Hermetia illucens, with good amount of chitin and proteins, represents a suitable diet replacement for laying hens. Little is known about insect diet effects on the microbial ecology of the gastrointestinal tract and bacterial metabolites production. In this study we investigated the effect of H. illucens larvae meal administration on cecal microbiota and short chain fatty acids (SCFAs) production in laying hens. 16S rDNA sequencing showed strong differences between cecal microbiota of soybean (SD) and insect diet (ID) groups both in type and relative abundance (unweighted and weighted beta diversity) of microbial species. In particular, Bacteroides plebeius, Elusimicrobium minutum, Alkaliphilus transvaalensis, Christensenella minuta, Vallitalea guaymasensis and Flavonifractor plautii represented the principal contributors of changes in gut microbiota composition of ID group . Of these, F. plautii, C. minuta and A. transvaalensis have the potential to degrade the chitin’s insect meal and correlated with the observed high levels of gut SCFAs produced in ID group. These microorganisms may thus connect the chitin degradation with high SCFAs production. Our results suggest H. illucens as a potential prebiotic by well feeding gut microbiota.Insects could be potential nutritional sources both for humans and animals. Among these,  Systema Naturae wrote: “Larvae assate in deliciis habentur” [roasted larvae are delicious] describing the larvae of Rynchophorous spp.Linneus in the 1735 work 1. Insects are considered part of the natural diet of chickens containing between 30% and 70% of protein on a dry matter basis, fats (about 35%), minerals and vitamins and, for this reason, protein-rich insects are a promising alternative to traditional protein sources, reducing environmental pollution and feed costs4. Insects are also much more efficient in converting feed to body weight than conventional livestock and research into rearing insects as food and feed on a large scale remains a priority despite the production system being still too expensive. Among the different species, the black soldier fly Hermetia illucens (Diptera: Stratiomyidae) seems to be very interesting as a sustainable alternative for food and feed and is considered a good candidate for mass production1. H. illucens is found in abundance and naturally occur around the manure piles of large poultry, pigs and cattle. As a component of a complete diet, H. illucens prepupae have been found to support good growth in chickens and other animals8. These larvae have natural antibiotics property modifying the microflora of manure, potentially reducing harmful bacteria, such as Escherichia coli 0157:H7 and Salmonella enterica in hen manure10. The black soldier fly contain chitin, a naturally occurring polysaccharide considered to be one of the most abundant biopolymers in nature11. Chitin is not degraded, absorbed in the small intestine and it can be fermented by the microbiota of the large intestine. Studies of humans and mice suggested that chitin may restore the compositional balance of the microbial community. In addition, chitin, or derivate, seems to exhibit anti-viral, anti-tumor, antifungal activities and antimicrobial properties and a bacteriostatic effect on Gram-negative bacteria, Escherichia coli, Vibrio cholerae, Shigella dysenteriae and Bacteriodes fragile12. Insect diet, indeed, may decrease the use of antibiotics in the poultry industry controlling the antimicrobial resistance and its adverse effects on human health1.The global demand for food, especially animal based protein sources, is drastically increasing. Therefore, finding new alternative protein sources is one of the most interesting goals for human and animal nutrition and health. In this case, insects as food and feed seem to be an interesting resource with many environmental and health benefits. About 1900 edible insect species are consumed around the world13, fermenting the diet ingredients to short chain fatty acids (SCFAs), as major products14. In chickens, the gut microbiota is still under observation and the predominant microbes in the duodenum, caecum and feces belong to Firmicutes (30–50%) and Bacteroidetes phyla (between 10 and 50%)16. Previous studies demonstrated that feed additives (prebiotics and probiotics) improve chicken gut functionality and consequentially the health status17. More recently, Marono et al. (2017) observed that H. illucens larvae administration has good effects on laying hen health status, reducing serum and eggs cholesterol and triglycerides levels18. These beneficial effects may be due to the chitin amount provided by diet. We hypothesized in this study that insect-based diet might modulates the gut microbiota and its principal metabolites, improving the health status in laying hens. To this aim, we deep sequenced the gut bacterial community and evaluated the SCFAs concentrations to assess the effect of H. illucens larvae meal administration in laying hens.The gut microbiota plays an important role in its vertebrate host facilitating the digestion of food or feed componentsH. illucens had no negative effects on laying hen health status as reported in the same experimental design previously published18 , all birds were clinically healthy, neither mortality or diarrhea or sickness signs were observed in the 2 groups throughout the entire experiment, indicating that 18 Table .Table 1FCecal microbiota analyses were performed in order to describe and compare the gut bacterial composition of two groups of laying hens fed with soybean and insect meal-based diet ; sequencing of V3-V4 regions of 16S rRNA gene from cecal samples generated 178,376 high quality reads assigned to a total of 1,461 operational taxonomic units (OTUs). A sequencing depth of 5,234 sequences/sample, with good’s coverage > 96.9% and clustered in 1,123 OTUs, was considered to elaborate the results and analyze the effect of insect meal-based diet on gut microbial communities with respect to SD.Insect meal administration significantly increased the diversity within microbial populations as indicated by a higher number of observed species and Shannon entropy increase in ID samples with respect to SD samples Fig. . Moreove19. Insect-based diet in ID group induced a significant increase of Elusimicrobia, Lentisphaerae (p < 0.05) and Cyanobacteria (p < 0.05 after FDR correction) and a decrease of Fusobacteria (p < 0.05 after FDR correction) compared to laying hens fed with a soybean-based diet , Bacteroidetes  and Proteobacteria (7.92 ± 1.31% in SD and 8.38 ± 0.47% in ID) were the most abundant bacterial phyla detected in both groups Fig.  in line iet Fig. . At genuiet Fig. . Furtheriet Fig. . Sequenc20, in order to obtain the bacterial species assignment of key genera with significant differences in relative abundance between SD and ID groups  slightly enriched in SD group. Indeed, β-N-acetylhexosaminidases (K01207) and N-acetylglucosamine 6-phosphate deacetylase (K01443), the key enzymes that cleave oligomers produced by chitinase, were highly represented in microbial communities of both hen groups, showing an increased count in ID microbial communities  analysises Table . The PICup Table .Figure 3F. plautii , C. minuta and A. transvaalensis  strongly correlated with high production of propionate, butyrate and with total SCFAs. In contrast, S. hippei (class of Gammaproteobacteria) and Phascolarctobacterium (unclassified member of Veillonellaceae), correlated negatively with propionate, butyrate, isovalerate and with total SCFAs concentrations  was applied to correlate the relative amount of each key variable in all samples . We found a strong clustering of ID away from SD groups, indicating that diet may drive all variables considered Fig. .The present study represents a comprehensive evaluation of microbial communities modulation and SCFAs production after insect larvae meal administration in laying hens.H. illucens had no negative effects on laying hen health status. Lower serum cholesterol and triglycerides levels and a reduction of feed intake and body weight were observed in the ID group with respect to SD group, as also reported by Marono et al. (2017)18. Moreover, ID group showed a lower cholesterol content in the egg yolks produced 24 where diet containing up to 30 g/kg chitosan reduced cholesterol egg yolk contents. In our study the inclusion of H. illucens larvae in hen diet, was shown to have an important effect in modulating the gut microbiota communities and their metabolites. Statistical analysis showed that the composition of the microbial community correlated significantly with SCFAs levels measured in the caeca during the different dietary administration.ed Table  accordin25. Moreover, comparing the ID and SD bacterial communities by beta-diversity analysis, the main differences within the two groups were due to a different assortment in bacterial species  and C. minuta were already reported in metagenomic studies as highly positively associated each with other, possibly in a mutualistic relationship26. These bacteria are currently under scrutiny, raising particular interest since their abundance was found to correlate with the lean host phenotype28. Moreover, Oscillospira is the only genus that increased also in the caecum during fasting in birds, fishes and mammals29. These evidences matched with the reduced body weight and the increase of Oscillospira in ID group.Overall, the new microbiota detected in ID group showed a higher microbial richness compared to SD group. The acquired microbial richness, in the symbiotic relationship with the host, may potentially provide further metabolic capabilities. Several studies, in fact, revealed that a rich bacterial community is associated with a healthy status, while a lower bacterial diversity, with reduction of metabolic, immunological and gut protective functions, is related to several diseasesies Fig. . The newOscillospira after this specific diet administration in laying hens is of considerable interest. Oscillospira is actually an enigmatic bacterial genus, butyrate producer that may contribute to the formation of secondary bile acids and known to prevent Clostridium difficile-associated infectious disease (CDAD) in humans31. Poultry represents a possible reservoir of C. difficile32 and our study suggests that H. illucens larvae administration may have a potential antimicrobial effect against C. difficile infection, by increasing the genus Oscillospira.The increase of 33. SCFAs play an important role in gut physiology, and especially butyrate, serves as energy source for colonic epithelial cells. In addition, SCFAs negatively affect the expression of virulence factors of bacterial pathogens13. In our study, total SCFAs, in particular propionate and butyrate, were higher in ID than in SD hens. This increase in SCFAs concentrations may be the result of modulation in the microbial population induced by the insect-based diet. Moreover, these bacterial metabolites also contribute to mineral uptake and provide extra energy to the birds34, adjusting, in turn, the intestinal microbiota ecosystem towards a host-friendly environment35. This effect may preserve the maintenance of the described new equilibrium induced by H. illucens larvae diet administration.The insect meal administration may affect also the levels of SCFAs, end products of bacterial fermentation. SCFAs, such as butyric, propionic and acetic acids, once absorbed into the blood, bio-regulate and