P for trend was .02. SP was not associated with any nutritional intake; however, the combined outcome of PE and SP was related to low crude calcium and potassium and energy‐adjusted calcium, potassium, and magnesium intakes . The same tendency was observed for GH. A prediction model that included crude calcium and potassium intakes performed better than a model without them. In conclusion, low dietary calcium, potassium, and magnesium were associated with higher HDP subtypes prevalence. The prediction model implied that crude calcium and potassium intakes might play a critical role in PE and SP pathogenesis.This study aimed to explore the relationship between dietary electrolyte intake and the prevalence of hypertensive disorders of pregnancy (HDP) subtypes. Our analysis included 19 914 pregnant women from the Tohoku Medical Megabank Project Birth and Three‐Generation Cohort Study. A food frequency questionnaire was used to estimate dietary calcium, potassium, sodium, and magnesium intakes. HDP was determined based on the medical records during regular antenatal care. Logistic regression analysis assessed the relationship between dietary electrolytes intake quintiles, and HDP subtypes with adjustment for basic characteristics. Dietary electrolyte intakes were applied for the prediction model. Of the cohort, 547 participants delivered with pre‐eclampsia (PE), 278 with superimposed PE (SP), and 896 with gestational hypertension (GH). PE was associated with low crude calcium intake  and  However, the evidence among studies is inconsistent. A meta‐analysis of 13 trials with 15 730 women suggested that calcium supplementation had a significant preventive effect on PE, especially among pregnant women with low baseline calcium intake.Further research examining the relationship between nutritional intake and PE is needed to identify high‐risk pregnant women and develop an optimal clinical strategy. This study investigated the relationship between dietary nutritional intake, particularly calcium, and the prevalence of the HDP subtypes. We also investigated whether dietary nutritional intake could help develop an accurate prediction model.22.1n = 335), had multiple pregnancies (n = 328), or had missing HDP diagnosis (n = 827) or food frequency questionnaire (FFQ) information (n = 1089). Therefore, this study analyzed 19,914 pregnant women. The ethics approval was obtained from the Ethics Committee of the Tohoku Medical Megabank Organization (2013‐1‐103‐1), and all participants provided written informed consent for study inclusion.The Tohoku Medical Megabank Project Birth and Three‐Generation (TMM BirThree) Cohort Study2.2The participants completed the FFQ during their gestation period  based on their eating habits during the year before the response date. We calculated dietary nutrition intake, including energy, protein, carbohydrate, fats, and electrolytes. The present study focused on calcium,We selected verified or possible risk factors for HDP as covariates, including age during antenatal care at 10–13 weeks of gestation,We previously developed a prediction model for PE and superimposed PE (SP) using competing risk model,2.3PE, SP, and gestational hypertension (GH) were identified based on the American College of Obstetricians and Gynecologists (ACOG) guidelines from 2002,We defined PE as systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg and proteinuria (≥2+ in the dipstick test) during at least one visit after week 20 of gestation in women with previously normal blood pressure. SP was diagnosed when women with CH developed proteinuria after week 20 of gestation. GH was defined as systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg, but no proteinuria during at least one visit after week 20 of gestation in women with previously normal blood pressure. These phenotypes were automatically diagnosed by an algorithm based on the medical record data and validated by a physician. Blood pressure measurement and dipstick test for proteinuria were undertaken as per usual protocol in clinics and hospitals and not standardized, as detailed elsewhere.2.4Dietary electrolytes intake with and without energy adjustment was divided into five percentile groups: category 1, <20%; category 2, 20%–39%; category 3, 40%–59%; category 4, 60%–79%; category 5, ≥80%. Category 5 was used as the reference. Basic characteristics and the prevalence of the HDP subtypes were compared among the electrolytes’ quintile groups without energy adjustment using analysis of variance for continuous variables and the chi‐squared or Fisher's exact test for categorical variables.We performed logistic regression analysis to assess the relationship between the dietary electrolytes intake and the prevalence of the HDP subtypes and PE+SP after adjusting for maternal age (older than 35 years or not), pre‐pregnancy BMI (over 25 kg/m2 or not), smoking status (yes or no), alcohol consumption (yes or no), educational attainment (over 12 years or not), DM, SLE, family history of HDP, family history of CH, parity with or without HDP, and conception by IVF. Pregnant women with CH were included in the population for PE+SP outcome analysis. CH was considered a risk factor for PE+SP and used as a covariate. Pregnant women with CH were excluded from the analysis of PE and GH, while pregnant women without CH were excluded from the analysis of SP. Multiple imputations with five imputed datasets were performed to address missing data in possible confounders using the mice package in R.We examined the relationship between dietary electrolytes intake and gestational age at delivery with PE+SP to develop the prediction model. We compared models with linear‐, quadratic‐, and broken‐stick‐transformed electrolytes using the Akaike information criterion (AIC) as a measure of goodness of fit. We selected the electrolyte transformation values according to their results and included them in the prediction models.10‐transformed multiple of the median (log MoM) values of MAP. Educational attainment, smoking status, alcohol consumption, and family history of CH were excluded. Gestational age at the last delivery and the inter‐birth interval in parous women were included in the prediction model based on the findings of a previous study.We performed a parametric survival time analysis that considered deliveries without PE+SP as censored observations, assuming gaussian survival curve distribution. Dietary electrolyte intake and basic maternal characteristics were included in our model. MAP, excluded from the logistic regression model, was included in the prediction model because it could be a strong mediator of PE+SP onset. We used the loghttps://www.r‐project.org).The probability of delivery with PE+SP before specific gestational ages  was divided into 10 equal‐size probability intervals, and calibration was assessed using a calibration plot with the confidence intervals (CIs) calculated based on 1000 bootstrap re‐samplings. We applied the five‐fold cross‐validation and calculated the area under the receiver operating characteristic curve (AUROC) for each fold. The mean of the five AUROCs (mAUROC) represented the model's performance. We used the bootstrap method to obtain the mAUROC distributions and the difference in mAUROCs among models and assessed 95% CIs. Statistical analysis was performed using R, Version 3.5.3  were nulliparous. The prevalence of PE was 3.5% and 2.6% for participants with a calcium intake of <251 mg/day and > 623 mg/day, and 3.3% and 2.5% for potassium intake of <1313 and >2676 mg/day, respectively Tables .P for trend, .02 and .03, respectively). SP was not related to any of the assessed nutritional electrolytes; however, PE+SP was related to crude calcium and potassium intake and energy‐adjusted calcium, potassium, and magnesium intake . The prevalence of GH was low in participants with low energy‐adjusted Na/K ratio and high in participants with low energy‐adjusted calcium intake . Furthermore, stratified by house damage, OR of energy‐adjusted magnesium intake (quintile 1 [<183 mg/day] versus quintile 5 [>243 mg/day]) on PE+SP was 1.45 (1.03–2.05) in the participants without house damage and 1.09 (.78–1.53) in the participants with house damage.Logistic regression analysis showed that low crude calcium intake was related to PE prevalence  was 46.5  weeks  in 2016.5Low dietary calcium, potassium, and magnesium were associated with a higher prevalence of various HDP subtypes. The developed prediction model implied that absolute calcium or potassium intake might play a key role in PE+SP pathogenesis.Hisashi Ohseto wrote the original draft. Hisashi Ohseto, Mami Ishikuro, and Taku Obara carried out the statistical analysis. Mami Ishikuro, Taku Obara, Keiko Murakami, Tomomi Onuma, Aoi Noda, Ippei Takahashi, Fumiko Matsuzaki, Fumihiko Ueno, Noriyuki Iwama, Masahiro Kikuya, Hirohito Metoki, Junichi Sugawara, and Shinichi Kuriyama were involved in the acquisition, interpretation of data, and the manuscript review process. All authors approved the submitted version.None.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Higher medication anticholinergic burden is associated with increased risk of cardiovascular disease and cognitive decline. A mechanistic pathway has not been established. We aimed to determine whether inflammation may mediate these associations.Participants were drawn from the European Prospective Investigation into Cancer, Norfolk cohort (40‐79 years at baseline). Anticholinergic burden score (ACB) was calculated at first (1HC) (1993/97) and second (2HC) (1998/2000) health checks. Fibrinogen and C‐reactive protein (CRP) were measured during 1HC and tumour necrosis factor alpha (TNF‐α) and interleukin 6 (IL‐6) during 2HC. Cross‐sectional associations between ACB and inflammatory markers were examined for both health checks. Prospective associations were also examined between 1HC ACB and 2HC inflammatory markers. Models were adjusted for age, sex, lifestyle factors, comorbidities and medications.In total, 17 678 and 22 051 participants were included in cross‐sectional analyses for CRP, and fibrinogen, respectively. Furthermore, 5101 participants with data on TNF‐α and IL‐6 were included in the prospective analyses. Cross‐sectionally, compared to ACB = 0, ACB ≥ 4 was associated with higher fibrinogen, beta  = 0.134 g/L , CRP 1.175 mg/L , IL‐6 0.593 pg/mL  and TNF‐α 0.137 pg/mL . In addition, a point increase in ACB was associated with higher levels of all markers. Prospectively, compared to ACB = 0, ACB ≥ 4 was associated with higher IL‐6(pg/mL) of 0.019  and TNF‐α (pg/mL) of 0.202% . A unit increase in ACB was associated with a significantly higher TNF‐α and IL‐6.Higher ACB was associated with higher inflammatory markers. Inflammation may mediate the relationship between anticholinergic medications and adverse outcomes. Higher anticholinergic burden from medications is associated with increased risk of cardiovascular disease and cognitive function decline.Using data from a large‐scale prospective cohort study from UK, we determined the cross‐sectional and prospective associations between the anticholinergic burden and inflammatory markers.Inflammation may mediate the relationship between exposure to anticholinergic medications and adverse outcomes through the “inflammatory reflex” or cholinergic anti‐inflammatory pathway.1Anticholinergic medications block the effect of the neurotransmitter acetylcholine by inhibiting muscarinic receptorsOlder people are at a particularly higher risk of anticholinergic complications due to (a) increased risk of polypharmacy including medications