The genetic diversity at major histocompatibility complex (MHC) in indigenous chicken breeds of India(Ghagus and Nicobari) in comparison with the White Leghorn (WLH) breed wasinvestigated by genotyping the MHC-linked LEI0258 marker. Altogether 38 allelesand 96 genotypes were observed among three breeds. The observed andeffective alleles were highest in Ghagus  followed by Nicobari  and WLH (10 and 2.2) breeds. The size of alleles ranged from 193 to 489 bp in Ghagus, 193 to 552 bp in Nicobari and 241 to 565 bp in the WLH breed. Thenumber of private alleles was also highest in Ghagus (18) followed byNicobari (8) and WLH (5) breeds. The most frequent allele was 261 bp in WLH(66 %), 343 bp in Nicobari (50.4 %) and 309 bp in the Ghagus (28.15 %)breed. Observed and expected heterozygosities were highest in Ghagus  followed by Nicobari  and WLH . The geneticdistance (Nei) between Ghagus and Nicobari breeds (2.24) was higher ascompared to that of Ghagus and WLH (1.23) and that between Nicobari and WLHbreeds (0.89). Association analysis revealed significant influence of MHCalleles on body weight, egg production in Ghagus and WLH breeds and antibodytitres to Newcastle disease vaccine in the Nicobari breed. These receptor genes are knownto play a significant role in innate immune competence. In another study, itwas demonstrated that expression of the TLR4 gene in lipopolysaccharide treated peripheral blood mononuclear cells wassignificantly higher in indigenous chicken breeds (Aseel and Ghagus) whencompared to improved (Dahlem Red and Broilers) chickens . Similarly, the expression of TLR4, TLR5 and TLR7 genes was significantly higher inindigenous breeds (Kadaknath and Aseel) than those of the WLH breed . General immune competence as assessed by cutaneous basophilhypersensitivity response against PHA-P and cytotoxic T lymphocytes (CD8Pastorella multocida) infection as mortality of the Nicobari breed was remarkably lesser ascompared to the improved Vanaraja cross . Therefore, it is evidentthat the genetic background of the chicken is known to influence the diseaseresistance and immune competence traits .Slow-growing indigenous chickens possess unique attributes like attractivemulticoloured plumage, hardiness, an ability to adapt to low input suboptimalrearing conditions and survive under harsh environments, broodiness,perceived desirable taste and flavour of meat and eggs, etc. Rearing ofindigenous chickens generates subsidiary income by utilizing minimum inputsand minimum human attention. It also helps in gender empowerment and socialupliftment of the rural/tribal people as mostly women and children areinvolved in rearing of indigenous chickens besides providing householdnutritional security. Further, they cater to the needs of consumers forcoloured birds and light-brown-shelled eggs in the niche market. Indigenouschickens are reported to be resistant to bacterial, protozoal, fungal andparasitic diseases as they were subjected to many years of natural selectionunder free-range or backyard systems of rearing . Previousstudies have reported the differences in immune competence traits betweenindigenous and improved chicken breeds owing to different geneticbackgrounds. Nicobari, a slow-growing indigenous breed was reported to berelatively resistant to viral diseases like Newcastle disease, Marek'sdisease  and infectious bursal disease . Furthermore, expression profiling ofvarious pattern recognition receptor genes in indigenous and White Leghorn(WLH) birds revealed the highest expression of Major histocompatibility complex (MHC) in chickens is known to play asignificant role in disease resistance, immune competence and productiontraits  including resistance to ectoparasites likenorthern fowl mites . Associations between the haplotypesof the MHC-B locus and responses to Rous sarcoma virus and resistance to avian coronavirus  were alsodemonstrated. MHC-linked LEI0258 alleles were reported to be significantlyassociated with production traits in WLH  andindigenous Khorasan chickens of Iran . Recently, itwas shown that LEI0258 alleles were associated with humoral and cellmediated immune response in broiler chickens .Alleles of the LEI0258 VNTR marker are associated with the serologically definedMHC B haplotypes . Various studies have used genotypingof the LEI0258 marker to assess the genetic diversity at MHC . In a study, Baelmans et al. (2005) detected completely unknown haplotypes using serological method infour lines of indigenous chickens of India, thus reflecting the potentiallyinteresting genetic pool at MHC. Therefore, the present study was carriedout with the objective of investigating the genetic variability at MHC inindigenous and WLH breeds of chicken and to study the association of allelesof LEI0258 on growth, production, immunity and survivability traits inindigenous chicken breeds in comparison with the improved WLH breed.22.1A total of 363 birds from three breeds of chicken were used for the presentstudy. Ghagus and Nicobari were the indigenous breeds of India, while a controlpopulation of White Leghorn (WLH) was the improved chicken breed. Breedingstocks of all three breeds were kept at the institute farm. Ghagus isphenotypically characterized by brown to red-coloured plumage, pea comb,white skin and brown eyes with high incidence of broodiness behaviour.Nicobari is small bird with brownish matt-coloured plumage and short legsthat originated in the Andaman and Nicobar islands of India . All three breeds were maintained as random bred closed populations.Chicks from three breeds were hatched simultaneously and reared on a floor(deep litter) up to 20 weeks of age, then in individual cages from 21 to 72 weeks ofage in an open-sided house. The details of rearing and management, healthcare andvaccinations of birds were described previously .2.2At 20 weeks of age, blood samples from female birds of 119 Ghagus, 125Nicobari and 119 WLH were collected from a wing vein of birds in 0.5 M EDTAanticoagulant, thoroughly mixed and stored at 2.3Alleles of the LEI0258 marker were identified through PCR. The nucleotide sequencesof primers used for amplification of the LEI0258 marker wereCACGCAGCAGAACTTGGTAAGG forward and AGCTGTGCTCAGTCCTCAGTGC reverse . Forward primer to amplify the LEI0258 marker through PCR waslabelled with 6-FAM dye. PCR was carried out using 0.13 2.4The exact size of alleles  of LEI0258 was determined by sequencing the PCR productsobtained using primers that bind just outside of the LEI0258 marker. Theprimers used to acquire entire region of the LEI0258 marker wereTCGGGAAAAGATCTGAGTCATTG forward (CAJF01F) and TGATTTTCAGATCGCGTTCCTC(CAJF01R) for the reverse direction . The PCR wasperformed in a final volume of 25 2.5The alleles were identified by their sizes. The observed , and natural antibody (NAb) titres to rabbitRBC (RRBC) at 20 weeks of age recorded in the three breeds previously  were used for the association study. The association ofLEI0258 alleles with various traits was determined using followingregression model3A total of 19 alleles (Table 1) were sequenced in bi-direction and aligned, andconsensus size was determined. It was observed that the sizes of allelesdetected by capillary electrophoresis (fragment lengths) and sequencing didnot match. Overall the difference in size ranged from 1 to 20 bp. There wasa pattern in the size difference. The smaller the size of alleles, the smaller thedifference in size was. The size difference of 1 bp was observed for 195, 262and 296 bp alleles. Likewise, the size differences were found to be 3 bp for 312 and 346 bp alleles, 4 bp for 361,373 and 385 bp alleles, 6 bp for 451 and 495 bp alleles, 13 bp for 552 bpallele, and finally 20 bp for 572 and 585 bp alleles. This pattern ofdifference in size was used for extrapolating the size of remaininggenotyped alleles . Similar to the findings of thepresent study, a size difference of 2 to 17 bp was reported by Mwambene etal. (2019). However, Han et al. (2013) reported a higher (1 to 65 bp)difference in the size of genotyped allele and that determined by thesequencing.Previous studies have reported two types of polymorphisms in the LEI0258marker. One is the number of repeats of the R13 and R12 motifs, and theother is the number of SNPs and indels upstream (Genetic diversity at major histocompatibility (MHC) in Ghagus, Nicobari andWLH breeds was studied by genotyping the MHC-linked LEI0258 marker. Overall 38alleles with size ranging from 193 to 565 bp and 96 genotypes wereobserved in three breeds . In Ghagus, a total of 23 alleles and 52genotypes were found. The size of alleles ranged from 193 to 489 bp. Themost frequent allele was 309 bp (28.15 %), and the least frequent alleleswere 453 and 458 bp (0.42 %). The most frequent genotypes occurring were 309/309, 309/357 and 309/445 bp, each with the proportion of 6.72 %. Atotal of 14 alleles and 27 genotypes were observed in Nicobari with allelesize varying from 193 to 552 bp. In this breed the most and least frequentalleles were 343 bp (50.4 %) and 354, 357 and 552 bp (0.40 %),respectively. The most frequent genotypes were on the order of 343/343(29.6 %), 261/343 (19.2 %) and 261/261 bp (8.0 %). In WLH, a total of10 alleles were seen with the most and least frequent ones being 261 bp(66 %) and 565 bp (0.42 %), respectively. The allele size varied from 241to 565 bp, and among the 17 observed genotypes, 261/261 (47.9 %), 261/403(9.2 %) and 261/552 bp (8.4 %) genotypes were the most frequentlyoccurring ones.Number of genotypes, number of observed alleles (Expected and unbiased expected heterozygosities were highest in Ghagusfollowed by Nicobari and WLH breeds (Table 2). As a result, significant. As compared to the reference (309 bp)allele, the 194 bp allele had a significant positive influence on body weightrecorded at 4 and 8 weeks of age. The 194 bp allele also had a significantpositive influence on shank length at 8 weeks of age, egg production andsurvivability up to 72 weeks of age. However, the 445 bp allele had asignificant negative effect on body weight recorded at 40 weeks of age. Thisallele also had a nonsignificant negative influence on body weight recordedat other ages. None of the alleles had a significant effect on age at firstegg, shank length at 16 weeks of age and immunity traits measured at 20 weeks of age in this breed.In the Nicobari breed, out of 16 alleles observed, 261 and 295 bp alleles had