promote mucosal growth via direct or indirect mechanisms in the gut, as well influencing metabolism systemicallyH. illucens larvae dietary administration may act as a potential prebiotic and chitin may be the key feature of this effect. The chitin content in the insect meal might serve as substrate for the gut microbiota, influencing the composition and the microbial fermentation metabolites. Chitin is the second most abundant polysaccharides in nature of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chicken has the acidic chitinase in stomach and in the intestine that breaks down chitin-containing insect in dimers of GlcNAc to produce chitooligosaccarides36. PICRUSt analysis revealed low levels of bacterial chitinase (K01183), slightly enriched in SD group. Interestingly, more than 4–fold increase of β-N-acetylhexosaminidases (K01207) was observed in ID microbial communities. β-N-acetylhexosaminidases (K01207) is the second main class of chitinolytic enzymes that degrade chitooligosaccharides formed by chitinases into monomers37. This enzyme is also attracting particular interest for oligosaccharide synthesis, but the sources of this enzyme are rare38 and the insect-diet fed bacteria might represent an important source of this exogenous chitinase. GlcNAc is deacetylated by N-acetylglucosamine 6-phosphate deacetylase (K01443), an enzymatic function increased in ID group, to glucosamine-6-phosphate and acetate39. Glucosamine-6-phosphate and acetate can be co-metabolized by cross-feeders bacteria to biosynthesize SCFAs42. Therefore, K01207 and K01443 enriched functions in microbial communities of ID group represent the key enzymes responsible for the degradation of chitin derivatives from chicken chitinase and higher SCFAs production. Of note, the principal contributors to K01207 and K01443 abundance were F. plautii (key genus Oscillospira), C. minuta and A. transvaalensis. Interestingly, we found a strong correlation between levels of F. plautii, C. minuta and A. transvaalensis with high production of propionate, butyrate and total SCFAs. Genus Alkaliphilus, isolated in soil and also in chicken, dismutates crotonate to acetate and butyrate and its relative abundance is influenced by diets45. Moreover, PICRUSt analysis for key enzymes involved in butyrate and propionate production revealed an enrichment of several KEGG orthologs in ID group 18 was probably promoted by increased of satiety. It has been suggested that SCFAs, derived from bacterial fermentation of dietary fibers, stimulate the production and secretion of satiety-related hormones associated with fed or fasted state (GLP-1 and PYY)46. In our study taxa of Oscillospira genus and C. minuta, highly correlated with production of butyrate and total SCFAs. These evidences matched with the reduced feed intake and consequently body weight registered in ID group. This effect is supported by the reported association of Oscillospira and Christensenella with lean host phenotype28. Furthermore, the insect-meal administration also increased the levels of propionate, produced as a result of fermentative activity of gut microbiota during fiber degradation. Propionate can penetrate the blood–brain barrier, produce widespread effects on neurotransmitters release (dopamine and serotonin) and affects mitochondrial fatty acid metabolism47. Recent findings from human and rodent studies suggest that propionate production may play an important role in attenuating reward-based eating behavior via striatal pathways, independently of changes in plasma satiety-related hormones48. On the other hand, it has also been suggested that propionate regulates appetite by stimulating GLP-1 and PYY secretion49. Finally, SCFAs serve as an immediate energy source when glucose absorption is decreasing in the small intestine, stabilizing glucose levels in blood50.The reduced feed intake during insect meal administration reported by Marono in vitro and ex vivo previous studies52. Our data strongly corroborate the hypothesis of possible beneficial effects of insect-based diet on global health of hens, even though further studies are necessary to decipher its precise impact on human and animal physiology.We hypothesized that insect-based diet and chitin content may have positive metabolic health effects, such as increasing satiety, lowering blood triglycerides and cholesterol levels. These effects may be associated with SCFAs derived by microbial degradation of insect meal, as also described in 1. Fermentable chitin, as a potential prebiotic, may determine a healthy and a well-fed microbiota in laying hens. How do the gut microbiota exerts its effect in the intestinal tract and throughout the rest of the body is actually a crucial question.Changes in the gut microbiota as a result of feeding with insect larvae meal are novel and highly significant in our study. Dietary substances like chitin, one of the principal biopolymer in insect meal, plus bacteria in the gut, may work together to produce SCFA molecules. Here the microbiota plays a key role in the coordination of polysaccharide degradation responsible for the increases in SCFAs concentrations, promoting both gut and overall health. Considering that insects are already part of the human and animal diet in many countries, their potential as nutritional source needs to be re-evaluated. Insects can partly replace the protein ingredients in the livestock and poultry industries. For this reason the aim for 2020 is to introduce farmed insects as ingredients for feed and foodWe believe that the beneficial effects on hens during insect diet administration, supported by here reported results, deserve further investigation, especially considering the current lack of knowledge and the growing worldwide interest in insect-based food for animals and humans.All hens were treated in accordance will Directive of the European Parliament of the Council on the Protection of Animals Used for Scientific Purpose and in agreement with the Institutional Animal Care and Use Committee of the University of Naples Federico II, D.lgs n. 26 04/03/2014. All experiments involving hens were approved by the Bioethical Committee of the University of Naples Federico II, under number of protocol: 2017/0017676.18 and obtained from a commercial hatchery, were randomly and equally divided in two different diet group (12 hens per group), each divided into 3 replicates of 4 hens each. The experiment was conducted on hens from 24 to 45 weeks old, housed in modified cages (800 cm2/hen), under controlled temperature and humidity conditions. The hens, visually inspected daily, had free access to feed and fresh water, that were daily distributed; the dark:light cycle was 9:15 hours. The experimental cages were surrounded by commercial hens flocks composed of birds of the same origin as those used in the experiments. The cages had the same dimensions and the same number of drinkers and feed hoppers to simulate commercial production conditions and three hens per cage were housed for each group.A total of 24 Lohmann Brown Classic laying hens, representing a subgroup of those employed in a previous studyad libitum with isoproteic and isoenergetic diets: Soybean meal (SD) group was fed a corn-soybean-based diet, while in the H. illucens larvae meal (ID) group, the soybean was completely replaced by a defatted meal of H. illucens larvae . The diets were formulated according to Lohmann Brown classic Management Guides (2013)53. Chemical characteristics of the two different diets , including the amount of chitin, are as reported by Marono et al. (2017)18. In particular, taking into account the average feed intake and the level of inclusion of the insect meal in the diet, the hens from ID group ingested around 1.02 g/d of chitin all along the trial.The two groups were fed et al., (2017)18  according to manufacturer’s instructions. DNA concentrations were measured fluorometrically using the Qubit dsDNA BR assay kit (Invitrogen) and quality was assessed by spectrophotometric measurements with NanoDrop (ThermoFisher Scientific Inc.). DNA samples were stored at −20 °C until processed for amplification.2, and 2 U FastStart High Fidelity PCR System (Roche Applied Science). PCR was performed using the following cycles conditions: an initial denaturation step at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 55 s and ended with an extension step at 72 °C for 5 minutes; products were visualized by electrophoresis on 1.2% agarose gel. After a purification step with Agencourt AMPure XP (Beckman Coulter Inc), the amplicons were indexed with 10 subsequent cycles of PCR using the Nextera XT Index Kit (Illumina). The second PCR was performed according to Illumina guidelines using the KAPA HiFi HotStart System. Amplicons were visualized using gel electrophoresis and subsequently cleaned as described above. Library sizes were assessed using a Bioanalyzer DNA 1000 chip (Agilent technologies) and quantified by Qubit. Normalized libraries were pooled, denatured with NaOH, then diluted to 10 pM and combined with 25% (v/v) denatured 10pM PhiX, according to Illumina guidelines. Sequencing run was performed on an Illumina Miseq system using v3 reangents for 2 × 281 cycles.Sequencing samples were prepared according to the protocol 16S Metagenomic Sequencing Library Preparation for Illumina Miseq System, with some modifications. The V3-V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 µL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl54 in order to assemble reads with an overlap of at least 40 nucleotides, and to retain high quality sequences (PHRED score ≥ 33) that were comprised between 400 and 500 bp. Filtered sequences were then processed with PRINSEQ55 in order to obtain FASTA and quality files for further analyses. Metagenomic analyses on the resulting data were conducted using Quantitative Insights Into Microbial Ecology 56. 16S sequences were used to pick OTUs at 97% of sequences similarity from Greengenes 16S gene database 57 with a closed reference-based OTU picking method. The GG database was used to taxonomically classify the identified OTUs and to compute their distribution across different taxonomic levels. Species and Clostridium cluster classification was performed using SPecies level IdentificatioN of metaGenOmic amplicons program (SPINGO version 1.3) with default parameters on a representative sequence of each OTU20. To avoid sample size biases in subsequent analyses, a sequence rarefaction procedure was applied using a maximum depth of 5,234 sequences/sample. To assess sampling depth coverage and species heterogeneity in each sample, alpha diversity metrics were employed on rarefied OTU table using Good’s coverage, Observed species and Shannon’s diversity indexes. A two-sample permutation t-test, using 999 Monte Carlo permutations to compute p-value, was performed to compare alpha diversities between sample groups. OTUs diversity among sample communities (beta diversity) was assessed by applying unweighted and weighted Unifrac distances and compared using the ANOSIM method58 with 999 permutations. Statistical differences in OTUs frequencies between SD and ID groups at different taxonomic levels were assessed using nonparametric Kruskal-Wallis test, with False discovery rate (FDR) correction for multiple testing. Finally, unweighted unifract distances and species level classification data were visualized by principal coordinate analysis (PCoA) biplots including the 10 most abundant bacterial species driving the samples clustering. Metagenomes of key species were predicted using PICRUSt based on normalized OTU table, corrected for multiple 16S rRNA gene copy number21. Kyoto encyclopedia of genes and genomes59 ortholog abundances of  chitinase [EC:3.2.1.14] (K01183), beta-N-acetylhexosaminidase [EC:3.2.1.52] (K01207) and N-acetylglucosamine-6-phosphate deacetylase [EC:3.5.1.25] (K01443) between groups were compared using two tailed Student’s t-test. Predicted contribution of each key bacterial species to K01207 and K01443 counts were computed using the script metagenome_contributions.py in PICRUSt and visualized with an Rscript in Microbiome Helper60.V3-V4 16S rDNA FASTQ paired-end reads were pre-processed with PEARet al., 2003).Cecal digesta samples (each about 5 ml) after dilution with oxalic acid , were subjected to short chain fatty acids (SCFA) analysis by gas chromatography  with OMEGAWAX 250 fused silica capillary column 30 m × 0.25 mm × 0.25 mm film thickness; analysis temperature, 125 °C; flame ionisation detector, 185 °C; carrier helium, 1.7 ml/min  was performed on the abundance of each key variable/sample  using JMP software .In this study results were considered statistically significant at p-value < 0.05. Significant differences were indicated in figures and tables by *p < 0.05, **p < 0.01, ***p < 0.001. Bar plots were created by using GraphPad Prism 6.0.Supplementary information


