Autoimmune hemolytic anemia (AIHA) is increasingly recognized as a strong risk factor for venous thrombosis. However, there are currently no guidelines on thromboembolism prevention and management during AIHA. Here, we describe the case of a patient with AIHA and pulmonary embolism and resume the current knowledge on epidemiology, risk factors, treatment, and pathophysiology of thrombosis during AIHA, as well as new therapeutic perspectives to prevent thrombus formation during AIHA. Autoimmune hemolytic anemia (AIHA) is a rare disease due to the presence of antibodies directed against antigens on the red blood cells (RBCs) surface. The estimated incidence in adults is 1 to 3 in 100,000 per year with a peak age at 60 to 70 years.Here, we report the case of a 75-year-old man who developed pulmonary embolism (PE) after being diagnosed with idiopathic wAIHA and discuss the current knowledge about thrombotic complications during AIHA.A 75-year-old man presented to the emergency room with severe shortness of breath. Symptoms had been increasing during the previous 4 weeks, no triggering factors were clearly identified. Dyspnea progressively worsened to a NYHA-III (New York Heart Association III) stage and was not associated with cough nor fever. He had chest pain only under effort, without palpitations, lower limb edema, or orthopnea. Three days prior to admission, he developed severe generalized weakness, dark-colored urine, and jaundice. His medical history was notable for well-controlled moderate chronic obstructive pulmonary disease  and for chronic ischemic heart disease for which he had undergone double coronary artery bypass graft in 2006.Vital signs on admission included temperature, 37°C; pulse rate, 85 bpm; blood pressure, 131/75 mm Hg; respiratory rate, 15/min; and oxygen saturation, 97% at ambient air.9/L (reference range: 20–120 × 109/L), total bilirubin of 154 μmol/L (reference range: 0–21 μmol/L), direct bilirubin of 13 μmol/L (reference range: 0–10 μmol/L), lactate dehydrogenase of 1,530 U/L (reference range: 135–225 U/L), and undetectable serum haptoglobin (reference range: 0.3–2.0 g/L). Direct antiglobulin test (DAT) was strongly (4 +) reactive for warm IgG1 and IgG3. Dipstick examination did not reveal the presence of hemoglobin in the urine. Peripheral blood smear was characteristic, showing a various degree of anisocytosis, polychromasia, microspherocytes, and few erythroblasts , absolute reticulocyte count of 614 × 10oblasts , and wAWorkup for underlying causes of wAIHA, including thoracic-abdominal computed tomography (CT) scan, serologic testing for cytomegalovirus (CMV), Epstein–Barr virus (EBV), HIV, viral hepatitis B and C, and screening for monoclonal gammopathy, was negative. Of note, a concomitant autoimmune condition was looked for and only low-titer (1/160) speckled antinuclear antibodies without antibodies to chromatin or extractable nuclear antigens were found. CT scan revealed PE at a right laterobasal segmental and several bilateral subsegmental emboli without evidence of right cardiac overload (no inversion of the right ventricle/left ventricle [RV/LV] ratio). Cardiac enzymes were within normal ranges and echocardiogram showed both normal size and function of LV and RV. No bilateral leg ultrasound was performed. Primary wAIHA with concomitant PE was diagnosed.Given the history of ischemic heart disease, the patient was transfused with 1 unit packed RBCs. High-dose oral prednisone (1.5 mg/kg per day) was prescribed. Because of ongoing hemolytic anemia, two intravenous steroid pulses (methylprednisolone of 125 mg) were administered additionally on day 9. Given the segmental location of one lesion and many thrombotic risk factors involved , anticoagulation with rivaroxaban  was prescribed. The patient was discharged home on day 13 with corticosteroid tapering and rivaroxaban during active hemolysis and for a minimum of 3 months.p < 0.001), 3.64 , and 3.83  for patients hospitalized for AIHA compared with controls in each cohort. Zöller et al analyzed the risk for developing PE in patients hospitalized for autoimmune disorders compared with the general Swedish population. They found an incidence ratio  of 11.07 (95% CI: 7.29–16.12) after less than 1 year of follow-up, 3.73 for 1 to 5 years of follow-up (95% CI: 2.58–5.22), 3.16 (95% CI: 1.87–5.00) for 5 to 10 years of follow-up, and an overall incidence ratio of 3.44 (95% CI: 2.74–4.26) for developing PE in patients hospitalized for AIHA.Among hospitalized patients, those admitted with an immune-mediated disease, including AIHA, are at an increased risk of subsequent VTE. Ramagopalan et al studied the rate ratio of VTE (deep vein thrombosis [DVT] and/or PE) of people admitted to hospital with immune-mediated diseases compared with three references cohorts of hospitalized patients, constructed by identifying the first admission for each individual with various other, mainly minor medical and surgical conditions.The risk of VTE occurrence among hospitalized adults with selected autoimmune diseases, including AIHA, was examined by Yusuf and colleagues in 2014.Amid studies of patients with AIHA,p = 0.024) and with a higher median lactate dehydrogenase level .p = 0.016).9/L  and total bilirubin level of 40 µmol/L or above  were predictive for VTE in a multivariate analysis.Classical thrombotic risk factors, which are utilized in clinical practice to decide when to install prophylactic anticoagulation in medical patients,Since none of these parameters has been validated in a prospective study, caution is needed in their use in clinical practice.In 2003, following three cases of fatal PE in patients with AIHA without anticoagulant prophylaxis, Hendrick conducted an audit of the effect of anticoagulant prophylaxis in acute exacerbations of severe autoimmune hemolysis, for which thromboembolic prophylaxis had been introduced on a case-by-case evaluation in 1992. By reviewing a small cohort (23 patients) of cases of AIHA hospitalized between 1986 and 2001, he found that 1 out of 21 patients with prophylaxis had VTE versus 5 out of 15 patients without prophylaxis.2+, and inhibition of stimulated adenyl-cyclase activity, thus promoting platelet aggregation.Multiple mechanisms have been suggested to explain the pathogenesis of the procoagulant condition associated with hemolysis . RBC me12receptor inhibitor. This was demonstrated, with cangrelor, in an in vitro study ran by Gremmel et al.Based on the pathophysiology of thrombus formation during AIHA, more targeted therapies to minimize the risk of bleeding are currently explored. Erythrocyte-derived ADP induces significant platelet activation that could be prevented by an ADP P2YTo date, no guideline preconizes the introduction of an antithrombotic prophylaxis nor the screening of VTE in patients suffering of AIHA.However, based on the scarce evidence in the literature and the postulated pathophysiology of thrombosis in hemolytic anemias, it seems reasonable to suggest a high level of suspicion for VTE in patients with AIHA flare and to prescribe anticoagulant prophylaxis while the patient is exhibiting frank hemolysis. Numerous questions are still unanswered. Should we screen every patient with AIHA for VTE? Could D-dimer levels help in this decision? Are there indicators for the need of thrombotic prophylaxis in AIHA? Which type of antithrombotic agent will suits best for prophylaxis and treatment, respectively? Which duration of anticoagulation is needed? Due to the rarity of AIHA, the responses will need prospective multicentric studies and time. For the time being, clinicians have to be aware of the increased thrombotic risk of AIHA patients and individualize management decisions.