with anticholinergic properties, (b) age‐related reductions in central cholinergic pathways and (c) decreases in the renal and hepatic clearance of drugs.Inflammation plays an important role in the pathogenesis of cardiovascular diseaseWe therefore hypothesised that increased anticholinergic burden leads to increased inflammation by blocking the central muscarinic‐dependent vagal activation. This may mediate the previously described relationships between anticholinergic burden and adverse outcomes. No previous investigations have assessed the relationship between antimuscarinic activity and inflammation in a population sample. In this study, we aimed to examine the cross‐sectional and prospective relationships between anticholinergic burden from medications and important inflammatory markers  in a large UK population‐based study.22.1The participants were drawn from 25 639 men and women who enrolled in the EPIC‐Norfolk study at baseline  between 1993 and 1998. The study protocol has been previously described.2.2During both health checks, trained nurses measured weight, height and body mass index (BMI) using standardized procedures. Participants also completed a health and lifestyle questionnaire that collected information on physical activity, smoking status, alcohol consumption, comorbid conditions and medications. The Food Frequency Questionnaire (FFQ) was used to measure participants' habitual dietary pattern during the previous year. The questionnaires were collected at the 1HC and after 5 years. Nonfasting venous blood samples were also taken. From each individual, ~40 mL of blood was drawn and serum, plasma, erythrocytes and buffy coat were aliquoted in plastic straws of 0.5 mL each. These straws were then heat‐sealed and stored under liquid nitrogen (−196 °C) in a centralized biobank. Figure 2.3Medications with anticholinergic properties were identified by searching the database for exact and similar entries for both generic and brand name drugs. A corresponding anticholinergic class/score was allocated to each medication: class 0 (none), class 1 , classes 2 and 3 . This was based on the criteria of the anticholinergic cognitive burden (ACB) scale developed by Boustani et al.2.4The inflammatory markers were the outcomes of interest: plasma fibrinogen, CRP, IL‐6 and TNF‐α. Fibrinogen (g/L) and CRP (mg/L) were available for a subsample of 1HC, and IL‐6 (pg/mL) and TNF‐α (pg/mL) were available for a subsample in 2HC.2.5Data were analysed using SPSS version 24.0 . Descriptive statistics were presented for the overall sample and by ACB score groups. Differences between ACB groups were assessed using the Pearson's chi‐squared test for categorical variables and analysis of variance (ANOVA) for normally distributed continuous variables or the Kruskal‐Wallis test for continuous variables that were not normally distributed.Linear regression analysis was performed to determine the cross‐sectional relationship of baseline/1HC ACB and 1HC inflammatory markers, fibrinogen and CRP. Linear regression was also performed to determine the cross‐sectional association of 2HC ACB and 2HC inflammatory markers IL‐6 and TNF‐α. The prospective relationship between baseline/1HC ACB data and 2HC inflammatory markers IL‐6 and TNF‐α was also determined.Four statistical models were constructed to assess the effects of potential confounding factors in a group sequential fashion. Model A was unadjusted, model B was adjusted for age and sex, model C was adjusted for age, sex and lifestyle factors  and, lastly, model D, the fully adjusted model, was adjusted for the variables age, sex, lifestyle factors, comorbidities , medications  and total cholesterol.3There were 25 639 participants that attended the 1HC. A total of 3588 participants were excluded from the fibrinogen analysis and 7961 individuals were excluded from the CRP analysis due to missing data. Therefore, a total of 22 051 and 17 678 participants were included in the fibrinogen and CRP analyses, respectively. In the 2HC, out of the 15 786 participants who attended, only 5101 men and women were included in the analysis for TNF‐α and IL‐6 after the exclusion of 10 685 participants with missing data for these variables. Figure All exclusions were made due to missing data. Participants that did not have a response (missing data) for diabetes mellitus, myocardial infarction, cerebrovascular accident, cancer and cholesterol were counted as not having the condition and were therefore not missing from the analysis. Similarly, those that did not respond for smoking status, alcohol consumption, NSAIDs and lipid‐lowering drugs were counted as not consuming these and therefore not missing from the analysis. In the 1HC, there were three participants with missing data for sex and 890 for fruit and vegetable consumption, whilst in the 2HC there were 1002 participants with missing data for physical activity and 3543 for fruit and vegetables.TNF‐α and IL‐6 were analysed together as they had similar amounts of missing data.3.1Tables There were significant differences between ACB groups for all variables considered except fruit and vegetable consumption. People in the higher ACB groups were older, had higher BMI and total cholesterol level, and a lower level of physical activity. NSAIDs and lipid‐lowering drug usage was more prevalent in higher ACB groups. In terms of comorbidities, high ACB score was associated with a greater percentage of individuals with a prior history of stroke, cancer, diabetes and myocardial infarction. Significantly higher mean fibrinogen and median CRP were observed in higher ACB groups compared to the ACB = 0 group.Supporting Information Table 3.2P value) an increase by 0.118  and 0.134  was seen. CRP was associated with the highest increase when the ACB score was compared to ACB = 0. There was an increase in CRP (mg/L)  by 0.765  and 1.175  when ACB = 1 and ACB ≥ 4, respectively. In the same way, when compared to ACB = 0, ACB = 1 and ACB ≥ 4 were associated with 0.073  and 0.593  increases in TNF‐α (pg/mL), respectively. However, IL‐6 (pg/mL) showed a decrease of 0.040  when comparing the reference category to ACB = 1. This was due to the reduced sample size. IL‐6 showed an increase of 0.593 pg/mL  when ACB ≥ 4 was compared to the reference category. These percentage increases get larger as the ACB score grows, therefore the relationship between ACB and inflammatory marker is a linear one. This is a cross‐sectional relationship due to inflammatory markers being collected at the same health check at which the ACB scores were collected.Table P < .001), CRP of 0.284 mg/L  (in 1HC), TNF‐α of 0.031 pg/mL  and IL‐6 of 0.112 pg/mL  (in 2HC), respectively.Table 3.3P = .319) and 0.019 , respectively, and in TNF‐α (pg/mL) by 0.012  and 0.202 , respectively.Table P = .013) and 0.076 pg/mL , respectively.Table 4We describe for the first time the association between anticholinergic burden and inflammation at a population level. Cross‐sectionally, increases in ACB scores were independently associated with significant increases in fibrinogen, CRP, TNF‐α and IL‐6. Prospectively, increases in ACB scores were independently associated with increases in TNF‐α and IL‐6. To the best of our knowledge, this is the first report which provides a possible mechanistic link through inflammatory pathways between anticholinergic medications and adverse longer‐term outcomes at a population level.Increases in such inflammatory markers in the long term can lead to adverse outcomes. The meta‐analysis by Kaptoge et alThe meta‐analysis by Yano et alThe results of our study alongside previous investigations linking markers of chronic inflammation with a variety of adverse incident outcomes allow, for the first time, the quantification of the contribution of inflammation in mediating the relationship between ACB and adverse outcomes. The inflammatory reflex may mediate the described relationships between anticholinergic burden and adverse outcomes. We hypothesised that an increased anticholinergic burden may lead to raised inflammatory markers due to the inflammatory reflex.Based on literature, our findings can be extrapolated that a 3‐point increase in ACB‐related chronic inflammation can be linked to an increase in relative risk of up to ~4% in cancer, cardiovascular disease and mortality. Furthermore, a previous studyIndeed, there may be plausible mechanisms other than chronic inflammation that may mediate the association between ACB from medications and adverse outcomes. Anticholinergic drugs have been found to suppress the parasympathetic control of heart rate, which is linked to an increased incidence of myocardial ischemia and tachyarrythmias that are known to increase the risk of embolic strokes and sudden cardiac death.Our study has several strengths. It benefited from using data from a large, prospective population‐based study. We were able to delineate both the cross‐sectional and prospective associations between ACB and markers of inflammation. The data were also prospectively collected, minimising recall bias. Additionally, a well‐validated ACB score was used, and we were able to control for a variety of sociodemographic and lifestyle factors, comorbidities and relevant medications.We acknowledge some limitations. As a volunteer study with long‐term follow‐up, a degree of healthy volunteer bias is possible. However, the baseline characteristics of the EPIC‐Norfolk participants are similar to other UK representative population samples.This study, along with future robust experimental studies, may change the anticholinergic burden paradigm by recognizing the chronic inflammatory state associated with anticholinergic medications as a therapeutic target in the era of targeted immune therapy for cardiovascular prevention. The landmark CANTOS trial used canakinumab to target the interleukin‐1β innate immunity pathway, leading to a significantly lower incidence of recurrent cardiovascular events.In conclusion, using data from a large‐scale prospective cohort study from the UK, we determined the cross‐sectional and prospective associations between the anticholinergic burden and inflammatory markers. Higher anticholinergic burden is significantly associated with higher inflammatory markers both cross‐sectionally and prospectively after multivariable adjustment. Furthermore, we underlined how chronic inflammation may be a previously unrecognized potential mechanism for the observed association between anticholinergic burden and adverse outcomes.The authors have no conflicts of interest to declare that are relevant to the content of this article.R.S., T.A.P. and P.K.M. conceived the study. R.S. performed the literature search and data analysis, and wrote the first draft of the paper. R.S., T.A.P. and P.K.M. verified the data. N.J.W. and K.‐T.K. are the PIs of the EPIC‐Norfolk cohort. All authors commented on previous versions of the manuscript and approved the final manuscript. P.K.M. is the guarantor.The Norwich Ethics Committee approved the study in view of the retrospective nature of the study.Informed consent was obtained from all individual participants included in the study.Supporting Information Table S1 Baseline sample characteristics for 17 678 men and women whose CRP level was measured in the EPIC‐Norfolk cohort  according to the total anticholinergic burden score groupsSupporting Information Table S2 Characteristics from second health check for 5101 men and women whose TNF‐α and IL‐6 levels were measured in the EPIC‐Norfolk cohort according to the total anticholinergic burden score groupsClick here for additional data file.