This study investigated the association between mild cognitive impairment (MCI) and physical function and bone health in older adults. MCI was associated with poor physical performance but not bone mineral density or bone microarchitecture.Cross-sectional study to investigate the association between mild cognitive impairment (MCI) and physical performance, and bone health, in a community-dwelling cohort of older adults.Cognitive function of 222 men and 221 women  was assessed by the Strawbridge questionnaire and Mini Mental State Exam (MMSE). Participants underwent dual-energy X-ray absorptiometry (DXA), peripheral-quantitative computed tomography (pQCT) and high-resolution peripheral-quantitative computed tomography (HR-pQCT) scans to assess their bone density, strength and microarchitecture. Their physical function was assessed and a physical performance (PP) score was recorded.p = 0.01). Higher MMSE score was associated with better physical performance in both sexes. Poorer cognitive function, whether assessed by the Strawbridge questionnaire, or by MMSE score, was not associated with bone density, shape or microarchitecture, in either sex.In the study, 11.8% of women and 8.1% of men were cognitively impaired on the MMSE (score < 24). On the Strawbridge questionnaire, 24% of women were deemed cognitively impaired compared to 22.3% of men. Cognitive impairment on the Strawbridge questionnaire was associated with poorer physical performance score in men but not in women in the unadjusted analysis. MMSE < 24 was strongly associated with the risk of low physical performance in men (OR 12.9, 95% CI 1.67, 99.8, MCI in older adults was associated with poor physical performance, but not bone density, shape or microarchitecture. Mild cognitive impairment (MCI) is a heterogeneous state between normal ageing and early dementia . The preEmerging data suggest bone mineral density (BMD) is reduced in severely cognitively impaired individuals, compared to cognitively normal individuals of a similar age . AlthougThe majority of evidence to date has been derived from studies set in long-term care/nursing homes and hospitals , 10–12 w2).The Hertfordshire Cohort Study (HCS) is a population-based UK cohort of older adults. Study design and recruitment have been described in detail previously . In brieAll assessments detailed below were performed in 2011/2012.Participants completed a Mini Mental State Examination (MMSE)  and StraHand grip strength was measured using a Jamar handheld dynamometer . Partici2) at the non-dominant hip using a Lunar Prodigy Advanced Scanner, UK. Positioning for all scans was completed in accordance with the manufacturer’s instructions. The effective dose to the subject during each scan was 0.009 μSv.A DXA scan was performed to measure BMC (g) and aBMD . A scout view was performed on the lower leg to place a reference from which measurement sites are determined. Two slices were taken in the radial scan (4 and 66%). Four slices were taken for the lower leg scan . Measurements were taken from both the radius and tibia total bone areas (TBAs), trabecular bone mineral density (tBMD), cortical bone mineral density (cBMD) and cortical bone area (cBA). Short-term measurement precision error was expressed as a coefficient of variation, ranged from 0.88%  to 8.8% , but was typically between 1 and 3%. These figures were obtained using images acquired from 20 volunteers who were part of the study and hence were representative of the study population. The effective dose to the subject during each scan was 0.01 μSv.Each participant had measurements of the non-dominant distal radius and distal tibia using HR-pQCT  except when it had previously been fractured in which case the dominant side was scanned. A total of 110 slices were obtained which represented a volume of bone 9 mm in axial length with a nominal resolution (voxel size) of 82 μm. Antero-posterior 2D scout views were performed to determine the region to be scanned. All scans were performed in keeping with the manufacturer’s guidelines and as described by Boutroy et al. .3), trabecular BMD , trabecular number , trabecular thickness , trabecular separation , cortical area , cortical density , cortical porosity  and cortical thickness . Short-term precision values for cortical and trabecular BMD have been shown to range from 0.3 to 1.2 [Image analysis was carried out using the standard manufacturer’s method which has been described in detail previously . Analysi3 to 1.2 . The effEthical permission for the study was granted by the East and North Hertfordshire Ethical Committees. All participants gave written informed consent.Study participants who were taking drugs that are known to alter bone metabolism (e.g. bisphosphonates) were excluded from the study. However, women who were taking hormone replacement therapy (HRT) were allowed to participate.z-scores (FY z-scores). Descriptive statistics for continuous variables were expressed as mean and standard deviation (SD) or median and interquartile range (IQR). Categorical variables were expressed as frequency and percentage (%). Differences between men and women were assessed using Student’s t tests, Mann-Whitney tests or Pearson’s χ2 tests, as appropriate. Linear and logistic regression analyses were used to examine the associations between being cognitively frail on the Strawbridge Cognitive Domain and MMSE score and physical performance, and bone outcomes. The regression analyses were undertaken with and without adjustment for the following lifestyle confounders: age, BMI, social class, smoker status, alcohol consumption, physical activity and dietary calcium intake, and, in women only, years since menopause and HRT use. Statistical analyses were performed using STATA, version 14.Variables were assessed for normality and transformed where necessary using the Fisher-Yates rank-based inverse normal transformation to create p < 0.001) and heavier (p < 0.001) than women but BMI did not differ significantly by sex.The characteristics of the study participants are displayed in Table The median (IQR) MMSE score was 28 (25–29) for both men and women. In the study, 11.8% of women were cognitively impaired on the MMSE (score < 24), compared to 8.1% of men. On the Strawbridge questionnaire, 24% of women were deemed cognitively impaired compared to 22.3% of men.p < 0.001). Gait speed was faster in men than in women: 0.78 vs 0.73 m/s (p = 0.006). The median time for the repetitive sit-to-stand test in men  was less than that for women , p = 0.05. Fifty-six (26.3%) women were unable to perform the Tinetti balance test for greater than 10 s, compared to 43 (20.1%) men (p = 0.13). One hundred and forty-one women (71.2%) had a physical performance score of 9 or less, compared to 116 men (56.9%) (p = 0.003).Men had a greater mean maximum grip strength compared to women, 36.1 and 21.3 kg, respectively , although we were unable to run adjusted models due to low numbers.In women, MMSE score was associated with gait speed, physical performance score and higher prevalence of low physical performance score (≤ 9) following adjustments for confounders. As in men, MMSE score was associated with physical performance score. However, following adjustment for confounders, this association became non-significant. In men, MMSE score < 24 was associated with poorer gait speed, repetitive sit-to-stand, modified Tinetti balance test and physical performance score. In men, MMSE < 24 was strongly associated with the risk of low physical performance . Few statistically significant relationships were found between DXA, pQCT or HRpQCT outcomes and either cognitive frailty, as assessed by the Strawbridge questionnaire, or MMSE score in both men and women .Bone mineral content (BMC) and bone mineral density (BMD) were higher in men than in women at both hip and spine (data not shown). Male mean radial (4 and 66% slices) and tibial  bone parameters as assessed by pQCT and HRpQCT scans were significantly greater than those in females  or maintaining a persistent physically inactive lifestyle is associated with greater decline in physical performance . FurtherIn this study, we demonstrated no association between MCI and bone mineral density, size or microarchitecture at the hip, radius or tibia. However, a previous study  has demoLongitudinal studies of the relationship between cognitive function and physical performance in subsets of patients have suggested a link. In 177 UK patients with moderate to severe cognitive impairment (MMSE 11–23) followed up for a 1-year period, many neuropsychological, physical and functional performance measures declined . BaselinMuch of the available literature reporting associations between cognitive impairment and physical performance have been performed in Asian populations –40. DecrOne of the limitations of this study is the lack of a standardised diagnostic tool for MCI. Therefore, by using the Strawbridge questionnaire and MMSE, we may have under-/overestimated MCI in our cohort. Additionally, our analyses are cross-sectional, and longitudinal data are now needed. Finally, appreciation that the Strawbridge questionnaire provides a subjective measure of cognitive impairment must be made. By contrast, one of the study’s strengths is the study population. The individuals recruited were a non-selective community-dwelling population who were born in Hertfordshire and continued to live there at the age of 60–75 years. HCS characteristic and mortality patterns have previously been demonstrated to be similar to those of England , thus geLongitudinal studies objectively assessing the full spectrum of cognitive impairment within a community-dwelling population would be beneficial to confirm our results. Additionally, it may identify which stage of cognitive impairment is associated with increased falls and reduction in bone mineral architecture.This study suggests mild cognitive impairment in elderly community-dwelling adults is associated with poor physical performance especially in men but not altered bone density or microarchitecture.


