Analyses of the control region sequences of European brown trout populations’ mitrochondrial DNA have revealed five main evolutionary lineages  mostly relating to the main water basins; however, the hybridization between lineages were increasingly reported. Due to the hydrogeography of Hungary, wild populations should theoretically belong to the Danubian lineage, however, this has not been verified by genetic studies.In our study multiple molecular marker sets  were used to investigate the genetic composition and population genetics of the brown trout populations in two broodstocks, six wild streams in Hungary and one Serbian population.The admixture of Atlantic and Danubian lineages in these populations, except the Serbian population with pure Danubian origin, was observed by control region sequences of mitochondrial DNA and PCR-RFLP markers in the nuclear genome, and one unpublished Danubian haplotype was found in Hungarian populations. A sex-specific marker revealed equal gender ratio in broodstocks and Kemence stream, whereas in other wild streams the proportion of female individuals were less than 50%. Structure and principal component analyses based on the alleles of microsatellite loci also revealed overlapping populations, however the populations were still significantly different from each other and were mostly in Hardy-Weinberg equilibrium.Stocking and migration can have a significant genetic impact on trout populations of wild streams, however there are no guidelines or common practices for stocking of small streams in Hungary, thus the genetic background of these populations should be considered when developing conservation actions. The family Salmonidae consists of three subfamilies , which include a diverse group of fishes with sixty-eight species . In the  is a native salmonid species in Eurasia  with poorly known stocking history and varying degree of angling pressure in the drainage system of the Danube in Hungary between 2011 and 2014 , Table 14)2SO4 , 200 µM dNTP mix, 250 nM for each primers, 1,5 mM MgCl2, 100 ng template and 1 U Taq polymerase (Fermentas). The following PCR cycles were used: 3 min preamplification denaturation at 94 °C, then 30–60 s at 94 °C, 30–90 s at annealing temperature  and 1 min at 72 °C for 35 cycles. As a final step, products were fully elongated for 5 min at 72 °C. The reactions were checked on 1% of agarose gels, containing 0.5 µg/ml ethidium bromide. The microsatellite fragments were analysed on the ABI 3130 sequencer with POP7 polymer based on the various dyes.The control region (CR) sequences of mitochondrial DNA (CR mtDNA), and PCR-RFLP of the same region (CR) as well as two nuclear genomic loci  were used to distinguish the Atlantic and Danubian alleles/haplotypes. In addition, five microsatellite loci were used to analyse the genetic composition of the populations. Amplification and digestion methods were used according to 434 on the amplified CR mtDNA fragment of Atlantic haplotypes and no cut represents in Danubian haplotypes. Endonuclease BslI also generates a single cut on Atlantic lineage-specific allele of amplified LDH fragment at the polymorphic site G353 and no cut represents in Danubian alleles (EU672412) and Danubian (EU672413) sequences in GenBank, endonuclease MspI generates no cut on Atlantic and a single cut at 212 bp on the Danubian lineage-specific allele of the SL sequences in GenBank. The alleles were separated on 2% agarose gels, containing 0.5 µg/ml ethidium bromide.Following the amplification, CRmtDNA fragments were digested with Fnu4HI (SatI) , LDH-C1 fragments with BslI  and the SL fragments with MspI (Thermo Fisher Scientific) restriction endonucleases. Endonuclease Fnu4HI generates a single cut at the polymorphic site C alleles . Based oFor the comparison of populations based on haplotypes of CR mtDNA, randomly chosen samples (at least 10%) of each sample group were analysed by sequencing of the same control region used in RFLP . For bi-AY185568) and Atlantic (AY185577) reference sequences (π) of the control region per populations were analysed in DnaSP 5.10.01 , effective allele number (Neff), observed (Ho) and expected (He) heterozygosity per locus and per population were calculated using GeneAlEX 6.5 . In caseDigested fragment-length analysis of CR mtDNA revealed the pure Danubian origin of the Serbian control population, whereas the Atlantic lineage were found in all Hungarian sites . There whttps://www.ncbi.nlm.nih.gov/) with accession numbers from MG751088 to MG751096. The analysis of sequence variation has revealed nine haplotypes   has not been described previously and was represented in only one individual of the KE stream. This haplotype also belongs to the Danubian group based on the median-joining network. The average divergence within the Danubian group was 0.0021, within the Atlantic group 0.0017 and between the groups 0.0112. The divergence of the newly found Da6 haplotype was 0.0013 from Da3, Da5 and 0.0027 from other Danubian haplotypes (The median-joining network  indicateplotypes . The medThe most abundant Danubian haplotype (Da1) appeared, beside the Serbian control, in the wild populations and the SZV broodstock. Da2 haplotype was detected in both hatcheries and one wild population while Da3 in one wild stream as a private haplotype with low frequency. Da4 haplotype was represented only in the hatcheries and Da5 haplotype was detected only in the SZV broodstock. The most frequent Atlantic haplotype (At1) was found in all populations with the exception of the Serbian control. In addition, two further Atlantic haplotypes were detected in the Hungarian populations, At2 haplotype was represented in the JO and KO streams as well as the LF broodstock 1, and the At3 Atlantic haplotype was found only in the JO stream as a private haplotype of this stock. The haplotype diversity of the sample set was 0.670 and the nucleotide diversity 0.005.Within nuclear markers, LDH and SL loci were analysed using digested fragment-length polymorphism. These loci also verified the pure Danubian origin of the Serbian control population, whereas varying proportions of both lineages were found in all Hungarian sites . In bothFIS values of nuclear PCR-RFLP loci indicated a significant excess of heterozygosity in LF broodstock 1 and KE stream at p < 0.01 , which were mainly related to OMM1064 and Ssa408uos loci. The highest number (11) of private alleles was revealed in the two Lillafüred broodstocks  and intermediate levels between Hungarian populations and the Serbian control (0.222–0.369) . Beside the Serbian control, BI and KO streams were the most divergent sites among the Hungarian populations. All FST values of microsatellite analyses showed a significant divergence among populations. In case of the nuclear PCR-RFLP markers only the Serbian control was significantly divergent from all other populations and was highly distinct from them (0.558–0.920). Within the Hungarian populations low and intermediate levels of significant genetic divergence were found (0.027–0.231) and again, the BI, KO and KE streams were the most divergent sites.Pairwise 2–0.369) . The meaThe Nei’s Da genetic distance based NJ tree of populations  also supAnalysis of all populations by STRUCTURE did not give a clear ΔK result, revealing two, three and five possible clusters of the sampled area. The highest probability of cluster numbers was at two, while the lowest at the five value . Figure The PCA analysis showed very similar, but more structured clustering. The individuals were separated to three main groups by the horizontal axes while the distribution of the population were continuous along the vertical axes . Both thAMOVA supports the results of STRUCTURE and PCA displaying a low variance among populations  while 90% of the variance was found within populations . These in turn are mainly related to the variance within individuals  and only low variance was found among individuals .Very little information is available on the origin of the current brown trout populations in Hungary and what is known comes mostly from anecdotal information. The reason for this is that prior to World War I, these watercourses were mostly neglected by anglers and aquaculturists who preferred the more typical salmonid waters of the Carpathian mountains in today’s Slovakia, Ukraine and Romania. The interest in the salmonid waters of the low mountain ranges of today’s Hungary has increased following the Paris peace treaty of 1920. The Lillafüred trout farm has been founded in 1932 and the first shipment of trout eggs has arrived from Traismauer, Austria and Kláštor pod Znievom, Czechoslovakia (today Slovakia) in 1933. This broodstock was later the main supply of fish stocked into various streams in Hungary. Stocking of fish from this broodstock has been documented in the Bán and Jósva streams investigated in this study .This study is the first genetic diversity assessment of wild brown trout populations of Hungary. In addition, it offers further information regarding the two Hungarian broodstocks. The sample sizes in the case of some populations are low, especially in the KO, BI and SRB populations. These were however limited by the circumstances of sampling, hydrogeographical features and the abundance of fish in the given watercourses. For instance, in the Bittva stream (population BI) only three locations