Furthermore, we found that the expression levels of tnfα and mpo were decreased either in pretreatment or cotreatment groups. In conclusion, our findings revealed that AA-induced nephrotoxicities can be attenuated by ZDW. Therefore, we believe that zebrafish represent an efficient model for screening AA-protective Chinese medicine.Zhibai Dihuang Wan (ZDW) is an eight-herbal formula of traditional Chinese medicine. Clinically, it regulated immune activity and was used to treat diabetes and renal disease. In this study, we aimed to explore the nephroprotective effect of ZDW in an aristolochic acid- (AA-) intoxicated zebrafish model. We used a green fluorescent kidney transgenic zebrafish to evaluate the nephroprotective effects of ZDW by recording subtle changes in the kidney. Our results demonstrated that ZDW treatment can attenuate AA-induced kidney malformations (60% for AA-treated, 47% for pretreatment with ZDW, and 17% for cotreatment ZDW with AA,  Zhibai Dihuang Wan (ZDW) is an eight-herbal formula of traditional Chinese medicine used to enrich kidney function. Clinically, it was used to treat chronic kidney disease and diabetic nephropathy and to regulate immunity, even was used to treat chronic renal insufficiency and relieve the symptoms –4. The lAristolochia or Asarum Aristolochia) and has been demonstrated to have some adverse effects in humans and other animals [Aristolochic acid (AA) is a natural compound which was found in some certain species of plants  has been described as a reliable model organism for toxicity study [wt1b:EGFP), in which the glomerulus (gl) and pronephric tubes (pt) were labeled by green fluorescent protein (GFP), has been effectively used in many toxicological studies [wt1b:EGFP). Subtle changes in pt and gl were easily observed by the detection of GFP signals or by staining with a well-known renal tubular differentiation marker, Na+/K+-ATPase α1 subunit (α6F) [Zebrafish  . The prowt1b:EGFP) strains [wt1b:EGFP) stain was provided by Dr. Englert  [Mature zebrafish of the wild-type  and Tg( strains  were maiGermany) . Fish maGermany) . Once th3CONHC6H4OH; Sigma) were purchased from Sigma Pharmaceutical Co. (USA). The powders of ZDW were purchased from SunTen Pharmaceutical Co. . ZDW are composed of 8 different herbs: Zhi Mu (Rhizoma Anemarrhenae), Huang Bai (Cortex Phellodendri), Shu Di Huang (Radix Rehmanniae Preparata), Shan Zhu Yu (Fructus Corni), Shan Yao (Rhizoma Dioscoreae), Fu Ling (Poria), Mu Dan Pi (Cx. Moutan), and Ze Xie (Rhizoma Alismatis). The detailed protocol for the preparation of ZDW powder was described in the previous study [Aristolochic acids  embryos at 48 hpf were compared with the kidneys of healthy embryos from the no treatment control group and subjectively classified as normal or malformed kidneys based on the observation of green fluorescence. The embryo displaying normal morphology in the regions of pt and gl was classified as the embryo with normal kidney, while the embryo which displayed defective morphology in either pt or gl was classified as the malformed kidney.For ZDW dose titration, AB strain embryos were collected, randomly divided into 50 per experimental group, and exposed to either water  or water containing ZDW  and/or 3 ppm AA. All embryos were cultivated in 6-well cell culture plates, and survival rates were determined at 48 hpf. For kidney malformation recording, the kidneys of drug-treated Tg and detected using an ABC staining system (Santa Cruz) and 3,3′-diaminobenzidine (DAB) as chromogens. Sections of 10 μm in thickness obtained from the pronephric regions were subjected to toluidine blue stain for counterstaining. For images, all live or stained embryos were examined under a microscope  equipped with Nomarski differential interference contrast optics and a fluorescent module having a GFP filter cube (Kramer Scientific), and the images were captured with a Canon digital camera .The procedures for embedding and cryosectioning were performed as previously described  except tcyclooxygenase 2 (cox2), tumor necrosis factor α (tnfα), and myeloperoxidase (mpo). The housekeeping gene β-actin was used as an internal control for normalization quantification.The experimental procedures of qRT-PCR have been previously described , 18, 19,n = 50 (number of tested embryos in each group), N = 3 (triplicate experiments)) of the embryos were alive at 48 hpf after exposure to 100 and 500 ppm of ZDW. However, the survival rate decreased to 76.9%  when the exposure dose was increased to 1000 ppm , exposed to the selected concentrations , and determined the survival rates at 48 hpf. Our results showed that 91.9% to 100% . The Tg(wt1b:EGFP) transgenic line, in which the gl and pt were labeled by GFP ,  Figures ″). Inter Figures ″, 5(e), tnfα and mpo in the AA-treated embryos was also significantly increased [cox2, mpo, and tnfα) was determined by RT-qPCR. As shown in tnfα and mpo in the AA-treated embryos were increased by 2.96 and 1.67 folds, respectively, compared with the no treatment control embryos (1 fold). In contrast, the expression levels of tnfα and mpo in the pretreatment embryos were decreased to 2.45 and 1.45 folds (0.9 and 1.04 folds in the cotreatment group), respectively, compared with the AA-treated embryos. However, ZDW treatment seems to have no obvious effects on the expressions of cox2. These results suggest that the attenuation of AA-induced renal malformation by either pretreatment or cotreatment of ZDW may be mediated by suppression of proinflammatory gene expression, such as tnfα and mpo.Our previous study showed that AA treatment caused kidney malformations in zebrafish, and the expression of proinflammatory genes ncreased . To furtκB and enhancing the activity of the ARE signaling pathway to ameliorate oxidative stress in the cultured cells [In this study, our findings reveal that AA-induced nephrotoxicity can be attenuated by cotreatment with ZDW. Previous studies have shown that ZDW possesses protective effects. For example, the study of the protective effect of ZDW on gentamycin-induced renal tubular cell apoptosis in mice suggests ZDW can reduce apoptosis and necrosis . Anothered cells . Thus, wPoria cocos), Zhi Mu (Rhizoma Anemarrhenae), Shu Di Huang (Radix Rehmanniae Preparata), Shan Zhu Yu (Fructus Corni), and Huang Bai (Cortex Phellodendri) [β, and TNF-α through inactivation of the NF-κB signaling pathway [It was shown that AA-induced nephrotoxicity is mainly due to inflammation , 19. In odendri) –24. For  pathway ; the aqu pathway –31. In a pathway , 33; Sha pathway , 34. TheIn summary, these observations support the pharmacological basis of ZDW as a traditional herbal medicine for the treatment of inflammation and its associated disorders. In conclusion, this strategy and the zebrafish model provide an efficient method for studying the nephroprotective effects of ZDW during zebrafish development.


Ovarian cancer (OC) is one of the most common gynecological malignant tumors worldwide, with high mortality and a poor prognosis. As the early symptoms of malignant ovarian tumors are not obvious, the cause of the disease is still unclear, and the patients’ postoperative quality of life of decreases. Therefore, early diagnosis is a problem requiring an urgent solution.We obtained the gene expression profiles of ovarian cancer and normal samples from TCGA and GTEx databases for differential expression analysis. From existing literature reports, we acquired the RNA-binding protein (RBP) list for the human species. Utilizing the online tool Starbase, we analyzed the interaction relationship between RBPs and their target genes and selected the modules of RBP target genes through Cytoscape. Finally, univariate and multivariate Cox regression analyses were used to determine the prognostic RBP signature.We obtained 527 differentially expressed RBPs, which were involved in many important cellular events, such as RNA splicing, the cell cycle, and so on. We predicted several target genes of RBPs, constructed the interaction network of RBPs and their target genes, and obtained many modules from the Cytoscape analysis. Functional enrichment of RBP target genes also includes these important biological processes. Through Cox regression analysis, OC prognostic RBPs were identified and a 10-RBP model constructed. Further analysis showed that the model has high accuracy and sensitivity in predicting the 3/5-year survival rate.Our study identified differentially expressed RBPs and their target genes in OC, and the results promote our understanding of the molecular mechanism of ovarian cancer. The current study could develop novel biomarkers for the diagnosis, treatment, and prognosis of OC and provide new ideas and prospects for future clinical research.The online version contains supplementary material available at 10.1186/s13048-021-00777-1. Ovarian cancer is one of the most common gynecological malignancies worldwide . AlthougGene expression is regulated at multiple levels in eukaryotes, for example, the epigenetic, transcriptional, and post-transcriptional levels, and so on. Currently, studies on regulation at the transcriptional and translational levels have been relatively thorough and in-depth, but the current understanding of post-transcriptional regulation is still not complete. RNA binding proteins (RBPs) are important components of post-transcriptional modification. They regulate the critical metabolic processes of RNA maturation, transport, stability, and degradation . In all,With the development of science and technology and the in-depth study of RBP by researchers, More and more studies have shown the involvement of RBPs in cancer occurrence. Compared with normal tissue, the expression level of RNA binding protein QKI is significantly lower in gastric cancer tissue. In vitro experiments also confirmed that overexpression of QKI could lead to inhibition of proliferation of gastric cancer cells . Argonauhttps://tcga-data.nci.nih.gov/tcga/). All data on 88 normal ovarian tissue samples were obtained from the GTEx data portal . Perl software was used to process the expression data of the normal group and the tumor group and then merge them. In addition, we