MicroRNAs (miRNAs) are endogenous, noncoding RNAs that regulate various biological processes including adipogenesis and fat metabolism. Here, we adopted a deep sequencing approach to determine the identity and abundance of miRNAs involved in fat deposition in adipose tissues from fat-tailed  and thin-tailed  sheep breeds. By comparing HiSeq data of these two breeds, 539 miRNAs were shared in both breeds, whereas 179 and 97 miRNAs were uniquely expressed in KS and TS, respectively. We also identified 35 miRNAs that are considered to be putative novel miRNAs. The integration of miRNA-mRNA analysis revealed that miRNA-associated targets were mainly involved in the gene ontology (GO) biological processes concerning cellular process and metabolic process, and miRNAs play critical roles in fat deposition through their ability to regulate fundamental pathways. These pathways included the MAPK signaling pathway, FoxO and Wnt signaling pathway, and focal adhesion. Taken together, our results define miRNA expression signatures that may contribute to fat deposition and lipid metabolism in sheep. Adipocytes are cells that form and store fat globules in the body. Adipose tissue deposits are located in different regions of the body, primarily in the form of subcutaneous fat or intramuscular fat in domestic animals. Though the volume of adipocytes varies from breed to breed and is influenced by diet, genetic factors are considered major determinants for the formation of adipocytes. Fat-tailed sheep, which exhibit distinctive large tails and hindquarters, comprise approximately 25% of the world's sheep population  and are MicroRNAs (miRNAs) are a set of small noncoding RNAs (~22 nt) that bind to partially complementary sequences on target mRNAs resulting in posttranscriptional regulation of gene expression . MicroRNWe have previously described specific changes of transcriptomic differences with respect to adipose tissues from the representative KS and TS breeds and identified a list of candidate genes underlying the phenotypic differences of fat/thin tails in sheep . In thisThe experiments were conducted following the guidelines of the Animal Ethics Committee at Northwest A&F University under the document 2011-31101684. The sampling procedures complied with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) Number 398 set by the Ministry of Science and Technology, China.Six adult individuals  from KS and TS breeds were randomly selected, respectively, and any two or more individuals with a traceable phylogenetic relationship were avoided in the sampling process. Adipose tissue biopsies were collected from the tails and stored as previously described .Total RNA from the mixed adipose tissues of six adult sheep was isolated using the RNAiso plus kit  according to the manufacturer's protocol. The small RNA libraries were prepared following Illumina® TruSeq™ Small RNA Sample Preparation protocol. The small RNA libraries were sequenced using an Illumina/Solexa 1 G Genome Analyzer System . The sequencing reads were deposited to the Sequence Read Archive, under accession code SRP093866.2(TS/KS)) of ≧1 and P value of ≦0.05. Scatter plots were used to demonstrate differentially expressed miRNA between the two sheep phenotypes.The raw miRNA sequencing reads were filtered and only the unique mapping reads were used for subsequent bioinformatics analysis, such as annotation and gene expression. The expression of miRNA in two samples (TS and KS) was normalized by transcripts per million (TPM) as previous description . After nhttps://doi.org/10.1155/2017/1857580). PCR-iQ5 system  was used to analyse miRNA genes expression level. The reaction mixture volume is 25 μL, containing 2 μL of cDNA (25 ng), 12.5 μL SYBR Premix Ex TaqTM II , 1.0 μL specific forward primer, 1 μL universal primer, and 8.5 μL water, the reaction program referenced to previous description [−ΔΔCt method was used to analyse the data. t-tests  was used to determine significance of the results using SPSS21. Graphpad Prism 6 was used to plot the graphs. All data were presented as mean ± SD and values of P < 0.05 were considered statistically significant.Total RNA from the mixed sheep tails (six from KS and six from TS) was isolated using a miRNA extraction kit . First strand cDNA was synthesised with reference to RevertAid First Strand cDNA Synthesis kit  manufacturer's instructions, using special stem-loop RT primers  (Additiocription . U6 snRN2(fold change)| > 1.The sequences of reads with poly(A) tracts were deposited to the Sequence Read Archive, under accession code SRP093860. The FastQC package was used to assess the quality of raw reads and then mapped to the sheep genome assembly Oar_v3.1 (GCA_000298735.1) using TopHat2 . Qualityα) for validation analysis.MicroRNA-mRNA integration analysis was performed on the mRNAs that were differentially expressed between the KS and TS breeds in our previous study , and theFor each miRNA with a significant association with its set of mRNA targets (based on the gene set enrichment test), the GO and KEGG terms were extracted for all of its mRNA targets. We reveal the functions significantly related to predicted target gene candidates of miRNAs used gene ontology (GO) analysis.P < 0.05 are considered as significantly enriched in target gene candidates. The KEGG indicates the main pathways in which the candidate genes are involved.The primary pathways of the candidate target genes were determined by KEGG pathway analysis. The formula used to calculate this is the same as that used in the GO analysis. Genes with The sequencing of two small RNA libraries from the KS and TS yielded 12 M counts of sequencing reads, respectively . All of There were 8,000,311 (67.53%) and 6,720,288 (56.68%) reads matched to the sheep genome sequence in KS and TS libraries, respectively. All clean reads were classified and annotated by tag2annotation software (developed by BGI), aligned against the Rfam database (Ver. 10.1) and the miRBase (Ver. 19.0). There were 11,599,356 and 11,603,988 total conserved miRNAs reads, and 0.77% and 0.81% of clean reads were identified as potential novel miRNAs from KS and TS libraries, respectively, and more information was given in the additional file .To identify conserved miRNAs in sheep, all sRNA sequences were mapped to known miRNAs in the miRBase 19.0 database. We presented a total of 815 miRNAs found in abundance in adipose tissues of sheep . Of 815 identified miRNAs, 539 miRNAs were expressed in both breeds, whereas 179 and 97 miRNAs were specifically expressed in KS and TS, respectively. The top ten abundant miRNAs from KS, TS, and both groups are listed in P < 0.05) and 276 miRNAs showed an increase expression (P < 0.05) in KS, compared to TS , 33 miRNAs showed significantly reduced expression .In order to validate the reliability of miRNA expression data, quantitative RT-PCR was conducted to assess the expression of six upregulated miRNAs  and four downregulated ones  among the differentially expressed miRNAs, using adipose tissues from both KS and TS groups. U6 was used as an internal control. Stable miRNA expression levels in both KS and TS tissues correlated well between qPCR and miRNA-seq data .In total, 55,354,542 and 51,410,666 clean reads were obtained from the TS and KS adipose tissues, respectively. The percentages of reads containing HQ clean reads, adaptors, N, low-quality reads, poly A, and clean reads were calculated. Approximately 99% of the clean reads passed the filter . After alignment by TopHat, 86.78% and 86.65% unique reads were found in Tibetan and Kazakhstan sheep, respectively . We identified 15330 (76.13%) known genes and 458 novel genes in Tibetan sheep and 15162 (75.29%) known genes and 459 novel genes in Kazakhstan sheep. Those expression levels were quantified by FPKM ≥ 0.05 .2(KS/TS)|). Compared with KS, 1931 DEGs were identified. Seven hundred and sixty-six of these were upregulated expression, and 1165 were downregulated . The scatter plot showed significant DEGs in the above comparisons between TS and KS (DEGs were selected out by FDR < 0.05 and FPKM fold change (|logS and KS .http://www.geneontology.org/) using Blast2GO [To determine the function of differentially expressed genes, all of the DEGs in this study were mapped to terms in the GO database . MicroRNA recognizes its target mRNA through binding to a seed sequence, which localizes on the 3′ untranslated regions (3′UTR) of target mRNAs . In an aα, which inhibits preadipocyte differentiation [http://bibiserv.techfak.uni-bielefeld.de/rnahybrid) and miRanda (http://www.microrna.org) and combining the results of the analyses, we presumed that miR-125a-5p regulates ESRRα translation. MiR-125a-5p was found to directly target the ESRRα 3′ UTR sequence (α-3′UTR WT) and also the mutated 3′ UTR (psiCHECK™-2-ESRRa-3′UTR-mutant). The two types were cotransfected with the miR-125a-5p mimics or negative control sample into 293T cells, respectively, and luciferase activity was measured after 48 h later. The luciferase activity of the miR-125a-5p group was significantly lower than that of the NC group (P < 0.01), whereas the mutated ESRRa 3′ UTR exhibited increased luciferase expression  using DAVID software and pathway enrichment using specific KEGG terms . We founNext generation sequencing approaches have become powerful tools to predict and identify the novel genes and small RNAs of livestock species and other organisms. Compared with other meat-producing livestock such as cattle and pig, fewer studies have focused on the role of miRNAs in the process of fat deposition in sheep, though there are a few omics studies screening miRNAs in sheep skin  and muscγ [In the present study, we were able to discover 35 novel miRNAs which have eluded previous efforts and identify 239 miRNAs that have not been previously annotated in sheep. Our analysis detected 815 differentially expressed miRNAs between the fat deposits of the two sheep breeds used here. Not surprisingly, a proportion of the miRNAs was found to be differentially expressed in our study had been previously reported to play a role in adiposity, adipocyte development/differentiation, and other metabolic disturbances in livestock, as well as adiposity and related disease risk in humans. This further highlights the importance of miRNAs in adipose tissue development and metabolism. Previous studies have shown an increase in miR-143 during human and murine preadipocyte differentiation, and the following experiment verified that it inhibited preadipocyte differentiation –32. miR-γ . The intγ , 37. miRγ , 39. miRγ , 41. miRγ , and othα [In addition to microRNA expression profiling, we performed miRNA-mRNA integration analysis using the miRNA and mRNA expression data from corresponding tissues in distinct sheep breeds and determined a limited number of putative target genes for the differentially expressed miRNAs. The integration analysis removed a large portion of false positive miRNA target genes and increased the precision of our predictions . Therefoα . This kiThe DAVID gene annotation analysis of the miRNA-associated targets indicated that the GO biological processes were enriched to modification processes, metabolic processes, signal transduction, and regulation of biological processes. This demonstrates the distinct roles of adipocyte development in the tail areas as the reserves of energy, in response to external or internal environmental conditions during the dry and cold seasons. KEGG pathway annotation of their putative miRNA-related targets revealed that the MAPK signaling pathway, focal adhesion, Pyruvate metabolism, FoxO signaling pathway, and TNF signaling pathway contribute to the formation of sheep fat/thin-tails phenotypes. Various tail phenotypes are unique for sheep compared to other animals in the word, interestingly, a mass of adipose depot in the fat-tail sheep breed and less in the thin-tail sheep breed. In a manner, this phenomenon is a form of energy storage in the fight against extreme conditions, and the function of tail adipose tissue is similar to subcutaneous adipose tissue of other animals according to the enrich pathways, which is associated with metabolism risk , 45.In conclusion, we identified a number of miRNAs that are differentially expressed between the fat-tailed and short-tailed sheep breeds. We further highlighted gene targets of related miRNAs that may be involved in regulating fat deposition and adiposeness, in sheep and other livestock. This occurs via the key signaling pathways including focal adhesion, Pyruvate metabolism, and the MAPK, FoxO, and TNF signaling pathway. Further studies are needed to verify the correlation between key miRNAs and their target genes by in vitro approach and elucidate the functional impacts that miRNAs serve during adiposeness. Our results also provide evidence for the interaction of miRNAs and genes in the regulation of obesity and metabolic syndromes, which suggests that this may serve as an animal model for human' obesity and metabolic syndromes researches.Additional file 1: Primer pairs used for this study. Additional file 2: The distribution of miRNAs in TS and KS separately. Additional file 3: The finding miRNAs list. Additional file 4: Novel miRNAs identified in sheep adipose tissues. Additional file 5: Filter Information for Reads. Additional file 6: Mapping Information. Additional file 7: Gene expression details for KS and TS. Additional file 8: List of the identified DEGs in fat tissues transcriptome in sheep. Additional file 9: TS vs KS DEGs genes pathway enrichment. Additional file 10: The detail correlation of miRNA-mRNA. Additional file 11: Predicated target genes GO Enrichment . Additional file 12: Predicated target genes pathway enrichment.