Haplotype and outlier‐based tests revealed a putative chromosomal inversion that contained three expressed genes and was nearly fixed in walleye assigned to Lake Winnipeg. These patterns exist despite the potential for high gene flow between these proximate Canadian lakes, suggesting that the inversion may be important for facilitating adaptive divergence between the two lakes despite gene flow. However, a specific adaptive role for the putative inversion could not be tested with the present data. Our study illuminates the importance of genomic architecture consistent with local adaptation in freshwater fishes. Furthermore, our results provide additional evidence that inversions may facilitate local adaptation in many organisms that inhabit connected but heterogenous environments.Differences in genomic architecture between populations, such as chromosomal inversions, may play an important role in facilitating adaptation despite opportunities for gene flow. One system where chromosomal inversions may be important for eco‐evolutionary dynamics is in freshwater fishes, which often live in heterogenous environments characterized by varying levels of connectivity and varying opportunities for gene flow. In the present study, reduced representation sequencing was used to study possible adaptation in  Sander vitreus) from three North American waterbodies, and found evidence of selection and a chromosomal inversion between two connected Canadian lakes. This inversion may underlie divergence between the Canadian lakes, local adaptation in an especially important fishery of one lake, and contributes to evidence that inversions may facilitate local adaptation in many organisms that inhabit connected but heterogeneous environments.Chromosomal inversions between populations may facilitate adaptation despite opportunities for gene flow. We used reduced representation sequencing on n=345 walleye ( Walleyen = 345 individuals from the three waterbodies described above. Population structure and demographic history were explored to provide context for signatures of selection. Simulations with EASYPOP were used to estimate historical migration rates between Lake Manitoba and Lake Winnipeg , a reduced representation approach, to genotype Balloux, . CandidaBalloux, , and out22.1n = 129, 51, and 55 respectively). Prior to sampling, a Portable Electroanesthesia System (Smith Root) was used to anesthetize fish following approved animal use protocols of Fisheries and Oceans Canada , the University of Manitoba (F2018‐019), and the University of Nebraska‐Lincoln (Project ID: 1208). Fin clips were stored in 95% ethanol. For Lake Manitoba fish, n = 50 fry from the 2019‐year class were sampled at Swan Creek Hatchery and stored in 95% ethanol. These fry were the offspring of wild fish from Lake Manitoba. From Cedar Bluff Reservoir, anal fin clips were taken from n = 60 sexually mature walleye . Fin clips were collected after gametes were harvested. Procedures for sample collection at Cedar Bluff Reservoir were approved by the Fort Hays State University Institutional Animal Care and Use Committee (Protocol #19‐0023). Fish were captured using seven 25‐mm‐mesh trap nets and four 76‐mm‐mesh gill nets (91.44 × 1.83 m panel per net). In total, n = 345 walleye were genotyped for the present study. See Figure Walleye anal fin clips from Lake Winnipeg individuals were collected by boat electrofishing in Spring 2017 and 2018 from spawning sites in the Red River and possible spawning sites near Matheson Island and in the Dauphin River, representing the south basin, channel, and north basin, respectively  to efficiently sequence a large number of individuals by separating RAD tag isolation and sequencing library preparation. BestRAD libraries were baited using protocols outlined in Euclide et al.  designed by Arbor Biosciences . Target loci for RAPTURE were selected based on minor allele frequency (>0.01) and alignment position along a draft walleye genome to retain one SNP approximately every 5,000 bp along every contig greater than 0.1 megabases. A total of 99,636 baits were thus selected. Read length was 150 base pairs with paired end sequencing and one capture reaction per library, where approximately 3.6 libraries were used for the present study . Bait‐captured libraries were then sequenced on a S4 NovaSeq 6000  at NovoGene .DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit  following manufacturer protocols. Restriction site associated DNA sequencing (RAD‐seq) library preparation was performed with the NEBNext Ultra DNA Library Prep Kit for Illumina  with the e et al.  using a Perca flavescens) genome with bwa v0.7.17 using bwa mem , gene diversity (HS), and inbreeding coefficients  for each population were calculated.Hierfstat v0.5–7 was used to find  Goudet, . Ninety‐NeEstimator v2.1 was used to estimate effective population size in 95% confidence intervals for each population using the linkage disequilibrium method and only comparing SNPs on different chromosomes . In EASYPOP, 1,000 simulated loci with two allelic states were used with free recombination allowed, using a KAM mutation model with mutation rate µ of 3.28x10−9 based on divergence between the blackfin icefish  and dragonfish (Parachaenichthys charcoti)  among walleye populations was analyzed via LD decay with PopLDdecay v3.40 . Significant candidate regions of selection were identified by analyzing 100 kilobase (kb) windows overlapping by 10 kb, in which at least three significant SNPs (q < 0.05) showing XP‐EHH were found. For visualization, only significant SNPs within candidate regions were highlighted (thus a significant SNP outside a candidate region would be de‐emphasized), using the R package ggman v0.99.0 with relative SNP positions to reflect distances between points in the reduced representation data (https://rdrr.io/github/veera‐dr/ggman/). Results for XP‐EHH were divided into three pairwise tests between each assigned population , and significant results for each pairwise test were further categorized into which population showed elevated XP‐EHH scores .Signatures of selection, where one genomic region has approached or reached fixation in one population compared to another, were explored with XP‐EHH using the R package rehh v3.2.1, with the markers previously described as phased and imputed after filtering for 90% present data  and measured in pairwise comparisons between each of three populations defined by Admixture. In addition, absolute allele frequency differences were analyzed between populations. VCFtools was used to calculate allele frequencies for each population. Minor alleles in Lake Winnipeg‐assigned individuals were used for comparisons across populations.Hierfstat v0.5‐7 was used to provide supporting evidence for selection at individual SNPs using 2.6r2), we calculated r2 using PLINK v1.9 for SNPs on chromosome 8 (‐‐r2 ‐‐ld‐window‐r2 0.05 ‐‐ld‐window 999999999 ‐‐ld‐window‐kb 30000) and generated a LD heatmap  was a putative inversion, we conducted the following analyses as suggested by Huang et al.  and Shi Jombart, . Third, Jombart,  but see Jombart, . Individn = 48 walleye in three sites in Lake Winnipeg, using previously published reads (NCBI SRA database accession #PRJNA596986)  values from the walleye transcriptome‐aligned data . 96,955 SNPs were called in STACKS, of which 63,882 were accepted as non‐paralogous SNPs after using HDPlot. Pruning non‐paralogous SNPs for linkage with PLINK left 46,342 SNPs for use for population structure and demographic reconstruction. The dataset of 63,882 SNPs was filtered for a maximum of 10% missing data and imputed and phased into a dataset of 18,601 SNPs for use with XP‐EHH. With the 46,342 pruned non‐paralogous SNPs and a method of moments estimation of relatedness, kinship estimates were 0.34 at the highest between any two individuals . No individuals were thus removed for relatedness. Synteny between the walleye and yellow perch genomes was high, indicating a large degree of concordance between the two genomes  Figure .3.2Admixture identified K = 2 populations based on the lowest cross validation error between K = 1 through 6, separating Canadian  and Kansas  populations of walleye   were each highest for the Cedar Bluff Reservoir population, with FIS approximately two times higher for Cedar Bluff walleye than the walleye in the northern lakes  was at approximately 2.8 kb. For Lake Manitoba, half‐decay was at approximately 4.2 kb, while moderate LD was at approximately 5.7 kb. For Cedar Bluff Reservoir, half‐decay was at about 1.9 kb, while moderate LD was at approximately 2.6 kb. And for unassigned individuals, half‐decay was at approximately 5.6 kb, while moderate LD was at about 8.0 kb. Individual pairwise r2, distance, and nonlinear regressions were visualized for each population in Figures For Lake Winnipeg, half‐decay was at approximately 2.4 kb, while moderate LD  were significant between Lake Winnipeg and Lake Manitoba Table . There wWith 63,882 SNPs accepted as non‐paralogous, two principal components (PCs) showed population structure in pcadapt, with PC1 showing 5.76% variance explained between northern and southern sites and PC2 showing 2.28% variance explained by Lake Winnipeg and Cedar Bluff Reservoir versus Lake Manitoba Figure . 345 SNPF’ST outlier SNPs , ETS homologous factor (EHF), and leucine‐rich repeat‐containing protein 4C (LRRC4C). Each of these genes had transcripts expressed in Lake Winnipeg walleye, with counts per million between 0.4 and 20 . Historic gene flow likely occurred in sporadic pulses, as we observed evidence of limited but present contemporary gene flow possibly facilitated by flooding in 2011 and 2014. These potential pulses of gene flow were consistent with population differentiation (FST) between the Canadian lakes that was lower in magnitude than each Canadian lake compared to a waterbody in Kansas, USA. Lake Winnipeg walleye showed unexpectedly strong signatures of selection on chromosome 8, with concurrent evidence for a chromosomal inversion between approximately 15.26 and 15.90 Mb. An inversion may have played a role in adaptive divergence between Lake Winnipeg and Lake Manitoba walleye  from individuals originating from each lake, based on admixture analyses  between two large lakes in Canada (Lake Winnipeg and Lake Manitoba), and comparatively stronger differentiation between the two lakes in Canada and the entirely stocked population in Cedar Bluff Reservoir, Kansas, USA of unknown origin. This relatively stronger differentiation between the Canadian lakes used here and the waterbody in Kansas was similar in magnitude to differentiation observed between walleye in Minnesota and Wisconsin (USA), differences which were attributed to Pleistocene lineages  out of 119 significant outliers (q < 0.05) along PC 2 in a pcadapt analysis are all from this small outlier region , the putative inversion was nearly fixed for the Lake Winnipeg genotype. Therefore, this putative inversion may play a role in walleye adaptation to Lake Winnipeg, but a specific adaptive role for the inversion could not be tested with the present data.PDHX, EHF, and LRRC4C. All three were expressed in Lake Winnipeg walleye, indicating at least some gene transcription activity within the putative inversion. While functional information from model species exists for each of these genes, their specific functional significance in Lake Winnipeg walleye is unknown. The specific life stage or environmental context in which the three genes within the putative inversion may have an effect on walleye biology is similarly unknown. Moreover, genes adjacent to the inversion may have functional consequences, and possibly in another group of walleye instead . That this putative inversion may have played a role in recent divergence between fish of two connected lakes, and that its frequency among likely admixed fish is consistent with a possible adaptive role in Lake Winnipeg walleye, indicates a potential importance for chromosomal inversions in freshwater fishes more generally. Inversions have been previously related to environmental adaptation, such as in freshwater emerald shiner (Notropis atherinoides) with high gene flow ; Data curation ; Formal analysis ; Investigation ; Methodology ; Visualization ; Writing – original draft (lead). Peter T. Euclide: Data curation ; Formal analysis ; Investigation ; Methodology ; Resources (supporting); Software (supporting); Validation ; Writing – review & editing . Jennifer D. Jeffrey: Conceptualization ; Formal analysis (supporting); Investigation ; Methodology ; Writing – review & editing . Yue Shi: Investigation ; Methodology ; Writing – review & editing . Jason R. Treberg: Conceptualization ; Funding acquisition ; Investigation ; Project administration ; Resources ; Supervision ; Writing – review & editing . Douglas A. Watkinson: Conceptualization ; Data curation ; Investigation ; Resources ; Supervision ; Writing – review & editing . Eva C. Enders: Conceptualization ; Data curation ; Funding acquisition ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Writing – review & editing . Wesley A. Larson: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Yasuhiro Kobayashi: Conceptualization ; Investigation ; Methodology ; Supervision ; Writing – review & editing . Ken M Jeffries: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration (lead); Resources ; Supervision (lead); Validation ; Visualization ; Writing – review & editing .https://doi.org/10.6084/m9.figshare.19610082.This article has been awarded Open Data, Open Materials Badges. All materials and data are publicly accessible via the Open Science Framework at Supplementary MaterialClick here for additional data file.


