state that during the COVID-19 pandemic, the medical information system has been hit by a storm of open-access pandemic-related manuscripts through preprint platforms, but also through accelerated review processes.To the Editor: We have read with interest the editorial by Škorić et al  in the Amaximize the efficiency of peer review, ensuring that key work related to COVID-19 is reviewed and published as quickly and openly as possible”  indicated that 60% of studies were preprints, meaning that they reported non-peer-reviewed information (However, unprecedented scientific efforts have generated a torrent of publications, many of which are not peer-reviewed. It is even possible that indexed articles with a digital object identifier did not undergo peer review. A Reuters analysis of some of the most important servers (Google Scholar, ormation .This raises some concerns. First, non-peer-reviewed preprints could be cited by peer-reviewed articles published in legitimate peer-reviewed journals, possibly leading to the spread of misinformation. Second, peer review quality may be compromised by rushing the process and assigning an excessive workload to peer reviewers, thus putting them under pressure and inducing psychological stress. Third, the overabundance of opinion papers and editorials hinders the discovery of valuable raw data and medical insight. Finally, some data, ideas, and content, preliminary and peer-reviewed, are constantly being disproved, outdated, and invalidated, which makes them candidates for corrections or retractions following post-publication peer review. There is a real possibility that uncontrolled and potentially misleading information will reach the general public, directly or via the media, leading to incorrect, sometimes fatal, responses to the pandemic. In this scenario with many ethical challenges, scientific progress could be hampered, allowing predatory journals and scholars to exploit open access and possibly compromise public health and academic integrity.Most of all, the spreading of misinformation “infodemic” through the social and traditional mass media poses a serious problem for public health systems. Restrictions imposed by policymakers and governments, such as lockdown and social distancing measures, are based on advice given by governments’ scientific advisory committees, which rely on scientific findings. However, the best scientific evidence available on COVID-19 is still scarce, making the decisions by governments susceptible to bias. Governments should not just base their decisions related to COVID-19 on scientific evidence , they sh