Objective. To determine the association between serum total alkaline phosphatase (TAP) and mortality in African maintenance haemodialysis patients (MHD). Patients and Methods. The study enrolled a total of 213 patients on MHD from two dialysis centers in Johannesburg between January 2009 and March 2016. Patients were categorized into a low TAP group (≤112 U/L) versus a high TAP group (>112 U/L) based on a median TAP of 112 U/L. Results. During the follow-up period of 7 years, there were 55 (25.8%) deaths. After adjusting for cofounders such as age, other markers of bone disorder, and comorbidity (diabetes mellitus), patients in the high TAP group had significantly higher risk of death compared to patients in the low TAP group . Similarly, serum calcium >2.75 mmol/L was associated with increased risk of death compared to patients within levels of 2.10–2.37 mmol/L . The HR for death in white patients compared to black patients was 6.88; 95% CI 1.82–25.88; P = 0.004. Conclusion. High levels of serum alkaline phosphatase, hypercalcaemia, and white race are associated with increased risk of death in MHD patients. Prior to the availability of commercial intact parathyroid hormone (PTH) assays, serum total alkaline phosphatase (TAP) measurements were used as one of the surrogate markers of high bone turnover that was utilized in the management of chronic kidney disease mineral and bone disorder (CKD-MBD) . SubsequAlthough the role of racial disparities in adverse clinical outcomes remains controversial and inconclusive, some studies have demonstrated survival benefits attributable to race in patients undergoing MHD , 9. In aTherefore, the aim of this study was to determine if there is a link between high serum alkaline phosphatase and mortality in African MHD patients.This study was a retrospective review of patients undergoing MHD from two dialysis centers in Johannesburg between January 2009 and March 2016. A total of 213 patients aged ≥ 18 years with available baseline line variables of interest were included. Exclusion criteria included patients with missing important data for analysis, being on dialysis for less than three months, having active or chronic liver disease, and having malignancies. In addition, we excluded Indian and mixed races to allow for a proper comparison between black and white patients. Retrieved data included patients' demographic characteristics, blood pressure measurements, duration on haemodialysis, comorbid disease, and medication history related to CKD-MBD. Determination of race was based on self-report by the participants.Patients were categorized into the low TAP group ≤112 U/L) versus the high TAP group (>112 U/L) based on median TAP level of 112 U/L. Secondary analysis involved exploring the relationship between race, other markers of mineral bone disorder, and primary outcome. In line with a previous study . Total alkaline phosphatase reference range is 53–128 U/L.Plasma albumin was measured by colorimetric (bromocresol green) method on a Cobas 6000 autoanalyzer .Other biochemical parameters were determined using routine laboratory techniques.Blood samples were generally collected predialysis at midweek with the exception of the postdialysis serum urea for kinetic modeling.F) + 0.251, where F = Kt/V × .Calculation of normalized protein catabolic rate was based on the formula , nPCR = t-test and Mann–Whitney U test for normally distributed and nonnormally distributed variables, respectively. One-way ANOVA and Kruskal-Wallis tests were used to compare normally and nonnormally distributed continuous variables across categories of serum calcium.Pearson's or Fisher's exact test was utilized for proportion comparisons. Continuous variables are presented as means ± standard deviations or median and interquartile range (IQR) as appropriate. Associations between serum alkaline phosphatase and other biochemical parameters were assessed by multiple linear regression analyses. The Cox proportional model was used to determine the crude and adjusted hazard ratios of death for different categories of serum alkaline phosphatase, calcium, PTH, phosphate, 25-OH vitamin D, and white versus black patients. Patients' demographic and baseline characteristics were compared between the low and high total alkaline phosphatase groups as well as white versus black patients, using an independent P value of less than 0.05 was considered statistically significant at the 95% confidence interval. All analyses were performed using STATA version 12 .A The study included two hundred and thirteen patients  undergoing MHD. The mean (±SD) of age, median dialysis vintage, and mean Kt/V were 54.5 ± 15.6 years, 24 months , and 1.44 ± 0.3, respectively. The majority of the patients were on three times weekly, 4 hr sessions of haemodialysis. Most of the patients were dialyzed with a dialysate calcium concentration of 1.50 mmol/L, which is usually modified based on serum levels of calcium. The blood and dialysis flow rates are generally 300–400 mls/min and 500 mls/min, respectively. However, these values varied according to patient's blood pressure and haemodynamic state. A native arteriovenous fistula was used in more than half of the study population (60.6%). Almost all patients (93.0%) were on ESAs.P = 0.02) ( = 0.02) .The characteristics of the patients across different categories of serum calcium levels are shown in P = 0.01).During a follow-up period of 7 years there were 57 (26.8%) deaths. After adjusting for cofounders such as age, other markers of bone disorder , serum alanine transaminase, 25-OH vitamin D, and comorbidity (diabetes mellitus), patients in the high TAP group had a significantly higher risk of death compared to patients in the low TAP group . Similarly, we found a significant association between race and mortality, in which white patients had an accentuated six fold increase in adjusted hazard ratio for death compared to black patients   had more than a sixfold increased risk of death compared to patients with normal calcium  . Figuresr2 = 0.04, P = 0.008), corrected calcium , and PTH . In multivariate regression analyses PTH and calcium remained significantly correlated with TAP, P = 0.006 and 0.04, respectively.Univariate linear regression analysis revealed a significant association between TAP and age  or smaller muscle mass and increased body fat in African American patients correlated less strongly with mortality than in whites . AdditioIn line with previous studies , 39, blaOur findings should be considered in the context of the following limitations. Firstly, the retrospective nature of this study could not allow us to make causal associations between markers of mineral bone disease and study outcome (death). In addition, the use of a single baseline laboratory measurement precludes the performance of time dependent Cox analysis to account for variations in the biochemical markers on the impact of death over a period of time. However, few studies have shown no significant difference between the baseline and time dependent Cox analysis .Secondly, the relatively small sample size precludes generalizability of our findings to African HD patients. Thus, there is a need for multicentre studies in Africa, to provide robust data on this important clinical entity (CKD-MBD) in African HD patients.Thirdly, similar to several observational studies we could not account for residual confounding variables. For instance, aside from diabetes mellitus, other comorbid conditions could not be ascertained. However, part of the exclusion criteria was to avoid patients with some coexisting conditions that are known as potential confounders.The strengths of this study lie in the heterogeneous nature of our study population (black and white patients) in an African setting which has allowed comparisons of data not only for Black Africans with Black Americans, but also between whites in Africa and USA/Europe. To our knowledge, this is the first study in Sub-Saharan Africa that has given important insights regarding the impact of serum alkaline phosphatase, calcium, and race on mortality in African MHD patients.In summary, high TAP, hypercalcaemia, and white race are associated with increased risk of death in MHD patients, thus, reaffirming the need to pay more attention to the two modifiable risk factors  in the management of CKD-MBD.