asignificant positive association with antibody titres to NDV vaccine at 20 weeks of age when compared to the reference allele (343 bp). However, noalleles were found to be significantly associated with growth, production,NAb titres to RRBC or survivability traits in this breed (Table 5).In WLH, the 403 bp allele had a significant negative influence on body weightrecorded at 16 weeks of age. The negative effect of this allele was alsoobserved on body weight recorded at a different age, but they were notsignificant. When compared to the reference allele (261 bp), 309 and 552 bpalleles were significantly negatively associated with the egg production upto 72 weeks of age. The 309 bp allele was also significantly negativelyassociated with survivability of hens up to 72 weeks of age. No allele wasfound to have significant effect on shank length, age at first egg orimmunity traits in this breed (Table 5).4Considering the number of alleles, genotypes and heterozygosity of the LEI0258VNTR locus, it was evident that genetic diversity at MHC in indigenousbreeds was higher than that of the WLH breed. Indigenous breeds were notsubjected to artificial selection, and only natural selection was operatingin their rearing environment to favour individuals with higher resistance tovarious diseases that prevailed in the field as MHC is linked with diseaseresistance, and hence the higher diversity was observed in indigenous breeds.Furthermore, the Ghagus breed was recently collected from its native tract (Sixgenerations back), while WLH was subjected to selection for higher eggproduction for last several generations, and hence, some degree ofinbreeding could have taken place in this breed. Previous studies alsoreported higher diversity in the MHC-linked LEI0258 locus in indigenous chickensof various countries. A high number of alleles of LEI0258 was reported ineight ecotypes  of Kenya , three indigenousbreeds  of Iran  and in 33 indigenousbreeds  of China . On the other hand, lessergenetic variability and diversity at MHC were reported in intensivelyselected commercial egg layer varieties  and commercialwhite egg layers  previously. Higher allelic diversityobserved in indigenous chicken breeds can act as a source of unique ornovel MHC haplotype types that could be helpful in deciding breedingstrategies for conservation and improvement of indigenous chickens forrearing in free-range or backyard systems of production.The most frequently observed allele was 261 bp among the three breeds. Thealleles that were common to the three breeds were 261 and 309 bp. Finding of thehigh frequency of the 261 bp allele in WLH (66.0 %) and to some extent inNicobari (22.8 %) breeds as compared to the Ghagus (7.6 %) breed couldbe explained by the fact that the 261 bp allele is known to be associated withthe B15 haplotype , and this haplotype is reported tobe linked with the high egg production . WLH is an egg-type improved breed which was selected for higher egg production, whileNicobari is known to have the highest egg production among indigenous breedsof India. Therefore high frequency of the 261 bp allele was observed in thesetwo breeds.The present study uncovered 12 unique/novel alleles  that were not reported previouslyeither by genotyping or by sequencing . The size of 403 and 565 bp alleles wereconfirmed by sequencing. However, the size of remaining alleles wasdetermined by extrapolating the genotyped sizes. Therefore, the precise sizeof these alleles needs to be confirmed by sequencing. Nevertheless, thefindings suggest the possibility of indigenous chickens  possessing the unique alleles of the LEI0258 marker. OnlyBW3 (194 bp), 13.1 (381 bp), 19 (539 bp) and 19.1 (552 bp) serologicallydefined B haplotypes could be correlated among the three breeds. These findingsare in conformity with the previous study, which reported that birds of Indian originhad completely unknown haplotypes, and only few serologically definedhaplotypes  could be detected in four lines of indigenouschickens of India , thus reflecting potentiallyinteresting genetic pool in indigenous breeds.Ghagus and Nicobari breeds deviated from HWE. The deviation from HWE occursdue to selection , missing genotypes, genetic drift,mutation, migration, population substructure, genotyping error, etc. . Genotyping error in this study was ruled out as sizes ofalleles were first determined on agarose gel electrophoresis, and the precisesize of all alleles were then determined on automated analyser, and theprocedures used were common to all three breeds. The possible reason fordeviation could be missing genotypes, population substructure or naturalselection observed in both indigenous breeds of chicken. Similar deviationfrom HWE was reported with high heterozygosity at MHC in chicken populationsoriginated in China . Similar to the findings of ourstudy, Mwambene et al. (2019) also reported significant deviation from HWEfor the LEI0258 marker in 6 out of 10 indigenous chicken ecotypes of Africa.It was opined that deficiency of heterozygosity (lesser The genetic distance (Nei) between Ghagus and Nicobari breeds was higher ascompared to Ghagus and WLH and that between Nicobari and WLH breeds.Mainland India and Andaman and Nicobar islands are separated by Bay ofBengal, and therefore there was least chance for genetic relatedness amongthese two indigenous breeds. Closer genetic relatedness observed betweenNicobari and WLH was somewhat unexpected, and this can be explained by thefact that the Nicobari breed was reported to have originated from the crossesof different exotic breeds such as WLH, Australorp, Plymouth Rock, etc., andindigenous fowl of the Nicobar group of islands .Therefore, closer genetic relationship was observed between Nicobari and WLHbreeds. Therefore, Ghagus was genetically distant from both Nicobari and WLHbreeds. Similar to the findings of the present study, Ahlawat et al. (2004)reported the high genetic similarity (0.80) between Nicobari and WLH breedsof chicken using RAPD analysis.Alleles of LEI0258 had a significant association with body weight, eggproduction and survivability traits in Ghagus and WLH breeds and antibodytitres to NDV in the Nicobari breed. Significant positive association of LEI0258alleles with body weight at 16 weeks of age was also reported in Tanzaniannative chickens , and negative association with bodyweight at 8 and 12 weeks of age was reported in Iranian Khorasan native chickens . Survivability of hens is an important economic traitwhich has bearing on production performance. A positive association of 194bp allele with egg production up to 72 weeks of age in Ghagus can beexplained from the fact that the survivability of hens with the 194 bp allele was alsosignificantly higher as compared to the reference allele (309 bp). Allele309 bp, which had negative association with survivability of hens up to 72 weeks of age, also had negative association with egg production up to 72 weeks of age. This might be an indirect association as hens which survivefor shorter/longer duration will have lesser/higher chances of producingless/more eggs. Egg production up to 72 weeks of age was highestin hens with the 261 bp allele , which has the highest frequencyin the WLH breed. Similarly, the survivability of hens having the 261 bp allele washighest as compared to other alleles  in the WLH breed.Significant association of LEI0258 alleles with the egg laying intensity traitwas also reported in Khorasan indigenous chickens of Iran . Another study reported the significant association ofalleles of the BF2 locus of MHC with early and late mortality in commercialbroiler line  suggesting the role of MHC haplotypes insurvivability trait of chickens. However, to our knowledge this is the firststudy to report the effects of LEI0258 alleles on duration of the survivabilityof hens during the 72-week laying period.In Nicobari, LEI0258 alleles (261 and 295 bp) were positively associatedwith antibody titres to NDV vaccine. Similar to the findings of the presentstudy, significant association of some of the LEI0258 alleles with antibodyresponse to NDV was also reported in Tanzanian chicken ecotypes  and broiler chickens . Thesefindings reiterate the importance of MHC in vaccine response to the NDV antigen.Among viral diseases, incidence of Newcastle disease is the highest innative chickens reared in backyard or free-range systems of production.Therefore, information on diversity at MHC helps in devising breedingstrategies for conservation and improvement of native chickens.Associations between LEI0258 alleles and survivability and immune traitsobserved in the present study can result from direct effect of MHC genes,which are known to influence disease resistance and immune competencetraits. Those of production traits may be due to indirect associationbetween survivability (longer duration of production) and egg production upto 72 weeks of age. Almost all associations of LEI0258 alleles withproduction traits were found to be breed specific. This is due to the factthat  no alleles were found to be common to allthree breeds and that reference alleles were different among the three breeds todetermine the effect of each allele in comparison to the reference allele.Therefore, allelic effects on various traits need to be studied breed/line-wise  and this is particularly relevant in indigenouschicken breeds due to prevalence of high diversity at MHC.5From the findings of the present study, it is concluded that the LEI0258 marker washighly polymorphic in indigenous breeds, and the number of alleles identifiedin each breed was different. Considering various parameters examined likenumber of observed and effective number of alleles, genotypes,heterozygosity, etc., it was evident that indigenous chicken breeds showedhigher genetic variability at the MHC region over WLH egg-layer birds.Furthermore, the indigenous breeds likely to have novel alleles of the LEI0258marker. Some of the alleles were found to have significant association withgrowth, production, survivability and antibody titres to NDV. Furtherstudies are required to explore the MHC diversity in indigenous chickensthrough sequencing of the LEI0258 marker for SNP and indel polymorphisms andalso to investigate the effects of novel alleles on variousimmune-competences, disease resistance and production traits using largenumber of samples particularly in indigenous chicken breeds.