were available for sampling  at a distance of several km from each other. Effective analysis of genetic mixing using low-marker number has been proved in several studies, as well as in case of similar marker set as we used . HoweverThe mixed origin of the broodstocks and genetic admixture of Atlantic and Danubian lineages in the wild brown trout populations were revealed using multiple marker sets. High proportions of Atlantic haplotypes and alleles were found in the Danubian basin. All Atlantic haplotypes found in this study were frequently detected in other countries  as well as from Austrian, German and Danish hatcheries that held breeders of Atlantic origin. Better performance of breeders with non-native alleles in hatchery conditions would explain their presence in all broodstocks . Their fFST values, as it was found in other studies, too  based on ies, too . This caOn the other hand, none of the analysed populations displayed significant deviation from the normal composition according to the results of Hardy–Weinberg equilibrium analyses. Only the PCR-RFLP marker loci in all broodstocks and a few streams showed a significant excess of heterozygosity, but in most cases the microsatellites also showed a tendency of heterozygosity excess. This could have been caused by anthropogenic effects (mixing of populations) or due to the low number of the available samples. These results refer to the post-stocking analyses of domesticated fishes in different rivers where fishes with poor performance were found . ResultsMore detailed evidence of the admixture was shown by MJ tree, STRUCTURE plot and PCA results, which implies to anthropogenic transfer of fish among populations. However the STRUCTURE Harvester could not clearly determine the number of the genetic clusters, the colour plots of STRUCTURE showed the most possible differentiations of populations with five clusters. The genetically clustered groups are dispersed over the populations that correspond with the outcome of the Median-joining network analysis of mitochondrial haplotypes. It was confirmed by PCA analyses, that also showed five genetic groups , but the classification of the individuals was more clear than in STRUCTURE. Because of these we recommend the usage of PCA analyses next to STRUCTURE. Although, we have used only a few microsatellites, the admixture among most streams and the cultured Szilvásvárad stock is evident. Only the geographically district Serbian population is separated (as it was expected), and the Apátkut, Kölöntés and the Lillafüred broodstock show lower overlaps with the genotype of the Szilvásvárad broodstock. However, all populations carry the elements of their ancient genetic backgrounds, which is shown by the relatively large number of private alleles (42),  and the fact that we could not identify non-admixed individuals. The identification of admixed and non-admixed (if any) individuals would be possible by increasing the number of analysed loci as it was illustrated by Currently no regulations exist in Hungary on stocking of natural watercourses with non-indigenous populations beyond the level of species. Although stocking is considered an important conservation activity of autochthonous populations , but theThe first comprehensive study of Hungarian brown trout cultured broodstocks and wild streams revealed genetic admixture of the lineages in these populations, which could derive from natural or anthropogenic sources. Currently, there are no guidelines or common practices for stocking of small streams; however, the genetic background of these populations should be considered when developing conservation actions.10.7717/peerj.5152/supp-1Table S1The number of base substitutions per site from between sequences are shown, analyses were conducted using the Tamura 3-parameter model. Da, Danubian haplotypes, At, Atlantic haplotypesClick here for additional data file.10.7717/peerj.5152/supp-2Table S2∗P < 005, ∗∗P < 0.01, ∗∗∗P < 0.001.Pop, population, N, number of samples, Na, number of alleles, Nma, mean number of alleles, Neff, effective allele number, Ar, Allelic richness, He, expected heterozigosity, Ho observed heterozigosity, Sign, significant deviation, NA, not available, ns, not significant, Click here for additional data file.10.7717/peerj.5152/supp-3Table S3Click here for additional data file.10.7717/peerj.5152/supp-4Table S4Click here for additional data file.10.7717/peerj.5152/supp-5Figure S1Click here for additional data file.10.7717/peerj.5152/supp-6Supplemental Information 1Click here for additional data file.


Groups of signals indicative of the source, and type (condensed vs. hydrolyzable) of tannin used in the manufacture are well resolved in the spectra of the finished leathers. These fingerprints are compared with those arising from leathers tanned with other common tanning agents. Paramagnetic chromium (III) tanning causes widespread but selective disappearance of signals from the spectrum of leather collagen, including resonances from acidic aspartyl and glutamyl residues, likely bound to Cr (III) structures. Aluminium (III) and glutaraldehyde tanning both cause considerable leather collagen signal sharpening suggesting some increase in molecular structural ordering. The 27Al-NMR signal from the former material is consistent with an octahedral coordination by oxygen ligands. Solid state NMR thus provides easily recognisable reagent specific spectral fingerprints of the products of vegetable and some other common tanning processes. Because spectra are related to molecular properties, NMR is potentially a powerful tool in leather process enhancement and quality or provenance assurance.Solid state  Tanning is an essential phase in one of civilization’s oldest processes, the transformation of hide and skins into leather , and vegNuclear magnetic resonance spectroscopy (NMR) is increasingly used in analysis and quality assurance of industrial samples of biological origin, such as fats, oils, and juices , materiain situ characterization of tannic substances in wood derived materials [Acacia mangium [13C-NMR spectra of industrial materials in which they are used in adhesives [NMR is useful in aterials , and chaaterials ,8,9. NMR mangium  and mari mangium , and has mangium ,13. Tanndhesives ,15, whicdhesives . NMR candhesives , with th13C-NMR spectral “window” between resonance frequencies of ca. 71 ppm and ca. 165 ppm which contains few signals from leather collagen protein but numerous signals from tannins. Tanning with other reagents has also been studied by NMR. Spectroscopic changes induced in model collagen peptides by Cr (III) [Solid state NMR has also been used to study leather ,19, and Cr (III)  and Al (Cr (III)  are inteHere we extend this work to a systematic study of the effects exerted by vegetable tanning and other common tanning procedures on the NMR characteristics of whole leather, leading to the identification of spectroscopic changes characteristic of some of these. The study also identifies effects that certain tanning methods exert on the protein structures in leather, which may hold clues to the molecular events underlying different processes. 13C-NMR spectral fingerprints. Most importantly each tannin spectrum is faithfully recapitulated in the spectra of the leathers tanned by them. Mimosa tannin is predominantly a prorobinetinidin polymer  [Spectra of leathers tanned with two types of condensed tannin, from mimosa and quebracho species, are shown in  B-ring) ,28 whileSimilarly, spectra of leathers tanned with two hydrolyzable tannins, from chestnut and tara, are shown in As in the condensed tannin cases, pure hydrolyzable tannin spectra reflect the chemistry of each ,33,34,351H to 13C (the standard 13C solid state observation technique) is used to enhance the inherently weak 13C signal, relating signal intensity to absolute molecular abundance is complicated by the fact that cross polarization efficiency is a function of molecular structure and must be established for different signals by examining their intensity dependence on cross polarization times. Although we have not carried out the necessary detailed studies it is nevertheless possible on the basis of a few simple assumptions to infer approximate leather collagen:tannin ratios from the data as presented here. If one assumes that cross polarization dynamics of non-protonated carbons are likely to be approximately similar to eachother, the well resolved signal from collagen amide carbons at ca. 175 p.p.m. is a measure of protein content, and the signal at ca. 160 p.p.m. (quaternary 7 and 8a carbons) is a measure of condensed tannin content. The ratio of the integrals of these two signals for both the mimosa and quebracho tanned materials is ca. 0.4 implying a molar ratio of collagen amino acid residues:tannin repeat units of ca. 0.2. Assuming an average molecular mass of 100 and 200 for collagen amino acid residues and tannin monomers, respectively, this molar ratio corresponds to a mass ratio of collagen:tannin of ca. 0.4. Quantification of hydrolysable tannins in leather is complicated by overlap between tannin carboxylate and ester carbons and collagen amide carbons, but tannin loadings seem to be quite similar to those resulting from the condensed tannin processes. In principle it is possible to measure leather:tannin ratios from solid state NMR spectra and thereby infer the tannin load in the product. When cross polarization  from 1H Once the details of cross polarization dynamics have been established for a given material, however, more accurate quantification would be straightforward. Quantification by NMR will probably prove a more robust measure of tannin loading than other spectroscopic, or extractive, approaches, and likely informative in studies correlating leather properties with tannin loading and type. NMR should also yield useful information about the molecular events underlying processes using multiple reagents such as chrome, tannins, and other metal salts and organic tanning agents. 