Labrus bergylta) for a better understanding of the basic biology of the intestinal functions and health in this stomach less species. During the trial, Ballan wrasse was fed either a reference diet, the reference diet supplemented with (i) a commercial prebiotic  expected to have beneficial effects, (ii) soya saponins (0·7 %) expected to induce inflammation or (iii) a combination of the prebiotics and the soya saponins to find a remedy for gut inflammation. Blood, intestinal tissue and gut content from four consecutive intestinal segments (IN1 – IN4) were collected. No significant differences in fish growth were observed between the four dietary groups. Saponin supplementation, both alone and in combination with prebiotics, increased weight index of IN2 and IN3 and decreased blood plasma glucose, cholesterol and total protein. Dry matter of intestinal content and activity of digestive enzymes were not affected by diet. Histomorphological analyses revealed a progressing inflammation with increased infiltration by immune cells particularly into the distal parts of the intestine in fish fed diets with saponins, both alone and in combination with prebiotics. Gene expression profiles obtained by RNA sequencing and quantitative PCR mirrored the histological and biochemical changes induced by the saponin load. The study demonstrated that Ballan wrasse gut health and digestive function may be markedly affected by feed ingredients containing antinutrients.A 5-week feeding trial was conducted in the cleaner fish Ballan wrasse ( Labrus bergylta) has, during the recent years, become a domesticated fish species due to its ability to feed on sea lice living on the surface of Atlantic salmon –3. Sea lice infestation has become the most important challenge for Atlantic salmon production in Norway and neighbouring countries,4 as a result of increased lice resistance against delousing chemicals. Ballan wrasse is considered as an effective therapeutic and preventive biological control agent against salmon lice,7. Although wild caught fish are still the main source of Ballan wrasse used in salmon production, farmed Ballan wrasse is increasingly being used. However, the challenges in this production are many, and several knowledge gaps must be filled. The present study addressed questions whether nutrients sources can secure or compromise gut health in Ballan wrasse. The observation of severe symptoms of gut inflammation in an earlier survey of gut health in cultivated Ballan wrasse demonstrated the urgency for better understanding of the general structure and physiology of the digestive tract of this species and relationships between diet composition and digestive and immune functions in the intestine. The present study aimed to strengthen knowledge on such aspects of the short and agastric digestive tract of the Ballan wrasse,10.The Ballan wrasse  containing a cell wall extract from baker’s yeast (Saccharomyces cerevisiae) and dried algae. It is claimed by the producer to maintain gastrointestinal integrity and stability, and to improve nutrient digestibility and intestinal health .Supplementing fish with prebiotics has been reported to enhance immune status of intestine, improve gut structure and disease resistance with possible direct or indirect modulatory effects on gut immune responsesThe saponins and the prebiotic were included in the diets alone or in combination, as tools for the study of mechanisms underlying gut function and health in Ballan wrasse. We hypothesised that soya saponins in diet would impair intestinal immunity, while the prebiotic would trigger immune reactions in the intestine and counteract the saponin effects when present in diet together.,19 or a mixture of the two (P + S), which might indicate whether gut inflammation can be prevented by functional ingredients. Together observations made on fish fed these four diets were expected to fulfil the aims of this study, i.e. to be able to improve diet composition and thereby health and gut function of the fish by strengthening knowledge of the general structure and physiology of the digestive tract of Ballan wrasse and relationships between diet composition and digestive and immune functions in the intestine.The experimental procedure of the present study composed of a 5-week feeding trial with four diets, i.e. a reference diet (Ref) and three experimental diets made by supplementation with a prebiotic (Pre) expected to improve gut health and function at a level suggested by the producer, soya saponins (Sap) at a level found in soybean meal and found to induce inflammation in Atlantic salmon,21.The feeding trial followed the Norwegian animal welfare act guidelines, in accordance with the Animal Welfare Act of 20 December 1974, amended 19 June 2009. The facility at Sunndalsøra, Nofima (division 60) got a permission granted by the Norwegian Food Safety Authority (FOTS ID 8060) to run the experiment. The decision was made on the basis of Regulation 18 June 2015 on the use of animals in experiments, §§ 6, 7, 9, 10 and 11. The 3R principles were used as a foundation for the design of the experiment, i.e. to ‘Reduce, Refine, Replace’, which means to minimise the use of experimental animals to a minimum, to keep the animals in such a way that the burden on the animals is as small as possible, and when possible, animal experiments are replaced by alternative methods. As effects of antinutrients and prebiotics on digestive functions and gut health, indicated by biochemical, histological and molecular responses, were the main goals of the present work, and effects of growth were not in focus, two tanks per treatment were considered sufficient for the generation of information on tank effects. The variables addressed have shown negligible tank effects from our previous work with antinutrient in diets for Atlantic salmon. Moreover, 5 weeks feeding was considered sufficient, as development of effects of saponins in Atlantic salmon takes only about 2 weeks to reach full inflammation pictures3 per tank (80 fish).The fish used for the trial had been reared for 1 year in a commercial farm  before transfer to the Nofima AS land-based research facility in Sunndalsøra, Norway. Upon arrival, the fish were distributed randomly to ten cylindrical flat-bottomed black tanks with a volume of 350 l, equipped with separate light sources and artificial kelp. The photoperiod was 24 h per day. A flow through system was used in the tank, with seawater pumped from the Sunndal fjord and filtered by drumfilters of approximately 20 µm. The water salinity was 32 ppt, and water temperature in tanks during the experiment was 14·5–17°C. All fish were individually tagged . Three experimental diets were made by supplementing the Ref diet with either a commercial prebiotic  (Pre), soyasaponin (0·7 %) (Sap) or the combination of prebiotics and soyasaponin (P + S). Feed was produced by extrusion (2 mm pellet size) at Nofima’s Aquafeed Technology Center in Bergen, Norway. Feed was distributed randomly to duplicate groups of fish per diet for 24 h with 10 min feeding intervals, using automatic belt feeders for the duration of the experiment. Each belt feeder (one per tank) was loaded with feed equivalent to 80 g feed/kg fish per day. Before loading to each belt automat, the 80 g of feed was soaked in 10 ml of water for 4–5 min to soften the feed. Feed intake was not recorded.One reference diet (Ref) containing high-quality marine raw materials was made . The die. Each segment was opened longitudinally, and the gut content was collected, snap frozen in liquid nitrogen and stored at –80°C before further processing. From the same intestinal segments, tissue samples were taken for RNA extraction  and histomorphological evaluation .The feeding trial ran for 5 weeks. At termination of the feeding trial, fish were randomly sampled from the tanks and fully anaesthetised and euthanised with 110 mg/l tricaine methane sulfonate . From six fish per tank, blood was sampled by venipuncture of the caudal vein. Blood was collected in vacutainers containing lithium heparin and stored on ice until centrifugation. Plasma was separated and immediately frozen in liquid nitrogen and stored at –80°C until analysis. After blood withdrawal, fish were killed by a blow to the head before organ sampling. Thereafter, fish were opened, and the viscera was removed from the abdominal cavity for dissection. The intestine was cleaned of mesenteric fat and divided into four segments (IN1–4) as previously describedS1–S5.Blood plasma was analysed for cholesterol, free fatty acids, total TAG, total protein and glucose at the Central Laboratory of the Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, according to standard medical procedures. The instrument used was a Siemens Atellica CH fra Siemens Healthineers. The procedures are available in Supplementary files  as well as an examination of the histomorphology of the intestine in wild Ballan wrasse that we describe in Zhou et al.Formalin-fixed tissue samples were processed using standard histological techniques and stained with haematoxylin and eosin (H&E). Examination was conducted blindly and in randomised order. The following histological characteristics associated with inflammatory reactions in the intestinal mucosa were evaluated: length and fusion of mucosal folds, cellular infiltration, width of the lamina propria and submucosa, enterocyte vacuolisation, nucleus position within the enterocytes and the relative number of goblet cells. The morphological assessment was guided by our experience with salmonid intestinal histopathology using the substrate benzoyl arginine p-nitroanilide  and a curve derived from standardised bovine trypsin solution.Trypsin activity and total bile salt levels were measured in pooled freeze-dried gastrointestinal contents from the four separate intestine segments (IN1–4). Trypsin activity was determined colorimetrically as described by Kakade and co-workersBile salt levels were determined using the enzyme cycling amplification/Thio – NAD method  in the ADVIA®1650 Chemistry System  at the Central Laboratory of the Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo.. Due to an analytical error, some data from IN3 were lost and are therefore not presented. Activities were calculated as mmol substrate hydrolysed per unit time in the whole tissue per kg body weight (enzymatic capacity). Protein was analysed using the Bio-Rad Protein Assay .The activity of the brush border membrane enzymes leucine aminopeptidase (LAP) and maltase was measured in tissue homogenates from IN1, IN2 and IN4 as described previously, in line with what is the case also in Atlantic salmon that shows distinct structural changes in this section upon feeding with diets containing saponins. Gene expression analyses were performed using n = 3 fish per tank (n = 6 fish per diet) fed the three experimental diets and the reference diet. Total RNA was extracted using a BioRobot® EZ1 and RNA Tissue Mini Kit  including a DNase treatment step according to the manufacturer’s instructions. RNA quantity and quality were assessed using a NanoDrop ND-1000 UV–vis Spectrophotometer . All samples had 260/230 and 260/280 ratios above 2·0 and 2·2, respectively, indicating high RNA purity. Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kit  were used for assessment of RNA integrity for the individual samples. The average RNA integrity number of all samples was 7·8 ± 0·5. RNA sequencing was performed by the Norwegian Sequencing Centre (www.sequencing.uio.no). Sequencing libraries were prepared with an automated NeoPrep platform (Illumina) using 90 ng total RNA input to the TruSeq Stranded mRNA Library Prep Kit (Illumina) and standard Illumina adaptors included in the kit. The libraries were sequenced using HiSeq4000 according to manufacturer instructions, generating paired end libraries with an average library size of 14·2 ± 1·4 million reads prior to mapping and remaining sequencing adaptors were removed using Cutadapt. Subsequent trimming and filtering were conducted using Sickle (https://github.com/najoshi/sickle) with a 40 bp minimum remaining sequence length, Sanger quality of 20 and no 5’ end trimming. The individual libraries were mapped to the Ballan wrasse genome , using the short read aligner TopHat2, and transcript abundances were estimated using the FeatureCounts. Differentially expression of genes (DEG)were analysed using the DEseq2 package, taking advantage of the 2 × 2 factorial design and enabelling analysis of combining effects between saponin and prebiotic. Features (genes) with row sums less than fifty were excluded prior to analysis of the results for differently expressed genes. RNAseq can be accessed through the Gene Expression Omnibus (GEO accession number GSE152475). An adjusted P value (P-adjust) of < 0·1 (Benjamini and Hochberg correction) was applied for further downstream analysis. Pathway and functional analysis of DEG was conducted using the ingenuity pathway analysis (IPA) software . Canonical pathway analysis, disease and biological function analysis and prediction of upstream regulators were conducted on the data set. While both canonical pathway analysis and biological function analysis predict affected biological events, the upstream regulator analysis identifies the cascade of upstream transcriptional regulators that can explain the observed gene expression changes. IPA results were filtered using P < 0·05 (Fisher’s exact test) and a Z-score > 2 or higher depending on the need for reducing the data set. While the p value filtering reflects the likelihood of a pathway/function being significantly affected by the treatment, the Z-score describes the likelihood of a directional association in our data set. Positive and negative Z-scores imply an increase or decrease in activity/activation of the enriched pathway, respectively.Gene expression profiling was conducted using samples from IN4, based on our previous finding that the distal intestine harbours the most active immune functions on separate RNA aliquots from all samples used for RNA sequencing. Complementary DNA synthesis, qPCR assays, primer design, optimisation and validation were carried out as described previously. For reference gene selection, we searched for stably expressed genes in the RNA sequencing data set by employing the following filter strategy: base mean > 200, log2fold < 0·01 and > 0·01. Three putative reference genes  were selected from the list, and gapdh2 was ultimately used as the only reference gene according to its stability in different diet groups. Target gene expression was normalised according to Muller et al.. Details of qPCR assays are given in In addition to RNA sequencing, quantitative real-time PCR (qPCR) was used as an independent gene expression profiling method to examine genes of interest in detail. Assays were carried out according to MIQE standards1/3 – IBW1/3) × (ΣD°)-1, where IBW and FBW are the initial and final body weights (tank means), and ΣD° is the thermal sum (feeding days × average temperature in °C).Thermal growth coefficient (TGC) was calculated as TGC = 1000 × (FBWThe specific growth rate (SGR) was calculated using the tank means for initial body weight (IBW) and final body weight (FBW) as follows: SGR = [(ln FBW – ln IBW) /number of days] × 100.3 *100, where FBW and FL are final body weight and final length.Condition Factor (CF) was calculated as CF = FBW⁄ FLOrganosomatic indices were calculated as percentages of the weight of the organ in relation to body weight.. Data that violated either normality of distribution of residuals or homogeneity of variance were subject to Box–Cox transformation.Data were processed using R  in the integrated development environment Rstudio . Tank effect was firstly examined by point plot followed by comparison of mixed-effect models. As tank effect in this study was not significant for any of the biomarkers, individual fish was used as the statistical unit. Data were then diagnosed for normal distribution of residuals and homogeneity of variance according to Kozak and PiephoP < 0·05.Two-way ANOVA was used for statistical evaluation with diet and gut sections as class variables. When the interaction of factors was significant, one-way ANOVA followed by Tukey’s test was applied to investigate effects of diets in individual gut segments. Kruskal–Wallis test was applied for those data that could not meet normal distribution of residuals, followed by Dunn’s multiple comparison test if significant. Differences were considered significant at Post hoc analysis for significant test results was conducted using the Chisq.post.hoc test (Fifer package in R). Differences were considered significant at P < 0·05.Differences in histological scores for the various evaluated morphological characteristics were analysed using ordinal logistic regression. When score differences were only two levels, statistical significance was assessed using the Fisher’s exact test. In accordance with the goals of the present work, presentation of the results are organised in two subchapters. The first reports results regarding the general physiology of the intestine of the Ballan wrasse, the second effects of the prebiotic and the saponins, alone and in combination..The histological observations showed that both branched and unbranched mucosal folds appeared in the intestine of Ballan wrasse, and the branching occurred in all regions of the intestinal tract. The branching was mostly simple bifurcation , and theTrypsin activity, analysed in the intestinal chyme, LAP and maltase activity in homogenates of the mucosa of the measured segments demonstrated digestive capacity throughout the intestinal tract, with a decreasing trend towards the distal part of the intestine . Bile saInitial and final weight and length, and condition factor (CF), TGC, and SGR are presented in Results for the blood plasma variables are presented in Adding the prebiotic alone (Pre) to the Ref diet did not alter the cellularity of the lamina propria  nor the In some individuals, marked infiltration of eosinophilic granular cells and increased rodlet cell numbers in the mucosal epithelium were observed in IN1 and IN2. However, the changes did not show relationship to dietary treatments.The results for maltase capacity along the intestine, observed for the sections IN1, 2 and 4, are presented in The Pre diet, compared to the Ref diet, elevated chyme bile salt levels significantly in IN2 and showed the same trend for IN1 and IN3 . NeitherFor dry matter and trypsin activity in the intestinal content, no diet effects were observed . The tryS6.As illustrated in histone h2a in gene regulation, mtrf1l in translation termination, l1td1 in stem cell function, mks1 in cell surface structure maintenance, jam3 in regulation of tight junctions, igkv4–1 in antigen binding, ly75 in antigen processing, itih3 in handling of oxidative stress, me2 in mitochondrial energy metabolism and hkdc in glucose metabolism. For the Sap treatment, in contrast, most of the top upregulated genes were involved in lymphocyte function and signalling as a response to infestation, such as neurl3, involved in protein degradation, the pro-inflammatory chemokines ccl11 and ccl4, pnp in purine metabolism, mov10b in RNA binding and the pro-inflammatory il8r1. A similar picture was observed for P + S for which neurl3, ccl11 and ccl4 were among the top abundant genes.The ten most upregulated genes (log2 fold change > 2) for fish fed the Pre diet relatively diverse in their biological function such as amdhd in histidine metabolism, blm involved in DNA damage response and ccer2 in microtubule bundling. Several of the most suppressed genes in fish fed Pre diet pointed towards cell development, differentiation and