When fat mass percentage and age were added to normalised jump power, 30% of TUG was explained in older men . Appendicular lean muscle mass percentage (ALM%) and age were the best determinants of TUG for older women . HR% explained 17–39% of 6MWD across all groups . In conclusion, in men, jump power was a key determinant for TUG, while in old women only it was the ALM%. As HR% was the most important determinant of 6MWD, motivational bias needs to be considered in the interpretation of this test.The aim of this study was to assess associations between performance in the timed up-and-go (TUG) and six-minute walk distance (6MWD) with physiological characteristics in young and old healthy adults. Thereto, we determined TUG, 6MWD, normalised jump power, centre of pressure displacement during 1-leg standing, forced expiratory volume in 1 s, percentage of age-predicted maximal heart rate (HR%) and height in 419 healthy young  and old  adults. Normalised jump power explained 8% and 19% of TUG in young ( The timed-up-and-go test (TUG)  was origIn addition to measures of balance, jump power normalised to body mass is strongly related to TUG and six-minute walk distance (6MWD), and more so than maximal force normalised to body mass ,18,19. IThe 6MWD has been used to predict maximal aerobic capacity in healthy middle-aged and older adults ,22 and iPrevious studies have been limited by small homogeneous populations and have assessed a limited number of variables to explain the TUG and 6MWD. Few studies have analysed whether the determinants of performance in the TUG and 6MWD differ between young and old men and women. Further investigation is therefore warranted in a larger multicentre cohort of healthy young and old men and women applying an integrative analysis of determinants of physical function across the lifespan to inform clinical practice. Specifically, the objective of this study was to examine associations between performance in the TUG and 6MWD with normalised jump power, balance, lung function, percentage of age-predicted maximal heart rate (HR%) and height in a large multicentre cohort of healthy young and old men and women. We hypothesised that normalised jump power and balance are determinants of performance in the TUG and that normalised jump power, lung function, and HR%  are dete2 were also excluded along with those undertaking competitive sports . Participants avoided strenuous exercise 48 h prior to the testing session and refrained from smoking two hours before the testing session. All studies were approved by the respective local ethical committees  and adhered to the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study and all participants were medically screened prior to participation.Four hundred and nineteen participants were recruited from the European multi-centre MYOAGE cohort . Partici2 (kg/m2).Standing height (m) was measured to an accuracy of one millimeter whilst body mass was measured to an accuracy of 0.1 kg. Body mass index (BMI) was calculated as mass/heightFollowing a 12-h overnight fast, body composition was assessed using whole-body dual-energy X-ray absorptiometry . DEXA scans were performed by a trained technician according to the manufacturer’s quality control procedures including daily calibration. After manual offline adjustment, whole-body lean mass and fat mass were calculated. Lean mass was calculated as: 2 and areas were demarcated as described previously [Appendicular lean muscle mass (ALM) was also given as ALM/heighteviously .n = 309), where postural sway was measured as centre of pressure displacement during one-leg standing with eyes open as measured from force platform data  [d).The majority of participants undertook a balance assessment  . The parFor the 6MWD, participants were instructed to complete as many 20-m laps (25-m in France) as possible in six minutes without running and the distance covered recorded . Verbal The TUG test involved getting up from a standard chair without armrests, walking around a cone 3 m in front of the chair and returning to the original sitting position as quickly as possible without running . The tesg) at a constant of 9.81 m/s2. Instantaneous vertical acceleration (a) was calculated and integrated to obtain instantaneous vertical velocity (v) and then power (P) was calculated from the instantaneous force and vertical velocity [Leg extension power was assessed with a maximal effort countermovement vertical jump on a force platform . Three maximal-effort countermovement vertical jumps were performed with a 1-min rest between efforts. Vertical ground reaction force was recorded throughout the movement at 1000 Hz. Maximum power was calculated using the following equations from the vertical force trace (Fz) and body mass of the participant (m) with acceleration due to gravity  was recorded in litres according to the criteria of the American Thoracic and European Respiratory Society [1 value was used in the final analysis.Participants completed three maximal spirometry tests whilst seated with their hips and knees flexed at 90° and wore a nose clip throughout the procedure. Participants were instructed to “blow into the mouthpiece as forcefully and as quickly as possible” and to continue blowing until no further air could be expelled . Spirometric pulmonary function was not undertaken at the Estonia site. Forced expiratory volume in 1 s (FEV Society  and the p < 0.1 were then selected for inclusion in multivariate regression models. Variables were entered into the models in order of the Pearson’s product–moment correlation coefficient (largest first). In age-group and sex-specific multivariate regression models, the most parsimonious model was identified as the model with the highest explained variance (R2adj), separately across young and old, men and women. Those body composition and physiological variables included in the most parsimonious model for any one of the separate subgroups  were then included together in a final multivariate regression model that was run in each of the four sub-groups to highlight the different outcomes for age and sex groupings. The level of statistical significance was set at p < 0.05. All analyses were performed using R .Differences in participant characteristics by age-group and sex were formally tested using two-way between-measures analysis of variances (ANOVAs) (2 age groups × 2 sexes). All participant characteristics, body composition, and physiological variables were then normalised to the average value of the young male population in each individual country to adjust for any systematic differences between countries. Data were formally tested and visually inspected for independence, linearity, normality of residuals, multi-collinearity and homoscedasticity to ensure suitability for entry into multivariate regression analyses. Relationships between each body composition and physiological variable and performance in the two physical function tests (TUG and 6MWD) were first evaluated using Pearson’s product–moment correlation coefficient stratified by age-group and sex. Those variables that had a bivariate correlation with statistical significance of p = 0.010), appendicular lean muscle mass (p = 0.001), appendicular lean muscle mass percentage (p = 0.009), ALM/height2 (p = 0.046), normalised jump power (p < 0.001), FEV1 (p < 0.001), and 6MWD (p = 0.032), which is reflected by a larger absolute age-related decline in men than women (p = 0.037), which is reflected by a larger age-related increase in men than women (an women . There wan women .p < 0.001), which was reflected by a larger age-related increase in fat mass percentage in men than in women.In a separate analysis, values were converted to a percentage of the sex-matched average . This showed that there were no significant age-by-sex interactions, indicating a similar age-related percentage decline in men and women. There was, however, a significant age-by-sex interaction for fat mass percentage  and older men , respectively, (p = 0.008), ALM% was a stronger single determinant and explained 13% of the variance in TUG for older women . Normalised jump power , fat mass (%)  and age  were the best determinants of TUG for older men , whereas ALM%  and age  were the best determinants of TUG for older women . There were no significant determinants of TUG performance for young women. Normalised jump power as a single determinant explained 8% and 19% of the variance in TUG performance for young men  (p < 0.001) and fat mass (%)  were determinants of TUG performance in older men and fat mass (%) was a determinant of TUG performance in older women .When all explanatory independent variables were combined in a separate multivariate regression model stratified by age and sex, there were no significant determinants of TUG performance for young men, and only height was a significant determinant of TUG performance for young women  . Normali2adj = 0.17 to 39, p < 0.001), (p < 0.001) and lean mass (%)  were the best determinants of 6MWD in young men, whereas HR%  and COP-MLd  were the best determinants of 6MWD in young women.HR% was the strongest single determinant of 6MWD across all young and old, men and women (R 0.001), . HR% (β p < 0.001), normalised jump power , lean mass (%)  and FEV1 , were the best determinants of 6MWD in older men . However, HR% , appendicular lean muscle mass percentage , FEV1  and age  were the best determinants of 6MWD in older women . HR% , and only HR%  and lean mass (%)  were significant determinants of 6MWD for young women (p < 0.001), lean mass (%)  and age  were determinants of 6MWD in older men whereas HR% , normalized jump power  and age  were determinants of 6MWD in older women.When all explanatory independent variables were combined in a separate multivariate regression model stratified by age and sex, only HR% was a significant determinant of 6MWD for young men  explains 10% of the variance for older men and ALM% explains 13% of the variance in older women. A greater proportion of lean mass is likely to contribute to increasing propulsive forces, which may result in an improved TUG, whereas a greater proportion of fat mass will likely hinder TUG due to the larger mass component that reduces propulsive forces relative to body mass . The comThe current results indicate that HR% explains 17 to 39% of the variance in the 6MWD across young and old, men and women. The HR% can serve as a measure of physical effort as a lower HR% is associated with a lower 6MWD, irrespective of sex and age. This indicates that the level of engagement with the test has a strong impact on the 6MWD, similar to previous observations .Interestingly there was a correlation with relative measures of lean mass and 6MWD for young men, older men and older women ,52, thatNormalised jump power explained an additional 4% of the variance in the 6MWD for older men only. Interestingly, normalised jump power was not a significant determinant of 6MWD in any other group. Lower limb power has shown a strong correlation with 6MWD in mobility-limited older individuals whereas aerobic capacity has shown no relation, suggesting that lower limb power may become increasingly important in limiting the 6MWD in populations with mobility impairments . The sig1 explains an additional 6% of the variance in 6MWD for older women and 5% in older men although not significant. This is noteworthy, as