obtained the overall survival and phenotype files. The targeting relationship between RNA-binding proteins and their target genes was obtained through the Starbase database (http://starbase.sysu.edu.cn/) [The level 3 gene expression profiles of 379 ovarian cancer patients were downloaded from the TCGA data portal (edu.cn/) .https://bioinfogp.cnb.csic.es/tools/venny/index.html) was drawn to identify differentially expressed genes. P < 0.05 and |Logfold change (FC)| > 1 were considered as the cutoff values of DEGs.Differentially expressed RBPs and RBP-Target genes were screened by comparing tumor and normal samples. The DEGs were identified with the R package limma , and a hP < 0.05.The R package clusterProfiler was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis . The ontThe RBP-Target gene network was retrieved from the Starbase database, and it was reconstructed and analyzed by MCODE and Cytohubba of Cytoscape software. The colors of the nodes in the PPI network reflected the gene expression level |LogFC|; the size of the node indicates the number of proteins that interact with the specified protein.P < 0.05 was set. We verified the overall survival of RBP in the Cox model through different GEO datasets on the Kaplan Meier plotter  [Survival analysis was performed using survival data from the TCGA database, and a threshold of alysis/) .P value < 0.05 was set as the cutoff). Next, prognostic RBP signatures were determined by multivariate Cox regression analysis. In multivariate Cox regression analysis, AIC had a minimum value of 2266.94 as the best cutoff point. The prognosis-related RBP signature is expressed as follows: risk score = (coefficient RBP1 × RBP1 expression) + (coefficient RBP2 × expression of RBP2) + … + (coefficient RBPn × expression RBPn). Finally, the R packages “survival” and “survminer” were used to explore the optimal cutoff for the risk score and draw the survival curve. In particular, the risk curve was performed by grouping the patients into high- and low-risk groups. The R packages “survivalROC” and “timeROC” were used to investigate the time-dependent prognostic value of the gene signature [We first screened for prognostic RBPs by univariate Cox regression analysis  and 95% confidence interval (CI) were calculated to determine the expression of RBPs associated with overall survival. Results were considered statistically significant at P < 0.05.Survival analysis was performed using survival data from the TCGA database and compared by log-rank tests with P < 0.05 and |LogFC| > 1), we identified 527 differentially expressed RBPs , we established a PPI network to show the interactions of the 557 target genes. MCODE analysis shows four modules from the core of the network. Module 1 consisted of 36 nodes and 583 edges, module 2 consisted of 17 nodes and 93 edges, module 3 consisted of 11 nodes and 38 edges, and module 4 consisted of 17 nodes and 53 edges. Of the 81 modular genes, 76 target genes were in the top 100 hub genes selected using CytoHubba. It should be noted that the genes in module 1 are extraordinarily closely related to each other. Next, we constructed the relationship network between RBP and RBP target genes in the module genes. We visualized the results . Interestingly, the model was observed that it can be used as an independent predictor of the prognosis of RBPs in OC patients (P < 0.05). We generated the formula: risk-score = (− 0.5676) * C2orf15 + (0.6076) * MRPL46 + (− 1.0251) * ZNF239 + (− 1.3075) * MRPL14 + (− 0.7560) * ISG20 + (− 1.2970) * LUC7L2 + (− 1.2124) * SRP9 + (0.5287) * PARP4 + (− 1.9149) * PAPOLA + (1.8874) * STRAP. According to the expression of these RBPs, we calculated the risk value of each case. Based on the median risk score, patients were divided into high- and low-risk groups. We plotted the patient’s survival curve  and survival state chart  . Similarly, for the analysis of the 3/5-year survival rate predicted by the model, we also plotted the R ROC curve and the AUC (> 0.7) analysis showing the sensitivity and accuracy (P < 0.05)   analysis of the model showed sensitivity and accuracy P < 0.0. Similarhttps://www.proteinatlas.org/) and immunohistochemical data showed that the expression levels of these genes  were significantly increased, and the antibody staining degree of these genes  was shallow in ovarian cancer tissue  can lead to the development of a variety of cancers. The up-regulation of IGF2BP2 activates the PI3K/Akt signaling pathway to promote the growth of pancreatic cancer cells . IGF2BP2In addition, based on univariate Cox regression analysis, multivariate Cox regression analysis, and survival analysis, a total of 10 key RBPS related to prognosis were identified, including C2orf15, MRPL46, ZNF239, MRPL14, ISG20, LUC7L2, SRP9, PARP4, PAPOLA, and STRAP. Studies have reported that MRPL46 , SRP9 2, PARP4 , and STRScreening methods for ovarian cancer mainly include transvaginal ultrasonography, histological examination and other traditional tumor markers, but these methods have certain clinical application limitations. For example, CA125 (a tumor-associated antigen) assay are less sensitive in the early stages and may be interfered under specific conditions such as menstruation or endometriosi s. It is vIn summary, we analyzed and discussed the biological function and prognostic value of differentially expressed RBPs and target genes in ovarian cancer by bioinformatics methods. These genes may be involved in the development, progression, invasion, metastasis, and progression of ovarian cancer. A prognostic model of 10 RBP-encoding genes and a PPI network based on 13 RBP and their target genes were constructed, which can be used as a reference for the prevention, diagnosis, and treatment of ovarian cancer. To our knowledge, this is the first report to construct an RBP-related prognostic risk model for ovarian cancer. Our findings will help to uncover the pathogenesis of ovarian cancer and develop new OC-specific biomarkers and therapeutic targets. It also provides some novel direction for the diagnosis and treatment of ovarian cancer.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


Malnutrition continues to affect under-five children in Africa to an overwhelming proportion. The situation is further compounded by the burden of sickle cell disease (SCD). However, association of SCD with stunting, wasting, and underweight in a nationally representative sample of under-five children remains unexplored. We aimed to describe prevalence of undernutrition by sickle cell status, to evaluate its association with growth faltering ascertained by anthropometric indices, and to explore mediating role of hemoglobin.Z-scores of length/height-for-age (HAZ), weight-for-height (WHZ), and weight-for-age (WAZ) were computed against the 2006 World Health Organization Child Growth Standards. We fitted logistic regression models to evaluate association of SCD with stunting, wasting, and underweight. Mediation analysis was performed to capture the indirect effect of and proportion of total effect mediated through hemoglobin level in SCD-anthropometric indices association.We availed data from the 2018 Nigeria Demographic and Health Survey (DHS) and the sample comprised 11,233 children aged 6–59 months who were successfully genotyped for SCD. The DHS employed a two-stage, stratified sampling strategy. SickleSCAN rapid diagnostic test was used for SCD genotyping. Prevalences of stunting, wasting, and underweight among children with SCD were 55.4% (54.5–56.4), 9.1% (8.6–9.7), and 38.9% (38.0–39.8), respectively. The odds of stunting were 2.39 times higher (adjusted odds ratio (aOR) 2.39, 95% CI: 1.26–4.54) among sickle children than those with normal hemoglobin. SCD was also significantly associated with underweight , but not with wasting . Association of SCD with all three anthropometric indices was significantly mediated through hemoglobin level: for SCD-HAZ, the adjusted indirect effect (aIE) was − 0.328 ; for SCD-WHZ, the aIE was − 0.080 ; and for SCD-WAZ, the aIE was − 0.245 .We presented compelling evidence of the negative impact of SCD on anthropometric indices of nutritional status of under-five children. Integration of a nutrition-oriented approach into a definitive SCD care package and its nationwide implementation could bring promising results by mitigating the nutritional vulnerability of children with SCD. Undernutrition continues to be entrenched in Africa. An estimated 149 million under-five children were stunted globally in 2018 and 39% of them lived in Africa. Wasting affected 49 million under-five children worldwide in 2018 and Africa was home to 28% of them. Africa is the only region that saw an escalation of the number of stunted children between 2000 and 2018 [z-score (WAZ) has been shown to significantly predict hospitalization in SCD [SCD and poor nutritional status of under-five children have several critical intersections. SCD mortality among African children is high: 50–90% of them reportedly die before 5 years of age . Nutritin in SCD . Therefon in SCD . Tacklinn in SCD  in NigerChildren with SCD become anemic due to chronic hemolysis and low hemoglobin (Hb) level attenuates anthropometric parameters of growth , 11. MorThis cross-sectional study was based on secondary analysis of nationally representative data from the Nigeria Demographic and Health Survey 2018. It was conducted from August to December 2018 by the National Population Commission (NPC) in collaboration with the Federal Ministry of Health, along with technical assistance from The DHS Program, a United States Agency for International Development (USAID)-supported project .A two-stage stratified sampling strategy was adopted where 37 administrative territories were split into urban and rural areas, yielding 74 sampling strata. From these strata, 1400 enumeration areas (EAs) were selected. Thirty households from each EA were selected with equal-probability systematic sampling and in a third of those 42,000 households children aged 6–59 months underwent genotyping and anthropometric assessment [z-scores, using the World Health Organization 2006 Child Growth Standards [z-score (HAZ) below − 2, wasting as weight-for-height z-score (WHZ) below − 2, and underweight as weight-for-age z-score (WAZ) below − 2. For further analysis, the three outcome variables were dichotomized: stunted versus non-stunted, wasted versus non-wasted, and underweight versus non-underweight, and coded binarily (“1” versus “0”).The outcome variables were three common anthropometric indices of nutritional status among under-five children: height-for-age, weight-for-height, and weight-for-age. These were standardized by constructing tandards . The dettandards . Three fWe used data from the Biomarker Questionnaire, Household Questionnaire, and Woman’s Questionnaire (DHS-7 version) to ascertain the explanatory variables selected according to existing knowledge mapped in a directed acyclic graph  with 95% confidence intervals (CI) are reported. Collinearity between categorical, explanatory variables was assessed by cross-tabulation with chi-squared test. If found dependent (P < 0.05), Goodman–Kruskal’s gamma (G-K gamma) was computed to examine the strength of association. Maternal education  and place of residence  were strongly associated with SES and, therefore, were not included in the logistic regression. Furthermore, addition of these two variables in the adjusted model did not change effect estimates considerably (< 5%).All statistical analyses accounted for the two-stage stratified sampling strategy employed in DHS with “svyset” commands in Stata version 14.0 . Sampling weights were applied to compensate for the unequal probability of being recruited and to obtain nationally representative estimates. Distribution of continuous, numerical data was checked by examining quantile-quantile plots. HAZ, WHZ, WAZ, and Hb levels were approximately normally distributed. Weighted frequencies and proportions were computed for categorical data. Difference in prevalences of stunting, wasting, and underweight by sociodemographic characteristics was evaluated with chi-squared test, and difference in mean was tested with one-way ANOVA. Statistical tests were two-tailed and For mediation analysis, we included children with sickle cell and Hb C traits (genotypes HbAS and HbAC) and those having normal hemoglobin (genotype HbAA) into one category versus children with SCD; converting the predictor into a dichotomous one. We tested three single-mediator models  for SCD-We retrieved standardized total, direct, and indirect effects, and proportion of the total effects mediated, both unadjusted and after adjusting for age, sex, and SES. Standard errors and CI were computed with bootstrapping (1000 repetitions). Mediation was assumed to occur if the indirect effect estimate, captured as average causal mediation effect (ACME), was statistically significantly different from zero. Following the recommendation by Hayes and Rockwood , we did The Nigeria DHS protocol was approved by the National Health Research Ethics Committee of Nigeria (NHREC) and the ICF Institutional Review Board. Enumerators obtained informed verbal consent prior to administering the questionnaires. Blood samples were collected from the children after acquiring consent from parents/guardians . We obtaSample characteristics are presented in Table Overall prevalences of stunting, wasting, and underweight were 38.8% (37.9–39.7), 6.8% (6.3–7.2), and 22.2% (21.4–23.0), respectively. The prevalence of stunting increased from 33.4% among children younger than 24 months to a peak of 45.4% among children aged 24–40 months. Contrarily, the prevalence of wasting was highest among children below 24 months of age and declined thereafter. More than one in five children younger than 24 months were underweight. Prevalences of all three forms of malnutrition were higher among the male children. The highest sex difference of 5.6 percentage points was observed for stunting prevalence: 41.5% among male versus 35.9% among female children. Children with SCD had the highest prevalences of stunting (55.4%) and underweight (38.9%).There was an ascending gradient of prevalence of all three forms of malnutrition from non-sickle cell (HbAA) children to those with SCD. Stunting was approximately 1.5 times more prevalent among children with SCD than their non-sickle cell peers (55.4% versus 38.2%). Underweight was roughly 1.8 times more prevalent among children with SCD compared with non-sickle cell children (38.9% versus 21.8%). The difference in wasting prevalence was statistically non-significant.A socioeconomic gradient of ascending prevalence from more to less affluent households was also observed for all three forms of malnutrition. The prevalence of stunting among children from the poorest households was more than 2.5 times higher than that among children from the richest households . Nearly one in 10 children from the poorest households were wasted. The prevalence of underweight was 23.9 percentage points higher among children from the poorest households than those from the richest households (35.4% versus 11.4%). Whereas 47.1% of rural children were stunted, 28.3% of their urban peers had stunting. Children of mothers with no education had a substantially higher prevalence of stunting (57.1%) than children born to mothers with higher educational attainment (22.1–40.7%). Wasting was more prevalent among children with uneducated mothers (9.5%) than children whose mothers had secondary education or above (5%). While the prevalence of underweight among children of mothers with secondary education or above was 12%, it rose to 35.2% among children with uneducated mothers. Approximately 43% of the anemic children were stunted and more than one in four anemic children were underweight. The prevalence of wasting among anemic children was 7.9% (Table z-scores when categorized by sickle cell status and children with SCD had the lowest means. Mean HAZ among children with SCD was 36.5% lower than non-sickle cell children (− 2.17 versus −1.59). Mean WAZ was 1.4 times lower among children with SCD in comparison to non-sickle cell children (− 1.63 versus − 1.13). While mean Hb level among children with SCD was more than 2 g/dL below than that of non-sickle cell children (8.04 g/dL versus 10.19 g/dL), mean Hb level of children with sickle cell trait was close to that of the latter (10.14 g/dL) . The odds of wasting were 72% lower  and that of underweight 14%  lower among those aged 41–59 months. Female sex was associated with lower odds of stunting , wasting , and underweight . SES showed an inverse association with the three forms of malnutrition. Children from the poorest households had more than fourfold higher odds of stunting compared with those from the richest households .In the unadjusted analyses, we found all three SCD-anthropometric indices associations to be significantly mediated through Hb level. The statistically significant, negative indirect effects of Hb level became further pronounced in the adjusted analyses for all three associations. The adjusted average causal mediation effect (ACME) ranged from − 0.328  for SCD-HAZ association to − 0.080  for SCD-WHZ association. The proportion of total effect mediated through Hb level was highest for SCD-HAZ association , implying that the effect of SCD on HAZ was decomposed into two parts: nearly 93% of the total effect was indirect, i.e., mediated through Hb level and approximately 7% was direct effect  in Tanzania, Cox et al. reported OR for stunting and underweight of 1.82 (95% CI 1.43–2.32) and 2.61 (95% CI 2.06–3.31), respectively, at enrolment. The mean age of the cohort was 10.1 years (range 0.5–48 years) [n = 455) [The magnitude of SCD’s association with stunting and underweight in our analyses was high with OR surpassing two. As the 2018 Nigeria DHS is the first DHS to implement SCD genotyping in a nationally representative sample , we coul8 years) . Conside8 years) . The OR n = 455) . The higInterestingly, we did not find a statistically significant association between SCD and wasting. This contradicts some of the previous studies , 23. Poon = 128) with median age of 21.1 months, mean HAZ and WAZ did not significantly differ from those of non-sickle cell children [n = 116). The mean age of cases in that study was 40.6 months (SD 16.79). The mean WAZ of − 1.63 (SD 1.16) among children with SCD found in our study is suggestive of a drastic left shift in weight-for-age trajectory compared with the WHO Child Growth Standards. An unitary increase in WAZ reduces hospitalization risk among children with SCD by 13% [The mean HAZ, WHZ, and WAZ of children with SCD in this study were significantly lower than their peers with HbAA genotype. The most severe deficit was observed for mean HAZ of children with SCD − 2.17, SD 1.47). This is appreciably lower than mean HAZ from children with SCD in Ilesa, Nigeria  ; Enugu, children . No signchildren  that rec, SD 1.47D by 13% . The lowz-scores of height, weight, and BMI as well as growth velocity in SCD [The mean Hb level of sickle children in our study  was similar to that found in a multi-country  analysis , and in y in SCD , 20. Oury in SCD , 40. Inty in SCD  are of rSome limitations of this study must be acknowledged for allowing careful interpretation of the findings. Causal inferences cannot be drawn from the findings of mediation analysis because of the lack of temporality in association. It is noteworthy that although clinical presentation takes time to fully develop, the genetic changes underlying SCD are present at birth. The rapid diagnostic test (SickleSCAN) used for SCD genotyping in the DHS showed diagnostic sensitivity and specificity of 85% and 98%, respectively, when compared with high-performance liquid chromatography . We did We presented compelling evidence of the association of SCD with stunting and underweight among under-five children in a setting burdened with childhood undernutrition and an under-five mortality rate of 132 per 1000 live births . MediatiAdditional file 1: Figure S1. Directed acyclic graph (DAG) for the association of sickle cell disease with anthropometric indices. Lines in green are potential causal paths and those colored red indicate potential biasing paths that need to be adjusted in the model.