13C-NMR spectrum of chromium (III) tanned leather is superimposed on that of untanned hide. The latter is very similar to that of pure collagen, and assignments [13C spectrum of the Cr (III) tanned leather is broadly similar, but it is notable that a number of signals in this spectrum are reduced in intensity relative to their counterparts in the native material. Cr (III), with three unpaired 3d electrons in its outer valence shell, is strongly paramagnetic. The large electron magnetic moment associated with the paramagnetic state can increase NMR relaxation rates, and/or shift the NMR frequencies, of nuclei which are close in space to the paramagnetic centre [Having shown that NMR was able to clearly recognize different vegetable tanning agents in final product materials, it was of interest to compare these leathers with leathers produced by processes which use other reagents. In ignments  of some c centre . In the Nevertheless it is significant that there is marked broadening of the envelope of signals centred at ca. 53 p.p.m.; this region contains signals from the α-carbons of acidic aspartate and glutamate residues (among others). Also strongly broadened is the shoulder centred at ca. 180 ppm to high frequency of the amide carbon signal, comprising signals from the carboxylate carbons of the same acidic residues . It is t27Al-NMR spectrum acquired from the Al (III) tanned material [27Al signal is close to that of aqueous hydrated Al3+ at 0 ppm, and the linewidth at half peak height is ca. 1.8 kHz. Neither the diamagnetic Al (III) ion nor the organic glutaraldehyde reagent produce the large changes in peak intensity caused by the paramagnetic Cr (III) ion. This is entirely to be expected because the ability of these diamagnetic species to exert long range effects on the NMR properties of the leather is much more limited than paramagnetic Cr (III). Nevertheless the response of the spectral linewidths of leather to tanning with either material is notable. Both tanned leathers exhibit signals which are generally much sharper than those of the parent untanned leather. In solid state NMR a significant cause of signal broadening is environmental heterogeneity resulting from partial or complete molecular disorder. Thus the signal sharpening seen with both processes is likely attributable to an increase in molecular order [via different underlying chemical processes. Spectra of leathers tanned with aluminium sulphate, and with glutaraldehyde, are compared with a spectrum of an equivalent sample of untanned material in material . The chear order  brought 27Al isotope  provide an extra NMR probe of the chemistry of the tanning process. The chemical shift of the 27Al signal argues for octahedral coordination by six oxygen ligands  coordination by four ligands) [27Al nucleus with a nuclear spin I = 5/2 means that the 27Al resonance lineshape is responsive to electric field gradients at the nucleus, which are a function of its bonded and non-bonded environment, particularly the nature and symmetry of the inner and outer coordination spheres. The lineshape is potentially amenable to modelling in terms of Al (III) coordination environment and the distribution of these environments [In the case of the Al (III) tanned leather the NMR properties of the ligands) . The quaronments  but we hIn the case of the glutaraldehyde tanned leather there are no clear signals attributable to the carbon atoms of the tanning reagent. There are several possible explanations; the incorporated glutaraldehyde molecules may be too mobile to cross polarize, their signals may be unobservably broad due to factors such as environmental heterogeneity, or the glutaraldehyde content required to produce a functional leather may simply be below the detection threshold of NMR. Much lower levels of reagent are used in the glutaraldehyde tanning process than in the vegetable tanning processes see , probablThe effects of these two reagents, one a metal ion and the other a bifunctional reactive organic dialdehyde, on the spectrum of leather collagen are rather different from those of the vegetable tannins. While the sharpening of signals induced by Al (III) and glutaraldehyde is quite general throughout the leather collagen spectrum, it is best appreciated by considering the envelope of signals centred at ca. 175 p.p.m. due to the collagen amide carbons. In the Al (III) and glutaraldehyde tanned materials it is possible to resolve three distinct signals from amide carbons of (in order of increasing resonance frequency) glycyl, prolyl/hydroxyprolyl, and other, residues. This resolution is not observed in spectra of the untanned or vegetable tannin tanned materials, in which these three signals are broadened into a single envelope. UK bovine hides were prepared for tanning using standard industrial processes, as follows: hides were soaked, washed, limed to remove hair, fleshed to remove unwanted flesh, connective tissue and fat, split to a substance of 3.0 mm, and weighed. All further processing percentages were based on the limed weight. The hides were then washed, delimed with ammonium sulphate and formic acid to a pH of 8.3, bated to remove unwanted inter-fibrillary proteins, washed again and pickled using salt, and formic and sulphuric acids to a pH of ca. 2.8 (chrome), 3.0–3.3  or ca. 4.5 (vegetable tannin extracts), and the penetration of pickle ensured to be complete. Hides were tanned with vegetable tannin extracts by adding 2% tannin and drumming for 30 minutes, followed by 10% tannin for 60 minutes, another 10% tannin (60 minutes) and a further 10% which was then drummed until penetrated. After tannage, a small percentage of EDTA (ethylenediamine tetraacetic acid) was added to sequester any iron present, followed by acidification with formic acid to a pH of 3.5. The leather was then given a light wash and dried (henceforth the leather is termed veg tanned). 2O3, 33% basicity), running for 2–4 hours until penetrated and then basifying with magnesium oxide or soda ash or sodium bicarbonate to a pH of 3.8–4.2, followed by draining and washing (the resulting leather is now referred to as wet blue).Chrome tanning was effected by adding 6–8% basic chromium sulfate . 13C-NMR spectra obtained using a Bruker AVANCE-400 9.4 Tesla wide bore spectrometer equipped with a standard dual channel broad band probe, at a magic angle spinning rate of 14 kHz and radio frequencies of 400.1 MHz (1H), 100.5 MHz (13C) and 104.2 MHz (27Al) respectively. 13C signal intensity was enhanced using standard cross polarization [1H π/2 pulse length 2.5 μs, 1H cross polarization field 70 kHz, 1H-13C cross-polarization contact time 2.5 ms, broadband TPPM15 decoupling during signal acquisition at a 1H field strength of 100 kHz, recycle time 2 s, typical number of scans accumulated per spectrum ca. 3,000). Chemical shifts were referenced to the methylene signal from solid glycine at 43.1 p.p.m. relative to tetramethylsilane at 0 p.p.m.. 27Al spectra were acquired using a rotor synchronized Hahn spin echo  and 0.2 s recycle delay, and no broadband decoupling, which had been established to have no effect on the 27Al-NMR signal characteristics. Chemical shifts were referenced to the resonance of aqueous ca. 1 M AlCl3 in ca. 1 M HNO3 at 0 p. p. m.. Dry leather samples were prepared for NMR by first cutting them into small pieces about 1 to 2 mm in size with a scalpel, and ball milling these for ca. 1 minute to a powder after freezing in liquid nitrogen, using a Sartorius Mikrodismembrator at 3,000 r.p.m.. Tannin powders were analyzed as received. All samples were packed into 4 mm outer diameter zirconia rotors  and rization  (CP) MASVegetable tanning agents all leave a distinctive spectroscopic signature in the tanned leather product by which the origin and type of the tannin used may be inferred. Several other commonly used tanning reagents also leave readily distinguishable spectroscopic fingerprints in leather products. Paramagnetic Cr (III) causes the loss of considerable signal intensity from the spectrum of the collagen proteins constituting leather without significantly changing the linewidths of remaining signals. The diamagnetic reagents Al (III) and glutaraldehyde cause considerable collagen signal sharpening indicative of an increase in collagen molecular order. It is to be expected that processes using mixtures of tanning reagents will leave equally distinctive fingerprints in the leather product. The fingerprint reflects not only the process chemistry but also underlying molecular mechanisms whereby tanning converts unprocessed leather into a commercial product. As such NMR potentially represents a powerful tool in understanding and improving tanning processes.