turnover, including anxa4, ccnb, daam2 and cdh2. A more uniform picture of genes responsible for nutrient digestion, transport and metabolism was evident among the most down-regulated genes in fish fed Sap and the P + S combination. In particular, reduced transcript levels of genes involved in fatty acid uptake and metabolism , lipoprotein synthesis  and sterol metabolism  were observed for fish fed Sap or P + S. Also, amino acid (slc1a4), peptide (slc15a1/pept1), starch (mgam) and vitamin A (rbp2) transporters were reduced by the Sap or the P + S.The top ten most down-regulated genes for fish fed the Pre diet belonged to diverse functional categories, such as S7 – S9). Following hierarchical clustering analysis of ‘Canonical pathways’ (Z-score < 2), ‘Disease and function’  and ‘Upstream regulators’ (Z-score < 6), the Sap group and the P + S combination group of DEG clustered together in all three analysis  suggesting that the expression fingerprint of the P + S was more similar to the Sap fed fish than to the Pre fed fish.Functional analyses of DEGs conducted by IPA software analyses revealed that several processes were affected by the individual dietary supplements (Pre and Sap) and that there was a significant interaction between prebiotics and saponin , nutrient absorption  and sterol metabolism (sqle and cyp51a1), and panel B includes genes related to immune regulation , cell proliferation (pcna), oxidative stress (cat), cell remodeling (mmp13) and mucin formation (fcgbp).Results from the qPCR analyses are illustrated in ctra gene coding for the principal precursor of the pancreatic proteases. In fish fed the Sap and P + S diets upregulation of was indicated for slc23a1, the vitamin C transporter, downregulation of one of the fatty acids transporters, cd36. Expression of fabp2, another fatty acid transporter, was downregulated only by the P + S diet. Fish fed the Sap diet exhibited a trend towards increased expression of genes involved in sterol metabolism (sqle and cyp51a1). The expression level of mrc1, a receptor mediating the endocytosis of glycoproteins by macrophages, was downregulated in fish fed all the three supplemented diets compared with the Ref diet.Regarding the genes related to the digestive processes (13A), fish fed the Pre diet showed expression not significantly different from fish fed the Ref diet, although the results seemed to indicate a trend towards upregulation of the fcgbp, did not show significant effects of any of the supplemented diets. A trend towards elevation of expression level of cd38, an enzyme synthesises cyclic ADP-ribose and NADP, as well as fcgbp, was observed in fish fed the Pre diet. Whereas, fish fed the P + S diet showed lower expression of cat, the catalase.Regarding the immune-related genes subject of qPCR analyses, most of them, including the mucin-forming-related gene, ,36. The reason for this may be the difference of fish weight, which was three times as high in the former studies as in the present, about 50–60g. On the other hand, trypsin activity and bile salt concentration of the chyme were at the same level. The same was true for the total capacity of brush border LAP and maltase. A difference between the present results and the previous was the capacities of the sections along the intestine. The explanation for this difference was most likely differences in the positioning of the division between IN1 and IN2 of the intestine, which in the two first studies were more proximal than in the present,36. The reason for this change was difficulties in defining the end of the first section, a bulb-like structure. In the latter study, the intestine was divided in four sections of similar length.The relative weights (organosomatic indices) of the intestinal sections of the fish, showing values decreasing along the intestine from 0·37 to 0·13 %, in sum comprising 1·1 % of body weight, were about half the values observed in our previous studies,36,37, i.e., the enzyme capacity in IN1 and IN2 was about 10 and 110 U/kg fish for maltase and LAP in the present study, about 10 and 50 U/kg fish in the previous ones. Also, trypsin activity and bile salt concentration in the intestinal content were similar. In the previous study, observations were not available for IN1. In IN2, the values in the present study were about 70 U and 40 mg/g dry matter, respectively, and in the previous study, the values were about 110 U and 40 mg/g dry matter.The results regarding the brush border enzyme capacity were quite similar in the present and the previous study,36,37. The results confirm that IN1 - 3 have similar absorptive capacity and that reabsorption of enzymes and bile salts mostly take as the chyme passes through IN4. Efficient absorption of nutrients in IN1 - 3 seems to be secured by retrograde movements while the feed is present in the intestine.The results for the distal most section indicated lower capacity than for the other sections, in the present as well as in the earlier studies,24,39–42. Since wide submucosa and lamina propria commonly exist in Ballan wrasse, it may not be appropriated to involve them as a sign of unhealthy status of intestine. Instead, increase of cellularity that composed of leukocytes would be the more appropriate marker of unhealthy intestine in Ballan wrasse, which has also been observed in our previous study in wild Ballan wrasse.At the histological level, Ballan wrasse possesses wide submucosa and lamina propria. Thickening of submucosa and lamina propria usually is associated with unhealthy status in fish intestine. Potentially, the influenced genes are involved in pathogen and disease resistance, and therefore, changes were not triggered without infestations, in spite that in other fish species,45 enhanced immunity was observed before pathogen challenge.The present study clearly indicated that supplementation with the prebiotics produced local effects in the gut on a molecular level, including modulation of innate and adaptive immunity. These responses were, however, not manifested in any clear histomorphological alternations in the gut. Previous studies indicate that prebiotics may strengthen disease resistanceSaccharomyces cerevisiae) extract and dried algae which contains functional ingredients such as mannan-oligosaccharides. The effects in fish would therefore be expected to be comparable to effects of mannan-oligosaccharides. In general, prebiotics appear to activate the innate immune system, directly or by enhancing the growth of commensal microbiota, but the effects seem to differ among different fish species. For instance, lysozyme expression was not altered in the present study, in line with observations in channel catfish  and European sea bass (Dicentrarchus labrax) fed mannan-oligosaccharides supplemented diets. These results, however, differ from effects observed in rainbow trout (Oncorhynchus mykiss) and red drum (Sciaenops ocellatus) in which dietary mannan-oligosaccharides supplementation caused increase lysozyme activity. Adaptive immunity can also be influenced by prebiotics. For instance, lymphocyte responses, including both T- and B-cell responses, were enhanced in European sea bass (D. labrax) fed concentrated mannan-oligosaccharides supplemented diet. The lymphocyte responses in Ballan wrasse, however, were not widely influenced by the supplemented prebiotics.The prebiotics used in the present study is a product of yeast  was particularly activated. It differentiates from CD4 + T cells together with regulatory T cells (Treg). It is demonstrated to participate in pathogen clearance and the balance of Th17 and Treg influence autoimmunity,64, and these may also be affected in the Ballan wrasse. Besides this, the type of B cells seems not to be IgM expressing, considering that igm expression showed no significant effect, but rather a decreasing trend. Instead, it might be IgT or IgD expressing B lymphocytes which, in earlier studies, have been found to be expressed in IN4 in Ballan wrasse.The fact that the gene expression profile for P + S clustered with that of Sap indicates that the modulatory effects of prebiotics were overridden by the challenging effects of the Sap. The responses to P + S differed from the responses to Pre in particular regarding intraepithelial lymphocyte infiltration. They were more similar to responses to Sap with enhanced lymphocyte responses. Based on the gene expression results, the type of lymphocytes particularly involved in the P + S treatment can be suggested. For instance, T helper cell 17 (T.However, present information regarding combination effects of prebiotics and soya saponins in fish intestine are not sufficient to conclude regarding the mechanisms underlying the combined effects of the two substances. Considering the properties of the prebiotics, it is highly likely that effects on gut microbiota were important factors. However, one study with Nile tilapia has indicated that prebiotics and saponins do not necessarily alter the composition of gut microbiotaAltogether the results of the present study indicate that the use of prebiotics should be done with care. Interaction with antinutrient effects, such as effects of saponins, might eliminate their potential beneficial effects and possibly potentiate challenging effects of the antinutrients.In the present study, the Ballan wrasse responded to dietary soya saponins in a similar way as Atlantic salmon. Observed responses included decreased cholesterol levels in blood, and clear alterations in the histomorphology and gene expression of the distal intestine indicative of a progressing inflammation with increased intraepithelial lymphocyte infiltration. The study also suggested that dietary supplementation of prebiotics had modulatory, potentially beneficial, effects on gut health and function. However, interaction with plant antinutrients such as saponins may prevent these effects.