Ongoing challenges in the swine industry, such as reduced access to antibiotics and virus outbreaks , have prompted calls for innovative feed additives to support pig production. Medium-chain fatty acids (MCFAs) and monoglycerides have emerged as a potential option due to key molecular features and versatile functions, including inhibitory activity against viral and bacterial pathogens. In this review, we summarize recent studies examining the potential of MCFAs and monoglycerides as feed additives to improve pig gut health and to mitigate feed pathogens. The molecular properties and biological functions of MCFAs and monoglycerides are first introduced along with an overview of intervention needs at different stages of pig production. The latest progress in testing MCFAs and monoglycerides as feed additives in pig diets is then presented, and their effects on a wide range of production issues, such as growth performance, pathogenic infections, and gut health, are covered. The utilization of MCFAs and monoglycerides together with other feed additives such as organic acids and probiotics is also described, along with advances in molecular encapsulation and delivery strategies. Finally, we discuss how MCFAs and monoglycerides demonstrate potential for feed pathogen mitigation to curb disease transmission. Looking forward, we envision that MCFAs and monoglycerides may become an important class of feed additives in pig production for gut health improvement and feed pathogen mitigation. Antibiotics have long been used in the swine industry for therapeutic purposes, as they are in human medicine, to prevent severe clinical disease and death . AntibioDespite these important capabilities, there are mounting concerns that antibiotic usage in food animals is contributing to the global problem of antibiotic-resistant bacteria across human and animal populations . These cin vivo MCT hydrolysis and subsequent release of biologically active MCFAs and monoglyceride derivatives to support pig growth and development  and improved feed efficiency ; Clostridium levels tended to decline in digesta of the small intestine, as compared to the control group without additive.Building on this work, Hanczakowska et al. investigated the effects of MCFA supplementation on piglet growth performance and tested 0.5% of an MCFA-containing feed additive mixture that included caproic, caprylic, capric, and lauric acids . The treP < 0.01) among piglets that received caprylic or capric acids or a combination thereof, as compared to the control group without MCFAs or antibiotics. In addition, mortality decreased and protein (P < 0.01) and fiber (P < 0.05) digestibility improved for pigs fed MCFAs. Furthermore, Clostridium perfringens levels in the ileum were reduced by each type of MCFA (P < 0.01), and capric acid caused significant increases in aerobic bacteria (P < 0.05) as well. It was also noted that capric acid supplementation led to improvements in the morphology of the mucosal epithelium in the ileum .Subsequently, Hanczakowska and colleagues investigated the effect of 0.2% caprylic acid and/or capric acid on weaned piglet growth performance, apparent digestibility of nutrients, intestinal microflora, and ileum structure . ADG wasP < 0.01). Although MCFA diets had a strong odor, they did not reduce feed intake. Cochrane et al. further investigated whether MCFAs could be an alternative to the antibiotic chlortetracycline (CTC) in nursery pig diets in a bacterial pathogen challenge model [E. coli, and fed a diet 1) without MCFAs or CTC, 2) with 0.036% CTC, or 3) with 1% inclusion of a 1:1:1 blend, 4) 12:48:40 blend, or 5) 4:54:38 blend of caproic, caprylic, and capric acids. It was determined that E. coli-challenged pigs that received one of the MCFA-containing diets exhibited similar FCR values to those receiving the antibiotic-containing diet.Gebhardt et al. also evaluated the dietary addition of MCFAs to nursery pig diets for growth promotion . They tege model . Pigs weP < 0.01). While most studies have focused on how MCFAs and monoglycerides can support early stages of pig growth and development, Zhang et al. have also investigated the effects of adding 0.15% or 0.3% total MCFA doses  to the diet of finishing pigs on growth, nutrient digestibility, and blood profiles [P < 0.05; similar performance to that of a 0.1% antibiotic cocktail of CTC, sulfonamide dimethazine, and procaine benzylpenicillin in a 1:1:1 ratio) and greater lymphocyte percentages (P < 0.05) and IgG concentrations (P < 0.05) in the blood of MCFA-treated pigs, as compared to the control and antibiotic-treated pigs. No changes in nutrient digestibility were observed. In addition to MCFAs and monoglycerides alone, MCFA additives have been tested in combination with other additives such as organic acids and probiotics.In addition, De Snoeck et al. investigated the effects of a 0.1% lauric acid/GML feed additive in the diets of weaning piglets on antibiotic use in farrow-to-finish pig farms . In compprofiles . The 0.3P < 0.01) [Escherichia coli levels in the digesta collected from the small intestine relative to the untreated control group (P < 0.01). Hanczakowska et al. also investigated the effects of adding 0.5% propionic and formic acids (1:1 ratio) together with 0.2% of either caprylic acid or capric acid or a combination thereof to the diet of weaning piglets [P < 0.05) and improved protein and fiber digestibility (P < 0.05), with no difference in FCR.Hanczakowska et al. investigated the effects of adding 1.5% fumaric acid together with 0.2% of either caprylic acid or capric acid to the diet of weaning piglets. Fumaric/caprylic acid supplementation led to the largest body weight gains; ADG was 276 g vs. 234 g in the untreated control group without additive ( < 0.01) . All tre piglets . CompareP < 0.001), increased nutrient digestibility (P < 0.001), and caused changes in fecal bacterial populations, as compared to an untreated control group. In particular, there were significant decreases in E. coli counts (P < 0.001) along with a tendency towards increased Lactobacillus counts (P = 0.06). In a separate study, Nguyen et al. also tested the effects of adding 0.1% or 0.2% of the same additive mixture on the growth performance of finishing pigs [P < 0.05) was noted in the treated group along with improved feed efficiency , greater IgG antibody concentrations (P < 0.05), and altered bacterial populations , as compared to an untreated control group.Upadhaya et al. further investigated how a combination of organic acids and MCFAs affects the growth performance of finishing pigs . A matriing pigs . IncreasP < 0.05), ADFI (P < 0.05), FCR (P < 0.05), and nutrient digestibility (P < 0.05), as compared to an untreated control group. Lan and Kim also investigated how a combination of 40% organic acids , 1.2% capric acid, and 1.2% caprylic acid along with 57.6% Kaolin (2SiO2·Al2O3·2H2O) aluminum silicate carrier affects the performance of sows and their piglets [P < 0.05) and the microbiota of sows and piglets, namely fecal Lactobacillus counts increased (P < 0.05) and E. coli counts decreased (P < 0.05) in sows and piglets.In follow-up work, Upadhaya et al. investigated how a combination of 40% organic acids , 1.2% MCFAs (capric and caprylic acids), and 58.8% vegetable oil carrier affects the growth performance of weaning pigs . The fee piglets . The resP < 0.05) in lactating sows along with higher white blood cell and lymphocyte concentrations in suckling piglets (P < 0.05), as compared to an untreated control group without additive. Fecal samples showed higher Lactobacillus levels and lower E. coli levels for both treatment levels (P < 0.05). In subsequent work, Lei et al. found that 0.2–0.4% of the same feed additive also improved growth performance of E. coli-challenged piglets [P < 0.05) compared to an untreated control group without additive. Lower incidence of diarrhea (P < 0.05) was also reported.In another study, Devi et al. incorporated 0.1% or 0.2% of a feed supplement, which contained 17% fumaric acid, 13% citric acid, 10% malic acid, and 1.2% capric and caprylic acids (in a 1:1 mixture), into sow diets and analyzed the effects on lactating sows and their piglets . The inc piglets . There wP < 0.05) and FCR  in pigs weaned at 21 d. This result was not observed in pigs weaned at 28 d. Organic acid/fatty acid supplementation also improved amino acid digestibility (P < 0.01), lowered inflammatory immune responses , and increased ileal Lactobacillus content (P < 0.05).Kuang et al. also tested the effects of dietary combinations of organic acids and MCFAs as a replacement for zinc oxide on growth, digestibility, and immunity of weaned pigs . A mixtuEnterococcus faecium) supplement on weaning piglet performance [9 colony-forming units of viable bacteria per kg of feed). It was determined that capric acid supplementation improved ADG during the first 70 d of life (P < 0.05), as compared to both the control and caprylic acid groups, while tending to reduce piglet mortality and also led to decreases in the length of the jejunum (P < 0.05) and mass of the cecum (P < 0.05) compared to the control group without additive. Both caprylic acid and capric acid exhibited antibacterial activity against E. coli (P < 0.05), while inclusion of the probiotic further enabled inhibition of Clostridium perfringens in the cecum and jejunum (P < 0.01).Aside from mixing MCFAs with organic acids, there have been attempts to combine them with probiotics as a means to ‘seed’ the gastrointestinal tract with beneficial microbiota. Hanczakowska et al. investigated the effect of MCFAs and/or a probiotic (formance . The dieEnterococcus faecium), on the growth performance of weaned pigs [P < 0.05), along with increased plasma glucose levels and nutrient digestibility (P < 0.05), as compared to the control group without additive.Devi and Kim also investigated the effects of adding 0.2% of a palm kernel oil-derived MCFA mixture, which consisted of caproic, caprylic, capric, and lauric acids , without or with an additional 0.01% probiotic (ned pigs . The proEnterococcus faecium), affected the growth performance of finishing pigs [P < 0.05). At the same time, there were negligible differences in blood parameters. Altogether, the findings demonstrate that MCFAs, by themselves and in combination with other additives, can exhibit positive effects on growth performance during different stages of the pig production process, an outcome that is likely related in part to improved gastrointestinal tract health.Lei et al. also investigated how a blend of matrix-coated organic acids and MCFAs, without or with a probiotic  could reduce coliform and Salmonella counts by > 99.99% (P < 0.01) [Another emerging area of opportunity is exploring the feasibility of MCFAs and monoglycerides to replace antibiotics and address infectious disease challenges in farm settings. So far, there has been interest in testing whether MCFA additives can curb  < 0.01) .Salmonella shedding and colonization in slaughter age pigs on a farm with high Salmonella prevalence [Salmonella levels in pre-slaughter feces (P < 0.01), in cecal contents (P < 0.05), and in the ileocecal lymph nodes (P < 0.01) after slaughter, as compared to the negative control group without additive. Thus, the feed additive was able to reduce Salmonella prevalence by around 50% and decreased Salmonella levels in cecal contents and in the lymph nodes by around 36% and 67%, respectively.Rasschaert et al. investigated the effect of MCFAs on reducing evalence . A feed E. coli-challenged piglets [P < 0.05) and interferon-γ (P < 0.05) at 9-h and 24-h post-challenge, along with reduced incidence of diarrhea.In addition, Ren et al. evaluated the protective effect of a mixture of organic acids and capric acid on the inflammatory immune response of  piglets . The mixSalmonella