Here we build on this limited body of work by investigating the efficacy of supplementing mdx mice with taurine postnatally at a time suggestive of when dystrophic symptoms would begin to manifest in humans, and when treatments would likely begin. Mdx mice were given either taurine (mdx tau), the steroid alpha methylprednisolone (PDN), or tau + PDN (mdx tau + PDN). Taurine (2.5% wt/vol) enriched drinking water was given from 14 days and PDN (1 mg/kg daily) from 18 days. Wild-type  mice were used as a control to mdx mice to represent healthy tissue. In the mdx mouse, peak damage occurs at 28 days, and in situ assessment of contractile characteristics showed that taurine, PDN, and the combined taurine + PDN treatment was ineffective at attenuating the force loss experienced by mdx mice. Given the benefits of taurine as well as methylprednisolone reported previously, when supplemented at close proximity to the onset of severity muscle degeneration these benefits are no longer apparent.An increasing number of studies have shown supplementation with the amino acid taurine to have promise in ameliorating dystrophic symptoms in the  Currently, steroids are the only effective treatment for Duchenne Muscular Dystrophy (DMD) ,2. Whilemdx mouse limb muscle, taurine levels have been found to be reduced both before and during the onset of severe dystropathology (21–28 days), before returning to endogenous levels as the pathology stabilises into adulthood (70 days) [mdx mouse.Taurine is an amino acid found ubiquitously in all mammalian cells, identified as vital for healthy muscle function and development ,7. In thlogy 21–2 days, be70 days) . The avamdx mice, increasing both muscle strength and the visible markers of muscle health [mdx mice. Understanding this would provide a better insight into the effectiveness of taurine as a nutritional supplement after diagnosis in DMD patients.In our previous work, prenatal supplementation of taurine was found to be effective at reducing the severity of dystrophic symptoms in peak damage 28 days e health . Howevermdx mouse, a recent study found that 4% taurine supplementation in chow from 14 days of age was sufficient to greatly reduce myofibre necrosis and markers of inflammation in 22 days mdx mice [mdx mice begins at approximately 21 days following an increase in activity; thus, mice were investigated at the very beginning of the degenerative period [While there is a limited body of work investigating taurine supplementation in the juvenile mdx mice ; howevere period , as oppoe period ,13.mdx mice. We further assess a combined treatment of tau + PDN in 28 days mice, with a dose similar to that previously reported by Cozzoli, et al. [To better understand the potential of taurine as a substance of therapeutic benefit to DMD, the current study compares the effectiveness of beginning administration of taurine 14 days postnatally with the steroid methylprednisolone (PDN) on peak damage 28 days mdx mice when supplemented at close proximity to the onset of severity muscle degeneration. This has important implications for the practical use of taurine as a substance of therapeutic benefit to DMD, as the same dosage of taurine given prenatally was found to be effective [Taurine, PDN, and the combined taurine + PDN treatment was ineffective at attenuating the force loss experienced by ffective . The finmdx and 10 wild-type  were used. Experimental animals were bred at the La Trobe Animal Research and Teaching Facility  using breeding pairs obtained from the Animal Resource Centre . The offspring of WT and mdx mice had access to standard rodent chow and water ad libitum, and were utilised for experimentation at 28 ± 1 days of age. The absence of dystrophin in mdx mice was confirmed by Western Blot, as previously described [mdx taurine (tau) and mdx taurine + steroid (tau + PDN) mice were supplemented with continuous access to taurine (2.5% wt/vol) enriched drinking water, as this dosage of supplementation has been previously demonstrated to elevate skeletal muscle taurine content in mdx mice [mdx PDN and mdx tau + PDN mice received a 1 mg/kg dose of alpha methylprednisolone  daily via intraperitoneal (I.P.) injection on alternate sides of the body until the day of experimentation at 28 days. This dosage has been identified previously as effective at increasing muscular strength in the mdx mouse [All procedures in this study were approved by the La Trobe University Animal Ethics Committee . Only male mice were used for experiments. A total of 26 dx mouse ,16. As r−1 Nembutal (Sodium Pentobarbitone) and kept unresponsive for the duration of the experiment with supplementary doses of Nembutal . The mouse was weighed, placed on a 37 °C heated pad, and the right leg was skinned to the waist taking care not to damage the fascia. Throughout the experiment, warmed physiological saline (0.9%) was applied to exposed muscle tissue. The distal tendon of the tibialis anterior (TA) muscle was isolated and secured with both a top and bottom knot as close to the myotendinous junction as possible using 5.0 surgical thread . The distal tendon was then severed, and the TA dissected free from the surrounding tissue keeping nerve and blood flow intact at the tibia below the tibial plateau. The sciatic nerve was exposed by gently parting the midline of the biceps femoris muscle above the knee joint. The mouse was secured on the heated platform (37 °C) of an in situ contractile apparatus  with a pin behind the patellar tendon and a foot clamp. The distal end of the TA was tied firmly to a lever arm attached to an isometric force transducer. The sciatic nerve was stimulated by two field stimulating platinum electrodes coupled to an amplifier.Mice were anesthetised with an I.P. injection of 10 µL·go) was first determined by eliciting twitch contractions by incrementally adjusting muscle length with a micromanipulator until a repeatable maximum peak twitch force was obtained. Muscle length at Lo was measured with precision digital callipers from the beginning of the distal tendon to the insertion of the TA at the base of the knee. Subsequently, the TA was stimulated at 100 Hz tetanic contraction, followed by a 2 min rest interval, and then twitch contraction. Comparable twitch forces pre and post 100 Hz stimulation indicated that the knots were both secure and unlikely to slip during the remaining protocol. If a decrease in twitch force was observed, the muscle was incrementally tensioned and stimulated between 2 min rest intervals until peak twitch force (Pt) was re-established. To establish the force frequency relationship, the TA was stimulated supramaximally (10 V) for 500 ms at 10, 20, 30, 40, 50, 80, 100, 150, and 200 Hz, with a 2 min rest interval in-between. Maximum isometric tetanic (Po) force was determined from the largest force produced during the force-frequency stimulation protocol. Maximum tetanic specific force (sPo) was determined as force per cross sectional area (CSA) as described by Lynch (DMD_M.2.2.005). Briefly, optimum fibre length (Lf) was first determined by multiplying Lo by the predetermined TA length to fibre length ratio of 0.6 [o = Po × (muscle mass/Lf × 1.06).The TA was contracted via square wave (0.2 ms) pulses at 10 V from the stimulator . Forces were converted to a digital signal and recorded by Dynamic muscle analysis 611A (DMA) . Optimum muscle length , and weighed. The mouse was then killed by cardiac excision.mdx; mdx, mdx tau, mdx PDN and mdx tau + PDN groups were performed using a one-way ANOVA of variance with individual stimulation frequencies in the force-frequency analysis, and individual time points in the fatigue and recovery analyses, with Sidak’s post-hoc analyses. All statistical analysis was performed using GraphPad Prism v 6. Significance was set at p < 0.05.All data are presented as mean ± standard deviation (SD) unless stated otherwise. Comparisons between WT and mdx mice , time to peak tension (TTP), and half-relaxation time (½RT) than the WT group  [A growing number of studies have reported the postnatal administration of taurine to be effective at reducing the dystropathology of 6 weeks) ,18,19,206 weeks) .mdx mice, elicited no improvement in in situ contractile strength or fatigue/recovery characteristics when assessed at 28 days of age , and found 8 days of taurine supplementation resulted in a 40% increase in hind limb muscle taurine content which was accompanied by 75% reduction myofibre necrosis, as well as a significant reduction in inflammatory markers [mdx mice [Most relevant to the current study is the work by Terrill, Grounds and Arthur , who sim markers . It is u markers . Considemdx mice , the impmdx mice  may reflMdx mice were supplemented with taurine in drinking water (2%) from 18 days of age with functional assessment taking place at 42 days of age (adult mice) following 24 days of supplementation. In that study, functional improvement was observed both in vivo when assessed by grip strength, and ex vivo in an isolated extensor digitorum longus (EDL) preparation. It is unclear if the natural regeneration of the mdx mouse may have facilitated the improvement in strength seen in that study [mdx mice during the damage period of ~21–28 days of age, but similar tissue taurine content is observed between mdx and WT mice both before and after this damage period  [mdx mouse, with particular emphasis from juvenile (~2 weeks) to adults (>6 weeks) mice in order to assess where/when taurine is the most efficacious.In a similar study, mice were assessed beyond the peak damage period 21–28 days of age . Mdx mic42 days) ,18,24. Tmdx mice receiving the PDN treatment from 18 days of age produced significantly greater peak twitch and maximum force than all other mdx groups, including the mdx tau + PDN group  and given a similar dosage of methyl-prednisolone , taurine (1 g/kg/day) or a combination of both for 4–8 weeks, in vivo forelimb grip strength was increased [mdx mice was shifted to more positive potentials, and the combined tau + PDN treatment was more efficacious than either treatment individually. This finding was not replicated here with a similar dosage