The MCC family of genes plays a role in colorectal cancer development through various immunological pathways, including the Th17/Treg axis. We have previously shown that MCC1 but not MCC2 plays a role in Treg differentiation. Our understanding of the genetic divergence patterns and evolutionary history of the MCC family in relation to its function, in general, and the Th17/Treg axis, in particular, remains incomplete. In this investigation, we explored 12 species’ genomes to study the phylogenetic origin, structure, and functional specificity of this family. In vertebrates, both MCC1 and MCC2 homologs have been discovered, while invertebrates have a single MCC homolog. We found MCC homologs as early as Cnidarians and Trichoplax, suggesting that the MCC family first appeared 741 million years ago (Ma), whereas MCC divergence into the MCC1 and MCC2 families occurred at 540 Ma. In general, we did not detect significant positive selection regulating MCC evolution. Our investigation, based on MCC1 structural similarity, suggests that they may play a role in the evolutionary changes in Tregs’ emergence towards complexity, including the ability to utilize calcium for differentiation through the use of the EFH calcium-binding domain. We also found that the motif NPSTGE was highly conserved in MCC1, but not in MCC2. The NPSTGE motif binds KEAP1 with high affinity, suggesting an Nrf2-mediated function for MCC1. In the case of MCC2, we found that the “modifier of rudimentary” motif is highly conserved. This motif contributes to the regulation of alternative splicing. Overall, our study sheds light on how the evolution of the MCC family is connected to its function in regulating the Th17/Treg axis. The Th17/Treg axis is crucial for both promoting and repressing colorectal cancer. Th17 and Treg belong to the CD4+ T cell population , they haMCC/MCC1  plays a significant role in colorectal cancer progression. MCC1 was found to be linked to the familial adenomatous polyposis susceptibility locus on chromosome 5q. Familial adenomatous polyposis is a colorectal cancer risk factor because hereditary precancerous colorectal polyps can evolve into colorectal cancer . RecentlIn this report, we conducted an extensive phylogenetic analysis of the MCC family to investigate its role in the context of the Th17/Treg axis function in colorectal cancer. We found a single MCC homolog in various invertebrate species, including Spirlai, Arthopoda, Cnidria, and Trichoplx. During the Cambrian explosion  and the two rounds of genome duplication (2R), two homologs, namely MCC1 and MCC2, emerged. The main building blocks of MCC1 in vertebrates are two domains of MCC-PDZ and a single EF-hand domain. This structure is also found in Oedothorax gibbosus (Gibbous dwarf spider) and is partially conserved in other investigated invertebrate species, hinting toward the functional conservation between MCC and MCC1. MCC2 sequences seem to have lost EF-hand domains. Our investigation indicates that the nearest homolog to the MCC family ancestral sequence homolog is a protein containing an EF-hand domain. Moreover, motif inspection suggests that the MCC1 and MCC2 families could be playing a primary role in cell cycle regulation. Additionally, we found that the motif NPSTGE, which is known to play a role in its interaction with KEAP1, is conserved in MCC1. Our findings suggest that MCC1 could be enhancing Treg differentiation by inhibiting the KEAP1 effect.Amphimedon queenslandica. However, Blastp’s E-value was lower than our threshold of 1 × 10−10. Thus, that sequence was not accepted. Two MCC homologs were found in all vertebrate species investigated. The bony fish seems to be the first vertebrate to possess a pair of MCC homologs. These results indicate that the divergence of the MCC1 and MCC2 homologs occurred at the 2R stage during the Cumbrian explosion. Our findings indicate that all vertebrate MCC1 sequences and MCC sequences in Cnidaria, Spiralia, and Arthropoda possess two MCC-PDZ domains. In contrast, all MCC2 sequences, as well as Tunicate and Trichoplax MCC sequences, feature only a single MCC-PDZ domain. It is noteworthy that the results of domain prediction for MCC1 and MCC2 exhibited variations among the three servers employed. The NIH server predicted the existence of unique SMC domains in certain MCC1 and MCC2 sequences that were investigated. Neither PFAM nor HMM servers identified this domain in the species studied. We investigated this line further by comparing MCC1 sequences against the SMC family sequences identified using the NIH server. However, the Blastp results did not indicate a significant homology between the two groups (MCC sequences and the SMC families). Therefore, we excluded the SMC domain from our subsequent analysis. However, experimental validation could shed light on this controversial aspect.Phylogenetic analysis indicates that two rounds of duplications resulted in the divergence of MCC1 and MCC2. We downloaded human MCC1 and MCC2 protein sequences from the GEO protein repository. We utilized the Blastp server to acquire homologous proteins for MCC1 and MCC2 among twelve species. We employed Seaview to perform the multiple sequence alignment using the MUSCLE algorithm A. After Our ancestral sequence reconstruction indicates that the MCC’s nearest-most ancient homolog is an EH hand domain-containing protein. We reconstruct the ancestral sequence of the MCC family using MegaX. The sequence reconstruction was based on our generated phylogenetic tree A. We seaA study of site-specific shifts in evolutionary rates following gene duplication was performed using Sequence Harmony. Multiple sites seem to play a primary role in the specificity of the divergence between MCC/MCC1 and MCC2. One of the important residues that seems to play a critical role in determining the functional specificity between the two groups is 737-N (Asparagine) . All theWe investigated the existence of differential motifs between the MCC1 and MCC2 sequences . Severalp-Value < 0.00001. (ii) Similarly, for the branch values, for MCC1 vertebrates, the ω value was 0.89, suggesting purifying selection, without strong evidence of statistical significance. Also, in the case of the MCC2 branch, the ω value was 0.64046. On the site level, we did not detect any sites that were subjected to significant positive selection   For the global model, we used the M0 model to estimate the possibility of positive selection over the whole tree. We found the ω value calculated using PAML for the M0 model to be 0.45728, , ω > 1) .Our study provides evidence supporting the ancient origin of the MCC family. In addition to MCC1 and MCC2 homologs in vertebrates, we identified a single MCC homolog in various invertebrate species  . SelectiOur results indicate a divergence of function between MCC1 and MCC2. We have found nine motifs conserved in MCC1, but not in the MCC2 family. Additionally, 14 motifs were specific only to MCC2. However, in the case of MCC1, these functional motifs were highly conserved in most of the species investigated. For example, Mod(r) is a motif encompassing 150 residues. It is conserved in various eukaryotic proteins investigated and is homologous to the Drosophila melanogaster Modifier of rudimentary “Mod(r)” proteins . Modr’s Our research suggests that MCC1 and not MCC2 could be contributing to Treg cells’ function in colorectal cancer. In our recent publication, using microarray analysis of the Th17/Treg pathway, we revealed that several genes that regulate the cell cycle are upregulated in Treg, but not in Th17 . MCC1 waStudy limitations: Our study provides valuable insights into the regulatory role of MCC1 in Treg differentiation by utilizing extensive RNA-seq data analysis. However, further experimental studies are warranted to expand upon these findings. In vitro assays could be conducted to investigate the impact of MCC1 overexpression or deletion on Treg differentiation. Additionally, in vivo studies using adoptive transfer in appropriate mouse models, such as Rag1-/-mice, could elucidate the effect of MCC on CD4+ T cell differentiation in the context of colorectal cancer genesis and development. These complementary experimental approaches will not only strengthen the evidence supporting the role of MCC in Treg function, but also provide valuable insights for potential therapeutic interventions targeting MCC in colorectal cancer.Pan troglodytes), House Mouse (Mus musculus), Common Wombat (Vombatus ursinus), Platypus (Ornithorhynchus anatinus), Red Junglefowl , Zebrafish (Danio rerio), Sea Squirt , Arthropoda, Spiralia, Cnidaria, Trichoplax, and Sponge (Amphimedon queenslandica) [−10 [This study aimed to examine the relationship between the molecular evolution of the MCC family and its functions. We reasoned that due to the diversity of this protein family, studying protein sequences rather than DNA sequences might be more informative. Furthermore, we selected 12 species and genera that span more than 500 million years to ensure that our analysis is a fair representation of the evolutionary history of the MCC family. BLASTP searches were conducted using human MCC1 and MCC2 protein sequences against the proteomes of Chimpanzee (landica) . Whenevelandica) . Sequencca) [−10 . ConservThe phylogenetic investigation was performed in three stages. First, MCC family amino acid sequences were aligned in Seaview using MUSCLE. After that, we employed PhyML to determine the best phylogenetic tree to represent the interrelationships among the MCC family homologs. We used the LG model with empirical values calculated for amino acid equilibrium frequencies . Invariahttp://elm.eu.org/ accessed on 30 September 2022 with a motif cut-off value of 100. We also conducted a motif search using www.genome.jp accessed on 3 October 2022 [We used Sequence Harmony and Multi-RELIEF to identify functional differences within the MCC subfamilies that may contribute to the disparity in function within the Th17/Treg axis. Sequence Harmony compared the two groups of sequences  to identify the variable amino acids and their distribution frequency. The positions, where the amino acid compositions differ between the two groups, were assigned low score values. A score of zero indicates distinct amino acids at that position, while a score of one signifies nearly identical amino acid compositions. Multi-RELIEF predicts the residue specificity of residues by performing two comparisons. The first comparison is between each sequence and its nearest homolog within the same group, and the second comparison involves comparing each sequence with its closest homolog in the second group. A residue is considered specific if it exhibits a high specificity score in at least one of the two comparisons. Additionally, we conducted an extensive motifs search using an ELM server ber 2022 .https://www.ebi.ac.uk/Tools/st/emboss_backtranseq, accessed on 5 September 2022) to estimate the cDNA of the investigated sequences [2 test [We utilized the maximum likelihood approach method to investigate the selection process that governed the MCC family’s evolution. We back-translated the downloaded protein sequences using the EMBOSS server (equences . After t [2 test .


