Meanwhile, niacin was negatively associated with poor cognition as assessed by the CERAD‐WL test, CERAD test, AFT, and DSST. An L‐shaped dose–response relationship between dietary niacin and cognitive function was observed in all participants (nonlinear p < .001). There were also interactions that existed in populations with different carbohydrate intakes and cholesterol intakes . These findings provide new evidence for the potential role of dietary niacin intake on cognitive function in the elderly.Recent studies have shown an inconsistent association between dietary niacin and cognitive function. And this remains unclear in the American outpatient population. The aim of this study was to assess whether there is an association between dietary niacin and cognitive performance in an older American population aged ≥60 years. A total of 2523 participants from the National Health and Nutrition Examination Survey (NHANES) 2011–2014 were enrolled. Cognitive function was assessed by the CERAD Word Learning (CERAD‐WL) test, the CERAD Delayed Recall (CERAD‐DR) test, the Animal Fluency test (AFT), and the Digit Symbol Substitution test (DSST). Cognitive impairment that meets one of the four scoring conditions listed above is defined as low cognitive function. Dietary niacin intake was obtained from 2 days of a 24‐h recall questionnaire. Based on the quartiles of dietary niacin intake, they were divided into four groups: Q1 (<15.51 mg), Q2 (15.51–20.68 mg), Q3 (20.69–26.90 mg), and Q4 (>26.91 mg). The stability of the results was assessed using multifactorial logistic regression, restricted cubic spline (RCS) models, and sensitivity stratified analysis. More than half of the participants had cognitive impairment (52.52%). In the fully adjusted model, niacin was associated with a significantly reduced risk of cognitive impairment in Q3 and Q4 compared with the Q1 group (OR: 0.610, 95% CI: 0.403, 0.921,  We derived a negative association between dietary niacin and low cognition. Our study may be able to provide some reference for dietary prevention and treatment strategies for people with cognitive hypoacusis, which has significant implications for the management of patients with impaired cognitive function. Additional details of the study sampling and exclusion criteria are shown in Figure https://wwwn.cdc.gov/nchs/nhanes/search/default.aspx. Everyone provided informed consent.Data from two consecutive National Health and Nutrition Examination Survey (NHANES) cycles from 2011 to 2014. The NHANES database is based on a stratified, multistage, and probability cluster designed and is mobilized by the National Center of Health Statistics of the Centers for Disease Control and Prevention  test and Consortium to Establish a Registry for Alzheimer's Disease‐Delayed Recall (CERAD‐DR) test, which are used to assess immediate and delayed learning of new verbal information (memory subdomains). The CERAD‐WL test has three consecutive tests CERAD‐WL1, CERAD‐WL2, and CERAD‐WL3. Participants were asked to read aloud 10 unrelated words, one at a time, and the order of the 10 words had to be changed for each learning trial. We averaged the three CERAD‐WL test scores to obtain the final score, which was determined to be cognitively impaired when it was less than 17. In contrast, on the CERAD‐DR test, participants had to recall the 10 unrelated words used in the first CERAD‐WL test after all cognitive performance tests were completed, a time approximately 8–10 min from the beginning of the word learning trial. Scores greater than 4 on the CERAD‐DR test were considered cognitively normal, otherwise, they were cognitively impaired. Participants on the DSST were asked to replicate the corresponding symbols in 133 boxes within 2 min, with scores greater than 33 considered cognitively normal. The AFT  system by trained dietary interviewers who are fluent in Spanish and English, and this fully computerized recall system is comprehensively composed of standard food‐specific questions and possible answers. Each MEC dietary interview room has a standard set of measurement guidelines. These tools can be used to help respondents report the volume and size of food consumed. Moreover, this set of measurement guidelines was specifically designed for use in the current NHANES setting and was agreed upon by experts during regular workshops to evaluate NHANES data collection .We considered complex sampling designs and sample sizes during data analysis  and 95% confidence interval (CI) for the relationship between niacin and the prevalence of cognitive impairment, and the categorical Q1 group of niacin (< 15.51 mg) was used as a reference. For the selection of variables used for adjustment, we selected statistically significant variables and combined them with clinical as well as covariates of covariance. We used both the unadjusted and adjusted models in the data analysis process. In Model 1, we adjusted for age, gender, ethnicity, marital status, income, educational levels, and alcohol use. We further adjusted for SBP, niacin supplement, drug of niacin, albumin, total cholesterol, and the covariates of Model 1 (Model 2). Furthermore, restricted cubic spline (RCS) regressions were performed at the 5th, 35th, 65th, and 95th percentiles of dietary niacin intake nodes to assess nonlinearity and validate the dose–response relationship between dietary niacin intake and cognitive function after adjusting for all variables in Model 2. Moreover, we performed a corresponding sensitivity stratification analysis to investigate the relationship between dietary niacin and cognitive function in populations with relevant covariate dietary variables. In addition, we also examined the relationship between low cognition and dietary niacin in the CERAD‐DR test, CERAD‐WL test, AFT, and DSST test scores, respectively.33.1In this study, we selected two continuous NHANES cycles (2011–2012 and 2013–2014) and focused on 2523 participants with completed interviews and MEC examination in the US (≥ 60 years). The baseline characteristics of the participants are summarized in Table 3.2p = .317), 0.610 , and 0.592 , respectively. Meanwhile, when niacin intake was greater than 20.69 mg, similar results as described above were observed between low cognition and niacin as demonstrated in the CERAD‐WL test, AFT, and DSST. Besides, Table p = .029).Table 3.3p for trend = .014, p for trend = .019) in Table p for nonlinearity = .112) and (p for nonlinearity = .795) in (c) and (d) of Figure p < .001). However, the dose–response relationship between sustained niacin intake and cognitive impairment showed a general trend of decreasing followed by a slow increase. Moreover, both cognitive depression by the CERAD‐WL test and by CERAD‐DR test showed a nonlinear relationship with dietary niacin , as shown in A and B in Figure In a restricted cubic spline regression analysis model fully adjusted for confounders, we found a linear trend between cognitive function and niacin intake in the AFT and DSST (3.4p for interaction = .031) and cholesterol intake (p for interaction = .005). In addition, we investigated the relationship between cognitive impairment and dietary niacin in different subgroups of the population as determined by the CERAD‐DR test, CERAD‐WL test, AFT, and DSST as shown in A, B, C, and D of Figure p for interaction <.05).We performed stratified analyses across dietary intake, gender and age, race, and education levels in Table 4p‐values for the interactions in these two subgroups were below .05, these results may not be clinically significant given the similar directionality of the multiple tests and associations. Meanwhile, we also found no association between niacin and cognitive function in the dietary subgroups of protein intake, energy intake, fiber intake, and sodium intake, these results further explain why these variables were not included in the model for adjustment in our study. In addition, the probability of low cognitive function found in this study appears to be higher than in several other studies conducted in different countries , but also with slower annual rates of cognitive decline. Subsequently, a randomized controlled trial ; methodology ; resources ; software ; validation ; visualization ; writing – original draft ; writing – review and editing . Long Yang: Data curation ; formal analysis ; software ; writing – original draft ; writing – review and editing . Yuan Yuan Liu: Validation ; writing – original draft ; writing – review and editing . Lei Jiang: Supervision . Jian Feng Huang: Supervision .The authors declare no conflict of interest.This study was supported by the National Center for Health Statistics Research Ethics Review Board, and the ethics approval number is Protocol #2011‐17 and Continuation of Protocol #2011‐17. You can find it at this website: NCHS Ethics Review Board Approval (cdc.gov).This study is an analysis of the publicly available NHANES data. Informed consent was obtained from NHANES participants by the National Center for Health Statistics Research Ethics Review Board.