Several studies have reported that diabetic persons have an increased risk for fractures than non-diabetes patients. The association between proximal humerus fractures and type 2 diabetes (T2DM) is unclear and some studies point to insulin treatment, hypoglycaemic episodes consequently to inadequate control of diabetes or, more recently, to an alteration of trabecular bone. We examined trends in the incidence of proximal humerus fractures, surgical procedures and outcomes among hospitalized patients aged ≥65 years, with and without T2DM in Spain, 2001–2013.This retrospective, observational study was conducted using the Spanish National Hospital Discharge Database to select all hospital admissions with proximal humerus fracture. We calculated incidences overall and stratified by diabetes status, year and sex. We analyzed surgical procedures, comorbidities, length of stay, in-hospital complications and in-hospital mortality.p < 0.001) and 2.9% among T2DM women vs. 1.7% in those without (p < 0.05). The use of open reduction of fracture with internal fixation and arthroplasty is increasing overtime and closed reduction with internal fixation is decreasing. Presence of T2DM in women was associated with higher in-hospital mortality . Comorbidities, in-hospital complications and older age were predictors of higher in-hospital mortality in both sexes.We identified 43,872 patients with proximal humerus fracture (18.3% had a T2DM diagnosis). Age-adjusted incidence rates elevated steadily over the study period for men and women with and without T2DM, independently of diabetes status, although we found a stable trend in the later years. Patients with T2DM had lower relative risk of proximal humeral fracture incidence: 0.87 (95%IC 0.82–0.93) for men and 0.97 (95%IC 0.95–1.00) for women. In-hospital complications were 4.0% of diabetic men vs. 2.6% in non-diabetic  contains supplementary material, which is available to authorized users. It is well known that the incidence of diabetes mellitus is growing steadily all around the world, especially in older people and it is becoming a very important public health problem , 2. SeveThe epidemiology and risk factors for proximal humerus fracture (PHF) have been previously described. –14.PHF represent 10% of all fractures in people age 65 years or over –6. TheseThe association between PHF and type 2 diabetes (T2DM) is unclear and some studies point to insulin treatment, hypoglycaemic episodes consequently to inadequate control of diabetes  or, moreRegarding the type of treatment used in PHF, several factors are important in determining the choices of treatment and include, among other, the pattern of the fracture (i.e. the number of segments), the displacement of these segments, patient characteristics , surgeon and implant availability, local traditions and guideline. . HoweverBased on national hospital discharge data, we compared trends in the incidence of PHF among hospitalized elderly patients with and without T2DM in Spain from 2001 to 2013. We focused on trends in the type of surgical interventions and also described and analyzed patient comorbidities, in-hospital complications, and in-hospital outcomes such as: length of hospital stay (LOHS) and in-hospital mortality (IHM).We performed a retrospective, observational study using the Spanish National Hospital Database (SNHDD) . We analThe SNHDD database compiles all the public and private hospital data, hence covering more than 95% of hospital discharges .The database includes patients’ variables , date of admittance, date of discharge, type of admission (elective or emergency admission), discharge destination , up to 14 discharge diagnosis, and up to 20 procedures performed during the admission , had higher mortality (2.3% vs 0.5%) and more IHC (3.2% vs 1.4%) and also had higher values of CCI. Overall women were more frequently treated with arthroplasty and men with non-operative treatment beside diabetes status .We found that 36.1% of patients were treated non-operatively whereas 63.9% of them had some type of surgical procedure: ORIF 25.7%, CRIF 22.3%, TSR 4.3% and PSR 11.6%. Non-surgical procedure were more frequent in men  and among those without the disease from 12.8% to 32.1% (p < 0.01). As is shown in Additional file p < 0.05).The most common surgical procedure used in all groups was ORIF, followed by CRIF and arthroplasty. We observed a significant increase in the use of ORIF; among diabetic men, it increased from 9.5% in 2001 to 33.1% in 2013 (p < 0.01). However, in those without T2DM IHM did not show a significant tendency (1.9% in 2001 to 1.3% in 2013). In non-diabetic men the median LOHS decreased significantly from 2001 to 2013.The IHM among men with T2DM decreased significantly over the study period, from 9.5% to 1.4% (p < 0.01). The use of CRIF decreased and the use of arthroscopy increased, in diabetic and non-diabetic women over time (p < 0.05). . Over the 10-year study period, LOSH for women with and without T2DM decreased significantly (p < 0.05).Diabetic women with PHF diagnosis reduceed their IHM significantly from 1.7% in 2001 to 1.0% in 2013 (p < 0.05).When we compared hospitalization outcomes between diabetic men and women we found almost double the crude IHM for men, compared to women, in the total study population. Among women those suffering T2DM had higher IHM than those without .p < 0.01) and in those with in-hospital complications .IHM was significantly higher in diabetic men and women with more comorbidities .Time trend analysis showed a significant decrease in mortality from 2001 to 2013 in diabetic men and women , but not in men .When we performed logistic regression on our whole study population to assess the influence of T2DM on IHM and after adjusting for all covariates, being diabetic and suffering a PHF was associated to higher IHM in women  for women and 4.5  for men . HoweverIn our study same patients with severe comorbidities, most frequently men, are considered for a non-operative treatment even if hospitalized. A possible explanation for this may be that, as upper arm fractures in men usually appear in cases of multiple injuries and they present higher mortality risk .IHC and CCI values were higher among men and women with T2DM, than in patients without T2DM. This result agrees with others that state that diabetic patients have more comorbidity and suffer more complications during hospitalization , 37. HowRegarding the type of procedure used over the study period, we describe changes in the treatment for these fractures, in line with other studies. Most PHF are non-displaced or minimally displaced and they were treated non-operatively, but this pattern is clearly declining because of the technical and medical advantages of surgical treatment, which has increased steadily and has currently reached a plateau in the last few years , 16, 38.Our results are consistent with other studies regarding the lower death risk seen in patients undergoing surgical treatment , but othOther studies have found a tendency to use hemiarthroplasty of the humerus head and total shoulder replacement to improve functional outcomes after treatment of comminuted (three or four segments) fractures, this seems to be also happening in Spain –9.The advantage seen for surgical treatment with respect to lower mortality risk, was not so clear for diabetic men, which could be explained, as we stated previously, by the more complex fractures seen in men. Another explanation could be, as our results also have found, non-operative treatment, even of marginally displaced PHF in elderly people, is an important aspect of treatment, which seems to be considered increasingly in multimorbid patients. Our results showed that hospitalized patients that did not receive surgical treatment presented worse clinical characteristics, with higher CCI values, more complications, were older and they were more frequently men, resulting in significantly higher in-hospital mortality. Further studies are needed to clarify this point.The strength of our investigation lies in its large sample size, its 13-year follow-up period and its standardized methodology, which has previously been used to investigate T2DM and its complications in Spain . NeverthPatients with T1DM were excluded because previous reports have shown that they have a different pattern of disease  and epidemiological characteristics than T2DM patients . FurtherOther limitation is the lack of information on the characteristics of T2DM therapy used, the control reached, duration and other comorbid conditions that could affect the results. The database that we used contained no information about drugs treatments (i.e. drugs for osteoporosis) and risk factors such as obesity or smoke which may have affected our outcome variables.As commented in the methods section patients who are treated at the emergency room or at out-patient clinic are not recorded in the SHNDD and therefore not included in our investigation. This may result in a selection bias, as those patients with less comorbidities and undergoing a non-operative treatment (immobilization) are more likely to be treated exclusively in the emergency room or as out patients and discharged without hospitalization. This can explain the very high proportion of PHF treated operatively (63.9%) in our study. However, it is also possible the existence of a bias by indication that would appear if the more advanced surgical treatment modalities were reserved for the more fit patients and similarly, patients with severe comorbidities were treated non-surgically.Beside these limitations the quality and validity of our dataset has been assessed and shown to be useful for health research .The incidence of PHF seems to be increasing in Spain. The incidence is lower among men with than without T2DM. T2DM is associated to higher IHC in both sexes. The use of ORIF and arthroplasty is increasing overtime beside T2DM status. Women with T2DM have higher IHM than those without the disease.Additional file 1: Figure S1.Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in men without T2DM (Spain 2001–2013). Graph showing the Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in men without T2DM (Spain 2001–2013). (TIFF 1794 kb)Additional file 2: Figure S2.Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in men with T2DM (Spain 2001–2013). Graph showing the Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in men with T2DM (Spain 2001–2013). (TIFF 1897 kb)Additional file 3: Figure S3.Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in women without T2DM (Spain 2001–2013). Graph showing the Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in women without T2DM (Spain 2001–2013). (TIFF 2051 kb)Additional file 4: Figure S4.Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in women with T2DM (Spain 2001–2013). Graph showing the Joinpoint analysis of age-adjusted proximal humeral fractures hospitalizations in women with T2DM (Spain 2001–2013). (TIFF 2373 kb)Additional file 5: Table S1.Procedures and outcomes of hospital discharges due to proximal humerus fracture among men and women with and without type 2 diabetes in Spain, 2001–2013. Table showing the procedures and outcomes of hospital discharges due to proximal humerus fracture among men and women with and without type 2 diabetes in Spain, 2001–2013. (PDF 52 kb)