Longitudinal information is needed on population-level incidence, prevalence and frailty progression to plan services to meet future population needs.retrospective open cohort study using electronic health records of adults aged ≥50 from primary care in England, 2006–2017. Frailty was calculated annually using the electronic Frailty Index (eFI). Multistate models estimated transition rates between each frailty category, adjusting for sociodemographic characteristics. Prevalence overall for each eFI category  was calculated.the cohort included 2,171,497 patients and 15,514,734 person-years. Frailty prevalence increased from 26.5 (2006) to 38.9% (2017). The average age of frailty onset was 69; however, 10.8% of people aged 50–64 were already frail in 2006. Estimated transitions from fit to any level of frailty were 48/1,000 person-years aged 50–64, 130/1,000 person-years aged 65–74, 214/1,000 person-years aged 75–84 and 380/1,000 person-years aged ≥ 85. Transitions were independently associated with older age, higher deprivation, female sex, Asian ethnicity and urban dwelling. Mean time spent in each frailty category decreased with age, with the longest period spent in severe frailty at all ages.frailty is prevalent in adults aged ≥50 and time spent in successive frailty states is longer as frailty progresses, resulting in extended healthcare burden. Larger population numbers and fewer transitions in adults aged 50–64 present an opportunity for earlier identification and intervention. A large increase in frailty over 12 years highlights the urgency of informed service planning in ageing populations. Frailty is already present in the population before age 65.Longer times spent in moderate and severe frailty suggest extended burden of disease.Frailty progresses more rapidly with increasing age, resulting in high prevalence.Frailty transitions are associated with increasing age, higher deprivation, female sex, Asian ethnicity and urban dwelling.Strategies to reduce the burden of frailty need to consider health inequalities.As populations age, healthcare systems must identify ways of meeting changing needs while managing growing demand . This isIt is estimated that 1.8 million people in the UK aged ≥60 were living with frailty in 2016 . InternaFurther evidence on frailty progression within the ageing population is needed from large-scale population studies designed for this purpose. This study addresses these evidence gaps using longitudinal analyses to explore expected transitions within the older population in primary care. This work builds on what is known about individual risk of frailty onset and progression.This study is part of a larger programme of work, which aims to explore and predict trends in onset, prevalence and progression of frailty, and the dynamics of frailty-related healthcare demand, outcomes and costs in the ageing population. This paper presents results relating to prevalence and transitions into and between frailty states in people aged ≥50 over 12 years. Transition probabilities reported here will inform the development of a predictive simulation model for use in estimating service demand and outcomes in the ageing population.Retrospective open cohort study using EHR from the Royal College of General Practitioners (RCGP) Research and Surveillance Centre (RSC) sentinel network, which at the time of the study collated routine primary care data from >500 GP practices in England and is nationally representative .Primary care patients, aged 50 years and above, registered at General Practitioner (GP) practices contributing to the RCGP RSC databank between 2006 and 2017 were eligible. The sample size was maximised to allow robust analysis by age and other sub-groups of interest. This was achieved by using retrospective data from the most recent complete year at the point of data extraction and all preceding years with the availability of study variables, a total of 12 years. The open cohort design enabled the addition of eligible patients who turned 50 or moved to a participating practice and were present on 1st January of a calendar year during the study period. Patients left the cohort by leaving participating practices or death. The cohort comprised 2,177,656 patients from 419 GP practices across England (previously described in ). PatienThe primary outcome of frailty was measured by calculating an eFI score  from elehttps://www.gov.uk/government/collections/rural-urban-classification); date of cohort entry; date of cohort exit (leaving a contributing practice or death). Age groups were chosen to reflect those commonly used in the frailty literature, allowing for exploration of the middle-aged to younger old and presentation of findings in groups relevant to service planners. The dataset was supplemented by linked data from the UK Office for National Statistics  and NHS Digital (ethnicity data). Ethnicity data were derived from both primary and secondary care data to minimise missing data according to the 16 categories in the NHS data dictionary (https://www.datadictionary.nhs.uk/data_elements/ethnic_category.html). To enable suitably sized categories for analysis, the categories were further aggregated into Asian, Black, Mixed/Other and White, where known.The RCGP RSC dataset included: age category ; sex; ethnicity; 2015 indices of multiple deprivation (IMD) quintiles ; income Frailty prevalence was calculated as per the eFI on January 1st for each calendar year, including all persons present in the cohort at that date, and stratified by age group and severity. Frailty incidence rates (new onset frailty of any severity) were calculated per 1,000 person-years across the 12-year study period.Continuous time multi-state Markov (MSM) models were used to estimate transition rates between states and identify determinants of frailty progression . VariablP values <0.05 were considered statistically significant and estimates are presented with 95% confidence intervals (CIs) where appropriate.An initial unadjusted MSM model was fitted to estimate average annual transition probabilities. Multivariable models were then fitted to assess the impact of the key sociodemographic variables of age group, sex, ethnicity , deprivation (grouped as the two most deprived quintiles versus the 3 least deprived) and rural/urban location on these transitions, in a forward selection process. The Akaike information criterion and likelihood ratio test were used to compare and choose between models. SAS version 9.4, R version 4.2.0 and Stata version 16.0 software were used for data manipulation and statistical analyses. The R msm package version 1.6.9 was used for the MSM modelling . P valueThe study was approved by the University of Southampton Research Ethics Committee (ref 46313) on 6 February 2019, the RCGP RSC Information Governance Panel on 24 January 2019 and NHS Digital’s Data Access Request Service (DARS) Independent Group Advising on the Release of Data (IGARD) panel on 19 April 2021.The cohort comprised 2,171,497 patients, contributing 15,514,734 person-years of data, with a median follow-up of 7 years (interquartile range 7 years).The average age of onset for frailty (any category) for patients who were fit at cohort entry was 69 years (Standard deviation (SD) 10 years). The overall frailty incidence rate was 47.1 cases per 1,000 person-years (95% CI 47.0–47.2). Crude incidence was higher in older age groups, female sex, Asian ethnicity, more deprived quintiles and people living in urban areas . Incidenn = 709,377) of the cohort over a median follow-up of 7 years. The average age of transition from fit to mild was 69 years (SD 10 years), fit/mild to moderate was 77 years (SD 10 years) and any category to severe was 81 years (SD 9 years).The analysis demonstrated at least one transition between frailty categories in 32.7% , with increasing prevalence within each age group as individuals aged. Our overall crude incidence rate of 47.1 per 1,000 PYAR is consistent with previous pooled estimates of 43.4 per 1,000 PYAR. The finding that at least one in 10 people aged 50–64 are already frail is noteworthy, as is the scale of change in prevalence within the study period, with significant increases in moderate to severe frailty in all age groups, tripling in the 50–64 age group and approximately doubling in all others. The estimated incidence in the younger age group was also higher than expected, at 31.8 per 1,000 PYAR.The multi-state model demonstrated an increasing speed of transitions with age, consistent with studies predominantly using phenotypic frailty assessments , 35. WitEarly onset in deprived groups, combined with the length of time that people spend in the severely frail state, suggests a long period of need associated with frailty and the importance of prevention across the life course to address inequity in frailty burden. Higher frailty onset and progression in people of Asian ethnicity explain differences in the prevalence of frailty with ethnicity observed in a London cohort [10], and suggest that tailored approaches for different communities may be important. The higher transition rate in people living in urban areas indicates that geographical considerations might also be important, in line with recent results from a small English cohort which suggested that coastal communities might be at higher risk . OverallThese findings are likely to be of particular importance when planning services, given that most frailty services are currently targeted at those aged 65 and above. Although high prevalence of severe frailty in older age groups is the focus of considerable policy and practice attention, our analyses demonstrate that absolute numbers of younger old people with mild and moderate frailty exceed those of older people with severe frailty. This suggests that population-level preventative