scheme; however, the differences in mouse age and functional assessment make direct comparisons unrealistic.The DN group . Howeverr groups , when tastrength B. No othx groups . Why thencreased . In thatmdx mouse, as seen previously in adult mice [PDN has been identified to increase muscle function and prolong ambulation in DMD patients on numerous occasions, albeit with side effects ,25,26. Iult mice . That a mdx mouse, potentially inhibiting specific pathways benefited by taurine. While no side effects manifested in the current study with methylprednisolone treatment, a worsened dystrophic state has been observed previously in 6 weeks old mdx mice following 3 weeks of the daily administration of the anabolic steroid nandrolone decanoate (0.15 mg/100 g body weight) [mdx mouse model of DMD.It is possible that the adverse effects of a potent steroid counteract any benefit of taurine in the 28 days  weight) . Ultimatmdx mice produced a significantly greater proportion of their maximum force at lower stimulation frequencies (10 and 20 Hz) compared to the WT (mdx mice have a more negative mechanical threshold for muscle contraction [mdx and the mdx tau, mdx PDN, and mdx tau + PDN groups, indicating that these treatments were ineffective at ameliorating the altered E-C coupling observed in the mdx mouse.The 28 days o the WT . These ftraction ,16,27, rmdx mice, there was no significant difference in fatigability across all groups (mdx mice that has been reported previously in older animals [While there was a trend for increased level of fatigue in WT mice at the end of the 180 s fatigue protocol compared to untreated l groups . Here, w animals ,31, whic2+, resulting in a greater amount of cross bridge cycling, which in turn results in a stronger contraction [Post tetanic potentiation was observed in all groups during the fatigue recovery period . This hatraction ,33.mdx mice. This is likely because the supplementation occurred in too close a proximity to the onset of severity muscle degeneration, as taurine has been found to be beneficial when supplemented prenatally. Findings further suggest that the benefit of taurine lies in its ability to reduce the initial onset of disease severity, as opposed to attenuating damage or downstream pathways once the pathology has started to progress. Similar to the DMD disease, skeletal muscles of mdx mice exhibit myofibre necrosis, varying fibre size, and an increase in centrally located nuclei. Despite the absence of dystrophin in skeletal and cardiac muscles, adult mdx mice do not exhibit the progressive characteristics of human DMD as necrosis and regeneration in skeletal muscles peaks around 3–4 weeks of age, and plateaus thereafter. Due to these significant differences in dystrophic pathology, any clinical implications cannot be confidently made until taurine is investigated in a pre-clinical model that has the full spectrum of DMD pathology, such as the mdx4cv/mTRG2 mouse model [When taurine was given at 14 days of age for 14 days, and PDN given at 18 days for 10 days, taurine, PDN, and the combined tau + PDN treatment were ineffective in a preclinical model of DMD at attenuating the force loss experienced by se model . In rega


This study investigated a rabbit model of autologous simple oral mucosal epithelium transplantation (SOMET) for limbal stem cell deficiency (LSCD). LSCD was created in the SOMET group and the Control group. In the SOMET group, oral mucosa harvested from the buccal region was treated with dispase, cut into small pieces, and placed on the exposed corneal stroma without using graft sutures, amniotic membrane, and/or glue. A soft contact lens was positioned and tarsorrhaphy was performed in both groups. Postoperative corneal neovascularization and fluorescein staining scores were evaluated by slit lamp microscopy in both groups. At 2 weeks postoperatively, eyes were excised and subjected to immunohistochemical staining for CK3, CK13, CK15, and p63. In the SOMET group, transplantation of oral mucosa led to complete recovery of LSCD, as indicated by low neovascularization scores, low fluorescein staining scores, and detection of stratified K3/K13-positive cells on the stroma at 2 weeks after surgery. In contrast, corneal epithelial defects persisted in the Control group at 2 weeks. SOMET achieved re-epithelialization of the corneal surface in this rabbit LSCD model. It is a simple technique that does not require culture and could be a promising option for ocular surface reconstruction in bilateral LSCD. When the limbus is damaged by chemical burns, Stevens-Johnson syndrome, autoimmune disease, or other insults, conjunctival invasion of the cornea occurs along with neovascularization and infiltration of fibroblasts, which can impair visual function by reducing corneal smoothness and transparency4. This situation arises because of limbal stem cell deficiency (LSCD). While patching with amniotic membrane may achieve recovery of LSCD in some circumstances5, an additional source of limbal stem cells (LSCs) is required for long-term maintenance of transparency in patients with complete LSCD7. Stem cell-based reconstruction of the ocular surface for LSCD has progressed from application of allografts such as conjunctival epithelial grafts9 and limbal transplantation11 to use of autografts for avoidance of rejection, including corneal limbal transplantation12 and cultured corneal epithelial sheet transplantation13 in patients with unilateral LSCD. In addition, bilateral LSCD has been treated by transplantation of cultured conjunctival epithelial cell sheets14 and cultured oral mucosal epithelial cell sheets19. Among these techniques, transplantation of cultured cell sheets reduces the amount of donor tissue required and allows early rehabilitation after surgery. However, this method has certain drawbacks, such as the need for expensive facilities and careful monitoring when manufacturing epithelial cell sheets, as well as legal restrictions.Corneal epithelial stem cells are localized in the basal layer of the corneal limbuset al. developed a tissue transplantation technique called simple limbal epithelial transplantation (SLET)20 and reported comparable results to those obtained after transplantation of tissue grafts or cell sheets21. The SLET technique has advantages for both patients and the medical system, since cell culture is not required and the risk of causing LSCD in the donor eye is minimized by reducing the donor tissue volume. Recently, allograft SLET was reported in patients who could not undergo autografting22. However, considering the risk of allograft rejection, it is worthwhile to develop an autograft-based corneal reconstruction method. We have previously performed simple conjunctival tissue transplantation for bilateral LSCD and confirmed a certain level of efficacy . However, patients with bilateral LSCD often also develop symblepharon, which results in insufficient conjunctival stem cells for reconstruction of the ocular surface. Therefore, methods based on transplantation of other autologous tissues are needed.Sangwan Accordingly, we investigated a novel technique for ocular surface reconstruction using uncultured oral mucosal epithelium and evaluated its efficacy in a rabbit model of LSCD.The harvested oral mucosa was composed of mucosal epithelium and subepithelial tissue. Two methods for removal of the subepithelial tissue from the epithelium were tested. (1) Blunt peeling with 2 pairs of forceps separated the tissue into two layers, which were mucosal epithelium Fig.  and submMucosal epithelium containing the entire basal layer Fig. , the surOne of the 4 eyes in the SOMET group was excluded from evaluation because the soft contact lens (SCL) dropped off and most of the grafts were lost by the first week after surgery. Figure After dispase treatment and removal of submucosal connective tissue, the oral mucosal epithelium was subjected to immunohistochemical examination. K3 and K13 were positive in all cell layers, except 3 to 5 layers of the basal cells, while K15 was positive in some basal cells and p63 was positive in the nuclei of most basal cells  and all four eyes in the autograft group (n = 4) showed no rejection at 120 days, suggesting good compatibility of dispase-treated oral mucosal epithelium with the ocular surface. In rats with LSCD created by alkali burns, transplantation of sutured autologous oral mucosal strips without using amniotic membrane was also reported to achieve favorable results25. While the graft become detached in one eye, re-epithelialization around the graft was observed from 2 to 5 days after surgery and was completed within 1 to 3 weeks in the remaining 6 eyes. This report suggested that direct transplantation of uncultured oral mucosal epithelium was effective for LSCD in rats. Clinically, grafted oral mucosa has been applied for reconstruction of the conjunctival fornix after enucleation of the eyeball and for reconstruction of the filtration bleb after glaucoma surgery26. In patients with ocular pemphigoid and severe fibrous symblepharon, sutured oral mucosa grafts were employed to reconstruct the conjunctival fornix, but most of the grafts failed within a 2-year follow-up period, presumably because fibrosis of the ocular surface was severe and preoperative/postoperative anti-inflammatory therapy was inadequate27. Conversely, there have been several reports of successful ocular surface reconstruction by direct transplantation of uncultured oral mucosal epithelium (attached with sutures and glue) to encircle the entire limbus in LSCD patients combined with amniotic membrane co-transplantation28 or temporary amniotic membrane transplantation29. In this study, we simplified the surgical technique and materials by omitting the use of amniotic membrane, graft sutures, and glue. We achieved ocular surface re-epithelialization and recovery of corneal transparency in the SOMET group, indicating that our simple technique was effective for LSCD. Many reports have been published about transplantation of cultured oral mucosal epithelium in animal and clinical studies31. There are three common points between our