To estimate recent trends in the prevalence of disposable e‐cigarette vaping in Great Britain, overall and across ages, and to measure these trends in the context of changes in smoking and vaping prevalence.The Smoking Toolkit Study, a monthly representative cross‐sectional survey.Great Britain.A total of 36 876 adults (≥ 18 years) completed telephone interviews between January 2021 and April 2022.Current e‐cigarette vapers were asked which type of device they mainly use. We estimated age‐specific monthly time trends in the prevalence of current disposable e‐cigarette use among vapers and inhaled nicotine use (vaping/smoking), smoking and vaping among adults.P‐value = 0.013): for example, the percentage of 18‐year‐old vapers using disposables rose from 0.4 to 54.8% , while it rose from 2.1 to 10.0%  among 45‐year‐old vapers. However, the overall percentage of people currently using any inhaled nicotine remained stable over time both among all adults  and among 18‐year‐olds . In 18‐year‐olds, vaping prevalence grew , and there was imprecise evidence for a decline in smoking . In 45‐year‐olds, there was relatively little change in vaping  or smoking prevalence .From January 2021 to April 2022, there was an 18‐fold increase in the percentage of vapers who used disposables, rising from 1.2 to 22.2% . Growth in disposable e‐cigarette vaping was most pronounced in younger adults (interaction Use of disposable e‐cigarettes in Great Britain grew rapidly between 2021 and 2022, especially among younger adults, but the overall prevalence of inhaled nicotine use was stable over time. Most young adult vapers in Great Britain now use disposable products. Early electronic cigarettes (‘e‐cigarettes’) were disposable products that were poor at delivering nicotine. Over time, new e‐cigarette types were developed to deliver nicotine contained in e‐liquid more effectively through rechargeable devices with refillable tanks or replaceable pods (e.g. Juul) . These dThe Smoking Toolkit Study (STS) is a monthly cross‐sectional survey that recruits a nationally representative sample of adults (≥ 18 years) in Great Britain. It uses a hybrid of population and quota sampling. Great Britain is divided into areas of approximately 300 households, which are stratified by region and demographic profile before being selected at random to be included on the interview list. In selected areas, interviews are performed with one individual per household until age, employment status and gender quotas are met. Raking is used to construct survey weights, adjusting data so that the demographic profile of the weighted sample matches that of Great Britain. This demographic profile is ascertained monthly using data from three sources: the 2011 UK Census, the Office for National Statistics mid‐year estimates and the annual National Readership Survey. Methods are described in detail elsewhere .n = 36 876) completed telephone interviews between January 2021 and April 2022, inclusive. University College London Ethics Committee provided approval for the study (0498/001), and participants gave oral informed consent. All methods were carried out in accordance with relevant regulations.Participants (All measures used were routinely collected in the STS. Smoking status was ascertained by asking participants which of the following applies to them: (i) ‘I smoke cigarettes (including hand‐rolled) every day’, (ii) ‘I smoke cigarettes (including hand‐rolled), but not every day’, (iii) ‘I do not smoke cigarettes at all, but I do smoke tobacco of some kind ’, (iv) ‘I have stopped smoking completely in the last year’, (v) ‘I stopped smoking completely more than a year ago’ and (vi) ‘I have never been a smoker (i.e. smoked for a year or more)’. Participants were told that this question referred to cigarettes and other kinds of tobacco, not e‐cigarettes or heat‐not‐burn products. Participants selecting (i) to (iii) were classified current smokers, (iv) and (v) former smokers and (vi) never smokers.Vaping status was assessed by asking participants whether they were currently using e‐cigarettes to cut down on the amount they smoke, in situations when they are not allowed to smoke, to help them stop smoking or for any other reason. Those who responded positively to any of these questions were considered current vapers.Current vapers were asked which type of device they mainly use. Those who responded, ‘a disposable e‐cigarette or vaping device (non‐rechargeable)’ were considered disposable e‐cigarette vapers. They could only choose one device type (the one they ‘mainly’ use).Participants were asked to provide their exact age in years. Those who refused to give their exact age were asked to select their age group from a list. For participants who only responded to the latter question (2% of respondents), exact age was imputed as the mean age within the age group they selected. Participants were also asked for their gender.Weighted logistic regression was used to estimate monthly time trends in the proportion of (i) adults and (ii) current vapers who use disposable e‐cigarettes, overall and for specific ages (using survey weights described earlier). For the overall analysis, models only included predictors for time. For the age‐specific analysis, models included time, age and their interaction as predictors—thus allowing for time trends to differ across ages. Both age and time were modelled continuously using restricted cubic splines with three knots . This allowed the relationship of prevalence with age and time to be flexible and non‐linear, while avoiding categorization . Age washttps://osf.io/km3x6/).Prevalence ratios (PR) for the change in prevalence across the whole time‐series (April 2022 versus January 2021) were presented, alongside 95% compatibility intervals (95% CIs) calculated using bootstrapping , 11. We Of the 36 876 eligible adults interviewed, 51.1% were women and the average age was 51.5 years [standard deviation (SD) = 18.6]. From January 2021 to April 2022, there was an 18‐fold increase in the percentage of vapers who used disposables, rising from 1.2 to 22.2% . Overall, the prevalence of disposable e‐cigarette use increased from 0.08 to 1.85% . For instance, prevalence of disposable use among 45‐year‐old vapers rose from 2.1 to 10.0% , whereas among 18‐year‐old vapers it increased from 0.4 to 54.8% .Growth in disposable e‐cigarette vaping was most pronounced in the youngest participants  was relatively stable during the study period (Table Most disposable e‐cigarette vapers were current (71.6%) or former smokers (18.8%), with few reporting never having smoked regularly (9.6%). The proportion of disposable vapers who also smoked was similar across ages, but it may have declined slightly over time .Harry Tattan‐Birch: Conceptualization‐Lead; formal analysis‐lead; investigation‐lead; methodology‐lead; writing – original draft‐lead; writing – review & editing‐lead. Sarah Jackson: Conceptualization; investigation; methodology; supervision. Loren Kock: Conceptualization; data curation; investigation; methodology. Martin Dockrell: Conceptualization; investigation; supervision. Jamie Brown: Conceptualization; data curation; investigation; methodology; supervision.Figure S1. Smoking prevalence across ages in Great Britain from 2021 to April 2022.Figure S2. Vaping prevalence across ages in Great Britain from 2021 to April 2022.Figure S3. Prevalence of inhaled nicotine use (smoking/vaping) across ages in Great Britain from 2021 to April 2022.Figure S4. Percentage of disposable vapers who currently smoke across ages in Great Britain.Figure S5. Percentage of disposable vapers who currently smoke across months from 2021 to April 2022 in Great Britain.Figure S6. Percentage of non‐disposable vapers who currently smoke across ages in Great Britain.Click here for additional data file.


Pinna nobilis is currently on the brink of extinction due to a multifactorial disease mainly caused to the highly pathogenic parasite Haplosporidium pinnae, meaning that the selection pressure outweighs the adaptive potential of the species. Hopefully, rare individuals have been observed somehow resistant to the parasite, stretching the need to identify the traits underlying this better fitness. Among the candidate to explore at first intention are fast‐evolving immune genes, of which toll‐like receptor (TLR). In this study, we examined the genetic diversity at 14 TLR loci across P. nobilis, Pinna rudis and P. nobilis × P. rudis hybrid genomes, collected at four physically distant regions, that were found to be either resistant or sensitive to the parasite H. pinnae. We report a high genetic diversity, mainly observed at cell surface TLRs compared with that of endosomal TLRs. However, the endosomal TLR‐7 exhibited unexpected level of diversity and haplotype phylogeny. The lack of population structure, associated with a high genetic diversity and elevated dN/dS ratio, was interpreted as balancing selection, though both directional and purifying selection were detected. Interestingly, roughly 40% of the P. nobilis identified as resistant to H. pinnae were introgressed with P. rudis TLR. Specifically, they all carried a TLR‐7 of P. rudis origin, whereas sensitive P. nobilis were not introgressed, at least at TLR loci. Small contributions of TLR‐6 and TLR‐4 single‐nucleotide polymorphisms to the clustering of resistant and susceptible individuals could be detected, but their specific role in resistance remains highly speculative. This study provides new information on the diversity of TLR genes within the P. nobilis species after MME and additional insights into adaptation to H. pinnae that should contribute to the conservation of this Mediterranean endemic species.The fan mussel  Pinna nobilis struggles to cope with mass mortality events that are likely caused by the protozoan parasite Haplosporidium pinna. As the congeneric species Pinna rudis and P. nobilis × P. rudis hybris are naturally resistant to the pathogen, we performed a comparative analysis on 14 toll‐like receptor loci, between groups of resistant and susceptible individuals. Our research highlights adaptive introgression and suggests that TLR‐7 might be involved in the resistance to H. pinnae.The endemic Mediterranean mussel  In most cases, host and pathogens have co‐evolved over long evolutionary periods, allowing populations to cope effectively with the usual periodic epizootics    is an iconic species living in Posidonia oceanica meadows, in the coastal fringes from 0 m to 60 m depth. Pinna nobilis plays a central ecological role, reducing the turbidity of the water as a filtering organism, sheltering a number of symbiotic species and providing a hard substrate for the development of many epibiotic species.The Mediterranean endemic pen shell P. nobilis was declared a protected species with the category of vulnerable (European Council Directive 92/43/EEC), mainly to counteract intensive captures and habitat loss. These conservation measures proved efficient in several regions, for instance in France, Croatia, Spain and Italy, where populations regained vitality  and a potentially high level of connectivity  since 2016, attributed to a multifactorial disease mainly conveyed not only to the protozoan parasite P. nobilis individuals surviving MMEs, primarily caused by H. pinnae, have been reported, and the phylogenetically related Pinna rudis, as well as the P. nobilis × P. rudis hybrids, seem to be naturally resistant to the parasite  from four distant geographical regions covering the largest part of all three taxa geographic ranges, sampled after epizootic diseases, in which H. pinnae was mainly involved. Therefore, we here described the genetic polymorphism and diversity at 14 predicted TLRs identified in the shotgun genome of P. nobilis  of the mantle tissue was quickly excised using biopsy forceps before the fan mussel closes its valves. The piece of tissue was transferred in a tube filled with 95% ethanol and stored at −80°C until use.No experimentation involving removal, dislocation or killing of Pinna nobilis, 1 as Pinna rudis and 4 as P. nobilis × P. rudis hybrids. These individuals were sampled during or after epizootic disease mainly due to Haplosporidium pinnae. Dead or moribund individuals at the time of sampling are termed ‘sensitive’ (S). For convenience, the others that were alive without obvious symptom of infection are termed ‘resistant’ (R) . Dead specimen presented