In order to obtain optimum packaging materials for peanut kernels, the effects of four types of packaging materials on peanut storage quality  were examined. The results showed that packaging materials had a major influence on peanut storage quality indexes. The color of the peanut seed coat packaged in the polyester/aluminum/polyamide/polyethylene (PET/AL/PA/PE) composite film bag did not change significantly during the storage period. Color deterioration was slower with polyamide/polyethylene (PA/PE) packaging materials than with polyethylene (PE) film bags and was slower in PE bags than in the woven bags. The use of PET/AL/PA/PE and PA/PE bags maintained peanut quality and freshness for more than one year and both package types resulted in better germination rates. There were significant differences between the four types of packaging materials in terms of controlling insect pests. The peanuts packaged in the highly permeable woven bags suffered serious invasion from insect pests, while both PET/AL/PA/PE and PA/PE bags effectively prevented insect infection. Peanuts stored in PET/AL/PA/PE and PA/PE bags were also better at preventing and controlling aflatoxin contamination. Peanuts often suffer from oxidation and aflatoxin contamination during storage  . A low tIn China, some hermetic storage experiments with peanuts have been carried out; however, the application of different packaging materials for long-term storage of peanuts has not been studied. Therefore, there is little empirical evidence available on what are the most suitable packaging materials for storage of peanuts. The objectives of this study were to investigate the effects of different packaging materials on maintaining the quality of peanuts and inhibiting aflatoxin production, and to obtain suitable packaging materials for storing peanuts for different lengths of storage times.2/(m2 day atm) and a water vapor transmission rate of 660 g/(m2 day atm), b) polyethylene (PE) pouches, 75 μm in thickness, having an oxygen permeability of 600 cm2/(m2 day atm) and a water vapor transmission rate of 10 g/(m2 day atm), c) polyamide/polyethylene (PA/PE) pouches, 90 μm in thickness, having an oxygen permeability of 47.6 cm2/(m2 day atm) and a water vapor transmission rate of 3.9 g/(m2 day atm), and d) Polyester/aluminum/polyamide/polyethylene (PET/AL/PA/PE) pouches, 99 μm in thickness, having an oxygen permeability of 2.0 cm2/(m2 day atm) and a water vapor transmission rate of 1.0 g/(m2 day atm). For our experiments, the woven and PE bags were procured from the local market, and PA/PE and PET/AL/PA/PE bags were obtained from Yinfeng Plastic Color Printing Co. Ltd., Luoyang, China. The oxygen permeability and water vapor transmission rate were determined according to GB/T 19789–2005 [The raw peanut kernels used in this study were produced in 2014 at Nanchang , and had an initial moisture content (m. c.) of 8%. The intact peanut kernels were placed in bags made from four different packaging materials: a) highly permeable woven bags, having an oxygen permeability of 5000 cm789–2005  and GB/T789–2005 , respectTen kilograms of peanuts were packaged in each bag and the four packaging material types were replicated fifteen times . Experiments were carried out during the period between December 2014 and December 2016. After 0, 6, 12, 18 and 24 months of storage, three separate identical samples (from three bags) were withdrawn from each packaging material type and subjected to chemical and sensory analysis. Samples were stored in Nanchang City Jiangxi Province of China, where the average temperature in summer is 27°C –34°C and the average relative humidity (r. h.) is 78.5%.The following indicators of quality were measured during storage: peanut color, acid value, germination rate (%), and prevention of contamination with insects and aflatoxins.The color of peanut kernels was measured using a Hunter Lab optical sensor colorimeter  and results expressed as color L* (lightness), a* (redness), and b* (yellowness) values. The results reported  are the means of twenty determinations.The acid value was determined using a titration method, by measuring milligrams of potassium hydroxide  used to neutralize 1 g of free fatty acids. The acid value was determined according to the official GBT5530-2005  method fThree samples of 100 seeds were removed from each packaging treatment. The seeds were bathed in a 1% bleach solution for 10 min and then rinsed three times under running tap water. Each sample was sowed in wet sand and placed in a climate incubator (28°C). After one week, the samples were removed and the number of seeds with at least part of the radical breaking through the seed coat was counted. Data was recorded as the percentage of the number of successfully germinated seeds out of the total number of peanuts sampled.Each sample had three, 50 mL subsamples removed to evaluate the level of peanuts damage by insects. The number of damaged and undamaged kernels, by insects in each subsample, were visually counted and separated. Subsamples of each were dried to 0% moisture by heating in an oven at 60°C for 5 days. The dry weight of the damaged and undamaged peanuts for each subsample was then determined. Relative percent damage was calculated using the following equation as described by Alonso-Amelot et al. (2011) :Xrel=[ was determined with High Performance Liquid Chromatography (hplc)  analysis, according to the official GB/T 5009.23–2006  method fTreatment condition effects on peanut coat color, acid value, germination rate (%), and prevention of insect and aflatoxin contamination were assessed using ANOVA. Post hoc comparisons were made between groups using LSD. Significant values were reported at the α = 0.01 level, unless noted otherwise. The ANOVA analyses included the time periods, but the statistical results are not shown in the figures or legends and tables.The seed color of samples in the woven bag (control) gradually changed color to dark red as storage time increased; L* parameters decreased significantly  with a parallel increase in a* and b* values after 24 months of storage . CompareThe changes in acid values for samples during 24 months storage are shown in P<0.001; PE: F = 248.77; d. f. = 4,14; P<0.001; PA/PE: F = 51.29; d. f. = 4,14; P<0.001). ANOVA comparison identified statistically significant differences among the four packaging treatments with regard to how much the treatments reduced peanut germination. The highest germination rates were observed in the PET/AL/PA/PE pouches , followed by the PA/PE pouches (51 ± 3.7%), the PE pouches (20 ± 5.1%), and finally the woven bags (0%) , an average of 96.5 out of every 100 peanuts successfully germinated. The wove, PE, and PA/PE treatment groups showed a statistically significant decline in germination rates after 24 months of storage relative to initial values  .P<0.001).During storage, in addition to oxidation and rancidity, the peanuts can become easily damaged by insect pests. Insect damage to the stored peanuts varied greatly with time in our experiment . At the In general, the presence of light and oxygen accelerated a decrease in L* parameters with a parallel increase in a* and b* parameters, resulting in a gradual darkening of product color during storage, which may be attributed to browning reactions due to oxidation of phenols . DarkeniThe results showed that the germination rate decreased rapidly (in woven bags) during storage under a high temperature and high humidity environment, even when the seed had been dried to a safe m. c. Storing peanut in the sealed foil bag had no effect on either the grain's m. c. or germination potential, and these values remained stable even after 12 months of storage. For tropical regions, using a barrier against water vapor transmission would prevent stored seed from absorbing water when humidity is high , which wP<0.01) levels of pest contamination in comparison to the other three packaging materials. The results showed that pest breeding was significantly affected by the barrier property of the packaging materials (P<0.01).When the oxygen concentration reaches 8%, the number of pests will be severely inhibited , and ourA. flavus and aflatoxin accumulation in moist maize can be controlled by placing the grain in a Purdue Improved Crop Storage bag. When the oxygen concentration is reduced to 5%, aflatoxin production is severely inhibited [A. flavus could grow and accumulate aflatoxin rapidly if exposed to a r. h. of 60%. Pore space r. h. in the woven and PE bags could increase to 60% r. h. easily in the south of China, because of the low barrier properties.Williams et al.  reportednhibited . Our resnhibited  showed tIn summary, this study examined the effects of four types of packaging materials on peanut quality, including peanut color, acid value, germination rate and prevention of insect and aflatoxin contamination. The results showed that the packaging materials had a notable influence on peanut storage quality indexes. Compared to woven and PE film bags, PA/PE and PET/AL/PA/PE bags with high barrier properties (moisture and oxygen barrier) can inhibit oxidative discoloration of the seed coat, prevent pest contamination and control aflatoxin production.Different storage bags can maintain the quality of peanuts for different lengths of time; therefore it is important to choose the correct packaging to store peanuts. The selection of peanut packaging materials can be determined according to the length of storage time and quality required. For peanut kernels that are to be used for only short-term storage (i.e. not more than six months), general PE bags can be used. If peanuts are to be stored for one year, good barrier properties of PA/PE composite film and PET/AL/PA/PE bags are recommended. If appearance and quality requirements are higher, and if the storage time is longer than one year, the strong barrier properties of PET/AL/PA/PE bags are required. Based on the present data, the optimum packages for storage of peanut kernels for a long period of time were the PET/AL/PA/PE pouches. When PET/AL/PA/PE pouches were stored at room temperature the product shelf-life was maintained for 24 months.


Anolis lizards are a model system for the study of adaptive radiation and convergent evolution. Greater Antillean anoles have repeatedly evolved six similar forms or ecomorphs: crown-giant, grass-bush, twig, trunk, trunk-crown and trunk-ground. Members of each ecomorph category possess a specific set of morphological, ecological and behavioural characteristics which have been acquired convergently. Here we test whether the semicircular canal system—the organ of balance during movement—is also convergent among ecomorphs, reflecting the shared sensory requirements of their ecological niches. As semicircular canal shape has been shown to reflect different locomotor strategies, we hypothesized that each Anolis ecomorph would have a unique canal morphology. Using three-dimensional semilandmarks and geometric morphometrics, semicircular canal shape was characterized in 41 Anolis species from the Greater Antilles and the relationship between canal shape and ecomorph grouping, phylogenetic history, size, head dimensions, and perch characteristics was assessed. Further, canal morphology of modern species was used to predict the ecomorph affinity of five fossil anoles from the Miocene of the Dominican Republic. Of the covariates tested, our study recovered ecomorph as the single-most important covariate of canal morphology in modern taxa; although phylogenetic history, size, and head dimensions also showed a small, yet significant correlation with shape. Surprisingly, perch characteristics were not found to be significant covariates of canal shape, even though they are important habitat variables. Using posterior probabilities, we found that the fossil anoles have different semicircular canals shapes to modern ecomorph groupings implying extinct anoles may have been interacting with their Miocene environment in different ways to modern Anolis species. As such, it stands to reason that more agile and mobile animals, such as fast moving arboreal species, would benefit from an enhanced sense of balance, granted through adaptation of canal morphology. Recent theoretical ,2, physiD. As sucThe vestibular system has, however, been little explored outside the Mammalia. While the semicircular canals are physiologically and anatomically homologous in all vertebrates, we cannot assume that the relationship between form and function in mammals will hold true for other taxa, especially given considerable morphological differences between mammals and other amniote groups. Extrapolation from mammals is particularly problematic for studies wishing to reconstruct the ecology of non-mammalian fossil species ,23–26. RAnolis lizards (Dactyloidae) represent a unique opportunity for furthering such research. Greater Antillean anoles originated ca. 65 Ma, diversifying throughout the Caribbean and neotropical mainland [Anolis, six ecomorph types have evolved independently on each of the four Greater Antilles islands , with each ecomorph encompassing a specific suite of anatomical (e.g. short versus long limbs), ecological (e.g. tree trunk versus branches), and behavioural  characteristics (reviewed in [mainland . Within iewed in ): trunk-iewed in .Anolis, and close phylogenetic relatedness, make it an excellent starting point for further understanding the morpho-functional relationship of the semicircular canals in squamates and beyond Mammalia in general. Here, we use 3D geometric morphometrics to quantify and investigate whether Anolis semicircular canal morphology is convergent among ecomorph groups. Specifically, we aim to test the hypothesis that anole species adapted to similar ecomorph niches have converged on similar semicircular canal morphologies, reflecting the sensory requirements of their shared ecological and behavioural habits. We also test the influence of phylogenetic relatedness, size, and head proportions on patterns of canal morphology, factors that may have an effect on vestibular system form [Anolis lizards and compare these to predictions based on external morphological traits [The recurrent and consistent adaptive radiations found in tem form ,28,35–39tem form . Furthertem form . We use l traits .2.2.1.et al. [The sample consists of 131 individuals representing 41 species of anoles originating from the four islands of the Greater Antilles: Hispaniola, Cuba, Jamaica and Puerto Rico. Species from Hispaniola are represented by multiple specimens including juvenile individuals (see further below). All six ecomorphs are represented by multiple species: CG (5), GB (6), Tr (3), TC (9) TG (4) and Tw (5) , with thet al. . All mod2.2.Various methods for measuring the complex structure of the three semicircular canals have been used to date. Traditional morphometric approaches, in the form of linear and angular measures of size and orthogonality, have been used extensively in the past ,6,7,11 waterialise Mimics® software. 