Simple and multiple linear regression were used to evaluate the correlation between tumour volume and HRQoL, controlling for potential confounders. No correlation was found between total somatostatin receptor expressing tumour volume and QLQ‐C30 summary score. Weak positive correlations were found between total tumour volume and the specific symptoms dyspnoea, diarrhoea and flushing. To the best of our knowledge, this is the first study to evaluate the association between total somatostatin expressing tumour volume and HRQoL. Our results indicate that, while tumour volume is weakly associated with symptom severity of the carcinoid syndrome, other factors might impact more on overall HRQoL.For patients with gastroenteropancreatic neuroendocrine tumours (GEP‐NET), health‐related quality of life (HRQoL) is important. Meanwhile, whether tumour volume is associated with HRQoL is unknown. Hence, the aim of this study was to assess if total somatostatin receptor expressing tumour volume is correlated with HRQoL in patients with metastatic GEP‐NET. Some 71 patients were included in the study. HRQoL and NET‐specific symptoms were assessed with EORTC QLQ‐C30 and EORTC GI.NET21. A summary score was calculated from the output of the QLQ‐C30. Total somatostatin receptor expressing tumour volume was retrospectively evaluated on somatostatin receptor imaging with positron emission tomography‐computed tomography ( Nevertheless, despite an occasional high tumour burden, 5‐year survival often exceeds 50%.While debulking surgery can be used to control symptoms of NET,68Ga‐DOTA‐TATE PET‐CT has been shown to have excellent sensitivity and specificity.68Ga‐DOTA‐TATE PET‐CT.While standard cross‐sectional imaging is helpful to depict tumour morphology, it does not convey information about expression of somatostatin receptors. For this purpose, in order to adequately assess patients with suspected NET, In summary, whether tumour volume predicts HRQoL in patients with GEP‐NET is unknown. If tumour volume is associated with HRQoL, decreasing tumour volume might improve HRQoL.2We hypothesised that, possibly through mechanisms of increased hormone levels, increased systemic metabolic demands and increased likelihood of gastrointestinal (GI) tract obstruction, increased tumour volume was associated with more symptoms and lower HRQoL. Therefore, our primary aim was to test whether total somatostatin expressing tumour volume, defined either as ∑SRETV or ∑TLSRE measured on PET‐CT images, was correlated with QLQ‐C30 summary scores in patients with metastatic GEP‐NET.To explore possible causative pathways of this relationship, our secondary aim was to test whether local tumour volume, that is, total SRETV within each anatomical site, was correlated with specific function scales or specific symptoms as defined by QLQ‐C30 complemented by GI.NET21.33.168Ga‐DOTA‐TATE/TOC PET‐CT within 1 year before or after the date of answering the questionnaires, and tumour‐modulating treatment  between 68Ga‐DOTA‐TATE/TOC PET‐CT and answering the questionnaires. All patients who gave their written consent and met none of the exclusion criteria were included in the current study. The study was approved by the Swedish Ethical Review Authority (DNR 2019–02378) and undertaken in accordance with the Helsinki Declaration.The patients included in this study were a subgroup of those included in a larger previous study of HRQoL in GEP‐NET. This previous study involved all patients alive on 1 September 2019 in the southern hospital region of Sweden, and whose histopathological diagnosis of well‐differentiated (G1‐G2) GEP‐NET had been established between 1 January 2000 and 31 December 2018. Details of the larger study have been published previously.3.2The following information was gathered from each patient's electronic medical record: age, height, weight, gender, date of diagnosis, primary tumour site, tumour grade and Ki67 at first histopathology, stage, most recent urine 5‐HIAA measurement, most recent chromogranin A, presence of SSA treatment, and previous tumour‐modulating therapy . Other comorbidities were also recorded, and based on these a Charlson comorbidity index (CMI) was calculated for each patient in accordance with methods described elsewhere.3.3The patients' HRQoL was evaluated with the cancer‐specific, generic questionnaire EORTC QLQ‐C30,To avoid type 1 errors due to multiple testing of different subscales, a QLQ‐C30 summary score was calculated according to the instructions of its authors.While validated and well‐used, the QLQ‐C30 does not cover the specific symptoms of NET. Therefore, a NET‐specific complementary instrument, the EORTC GI.NET21,3.468Ga‐DOTA‐TATE was used for somatostatin receptor imaging until 2019. During 2019 there was a shift in production to 68Ga‐DOTA‐TOC at Skåne University Hospital. Both 68Ga‐DOTA‐TATE and 68Ga‐DOTA‐TOC were prepared according to established techniques.The scans were performed using a Discovery MI or Discovery D690  PET‐CT system. 3.568Ga‐DOTA‐TATE/TOC within the timeframe allowed by the inclusion criteria, the most recent scan was chosen in terms of the date the questionnaires were answered. Images were analysed retrospectively by AG and ET. Semi‐automatic segmentation of tumours was performed using the software Hermes . SRETV was defined as tumour volume  with uptake higher than 50% of maximum standard uptake value (SUVmax) in a volume of interest (VOI). TLSRE was defined as the product of SRETV and mean SUV (SUVmean) per lesion. Pathological uptake of 68Ga‐DOTA‐TATE/TOC was considered significant for tumour segmentation if SUVmax was >3 and did not correspond to physiological uptake. A relatively high normal background uptake in the liver meant that manually drawn VOIs were often needed to avoid physiological uptake, as previously described.If a patient had had more than one 3.6liver) and ∑SRETV was calculated for each patient.Continuous variables involving EORTC QLQ‐C30 and GI.NET scores are presented using descriptive statistics , while categorical variables are presented as frequencies and percentages. As it has been suggested that a score below 50 for QoL/function scales or above 50 for symptom scales denotes significantly impaired HRQoL, the number of patients with scores above or below 50, respectively, was counted for each function/symptom scale. To describe tumour distribution in the cohort, median SRETV was calculated for all patients with a tumour in each respective anatomical site. Since the SRETV in the liver was disproportionately higher than in the other anatomical sites, and in order to quantify the distribution of tumour volume in each patient further, a ratio between SRETV in the liver  tumour directed treatment. The same procedure was then performed with log∑TLSRE as the independent variable.Since the distributions of ∑SRETV and ∑TLSRE were found to be highly skewed , they were transformed to their natural logarithms, log∑SRETV and log∑TLSRE. Simple linear regression was then performed between the dependent variable QLQ‐C30 summary score and log∑SRETV. To control for possible confounders, we performed multiple linear regression between QLQ‐C30 and log∑SRETV with adjustment for age, Charlson CMI and SSA treatment. To explore whether previous major surgery using Whipple's procedure would interfere with the results, a sensitivity analysis was made which excluded these patients. Another sensitivity analysis was carried out which excluded all patients with r > 0.2 was chosen to separate weak correlation from no correlation.To analyse the secondary aim, a correlation table was made with Pearson's correlation coefficients between logSRETV in each anatomical site and each function scale/symptom scale from EORTC QLQ‐C30 and GI.NET21. As suggested in previous literature,4Between 1 January 2000 and 31 December 2018, 806 unique patients received a histopathological diagnosis of GEP‐NET in the Skåne healthcare region. Of these, 561 patients were excluded from the analysis by one exclusion criterion or more, as follows: where NET had been found incidentally during the resection of another cancer, or if there was synchronous inoperable colorectal cancer, synchronous inflammatory bowel disease or a localised tumour where endoscopic excision or appendectomy had sufficed as treatment. Between 2 September 2019 and 27 September 2019, the remaining 245 patients were invited by normal post to participate in the study. Of these, three declined to participate, and 10 had to be excluded as they had emigrated or their address was unknown. A further 67 patients did not respond. The remaining 165 patients returned the completed instruments and signed consent forms, resulting in a response rate of 67%. For the present study, a further 94 patients were excluded: some 92 due to non‐metastatic disease, one due to chemotherapy between scan and questionnaire and one subsequently declining to participate. The final cohort included 71 patients.4.1n = 24), disease‐related worries (n = 25) and muscle/bone pain (n = 16). Some 61 patients (86%) had small intestine as their primary tumour site. Of the 71 patients in the cohort, some 42 patients had undergone small bowel resection, and 23 patients right‐sided hemicolectomy or ileocaecal resection.Patient characteristics are displayed in Tables 4.268Ga‐DOTA‐TATE/TOC. Of these, the median SRETV was 18.8 ml. Meanwhile, there was considerable variability of SRETV in the liver, with an IQR of 10.6–112.7 ml. In comparison, median SRETV in patients with evidence of NET in the mesenteric lymph nodes or NET in the GI tract was only 1.6 and 1.4 ml, respectively. For the 40 patients with liver metastases, the median ratio of SRETVliver/∑SRETV was 0.93 (IQR 0.66–0.99). This suggests that for a significant number of patients in the cohort, tumour volume in the liver made up a substantial part of total tumour volume. For details, see Table Median ∑SRETV was 9.6 (interquartile range [IQR] 1.7–45.4) ml. The maximum value was 539 ml, indicating highly skewed data. Some 40 patients (56%) had visible liver metastases of NET on 4.3p = .88). Multiple linear regression with adjustment for age, Charlson CMI and treatment with SSA showed a similarly nonsignificant result, with a beta‐coefficient of 0.39 . Sensitivity analyses which excluded the six patients who had undergone pancreaticoduodenectomy, or the 15 patients with more than 6 months between answering the questionnaire and undergoing 68Ga‐DOTA‐TATE/TOC, did not change these results. Subgroup analysis of patients according to grade and recent diagnosis showed the same results  and muscle/bone pain (MBP), were correlated with increased SRETV in different anatomical sites: DI was most correlated with SRETV in the GI tract (r = 0.31), SRETV in the liver (r = 0.28), SRETV in other lymph nodes (r = 0.20) and SRETV at other unspecified sites (r = 0.25). Conversely, MBP was only weakly correlated with SRETV in the mesenteric lymph nodes (r = 0.21). Disease‐related worries (DRW) were not correlated with SRETV in any anatomical site. Total tumour volume, ∑SRETV, showed weak positive correlation with dyspnoea (r = 0.21), diarrhoea (r = 0.23), endocrine dysfunction (r = 0.33) and information (r = 0.27), and weak negative correlation with weight gain (−0.27).Table 568Ga‐DOTA‐TATE/TOC in patients with metastatic GEP‐NET, measured as either ∑SRETV or ∑TLSRE, was associated with HRQoL. Linear regression, with both a simple unadjusted model and a multiple adjusted model, indicated no correlation between either ∑SRETV or ∑TLSRE and EORTC QLQ‐C30 summary scores. Subgroup analysis and repeated sensitivity analysis which excluded possible confounders did not alter these results. However, results from the secondary analysis could indicate a correlation between ∑SRETV and the specific symptoms of diarrhoea, flushing and dyspnoea. To our knowledge, these findings have not previously been reported.In the present study, we