heavy lesions in the digestive gland connective tissue and signs of degenerative process in epithelial tissue. Moribund specimen presented half‐open valves and a retracted body  , following the manufacturer's instructions. Concentrations were determined by measuring the absorbance at 260 nm using Quantus (Promega) with quantifluor reactant. DNA integrity was assessed on an agarose gel at 1%.DNAs were sequenced by Genewiz using Illumina sequencing generating paired‐end (PE) reads (2× 150 bp). The PE reads were processed as follows: PE reads were first filtered using Trimmomatic v0.36 in order to remove adapters and low‐quality leading and trailing bases (parameters: PE ILLUMINACLIP:TrueSeq‐PE.fa:2:30:10:4 LEADING:3 TRAILING:3). Mate‐pairs were then filtered using Fastp which resulted in PE reads flagged as MP and UNMERGED, which were concatenated (parameters: ‐t 4 ‐f 4 ‐T 4 ‐F 4 ‐c ‐g ‐x ‐m ‐‐include_unmerged). Finally, mate‐paired reads were filtered again using Trimmomatic v0.36 as described above including a filtering if the average base quality dropped below 20. Finally, reads of <60 nucleotides in length were discarded (parameters: PE ILLUMINACLIP:TrueSeq‐PE.fa:2:30:10:4 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 AVGQUAL:20 MINLEN:60). FastQC was run at each step.2.3Q > 40) with a read length higher than 150 nucleotides were aligned against the reference shotgun genome, as well as the partial sequences of Haplosporidium pinnae coding for the small subunit ribosomal RNA (LC338065), Mycobacterium sp. coding for hsp65 (AY379077) and ITS (MN637877), Vibrio sp. coding for ATPaseA (MN201557), Perkinsus mediterraneus, P. atlanticus and P. marinus coding for the small subunit ribosomal RNA , using bwa‐mem , for both P. nobilis and P. rudis. Consensus sequences were used to model the 3D structure, using I‐TASSER , and to draw a phylogenetic tree depicting the relationships between TLR proteins based on multiple sequence alignments, using MEGA 11 and visualized using iTOLv6 , the average number of nucleotide differences among all alleles (k), the nucleotide diversity (π), the number of nonsynonymous (dN) and synonymous (dS) changes. For each Pinna nobilis TLR, median‐joining haplotype networks were drawn using PopART (http://popart.otaga.ac.nz).Neutral microsatellites and TLR haplotypes were aligned with MEGA11, using CLUSTAL and MUSCLE (codon), respectively. Singletons of TLR sequences were removed. These alignments were used in DNAsp , allelic richness based on a sample of eight individuals (AR), observed and expected heterozygosity  and calculate estimates of FIS, with 10,000 genotype randomizations performed among samples to test for significance. Then, significant deviation from the Hardy–Weinberg equilibrium (HWE) was tested using the randomization procedure  implemented in ARLEQUIN v3.5.1.2  among each population. The corrected significance threshold for multiple tests was set using the Benjamini–Hochberg correction procedure , accessible online from the DataMonkey server (https://www.datamonkey.org/) , and with n.pca = 6; n.da = 4.First, the evolutionary pressures acting on each TLR locus were assessed by a McDonald and Kreitman test and by estimating the Tajima's and Fu and Li′s 33.1Pinna rudis and hybrids  were always alive, without signs of disease, and no trace of H. pinnae could be detected in the genomic data. Sixteen P. nobilis did not present any obvious sign of disease (i.e. termed resistant ‘R’) of which eight were infected by H. pinnae, while 20 P. nobilis were dead or presented obvious signs of disease (i.e. termed sensitive ‘S’), of which 17 were infected by H. pinnae. Four French P. nobilis could not be defined as resistant (i.e. termed undefined ‘U’) since we only have the suspicion that H. pinnae affected the whole population  of 0.771 ± SD 0.166. Overall, one or a few haplotypes predominate at each TLR locus  and was mostly higher at the extracellular domain (mean = 2.1 ± SD 2.1) compared with that of the intracellular domain (mean = 0.6 ± SD 0.6), which reveals a greater polymorphism in the protein region harbouring binding sites. The observed mean number of translated proteins was 11 ± SD 6.2 (Table The dHd = 0.714 and S/1000 nt = 9.4). However, for dynein, the dN/dS ratio was 0.2, that is three and seven times lower than the dN/dS ratios observed at intracellular domain and at the full‐length sequence of TLR, respectively.The polymorphism observed at TLR loci was found like that observed for the nonimmune comparative control gene  of positive selection acting on TLR‐7, TLR‐4 (i.e. Contig 473) and TLR‐3. The Fu and Li′s D values were significantly positive for most of the TLR, evidencing overrepresented haplotype variants in the population, that may have been selected in the past .Considering full‐length sequences, 7 TLR presented a d3.4.3Overall, we observed clear star‐like phylogenies, with few high‐frequency nodes from which derive eventually rare haplotypes, which could be consistent with a population expansion model and/or a recent positive selection Figure . This paP. nobilis and P. rudis TLR loci, the networks demonstrate a clear differentiation of naturally resistant haplotypes, separated by numerous mutational steps from sensitive, resistant or undefined ones. On the contrary, resistant and sensitive P. nobilis were evenly distributed across the nodes and branches, highlighting that there was no specific lineage associated with a particular phenotype, hence, no network evidenced strong purifying selection .Before the analysis using FUBAR, we assessed the putative recombination sites within TLR sequences, using GARD. A total of eight recombination sites were detected across six TLR sequences, mostly consisting in one recombination site detected in four TLR sequences and two recombination sites in TLR‐7 and a protein toll‐like (P. nobilis and P. rudis), FUBAR identified 395 sites (26.6%) putatively under selection, with a posterior probability of .9. Most of these (95%) were negatively selected sites, and most of the positively selected sites (47 out of 53 sites) were found at the extracellular domain of the proteins . On the contrary, the groups of resistant and sensitive phenotypes overlapped, though few resistant individuals were remotely distributed from the others , eight SNPs of TLR‐4  and the snp1030 of TLR‐1, presented above.When the entire set of SNPs was used in the DAPC, almost all SNP of TLR‐4 , one SNP of TLR‐6  and one SNP of TLR‐1  mainly contributed to the cluster according to axis 1, which separate the group of naturally resistant individuals from other groups. The distribution of individuals according to axis 2, which separated P. nobilis groups only. Both axes explained a large part of the genetic variance, and the clustering was mainly driven by 13 SNPs belonging to the two TLR‐4 and the two TLR‐6. Among these SNP, only two were positively selected . Noteworthy, whatever the SNP contributing to the clustering, their overall contributions were weak, and no SNP could clearly segregate the resistant and sensitive groups.Afterwards, the selected SNPs were used to maximize the clustering of 3.5P. nobilis and P. rudis consensus TLR‐7 proteins. The software evidenced, overall, similarly shaped proteins and few 3D changes, such as the structure around the amino acids at position 733 , while that of P. nobilis, situated between amino acids 369 and 443, binds the XG1 ligand . Figure I‐TASSER was used for predicting the 3D model structure of 3 Figure . Two binPubChem 19,603, whP. rudis and P. nobilis TLR‐7 consensus sequences reveals 30 amino acids differences of which 12 are associated with obvious physicochemical changes. Four of those are situated in or near the second binding sites of P. rudis and P. nobilis TLR‐7, respectively, and noteworthy two were also found under positive selection. Significant physicochemical changes due to amino acid substitutions could also be observed outside binding sites and even at the intracellular domain , but finally died, which may be attributed to other pathogens or factors as well. During MMEs, no regular monitoring of disease progression at the individual level could be done, so it was not possible to clearly compare the level of disease at the specimen level, nor the interindividual stress response over the course of infection. Given our samples and the lack of standardized sampling protocol, such an assessment could not be made neither. As a consequence, strictly speaking, these P. nobilis cannot be obviously defined as either resistant or tolerant, precisely to H. pinnae infection, despite the eventual inability to survive. In our analysis, for convenience, these P. nobilis were considered resistant.Here, the P. nobilis. Additionally, three individuals of P. nobilis carrying the original full‐length TLR‐7 of P. rudis appeared as naturally resistant as the hybrids and P. rudis (i.e. no infection detected), suggesting that TLR‐7 was indeed probably involved in disease resistance.Although we cannot exclude some assignment errors in the resistant group, we assumed that field observations were nevertheless good indicators of signs of resistance. At the molecular level, we were able to demonstrate an obvious difference between the sensitive and resistant groups since introgression at the TLR‐7 locus was observed only in the group of resistant P. nobilis of our dataset, this result appears highly consistent with the recent research evidencing that TLR‐7 can efficiently hamper the development of intracellular protozoan parasites  and TLR‐4/2/13 , all coding DNA sequences of our contigs consisted in one large exon, a feature that has been reported in other molluscs  and two that aligned a high proportion of the same reads, which would have strongly biased the analysis.4.2D overall indicated that the diversity, at most TLR loci, was made of few high‐frequency haplotypes and a few to high number of rare alleles. The number of haplotypes and the level of polymorphism were similar to that reported in several vertebrate and invertebrate species  at site 2. Both binding sites are situated at either side of a Z‐loop region which is also involved in the ssRNA recognition. Recent research evidenced that the first site was essential for TLR‐7 activation, while the second one only contributes to ssRNA‐induced activation. The TLR‐7 is thus synergistically activated in response to guanosine and ssRNA  ; data curation (lead); formal analysis (lead); funding acquisition ; investigation (lead); methodology (lead); project administration (lead); resources ; software (lead); supervision (lead); validation ; visualization ; writing – original draft (lead); writing – review and editing . Ioannis A. Giantsis: Formal analysis ; resources ; validation ; writing – review and editing . Maite Vázquez Luis: Formal analysis ; resources ; validation ; writing – review and editing . Fabio Scarpa: Formal analysis ; resources ; validation ; writing – review and editing . Mathieu Foulquié: Formal analysis ; resources ; validation ; writing – review and editing . Jean‐Marc Prévot: Software ; writing – review and editing . Marco Casu: Resources ; validation ; writing – review and editing . Athanasios Lattos: Resources ; validation ; writing – review and editing . Basile Michaelidis: Resources ; validation ; writing – review and editing . Daria Sanna: Resources ; validation ; writing – review and editing . José Rafa García‐March: Resources ; validation ; writing – review and editing . José Tena‐Medialdea: Resources ; validation ; writing – review and editing . Nardo Vicente: Resources ; validation ; writing – review and editing . Robert Bunet: Conceptualization ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; validation ; writing – review and editing .This work was supported by the University of Toulon and Toulon Provence Méditerranée (TPM) related to the PINORES project, the University Institute of Technology of the University of Toulon under the grant ‘CARTT’ and by the European Union's LIFE programme through the project LIFE PINNARCA (NAT/ES/001265). Fabio Scarpa, Marco Casu and Daria Sanna acknowledge the support of NBFC to the University of Sassari, funded by the Italian Ministry of University and Research, PNRR, Missione 4, Componente 2, ‘Dalla ricerca all'impresa’, Investimento 1.4 Project CN00000033.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Appendix S1Click here for additional data file.