strategies are needed and could have more impact than a focus on severe frailty. It is also important to note that our analysis demonstrates that older age groups transition to higher levels of frailty faster than middle-aged adults. A population-level approach to prevention of frailty or slowing of frailty progression earlier in the life course is therefore likely to be a key strategy for long-term reduction of population morbidity, disability and service use. Further work is necessary to understand points in the frailty trajectory where intervention will have most impact at population level. Although it has been shown that people with more severe frailty have higher healthcare costs , analysiThis analysis suggests a higher prevalence in people aged 50–64 than reported elsewhere utilising different frailty indices and data sources , but othThe calculation of the eFI score depends on the quality and completeness of the EHR data, which can be influenced by policy and practice conventions. Increasing prevalence could have been driven by more complete coding of eFI deficits, although methods of calculating the eFI in English general practice were introduced in 2016, so would be unlikely to have affected coding during the study period. Similarly, Quality and Outcomes Framework coding, introduced during this time period, might have increased recording of diseases that contribute to the eFI score. However, no sharp changes in prevalence that might suggest coding impact were observed. The use of the most recent address in identifying urban/rural location could mask selective migration between residential areas and types driven by frailty status.The eFI is a cumulative measure based on conditions recorded in the EHR; although reversals are possible, in clinical practice it is uncommon for clinicians to remove codes from the record, other than for medications (polypharmacy). For this reason, we found few improvements in frailty status over time. The eFI has, however, been shown to demonstrate progression in frailty in longitudinal studies , 23, a pEthnicity data were incomplete within primary care, so were supplemented with linked secondary care data. The two data sources used different ethnicity categories, necessitating broad aggregation of ethnicity groups. Categories were further collapsed for the MSM due to computational demand. Despite this, the model demonstrated that ethnicity was an independent factor in transitions, suggesting that this is an area worthy of more detailed study.This study is unique in exploring frailty transitions in an ageing population, including people aged ≥50, and demonstrates frailty is already prevalent before age 65. It provides new evidence on the rate of decline within an ageing cohort, demonstrating frailty transitions were independently associated with both older age and socioeconomic factors. We provide new evidence about high prevalence of frailty within the ageing population and the social inequity in patterns of frailty. Slower transition rates in middle age and earlier onset in some groups might present an opportunity to reduce health disparities through for earlier identification and multisectoral intervention to slow progression and reduce care needs.aa-22-1458-File002_afad058Click here for additional data file.


Diabetes causes impaired microarterial blood flow, demyelination and neuronal damage, which may lead to cochlear damage and vestibular malfunction. Vestibular evoked myogenic potentials (VEMP) is a simple, reproducible test. Cervical and ocular vestibular evoked myogenic potentials (cVEMP and oVEMP) can be explored in the saccadic-spinal and utriculo-ocular pathways in regular clinical practice.To evaluate possible vestibular evoked myogenic potential (VEMP) abnormalities in patients with diabetic peripheral neuropathy.89 patients with Type 2 Diabetes in the present study consisted of three groups: 29 patients with no peripheral neuropathy (NDPN group), 26 patients with asymptomatic neuropathy (SDPN group), 34 patients with symptomatic neuropathy (DPN group). Meanwhile, 42 healthy subjects were recruited as controls. The clinical characteristics , and illness duration), as well as lipids (including triglyceride (TG), cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)), uric acid, fasting blood glucose (FBG), and glycated hemoglobin (HbA1c) were compared among the four groups. Four groups were assessed using two vestibular tests including oVEMP and cVEMP. Latency and amplitude parameters were analyzed from VEMP plots.https://fyey.shinyapps.io/VEMP_Model/ ).The latency of n10, p15 (oVEMP), p13, n23 (cVEMP) were significantly prolonged in the SDPN and DPN groups compared with the control and NDPN groups (p < 0.01), whereas latencies were similar in NDPN and the control groups. The amplitudes were not significantly different (p > 0.05). oVEMP latency p15 and cVEMP latency  were positively correlated with HbA1c, FBG, and illness duration, and oVEMP latency n10 was positively correlated with HbA1c and FBG. A nomogram, including FBG, HbA1C, HDL, TG, TC, LDL and group, was constructed to predict VEMP parameters and p13 was found to be independently associated with diabetic subgroups. Receiver operating characteristic curve (ROC) analysis showed good accuracy in predicting p13 in this nomogram. A user-friendly website has been created to facilitate the application of this prediction model ( Patients with diabetic peripheral neuropathy may have vestibular dysfunction. VEMP may be useful in assessing vestibular impairment in diabetic patients. Vestibular evoked myogenic potential (VEMP) is an objective method for assessing the otolithic apparatus and vestibular nerve conduction pathways in the vestibular system, such as the utricle and saccule, and is a myogenic potential recorded after stimulation of the otolithic apparatus by high-intensity acoustic signals . DependiDiabetes mellitus is a metabolic disorder of multiple etiologies, characterized by chronic hyperglycemia and long-term complications . In ChinThe detection of vestibular function in diabetics has received attention due to the increasing recognition of the impact of vestibular impairment on imbalance and falls in diabetics. Diabetic patients may develop vestibular dysfunction in the absence of significant vestibular symptoms . Many reThis study was conducted at the Second Hospital of Fujian Medical University , in accordance with the guidelines of the Declaration of Helsinki, and was approved by the Ethics Committee of the Second Hospital of Fujian Medical University [Ethics Approval No.: Lun Audit (Research) No. 213 of 2021].89 patients with type 2 diabetes who visited the Second Hospital of Fujian Medical University from April 2021 to November 2021 were enrolled in this study, including 57 males and 32 females with a mean age of (53.0 ± 11.9 years). There were 42 cases in the healthy control group, including 23 males and 19 females, with a mean age of (52.3 ± 5.6) years. , including nerve conduction velocity measurement, cVEMP test, and oVEMP test. All subjects were tested in a quiet environment.All enrolled patients were examined by a neurologist specializing in nerve conduction velocity. Diabetic peripheral neuropathy was diagnosed using the diagnostic criteria of the Expert consensus on the diagnosis and management of diabetic neuropathy (2021 Edition) . DiagnosThe diagnostic criteria for diabetic peripheral neuropathy include the following three points: (1) definite presence of diabetes; (2) presence of clinical and/or electrophysiological evidence of peripheral neuropathy; and (3) exclusion of other causes of peripheral neuropathy . The cli[13]]]13]]. The presence of cVEMP was confirmed when reproducible short-latency biphasic waveforms appeared at certain latencies. The latencies of p13 and n23 were measured. Typical cVEMP waveforms were bidirectional, with the positive wave latency near 13 ms, labeled p13, and the negative wave appearing near 23 ms, labeled n23. The recorded EMG activity was corrected, which reflects the reflected potential originating from the saccule. (2) oVEMP test: The recording electrode was located just below the lower lid margin of each eye in line with the pupil. Subjects were asked to gaze maximally upward by looking at the target point located 60–70 cm from the eyes with a visual angle of 25°, and oVEMPs were recorded from the contralateral extraocular muscles [VEMP test was executed using a Keypoint  with the following settings and equipment: 2 channels; The velocity is 10 ms/D; sensitivity 0.2 mV/D; Filters range from 5 Hz to 5 kHz; click sound (300–500 stimuli for 0.1 ms at 5 Hz each); single-channel calibrated stereo headphones and surface electrodes. Consecutive runs were performed to confirm the reproducibility of biphasic waveform for cVEMP and oVEMP. Conversely, cVEMP and oVEMP responses were absent when reproducibility was lacking. The presence of a VEMP reaction was evaluated, and the parameters included latencies (ms) and amplitudes (mV). (1) cVEMP test: The patient was placed in the supine position. Two active electrodes were placed on the upper two thirds of the bilateral sternocleidomastoid (SCM) muscles with the reference electrode on the sternoclavicular joint. Subjects were instructed to lift the head from the pillow so that the sternocleidomastoid muscle contracted and maintained a muscle tension greater than 50 uV. Thereafter, the examiner adjusted the headset stimulus to 135 dB SPL for an average of approximately 300–500 repetitions until a steady waveform was obtained and labeled p13, n23  muscles . The n10 muscles .p-value < 0.05 was considered to indicate statistical significance.The data was analyzed using SPSS software(Version 26) and R (version 4.2.2). The quantitative results were presented with the mean ± standard deviation. Data between