could not confirm that somatostatin expressing tumour volume on It might seem intuitive that increased tumour volume would lead to a proportionate decrease in HRQoL through the causal pathway where tumour volume causes symptoms, which causes decreased HRQoL. Our findings contradict this notion. A potential explanation may be that the disease in most patients in our cohort was stable, and most symptoms were not new or worsening. Adaptation level theoryliver and some symptoms of carcinoid syndrome , might indicate that although tumour volume does not affect HRQoL in a general sense, it still affects the extent of symptom severity. Since total SRETV in the majority of patients in this study consists mostly of liver metastases, this is consistent with the general biological behaviour of functioning NETs, which require metastasis to the liver for symptoms of carcinoid syndrome to develop. It is also consistent with the findings of Tirosh et al., which indicate a positive correlation between tumour volume assessed by 68Ga‐DOTA‐TATE and 24‐h urine 5‐hydroxyindoleacetic acid (U 5‐HIAA) in patients with small‐intestinal NET .HRQoL is a multifaceted concept which consists of patient‐reported somatic, psychological and social aspects.The findings of Vinik et al.,One strength of our study is its population‐based design, which should minimise selection bias. Equally, compared to previous studies measuring tumour burden, the method of measuring SRETV and TLSRE described here is more detailed and, in terms of total metabolic demand and total hormone‐producing ability, biologically more coherent.18F‐FDG PET‐CT, where tumours with necrotic cores and lesions with low uptake relative to the background could be under‐ or overestimated.68Ga‐DOTA‐TATE/TOC might also have a modest impact on an individual basis. Nevertheless, primarily because all tumours were grade 1 or grade 2 and only one patient had Ki67 above 15%, we do not judge these limitations to be significant enough to affect the overall outcome of the study. Other limitations include the cross‐sectional, retrospective design, the small sample size and the fact that participation in the study was voluntary. The latter could have led to a problem of nonrandom missing data due to selection bias, by including more (or fewer) healthy patients than the general population of patients with GEP‐NET. Since medical records were inaccessible for the patients who were not included in the study, there was no opportunity to investigate whether this had been an issue in the present cohort.Meanwhile, some limitations should be noted in terms of measurements of tumour volume. At the present moment, no validated method is available for measuring SRETV, but it can be measured in a number of ways. We chose a method that has been described before, and which has also been shown to have potential prognostic value.In summary, although a weak correlation was noted between some symptoms of carcinoid syndrome and tumour volume, this study showed no correlation between overall HRQoL and total tumour volume in patients with metastatic GEP‐NET. Our results could point to the importance of coping strategies in patients with incurable malignant disease, and may provide hypotheses for further research in this area.The senior author has received research grants from IPSEN, and consultancy fees from Medtronic, outside this study. AS has received speaker and consultancy fees from IPSEN, Novartis, Sam Nordic and Spago Nanomedical, none of which are related to this study. The other authors declare no conflicts of interest.Håkan Ohlsson: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; validation; visualization; writing – original draft; writing – review and editing. Anni Gålne: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; validation; writing – original draft; writing – review and editing. Elin Trägårdh: Conceptualization; data curation; funding acquisition; investigation; methodology; resources; software; supervision; writing – review and editing. Marlene Malmström: Conceptualization; methodology; supervision; validation; writing – review and editing. Anna Sundlöv: Conceptualization; methodology; supervision; validation; writing – review and editing. Martin Almquist: Conceptualization; formal analysis; funding acquisition; methodology; resources; software; supervision; writing – review and editing.https://publons.com/publon/10.1111/jne.13139.The peer review history for this article is available at Table S1. Supplement.Click here for additional data file.


Solanum stoloniferum Schltdl.) accessions and cultivated potato (Solanum tuberosum) to aphid (Myzus persicae Sulzer) herbivory.Plant resistance to insects can be reduced by crop domestication which means their wild ancestors could provide novel sources of resistance. Thus, crossing wild ancestors with domesticated crops can potentially enhance their resistance against insects. However, a prerequisite for this is identification of sources of resistance. Here, we investigated the response of three wild potato (Diaeretiella rapae (M'Intosh) was significantly attracted to volatiles from CGN18333. Volatile analysis showed that wild accessions emitted significantly more volatiles compared to cultivated potato. Principal component analysis (PCA) of volatile data revealed that the volatile profiles of wild and cultivated potato are dissimilar. β‐Bisabolene, (E)‐β‐farnesene, trans‐α‐bergamotene, d‐limonene, ‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT), and p‐cymen‐7‐ol were the main volatiles contributing to the emitted blends, suggesting possible involvement in the behavioural response of both M. persicae and D. rapae.Results revealed that there was a significant reduction in aphid survival and reproduction on wild potato accessions  compared to cultivated (Desiree) potato plants. A similar trend was observed in olfactometer bioassay; the wild accessions had a repellent effect on adult aphids. In contrast, among the tested wild potato accessions, the parasitoid Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Our findings show that the tested wild accessions have the potential to be used to breed aphid‐resistant potatoes. This opens new opportunities to reduce the aphid damage and to enhance the recruitment of natural enemies. © 2022 The Authors.  Our study found evidence of aphid resistance in wild potato accessions. Aphid performance was significantly lower on wild potato and their volatiles were repellent to aphid and attractive to parasitoids. To this end, we hypothesized that wild potato accessions better respond to aphid herbivory than commercial potato cultivars. To test this hypothesis, we performed growth assays using peach potato aphids, Myzus persicae (Sulzer), as a model herbivore, observing their reproduction and survival on wild and cultivated potato. Myzus persicae is one of the most deleterious polyphagous aphids that can damage plants directly through feeding and honeydew deposits, or indirectly by transmitting plant viruses.M. persicae and its common endoparasitoid [Diaeretiella rapae (M'Intosh)] was investigated in an olfactometer bioassay. To correlate insect behavioural response with the emitted VOC profiles, volatile entrainment was conducted and the collected VOCs from wild and cultivated potatoes were analysed and compared.In the present study, we aimed to identify the insect resistance level of wild potato  used in experiments were obtained from the Wageningen Centre for Genetic Resources (CGN), Wageningen University, The Netherlands, while S. tuberosum Desirée tubers  were purchased from Sainsbury's supermarket, UK. All plants were grown under controlled environment conditions  in a growth chamber . Potato plants were grown individually in 7.5 cm pots in John Innes No. 2 compost . Four‐five week old plants were used for the experiments.Wild potato, 2.2Myzus persicae aphids, originally obtained from Harper Adams University , were reared in the insectary in the Centre of Applied Entomology and Parasitology (CAEP) at Keele University, UK. Myzus persicae clone O was reared on Pak choi Brassica chinensis, commonly known as Chinese cabbage, in Bugdorm cages  under controlled conditions . The aphid parasitoid D. rapae (obtained from Harper Adams University) was reared on Pak choi plants infested with M. persicae. To rear parasitoids, mummies of D. rapae, attached to plant leaves, were introduced to cages containing fresh Pak choi plants infested with M. persicae and kept under controlled condition . Upon emergence, parasitoid adults were provided with honey solution (1:1 in water) as food. Only female parasitoids were used in experiments and they were 2–3 day old and mated.2.3M. persicae was assessed on the wild and cultivated potato species. There were two separate series of experiments with different plants; the first series recorded observations after 48 h and the second recorded observations after 96 h. Fresh plants and aphids were used in both observations in each experiment. In each replicate, ten adult alate M. persicae were placed in a clip cage , which was attached to the lower surface of plant leaves as described in Sobhy et al.Performance of 2.4et al.,Plants volatiles were collected following the procedure described by Ali 2.5M. persicae and D. rapae to potato VOCs was investigated using a perspex four‐arm olfactometer in a controlled environment room  as described in Ali et al.M. persicae or female D. rapae was introduced, which was connected to a low‐pressure air pump. Air was pulled from the centre of the olfactometer by a vacuum pump with a layer of muslin to prevent access by the tested insect during the bioassays. All replicates were carried out under uniform illumination. The olfactometer arena was split into five areas; four areas by each arm (one VOC treatment and three solvent control arms) and a central area.The behavioural response of alate 2.6−1. Automated injections of 1 μL were made using a G4513A autosampler (Agilent Technologies) in splitless mode (285 °C), with oven temperature programmed from 35 °C for 5 min then at 10°C min−1 to 285 °C. Compounds were identified according to their mass spectrum, linear retention index relative to retention times of n‐alkanes, and co‐chromatography with authentic compounds.Volatile analysis was carried out on a 7820A GC coupled to a 5977B single quad mass selective detector . The gas chromatograph was fitted with a non‐polar HP5‐MS capillary column (30 m × 0.25 mm × 0.25 μm film thickness) coated with (5%‐phenyl)‐methylpolysiloxane (Agilent Technologies) and used hydrogen carrier gas at a constant flow rate of 1.2 mL min2.72.7.1P < 0.05).Differences in the mean number of live adult aphids and produced nymphs on wild  and cultivated (Desiree) potato plants were compared at two time‐points (48 and 96 h) by one‐way analysis of variance (ANOVA). Prior to analysis, data were examined for a normal distribution using the Shapiro–Wilk test. Comparisons among means were performed using Holm–Sidak method . In this analysis, the time spent by the tested individuals in treated and the average of three control arms in the four‐arm olfactometer were compared.Data on the behavioural response of 2.7.3F‐test) of variances was performed to investigate whether the concentrations of individual volatile compounds differed between wild and cultivated potato plants. All univariate analyses were performed using SigmaPlot 12.3 .To visualize the overall differences in volatile profiles emitted from wild  and cultivated (Desiree) potato plants, a principal component analysis (PCA) was performed using the concentrations of the detected volatiles as dependent variables. Loading and score plots were derived after mean‐centring and log transformation of volatile data. VOC visualization was done using the MetaboAnalyst online tool suite.33.1M. persicae surviving on wild potato accessions , particularly on CGN18333 and CGN22718. The same