irrespective of physical activity level FEV1 shows an age-related decline that may limit aerobic capacity with advancing ageing [1 may be particularly important when considering that in older adults declines in resting lung function can occur with no changes in maximal heart rate over a six year period [FEVg ageing . The ager period .The strength of this study was that healthy young and older volunteers were recruited and our work thus represents the determinants of performance during healthy ageing. In addition, this study had a cross-sectional design and can only highlight associations between physical function tests and physiological variables. To establish a causal effect of the measured variables on physical function, intervention studies are necessary. The multi-centre design may have meant that testing protocols were not exactly identical at different sites. To minimise this effect, (1) staff at all sites were trained to deliver the same protocols by the same experienced researcher and all equipment was calibrated according to the manufacturer’s requirement and (2) all data from a study centre were normalised to the average data from the young men in that centre.1 and age, 53 to 60% of the variance in 6MWD was explained in older men and older women respectively. As HR% was the most important determinant of 6MWD, motivational bias needs to be considered in the interpretation of this test. It is important to consider that individuals with low muscle power or individuals demonstrating a low level of effort are at risk of lower functional performance in a clinical setting.In conclusion, in men, jump power was a key determinant for TUG, while in old women, it was only the ALM%. The largest single determinant of the 6MWD was HR%, which explained 17 to 39% of the variance in 6MWD across young and old men and women. When HR% was combined with normalised jump power (older men only), relative measures of body composition, FEV


Thygeson’s superficial punctate keratitis (TSPK) is a rare and still poorly understood disease of the ocular surface, responsible for recurrent episodes of photophobia and eye pain. While TSPK is considered as a benign condition, a subset of patients has frequent recurrences or even chronic disease, two situations in which there are currently no therapeutic guidelines. We used a preexisting Facebook TSPK patient support group to assess the clinical journey and the burden of disease of TSPK.An online survey was sent to the patient support group. The first part of the questionnaire gathered information on demographics and the patient’s clinical journey . The second part focused on quality of life (QoL) using the Ocular Surface Disease-QoL (OSD-QoL) questionnaire. Seventy-two patients out of 595 members of the support group completed the questionnaire during the 3-months study period. Eighty percent of patients developed symptoms before 30 years old, and 47% reported a delay in the diagnosis above 1 year. Sixty percent of patients reported over 5 RE yearly, and 18% of RE lasted more than 3 months. Forty percent of all patients used cyclosporine eyedrops (50% of those with > 5 episodes/year) and it was perceived as effective by 72% of these patients. The impact on daily life activities was judged as severe by 22% of patients, while 38% reported reduced professional activity and 80% were deeply saddened by their eye condition.TSPK patients may present with frequent recurrences and/or chronic disease, that result in a severe impact on QoL, and an off-label use of topical immunomodulatory eye drops, suggesting the urgent need for controlled studies. The utility of using social networks for rare ophthalmic disease research includes, faster data collection, data from patients across the globe, and also raises relevant questions about their real needs. Thygeson’s superficial punctate keratitis (TSPK) is a rare ocular surface disease, characterized by recurrent episodes of photophobia and foreign body sensation, associated with multiple white–grey superficial epithelial lesions, without stromal involvement and mild or absent conjunctival hyperemia –3. The eTSPK seems to affect more females than males, age of onset is usually in the third decade of life, but can affect people of all ages, from 2 years old to 71 years of age –10. HoweThe assessment of quality of life (QoL) of patients with TSPK has not been reported in the literature even though recurrent episodes of keratitis could have a significant impact both professionally and socially .Patient support groups formed through online social networks offer a new opportunity for patients with rare diseases to exchange information worldwide on their condition. Facebook is the most popular online social network. This online social media platform was founded in 2004, has more than 2.4 billion active monthly users .Physicians and scientists can therefore take the opportunity to reach out to patients worldwide through online patient support groups, to collect data on rare diseases, across the global demographic –17 but, This non-interventional study involved the evaluation of results from an online survey, which was sent to a Facebook support group of patients with TSPK . The queThe questionnaire was divided in two parts. The first part used 29 questions to gather data on demographics, the clinical journey of patients including diagnostic modalities, symptoms, duration and frequency of recurrent episodes, efficacy and tolerance of treatments. The full questionnaire is available online  completed the questionnaire from November 2018 to January 2019. There were 58 (81%) females and 14 (19%) males, aged 30 ± 13 years (range 5–59 years). The majority of patients were Caucasian (75%) and living in North America (64%)  declared a history of inflammatory or autoimmune disease such as psoriasis, systemic lupus erythematosus, Sjögren’s syndrome, Hashimoto's thyroiditis or Crohn's disease. None of the patients reported myasthenia gravis, coeliac disease or type 1 diabetes mellitus.. Therapeutic contact lenses had been used by 36% of patients and considered efficacious by 68% of these patients whereas lubricants were considered not efficacious by 48% of patients , 7 patients were treated with immunomodulatory eyedrops  and 6/7 patients reported moderate to complete efficiency.Overall, the average OSD-QoL score was 55.8 ± 17.5. The impact on daily life activities was moderate for 53% of patients and severe for 22% of patients. Impact on professional and physical activities was moderate to severe for 66% of patients and 38% had to reduce their professional activity. Seventy-eight percent and 54% of patients reported that their eye problems were not acknowledged or barely acknowledged by other people or clinicians respectively. Sixty-eight percent of patients feared never being cured or have greater impairment over time. 80% reported being deeply saddened by their eye condition  . ResponsIn summary, we collected important and new data on the patient’s clinical journey and the burden of disease of TSPK, a rare and still misunderstood ocular disease. The majority of participants suffers from frequent recurrences and/or chronic disease is common and results in a severe impact on QoL. Additionally, there is frequent off-label use of topical immunomodulatory eyedrops, suggesting the still unmet need for controlled studies to standardize therapeutic protocols. This study also highlights the potential use of social networks for research in rare ophthalmic diseases for rapid collection of data from a diverse sample of patients and thus raises relevant questions about their real needs.Additional file 1: Online questionnaire for Thygeson superficial puncate keratitis patients.Additional file 2: Supplementary table 1: Patients demographics. Supplemetary table 2: Patient-reported treatment efficacy. Supplementary table 3: Patient-reported treatment tolerance. Supplementary table 4: Distribution of patients according to their answers to the OSD-QoL items.


After extensive acute injuries, this phenomenon substantially limits the possibility of lost function recovery and, in case of chronic injury, it leads to pathological remodeling of organs affected. Managing outcomes of damaged tissue repair is one of the main objectives of regenerative medicine. The first priority for reaching it is comparative investigation of mechanisms responsible for complete restoration of damaged tissues and mechanisms of scarring. However, human body tissues that undergo complete scar-free healing are scarce. The endometrium is a unique mucous membrane in the human body that heals without scarring after various injuries, as well as during each menstrual cycle . We hypothesized that absence of scarring during endometrial healing may be associated with tissue-specific features of its stromal cells (SCs) or their microenvironment, since SCs transform into myofibroblasts—the main effector link of scarring. We found that during healing of the endometrium, soluble factors are formed that inhibit the transition of SCs into myofibroblasts. Without influence of these factors, the SCs of the endometrium undergo transformation into myofibroblasts after transforming growth factor β1 (TGF-β1) treatment as well as the SCs from tissues that heal by scarring—skin or fat. However, unlike the latter, endometrial SCs organize extracellular matrix (ECM) in a specific way and are not prone to formation of bulky connective tissue structures. Thus, we may suggest that tissue-specific features of endometrial SCs along with effects of soluble factors secreted in utero during menstruation ensure scar-free healing of human endometrium.Besides certain exceptions, healing of most tissues in the human body occurs  Formation and expansion of fibrous tissue known as fibrogenesis occur at the site of injury and in adjacent tissues. In the human body, it is an integral feature of normal physiological response to damage .Despite its obvious helpfulness from the survival point of view, fibrogenesis poses a challenge for modern healthcare. Human organs undergoing fibrosis after the damage of different kinds are subject to further pathologic remodeling and loss of function resulting in chronic disease. Besides, fibrotic tissue limits the mobility of surrounding tissues and creates an obstacle to the formation of new functional structures at the lesion site. Hence, establishment of molecular mechanisms of fibrogenesis may also be important for the development of methods that prevent fibrosis and stimulate the recovery of normal structures after damage. Its challenging nature and complexity drive multiple studies in the field, and in spite of significant advances, effective prevention of fibrosis in humans is still to be reached . The facThe human body does have a limited number of structures able to respond to different kinds of injuring events preferably by regeneration. These include the endometrium—the internal mucous layer of the uterus reaching 10–15 mm thickness in adult healthy women. The endometrium has a remarkable ability to regenerate after damage without scarring. This occurs both after surgical scraping of endometrium known as curettage and after its full-thickness section during resectoscopic tumor removals . MoreoveHomo sapiens is one of the few species that possess an interesting feature relying on endometrial regeneration. That can be described as a physiological renewal of endometrium via its self-damage—namely, menstruation, which swiftly transits to rapid healing without fibrosis and ends up by the full recovery of its structure in every menstrual cycle. The latter is high in number and in healthy women may account for up to 400 episodes of endometrial self-damage and regeneration without scarring.A comprehensive review of menstruation mechanisms is provided by We paid attention to stromal cells (SCs) of the endometrium as far as in most tissues, SCs are major effectors of fibrogenesis . At the Damaged tissues have a high concentration of TGF-β1: it is released from activated platelets, produced by recruited monocytes and macrophages; eventually, it is secreted by cells of damaged tissue especially under mechanical stress . TGF-β1 via polymerization of actin with inclusion of α-SMA providing the high contractile ability of myofibroblasts. Stress fibers are assembled around focal adhesion contacts that mediate interaction of cytoskeleton with ECM via talin, vinculin, and a repertoire of integrins (An increased production and decreased degradation of extracellular matrix (ECM) shift the balance toward its deposition . At the ntegrins .Thus, the impact of TGF-β1 released after damage may result in a stable transition of SCs to myofibroblasts, but due to unknown reasons, this does not seem to occur during the healing of the endometrium . We suggIsolated SCs were cultured using uncoated culture dishes or plates  in an appropriate growth medium according to a previously performed experimental selection:•EndoSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12  + 10% fetal bovine serum .