groups were analyzed by one-way ANOVA, and multiple comparisons were executed by LSD method. The elicited rates of oVEMP and cVEMP testing were compared in two groups by chi-square test. Correlation coefficient and prediction capability were assessed by Pearson correlation analysis and ROC analysis, respectively. The “ggplot2” and “Regplot” R packages were used to draw figures. DynNom: Visualising statistical models using dynamic nomograms. A The demographic features and hematological examination of the groups are shown in Table 2 = 1.54; cVEMP: χ2 = 2.70, P > 0.05).A total of 84 ears were used in healthy controls, of which 80 ears elicited oVEMP (elicitation rate of 95.2%) and 82 ears elicited cVEMP (elicitation rate of 97.6%). Diabetic subjects had a total of 178 ears, of which 160 ears elicited oVEMP (elicitation rate of 90.1%) and 163 ears elicited cVEMP (elicitation rate of 91.6%). There was no significant difference in oVEMP and cVEMP elicitation rates in healthy controls compared with diabetic subjects . If you press Predict, the Graphical Summary will indicate ERROR: An error has occurred. Check your logs or contact the app author for clarification”, then press “Quit”.We used a Cox regression model to determine the factors that affect the VEMP parameters of patients with Type 2 Diabetes to construct a nomogram. FBG, HbA1C, HDL, TG, TC, LDL, and group were included in the nomogram. The nomogram is displayed in Fig. In this study, vestibular function was assessed in patients with type 2 diabetes. The results indicated that the latency  was prolonged in patients with the SDPN group and DPN group, compared to the NDPN group and control group. The latency was correlated with FBG, HbA1c, and duration of illness. VEMP abnormalities are present in patients with diabetic peripheral neuropathy.The maintenance of the body’s balance posture involves the combined action of the vestibular organs, visual senses, and proprioception . The patThe detection of diabetic vestibular function has received attention as the impact of vestibular impairment on balance and falls in diabetic patients has been progressively recognized. Different tests, such as Pure Tone Audiometry, Video Head Impulse Test, VEMP, are used to assess vestibular function. The VEMP is a short-latency evoked potential that allows non-invasive assessment of the vestibular-collic and vestibular-ocular pathways. It is divided into cVEMP and oVEMP. The cVEMP assesses the saccule, inferior vestibular nerve, and sternocleidomastoid muscle. oVEMP assesses the utricle and superior vestibular nerve function . VEMP evRecently, the function of the vestibular system in diabetics has been investigated using VEMP. Data on VEMP in diabetics is still limited. In a previous report by Kalkan et al. , latenciVEMP appears to be more significant in the detection of subclinical lesions. In the present study, subjects with vestibular symptoms were excluded to investigate vestibular involvement in patients with diabetic peripheral neuropathy without significant vestibular symptoms. Rigon et al.  reportedCurrent studies on the effects of diabetes duration and glycemic control on vestibular function are inconsistent. Agrawal et al.  concludeDiabetic microvascular complications have so far been defined as retinopathy, nephropathy and neuropathy. Polyneuropathy and retinopathy in diabetes mellitus lead to an increased risk of falls due to gait unsteadiness among many patients. However, the risk of falls and dizziness in DM patients should also take into account the contribution of damage to the vestibular end organs. Vestibular function impairment as an important complication of diabetes mellitus, leading to balance dysfunction in diabetic patients and severely affecting the quality of life. The results of oVEMP and cVEMP assays may be affected by metabolic factors. By studying the characteristics of vestibular function changes in diabetics, it is beneficial to increase the understanding of vestibular impairment in diabetics, provide a basis for early detection and rehabilitation of vestibular impairment in diabetics, reduce the incidence of falls in diabetics, and improve quality of life.


Background and objectivesSleep quality and nicotine addiction are important public health issues with significant negative impacts on individual well-being and the performance of healthcare professionals. This study aims to determine the prevalence and association of nicotine dependence and poor sleep quality among residents and fellows enrolled in the Lebanese University.MethodsA cross-sectional study using the snowball-sampling technique was conducted in Lebanon between January and March 2023. Data were collected through an online survey that included information on socio-demographic characteristics, nicotine dependence, and sleep quality. A total of 350 residents and fellows were included in the study. Bivariate analysis and multivariable logistic regression were carried out to identify the factors associated with sleep quality. Adjusted odds ratio and 95% confidence intervals were reported.ResultsOne quarter (25.1%) of the residents and fellows were smokers; among them, 44.3% smoked I Quit Ordinary Smoking (IQOS), 14.8% smoked cigarettes, 10.2% smoked waterpipe (WP), 12.5% smoked cigarettes and WP, and 18.2% smoked IQOS and WP.adj = 12.58 with 95% confidence interval [CI] of 7.07-22.36; p-value <0.001). In addition, smoking a combination of two types of tobacco products (cigarettes with WP or IQOS with WP) posed the highest risk of poor sleep quality, with an adjusted odds ratio of 31.54 .According to the Pittsburgh Sleep Quality Index (PSQI), 34.3% of participants had poor sleep quality. Smokers had 12.5 times higher odds of experiencing poor sleep quality compared to non-smokers .Elevated Fagerström Test for Nicotine Dependence (FTND) and Lebanon Waterpipe Dependence Scale (LWDS-11) scores indicated an increased risk of poor sleep quality (adjusted odds ratio ORConclusionOur study found a significant association between nicotine dependence and poor sleep quality among medical residents and fellows, with smokers being more susceptible to sleep disturbances. The high prevalence of IQOS smoking among medical residents and fellows in Lebanon highlights the urgent need for comprehensive research investigating the effects of heated tobacco products. Furthermore, our study reveals a critical insight into the potential additive effects of nicotine, suggesting that the concurrent use of multiple tobacco products may further elevate the risk of poor sleep quality. Recognizing the implications of our findings, it is imperative to develop targeted interventions and educational programs that promote healthier sleep habits and facilitate smoking cessation among medical residents and fellows. The global prevalence of tobacco smoking and nicotine addiction has been a persistent concern, giving rise to a myriad of health-related challenges ,2. It isIn addition to its association with several pathologies, especially cardiovascular diseases and cancers, nicotine dependence can have negative impacts on cognitive function and job performance, with several studies linking tobacco use to decreased concentration, impaired memory, and slower reaction times ,3. TheseNicotine addiction can also disrupt the normal sleep cycle by altering the levels of neurotransmitters in the brain, such as acetylcholine, dopamine, and serotonin which are responsible for regulating sleep and wakefulness . A studyThe impact of poor sleep quality and nicotine addiction on the health and cognitive function of physicians has been well documented. Vela-Bueno and his colleagues found that physicians with poor sleep quality were more likely to report lower levels of job satisfaction and higher levels of burnout ; similarThere are various mechanisms through which smoking and nicotine dependence can impact the quality of sleep. Nicotine acts on the central nervous system (CNS) as a stimulant that interferes with the natural sleep-wake cycle of the body resulting in increased time taken to fall asleep. It can also elevate heart rate and blood pressure, making it challenging to initiate and maintain sleep . AdditioA multitude of tobacco products are widely available, with cigarettes, waterpipes (WPs), and IQOS smoking being the most commonly used among the Lebanese population. WP smoking can be a significant source of nicotine, as a single session with unflavored tobacco can contain the equivalent nicotine of 70 regular cigarettes . On the Unlike previous studies that focused solely on nicotine addiction or sleep quality among medical students, and typically included only the first three years of academic medicine, our study aims to investigate the prevalence of both poor sleep quality and nicotine addiction, their association as well as the potential moderating effects of age, gender, coffee consumption, medical year, physical activity, years of smoking, and the type of tobacco product used.Study design and populationA cross-sectional study was conducted at the Faculty of Medicine at the Lebanese University between January and March 2023 using an online questionnaire. All residents and fellows enrolled at the Lebanese University for the academic year 2022-2023, working in different hospitals in Lebanon, and who agreed to participate in the study were included. However, the following participants were excluded from this study: being pregnant or lactating, having a known history of sleep disorders such as insomnia, parasomnia, obstructive sleep apnea, restless legs syndrome, or narcolepsy, having any type of addiction other than nicotine, and currently suffering from an ongoing illness.Sample size calculationThe minimum sample size was calculated using an online minimum sample size generator. Assuming that the number of fellows and