Postcardiotomy RV dysfunction is an under‐recognized cause of acute kidney injury (AKI). Insertion of a percutaneous right ventricular assist device (RVAD) reduces central venous hypertension and congestive nephropathy by augmenting cardiac output. In selected patients, percutaneous RVAD insertion may improve renal function and obviate the need for long‐term dialysis. In the setting of right heart failure, rising CVP is a central cause of renal dysfunction. Increasing backpressure leads to an increase in renal venous pressure, perpetuating AKI via a reduction in glomerular filtration rate (GFR).The following is a case series of three patients with postcardiotomy AKI who underwent percutaneous RVAD placement and had subsequent return of meaningful renal function while avoiding long‐term renal replacement therapy.2We present the cases of three patients who underwent treatment for ACC/AHA Stage D heart failure with reduced ejection fraction: two with left ventricular assist device (LVAD) insertion and one with orthotopic heart transplant. Postoperatively, these patients developed renal dysfunction with clinical findings of RVF. In each case, percutaneous RVAD insertion resulted in renal recovery as evidenced by improvement in serum creatinine and urine output. The patients were weaned from right‐sided cardiac support and the RVADs were able to be removed. None of the three patients required long‐term renal replacement therapy.33.1A 73‐year‐old man with a history of coronary artery disease, hyperlipidemia, hypertension, and heart failure with reduced ejection fraction secondary to ischemic heart disease who was admitted to the Cardiovascular Intensive Care Unit (CVICU). He was listed for cardiac transplantation as Status 1 due to inability to wean from percutaneous RVAD and LVAD support. The patient underwent successful orthotopic heart transplantation. On post‐transplant day 1, he had adequate filling pressures with a CVP of 10 mmHg , creatinine increase from 0.7 to 1.2 mg/dL, and a notable decline in urine output from 1.2 cc/kg/h the day prior to less than 0.5 cc/kg/h (reference range: 1–2 cc/kg/h). The patient's vasopressor requirements increased throughout the day, his pulmonary artery pulsatility index (PaPi) decreased to 0.83 mmHg , and his CVP rose to 20 mmHg prompting a return to the operating room for further exploration.Intraoperatively, it was found that the dysfunction was partially related to oversizing and edema of the graft. Ongoing RV dysfunction prompted insertion of a percutaneous RVAD via the right internal jugular vein. Shortly after returning from the operating room, the patient had continued low urine output. The RVAD flows were initially ~5 L at 4000 RPM in the operating room but decreased to ~3.5 L in the CVICU. Urine output and RVAD flows did not improve with additional resuscitation. The patient began exhibiting clinical signs of superior vena cava syndrome, and he was promptly returned to the operating room for cannulation of the subclavian and femoral veins to reduce central venous congestion. Within 24 hours, his urine output increased significantly to 2.3 cc/kg/h and 3.9 cc/kg/h by 48 h. The RVAD was weaned, and on postplacement day 6, it was removed. By this time, his creatinine had decreased to 1.24 mg/dL and his urine output remained robust at 1.7 cc/kg/h  revealed adequate RV function and PaPi of 1.5.By POD 10, the patient had been weaned to a single inotrope and was being diuresed. It was noted that although his PaPi scores were previously consistently above 1.0, they had dropped to 0.94 with a rising CVP to 16 mmHg. On POD 12, the patient's serum creatinine had risen from 1.45 mg/dL to 1.89 mg/dL. Left ventricular assist device speed was increased for additional left ventricular offloading. The patient's creatinine continued to rise, up to 3.51 mg/dL on POD 15. Urine output dropped precipitously to 0.1 cc/kg/h during this time. The decreased PaPi and worsening kidney injury were concerning for ongoing RV dysfunction. The decision was made to proceed with percutaneous RVAD insertion via the right internal jugular vein. He was concurrently started on continuous renal replacement therapy (CRRT) due to persistent periprocedural hyperkalemia. Overnight, the patient's urine output improved to 0.7 cc/kg/h while his creatinine improved from 3.51 mg/dL to 2.71 mg/dL. He was weaned from CRRT within 24 h. By 48 h, his creatinine decreased to 2.29 mg/dL and urine output was 1.9 cc/kg/h. The RVAD remained in place for 13 days, and by decannulation, the patient had a urine output of 1 cc/kg/h, a serum creatinine of 1.26 mg/dL, and a CVP of 10 mmHg , and mean arterial pressure (MAP) without vasopressor assistance. The decision was made to insert a percutaneous RVAD to decompress the right ventricle and attempt to alleviate his presumed cardiorenal syndrome. Right ventricular assist device insertion allowed for significantly increased LVAD speeds improving CO. His renal function slowly improved Figure , and the4Presented is a case series of three patients who developed AKI following LVAD insertion or heart transplant and who were able to avoid long‐term dialysis with temporary percutaneous RVAD insertion. Acute kidney injury remains one of the most common complications following cardiac surgery with an incidence of approximately 40%.As previously stated, LVAD implantation and cardiac transplantation carry a particularly increased risk of postoperative kidney injury. Anywhere from 5.7% to 19.7% of these patients require some form of renal replacement therapy, which is associated with worse long‐term prognosis, morbidity, and mortality.There are limited interventions that serve to reliably prevent AKI following cardiac surgery. Intensivists use a variety of physical examination findings, invasive monitoring catheter data, laboratory testing, and point‐of‐care ultrasound imaging to attempt to assess fluid status and intravascular volume. Goals include maintaining low CVP  and adequate MAPs to optimize end‐organ perfusion. These are accomplished through judicious fluid resuscitation, hemodynamic support including inotropes and vasopressors, and diuresis to minimize vascular congestion. Once renal dysfunction occurs, it can potentiate the decline of other inter‐related organ systems. These three cases highlight the potential impact of early RVAD support in combating this complication. Despite the well‐described relationship of renal failure in the setting of RV dysfunction, there are little published data regarding the impact of temporary RV support in preventing permanent kidney dysfunction. Moayedifar et al. published a retrospective single‐institution study in 2021, which assessed the effect of temporary RVAD implantation in terminal heart failure patients who underwent LVAD implantation.Right ventricular failure remains a significant cause of morbidity and mortality following cardiac surgery. There are several factors that make the right ventricle more susceptible to failure compared with the left ventricle. The right ventricle is anatomically the more anterior of the two ventricles and thus is more susceptible to myocyte damage from thermal injury during cardiopulmonary bypass. In addition, retrograde cardioplegia may not adequately perfuse the myocardium of the right ventricle. The presence of right coronary artery disease can limit adequate protection during antegrade cardioplegia.This case series demonstrates a potential relationship between percutaneous RVAD insertion and amelioration of renal dysfunction in patients undergoing durable LVAD implantation or heart transplantation. We are cautiously encouraged by the results, which we feel highlight the importance of recognizing subtle clinical signs of RVF and initiating early right ventricular mechanical support. A prospective study is currently planned to assess the rate of renal recovery in patients who undergo percutaneous RVAD insertion to investigate its ability to potentially abate the need for renal replacement therapy.Mami Sow: Conceptualization; data curation; investigation; methodology; writing – original draft; writing – review and editing. Benjamin D Seadler: Investigation; methodology; writing – original draft; writing – review and editing. Sonal R. Chandratre: Conceptualization; writing – review and editing. Abhilash Koratala: Conceptualization; writing – review and editing. Samuel F. Carlson: Conceptualization; data curation; investigation; writing – review and editing. Lyle D. Joyce: Writing – review and editing. Takushi Kohmoto: Writing – review and editing. Lucian A Durham: Writing – review and editing. David L. Joyce: Conceptualization; supervision; writing – review and editing.None.David L. Joyce MD, MBA, is a member of the steering committee for the THEME registry. There are no other conflicts of interest to disclose.Written informed consent was obtained from the patients to publish this report in accordance with the journal's patient consent policy.


Large atypical nuclei with scant cytoplasm were found to exist using histopathological analysis. No metastasis was observed in the tumour‐bearing mice. A combination of CsA, ketoconazole and cyclophosphamide can be used to suppress the immune system in BALB/c mice and induce tumours with significant size.Dealing with nude mice, which lack thymus and therefore are sensitive to unsterile conditions, needs special care and laboratory conditions. For preclinical studies, especially tumour imaging purposes, in which therapeutic properties of drugs or therapeutic compounds are not studied, mice with normal immune system can be a favourable alternative if they carry tumours of interest. In the current study, we introduce an optimized protocol for induction of human tumours in BALB/c mice for preclinical studies. Immune system of BALB/c mice was suppressed by administration of cyclosporine A (CsA), ketoconazole and cyclophosphamide. The tumours of MDA‐MB‐231, A‐431 and U‐87‐MG human cancer cells were induced by subcutaneous injection of the cells to the immunosuppressed mice. Tumour size was calculated weekly. Histopathological and metastatic analyses were performed using haematoxylin and eosin staining. The combination of the three drugs was found to suppress immune system and decrease the numbers of white blood cells, including lymphocytes. At the eighth week, tumours with a dimension of approximately 1400 mm For most in vivo studies, the use of nude mice is unavoidable—where the animal immune system should be inactive to ensure that the drug (or compound) of interest can affect tumour cells. For imaging purposes, in which tumour growth suppression is not the goal of study, laboratory animals with normal immune system will be of great interest, if they carry tumours of the cells of interest. In such cases, results may be more consistent to real conditions when compared to those obtained with nude mice. Compared to nude mice, animals with normal immune system are not highly sensitive to unsterile conditions and do not need strict care during study. Considering such limitations of nude mice, numerous researchers have proposed various immunosuppression regimens for the development of drug‐induced immunosuppression.Tolypocladium inflatum. It is a lipophilic molecule, which can readily cross the blood–brain barrier.Cyclosporine A (CsA) is an immunosuppressive peptide drug produced by the fungus Since 1978, CsA has been frequently used both clinically and experimentally.In our previous work, we induced tumours of A‐431, U‐87‐MG human cancer cell tumours in BALB/c mice,22.1Cell culture medium, Dulbecco's modified Eagle medium (DMEM), was purchased from Gibco . Similarly, foetal bovine serum (FBS) was obtained from Gibco (#26140). Trypsin (#S‐8064), and penicillin/streptomycin (Pen/Strep) (#S‐8062‐5ML) were purchased from DENAzist Asia. Trypan blue was purchased from Merck. CsA, ketoconazole, and gentamicin were purchased from Sigma‐Aldrich. Cyclophosphamide (CP) (Endoxan) was purchased from Baxter. Co‐amoxiclav (Clamox) was purchased from Daana Pharma Co.2.2BALB/c female mice weighing 20 ± 2 g (4–6 weeks old) were used in this study. The mice were initially kept in individually sized standard cages with a relative humidity of 60 ± 5% and a constant temperature of 25 ± 2°C. Access to autoclaved water and rodent food was available to all mice at all times. All procedures for anaesthesia/euthanasia and animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC). Ethics approval for this study was obtained from the Golestan University of Medical Sciences (ethics registry