•DermSCs in DMEM low glucose  + 10% FBS .•AdipoSCs in AdvanceStem Medium  + 10% AdvanceStem Supplement .The medium was changed every 2–4 days, and when confluence reached 80–90%, cells were detached and passaged at 1:3–1:4 ratio. Trypsin-EDTA (0.05%) solution  was used for detachment of cells from the culture plastic. Obtained suspension was transferred into a tube with 5 ml of complete growth medium and centrifuged at 200 g for 5 min, then supernatant was removed, and the cells were resuspended in growth medium and seeded to clean culture dishes.Adipose tissue samples were obtained from three healthy donors chosen from inpatients of Lomonosov Moscow State University Medical Centre. Tissue sample was placed into a tube with Hanks’ balanced salt solution  containing 500 U/ml of penicillin and 500 μg/ml of streptomycin  and transported to the laboratory. Obtained material was minced to a homogeneous state with sterile surgical scissors under a laminar hood and placed into a tube. The homogenate was then subjected to enzymatic treatment in AdvanceStem Medium  with a mixture of collagenase I  and dispase  added in a volume equal to the minced tissue suspension. Enzymatic digestion lasted 60 min at 37°C with intense shaking every 5–10 min. After digestion, an equal volume of AdvanceStem Medium  with 10% AdvanceStem Supplement  was added to the resulting mixture to inactivate enzymes. After subsequent centrifugation at 200 g for 10 min, the supernatant and floating fraction of adipose tissue were discarded and the pellet was resuspended in complete growth medium  with 10% AdvanceStem Supplement . The obtained suspension was filtered through nylon strainers with pore size of 100 μm  to remove debris. Filtrate was centrifuged , supernatant was removed, and the pellet was resuspended in complete growth medium followed by transfer to an uncoated cell culture dish of a suitable size. After overnight culture, the medium was changed and isolated AdipoSCs were cultured as described above (see section “Cell Culture”).3 that were placed into a tube. Equal volume of DMEM low glucose containing a mixture of type I collagenase  and dispase  was added to the cut skin. Enzymatic digestion lasted 60 min at 37°C with intense shaking every 5–10 min. After digestion, an equal volume of complete growth medium [DMEM low glucose  with 10% FBS ] was added to the resulting mixture to inactivate enzymes. Obtained suspension was centrifuged at 200 g for 10 min, and supernatant was removed. The pellet was resuspended in complete growth medium and transferred to a cell culture dish of suitable diameter. The next day, the growth medium was changed and isolated DermSCs were cultured as described above (see section “Cell Culture”).Human skin samples were obtained from three healthy donors. Every sample was cut with sterile scissors in a laminar box to pieces of no more than 0.25 cmMenstrual discharge was collected from healthy volunteer female donors using a menstrual cup  for 6–8 h during the second day of menstruation. Obtained discharge was transferred into a sterile 50 ml tube, and biomaterial was delivered to the laboratory at + 4°C within 2–3 h. Immediately after delivery, menstrual discharge was diluted twofold by serum-free DMEM/F12 , gently resuspended and centrifuged for 20 min at 300 g . The supernatant [menstrual discharge serum (MDS)] was collected with a 25 ml syringe with a needle into a separate 50 ml tube and left until the centrifugation step described below.3, Paneco, Russia) in a 50 ml tube. The tube was allowed to stand for 10 min for sedimentation of blood clots and large pieces of tissue into the thickness of the Ficoll. Then, the tube was carefully transferred to a centrifuge  avoiding phase mixing.The cell pellet was resuspended in four volumes of dilution buffer consisting of 1% FBS  in HBSS  with 5 mM ethylenediaminetetraacetic acid . Then, it was intensively pipetted with a 25 ml serological pipette tip and carefully layered on 13 ml of Ficoll . After centrifugation, the supernatant from the MDS tube was collected with a 25 ml syringe with needle and filtered through a 0.22 μm filter , and the obtained liquid was designated as 25% MDS .Cell Culture). The cells were successfully isolated from 10 samples, and three cultures were chosen for subsequent experiments.After centrifugation, 3/4 of the supernatant were discarded. Residual supernatant, cells, and small pieces of tissue were collected from the Ficoll surface into a 15 ml tube using a 10 ml pipette, diluted to a volume of 14 ml by dilution buffer and centrifuged for 10 min at 300 g. Supernatant was discarded, pellet was resuspended in 2 ml of complete growth medium [DMEM/F12  with 10% FBS ] and transferred to a 35 mm cell culture dish for 1 h. Thereafter, the excess of sedimented, but non-adhered, cells was detached by gentle manual shaking of the dish, and the medium was changed. After 24 h, the medium was changed again and isolated EndoSCs were cultured as described above  was collected into tubes containing coagulation activator  from the cubital vein of 15 healthy volunteer female donors at the second day of menstruation. After completion of blood clotting, tubes were centrifuged for 20 min at 300 g. Blood serum (BS) was collected with a 10 ml syringe with a needle, diluted four times with DMEM/F12 , filtered through a 0.22 μm filter  to comply with MDS isolation protocol, then frozen in liquid nitrogen and stored at −20°C. The resulting liquid was used for further experiments as 25% BS. Before the experiments, BS samples obtained from 15 donors were thawed and mixed into one stock solution, which was aliquoted, frozen again in liquid nitrogen, and stored at −80°C until use.For immunophenotyping of SCs, the MSC Phenotyping Kit  and antibody to CD31  with corresponding control  were used according to the manufacturer’s instructions. Cells were washed with Versene solution , detached from plastic with 0.05% trypsin-EDTA solution , and after trypsin inhibition by complete growth medium, they were aliquoted in four test tubes and centrifuged for 10 min at 300 g. Pellets were then resuspended in flow cytometry buffer [1% bovine serum albumin  in HBSS ] and incubated for 10 min at 4°C with antibodies to CD31 or cocktail of antibodies to CD73, CD90, CD105, CD14, CD20, CD34, CD45 from the kit or corresponding control antibodies at a dilution provided by the manufacturer. After labeling, cells were diluted by 1 ml of flow cytometry buffer, centrifuged for 10 min at 300 g, then supernatant was discarded, and cells were resuspended in 500 μl of flow cytometry buffer. Cells were detected using BD LSR Fortessa flow cytometer , and results were analyzed using the FlowJo software .Cell differentiation was performed using StemPro Adipogenesis Differentiation Kit  and StemPro Osteogenesis Differentiation Kit  according to the manufacturer’s instructions. SCs were seeded in 12-well plates at a density of 100,000 cells per well and cultured during 2–3 days until reaching 100% confluency, and growth media have been replaced by differentiation induction media. For adipogenic differentiation, the medium was changed every 2–3 days, and cells were fixed with 4% formaldehyde  for 30 min on day 14; lipid droplets were stained with Oil Red O solution from Mesenchymal Stem Cell Adipogenesis Kit  according to the manufacturer’s instructions. For osteogenic differentiation, the medium was changed every 3–4 days, and cells were fixed with 4% formaldehyde for 30 min on day 21; mineral deposits were stained by Alizarin red solution from Mesenchymal Stem Cell Osteogenesis Kit  according to the manufacturer’s instructions. Visualization and image acquisition were performed on an inverted Leica DMi8 microscope with a DFC7000T camera, and images were processed in Fiji package  based on∗ 103 SCs were formed by centrifugation at 300 g for 10 min in 15 ml tubes . Then, 1.5 ml of differentiation medium from the kit was gently added atop, and the caps of the tubes were replaced with those containing an air filter. Full culture media specific for every SC type were used as control media. After 4 days, pellets transformed to spheroids, and from this point, the media were changed two times a week for 17 days. At the end of the culture period, spheroids were frozen in OCT medium and were sectioned on Leica CM1850 cryotome. Up to 10 sections per 10 microns were obtained from every specimen and were placed on a separate slide. Then, prepared sections were fixed in 4% formaldehyde followed by rinse in PBS for 15 min. Slides with the differentiated cells and slides with the corresponding controls were stained in parallel with Alcian blue  according to the following protocol:Cell differentiation was performed using StemPro Chondrogenesis Differentiation Kit . Pellets consisting of 350 1)Incubation in 3% acetic acid for 3 min2)Incubation in 1% Alcian blue solution for 30 min3)Washing with an excess of 3% acetic acid (40 dips)4)Washing in running tap water for 10 min5)Washing in distilled water (10 dips)6)Dehydration and clearing: 1 min 70% alcohol, 1 min 96% alcohol, 3 min xylene7)Embedding with… DAKO Mounting Medium Visualization and image acquisition were performed on Leica DM6000 microscope with a DFC420 camera, and images were processed in Fiji package  based onTGF-β1 concentration measurement was performed using Human/Mouse/Rat/Porcine/Canine TGF-β1 Quantikine ELISA Kit  according to the manufacturer’s instructions. Samples containing 10% solution of MDS, BS, or FBS in DMEM/F12 (similar to solutions used for cell cultivation in our experiments) were centrifuged, and supernatants were stored at −80°Ñ. Immediately before use, samples have been activated to transform latent TGF-β1 to the immunoreactive form. For this purpose, 20 μl of 1 M HCl solution was added to 90 μl of each sample, and then it was neutralized by addition of 20 μl of 1.2 N NaOH, 0.5 M HEPES. The final pH was checked in obtained activated samples, so that it was within the pH 7.2–7.6 range. Then, some of these activated samples were additionally diluted for two or five times with appropriate calibration diluent, if needed. Further steps were carried out using the manufacturer’s instructions for the kit. For each measurement procedure, the calibration curve was built using TGF-β1 standard solutions. Each sample and standard measurement was performed in duplicate. The dilutions as a result of sample activation procedure (1.45-fold) and further two or fivefold dilutions were taken into consideration in the final calculation of TGF-β1 concentration in the samples.SCs were grown in a 24-well plate to 100% confluence, and medium was changed to one of the following or a corresponding control:•DMEM/F12  + 10 ng/ml TGF-β1  + 100 μM magnesium ascorbil phosphate  and control DMEM/F12 + 100 μM MAP were used to assess the fibrogenic effect of TGF-β1. MAP was added to serum-free medium as a coenzyme required for collagen I deposition .