3D landmarks were derived from the centreline of the semicircular canals, calculated using the ‘medcad’ module in Mimics®, and then manually adjusted to optimize the position of the centreline through the lumen. This centreline was then split into four segments—the three canals  and the crus commune (a)—and exported as 3D coordinates describing a curve. The centreline curves were then resampled using the Resample executable [b).Specimens were µCT scanned using a variety of imaging systems and settings  and the semicircular canals manually segmented and 3D rendered using M commune a—and expecutable  so that 2.3.geomorph v. 2.1.4 [n = 131) and a phylogenetic subset (n = 41). Since the full dataset represented ontogenetic series of each species, the phylogenetic subset was represented by the largest adult male from each species paired with the phylogeny of Gamble et al. [c). As a previous study found significant sexual dimorphism in some Anolis species [Landmark coordinate data were aligned by Procrustes superimposition, allowing semilandmarks to slide along their tangent directions in order to minimize bending energy ,48 usingv. 2.1.4 . The rese et al.   was performed on the phylogenetic subset dataset to visualize semicircular canal shape variation among all species. Eigendecomposition of this PCA was performed using only modern taxa (including unique species); fossil specimens were later projected into this morphospace by matrix multiplication with the PCA eigenvectors. MANOVA was conducted to assess which PC axes significantly separated ecomorph groups. The phylogeny  was projtatistic ,54 with . 0.5-10 ,56. Cent. 0.5-10 . To visugeomorph, with pairwise comparisons tested using ‘advanced.procD.lm’ [. These functions perform statistical assessment of the terms in the model using Procrustes distances among specimens, rather than explained covariance matrices among variables, and are thus suitable for multivariate datasets [To determine whether ecomorphs occupy different regions of morphospace , and to test the effect of size and head proportions on canal shape, analysis of covariance (ANCOVA) and phylogenetic generalized least squares (PGLS) were performed on the phylogenetic dataset using the ‘procD.lm’ and ‘procD.pgls’ functions respectively  of geomorocD.lm’ . These fdatasets ,58. Thredatasets  and J. BMorpho v. 2.3.0 [n = 40) was less than the number of individual specimens (n = 99) [p < 0.05) of within-group distances, we reject the null-hypothesis that it belongs to that ecomorph group [Finally, a canonical variate analysis (CVA) with cross-validations was run using the ‘CVA’ function of the package v. 2.3.0  to explo(n = 99)  and to r(n = 99) . The pos(n = 99) –65. If tph group ,65–68. Fph group . To visuph group .3.3.1.a,b) shows that PC1 (40.2% of variation) largely represents changes in anterior and lateral canal morphology. Moving from PC1 positive to PC1 negative, there is a trend for the canals to become more rounded and anterodorsally shortened. PC2 (11.0% of variation) represents moderate changes in all three canals, with a PC2 positive to PC2 negative shift showing rounding of the anterior-most section of the anterior canal, more torsion (out-of-plane curvature) of the lateral canal, and less torsion of the posterior canal. PC3 (10.5% of variation) represents changes in the anterior and posterior canals, with a transition from PC3 positive to PC3 negative showing increased curvature and deepening of the posterior canal and reduction of the lateral aspect of the anterior canal. MANOVA results show that ecomorphs at not distinct on PC1, but they do significantly separate over subsequent PCs .PCA a,b showsVisually, there is significant overlap between ecomorph groupings along PC1 and PC2, with GB anoles occupying most of PC1. PC3 separates the three ‘trunk’ ecomorphs from the Tw and CG ecomorphs. When the shape data were corrected for size and phylogeny, the PC morphospace is minimally altered. The unique species are widely distributed across morphospace, overlapping with most ecomorph grouping (PC2 versus PC1) and falling outside the variation enclosed by the ecomorphs (PC2 versus PC3). All fossil specimens fall along the positive end of PC1, in the GB area of morphospace, which represents flattening and anterodorsal elongation of the anterior canal. Furthermore, three fossil specimens  overlap with multiple ecomorph groupings along PC3. Fossil specimens M-525 and OAAAA appear to fall beyond the morphologies of all living taxa sampled.3.2.K = 0.58), though permutation found this influence to be greater than expected from random (p = 0.0034). Mapping of the phylogeny onto morphospace  of canal shape. Multivariate pairwise post hoc tests found all ecomorphs to be significantly different (p < 0.05) from one another except Tr and TG . A weak but significant relationship also exists between canal centroid size and canal shape  and their interaction , as well as head proportions and canal shape  and their interaction  . This reduction in the correlation coefficient indicates an interaction between phylogeny and ecomorph and that ecomorph groupings are not entirely independent of phylogeny. PGLS  and an interaction between ecomorph and centroid size . Head proportions remains weak, yet significant , though PGLS reveals a much stronger interaction between head proportions and ecomorph . There was no significant relationship between either perch height or diameter and canal shape, with and without phylogenetic correction (tables ANCOVA  found th< 0.001) . Further< 0.001)  found ecny. PGLS  also rev3.3.p < 0.0001), with a cross-validation accuracy of 87.9% . On the extreme negative end of canonical function 1  are the Tw and CG ecomorphs. This region of morphospace is characterized by greater out-of-plane curvature of the anterior canal. The two groups, however, occupy opposite extremes of CV2 . CGs occupy the extreme positive end of CV2 and, when compared with Tw species, display greater dorsoventral curvature and reduced torsion of the posterior canal, and lengthening of the crus-commune. GB and the three ‘trunk’ ecomorphs occupy the central and positive region of CV1, characterized by reduced anterior canal curvature, but they do separate along CV2 and CV3 (b). TC and GB species occupy a more positive region along CV2, displaying greater dorsoventral curvature of the anterior canal, reduced torsion of the posterior canal, and longer crus-commune. TC and GB species separate along CV3 with the TC ecomorph occupying the positive end of CV3 indicating greater curvature of the lateral canal and reduced curvature of the posterior canal. The remaining ‘trunk’ ecomorphs separate along CV2, with the TG species occupying a more negative region of CV2, representing reduced torsion of the canals in general.The CVA biplots  and postn 1 CV1, a are thes of CV2 a,b. CGs  and CV3 b. TC andA. argenteolus falls within the 95% CI of TC (p = 0.139); (ii) the sole A. bartschi specimen falls within the 95% CI of TC (p = 0.895); (iii) out of five A. christophei specimens, one falls within the 95% CI of TG (p = 0.223), one in TC (p = 0.151), and one in GB (p = 0.073); (iv) out of five A. etheridgei specimens, two fall within the 95% CI of TG ; (v) out of five A. monticola specimens, three fall within the 95% CI of TG  and one in Tr (p = 0.103); (vi) the only A. reconditus falls within TG (p = 0.124); and (vii) out of four A. rimarum specimens, one falls within the 95% CI of GB (p = 0.478) and one in TC (p = 0.059). These results indicate that unique species display greater variation in semicircular canal shape than what is encompassed by the ecomorph categories. Our log-likelihood calculations do, however, assign all unique species to the defined ecomorphs, although these assignments are also inconsistent within and between species .The unique species (those that have not been assigned to any of the ecomorphs in previous studies) are spread throughout morphospace , with onet al. [All the fossil specimens fall either outside or just on the margin of the 95% CI of the modern ecomorphs, much like the unique species . Our poset al. : OAAAA, 4.4.1.Anolis species have convergent semicircular canal morphologies, allowing them to navigate similar ecological niches. Of the covariates we tested , we found ecomorph grouping to be the best determinant of canal shape, even when phylogeny is accounted for . While CV1 and 2 do not discriminate all groups, all axes of variation must be considered to properly establish group separation [post hoc tests do not fully align is uncertain—both statistical tests use similar non-parametric methods, though use different distant measures . Of alla): CG and Tw ecomorphs are the two groups that generally run the least, but also have to negotiate extremely complicated 3D environments. In mammals, increasing out-of-plane torsion of a canal may increase sensitivity to rotations out of the canal's major plane of motion detection [Both CG and Tw ecomorphs display more torsion of the anterior canal than those of the other ecomorphs, separating significantly along CV1 a: CG andetection ; thus suetection  and lengetection –72, requetection ,73 and cAnolis skull morphology using geometric morphometric techniques have established differences in skull shape between ecomorphs [Generally, however, the ecomorphological signal we found does not fully explain variation in semicircular canal shape. Analysis of canal shape and perch height and diameter returned non-significant results tables  and 2, dcomorphs ,74. Perh4.2.et al. [We found a small yet significant relationship between phylogenetic relatedness and semicircular canal morphology  and 2. Tet al.  concludeAnolis and our results also show that it covaries with ecomorph . This interaction suggests differences among the allometric shape trajectories of the six ecomorph groups. Previous studies have found that canal size appears to scale with negative allometry, such that smaller animals have relatively larger canals [Skull length and width  is correlated with semicircular canal morphology in r canals ,9,37,38.r canals  and that4.3.Anolis species enabled us to explore the palaeoecology of fossil taxa. Using posterior  probabilities, we find that the semicircular canal shapes of all five fossils are significantly different from modern ecomorph groupings  probabilities, we were unable to assign fossil anoles to modern ecomorph groups based upon semicircular canal shape. Our results indicate that the semicircular canals of these extinct anoles are morphologically different from modern Anolis ecomorphs, suggesting fossil taxa may have been interacting with their Miocene environment in different ways to modern Anolis species.Here we demonstrate that the classic ecomorph definitions of