Typhimurium and enterotoxigenic E. coli, in weanling piglets [Salmonella spp. levels in the cecum  and a tendency towards decreased enterobacteria and total coliform levels in the ileum and colon . The feed additive also tended to modulate colonic microbiota in both models, while there were no marked changes in intestinal fermentation products, serum pro-inflammatory mediators, or histological parameters.Recently, López-Colom et al. also investigated the efficacy of an MCFA salt-containing feed additive against two enteric pathogenic challenges,  piglets . The addTogether, the data support that MCFAs can at least curb certain types of bacterial infections in pigs and additional investigations are warranted, especially against viral infections such as PEDv and ASFv.The gastrointestinal tract plays an important role in facilitating nutrient digestion, absorption, and metabolism along with critical immune functions. It has been reported that MCFAs and monoglycerides can improve immune health-related parameters and such findings have motivated deeper investigation of the mechanistic underpinnings.in vivo , Enterobacteriaceae (P < 0.001), clostridial clusters I (P < 0.001) and IV (P = 0.019), Lactobacillus johnsonii (P < 0.001), and Lactobacillus amylovorus (P < 0.001).There has also been interest in understanding how MCFA additives affect gut-related microbial populations, which are intimately linked with proper immune function. For example, Zentek et al. investigated how MCFAs  affect gastrointestinal microbial ecology and bacterial metabolites in the digestive tract of weaning piglets . Notablyin vivo [While MCFAs and monoglycerides have proven to be useful feed additives, there has also been exploration of different delivery strategies to encapsulate them in order to slow down absorption, guide delivery to targeted regions of the gastrointestinal tract, and thus potentially enhance functional performance. One effective strategy involves using MCTs as the feed additive because they protect the MCFAs and monoglycerides from rapid absorption while the active compounds can be gradually released by lipases in vivo , 71, 92.Salmonella typhimurium in infected piglets [Another emerging approach involves delivering free MCFAs and monoglycerides that are either coated with a protective layer or encapsulated within a carrier. For example, Boyen et al. investigated whether a 0.31% dose of coated caprylic acid  could decrease fecal shedding of  piglets . At 2 d P ≤ 0.001) in the stomachs of piglets receiving an MCFA supplement, although the highest concentrations were only present at the beginning of the gastrointestinal tract due to efficient absorption. There was no significant difference in the results obtained for uncoated or coated MCFA supplements. In addition, dietary supplementation with uncoated or coated MCFA similarly influenced bacterial populations in the stomach, as indicated by the aforementioned increases in the cell numbers of eubacteria and Enterobacteriaceae, along with increased ammonia concentrations in the distal small intestine (P < 0.001). The authors also noted that coated MCFA supplementation tended to cause the most distinct changes in gut microbiota, although further investigation is warranted to determine whether that effect is due to the coating material itself or due to slower release of the fatty acids.Zentek et al. further examined the distribution of caprylic and capric acids, either uncoated or coated with vegetable fat and lecithin, within the digestive tract of piglets and resulting effects on gut microbiota and bacterial metabolic products . The pigP < 0.05), and it demonstrated similar performance levels to that of an in-feed antibiotic blend (0.003% tiamulin and 0.004% lincomycin) or 0.15–0.25% zinc oxide. Nutrient digestibility was also highest in the group that was fed the eucalyptus-MCFA mixture (P < 0.05). Recently, Omonijo et al. developed microparticles that are intended for effective delivery of essential oils and MCFAs to the pig intestinal tract [In addition, Han et al. investigated the use of a micro-encapsulated eucalyptus-MCFA dry powder feed additive (supplemented at ~ 0.1% of the diet) to promote piglet growth . The powal tract . Thymol al tract , warrantP < 0.01) and reduced mortality, as compared to the control group without additive. There was also improved total antioxidant capacity (P < 0.01) and reduced oxidative stress biomarker levels (P < 0.01) compared to pigs receiving the control diet and pigs receiving a diet with 0.04% antibiotic (Amoxicillin) [In another example, Giorgi et al. investigated whether laurate calcium soap – a saponified version of lauric acid with calcium – could improve the growth performance and health of post-weaning pigs . The moticillin) .Feed is known to be a vector that is capable of delivering infective levels of some, but not all, pathogens . Thus, when MCFAs and monoglycerides are delivered as feed additives, they can also play an important role in feed pathogen mitigation by inhibiting infectious pathogens, such as viruses and bacteria, that might be present in the feed and would otherwise remain viable in the feed matrix for extended periods of time . In effeP < 0.05). Subsequent inoculation of piglets with PEDv-contaminated choline chloride ingredient caused infection, as indicated by detectable PEDv in the small intestine, viral shedding in feces, mild diarrhea, and anatomical changes. By contrast, all piglets inoculated with MCFA-treated, PEDV-contaminated feed ingredients showed no evidence of PEDv infection and the MCFA blend performed equally as well as formaldehyde in the piglet inoculation studies. In another study, Cochrane et al. investigated the effects of including 2% of the same MCFA blend as an additive in four different feed matrices – feather meal, avian blood meal, porcine meat and bone meal, and poultry by-product meal – to inhibit Salmonella Typhimurium [Salmonella colonies by > 99% compared to the negative control group without treatment (P < 0.05) and demonstrated similar performance to a formaldehyde-treated test group.A prominent example is PEDv. Dee et al. investigated the effectiveness of a 2% MCFA blend of caproic, caprylic, and capric acids (1:1:1 ratio) to inhibit PEDv contamination of various classes of swine feed ingredients . It was himurium . It was P < 0.05). The MCFA feed pathogen mitigation strategy also protected pigs, which consumed PEDv-contaminated feed, against PEDv infection, as indicated by the lack of PEDv in fecal swabs and cecum content. While MCFAs were effective at feed pathogen mitigation, 1–2% amounts of various oils  and 0.3% of FRA® C12 Dry , the latter of which is composed of GML and additional monoglycerides, did not inhibit PEDv in feed to a significant extent. Coconut oil did, however, delay PEDv infectivity in pigs. Fatty acid profiles of the coconut and palm kernel oils used in the study indicated high fractions of lauric acid (> 45%) while there were appreciably smaller fractions of caprylic and capric acids . These data point to the likely importance of including a sufficient amount of C6-C10 fatty acids for effective feed pathogen mitigation. On the other hand, canola and soy oils did not contain MCFAs and were ineffective.Cochrane et al. have also systematically studied the mitigating effects of individual types of MCFAs and combinations thereof on PEDv-contaminated feed samples . Test saP < 0.05), thus demonstrating that the MCFAs appear to remain stable and functionally active once incorporated into the feed.In related work, Gebhardt et al. also quantified the long-term feed pathogen mitigation effects of MCFAs on PEDv contamination following feed manufacture . The feeAltogether, these findings demonstrate that MCFAs can serve as the basis for effective feed pathogen mitigation strategies. Such mitigation strategies might also be useful for preventing transmission of ASFv, which is capable of spreading via feed .Lipid Selection: Most studies concentrate on a relatively narrow range of MCFAs and further exploration of monoglyceride derivatives is warranted, especially since they can be more biologically potent and palatable in some cases.Delivery Strategies: Water-deliverable formulations of MCFAs and monoglycerides  would bRespiratory Disease Support: We anticipate that medically important antibiotics will continue to be used for treating clinical diseases in pigs, especially respiratory infections . The devMCFAs and monoglycerides can be useful for aiding numerous production issues such as growth, pathogen control, gut and immune health, and feed pathogen mitigation, and there is a growing molecular-level basis to explain these performance results. Based on the latest evidence, the data support that MCFAs and monoglycerides are typically useful in the dose range of 0.2–1% for supporting pig growth performance and gut health and in the range of 1–2% for feed pathogen mitigation. These functions are not mutually exclusive and it will be advantageous to find optimal combinations and concentrations of MCFAs and monoglycerides that support both functions. Ultimately, further validation of the results described herein and continued work in this direction are needed through eventual testing in commercial operations, where pig population density within a room and on-site is typically much greater, and where challenge by enteric and/or respiratory pathogens is normally more daunting than in academic facilities. We also see a few additional areas where further work would be beneficial to advance the field, including:


Solanum berthaultii is a wild species belonging to Solanaceae family. The complete chloroplast genome of S. berthaultii was constituted by de novo assembly using a small amount of whole genome sequencing data. The chloroplast genome of S. berthaultii was 155,533 bp in length and consisted of 25,593 bp of a pair of inverted repeats, 18,372 bp of small single copy and 85,975 bp of large single-copy regions. 158 genes were annotated including 105 protein-coding, 45 tRNA, and 8 rRNA genes. Maximum-likelihood phylogenetic analysis with eight Solanaceae species revealed that S. berthaultii is most closely grouped with S. tuberosum. Solanum berthaultii, a wild diploid species originating from Bolivia, South America is a relative to potato (S. tuberosum). It was identified as a source of resistance to several important pathogens such as Phytophthora infestans, Potato virus Y etc. and used for potato breeding  was provided by Highland Agriculture Research Institute, South Korea. An Illumina paired-end (PE) genomic library was constructed with total genomic DNA according to the PE standard protocol  and sequenced using an Illumina HiSeq2000 at Macrogen (http://www.macrogen.com/kor/). Low-quality bases with raw scores of 20 or less were removed and then approximately 2.5 Gbp of high-quality of PE reads were assembled by a CLC genome assembler   as the reference sequence  was 155,533 bp in length including 25,593 bp inverted repeats (IRa and IRb) regions separated by small single copy (SSC) region of 18,372 bp and large single copy (LSC) region of 85,975 bp with the typical quadripartite structure of most plastids, and the structure and gene features were typically identical to those of higher plants. A total of 158 genes with an average size of 583 bp were annotated including 105 protein-coding genes with an average size of 765 bp, 45 tRNA genes, and 8 rRNA genes. An overall GC content was 37.88%.The complete chloroplast genome of S. berthaultii and those of seven species including S. lycopersicum (Daniell et al. Capsicum annuum (Jo et al. S. berthaultii belonged to the same clade in Solanum species as expected and interestingly it was most closely grouped with S. tuberosum (Phylogenetic analysis was performed using chloroplast coding sequences of uberosum .