•10% BS in DMEM/F12 or 10% MDS in DMEM/F12 was used as tested media.•10% FBS  in DMEM/F12 was used as a control media to assess the effects of MDS and BS in a model of fibrogenesis.•BS was used as negative control for soluble factors derived from the endometrium and contained in MDS.Electrophoresis and Western Blotting) or fixed with 4% formaldehyde  for immunofluorescent labeling of extracellular collagen I and ED-A fibronectin (see section “Immunofluorescent Labeling”).The medium was changed every 48 h to maintain the concentration of its active components. At 96 h of the experiment, cells were either lysed by Laemmli buffer  to analyze collagen I and ED-A fibronectin content by Western blotting (see Electrophoresis and transfer were performed using devices and reagents from BioRad. Electrophoresis was performed according to a method described by 2) after normalization to loading control and corresponding control sample.After electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane . Western blotting was performed according to the method described by Immunofluorescent labeling was performed directly in wells of culture plates. For confocal microscopy, we used special dishes with thin plastic bottoms . Cells were fixed with 4% formaldehyde  during 10 min, then permeabilized for 10 min by 0.02% Triton-X100  for α-SMA and vinculin labeling. For collagen I and ED-A fibronectin labeling, permeabilization stage was omitted. Blocking was done with 10% goat serum  solution in HBSS  during 1 h. Overnight incubation with the first antibodies to α-SMA , vinculin , collagen I , or ED-A fibronectin  was performed at 4°C. Incubations with the corresponding secondary antibodies  lasted 2 h at room temperature. The nuclei were labeled with 4′,6-diamidino-2-phenylindole . After each step, except for blocking, the wells were washed three times with phosphate buffer saline (PBS). Upon completion of labeling, PBS remained in the wells and samples were analyzed by microscopy.Visualization and image acquisition were performed on a Leica DMi8 inverted fluorescence microscope with a DFC7000T camera. Confocal images were obtained on a Leica TCS SP5 confocal microscope, and water was used as an immersion medium. Image processing and analysis were completed using Fiji. Coloc2 plugin was used for assessing signal colocalization, 3D viewer plugin for 3D reconstruction, and 3D Object counter plugin for the evaluation of matrix protein levels of localization.3, 80 × 103, or 160 × 103) was placed in a 1.5 ml tube followed by centrifugation for 5 min at 200 g. The supernatant was discarded, the cells were resuspended in 400 μl of DMEM low glucose + 10 ng/ml TGF-β1  or DMEM low glucose  as corresponding control, then 200 μl of pork collagen  was added. Obtained suspensions were normalized with 1 M NaOH  and transferred into wells of a 24-well plate (500 μl in each well). The collagen in wells polymerized at room temperature for 30 min and then 400 μl of DMEM low glucose were added atop. After that, collagen discs were detached with a pipette tip from the walls of the wells to induce flotation and let contraction. Disc flotation was visually checked every 24 h, and the disc area was measured in photographs taken at 24, 48, 72, and 96 h after detachment. To assess collagen disc contraction, we calculated the percentage of collagen disc area relative to the plate well area (collagen disc area/well area × 100%). We designated the resulting value as “disc size.” At each time point, for each donor, the mean size of the discs with TGF-β1 and without TGF-β1 was calculated to plot the dynamics of mean disc size. Mean disc size for samples treated by TGF-β1 was estimated in each time point and compared vs. the corresponding control with evaluation of statistical significance for each type of SC.SCs were detached from the culture plastic as described above and centrifuged for 5 min at 200 g. The supernatant was discarded, cells were resuspended in PBS and counted, and the required number of cells . Kolmogorov–Smirnov test was used to evaluate normality of data distribution. To assess the ability of EndoSCs for myofibroblast transition, we used TGF-β1 as inducer and compared them vs. SCs from human dermis (DermSC) and subcutaneous fat (AdipoSC) in culture models of fibrogenesis and contraction. Prior to this comparative analysis, we investigated the compliance of isolated cells to previously described criteria of multipotent mesenchymal stromal cells (MSCs)  to suppon = 3 for each source) were positive for MSC markers  and negative for endothelial, immune, and hematopoietic cell markers   . We foun0, CD45) .After induction of adipogenic, chondrogenic, or osteogenic differentiation at passages 3–5, we found that independently of tissue source, all cultures treated by specific differentiation media accumulated lipid droplets, glycosaminoglycans or showed mineral deposition at respective endpoints , which rWe conclude that EndoSCs, DermSCs, and AdipoSCs matched accepted criteria of MSC and could be compared in further experiments as belonging to a common cell type.EndoSC, DermSC, and AdipoSC were taken at passages 3–5 and grown to confluence prior to modeling of fibrogenesis. At Day 0 of the experiment, we changed the medium to DMEM/F12 with TGF-β1 (10 ng/ml) supplemented with MAP (100 mM) to support ECM deposition as previously described . After tTreatment with TGF-β1 increased the level of phosphorylated SMAD2 (pSMAD2), indicating activation of canonical TGF-β1 signaling pathway in all cultures including EndoSC . Thus, SBesides TGF-β1 signaling measurement, we used immunoblotting to evaluate level changes of ED-A fibronectin and collagen I in SC cultures in response to TGF-β1 . StatistAs far as immunoblotting of total culture lysates fails to discern between intracellular and extracellular ECM components, we addressed immunofluorescence studies. To visualize extracellular deposition of collagen I and ED-A, we omitted permeabilization stage during immunofluorescent labeling.As seen in In general, TGF-β1 increased production and deposition of ECM proteins in EndoSC as well as in DermSC and AdipoSC in a similar manner. However, the observed pattern of collagen I and ED-A fibronectin deposition was peculiar in EndoSC. Indeed, analysis of merged images  suggestep-value of 0.05  and the reduction of α-SMA-positive stress fibers (immunofluorescence) compared to BS . VinculiAltogether, we suggest that soluble factors within MDS counteract transition of SCs to contractile myofibroblasts in response to profibrotic factors.To assess the influence of local factors produced during menstruation on ECM production by SCs, we evaluated collagen I and ED-A fibronectin after treatment with MDS or BS. Cells were cultured for 96 h in DMEM/F12 supplemented by 10% of either MDS or BS. Treatment with 10% FBS was used as the baseline culture condition control. Western blotting demonstrated that in all SC cultures, MDS decreased the amount of non-aggregated, full-length (approximately 130 kDa) type I collagen molecules compared to BS . StatistImmunofluorescent labeling without permeabilization demonstrated enhanced extracellular deposition of collagen I and ED-A fibronectin in all studied SCs after treatment with either MDS or BS . This doFinally, we found that MDS, but not BS, induced anisotropic deposition of collagen I  with uneAnalysis of merged images after labeling for collagen I and ED-A fibronectin showed anisotropic deposition of collagen I in DermSC and AdipoSC treated by MDS, but not BS. In areas with high cellular density, the fibrillar structure of collagen I was enhanced, while in EndoSC cultures, we did not observe this feature . AbovemeZ-axis level  these soluble factors. First of all, we evaluated TGF-β1 content in the obtained serum samples and found that MDS contained about 95 ng/ml TGF-β1, which is enough to induce transformation of SCs into myofibroblasts according to n in MDS .in utero EndoSC might be exposed to regulatory stimuli that prevent this process and ensure healing of the endometrium without fibrosis, unlike other soft tissues.Taking into account the vivid ability of EndoSC to myofibroblast differentiation in response to TGF-β1, we assumed that Our data about effects of MDS on SCs confirmed our assumption about the presence of soluble factor(s) that may suppress transition to myofibroblasts. Together with the results mentioned above, this suggests that soluble MDS components are indispensable for endometrial scar-free healing. Otherwise, the latter outcome would be unlikely given the high TGF-β1 content we found in MDS and pronounced mechanical stress due to contractions of uterine smooth muscles during menstruation and after labor .Despite the fact that the nature of regulatory stimuli that determine the antifibrotic effect of MDS in SCs remains highly enigmatic, we can assume that their main targets are downregulation or modulation of mechanical stimulus reception (mechanosensing) and response to ones. In our experiments, we found that under the influence of MDS containing soluble factors secreted during menstruation, the formation of focal adhesion contacts, as well as levels of α-SMA and vinculin, is suppressed. In SCs, mentioned molecular targets comprise a system necessary for mechanosensing, mediating resistance to mechanical forces and subsequent transformation into myofibroblasts . DecreasSoluble factors formed during menstruation suppressed the transition to myofibroblasts not only in endometrial SCs. This phenomenon was also observed when MDS was added to cultures of SCs isolated from fat and dermis, which excludes tissue-specific suppressive action of MDS components on myofibroblast transition. Once putative antifibrotic factors in MDS are identified, this will provide ground for a hypothetical possibility of their application to suppress fibrogenesis not only in the uterus but also in other organs and tissues.Suppression of new myofibroblasts formation can lead to decreased fibrosis or scarring. However, to end up with regeneration, healing must involve morphogenetic processes in the damaged area to induce replacement of functional