code: IR.GOUMS.REC.1398.001).2.32. Semi‐confluent cells  were trypsinized by using 0.20% trypsin to detach the cells. Cells were centrifuged at 2800 rpm for 5 min, re‐suspended and washed in DMEM medium. After washing, cells were again re‐suspended in DMEM medium. The cells were counted and viability was determined by trypan blue exclusion test. A supply of live cells was kept on ice and injected immediately.All human cancer cell lines  were obtained from the Pasteur Institute of Iran. The cell lines were maintained in DMEM medium supplemented with 10% FBS, 1% Pen/Strep, and 25 μg/mL gentamicin. The cells were grown in T75 flasks (SPL Life Sciences) and maintained in a standard tissue culture incubator at 37°C with 5% CO2.4Healthy BALB/c female mice were divided into 10 groups (eight mice in each group). Groups 1–3 were administered 20 mg/kg CsA; Groups 4–6 were administered 30 mg/kg CsA; and Groups 7–9 were administered 40 mg/kg CsA by the intraperitoneal route on seven subsequent days. Groups 1, 4 and 7 were administered 5 mg/kg ketoconazole; Groups 2, 5, and 8 were administered 10 mg/kg ketoconazole; and Groups 3, 6 and 9 were administered 15 mg/kg ketoconazole by the oral route every day for 7 days. Groups 10 and 11 were administered 30 mg/kg CsA and 10 mg/kg ketoconazole, respectively. No treatment was given to Group 12 (control).Autoclaved water and rodent food pellets were provided for the mice. The animals were given Co‐amoxiclav (0.1 μg/mL) by drinking water during the experiment. After completion of the study, haematology was carried out to determine total white blood cell (WBC) and lymphocyte count to confirm immunosuppression. In groups of mice with the highest levels of immunosuppression, CP was subcutaneously given at a dose of 30 mg/kg on 1 day before tumour cell injection.2.5Under ketamine‐xylazine anaesthesia, blood samples were taken from the submandibular vein of each animal and placed in a heparinized 1.5‐mL microcentrifuge tube. Then, total WBC and lymphocyte counts were performed in an automated haematology analyser (Sysmex XP‐300).2.66 either A‐431, U‐87‐MG or MDA‐MB‐231 cells) was subcutaneously administered to each mouse into the right hind flank region. Tumour growth was observed at the injection site. In order to estimate tumour volume by an external calliper, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined while mice were conscious. Tumour volume was calculated by the modified ellipsoidal formula: V = ½ (Length × Width2).In the present study, immunosuppressed female BALB/c mice were used. After shaving the body hairs of the mice, 100 μL FBS free culture medium containing cells  was used to observe the slices.2.8anova followed by post hoc Bonferroni correction was applied to determine the significance of differences among groups. p ≤ 0.05 was considered to be significant.All data are presented as mean ± SD. One‐way 33.1Compared to the control mice, the combination of CsA and ketoconazole drugs significantly suppressed the immune system in BALB/c mice. Figure 3.26 cells from each cell line  were injected subcutaneously into the right hind flank region of immunocompromised mice. The mean tumour volume for each week following tumour induction is depicted in Figure 3, respectively. The tumour volume then started to shrink in some animals. Figure Approximately 8 × 103.3Histopathology experiments proved that a malignant tumour was present. Tumours were removed, sectioned and stained using the common haematoxylin and eosin method. Figure 3.4No metastases were found in the liver and kidney using microscopic histopathologic evaluation in all the 12 groups Figures  and 6.4Anti‐cancer drugs are ussualy evaluated on animal xenograft models after in vitro studies.All anti‐cancer agents that have received clinical approval have been assessed using traditional preclinical research models. The process of conducting xenograft studies can be extremely complicated, starting with the choice of the best animal model, selection of the tumorigenic cell line, administration strategy, dosing and tumour growth rate analysis.Numerous immunosuppressive drugs, including CsA, tacrolimus, ketoconazole, CP, azathioprine, glucocorticoids and methotrexate, are used alone or in combination to prevent the rejection of human tumour xenografts in murine models.There are a number of reports on immune system suppression using chemical compounds as a single agent or in a combination. Fayzullin et al. used a combination of CsA (30 mg/kg) and ketoconazole (10 mg/kg) to induce immunosuppression in BALB/c mice.In this study, CsA, ketoconazole and CP drugs were used to improve an immunosuppression protocol in order to induct human tumour xenografts in mice. Ketoconazole inhibits fungal cytochrome P450 and blocks the production of ergosterol from lanosterol.A significant reduction in the total WBC and lymphocyte counts in mice receiving CsA and ketoconazole indicates that immune is significantly suppressed. Metastatic potential studies using histopathology revealed metastatic cases neither in liver tissues nor in kidney tissues.Thus, using a multimodal treatment with three drugs including CsA, ketoconazole and CP seems to be effective in inducing human tumours of the cells  cells in BALB/c. All three types of xenografts kept growing tumour volume for 8 weeks after tumour induction.Abolfazl Amini: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; software ; validation ; writing – original draft ; writing – review and editing . Gholamreza Mesbah: Data curation ; investigation ; methodology ; resources ; validation ; visualization ; writing – original draft ; writing – review and editing . Fatemeh Tash Shamsabadi: Data curation ; formal analysis ; validation ; writing – review and editing . Mohammad Ali Zeyghami: Conceptualization ; data curation ; formal analysis ; methodology ; validation ; visualization ; writing – review and editing . Yaghoub Safdari: Supervision ; writing – review and editing .This project was supported by Golestan University of Medical Sciences (grant number: 110121).The authors confirm that there are no conflicts of interests.Not applicable.


Pennisetum silages. A control (CK – no additives) and seven treatments were tested, namely, Lactiplantibacillus plantarum (LP), Lentilactobacillus buchneri (LB), propionic acid (PA), calcium propionate (CAP), LP + LB; LP + PA and LP + CAP. In comparison with CK, all treatments increased the contents of crude protein and lactic acid, decreased the content of butyric acid, and altered the bacterial communities of the silage. Except for the CAP and LP + CAP treatments, the additives decreased pH and the ammonia nitrogen:total nitrogen (NH3-N:TN) ratio. The results of principal component analysis revealed that the PA, LP + PA and LP + LB treatments ranked as the top three silages. The PA and LP + PA treatments exhibited higher water-soluble carbohydrate content, but lower pH, and NH3-N:TN ratio than the other treatments. With the PA and LP + PA treatments, the relative abundances of Lactobacillus and Enterobacter decreased, and of Proteobacteria and Delftia increased, while the carbohydrate metabolism of the microorganisms improved. The LP and LB treatments reduced the Shannon and Simpson diversities. In the beta diversity, PA and LP + PA separated from the other treatments, indicating that there were differences in the composition of bacterial species. The relative abundance of Lactobacillus increased in the LP and LB treatments and of Leucanostoc and Weissella increased in the CAP and LP + CAP treatments. In summary, the addition of L. plantarum, L. buchneri, propionic acid, calcium propionate, and their combinations improved fermentation quality, inhibited harmful bacteria and conserved the nutrients of hybrid Pennisetum.The aim of this study was to compare the effect of different additives on nutritional quality, fermentation variables and microbial diversity of hybrid  Pennisetum (Pennisetum americanum × Pennisetum purpureum) has been incorporated into animal feed and has great potential to be used for ruminants. It is distributed widely in southern China, and is characterized by low cultivation input, high biomass, and strong stress tolerance  count  count . BacteriB) count . The LABB) count . Lactipl rapidly . Lentilaeed loss . Propioneed loss . Due to eed loss . The aboPennisetum was harvested in Fujian Province  in May 2021 by manually mowing at 8–10 cm above ground level, and was transported to the laboratory immediately. The Pennisetum was spread out evenly, and air-dried for 6 h, resulting in a dry matter (DM) content of 181.3 g/kg fresh weight (FW), and was chopped into 1–2 cm lengths using a paper cutter. The following were added to the Pennisetum: (1) distilled water (CK); (2) L. plantarum ; (3) L. buchneri ; (4) propionic acid ; (5) calcium propionate ; (6) LP + LB; (7) LP + PA; and (8) LP + CAP. Each additive was dissolved in 10 mL sterile water and sprayed evenly onto the surface of the Pennisetum . Subsequently, 400 g of the sprayed hybrid Pennisetum samples were placed in a polyethylene bag (248 mm × 344 mm), with 3 replicates for each treatment. The bags were vacuum sealed, and ensiled at room temperature of 26°C for 30 or 60 days.Hybrid After ensiling, DM content of the silage was determined by oven drying at 65°C for 48 h, and the oven-dried samples were sieved through a 0.425 mm screen. The content of water-soluble carbohydrates (WSC) was determined by anthrone sulfuric acid colorimetry ; total n3-N) was determined by phenol sodium hypochlorite colorimetry . Ammonia nitrogen  for sequencing. The library was paired-end sequenced based on the Illumina Novaseq sequencing platform resulting in a complete microbial community.After 60 days of ensiling, a sample of each treatment was stored at −80°C for microbial diversity determination. High throughput sequencing was performed in triplicate, and total DNA was extracted using the CTAB/SDS method to check DNA concentration and purity with a 1% agarose gel. The 16S rDNA gene of the bacterial V3 ~ V4 hypervariable region was obtained by primer sequences 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTAC HVGGGTWTCTAAT) . PCR ampRaw fastq files were demultiplexed and quality filtered using Trimomatic, and further merged by FLASH software. Uparse software (Uparse v7.0.1001)The data of silage quality were analyzed by the GLM program in SPSS software  with ensiling time, additive treatment and their interaction. The principal component analysis used SPSS software. The eigenvalues of the variance matrix, the variance contribution rate and the weight coefficient of each factor were calculated to generate the principal component equation. The principal component comprehensive score was calculated by standardizing the original data into the principal component equation .Pennisetum silage were affected by ensiling time, additives and their interactions (p < 0.05) DM content, while the LP + LB, PA, LP + PA, CAP and LP + CAP treatments had greater (p < 0.05) DM contents. The loss of DM from silage was due to the breakdown of nutrients, as aerobic microbes converted carbohydrates to water, carbon dioxide, and heat (The DM and WSC contents of hybrid ractions . When coand heat . In the and heat . Propionand heat .p < 0.05) with each additive than in CK. Previous studies reported that propionic acid inhibited Clostridia and Enterobacteria effectively, as these bacteria were poor acid resistant bacteria, and reduced protein breakdown (p < 0.05) of CP content with the LB treatment most likely involved propionic acid. L. buchneri produces 1,2-propanediol from sugars and then propionic acid in the metabolic process, resulting in a bacteriostatic effect  at 60 days than at 30 days of ensiling. The WSC serves as an energy source for microorganisms and its consumption implies microbial activity (p < 0.05) with the LP + LB treatments. Similarly, L. plantarum, L. buchneri and L. casei was reduced after 60 days of ensiling. When ensiled for 30 days, the PA and LP + PA treatments had a lesser (p < 0.05) NDF content than CK. This could be due to the increase in total organic acids after the addition of PA, which could hydrolyze digestible cell walls  than for CK. L. plantarum, which is regarded as the most commonly used homofermentative LAB, has the ability to reduce pH rapidly  lactic acid content than CK. The LP treatment had the lowest pH and highest lactic acid content of all treatments. It is likely that lactic acid has a lower pH than other organic acids and plays a vital role during fermentation (p < 