method and the techniques used in these other studies: 1) transplantation of oral mucosal epithelial stem cells, 2) reconstruction of bilateral LSCD, and 3) expression of the same proteins as native oral mucosal epithelium (CK3 and CK13) by the grafted epithelium. Transplantation of cultured oral mucosal epithelium may have the advantage of an anti-inflammatory effect because the cultured epithelial sheet covers the entire cornea and protects it after surgery. At hospitals with tissue culture facilities, transplantation of cultured oral mucosal epithelium is undoubtedly a good choice for binocular LSCD. However, our SOMET technique has the advantage of providing easy to access treatment for LSCD patients because it can be performed in hospitals where cell culture facilities and amniotic membrane are unavailable.Because severe LSCD accompanied by symblepharon is often bilateral, attention has been focused on autologous cell sources other than the corneal limbus, and the oral mucosa has been investigated as an option for ocular surface reconstruction26. Clinical studies of SLET have also shown that limbal epithelial tissue grafts was observed for approximately 6 months postoperatively20. Accordingly, it can be suggested that dispase treatment of oral mucosal epithelium before grafting contributes to tight adhesion to corneal stroma and faster recovery of visual acuity compared with previous methods26.We treated the oral mucosal tissue with dispase before transplantation, and we used a SCL bandage and tarsorrhaphy to promote efficient attachment of undifferentiated cells in the mucosal epithelium to the denuded corneal stroma. Dispase treatment of the mucosal grafts completely removed any residual unwanted submucosal connective tissue Fig.  without 34. In one rabbit from the SOMET group, loss of the SCL led to graft failure and re-epithelialization was not observed at 2 weeks after surgery. Because we did not use sutures or glue, the SCL bandage is essential for stable fixation of the grafts. In animals, tarsorrhaphy is critical for maintaining the SCL in situ, but it is too invasive in humans. As a substitute for tarsorrhaphy, an eye patch may be effective when this procedure is applied clinically. We did not utilize amniotic membrane in our SOMET procedure. Amniotic membrane has previously been used in a wide variety of ocular surface reconstruction techniques, such as amniotic membrane transplantation5, limbal transplantation11, cultured corneal epithelial sheet transplantation13, cultured conjunctival epithelial cell sheet transplantation14, COMET19, and SLET20. However, we found that using fibrin glue with human amniotic membrane induced severe ocular surface inflammation after transplantation in rabbits . It is unknown whether this occurred because the fibrin glue and amniotic membrane grafts were derived from humans and not rabbits, but the present results suggest that ocular surface reconstruction without amniotic membrane may be an option for LSCD.In patients with corneal erosion, a SCL bandage has been reported to be effective for reducing pain and foreign body sensation, promoting epithelialization, and preventing chronic inflammation and neovascularization31. Markers of undifferentiated epithelial cells, P6335 and K1536, were present in the basal layer of graft specimens from the SOMET group  were assigned to a simple oral mucosal epithelium transplantation (SOMET) group (n = 4) or a Control group (n = 4). General anesthesia was induced by intramuscular injection of xylazine (5 mg/ml) and ketamine (50 mg/ml). In all rabbits, the right eye was used for this study. Evaluation was performed at 1 and 2 weeks after surgery, and ocular specimens were excised at 2 weeks postoperatively after the rabbits were killed by intracardiac injection of sodium pentobarbital . Animals were treated in accordance with the animal experiment protocol approved by the Animal Experiment Committee of the University of Tokyo and the ARVO Statement for the Use of Animals Research in Ophthalmic and Vision Research.30. The epithelium was washed with saline after dispase treatment.After the oral cavity was disinfected with povidone iodine, the mucosa was grasped with hooked forceps and a 3 × 4 mm piece of mucosal tissue was excised with spring scissors. Separation of the oral mucosal epithelium from the submucosal tissue was compared between pulling or peeling with two pairs of forceps and treatment with 1.2 IU of dispase at 37 °C for 40 minutes, as described elsewhere2 incubator at 37 °C for 3 weeks. The medium was exchanged after the first week, every other day during the second week, and every day during the third week. In order to promote tissue adhesion, sufficient medium was added to completely cover the surface of the mucosal tissue for the first week.The separated oral mucosal epithelium was cultured in DMEM/Ham’s F-12 containing L-glutamine, Phenol Red, HEPES, and sodium pyruvate  supplemented with B27, EGF and penicillin-streptomycin-amphotericin b Suspension  in a COTo create a rabbit model of LSCD, the conjunctiva and subconjunctival tissue were excised to 5 mm outside the limbus and the corneal epithelium including the limbus was completely removed for 360 degrees in one eye under anesthesia. This procedure was done in 8 animals to create LSCD. In the SOMET group (n = 4), the harvested oral mucosal epithelium was cut into 8 to 10 pieces with scissors and treated with dispase, as described above. After washing with saline, pieces of the treated epithelium were arranged on the denuded corneal stroma with the epithelial surface upward ; Grade 1, neovascularization ≤2 mm (1); and Grade 2, neovascularization >2 mm (score = 2). Epithelialization was scored as follows: Grade 0, no fluorescein staining of the cornea (score = 0); Grade 1, staining <1/4 (score = 1); Grade 2, staining from 1/4 to 1/2 (score = 2); and Grade 3, staining >1/2 (score = 3). If the SCL fell off before follow-up examination, the eye was excluded from evaluation. Scores were analyzed by the Mann-Whitney U test. Following evaluation at 2 weeks after surgery, all treated eyes were excised and frozen blocks were prepared for immunohistochemical examination.Samples were fixed in 4% paraformaldehyde. Paraffin blocks of cultured cell sheets and frozen blocks of the ocular specimens were prepared. The frozen blocks were cut into 5 μm sections on a cryostat, while the paraffin blocks were cut into 4 μm sections. Then the sections were washed with phosphate-buffered saline (PBS) and blocked with 10% goat serum / PBS for 30 minutes at room temperature. The primary antibodies were K3 , K13 , K15 , and p63 . Each antibody was diluted with 0.1% bovine serum albumin (BSA) / PBS and added to the sections for overnight incubation at 4 °C, followed by three 5-min washes with PBS, incubation for 90 minutes at room temperature with the goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 647 , and three further 5-min washes. Subsequently, the sections were mounted in medium with 4′,6-diamidino-2-phenylindole (DAPI).P < 0.05 was considered to indicate statistical significance. All analyses were carried out by using JMP® Pro 14.1.0 software.Results are expressed as the mean ± standard deviation. The Mann-Whitney U test was used to compare the two groups and


The acetoclastic methanogen Methanosaeta was completely replaced by Methanosarcina. The propionic acid degrading genus Syntrophobacter was replaced by yet unknown propionic acid degraders. The butyric acid degrading genus Syntrophomonas and hydrogenotrophic Methanomicrobiaceae were hardly affected. We hypothesized that the ammonia sensitivity of the initially dominating taxa Methanosaeta and Syntrophobacter led to a stronger inhibition of the acetic and propionic acid degradation compared to butyric acid degradation and hydrogenotrophic methanogenesis, which were facilitated by the ammonia tolerant taxa Syntrophomonas and Methanomicrobiaceae. We implemented this hypothesis into a multi-taxa extension of ADM1, which was able to simulate the dynamics of both microbial community composition and VFA concentration in the experiment. It is thus plausible that the effect of ammonia on VFA degradation strongly depends on the ammonia sensitivity of the dominating taxa, for syntrophic propionate degraders as much as for acetoclastic methanogens.Ammonia inhibition is an important reason for reactor failures and economic losses in anaerobic digestion. Its impact on acetic acid degradation is well-studied, while its effect on propionic and butyric acid degradation has received little attention and is consequently not considered in the Anaerobic Digestion Model No. 1 (ADM1). To compare ammonia inhibition of the degradation of these three volatile fatty acids (VFAs), we fed a mixture of them as sole carbon source to three continuous stirred tank reactors (CSTRs) and increased ammonium bicarbonate concentrations in the influent from 52 to 277 mM. The use of this synthetic substrate allowed for the determination of degradation efficiencies for the individual acids. While butyric acid degradation was hardly affected by the increase of ammonia concentration, propionic acid degradation turned out to be even more inhibited than acetic acid degradation with degradation efficiencies dropping to 31 and 65% for propionic and acetic acid, respectively. The inhibited reactors acclimatized and approximated pre-disturbance degradation efficiencies toward the end of the experiment, which was accompanied by strong microbial community shifts, as observed by amplicon sequencing of 16S rRNA genes and terminal restriction fragment length polymorphism (T-RFLP) of  Biogas production is an important renewable energy source and organic waste treatment technology Plugge, . For nitSeveral, partly contradicting theories have been presented on ammonia inhibition in anaerobic digestion. A major controversy is whether acetoclastic or hydrogenotrophic methanogens are more strongly inhibited, with experimental evidence for both cases , and followed VFA degradation efficiencies and microbial community dynamics over time. Deliberate ammonia inhibition was induced by increasing ammonium bicarbonate concentration in the substrate in two reactors while the third reactor remained unchanged as a control. Furthermore, we added HCl to one of the reactors to reduce the pH and thus the share of free ammonia, which has been shown previously to successfully alleviate ammonia inhibition  with working volumes of 6 L were operated in parallel at 37°C at a hydraulic retention time (HRT) of 5.5 d. All CSTRs were fed continuously with a synthetic, liquid substrate containing a mixture of VFAs as the only carbon source  with a total concentration of 37.2 gCOD L−1 in a mineral medium containing all necessary trace elements, macronutrients, and vitamins , biogas production rate, pH, VFA concentrations, total solids content (TS), and volatile solids content (VS) were determined as described previously  to reduce the potential error of VFAs and ammonia in the reactor sample.Three CSTRs (REi (t) for each individual VFA i were calculated for each sampling date based on mass balances over 1 day considering the VFA input via the influent over the last day Si,in/HRT·Δt, the VFA output via the effluent over the last day ΔSi, out (t), the amount of VFAs in the reactor at the sampling date minus the amount at the previous day ΔSi(t) and, for acetic acid degradation only, the production of acetic acid from propionic and butyric acid oxidation over the last day ΔSi, p (t). VFA mass degraded during 1 day is then given by Si,in/HRT.