structures . SCs areProduction of matrix proteins by SCs is one of their main morphogenetic features that is necessary for spatial organization of cells in the forming tissue . Our comSpecific features of ECM composition and organization by SCs regulate their functional state through feedback mediated by integrin signaling and form a unique topography of connective tissue and determine its interaction with other cell types . Thus, pDanio rerio (Another morphogenetic feature of EndoSC is their inability to form “hills,” which are, actually, bulky connective tissue structures. Formation of such structures occurs during healing in the early stages during granulation tissue growth . In mostio rerio . HoweverTissue-specific differences in the morphogenetic properties of SCs described above became more pronounced under the influence of MDS and its soluble factors. Indeed, MDS significantly accelerated the formation of “hills” in AdipoSC and DermSC but did not induce their appearance in EndoSC cultures; in contrast, soluble factors of peripheral BS did not show this effect. In addition, MDS induced the formation of a prominent fibrillar network of collagen I in the “hills,” which suggested a higher degree of matrix maturity, which is a characteristic of formed connective tissues .in vivo, but intriguing assumption could be made: possibly, different tissue-specific regenerative programs are regulated and induced by similar regulatory stimuli. Thus, soluble factors formed during menstruation induce a tissue-specific morphogenic program in different SCs. Our data on cellular models may leave space for dispute on how this effect is realized From the point of view of the physiology of the healing process of the endometrium, we can assume that the factors contained in the menstrual discharge prevent transition of SCs into myofibroblasts and activate their tissue-specific morphogenetic program. These two components are likely to ensure the rapid regeneration of this tissue and its subsequent preparation for the realization of reproductive function.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Lomonosov Moscow State University. The patients/participants provided their written informed consent to participate in this study.VT and PM: study supervision and funding acquisition. RE study concept. PM and RE: study design and wrote the manuscript. RE: microscopy, immunofluorescence, MDS, BS, and EndoSC isolation, cell culture, fibrogenesis assay, data analysis. MK: electrophoresis and western blotting. NA and OG: isolation of DermSC and AdipoSC. NA: cell culture. PN and ME: protein assays and ELISA. NB: collagen disc contraction assay. OG: cell differentiation. DD: flow cytometry. All authors contributed for the development of this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Danio rerio is a promising model organism to study EP300 related diseases and drugs; however, the effect of EP300 duplicates derived from teleost-specific whole genome duplication should not just be neglected.EP300 is a conserved protein in vertebrates, which serves as a key mediator of cellular homeostasis. Mutations and dysregulation of EP300 give rise to severe human developmental disorders and malignancy. D. rerio and Oryzias latipes and found that in either species the faster evolving copy had more short helixes.In this study, we obtained EP300 protein sequences of representative teleosts, mammals and sauropsids, with which we inferred a highly supported maximum likelihood tree. We observed that Ep300 duplicates (Ep300a and Ep300b) were widely retained in teleosts and universally expressed in a variety of tissues. Consensus sequences of Ep300a and Ep300b had exactly the same distribution of conserved domains, suggesting that their functions should still be largely overlapped. We analyzed the molecular evolution of Ep300 duplicates in teleosts, using branch-site models, clade models and site models. The results showed that both duplicates were subject to strong positive selection; however, for an extant species, generally at most one copy was under positive selection. At the clade level, there were evident positive correlations between evolutionary rates, the number of positively selected sites and gene expression levels. In Ostariophysi, Ep300a were under stronger positive selection than Ep300b; in Neoteleostei, another species-rich teleost clade, the contrary was the case. We also modeled 3D structures of zf-TAZ domain and its flanking regions of Ep300a and Ep300b of Collectively, the two copies of Ep300 have undoubtedly experienced directional divergence in main teleost clades. The divergence of EP300 between teleosts and mammals should be greater than the divergence between different teleost clades. Further studies are needed to clarify to what extent the EP300 involved regulatory network has diverged between teleosts and mammals, which would also help explain the huge success of teleosts. Cells under changing external environments need to regulate their transcriptions to maintain internal homeostasis, during which lysine acetylation plays a key role in connecting external signals and downstream regulations . Tens ofDanio rerio (zebrafish) is a useful model organism to study genetic diseases and test new drugs due to its transparent embryos and fast growing speed [ng speed –14. Howeng speed –17. It wng speed . Howeverng speed , 20. As ng speed . It was ng speed . On the ng speed , which wng speed –26. If Eng speed . HoweverIn this study, we focused on the molecular evolution of Ep300 in teleosts. We are particularly interested in the way and the extent of divergence of the retained duplicates in different teleost clades, which were explored through analyses of molecular evolution of Ep300 duplicates based on diverse evolutionary models, tissue expression profiles and protein structures.Sinocyclocheilus anshuiensis, Carassius auratus (goldfish), Austrofundulus limnaeus and Oncorhynchus kisutch  had 3 ~ 4 copies, which was in accordance with the fact that their respective ancestors underwent recent genome duplications [Erpetoichthys calabaricus (reedfish) and Lepisosteus oculatus (spotted gar), had only one copy. We also conducted tblastn search against RefSeq genomes of 19 fish species that have available assembled chromosomes and found no evidence of any species to have additional copies that might originate from small-scale duplications (SSDs)  and Paramormyrops kingsleyae  across sites and branches. Still, their results were accordant: in each comparison, a higher ω2 in CmC result was accompanied by a higher mean ω value in RELAX result   and RELAlt Table . Both duer Table . In fourTo get a thorough exploration of positively selected sites of Ep300a and Ep300b, we used MEME  to detecD. rerio and O. latipes  using I-TASSER suit. All four best models of respective sequences had significantly greater structure density (by the number of decoys) than respecti,ve lower-rank models; three of them even had TM-score greater than 0.5 , the region corresponding to zf-TAZ domain was characterized by long α-helixes, further confirming the credibility of the best models , O. latipes, Esox lucius (northern pike) and Oreochromis niloticus (Nile tilapia); ep300 of one holostean fish, L. oculatus, which had not been affected by the teleost-specific WGD event; and Ep300 of Mus musculus (house mouse) as control. In three teleosts , tissue expression profiles of ep300a/ep300b did not correlate with ep300 of L. oculatus, neither individually nor on average , suggesting that their functions have not sufficiently diverged yet. Compared to teleosts and M. musculus, ep300 gene of L. oculatus was highly expressed in a smaller subset of tissues. According to the PhyloFish database, the quality of sequencing data of L. oculatus was not significantly inferior to that of other fishes  where one copy evolved faster than the other  might still play subtle but important roles in development [D. rerio had undoubtedly experienced subfunctionalization. On the other hand, we observed that the faster evolving copy of Ep300a/Ep300b generally contained more positively selected sites and more structural innovations (short helixes) in the most intensively selected regions , mammals (NCBI:txid40674) and sauropsids (NCBI:txid8457), respectively. The protein sequences  of the above three species were used as query sequences to conduct blastp search against nr database of their respective clade, with the max target sequences set to be 20,000 and expect threshold to be 1e-5.To obtain homologs of EP300 from interested species . The remaining hits would be selected based on gene ids: for each gene id, if none sequence had the status “VALIDATED”, the top hit would be selected; if at least one sequence had the status “VALIDATED”, the top hit of them would be selected.To extract sequences of interested species from the blastp results, we first filtered non-wanted hits: if the word “p300” did not appear in the description of hit sequence or if the source organism was not interested, this hit would be ignored. Then we extracted the NCBI gene id, sequence status  and respective nucleotide sequence accession number of each hit from the file “gene2accession”  into the EP300 sequences to serve as outgroup. After that, the protein sequences were subject to multiple sequence alignment by MAFFT [Based on the above information  [To get a consensus sequence of the full sequence set, we extracted the most frequent residue of each column  from the trimmed protein sequence alignment. For smaller sequence sets, e.g. a set only contained mammals’ sequences, the sequences would be aligned with MAFFT first. Then the alignment would be trimmed to the average length of the original sequences . The conserved domains and their exact locations within consensus sequences were predicted by the NCBI online tool CDD search (psb.cgi) .D. rerio (Genbank accession No.: XP_021335970.1 and XP_009297687.1) and O. latipes (Genbank accession No.: XP_023805552.1 and XP_011476788.1). For Ep300a and Ep300b of D. rerio, the top 500 aa were used as query sequences; for Ep300a of O. latipes, the top 550 aa were used as query sequence; for Ep300b of O. latipes, the top 520 aa were used as query sequence. The best models were visualized using PyMOL [We used I-TASSER suit (version 5.1)  to modelng PyMOL . We usedep300a/ep300b of D. rerio, A. mexicanus, O. latipes and E. lucius and ep300 of L. oculatus, the nucleotide sequence of each gene was used as a query to search against PhyloFish database [ep300a/ep300b of O. niloticus and Ep300 of M. musculus in different tissues were obtained from the NCBI gene database with their respective gene ids as queries  were calculated using the pearsonr function of scipy.stats module [To get gene expression data of database . The bess module .Additional file 1: Lineage information of selected species.Additional file 2: Detailed information of selected sequences.Additional file 3: Tblastn search results against RefSeq genomes of 19 fishes.Additional file 4: Maximum likelihood tree of EP300.Additional file 5: Bayesian tree of EP300.Additional file 6: Supplementary information and results.Additional file 7:ω ratios of fishes reported by aBSREL test.Additional file 8: Multiple alignment of EP300 sequences and original tree files.Additional file 9: Multiple alignment of four sequences used for structure modeling.