was also true after 96 h .There was a significant reduction in nymph production on wild potato accessions across both time points compared to cultivated potato . In particular, the accessions CGN18333 and CGN23072 had a significant repellent effect on M. persicae with P values of 0.013 and 0.018, respectively .In contrast to oid Fig. . Accessi3.3F3,12 = 61.20; P < 0.001; Fig. Gas chromatography–mass spectrometry (GC–MS) analysis of headspace collections from wild  and cultivated (Desiree) potato plants revealed 23 detectable VOCs in seven functional classes , ‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT) (0.272), and p‐cymen‐7‐ol (0.255), whereas the major loadings of PC1 were for β‐bisabolene (0.293), (E)‐β‐farnesene (0.288), and trans‐α‐bergamotene (0.285). This suggests that these VOCs, shown to contribute to PC1 and PC2, may impact the behaviour response of both M. persicae and D. rapae.PCA of the VOCs showed that the first two principal components (PCs) accounted for 54.3% of the total variation in the volatile data . Of these previously reported species, S. palustre was the most resistant to M. persicae as it contained the highest number of glandular hairs, which is one of the aphid resistance factors in Solanum.Macrosiphum euphorbiae Thomas) on wild Solanum berthaulti.There was a significant difference in the susceptibility of wild and cultivated species of S. berthaultii was very repellent to M. persicaeE)‐β‐farnesene, from trichomes. In our study a mixture of volatiles was released by S. stoloniferum. Conversely, the olfactometer studies showed that wild accessions also help in recruiting natural enemies as the parasitoid D. rapae spent more time in arm treated with volatiles from wild plants of accession CGN18333. In contrast, parasitoids spent significantly less time on Desiree plants. Chemical analyses revealed that wild accessions released higher amounts of volatile compounds compared to cultivated potatoes. Consistent with our findings, it has been shown that commercial cultivars have lost the ability to produce certain key volatiles in response to herbivory, which thereby negatively affected natural enemy recruitment.In olfactometer experiments, aphids were repelled by the odours of accessions CGN18333 and CGN23072. Similarly, the wild potato E)‐β‐farnesene, trans‐α‐bergamotene, d‐limonene, TMTT, and p‐cymen‐7‐ol were produced in larger amounts by the wild potato accessions, and it is likely that these compounds played a role in repelling aphids and attracting their natural enemies. Previous work has shown an increased emission of β‐bisabolene,E)‐β‐farnesene,trans‐α‐bergamotene,d‐limonene,p‐cymen‐7‐olM. persicae when tested individually.E)‐β‐farnesene functions as an alarm pheromone and also serves as a host finding kairomone for aphid natural enemies.Multivariate analysis of volatile data revealed that β‐bisabolene, (Previous studies reported that plant secondary metabolites could provide a way to enhance plant resistance5S. stoloniferum accessions tested. The very low aphid survival observed suggests that toxic phytochemicals were present. Although the potential of crop wild relatives as sources of novel resistance to insect pests has been extensively studied, the progress of transferring resistance traits from wild cultivars is still slow. This is attributed to difficulties in identifying the key secondary metabolites that determine resistance and the genes encoding their production. Thus, identification of the bioactive compounds and genes encoding resistance will be important topics for future studies. To retain the marketable quality required, it will be also important to test if bioactive compounds are harmful to humans or if they affect the taste of the potatoes. Morphological differences could be seen between the Solanum species which were used in experiments; wild accessions had smaller leaves compared to Desiree. Although there could be some relation between aphids and leaf size it is unlikely to explain the high mortality observed in the current study which is more likely due the presence of toxic phytochemicals. The current research findings open up the prospect of breeding for aphid resistance by crossing cultivated and wild potatoes.The current study shows that there are promising sources of direct aphid resistance in the JA, ISS and TJAB conceived the ideas and designed the experiments. JA performed the experiments and collected the data. ISS performed the GC chemical analysis and interpreted the volatile data. JA, ISS and TJAB wrote the manuscript. All authors contributed critically to the drafts and gave final approval for publication.The authors declare that they have no conflict of interest.


The development of novel devices and the favorable results of several randomized clinical trials have allowed for the rapid expansion of transcatheter aortic valve implantation (TAVI) to elderly patients with aortic stenosis across all risk categoriesInternational Classification of Diseases, Tenth Revision, Clinical Modification (ICD‐10‐CM) codes: 02RF37H, 02RF37Z, 02RF38H, 02RF38Z, 02RF3JH, 02RF3JZ, 02RF3KH, and 02RF3KZ. Patients with age ≤18 years (n=22), cirrhosis (n=760), end‐stage renal disease (n=2136), do‐not‐resuscitate status or palliative care involvement (n=383), and cancer (n=1952) were excluded with reference to previous trials. Patients with missing data (n=12) were also excluded. Research participation status was identified using ICD‐10‐CM code Z00.6,The data that support the findings of this study are available from the corresponding author upon reasonable request. The Nationwide Inpatient Sample is the largest publicly available all‐payer inpatient health care database in the United States and did not require ethical approval. All patients who underwent TAVI between 2013 and 2019 (n=56 648) were identified from the Nationwide Inpatient Sample using the following The primary outcome in this study was in‐hospital mortality. A multilevel logistic regression analysis accounting for strata and hospital clustering was performed to examine the association between clinical research participation status and in‐hospital mortality. A sensitivity analysis was performed in the propensity score‐matched cohort, which was generated by 1:1 nearest‐neighbor matching using a caliper width of 0.01 adjusting for baseline characteristics. Variables for these analyses were chosen on the basis of clinical relevance and the previous workBetween 2013 and 2019, 51 383 patients undergoing TAVI met the inclusion criteria: 14 311 (28%) research participants and 37 072 (72%) nonparticipants. Baseline patient characteristics are shown in P<0.001). Regarding TAVI‐related complications, permanent pacemaker implantation, cardiac arrest, cardiogenic shock, acute kidney injury, respiratory failure, vasopressor requirement, and requiring mechanical circulatory support were less frequent in research participants than in nonparticipants. Furthermore, research participants had higher total inflation adjusted costs than nonparticipants.Crude in‐hospital mortality after TAVI was lower in research participants than in nonparticipants . In nonparticipants, crude in‐hospital mortality after TAVI also decreased from 3.6% in 2013 to 1.0% in 2019 (Cochran‐Armitage trend P<0.001).Crude in‐hospital mortality after TAVI in research participants decreased from 4.4% in 2013 to 0.6% in 2019 (Cochran‐Armitage trend P<0.001). The propensity score‐matched cohort gave similar results in terms of in‐hospital outcomes (After adjustment for baseline characteristics, adjusted odds ratio of in‐hospital death was significantly lower in research participants than nonparticipants (odds ratio, 0.72; 95% CI, 0.60–0.88; outcomes .There are several possible explanations for the significant relationship between research participation status and in‐hospital mortality after TAVI in this study. First, physicians or hospitals participating in clinical research may have high‐level experience and provide cutting‐edge care. Previous studies demonstrated an inverse volume‐outcome relationship of TAVI.Several limitations should be acknowledged. First, details on individual trials were lacking, such as inclusion and exclusion criteria and the reason for research participation. The interest of researchers may not be limited to TAVI. Second, the Nationwide Inpatient Sample database did not contain information regarding echocardiographic parameters, the Society of Thoracic Surgeons risk scores, frailty scores, and medication use. The impact of research participation status on outcomes should be further evaluated in patients with similar risk scores and frailty.In conclusion, although our findings are subject to unmeasured confounders and selection bias for patients with favorable characteristics,None.None.


Helicobacter pylori eradication. We explored whether this was an appropriate first‐line treatment.To date, no interventional trial has assessed the efficacy and safety of vonoprazan and high‐dose  amoxicillin dual therapy in terms of H. pylori‐positive patients lacking any eradication history were treated with vonoprazan 20 mg twice daily and amoxicillin 500 mg four times daily (qid) for 7 days. Eradication was evaluated using a stool H. pylori antigen test. We evaluated safety using patient questionnaires. This study was registered in the jRCT database (jRCT031200128).This prospective, dual‐center, single‐arm interventional study was performed in Japan. Twenty n = 20) and 94.4%  respectively. No significant adverse event was recorded.The intention‐to‐treat and per‐protocol eradication rates were 90%  amoxicillin dual therapy. No significant adverse event was recorded. Helicobacter pylori (H. pylori) infections induce gastric cancer via several mechanisms,H. pylori in our country is the triple regimen with VPZ 20 mg bid, amoxicillin (AMPC) 750 mg bid, and clarithromycin (CAM) 200 or 400 mg bid for 7 days (VAC regimen). We also showed the VAC‐regimen was superior to 7‐day proton pump inhibitor (PPI)‐based triple therapy (with AMPC and CAM) in terms of eradicating CAM‐resistant H. pylori.H. pylori afforded by the VAC‐regimen was 82.9% ,et al. retrospectively compared VPZ and AMPC dual therapy  and a first‐line VAC‐regimen; the eradication rates were 92.9% (95% CI 82.7–98.0%) and 91.9% (95% CI 80.4–97.0%) (p = 0.728), respectively and the eradication rate by the dual therapy was not inferior to that of the triple therapy.et al. conducted a multicenter randomized trial comparing VPZ and low‐dose AMPC dual therapy ;  to a first‐line VAC therapy and VA‐dual was non‐inferior to VAC‐triple in the per‐protocol (PP) analysis, but not in intention‐to‐treat (ITT) analysis.Vonoprazan (VPZ) is the first clinically available potassium competitive acid blocker and can be the more potent gastric acid inhibition in comparison with PPIs. Recent most popular standard regimen for eradication of H. pylori eradication. We explored whether it was appropriate to proceed to an RCT comparing the new dual therapy and the VAC‐regimen.As a result of considering a therapy to improve the eradication rate by modifying the dual therapy, we thought that the modification of the AMPC dose from 750 mg bid to 500 mg qid (4 times daily) is reasonable because the effects of AMPC depend on the percentage time for which the minimum inhibitory concentration is exceeded (%Time > MIC)https://jrct.niph.go.jp/latest‐detail/jRCTs031200128) in September 2020.This was an open‐label, dual‐center, single‐arm interventional study of high‐dose AMPC dual therapy (with VPZ). After this assessment, we plan an RCT comparing this VPZ dose to high‐dose AMPC dual therapy and 7‐day triple therapy with VPZ, AMPC, and CAM. This study was conducted in accordance with the Clinical Trials Act of Japan and the Declaration of Helsinki. The study was conducted by the Yokohama City University Hospital and Yokohama Minami Kyousai Hospital. The study was reviewed