0.05) than at 60 days of ensiling in all treatments, except for the PA treatment. Concomitantly, there was a decrease in acetic acid content, which implied that homolatic fermentation dominated. L. plantarum enhanced the quality of alfalfa silage. At 30 days of ensiling, the LP + LB, PA, and LP + CAP treatments had greater (p < 0.05), but the LP, LB and CAP treatments (p < 0.05) had lesser concentrations of propionic acid than CK. The increase in propionic acid content was likely a result of lactic acid being consumed by L. buchneri (p < 0.05), which could be attributed to the dissociation of corresponding organic acid salts (p < 0.05) in the LP, PA, LP + PA and CAP treatments than in CK. The production of silage undergoes dynamic enzymatic and microbial processes of which the degradation of proteins is one of the most crucial stages (3-N:TN ratios in the PA and LP + PA treatments were lesser (p < 0.05) than in the other treatments, probably because the lower pH inhibited the activity of the protease  than in CK. L. plantarum inhibits protein degradation through its effect on enzymes and microorganisms  with DM content, but negatively (p < 0.05) with NDF and ADF contents (p < 0.05) with lactic acid content and negatively (p < 0.001) with butyric acid content (p < 0.05), pH (p < 0.01) and the NH3-N:TN ratio (p < 0.001). Moreover, the WSC content was correlated negatively with pH as a result of the lactic acid produced by LAB, with WSC as a substrate (p < 0.05) between pH and the NH3-N:TN ratio, and this correlation was stronger after 60 days of ensiling. Generally, NH3-N accumulates continuously during fermentation  not only reduces the loss of original information, but also simplifies multiple related indicators into independent components, and, subsequently, assesses the indicators based on the difference in principal component scores . In the ed 89.7% , 4. SimiWe concluded that the lower composite scores indicated better silage quality according to the composite of each original index and the proportion of principal components. Therefore, the top three treatments were LP + PA, PA, and LP + LB.p < 0.05) in all treatments, except for the PA and LP + PA treatments. When the pH of the silage increases by 0.5 after aerobic exposure, it could be regarded as aerobic deterioration (p < 0.05) than that of CK during aerobic exposure. During aerobic exposure, the abundance of LAB in the CK, LP, LB and LP + LB treatments displayed an increasing trend between days 0 and 6 and then a decreasing trend between days 6 and 9 (p < 0.05), while the abundance of LAB in the PA, LP + PA, CAP and LP + CAP treatments displayed an increasing trend (p < 0.05). When the number of yeasts exceeded 5 log10 cfu/g FW, the silage was prone to aerobic spoilage (p < 0.05) than in other treatments, while the pH remained stable, and was lower (p < 0.05) than in other treatments on day 9, which improved the aerobic stability of the silage. The LB treatment failed to inhibit the proliferation of yeast, resulting in an increase (p < 0.05) in pH during aerobic exposure. It is likely that the undesirable microorganisms used the high content of residual lactic acid as a substrate after aerobic exposure (The changes in pH and relative abundances of microorganisms during aerobic exposure after 60 days of ensiling are presented in ioration . In the spoilage . In thisspoilage . The actspoilage . In thisexposure .p < 0.05) than the other treatments, while the Chao 1 index was greater (p < 0.05) and the ACE were lesser (p < 0.05) in the LP treatment than CK. Compared to CK, Shannon indices of LP and LB and the Simpson index of LP + CAP were lesser (p < 0.05), while the Shannon index of LP + CAP and the Simpson indices of LP, LB and LP + LB were greater (p < 0.05) than CK. The Shannon indices of LP + LB, LP + PA and LP + CAP were greater but the indices were lesser (p < 0.05) than the LP treatment (p < 0.05). The change in alpha diversity among silages was caused by the dynamic response of microorganisms . According to PCoA, CK and LP + LB were clustered in the second and third quadrants, LP and LB in the third quadrant, PA and LP + PA mainly in the fourth quadrant, and CAP and LP + CAP mainly in the second quadrant. In addition, PA and LP + PA were separated from the other treatments, indicating that there were differences in the composition of species; whereas, CK was relatively close to LP, LB, and LP + LB, indicating that the composition of species was similar among these treatments. These results demonstrated that different additive treatments had significant effects on the bacterial community of hybrid Pennisetum silage  and of Proteobacteria was greater (p < 0.05) in PA and LP + PA than in CK, while there was no difference (p > 0.05) in the other treatments. The relative abundance of Cyanobacteria in the LP and CAP treatments was lesser (p < 0.05), and in the LP + PA treatment was greater (p < 0.05) than in CK. Cyanobacteria is often found in tropical herbage and could be replaced by Lactobacillus and Enterobacteria after fermentation, but further studies are needed to determine their roles in silage production . The relative abundance of Actinobacteriota in LB was lesser (p < 0.05), while of Actinobacteriota in CAP and LP + CAP was greater (p < 0.05) than in CK. When mixed with L. plantarum, the relative abundances of Firmicutes and Cyanobacteria in LP + PA were greater (p < 0.05), while the relative abundance of Proteobacteria was lesser (p < 0.05) than in LP. The relative abundance of Actinobacteriota in the LP + PA and LP + CAP treatments was greater (p < 0.05) than in the LP treatment. It was reported that Actinobacteriota had the potential of bioremediation to degrade pesticides and heavy metals  was greater (p < 0.05), but in PA (46.8%) was lesser (p < 0.05) than in CK. The relative abundance of Weissella in the LP, LB, PA and LP + PA treatments was lesser (p < 0.05) than in CK (12.7%). Weissella belongs to the heterofermentative bacteria and consumes WSC to produce a mixture of lactic acid and acetic acid (Delftia in the PA and LP + PA treatments was greater (p < 0.05) than in CK. Delftia is often present in soil and plants and promotes the growth and bioremediation of plants  than in CK. Enterobacter was one of the dominant bacteria during ensiling, especially in silage treated with LAB. However, Enterobacter was unwanted due to nutrient loss caused by acetic acid fermentation (Enterobacter bacteria in silage were considered non-pathogenic (Weissella and Pediococcus in LP + CAP were greater (p < 0.05) than in LP, while the relative abundances of Delftia and norank_o__Chloroplast in LP + PA were greater (p < 0.05), but the relative abundance of Enterobacter in LP + PA and LP + CAP was lesser (p < 0.05) than in CK. The relative abundance of Methylobactere-methylorubrum in LP + CAP (1.42%) was greater (p < 0.05) than in the other treatments. Methylobactery-methylorubrum is a gram negative, rod-shaped, strictly aerobic bacteria that can utilize methanol and other reduced one-carbon compounds via the serine pathway  . Proteob high pH , and the high pH .p < 0.05) than in CK, which most likely was due to carbohydrate metabolism. The metabolism abundances of the CAP, LP + CAP, PA and LP + PA treatments were greater than CK (p < 0.05), which might be due to the response of microorganisms to long-term acid stress in silage , and negatively with CP   and Delftia , and negatively with Pediococcus  and Weissella . WSC could be used by microorganisms as a substrate, so it was not surprising that WSC correlated negatively with microorganisms , Pantoea , and Microbacterium  were correlated negatively with CP. Previous studies had shown that certain Enterobacter were proteolytic, which could cause the loss of protein from silage  and positively with acetic acid , propionic acid , butyric acid , pH  and NH3-N:TN . Results were consistent with those of Weissella. Furthermore, Pediococcus was correlated positively with NH3-N:TN  and acetic acid . Several studies indicated that Pediococcus possessed probiotics properties  . Moreoverganisms . In addim silage . In thisoperties . Accordiosystems . It was charides . HoweverPennisetum silage by increasing crude protein and lactic acid contents and inhibiting the growth of undesirable bacteria. Principal component analysis revealed that the silage quality of the PA, LP + PA and LB + LP treatments ranked as the top three of the seven treatments. The synergistic effect of L. plantarum combined with L. buchneri improved the quality of silage more so than any one of them alone. The addition of propionic acid was very beneficial, as it increased the relative abundance of Delftia, inhibited the activity of Enterobacter, maintained pH, butyric acid and the NH3-N:TN ratio at low levels and reduced the contents of NDF and ADF. In summary, L. plantarum, L. buchneri, propionic acid, calcium propionate and their combinations could improve the silage of hybrid Pennisetum, which would mitigate the shortage of feed for livestock.Additives affected the quality of hybrid https://www.ncbi.nlm.nih.gov/, PRJNA946341.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: QF, JuZ, and WL: writing—original draft, formal analysis, and data curation. AD and CG: writing—review and editing. YZ: formal analysis and data curation. FY and JiZ: funding acquisition, supervision, and writing—review and editing. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (42075116 and 32101418), the Fujian Provincial Outstanding Youth Fund Projects (2023J01313545), and the Fujian Provincial Subsidy Project for Science and Technology Special Commissioner (2022S2071).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Alanine aminotransferase (ALT) is an enzyme whose activity became the principal biomarker for liver disease. In the current study, we aimed to determine the prevalence of abnormal ALT, as a surrogate of nonalcoholic fatty liver disease (NAFLD) and its associated determinants using different criteria among Tehranian subjects between 2018 and 2022.This is a cross‐sectional study on 5676 Tehranian individuals aged 20–70 years. The weighted prevalence of abnormal ALT was calculated using both the National Health and Nutrition Examination Survey in the United States  and the American College of Gastroenterology (ACG) guideline  thresholds. Moreover, uni/multivariable logistic regression analysis was performed to find the determinants of abnormal ALT.The weighted prevalence of abnormal ALT was 12.8%  and 22.5%  based on US‐NHANCE and ACG criteria, respectively. Our results showed every decade increase in age decreased the risk of abnormal ALT by 32%. We also found that generally male gender, being overweight/obese, central adiposity, TG ≥6.9 mmol/L, non‐HDL‐C ≥3.37 mmol/L, lipid‐lowering medications, pre‐diabetes/T2DM were associated with abnormal ALT using different cutoff points. Moreover, among men resting tachycardia (≥90 beats per min), hypertension, and females past‐smoker were also found as other determinants of abnormal ALT.High prevalence of abnormal ALT among non‐elderly Iranian adults, especially among men, necessitates immediate multifaceted strategies by policymakers to prevent potential complications caused by NAFLD. The weighted prevalence of abnormal ALT according to ACG guideline was significantly higher compared to US‐NHANCE suggested threshold. Considering US‐NHANCE suggested threshold the weighted prevalence (%) (95% CI) of abnormal ALT was 18.0 (16.6–19.8), 7.6 (6.7–8.7), and 12.8 (11.9–13.8) in males, females, and total population, respectively. Also, individuals with abnormal ALT were more likely to be younger . Considering the ACG guideline, the weighted prevalence (95% CI) of abnormal ALT was 27.3 (25.5–29.2), 17.7 (16.3–19.3), and 22.5 (21.3–23.7) in males, females, and total population, respectively. ALT also exists in other tissues including, the kidney, heart, pancreas, muscles, adipose tissues, intestines, colon, prostate, and brain with a much lower concentration than the liver.Based on the data from the National Health and Nutrition Examination Survey (NHANES), the prevalence of elevated ALT raised from 8.9% in 2002 (ALT >43 IU/L for both genders) to 11.4% in 2012  in the U.S adults.In addition to age and gender, abnormal ALT is associated with general and central adiposity and other cardiometabolic components including high blood pressure,Since the current first‐line management of NAFLD rely on increasing physical activity levels, nutritional care, and lifestyle modification, early screening with detection of