residents for the academic year 2022-2023 at the Faculty of Medical Sciences was estimated at 440, the required calculated sample size was 206 with a confidence level of 95%, a 5% margin of error, and 50% of expected prevalence. Data collection and instrumentationData were collected using an online survey. The questionnaire was administered via Google Forms, and a direct link was sent in both English and Arabic languages to approximately 440 participants via social media using the snowball technique. We collected a total of 360 responses, of which 10 were excluded for ineligibility.The questionnaire was divided into four sections. The first section included baseline characteristics including age, gender, medical year, coffee consumption, physical activity, number of shifts covered, smoking status, years of smoking, and tobacco products. The smoking status included two groups: smokers (those who currently used tobacco products at least once in the past 30 days) and non-smokers .The second section included the Fagerström Test for Nicotine Dependence (FTND), a widely used tool to assess the severity of nicotine addiction . It contIn the third section, the Lebanon Waterpipe Dependence Scale (LWDS-11) was used as it is specifically designed to assess the level of dependence on WP smoking, especially among the Lebanese population. It includes 11 items measured on a 4-point Likert scale ranging from 0 to 3. High WP dependence was defined as having a score of LWDS-11 ≥10 . In a stFinally, the last section included the Arabic Pittsburgh Sleep Quality Index (PSQI) which is a reliable and valid tool that measures sleep quality over the last one-month period. It consists of 17 self-rated questions that are subsumed within seven component scores: (1) subjective sleep quality, (2) sleep latency, (3) sleep duration, (4) habitual sleep efficiency, (5) sleep disturbances, (6) use of sleep medications, and (7) daytime dysfunction due to sleepiness. Each component was scored 0-3. A global score can be obtained from the summation of the seven components that range from 0 to 21 with higher scores denoting poorer sleep quality ,19. The Pilot studyThe survey was pilot tested in a sample of five residents to check the clarity and readability of all items. They did not report any problems in understanding the questionnaire. On average, the survey was completed within approximately 10-15 minutes. The data from the pilot study were removed from the final analysis.Statistical analysisStatistical analysis was carried out using the Statistical Package for the Social Sciences (SPSS) version 22 . Descriptive statistics were reported using means and standard deviations (SD) for continuous variables and frequency with percentages for categorical variables. Statistical bivariate analysis was performed using Pearson’s chi-square test for categorical variables and an independent student t-test for continuous variables.Forward stepwise binominal logistic regression was used to determine the predictors of sleep quality. Adjusted odds ratio and 95% confidence intervals were reported. The logistic regression model was reached after ensuring the adequacy of our data using the Hosmer and Lemeshow test. A p-value <0.05 was considered statistically significant.Characteristics of the study participantsThe baseline characteristics of our study population are shown in Tables The majority of participants (80.6%) reported that they drank one cup of coffee per day, while 2% drank 2-3 cups per day, and 17.4% did not drink coffee at all. One quarter (25.1%) of the participants were smokers, among them 44.3% smoked I Quit Ordinary Smoking (IQOS), 14.8% smoked cigarettes, 10.2% smoked WP, 12.5% smoked cigarettes and WP, and 18.2% smoked IQOS and WP. Participants who smoked cigarettes or IQOS  completed the FTND score, the overall mean FTND score was 5.30 ± 1.60. Similarly, those who smoked WP  completed the LWDS score, and the overall mean LWDS score was 14.91 ± 6.58.In our sample, the internal consistency index obtained by means of Cronbach’s alpha coefficient was 0.71 for the FTND score, 0.93 for the LWDS-11 score, and 0.92 for the PSQI score.Table The association between the characteristics of the participants and sleep qualityTable Our results showed that post-graduate year, smoking status, and type of tobacco products are highly associated with sleep quality .Moreover, we found that participants who had exhibited a high mean FTND (5.77 ± 1.47) score and high mean LWDS score (16.26 ± 6.11) had poor sleep quality compared to those with mild nicotine dependence .Our findings reveal no statistically significant association between sleep quality and age, gender, daily consumption of coffee or caffeinated drinks, physical activity, number of night shifts covered over the last month, and years of smoking .Factors associated with poor sleep qualityResults from the logistic regression showed that smoking status, type of tobacco products, FTND, and LWDS scores were significantly associated with poor sleep quality. Smokers had 12.5 times higher odds of experiencing poor sleep quality compared to non-smokers . Among smokers, the combination of two types of tobacco products (cigarettes with WP or IQOS with WP) posed the highest risk of poor sleep quality, with an adjusted odds ratio of 31.54 . Smoking only IQOS was also associated with poor sleep quality, with an adjusted odds ratio of 11.43 . Our results also showed that PGY2, PGY3, and PGY4 had poor sleep quality compared to PGY1  . Furthermore, linear regression analyses showed that poor sleep quality was positively associated with nicotine dependence . Our results among smokers also showed that cigarettes and WP or IQOS and WP smokers experienced poor sleep quality compared to cigarette smokers .To the best of our knowledge, this study represents the first examination of the prevalence of poor sleep quality and nicotine addiction, as well as their association, among Lebanese residents and fellows. Additionally, it addresses a significant knowledge gap regarding the prevalence and health implications of IQOS smoking, which remains understudied in Lebanon and the wider Arabic world, despite its popularity among young individuals. Given the absence of stringent smoking restrictions and tobacco advertising regulations in Lebanon, it becomes imperative to conduct research starting with future physicians who play a crucial role in guiding and educating the general population about the multifaceted risks associated with tobacco smoking ,21.The smoking prevalence among our study population was approximately 25%, which aligns with the prevalence reported by Chidiac et al. in their 2016 study among Lebanese medical students . FurtherInterestingly, we have found that individuals who used a combination of two tobacco products exhibited higher odds of having poor sleep quality in comparison to those using a single type of product, indicating a possible additive effect of nicotine on sleep quality, particularly among individuals using multiple tobacco products. Notably, no previous studies have investigated the specific impact of smoking one versus two types of tobacco products, underscoring the novelty and significance of this study in shedding light on this relationship.Regarding the type of tobacco product used, it is surprising to find that IQOS smoking was more prevalent than cigarettes and WP. IQOS represents a relatively new and increasingly popular tobacco product in Lebanon, yet no prior studies have examined its prevalence in the country .These results emphasize the need for interventions targeting smoking cessation among medical residents. By quitting smoking, residents can improve their overall well-being, sleep quality, and job performance within the hospital setting, thereby reducing the risks of errors and mismanagement caused by sleep deprivation .Our study has some limitations. Due to the snowball sampling strategy, the findings did not represent all the Lebanese residents and fellows and therefore we cannot generalize our results, and it is merely impractical to extrapolate these findings to other Lebanese residents and fellows with different demographic characteristics. However, we intended to choose students from the Lebanese University, which is the only public university in Lebanon that encompasses students from all regions of Lebanon, and it has experienced a significant increase in enrollment over the years . In factMoreover, the reliance on self-reported questionnaire subjected the study to information bias. Although a validated instrument was used for assessing sleep quality, objective methods of measuring sleep quality such as actigraphy and polysomnography may strengthen the results.Finally, the cross-sectional nature of the study can only demonstrate association and not a cause-effect relationship. Therefore, it is not possible to ascertain the causality or temporal relationship concerning the pathways of association between quality of sleep and nicotine dependence which is likely to be bidirectional. Despite the limitations identified, we believe that the study addresses a major health problem that challenges physicians in Lebanon. In conclusion, this study highlights a significant association between nicotine dependence and poor sleep quality among medical residents and fellows, indicating that smokers are more prone to experience compromised sleep compared to non-smokers. The notable prevalence of IQOS smoking among medical residents emphasizes the necessity for additional research on the effects and safety of alternative tobacco products. Moreover, this study suggests that using multiple types of tobacco products may increase the likelihood of poor sleep quality, suggesting a potential additive effect of nicotine on sleep disturbances that warrants further investigation in future studies.