and approved by the Yokohama City University Certified Institutional Review Board and was registered with the Japan Registry of Clinical Trials (jRCTs)  as jRCTs031200128 , rapid urease test (biopsy sample), or histological diagnosis (biopsy sample) as H. pylori infection. This definition is in accordance with the guidelines for H. pylori in Japan, and clinically these diagnostic methods are generally used in Japan.H. pylori antibody test, which was reported with sufficient sensitivity and specificity for diagnosis. The anti‐H. pylori test was performed by an external agency. Endoscopy was performed within 1 year in all patients. Patients who suspected H. pylori infection by endoscopy, including gastritis, was conducted the above‐mentioned H. pylori infection diagnosis according to national insurance‐covered medicine regulation procedure.We included male and female adults (aged >20 years) with The exclusion criteria were an allergy to VPZ or AMPC; lactation or pregnancy; infectious mononucleosis; the use of atazanavir, rilpivirine, pimozide, ergotamine, suvorexant, lomitapide mesylate, tadalafil, ticagrelor, ibrutinib, asunaprevir, ivavradine, or venetoclax; the use of colchicine to treat liver or renal dysfunction; severe dysfunction of the liver, kidneys, or heart; and disqualification by study physicians. Written informed consent was obtained from all patients.Patients received high‐dose AMPC dual therapy with VPZ: VPZ 20 mg twice daily (bid) and AMPC 500 mg four times daily (qid) for 7 days. The use of other antibiotics or PPIs was prohibited. The primary eradication rate was the (objective) outcome; the study was open‐labeled.H. pylori susceptibilities were assessed by culture and agar dilution of endoscopic gastric biopsy samples as described previously.H. pylori eradication rate determined using the Full Analysis Set (FAS); stool antigen tests were performed at least 8 weeks after the end of the therapy. The FAS was defined as all patients who commenced eradication therapy except those who did not meet the study criteria. Patients lost to follow‐up or who did not undergo H. pylori stool antigen tests were regarded as treatment failures. The secondary endpoints were the eradication success rate determined using the per‐protocol set (PPS), and safety. All patients were told to stop VPZ, PPIs, and histamine‐2 blockers for at least 2 weeks before the H. pylori stool antigen test. H. pylori stool antigen test was performed by the hospital's internal inspection units as components of regular medical care.We checked the efficacy and safety of the regimen to assess whether to continue with an RCT. The primary endpoint was the first‐line Safety was physician‐evaluated using the Common Terminology Criteria for Adverse Event ver. 5.0 (CTCAE). Also, patients completed adverse effect questionnaires (AEQs) that we have used previously.We chose a sample size of 20. If the upper 95% CI was <80%, we would not proceed to an RCT. This corresponds to fewer than 12 successes. The 80% was set based on unacceptable eradication and a clinically meaningful 10% decrease from 90%, which is the eradication rate of the standard regimen.We calculated eradication rates with 95% CIs. We assessed whether the upper 95% CI was <80%. We calculated CTCAE and AEQ scores. All statistical analyses employed SPSS ver. 28. All authors reviewed the data and approved the final manuscript.H. pylori infections without any eradication history received high‐dose AMPC dual therapy with VPZ. Two patients were excluded from PP analysis. One was lost to follow‐up after the completion of eradication therapy. The other was excluded because of a delayed stool antigen test (which indicated successful eradication).We registered the study in September 2020 and enrolled patients from November 2020. The last follow‐up date was September 2021. Twenty patients with H. pylori culture, and anti‐H. pylori antibody (serum), or rapid urease test. All patients did not have comorbidities generally treated with antibiotics or antibiotic use at the recruitment. In six cases , AMPC‐, CAM‐, metronidazole (MNZ)‐, and STFX‐resistance data were collected prior to eradication. The AMPC resistance rate was 0% (0/6), the CAM rate was 33.3% (2/6), the MNZ rate was 0% (0/6), and the STFX rate was 50% (3/6).The baseline characteristics of all patients are summarized in Table n = 20) in the FAS population and 94.4%  in the PPS population. The upper 95% CIs exceeded 80%; we will now proceed to an RCT comparing the VA‐dual and VAC‐regimens.The eradication rates were 90% . All adverse events were mild (CTCAE grade 1). The 11 adverse events were 15% abdominal fullness, 15% nausea, 10% abdominal pain, 5% urticaria, 5% diarrhea, 5% constipation, 5% anorexia, 5% vomiting, and 5% fatigue. All adverse events resolved spontaneously. No patient was hospitalized. The AUQ scores are shown in Table H. pylori eradication. The eradication rates were good (90–95%) in both the FAS and PPS populations. Only CTCAE grade 1 adverse events were noted; these disappeared after the completion of eradication therapy.To the best of our knowledge, this is the first interventional study to assess the efficacy and safety of VPZ 20 mg bid and AMPC 500 mg qid 7‐day dual therapy in terms of first‐line et al. reported a systematic review with meta‐analysis of high‐dose PPI‐amoxicillin dual therapy, and AMPC tid had an unacceptable eradication rate of 73%, whereas AMPC qid showed a more effective eradication rate of 87%.In terms of AMPC dose and frequency, as shown in Table We think PPI‐based dual therapy regimens with 500 mg tid or 1000 mg bid AMPC should not replace triple therapy regimens, because, randomized trials have revealed the superiority of triple, as compared to dual therapies.H. pylori cannot survive for 1 week on the agar plates containing AMPC at pH 7.In terms of the regimen period, we chose 7 days based on Furuta and Suzuki's results. About the reason for 7 days settlement, VPZ enables the intragastric pH to reach around 7 within 3‐4 h and usually, AMPC‐sensitive In terms of study design, the Furuta work was retrospectiveIn terms of antibiotics resistance results, we detected no AMPC resistance in this study Table . The AMPH. pylori can enter a growth phase, and become more susceptible to AMPC.H. pylori.Regarding the mechanism by which the regimen in this study is effective, we believe that both adequate acid blockade and pharmacologically correct AMPC dosing are key points. VPZ inhibits H+/K+ ATPases rapidly, strongly, and stably compared to PPIs. The intragastric pH increased to over pH 4.0 within 4 hH. pylori. Our results are meaningful in regions or countries (including Japan) with very low AMPC‐resistance.Our study had certain limitations. First, this was a single‐arm interventional study exploring whether to continue to a non‐inferiority RCT comparing 7‐day VPZ 20 mg bid and AMPC 500 mg qid dual therapy with 7‐day triple therapy including VPZ 20 mg bid. Our sample size was small; our planned RCT will feature non‐inferiority testing. Second, AMPC‐resistance information was obtained for only 30% of cases (rate: 0%). However, our recent review showed that AMPC‐resistance is generally very low in JapanIn summary, this dual‐center, single‐arm interventional study revealed a good eradication rate, and treatment was safe. We now proceed to an RCT comparing 7‐day VPZ 20 mg bid and AMPC 500 mg qid dual therapy to VPZ‐based triple therapy with AMPC and CAM. A 7‐day VPZ 20 mg bid and AMPC 500 mg qid dual therapy may be better than a 7‐day VPZ 20 mg bid and AMPC 750 mg bid dual therapy.All participants provided written informed consent prior to study enrolment.


A 9‐year‐old Quarter Horse gelding was presented for lethargy, decreased appetite, polyuria and polydipsia (PU/PD), and severe muscle wasting suggestive of immune‐mediated myositis.The horse displayed lethargy, fever, tachyarrhythmia, inappetence, PU/PD, and severe epaxial and gluteal muscle wasting. Clinicopathologic findings were consistent with previously reported cases of systemic calcinosis in horses, including increased muscle enzyme activity, hyperphosphatemia, increased calcium‐phosphorus product, hypoproteinemia, and an inflammatory leukogram. A diagnosis of systemic calcinosis was established by histopathologic evaluation of biopsy specimens from skeletal muscle, lung, and kidney.Symptomatic treatment was complemented by IV treatment with sodium thiosulfate to reverse calcium‐phosphate precipitation in soft tissue and PO aluminum hydroxide to decrease intestinal phosphorus absorption and serum phosphorus concentration.This is the first report in the veterinary literature of an antemortem diagnosis of systemic calcinosis in the horse that was successfully treated and had favorable long‐term outcome. BSAbody surface areaFEfractional excretionIMMimmune‐mediated myositisMYH1myosin heavy chain 1PTHparathyroid hormonePU/PDpolyuria/polydipsiaRANK‐Lreceptor activator of nuclear factor‐kappa B ligandRIreference intervalSGspecific gravity1Systemic calcinosis in horses is a rare syndrome characterized by heterotopic calcification of soft tissues including skeletal muscle, heart, kidneys, lungs, and occasionally blood vessels.Inflammation may be a key factor in the development of systemic calcinosis through synergistic effects of cytokines on receptor activator of nuclear factor kappa B ligand (RANK‐L)‐mediated osteoclast formation and bone resorption.2A 9‐year‐old Quarter Horse gelding was presented to Iowa State University's Lloyd Veterinary Medical Center with a 2‐week history of lethargy and decreased appetite, a 5‐day history of polyuria and polydipsia (PU/PD) and a 2‐day history of acute, severe muscle wasting. The horse had been kept individually on pasture, had no contact with other horses, and had no prior health concerns. Annual vaccinations for Eastern and Western equine encephalitis, West Nile virus, and tetanus had been given 10 months before presentation. One dose of ivermectin (200 μg/kg PO) was administered at the time of onset of lethargy.On admission, the horse weighed 491 kg, had a body condition score of 2‐3/9, and severe epaxial and gluteal muscle atrophy suggesting IMM. Lethargy, fever (39.5°C), tachypnea (44 breaths/min), and tachyarrhythmia (50‐100 beats/min) were present. The hair coat was dull and pitting edema of the ventral thorax was evident. Other abnormalities included mild dehydration and PU/PD with hyposthenuria (specific gravity [SG] 1.003), aciduria (pH 6.5) and microhematuria.A CBC disclosed leukocytosis , mature neutrophilia , and hyperfibrinogenemia . Serum biochemistry indicated hyponatremia , hypochloremia , hypomagnesemia , hypoglycemia , hypoproteinemia , hypoalbuminemia , and mild hyperphosphatemia . Creatine kinase activity , aspartate aminotransferase activity , and BUN concentration  were mildly increased. Serum creatinine concentration  was within normal limits. Initial differential diagnoses included an infectious process with secondary IMM and myocarditis, ionophore toxicity, early renal failure, and diabetes insipidus.Thoracic and abdominal ultrasonography were within normal limits except for increased echogenicity of both kidneys. Transrectal ultrasound evaluation of the left kidney showed hyperechogenicity of the capsule, cortex, and medulla with hyperechoic stippling along echogenic strands in the medulla Figure . Thoraci2,70.1 mm Hg; RI, 85‐95 mm Hg) and serum cardiac troponin I concentration was increased 2/dL2; RI, ≤65 mg2/dL2).On day 2 of hospitalization, pyrexia had resolved but tachypnea and tachyarrhythmia persisted. Arterial blood gas analysis identified hypoxemia  to treat suspected IMM. Because of further increased CK activity with a peak of 1515 IU/L on day 5, the dosage was increased to 0.08 mg/kg IV q24h. Serum vitamin E and selenium concentrations were within normal limits, ruling out nutritional myodegeneration. Because systemic