KLOTHO gene expression in mice led to a syndrome resembling human ageing. This study evaluated three KLOTHO polymorphisms, namely G395A, C1818T, and C370S, in an elderly population (mean age of 73 years) and their associations with ageing-related outcomes  and mortality. Estimated glomerular filtration rates (eGFR) was lower in subjects with 1818TT (P = 0.047) and 370SS (P = 0.046) genotypes. The 1818TT genotype (P = 0.006) and 1818T allele were associated with higher frequency of myocardial infarction (MI) . The 370SS genotype was associated with lower stroke frequency (P = 0.001). MI (OR 3.35 [95% CI: 1.29–8.74]) and stroke (OR 3.64 [95% CI: 1.48–8.97]) were associated with mortality. Regarding MI, logistic regression showed 1818T allele was a risk factor for death-related MI , while 370C was protective . Regarding stroke, the 395A and 370C alleles were protective factors . This is the first study to determine potential associations between common ageing-related outcomes/mortality and KLOTHO polymorphisms. The 1818T allele was a risk factor for MI-related death. The 395A and 370C alleles were protective factors for stroke-related death in elderly from community.Defective  This trend comes with significant social consequences: communities with higher percentages of elderly people present higher disease rates  and increased dependency of residential and hospital care1. Under these circumstances, several studies directed to basic health fields decided to look for a deeper comprehension of human aging to subsequently focus on intervention strategies3.The percentage of elderly people is increasing more than any other age group worldwideKLOTHO gene was discovered in 1997, when a study revealed that its defective expression in mice led to a syndrome that resembles human ageing, including a short lifespan, arteriosclerosis, and osteoporosis4. Its products exist as a secreted protein5 or a transmembrane protein expressed predominantly in the distal renal tubules, choroid plexus, and pituitary gland4. Their function in humans, however, remains unknown. The secreted form suppresses oxidative stress and growth factor signalling, including insulin/IGF-1 signalling, all effects associated with longer life span6.The KLOTHO mutations and single-nucleotide polymorphisms (SNPs) have been described in the humans. Nonetheless, the effects of each SNP in human ageing are still unclear. In fact, studies in human populations have sought for associations between aging, longevity, and some of such variants, including the most common SNPs G395A and C1818T7 and the functional Klotho variant KL-VS. This last variant comprises six polymorphisms in linkage disequilibrium, two of which resulting in amino acid substitutions: F352V and C370S6. Associations between these variants and age-related outcomes were demonstrated in different populations, mainly in Asians9.At least 10 10. Hence, the present study seeks to evaluate three KLOTHO gene polymorphisms in an elderly population from the São Paulo Ageing & Health Study (SPAH) and their potential association mainly with mortality, but also with the prevalence of major ageing-related outcomes, including osteoporosis, osteoporotic fractures , sarcopenia, kidney function (estimated glomerular filtration rate [eGFR]), and cardiovascular events .There is only one publication regarding KLOTHO polymorphisms and age-related comorbidities in a Brazilian populationThe demographic, anthropometric and clinical data of the 601 subjects are shown in Table angina pectoris, myocardial infarction or stroke.Of the 601 subjects, 80 (13.3%) experienced cardiovascular events, including Only 27 (4.5%) subjects had a history of MI. The analysis of the C1818T SNP revealed that the 1818TT genotype was associated with a higher frequency of MI (P = 0.006) (Table Thirty (5%) stroke events occurred in the studied population. The 370SS genotype was associated with a lower outcome frequency (P = 0.001) while single-allele analysis detected no significant differences Table .Three hundred and forty-six (57.6%) subjects presented with abdominal aortic calcification score >0 visible on radiographic assessment. However, none of the three polymorphisms were associated with this outcome Table .Subjects with the 1818TT and 370SS genotypes featured lower eGFR   deaths among the 601 participants, mostly related to cardiovascular events , followed by pulmonary non-neoplastic causes  , non-ischemic heart failure , infections (excluding pneumonia) , cancer , and other causes, including pulmonary thromboembolism, Alzheimer’s Disease and kidney failure . The mean age of the deceased was 79.7 years, with a standard deviation of 6.8 years.MI and stroke were the only variables associated with mortality. They were therefore analysed by logistic regression, which was adjusted by sex, BMI and age considering the average life expectancy of the Brazilian population during the data collection period. No association was observed concerning other outcomes, including eGFR.MI was independently associated with death, with an Odds Ratio (OR) of 3.35 [95%CI: 1.29–8.74]). The presence of 1818T was a risk factor for MI-related death OR: 4.29 [95%CI: 1.60–11.52]; P = 0.003), whereas both the presence of 370C and the female sex were independent protective factors  , and multiple logistic regression found that 395A and 370C alleles were protective factors   than those with the CC genotype, particularly in younger Korean subjects (<60 years)8. Analysis of an Iranian population, in turn, revealed a protective effect of the 1818CC genotype for age-related occurrence of CAD combined with hypertension in the subgroup older than 57 years12. The only study conducted in a Brazilian population so far, however, did not analyse the C1818T polymorphism in the mentioned setting10.In this community population, cardiovascular events were the main cause of death, and, as expected, the female sex was a protective factor for MI-related death. The 1818T allele was associated with a higher myocardial infarction frequency and, according to the logistic regression analysis, constitutes a risk factor for MI-related death. Most studies analysed this polymorphism regarding cardiovascular disease using coronary angiography19, and a Spanish study20 found that it was a predictor of diabetes. On the other hand, regarding C370S, studies from Massachusetts, USA revealed no association between C370S and cardiovascular disease  in young11 and older patients16. Moreover, there are other evidences tying Klotho to cardiovascular events, even though not related to its polymorphisms, but to its serum and tissue expressions. In fact, a Brazilian community-dwelling elderly population showed association between serum Klotho levels and history of myocardial infarction10. A Spanish study found association between the vascular levels of Klotho expression and presence of cardiovascular disease and cardiovascular risk factors. Indeed, individuals with coronary artery disease had a significantly lower expression of Klotho in aortic samples20.The other two KLOTHO polymorphisms analysed (G395A and C370S) were not associated with MI. Regarding G395A SNP, previous studies showed an association with CAD mainly in Asian population22. The C1818T SNP was also studied in Indian population, but was not associated with ischemic stroke21. Furthermore, in Korean women, the G395A SNP A allele was related to cardioembolic stroke23.In the present study, stroke was an important cause of cardiovascular death. The multivariate analysis indicated that the S allele of the C370S SNP and the A allele of the G395A SNP were protective factors for stroke-related death, whereas sedentary behaviour was identified as a risk factor for this outcome. Prior data have demonstrated that homozygous 370CC or 370SS individuals have a higher risk for stroke, while heterozygotes display a lower riskThe present study found no associations between vascular calcification and Klotho polymorphisms in our community-dwelling elderly subjects. Of note, this issue had never been investigated in humans.KLOTHO polymorphisms in subjects without chronic kidney disease. Our results showed that 1818TT and 370CC genotypes were associated with lower eGFR. Analysis of two Chinese populations (Uygur and Kazak) with different longevities revealed that such genotypes were associated with higher serum creatinine in the Kazak population, which has shorter longevity5.Renal dysfunction has been related to Klotho protein, but there are few studies correlating kidney function to 25 and osteoblasts26, in addition to promoting osteoclast differentiation24. Data from previous studies are controversial, showing that the CS/SS genotypes (C370S SNP) were associated with higher BMD, but not with osteoporotic fractures in Spanish women27 and young/middle-aged Spanish men28. Moreover, postmenopausal white and Japanese women with the G395A SNP GG genotype tended to have lower BMD29, while A allele carriers among Japanese women had higher BMD30. The same association was found with T allele carriers of the C1818T SNP in postmenopausal women30, a finding not observed in another report9.No association between Klotho polymorphisms and BMD and fractures were found in the present study. In contrast, data from literature have speculated that Klotho may have an effect on osteocytesKLOTHO polymorphisms had not been previously studied before, so the lack of such associations revealed by our study consists in negative but novel information.Potential associations between sarcopenia and 32. Such findings prompted research about those variants and mortality itself, however only in chronically ill populations, such as patients in haemodialysis34 and IgA nephropathy35. Our study is the first one to examine KLOTHO SNPs and mortality in elderly community-dwelling subjects and two polymorphisms were associated with the main events that led to death, i.e., stroke and myocardial infarction.Klotho polymorphisms have been previously associated with longevity  in different populations36. The subjects were invited to participate in a second survey from 2010 to 2012, which provided data regarding mortality37. Full details pertaining to the study population, assessments, and procedures have been previously reported elsewhere37.This study