Δt + ΔSi,p(t) − ΔSi,out(t) − ΔSi(t). Based on a constant reaction volume, the masses of each component can be calculated based on the respective concentrations in the influent and reactor digestate so that degradation efficiency can be expressed as:fac,pro and fac,bu being the stoichiometric conversion factors of propionate oxidation (1 mol acetate per mol propionate) and of butyrate oxidation (2 mol acetate per mol of butyrate), respectively. Between sampling dates, VFA concentrations were linearly interpolated. E values of 1 correspond to a complete degradation in steady state. E values can reach values higher than 1 when accumulated VFAs are degraded (ΔSi(t) < 0).The degradation efficiencies Reactor samples were taken using 5 mL pipetting tips that were cut at the top to prevent the exclusion of aggregates of microorganisms and thereby a biased community analysis. Samples were centrifuged at −7°C, 15,000 × g for 2 min and the supernatant was discarded. The low centrifugation temperature and short duration did not lead to sample freezing and were chosen in order to prevent RNA degradation. Pellets were stored at −80°C prior to RNA extraction and −20°C prior to DNA extraction. NucleoSpin® Soil Kit  was used to extract DNA from the pellets. The quality and quantity of purified DNA were determined by agarose gel electrophoresis and by NanoDrop ND 1000 spectral photometer . DNA was stored at −20°C. The ZR Soil/Fecal RNA MicroPrepTM Kit  was used for RNA extraction. DNA was removed using the DNA-free™ DNA Removal Kit . The quality and quantity of extracted RNA were determined by Qubit® Fluorometer 3.0  with the high sensitivity Qubit® RNA HS Assay Kit. RNA was converted to cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit . The resulting cDNA was stored at −20°C. The concentration of cDNA was quantified with a NanoDrop® ND-1000 spectral photometer .mcrA gene amplicons as described previously  was used as restriction enzyme.T-RFLP was used to analyze the methanogenic community composition based on http://bbmap.sourceforge.net/). Merged reads were processed using the QIIME 1.9.1 Virtual Box release . Relative abundances of 16S rRNA genes were converted to relative genome abundances using the average 16S rRNA gene copy number per genome taken from the rrnDB database (version 5.2).Amplicon sequencing of 16S rRNA genes was used to analyze the bacterial community compositions. PCR amplification using the primers 341f (CCT ACG GGN GGC WGC AG) and 785r (GAC TAC HVG GGT ATC TAA KCC)  were replaced by two competing populations: Methanosarcina  and Methanosaeta . Accordingly, the differential equation for acetic acid was adapted to Equation 2c. The original ADM1 inhibition functions  and Methanosaeta (i = ac2).The acetoclastic methanogens (Xpro) were replaced by two competing populations: an unknown taxon  and Syntrophobacter  following the same approach as described above for the acetoclastic methanogens  were approximated based on simulation results for steady state concentrations for the inorganic nitrogen concentration of 52 mM in the influent , Xpro1(t = 0), KS,ac1, KS,pro1) and ammonia inhibition constants of all acetic acid and propionic acid degraders  were estimated using the solver fmincon  in Matlab R2014b (Mathworks) minimizing the sum of squared error between experimental and simulation results, first for propionic acid concentration and second for acetic acid concentration.Only parameter values for acetic acid and propionic acid degraders were changed compared to the benchmark model parameter values by Rosen and Jeppsson . The ini−1 and pH values of maximal 7.35. Increasing the ammonia concentration in the influent to 277 mM in RNH3 and RNH3, HCl starting on day 21 was followed by an accumulation of VFAs and decline in methane production rates. The strongest accumulation was observed for acetic acid with up to 8.86 g L−1 (RNH3 on day 42). Propionic acid accumulated up to 1.63 g L−1 (RNH3 on day 49) and butyric acid showed the smallest accumulation with up to 0.52 g L−1 (RNH3 on day 42). On day 55, acetic, propionic, and butyric acid concentrations had declined to 0.85, 1.26, and 0.02 g L−1 for RNH3 as well as 0.69, 0.48, and 0.01 g L−1 for RNH3, HCl, respectively. This decline was accompanied by increases in pH values to 7.7 and 7.53 for RNH3 and RNH3, HCl, respectively. Starting on day 73, after HCl was omitted from the medium of RNH3, HCl, the pH value in RNH3, HCl rose to 7.7 toward the end of the experiment, equal to RNH3, while total VFA concentrations remained at about 1 gCOD L−1. Hydrogen partial pressures in the biogas were hardly affected by the increasing ammonia concentration and remained below 17 ppm in all CSTRs between day 21 and 55.On day 21, after 3 HRTs under low ammonia concentrations of 52 mM, all three CSTRs  and the hydrogenotrophic Methanomicrobiaceae (25–30%). Methanosarcina, which can perform both acetoclastic and hydrogenotrophic methanogenesis, reached a relative abundance of up to 13% , Syntrophobacter (13–16%), Thermovirga (15–22%), and Blvii28 wastewater-sludge group (6–38%).On day 21, 96 to 110 OTUs were detected in the three CSTRs. All CSTRs were dominated by the OTUs NH3 and RNH3, HCl showed strong changes. Syntrophobacter, Thermovirga and Blvii28 wastewater-sludge group decreased in relative abundance to < 0.1% of all bacterial reads in both RNH3 and RNH3, HCl toward the end of the experiment  strongly increased in relative abundance shortly after the ammonia inhibition in RNH3 and decreased again toward the end of the experiment. A similar behavior was observed for Parcubacteria in RNH3, HCl. Syntrophomonas remained one of the most abundant bacterial genera throughout the experiment in all CSTRs.After the increase in ammonia concentration, the community composition of Rt Figure . AminobaNH3 and RNH3, HCl, respectively, compared to 13 and 17%, respectively, on day 79.Our primer pair also led to the amplification of 16S rRNA genes from methanogens despite the lower coverage of archaea compared to bacteria . Only acetic acid accumulation was predicted, but not the acclimatization toward the end of the experiment. After implementing our model modifications, i.e., second populations for acetic and propionic acid degradation, respectively, the simulations represented the observed VFA concentrations better . After the increase of ammonia concentration in the influent on day 21, the ammonia sensitive populations in the simulations  became strongly inhibited and were washed out, while accumulations of 8.2 and 2.5 gCOD L−1 occurred for acetic and propionic acid, respectively. At the same time, ammonia tolerant populations  increased in relative abundance , acetoclastic methanogenesis commonly dominates over SAO  lead to a process recovery after the increase in VFA concentrations following ammonia inhibition because only one taxon of each functional group is part of the original structure. For example, only one taxon is capable of acetoclastic methanogenesis. As a consequence, our simulations using the original ADM1 structure could only lead to no VFA accumulation, to a constant elevated VFA concentration, or process breakdown. However, our experimental data clearly showed an increase in VFA concentration due to ammonia inhibition, followed by a decrease in VFA concentration toward the end of the experiment after the microbial community adapted to the new conditions. Therefore, the addition of at least a second taxon of acetic and propionic acid degraders was necessary in ADM1 to reflect the reactor performance dynamics as a consequence of the microbial community dynamics.ac,1, Xac,2, Xpro,1, and Xpro2, and the initial concentration and half saturation constants of Xac,1 and Xpro,1 compared to the benchmark model parameter values by Rosen and Jeppsson  compared to the ammonia tolerant populations . This reflects the higher substrate affinities previously observed for Methanosaeta compared to Methanosarcina , i.e., decreasing the ammonia sensitivity of these populations, did not change the simulation results. This indicates that higher ammonia concentrations are necessary to unambiguously determine these parameter values .The datasets generated for this study can be found in the Supplementary Material FB, HS, DP, SK, and FC designed the experiments. FB performed the experiments and simulations and analyzed the reactor and T-RFLP data. SW and FB developed the ADM1 model extension. DP performed the 16S rRNA gene sequencing data analysis. HH contributed to the discussion of the results. All authors contributed to the preparation of the manuscript. All authors read and approved the manuscript.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.