calcification was suspected, biopsy samples were collected from the gluteus medius muscle and lung on day 2, and liver and kidney on day 3 of hospitalization. Histopathologic evaluation identified a degenerative and mineralizing myopathy, diffuse pulmonary mineralization, and subacute to chronic purulent tubulointerstitial nephritis with focal necrosis and mineralization. A core liver specimen had no microscopic lesions. The pulmonary calcification appeared to be purely metastatic with no evidence of another cause of tissue damage or inflammation. Bronchi were not present within the biopsy sample but diffuse mineralization of the alveolar wall was observed Figure . Skeleta2/dL2), sodium thiosulfate  was added to the treatment regimen on day 4 of hospitalization because of its proposed anti‐calcification properties in human patients with extraosseus calcification. Additionally, aluminum hydroxide  was administered beginning on day 5 to decrease dietary phosphorus absorption and the calcium‐phosphorus product. Hematologic parameters had improved with persistent mild mature neutrophilia (7200/μL) and hyperfibrinogenemia (500 mg/dL). At that time, antimicrobial and anti‐inflammatory medications were changed to sulfamethoxazole‐trimethoprim  and pentoxifylline , respectively.Given the multifocal calcification and further increase in calcium‐phosphorus product , whereas serum chloride concentration and CK and AST activities approached normal limits.Tachypnea resolved within 3 and tachyarrhythmia within 6 days after admission, and repeat blood gas analysis was within normal limits. Sequential serum biochemistry analyses showed normalization of serum phosphorus concentration on day 8 (4.2 mg/dL) and hypophosphatemia (2.0 mg/dL) on day 10, with normal calcium‐phosphorus product of 53 and 25 mg%).Polyuria and polydipsia were persistent features with initial water consumption of approximately 181 mL/kg/day that decreased to 119 mL/kg/day over 10 days of hospitalization. Serum creatinine concentration remained within normal limits whereas BUN peaked on day 3 (26 mg/dL) before returning to normal. Fractional excretion (FE) of sodium in urine was measured on day 4 to assess renal tubular function because it may be a more sensitive diagnostic tool to document early renal dysfunction in the absence of azotemia.6/μL) was the only abnormality on a CBC. The horse had a good appetite and attitude and was active during limited pasture turnout. Eleven weeks after initial presentation epaxial and gluteal muscle atrophy had markedly improved  had improved whereas urinalysis remained within normal limits with a SG of 1.027. Thirty weeks after the initial presentation, epaxial and gluteal muscle atrophy had resolved and biopsy of the gluteus medius muscle identified no histopathologic abnormalities. The gelding continued to do well at home and a CBC and serum biochemistry profile 3 years after the initial presentation were within normal limits. Genetic testing 8 years after initial presentation identified homozygosity for a myosin heavy chain 1 (MYH1) mutation indicating increased susceptibility to IMM.Five days after discharge, the horse was readmitted and hospitalized for 10 days because of decreased appetite, intermittent bruxism, and mild signs of colic that responded to treatment with omeprazole . Additional doses of 10% sodium thiosulfate were given 1 day and 4 days after readmission. Polyuria, polydipsia (102‐118 mL/kg/day), isosthenuria , and mild microhematuria persisted, whereas FE of sodium (0.147%) remained normal. Activities of CK and AST had normalized at readmission and 1 week thereafter, respectively. Five weeks after initial presentation, urinalysis was within normal limits with hypersthenuria , indicating that the kidneys were actively concentrating urine, and no evidence of hematuria. Water intake had normalized at approximately 80 mL/kg/day. Anemia ,Early diagnosis in our case allowed targeted treatment aiming at halting and reversing soft tissue calcification by PO administration of phosphate binders and IV administration of sodium thiosulfate. Sodium thiosulfate has been used in the treatment of pathologic calcification in humans, including tumoral calcinosis, characterized by deposition of calcium phosphate microcrystals in periarticular tissues,MYH1 gene, coding for the fast skeletal myosin heavy chain type 2X that predisposes to IMM, has been identified in Quarter Horses and related breeds.MYH1 heterozygotes, homozygotes appear to be more commonly affected with IMM and display hypercontractility of type 2X fibers, which may trigger an auto‐immune response via cellular damage and exposure of affected myosin.The use of glucocorticoids in the treatment of systemic calcinosis is controversial given their pro‐osteoclastic effects. Glucocorticoids increase expression of RANK‐L and prolong osteoclast life span resulting in enhanced bone resorption and possibly worsening of hyperphosphatemia.Authors declare no conflict of interest.Gentamicin sulfate, aluminum hydroxide and sodium thiosulfate was used off‐label.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.


Hypophosphatemia is a less known complication of parenteral iron use, particularly after the use of certain iron formulations. We report the case of a young male with inflammatory bowel disease and iron deficiency anemia, who developed severe symptomatic hypophosphatemia after his third exposure to iron carboxymaltose with no evidence of the same occurring upon prior exposures to the compound. Investigations revealed serum phosphorous levels of 0.7 mg/dl, corrected serum calcium of 8–9.5 mg/dl, alkaline phosphatase of 50 U/L (38–126), 25 hydroxy vitamin D level of 40.2 ng/ml, and intact PTH elevated to 207 pg/ml. Urine studies indicated renal phosphate wasting. Presentation was not in keeping with refeeding syndrome. Intact fibroblast growth factor 23 level, measured after the initiation of treatment was within the normal range at 179 RU/mL (44–215). 1,25 dihydroxy vitamin D level, also measured after the initiation of treatment, was normal at 26.3 pg/ml (19.9–79.3). The patient was treated with calcitriol and aggressive oral and intravenous phosphorous repletion. Symptoms then resolved and the patient was discharged on an oral regimen. This phenomenon is postulated to occur due to an increase in the level and activity of FGF23 and decreased cleavage of the same, due to anemia as well as use of specific iron formulations. This is the first instance, in our literature review, of this complication known to occur, not after initial exposure to an implicated iron formulation but occurring on subsequent exposure. Hypophosphatemia is a lesser-known side effect of intravenous iron repletion, particularly with certain formulations of iron including saccharated ferric oxide and ferric carboxymaltose . First d2. Examination was unremarkable and no focal sensorimotor deficits were noted. Chvostek's and Trousseau's signs were negative.A 35 year old male with a past medical history of Crohn's disease with associated malnutrition  as well as iron deficiency anemia  was admitted after the detection of severe hypophosphatemia (serum phosphorus-0.7 mg/dl), two weeks after receiving 685 mg of parenteral ferric carboxymaltose. Patient's treatment for Crohn's disease had been started 4 years ago. The patient reported no symptoms at presentation but endorsed minimal tingling and cramping in fingers of bilateral upper extremities two days after presentation. No other sensorimotor complaints were endorsed. No cardiovascular or respiratory symptoms were endorsed. The patient reported no change in appetite or diet or no recent alteration in food intake, endorsed good oral intake and no recent weight loss. General examination was notable for Body Mass Index (BMI) of 16.14 kg/mInvestigations revealed serum phosphorous levels of 0.7 mg/dl, corrected serum calcium of 8–8.5 mg/dl which rose to 9–9.5 mg/dl after Vitamin D supplementation was started, alkaline phosphatase of 50 U/L (38–126), with bone specific alkaline phosphatase level, measured using ELISA, of 8.5 mcg/L (4–27). 25 hydroxy vitamin D level measured using ELISA was 40.2 ng/ml, and intact PTH was elevated to 207 pg/ml. 24 hour urine phosphorous level was 1.2 g and TMP/GFR (tubular maximum reabsorption of phosphate per glomerular filtration rate) was 1.39 mg/dl  with fractional tubular excretion of phosphate of 31.82%, suggestive of renal phosphate wasting.1,25 dihydroxy vitamin D level, measured by RIA, was 26.3 pg/ml (19.9–79.3); although, this was measured after calcitriol supplementation was initiated. Intact fibroblast growth factor 23 level was measured using ELISA to be 179 RU/mL (44–215) although the level was obtained 8 days after initiation of treatment.Interestingly, the patient had received infusions of the same formulation in doses of 675 mg and 700 mg in 2018 and 750 mg in 2017 with no documented evidence of consequent hypophosphatemia (Serum P 3.3–3.7 mg/dl). The patient also received iron dextran and ferrous gluconate in the 2 months prior to the described instance with no resulting hypophosphatemia.The patient was started on calcitriol 0.25 mcg daily which was then increased to 0.5 mcg daily to achieve phosphate repletion while ensuring normalized serum calcium levels. Corrected serum calcium levels rose to 9–9.5 mg/dl after this supplementation was started. PTH level, which was likely elevated secondary to mild hypocalcemia, was slated to be monitored upon outpatient follow up.Aggressive phosphate supplementation was initiated through oral and parenteral routes due to severity of hypophosphatemia and likely coexistent malabsorption in the setting of Crohn's disease. Symptoms subsided after repletion was started.The patient's phosphorous level rose to a maximum of 2.2 mg/dl during the hospitalization (day 24 post infusion) and the patient was discharged on sodium phosphate 500 mg four times daily and calcitriol 0.5 mcg daily with close follow-up scheduled.Hypophosphatemia has been increasingly reported following therapy with parenteral iron formulations recently . CertainP < 1 mg/dl) [The occurrence of de novo hypophosphatemia is reported with varying incidences across studies, but most studies reported an incidence between 18.5% and 50% with about a third to half of patients developing severe hypophosphatemia (1 mg/dl) –8. Sever1 mg/dl) .The presence of coexistent inflammatory bowel disease or other causes of malabsorption, low body weight, and African American race, as seen in our patient have been shown to increase the risk of developing hypophosphatemia –8.The refeeding syndrome was ruled out as the patient had no prior history of decrease followed by increase in dietary intake, no other electrolyte abnormalities, no episodes of hypoglycemia and hypophosphatemia resolved with no alteration in feeding rate while in the hospital. In addition, Ustekinumab has not been associated with hypophosphatemia.Hypophosphatemia is caused due to an increase in the amount of intact fibroblast growth factor 23 (FGF 23) in circulation , 6. FGF α1) [Iron deficiency and the resulting anemia increase the levels of iFGF23 through an increase in the level of hypoxia inducible factor alpha 1 (HIF-α1) . Howeverα1) , 10. Ferα1) , 10. In α1) . The levThe timeline for development of hypophosphatemia indicates that the lowest levels of serum phosphorus were noted at week 2 and increased in the following weeks . The timHypophosphatemia is a little known but common adverse effect with the use of certain preparations of intravenous iron, especially in patients with antecedent risk factors, must be anticipated and treated in order to avoid osseous and systemic complications. In addition, this may develop upon subsequent exposures to a particular intravenous formulation even if not encountered after first exposure, like in our patient, and must therefore not be ruled out in these situations.