The fraction of alveoli with each phenotype is reflected in the proportional changes in pulmonary system elastance at positive end expiratory pressures of 0, 3, and 6 cmH2O.Mechanical ventilation is an essential lifesaving therapy in acute respiratory distress syndrome (ARDS) that may cause ventilator-induced lung injury (VILI) through a positive feedback between altered alveolar mechanics, edema, surfactant inactivation, and injury. Although the biophysical forces that cause VILI are well documented, a knowledge gap remains in the quantitative link between altered parenchymal structure , pulmonary function, and VILI. This information is essential to developing diagnostic criteria and ventilation strategies to reduce VILI and improve ARDS survival. To address this unmet need, we mechanically ventilated mice to cause VILI. Lung structure was measured at three air inflation pressures using design-based stereology, and the mechanical function of the pulmonary system was measured with the forced oscillation technique. Assessment of the pulmonary surfactant included total surfactant, distribution of phospholipid aggregates, and surface tension lowering activity. VILI-induced changes in the surfactant included reduced surface tension lowering activity in the typically functional fraction of large phospholipid aggregates and a significant increase in the pool of surface-inactive small phospholipid aggregates. The dominant alterations in lung structure at low airway pressures were alveolar collapse and flooding. At higher airway pressures, alveolar collapse was mitigated and the flooded alveoli remained filled with proteinaceous edema. The loss of ventilated alveoli resulted in decreased alveolar gas volume and gas-exchange surface area. These data characterize three alveolar phenotypes in murine VILI: flooded and non-recruitable alveoli, unstable alveoli that derecruit at airway pressures below 5 cmH Mechanical ventilation is required for major surgery and resuscitation and in critical emergencies such as acute respiratory distress syndrome (ARDS) , but thiChanges in the lung structure–function relationship during VILI are currently understood through the lens of organ-scale imaging techniques such as CT . HoweverTo address this knowledge gap, we used design-based stereology to measure parenchymal structure over a range of air inflation pressures in a mouse model of VILI. The state of the pulmonary surfactant system was assessed by measuring minimum surface tension and surfactant composition. Organ-scale mechanical function was determined with the forced oscillation technique (FOT) to quantify stiffness, airway resistance, and tissue damping at different levels of lung inflation. Together, these data define the structure–function relationship in murine VILI.via 0.8 mg/kg pancuronium bromide administered at the onset of mechanical ventilation. Alternating doses of 50 mg/kg ketamine and 50 mg/kg ketamine with 8 mg/kg xylazine were administered at 30-min intervals with 150 μL IP 5% dextrose lactated Ringer’s solution. Fluid volume from the anesthetic was 10 μL/g. The electrocardiogram was monitored to assure that the rodents maintained a deep plane of anesthesia during paralysis.Eight to ten week old female BALB/c mice  weighing 17.1–22.3 g were studied under Institutional Animal Care and Use Committee (IACUC)-approved protocols. Mice were anesthetized with an intraperitoneal (IP) injection of 100 mg/kg ketamine and 16 mg/kg xylazine, tracheostomized with a 18 ga metal cannula, and ventilated using a flexiVent small animal ventilator . Respiratory drive was suppressed Mechanical ventilation experiments to generate tissue for structural analysis were conducted at the University of Vermont and approved by the University of Vermont Institutional Animal Care and Use Committee (IACUC #14-056). Bronchoalveolar lavage fluid for surfactant analysis was collected under identical ventilation conditions by the same investigator at the University of Colorado Denver in experiments approved by the University of Colorado Denver Anschutz Medical Campus, IACUC (#00230).2O (baseline ventilation). Recruitment maneuvers (RM) consisting of a 3 sec ramp to 30 cmH2O followed by a 3 sec breath hold were applied to reopen regions that may have collapsed during the surgical procedure. Following the stabilization period, lung function was assessed. Testing consisted of an RM and a dynamic pressure-volume (PV) loop that ramped airway pressure (Paw) from 0 to 37.5 cmH2O, held pressure for 3 sec, and ramped pressure back to zero over 3 sec. Derecruitability tests were then performed at PEEP = 6, 3, and 0 cmH2O, denoted by All animals were ventilated for a 10-min stabilization period with a delivered tidal volume (Vt) = 10 ml/kg at a respiratory rate (RR) = 200 breaths/min and a positive end expiratory pressure (PEEP) = 3 cmHse model  to deter2O, RR = 50 breaths/min, inspiratory:expiratory ratio of 1:2, and PEEP = 0 cmH2O. This Pplat was selected to generate severe VILI in an experimentally tractable timeframe without causing any pneumothoraces. At the end of each block a PV loop was recorded followed by two FOT measurements separated by 10 s of baseline ventilation. These 5-min ventilation blocks were applied until H doubled, at which point the lung function assessment was repeated prior to harvest for either morphometry or surfactant analysis. The mean duration of injurious mechanical ventilation was 87.3 min with a standard deviation of 17.5 min. Including with the 10 min stabilization period and two 13 min lung function assessments, the mean duration of ventilation for the VILI group was 123.3 min.The VILI group was then subjected to repeated blocks of ventilation consisting of 4 min 45 s of injurious ventilation with a plateau pressure (Pplat) = 37.5 cmHg at 4°C for 5 min. A total of 100 μl of the cell-free supernatant for each animal was separated, and the remainder of the BALF from three mice in the same treatment group was combined and centrifuged at 40,000 g. The ultracentrifuged supernatant containing the surfactant small aggregate (SA) was stored at −80°C for later analysis. The surfactant pellet, containing the large aggregate (LA) fraction, was washed twice by resuspending the pellet in 1 ml saline and centrifuging at 40,000 g. Finally, the LA pellet was resuspended in 100 μl saline and stored at −80°C for later analysis.Bronchoalveolar lavage fluid was collected by instilling and suctioning back 1 ml warm normal saline three times and then centrifuging at 700 ex vivo using a pulsating bubble surfactometer  as described  to maintain surface tension effects and allow comparison to the lung function data . The fixred rats  where ne4 in 0.1 M cacodylate buffer for 2 h and then overnight in half saturated aqueous uranyl acetate, dehydrated in a graded acetone series, and embedded in glycol methacrylate  to avoid tissue shrinkage  for Vv(par/lung) was 100% at 5× magnification. The parenchyma was then subdivided into volume fractions of alveolar airspace [Vv], alveolar duct airspace [Vv(ductair/par)], and non-air material [Vv(non-air/par)] that include tissue and airspace edema fluid. This assessment was performed at 20× magnification and SF = 11%. Fields of view were selected for analysis using systematic uniform random sampling whereby the morphometry software selected a random starting location and images were then automatically sampled on a 1,200 × 1,200 μm grid over the entire tissue section. This approach blinds the investigator to the selection of the images used for analysis, thus removing the potential for bias. The non-air material in the parenchyma [Vv(non-air/par)] was then analyzed at 40× magnification  to determine the volume fractions of septal tissue of patent alveoli [Vv], and airspace edema [Vv(edema/par)]. The volume of each compartment (demarcated with V in place of Vv) was determined by multiplying the volume fractions by the volume of the reference space. The surface area per volume available for gas exchange [Sv] was estimated at 40× magnification  by counting line intersections with aerated septal tissue that was not covered with alveolar edema. The gas exchange surface area [S] was determined by multiplying Sv by the parenchymal volume [V(par)] and the mean septal thickness defined as τ(sep) = 2 V/S. The physical disector method . The total number of alveoli is then N = N + N(flood), the number-weighted average alveolar volume Vn = V/N, and the alveolar surface area to volume ratio S/V = S/V.A cascade sampling design was used starting with the volume fraction of parenchyma in the whole lung [Vv(par/lung)] that we define as areas contributing to gas exchange and excluding airways, vessels outside the septal walls, and peribronchiolar tissue. This, and all volume fractions, were measured using point counting. The percentage of the tissue section analyzed  then an ANOVA was performed followed by pairwise comparisons using the lsmeans package . A Shapiro–Wilk test was used to assess normality. If the data were not significantly different from the normal distribution ( package  and Tukey = α tβ to the time series of measurements as we have previously described (t), and whether the measurement was recorded before or after VILI (Injury). The intercept for each animal was included as a random effect and the Akaike information criterion (AIC) was used to determine the most appropriate model. Temporal autocorrelation was accounted for with a first-order autocorrelation structure for the continuous time covariate grouped by animal, derecruitability test PEEP, and injury status  (NLME package in R) on the natural logarithm of the time and parameter values. This is equivalent to fitting the equation escribed . Fixed ey status .t (Pulmonary system elastance following an RM in the uninjured lungs . VILI (Fnjury (t . Followiith PEEP  in both  by VILI .2O (right column) down to airway pressures of 2 cmH2O (left column). Note that these airway pressures are applied in open-chested mice, and the contributions of the chest wall are discussed below. By contrast, the VILI lungs exhibited diffuse alveolar edema  and retraction of the alveolar septa  at Paw = 5 and 10 cmH2O. At Paw = 2 cmH2O, the injured parenchyma tended to consolidate into patches of flooded and collapsed alveoli with air remaining in the ductal space. These injured regions were interspersed between areas of parenchyma that appeared normal.The changes in lung function are associated with structural alterations in the perfusion-fixed lung tissue. Alveoli in the Control lungs ] is defined as the ratio of these two quantities and indicates a modest stiffening of patent alveoli with injury  at low Paw in VILI suggests that the alveolar geometry becomes more distorted in the injured lung. Injurious ventilation did not change V   in conjuh injury . By conth injury , leadingh injury  that wash injury  was signjury   or τ . Little , total) , and V at Paw = 5 and 10 cmH2O that was significantly less than in the Controls. Reducing the inflation pressure to 2 cmH2O in the VILI animals led to a significant reduction in aerated alveoli. Flooded alveoli [N(flood), 2O where there is a striking decrease with VILI.The number of ventilated alveoli [N, 2O. Note that the mean pressure of the derecruitability tests is approximately 2–3 cmH2O greater than the onset pressures so that the mechanics and structure are measured at roughly the same pressure. Elastance is strongly correlated with the number of patent alveoli (R2 = 0.78), and the form of the regression was selected based on our prior computational simulations where elastance increases with the inverse of the lung open fraction  content  signific protein , which lThe macroscale pressures and flows applied at the trachea during mechanical ventilation result in microscale parenchymal injury . The res2O immediately following an RM in VILI compared to controls   at all pressures . Less exVn , becauseVn . BleomycVn  to thoseVn . The latVn , which m2O, indicating that the alveoli themselves had become less spherical and the alveolar septa possibly more convoluted. This is generally what one might expect in a deflating alveolus  that remain aerated at low Paw. Our microscale analysis shows that the mechanics of these open alveoli are not entirely normal since Vn and S/V are altered in VILI, but these alterations are secondary contributors to organ-scale mechanics when compared to the loss of ventilated alveoli. These three alveolar phenotypes have different dynamic behaviors that are reflected in the time- and pressure-dependence of H. It is important to note that while these three alveolar phenotypes are generally stable there may be variability between respiratory cycles. In vivo confocal microscopy shows that flooded alveoli are typically stable but may occasionally expel their edematous contents and become re-aerated  that is indicative of parenchymal overdistension. Taken together, these data suggest that the optimal PEEP based on lung elastance (or driving pressure) for these VILI mice lies between 3 and 6 cmH2O to recruit the recruitable alveoli (Zone R) without causing tissue overdistension by applying excessive PEEP in a futile attempt to recruit the Zone D alveoli that, in our VILI model, are flooded with edema.The correlation between alveolar recruitment and elastance  may prov 3 cmH2O . By cont2O the VILI-induced changes in G were relatively less than those in H. This contrasts with findings in open-chest but otherwise healthy mice in which tissue hysteresivity = G/H remained constant with lung volume  flooded alveoli that cannot be recruited at any pressure, (2) unstable alveoli that are open at high pressures but readily collapse as pressure is reduced, and (3) relatively normal alveoli that remain open at low pressures. The relative proportions of each phenotype can be estimated using derecruitability tests that quantify time-dependent alveolar derecruitment as a function of PEEP. These correlations between lung structure and function could provide insights into optimally lung-protective ventilation strategies.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by University of Vermont, Institutional Animal Care and Use Committee and the University of Colorado Denver Anschutz Medical Campus, Institutional Animal Care and Use Committee.BS, GR, KH, LK, MO, RH, and JB contributed to the conception and design of the study. BS, GR, AC, CM, KO, CC, and MN acquired the experimental data. BS, KH, RH, and JB analyzed and interpreted the data. BS drafted the manuscript. CM, KO, RH, and JB wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.