was based on data from the São Paulo Ageing & Health (SPAH) Study, which surveyed 65-year-old and older individuals living in a community in the Butantã district, located on the western zone of the city of São Paulo. The first survey took place from 2005 to 2007, and provided the baseline dataOne hundred and twenty-five participants were interviewed on the first survey; 725 (70.73%) attended the second evaluation, and 601 of them (381 women and 220 men) had blood samples collected for DNA extraction and were included in the genotype-based analyses. Fifty-one of the analysed subjects (8.49%) died from 2005 to 2012.This study was approved by the local ethics committee (Comissão de Ética para Análise de Projetos de Pesquisa do HCFMUSP [CAPPesq] #426/06), and all the participants provided written informed consent. All the methods were carried out in accordance with relevant guidelines and regulations stated by Declaration of Helsinki.Each subject was interviewed by a physician and responded to a standard questionnaire designed to gather information about lifestyle, health behaviours, and medical history, including age, smoking status, physical activity (ability to perform daily chores), alcohol consumption (at least 3 units per day), previous clinical fragility fractures, history of falls during the past year, and history of myocardial infarction or stroke.38, and serum creatinine concentration, determined using standard automated laboratory methods. Estimated glomerular filtration rate (eGFR) was calculated according to the Modification of Diet in Renal Disease (MDRD) formula40. The second interview provided data concerning mortality.Baseline measurements included height and weight, and were carried out using standard protocols to calculate the body mass index (BMI) and the appendicular skeletal muscle mass (ASM). Baseline laboratory and imaging evaluations included thoracic and lumbar spine radiographs, bone mineral density (BMD) measurement41. The least significant change was calculated with 95% confidence to be 0.033 g/cm2 for anteroposterior spine, 0.047 g/cm2 for femoral neck, and 0.039 g/cm2 for total hip. BMD characterization was performed according to the WHO classification: normal, T-score ≥ −1; osteopenia, T-score < −1 and > −2.5; and osteoporosis, T-score ≤ −2.5 in the spine, femoral neck, or total hip41.Bone Mineral Density was measured using dual-energy X-ray absorptiometry  at the lumbar spine, femoral neck, and total hip. All BMD measurements were performed by the same experienced technologist, following the recommendations by the International Society for Clinical Densitometry (ISCD)42.Osteoporotic fracture was defined as a fracture occurring at sites characteristic of bone fragility  on fifty-year-olds and older. Fractures occurring in the face, skull, ankles, elbows, and fingers were not considered osteoporotic fractures43.Standard lateral thoracic and lumbar spine radiographs were taken using a 40-in tube-to-film distance centered at T7 and L2. All images provided good visibility of all vertebrae from T4 to L4, and vertebrae could be reliably identified. Two experienced readers independently performed the identification of vertebral fractures. Each of the T4–L4 vertebral images was evaluated to detect fractures, and nonvisible vertebrae were excluded. The agreement rate between the readers, which was established on a random subsample of 60 radiographs, was 96% with a kappa coefficient of 0.82. A consensus was reached between the readers for any difference of interpretation. Vertebral fractures were classified using the Genant semiquantitative approach44. Linear regression was used to determine the relationship between ASM relative to height (in meters) and fat mass (in kilograms). The residuals of the regression were used to identify those with ASM lower than the predicted value (given by an equation derived from the model), for an individual fat mass. A positive residual would indicate a relatively muscular individual, whereas negative values would indicate relatively sarcopenic individuals. In men, the equation resultant from the model was as follows: ASM (kg)  =   −20.67  +  22.48 × height (m)  +  0.177 × fat mass (kg); and, in women: ASM (kg) = −14.51 + 17.27 × height (m) + 0.20 × fat mass (kg). The 20th percentile of the distribution of residuals was used as the cut point for sarcopenia according to ASM adjusted for fat. The cut-off corresponded to a residual of −2.06 in men45, and −1.45 in women46 in our Brazilian sample.Appendicular skeletal muscle mass (ASM) was measured by DXA: ASM relative to height and total fat mass were assessed. Relative ASM was derived by adjusting for fat mass in addition to height47. This resulted in an abdominal aortic calcium score (AACS) ranging from 0 to 24. The reproducibility of AACS blind assessment was analysed using the Wilcoxon test for sum score as continuous and categorized data. In both cases, the P values were higher than 0.10 , confirming appropriate level of reproducibility.Lateral lumbar spine radiographs were acquired to quantify aortic calcification, besides assessing vertebral fractures. Calcium deposits were considered to be present if the densities were visible in an area that was parallel to the lumbar spine and anterior to the lower part of the spine. These calcific densities were graded on a 0 to 3 scale at each lumbar vertebrae segment: 0 denotes no aortic calcific deposits; 1 denotes small scattered calcific deposits that occupies less than one third of the longitudinal wall of the aorta; 2 indicates that one third or more but less than two thirds of the longitudinal wall of the aorta is calcified; and 3 indicates that two thirds or more of the longitudinal wall of the aorta is calcifiedDuring the interview, when a past history of myocardial infarction (MI) or stroke was identified, the participant’s medical records  were reviewed. Stroke was defined as a sudden focal neurological deficit of presumed vascular origin that persisted for more than 24 hours. MI was defined as ischemic symptoms, development of diagnostic Q waves on the electrocardiogram, electrocardiographic changes indicative of ischaemia (ST segment elevation or depression), or coronary artery intervention.Mortality data of the original SPAH cohort was gathered from the date of the baseline examination through December 31, 2012. They were collected from the publicly available death certificates ascertained by the Programa de Aprimoramento das Informações de Mortalidade no Município de São Paulo . The underlying causes of death were coded according to the 10th revision of the International Classification of Diseases (ICD-10). Cardiovascular mortality was defined as ICD-10 codes I00 to I99 and non-cardiovascular mortality as all other causes of death.48, and was stored at −70 °C prior to analysis. The TaqMan allelic discrimination method was employed for genotyping, using specific probes labelled with VIC and FAM .Genomic DNA was isolated from peripheral blood leukocytes using the salting-out methodology50. Such SNPs comprised C1818T (rs564481), a silent mutation30 located in exon 4 of KLOTHO30; G395A (rs1207568), located in the promoter region of KLOTHO30 and possibly associated with its expression and function30; and C370S , located in exon 251. C370S is one of the SNPs that compose the functional Klotho variant KL-VS, which impairs the protein function by influencing its trafficking and catalytic activity52.The analysed SNPs were chosen based on previous literature data that suggested possible associations with ageing and functional significanceAll SNPs were genotyped using commercial TaqMan assays  with TaqMan Genotyping Master Mix and were measured using a StepOne plus detection system (Applied Biosystems). Cycling conditions included a denaturation step at 95 °C for 10 minutes followed by 40 cycles of denaturation at 92 °C for 15 seconds and annealing for 1 minute at 60 °C. To assess the genotyping quality, the polymorphisms were analysed twice in 5% of all samples, yielding a 98% concordance.KLOTHO SNPs, we selected the most clinically relevant ageing-related outcomes associated to them. As discussed above, there lacks data correlating mortality to KLOTHO SNPs, thus mortality is a hard outcome that was chosen to be one of this studies cornerstones. Potential causes for death, as cardiovascular events, and outcomes related to healthy ageing that were previously studied in relation to these and other KLOTHO SNPs  were chosen according to previous literature, also debated on Discussion.Following the evidence-based choice for studying each of these three 2. Continuous variables were expressed as mean ± standard deviation for parametric data or median and quartiles for non-parametric data. When comparing three or more groups, nonparametric data were analysed using the Kruskal-Wallis test adjusted by the Bonferroni post-test, while the Mann-Whitney test was applied for comparisons between two groups. In the multivariate analysis, Logistic Regression, results were expressed as odds ratio and 95% confidence interval. Hosmer–Lemeshow goodness-of-fit test was applied to evaluate the calibration of logistic regression model and to check the importance of the discrepancy between observed and expected for Myocardial Infarction and Stroke. Forward stepwise selection procedure was used to select the variables significantly related to Myocardial Infarction and Stroke, as assessed by the Likelihood Ratio test. Odds ratios with 95% confidence intervals were calculated for statistically significant variables. A P-value <0.05 was considered statistically significant. The genotype distribution and allele frequencies of the C1818T and G395A SNPs were consistent with the Hardy-Weinberg equilibrium , whereas the ones associated with C370S were not (χ² = 3.90 with P = 0.05). However, since the number of subjects that would have to be excluded from the analysis to adjust it was higher than 5 to 10%, all the data were analysed to avoid additional bias53. Furthermore, for the allelic analyses, the number of patients was not duplicated as not to create bias from the duplication of phenotypic traits.Categorical data were expressed as absolute frequency (n) and relative frequency and analysed by Pearson χSupplementary information.