abnormal ALT could be useful for a variety of noncommunicable diseases such as metabolic syndrome, type‐2 diabetes (T2DM), hypertension, and cardiovascular disease.22.1n = 15,005) and the second , and it is planned to continue for at least 20 years with a tri‐annual interval design . The design and methodology of the TLGS have been reported elsewhere.n = 34), pregnant women (n = 16), and without complete information on other confounders including body mass index (BMI), waist circumference (WC), hypertension, diabetes, TG, HDL‐C, TC, physical activity, smoking, and creatinine (n = 308), 5318 individuals  were eligible for the current study . This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the The Institutional Review Board (IRB) of the Research Institute for Endocrine Sciences (RIES), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Written informed consent was obtained from all subjects.The Tehran Lipids and Glucose Study (TLGS) is a community‐based longitudinal study performed on a Tehranian urban population aged ≥3 years to determine the prevalence and incidence of non‐communicable diseases (NCD) and related risk factors. Volunteer recruitment was done in two different phases, the first , history of cardiovascular disease (CVD), medication history, education levels, and smoking habits. The subject's anthropometric parameters were measured with light clothing and without shoes. Weight was measured using a digital scale . Height was measured with a tape meter in a standing position and shoulders in normal alignment. WC at the umbilical was measured by an unscratched tape meter. After 15 min of rest, systolic and diastolic blood pressures  were measured twice on the right arm  by a standardized mercury sphygmomanometer , and blood pressure was considered a mean of these measurements.For all subjects after 12–14 h of overnight fasting, a venous blood sample was collected. Fasting plasma glucose (FPG) was measured using an enzymatic colorimetric method with glucose oxidase. TC was assayed using the enzymatic colorimetric method with cholesterol esterase and cholesterol oxidase. Measurement of HDL‐C was done with a homogeneous method (HDL‐C Immuno FS) in which non‐HDL‐C was removed using antibodies against human lipoproteins, and then HDL‐C was measured by the enzymatic colorimetric cholesterol assay. TG was assayed using an enzymatic colorimetric method with glycerol phosphate oxidase. The intra‐ and inter‐assay coefficients of variation (CVs) both were 2.2% for glucose. For both TC and HDL‐Cholesterol, intra‐ and inter‐assay CVs were 0.5% and 2%, respectively. Intra‐ and interassay CVs were 0.6% and 1.6% for TG, respectively. Serum creatinine (SCr) levels were assayed by the kinetic colorimetric Jaffe method. ALT was measured by an optimized UV‐test according to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) on a photometry system; their intra‐ and interassay CVs were 2.2% and 3.8% for ALT, respectively. Analysis was performed in the TLGS research laboratory on the same day as blood sampling using commercial kits  and a Selectra 2 auto‐analyzer . To monitor the quality of measurements, lyophilized serum controls in two different normal and pathologically high concentrations  were used. To assess the repeatability of all assays, in the first run, at least eight repeated measurements were performed and followed by repeated measurements on consequent days. Intra‐assay CV was calculated using data from the first run and interassay CV was calculated using data from all runs. Furthermore, the TLGS laboratory participates in an external quality assurance program. Laboratory analyses of the study samples were performed when both internal and external quality control measures met the acceptance criteria.2.32). BMI was categorized into three groups: 1. <25 kg/m2 (as reference); 2. 25–30 kg/m2, and ≥30 kg/m2. Abdominal obesity was defined as WC ≥95 cm as recommended by The Iranian National Committee of Obesity and based on multiple cross‐sectional and prospective studies.2 for >3 months.κ is 0.7 for females and 0.9 for males and α is −0.329 for females and −0.411 for males.Age groups were defined as; 20 to <30 years (as reference), 30 to <40 years, 40 to <50 years, 50 to <60 years, and ≥60 years in both genders. BMI was calculated as weight (kg) divided into the square of height  data 2.5t‐test for continuous variables, chi‐square test for categorical variables, and Mann–Whitney test for skewed variables. The weighted prevalence of abnormal ALT was calculated using both US‐NHANCE and ACG thresholds. Data were weighted directly to the 2016 urban population of Tehran,Baseline characteristics of the study population were shown as mean (standard deviation: SD) and number (%) for categorical variables. For covariates with a skewed distribution, the median (interquartile range: IQR) was reported. A comparison of baseline characteristics between normal and abnormal ALT according to two definitions was done by student's p‐value less than 0.2 in the univariable analysis were selected to enter the multivariable model. We evaluated the effect modification of gender and each variable in a multivariable model. All analyses were performed in each gender separately in addition to the total population. All analyses were conducted using STATA version 17 SE , and a two‐tailed p < 0.05 was considered significant.The association of different categorical variables with abnormal ALT (for both US‐NHANCE and ACG definition) was assessed by calculating multivariable‐adjusted odds ratios (ORs) with a 95% confidence interval (CI) using binary logistic regression analysis. A univariable analysis was done for each potential covariate including gender , age categories, marital status, education levels, smoking status, physical activity groups, BMI categories, abdominal obesity, glucose tolerance status, blood pressure categories, CKD, high TG, low HDL‐C, non‐HDL‐C categories, lipid‐lowering medications, a steroid medication, FH‐DM, and history CVD. Then, those covariates with a 3The study population consists of 5318 participants  with a mean age (SD) of 45.2 years (12.8). The baseline characteristics of participants by ALT levels for both defined thresholds are shown in Table In the total population, the weighted mean (95% CI) value of ALT level for individuals aged 20–30 years was significantly higher compared to those ≥60 years  Table . As show.7–24.0 v2 (overweight and obese), non‐HDL‐C ≥3.37 mmol/L, FPG ≥5.55 mmol/L (prediabetes and T2DM), high TG, and lipid‐lowering medications were significantly associated with an increased level of abnormal ALT. Moreover, the results show that a heart rate ≥90 beats per min increased the odds of abnormal ALT (US‐NHANCE criteria) with the odds of 1.37 (1.06–1.76) while central obesity increased the odds of abnormal ALT (ACG criteria).Results of the uni‐ and multivariable logistic regression analysis for abnormal ALT using different definitions are illustrated in Tables 2, FPG ≥5.55 mmol/L (prediabetes and T2DM), and high TG were significantly associated with an increased level of abnormal ALT, and hypertension shows a significant association using US‐NHANCE threshold [1.50 (1.10–12.03)]. Heart rate ≥ 90 beats per min increased the odds of abnormal ALT using ACG guideline [1.54 (1.11–2.14)] (Tables Gender‐split multivariable‐adjusted ORs (95% CI) of potential determinants for elevated ALT for different criteria are presented in Tables ] Tables  and S4. ] Tables  and S5.To show the robustness of our results, a series of sensitivity analyses were conducted. First, a sex‐specific cut point of elevated WC using the national survey of risk factors for noncommunicable diseases of Iran  was considered.4In a population‐based study of middle‐aged Tehranian residents, the weighted prevalence of abnormal ALT ranged from 12.8% to 22.5% applying US‐NHANCE and ACG criteria, respectively. We also found that generally younger age, male gender, being overweight/obese, central adiposity, TG ≥6.9 mmol/L, non‐HDL‐C ≥3.37 mmol/L, lipid‐lowering medications, and pre‐diabetes/T2DM were associated with abnormal ALT using different definitions. Moreover, among men resting tachycardia, and hypertension, and for females, past‐smoker were also found as another determinant of abnormal ALT.Our reported prevalence of abnormal ALT in the current study was generally higher than those reported in developed countries, estimated at 11.2% in the Australian populationSeveral cross‐sectional studies have investigated the association of abnormal ALT with gender and the majority of them showed that male gender is an independent risk factor for ALT elevation. Our results showed that the male gender had higher odds of having elevated ALT according to different thresholds, similar results were found in the Australian and Mexican populations.With increasing age, the prevalence of abnormal ALT declines significantly among the Tehranian population, an issue that was addressed in previous studies.2 and central obesity were associated with high ALT .The prevalence of abnormal ALT is higher among overweight and obese individuals using both US‐NHANCE and ACG thresholds. We also observed that overweight increase the odds of abnormal ALT by 60% and 40% using US‐NHANCE and ACG threshold, receptively; for obesity, this was found to be more than twofold, similar to those found by Gutiérrez E et.al.Prediabetes/diabetes was associated with abnormal ALT after adjustment for the BMI, WC, TG, non‐HDL‐C, hypertension, and FH‐DM. Several studies in Iran,Considering lipid profiles, TG ≥6.9 mmol/L, non‐HDL‐C ≥3.37 mmol/L, and lipid‐lowering medications were independently associated with abnormal ALT in the total population. A gender‐specific analysis shows that high triglyceridemia was associated with abnormal ALT only among males, while the last two variables were associated only with females. In previous Iranian studies, hypertriglyceridemia was associated with elevated ALT in both genders.The influence of hypertension on abnormal ALT was observed only among men using the US‐NHANCE threshold. This positive association among men was reported by the CHIEF StudyOur study showed positive associations between abnormal serum ALT levels and heart rate ≥90 beats per minute in the total population using US‐NHANCE threshold and among males in ACG guideline definition even after multiple adjustments with metabolic risk factors as well as physical activity levels that did not remain as an independent factor. This significant association was reported by Laine et.alSeveral limitations in the current study need to be acknowledged. First, since it was a cross‐sectional study we were not able to determine the causal effect of metabolic factors on abnormal ALT. Second, we did not measure markers of hepatitis B and C infectious as well as other liver enzymes such as γ‐glutamyltranspeptidase or common medications that may cause elevated ALT including antibiotics, antiepileptics, nonsteroidal anti‐inflammatory agents, anti‐tuberculosis drugs, anti‐retroviral treatment for HIV, biologic agents such as anti‐tumor necrosis factor drugs, and some cancer chemotherapeutic agents. Third, despite widespread adjustment in our study, unmeasured confounders such as alcohol consumption may explain part of this association. Alcohol consumption is a great risk factor for liver damage and it's estimated that about 35% of alcohol drinkers developed early liver disease; the issue that might affected by the individual gut microbiome.5In conclusion, we found that out of every 100‐person, about 18–27 males and 8–18 females have abnormal ALT. We also observed male sex and younger age showed significant risk factors for abnormal ALT among the Tehranian population. Obesity, prediabetes/T2DM, hypertriglyceridemia, high non‐HDL‐C in both genders, as well as resting tachycardia and hypertension for men and past‐smoker for women had the most consistent association with abnormal ALT. Hence, the high prevalence of abnormal ALT among non‐elderly Iranian adults, especially among men, necessitates immediate multifaceted strategies by policymakers to prevent potential complications caused by NAFLD.SA, FH conceived and planned the study. A.AM and FA cooperated in data gathering. SA conducted the analyses. SA, MT, DM, and FH, developed the first draft of the manuscript. All authors have read and approved the final manuscript.This study was supported by the Shahid Beheshti University of Medical Sciences, Tehran, Iran [grant no. 02‐43004160].None declared.Table S1Click here for additional data file.


Attentional control deficit has been proposed as one of the reasons for lower arithmetical performance in people with high math anxiety (HMA). Previous research trying to discern whether this deficit concerned proactive or reactive use of attentional control has been criticised because the methodologies used were mostly suited to investigating reactive control only. The aim of this study was to investigate proactive control in HMA individuals in a classical Stroop task. Twenty HMA and 20 low math-anxious individuals (LMA) named the ink colour in which congruent and incongruent colour words as well as X strings  were presented. The HMA group was slower than their LMA peers in the congruent and incongruent conditions only. Furthermore, HMA individuals showed a higher interference effect. Last, only LMA participants showed a facilitatory effect of the congruent condition. These results are interpreted as indicating the presence in the HMA individuals of a task conflict between the task to perform (ink naming) and an irrelevant task triggered by the stimuli (word reading). Task conflict is evident only when proactive control, responsible for maintaining the current goals, is too weak to solve the competition between tasks. Therefore, this study confirms that HMA individuals find it difficult to implement attention proactively. Math anxiety is frequently described, using the classical definition given by Richardson and Suinn , as a feACT claims that anxiety impairs the efficiency of central executive functions related to attentional control: inhibition, understood as the capacity to prevent irrelevant stimuli or responses from harming performance, and shifting, or the capacity to flexibly change the allocation of attention to the current relevant task or stimuli , this deficit in attention control might lead them to pay excessive attention to their ruminations on their poor performance or abilities, or to bias attention towards external mathematically related stimuli, which are perceived as threatening  displayed in different ink colours and have to name the colour in which words have been presented while ignoring the meaning of the printed word, even though it is automatically processed. Most studies focus on the interference effect, that is the longer reaction times (RTs) for incongruent trials (e.g. saying “blue” when faced with the word “green” printed in blue) compared to neutral ones (XXXX string printed in blue). Interference is thought to be caused by an information conflict: although we pay attention to the colour, the meaning of the word is also processed and competes for selection. A facilitation effect is also sometimes found: participants are faster when saying “blue” faced with the word “blue” printed in this colour than when faced with a blue string of Xs. However, this effect is usually less robust and sometimes even disappears or transforms in reverse facilitation (RF), with neutral stimuli leading to faster responses than congruent ones. This has led some authors  effect and a smaller or even reverse facilitation  effect.t (38) = 15.40, p < 0.001.Forty students from the University of Barcelona took part in this experiment. They were selected on the basis of their extreme scores in the Shortened Mathematics Anxiety Rating Scale (sMARS)  = 1.49, p = 0.14, and gender distribution . Lastly, given that trait anxiety has been shown to affect cognitive control  < 1. All students signed an informed consent and received monetary compensation. This experiment is part of a research project that has been approved by the Ethics Committee of the University of Barcelona. See Table In contrast, both groups were comparable in terms of age, The Spanish version of this test was used .Only the trait anxiety scale (STAI-T) was used. Participants must answer how they feel in “general” using a four-point Likert scale ranging from 0  to 3 , on 20 items describing different emotions. The Spanish version of the scale , red (rojo), green (verde) and yellow (amarillo). Each ink colour was presented under three different conditions: in the congruent one, the colour was used to display its own colour name. In the incongruent one, any of the other three colour names was presented. In the neutral condition, a string of four Xs was displayed. There were 240 trials in which each ink colour was paired in the same number of trials with each colour word and with the neutral sequence, leading to 20% congruent, 20% neutral, and 60% incongruent trials. The fact that the majority of trials were incongruent is considered to favour the use of proactive control  were displayed until the participant named their colour or for a maximum of 2000 ms. A blank screen lasting 500 ms was displayed between trials. Vocal responses were recorded from the onset of the stimuli. E-prime 2  was used to present the stimuli and record the answers’ latencies.2.65% of the responses were removed before the analyses because they contained verbal disfluencies or nonverbal sounds that triggered the voice key. An ANOVA was conducted on the median latencies of correct trials, with condition  as the within-subject variable and group (HMA and LMA) as the between-subject factor. See Fig. F = 92.96, p < 0.001, ηp2 = 0.71, and a significant condition x group interaction, F = 4.11, p = 0.02, ηp2 = 0.10, while the difference between groups was only marginally significant, F = 3.76, p = 0.06, ηp2 = 0.09. Low math-anxious participants were significantly faster than their highly math-anxious peers in the congruent trials, t(38) = 2.33, p = 0.02, and answered marginally faster in the incongruent condition, t(38) = 1.93, p = 0.06, but the two groups did not differ in the neutral condition, t(38) = 1.29, p = 0.20. Given that we had specific predictions for group differences in the facilitatory and interference effects, we ran new ANOVAs in which we analysed them separately. Regarding the facilitation effect, congruent and neutral stimuli did not differ, F < 1. However, there was a marginal effect of math anxiety, F = 3.57, p = 0.06, ηp2 = 0.08, as well as a significant condition x group interaction, F = 8.82, p = 0.005, ηp2 = 0.18. When looking at each group separately, standard facilitation for congruent trials was found in the low math-anxiety group, t(19) = 3.11, p = 0.006. There was a trend towards reverse facilitation for HMA participants , although it did not reach significance, t(19) = 1.28, p = 0.21.There was a main effect of condition, F = 117.14, p < 0.001, ηp2 = 0.75, as well as a significant condition x group interaction, F = 4.07, p = 0.05, ηp2 = 0.10. Although both groups showed an interference effect (t(19) = 9.04, p < 0.001 and t(19) = 7.35, p < 0.001, for LMA and HMA groups, respectively), the size of this effect was larger for the HMA group (t(38) = 2.01, p = 0.05, 58 vs. 85 ms). Latencies of the two groups did not differ, F = 2.84, p = 0.10, ηp2 = 0.07.Regarding interference, a main effect of condition was found, F = 21.75, p < 0.001, ηp2 = 0.36. Accuracy was lower for incongruent than for congruent, t(39) = 5.18, p < 0.001, or neutral trials, t(39) = 4.56, p < 0.001, and the latter two conditions did not differ, t(39) = 1.60, p = 0.11. Neither the group effect nor the interaction were significant , and accuracy was not further analysed.Accuracy was in general very high , although it decreased slightly in the incongruent trials (96%). The same ANOVA as used in the latencies analysis was performed and it confirmed a significant effect of condition, It has been suggested that individuals suffering from math anxiety might have a deficit in attentional control that would make them more vulnerable to distractions. Recent studies on attention have made a further distinction between proactive and reactive attention. Anxiety has been named as one of the factors that might weaken proactive control but previous attempts at studying it in math anxious populations have been criticised for not using the most suitable methods. On this occasion, we used a classical Stroop task and measured task conflict, as it is a widely accepted method for investigating proactive control. We found that our two anxiety groups behaved similarly in the neutral condition. In contrast, HMA individuals were slower than their LMA peers in the congruent and  in the incongruent trials, that is, the conditions in which a conflict arises between the task to perform (naming the colour) and the reading task strongly associated with the colour words. Interference was found in both groups, but HMA participants suffered it to a greater degree. Last, facilitation was found in the LMA group only. Altogether, our data points toward the existence of a task conflict in math anxious individuals, and therefore, an attenuation in their proactive implementation of attentional control.Our results fit nicely with the predictions of the ACT (Eysenck et al., DMC Braver,  specifieWe cannot conclude without mentioning a puzzling finding that is repeatedly reported in the literature and was replicated here. It is usually considered that anxious individuals behave differently from their peers because feelings and ruminations related to their anxiety deplete some of the central executive resources and prevent them from applying attention proactively. Yet, HMA individuals have shown a deficit in attentional control in many tasks that did not contain mathematical or numerical stimuli, and in principle, should not have caused anxiety: reading non-mathematical words (Hopko et al., In summary, HMA individuals showed the presence of task conflict in conditions in which distractor words were presented. Given that task conflict is considered as an indicator of a deficit in proactive control, our data confirm that HMA individuals have difficulty applying attention to achieve their goals in an anticipatory and sustained way.