Background and objectives: To compare the long term clinical outcomes, range of motion (ROM) and strength of two different postoperative rehabilitation protocols after arthroscopic rotator cuff repair (RCR) for full-thickness rotator cuff (RC) tears. Materials and Methods: Patients undergoing RCR were divided into two groups. In 51 patients (56 shoulders), rehabilitation was performed without passive external rotation, anterior elevation ROM, and active pendulum exercises in the first 2 weeks after surgery (Group A). In 49 patients (50 shoulders) aggressive rehabilitation was implemented, with early free passive external rotation, anterior elevation ROM, and active pendulum exercises were allowed from the day after surgery (Group A). Results: No statistically significant differences were found in clinical scores, muscle strength, passive forward flexion, passive and active internal/external rotation between the two groups. However, the mean active forward flexion was 167.3° ± 26° (range 90–180°) in group A and 156.5° ± 30.5° (range 90–180°) in group B (p = 0.04). Conclusions: A statistically significant difference between the 2 groups was found in active forward flexion ROM, which was better in patients of group A. Lesions of the rotator cuff (RC) are common , especiaThe management of full-thickness RC tears is often surgical. Various arthroscopic operative techniques allow good clinical and functional outcomes. Despite advances in arthroscopic techniques ,19,20,21Various surgical and nonsurgical factors may affect whether a tendon will successfully heal after repair ,25,26,27Early rehabilitation plays a critical role to obtain good results after RC repair. This is recommended for preventing postoperative stiffness but can be considered one of the factors that may affect clinical, functional and structural outcomes ,30,31,32Only a few studies have compared clinical and functional outcomes, as well as strength, of two different early rehabilitation protocols after rotator cuff repair (RCR) for full-thickness RC tear.The purpose of the present investigation was to compare the long term clinical outcomes, ROM and strength between two different early rehabilitation protocols after arthroscopic RCR for full-thickness RC tears.All participants were recruited at the University Hospital Campus Bio-Medico of Rome. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Campus Bio Medico of Rome (Prot 37/16 OSS ComET CBM date 14-9-2016). Authors declare that the investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013.Patients were included in the study if they presented the following conditions at the time of surgery: RC tears diagnosed on clinical grounds; no episodes of shoulder instability; no radiographic signs of fracture of the glenoid or the greater or lesser tuberosity; magnetic resonance imaging (MRI) evidence of RC tear; duration of symptoms of at least three months; inadequate response to non-operative management . Patients with partial or massive RC tears were excluded, while patients with biceps lesions or tendinopathy were included in the study.Patients were excluded if they presented the following conditions at the time of surgery: inflammatory joint disease; prior surgery on the affected shoulder; labral pathology amenable to surgical repair; degenerative arthritis of the glenohumeral joint; symptomatic arthritis of the acromioclavicular joint; RC arthropathy.At the time of the last follow up, patients were also excluded if unable to complete questionnaires because of language problems or cognitive disorders.Materials and Methods should be described with sufficient details to allow others to replicate and build on published results. Please note that publication of your manuscript implicates that you must make all materials, data, computer code, and protocols associated with the publication available to readers. Please disclose at the submission stage any restrictions on the availability of materials or information. New methods and protocols should be described in detail while well-established methods can be briefly described and appropriately cited.Research manuscripts reporting large datasets that are deposited in a publicly available database should specify where the data have been deposited and provide the relevant accession numbers. If the accession numbers have not yet been obtained at the time of submission, please state that they will be provided during review. They must be provided prior to publication.Interventional studies involving animals or humans, and other studies require ethical approval and must list the authority that provided approval and the corresponding ethical approval code.100 patients (106 shoulders) followed two types of postoperative rehabilitation after arthroscopic RC repair. 49 patients (50 shoulders) underwent limited rehabilitation, in which early free passive external rotation, active elevation ROM, and active pendulum exercises were restricted (Group A), while in the remaining 51 patients (56 shoulders) the rehabilitation protocol performed included early passive free ROM and pendulum exercises (Group B). For all 100 patients assigned to one of the two treatments, an average of 4 ± 1-year results were available.At the time of surgery, all patients underwent MRI scans and oblique coronal, oblique sagittal and axial T2-weighted spin-echo MRIs (repetition time (RT): 3200 ms; echo time (ET): 85 ms). Furthermore, a pre-operative assessment using standard radiographs  was performed in all patients.Two orthopedic surgeons blindly performed all the measurements. All patients were evaluated for age; sex; arm dominance; history of trauma; location of the RC tear; dimension of the RC tear; biceps tendon lesion or tendinopathy; type of treatment of biceps tendon; number of suture anchors used for RC repair.A modified UCLA  shoulderThe assessment of postoperative ROM was performed using a standard universal goniometer with the scale marked in one-degree increments. The measurements of supine passive forward elevation , passive internal and external rotation (90° abduction) were obtained using standard measurement . PatientThe mean follow up was 4 + 1 years ranging from 1–10 years. There were 46 male and 54 female, with a mean age of 56.9 + 9.4 (range 29–76 years).Demographic and surgical details of the patients enrolled in the study are shown in Patients underwent brachial plexus block , and were placed in a lateral decubitus position. The arm was suspended at approximately 45° of abduction and 20° of forward flexion. The distraction of the shoulder joint was accomplished with 4.5 to 6.5 kg of traction. Diagnostic arthroscopy was then performed to evaluate the extent of the RC tear, any lesions of the biceps tendon, and other associated lesions. To control bleeding, we used radiofrequency, adrenalin admixture to the irrigation fluid, and asked the anesthesiologist to lower the systolic blood pressure to 90 mm Hg, if possible. An arthroscopic pump maintained fluid pressure at 40 mmHg, increasing it temporarily on demand. A subacromial decompression, consisting of the release of coracoacromial ligament, anteroinferior acromioplasty, and subacromial bursectomy was performed in the presence of a type III acromion, acromial spurs or according to surgeon decision. The lateral portal was used to mobilize the RC back to its bony insertion. Using a burr through the lateral portal, the footprint of the greater tuberosity was abraded. The RCR was performed placing one or two rows of suture anchors double-loaded just in the lateral aspect of the footprint. The number of suture anchors and of suture rows varied with the size of the tear, the type of repair techniques and the surgeon decision. At the end of surgical procedure, a drain was left in situ in all patients, and removed 24 h after surgery.The postoperative rehabilitation regimen was different between the two groups.http://www.moonshoulder.com/impactstudy.html). Isoinertial strengthening and rehabilitation of the RC, deltoid and scapular stabilizers were initiated 10–12 weeks after the operation. Sports activities, heavy manual work, and overhead activities were allowed after the OSS became >20, which occurred 6 months after surgery.Patients in group A  performed passive assisted shoulder stretching exercises with limitation of ROM. Passive exercises allowed for the first 2 weeks from the operation included only passive forward flexion up to a tolerable range, while passive external rotation exercises and active pendulum exercises were restricted. On the other hand, active elbow flexion and extension were allowed. Patients were instructed to perform exercises 5 times a day, with 10 repetitions of each movement each time. Patients were allowed to do this at home, only when they demonstrated to the authors complete autonomy in exercise execution. Exercises were performed by patients at prearranged hours . Each session lasted about 30 min. The rest time was 1 minute. The arm was supported during rest, using a sling with an abduction pillow for 6 weeks. After 2 weeks from the operation, passive external rotation up to 30° ROM and pendulum exercises were introduced. Active assisted exercises and muscle strengthening exercises were introduced after 6 weeks from the operation according to a validated postoperative protocol  followed an aggressive assisted rehabilitation protocol. They had the same protocol of group A except for passive forward flexion, active pendulum exercises, and passive external rotation up to 30° that were allowed from the day after surgery. Overhead stretching was restricted until 6 weeks post-operatively. Isoinertial strengthening and rehabilitation of the RC, deltoid and scapular stabilizers were initiated 10–12 weeks after the operation, as previously described.http://www.moonshoulder.com/impactstudy.html).Rehabilitation was continued for 6 months in both groups according to a validated postoperative protocol  for Mac.t-test for continuous variables and the x2 test for categorical variables. Significance was set at p < 0.05, and the confidence level was 95%.Statistical analysis was performed considering the following outcomes: total modified UCLA shoulder score; total Wolfgang criteria shoulder score; total OSS. We considered also passive and active ROM, strength of anterior elevation, internal rotation, and external rotation ROM. We analyzed the following independent variables: age; sex; arm dominance; history of trauma; dimension of the RC tear; biceps tendon rupture or tendinopathy; type of treatment of biceps tendon; number of anchors used for the RC repair. Comparison between the two groups for each independent variable was carried out with the Student’s No patients reported postoperative complications. We report clinical and functional results at an average follow up of 4 ± 1 years (range: 1–10 years).No statistically significant differences were found in clinical scores between the two groups .p = 0.04), resulting statistically significant  in group A and 156.5° ± 30.5° (range 90–180°) in group B  were not statistically significant.Our study was conducted on 2 groups of patients whose characteristics were homogeneous for all variables considered, confirming that the sample chosen for the study was appropriate. We decided to include only patients with small, medium or large RC tears, excluding patients with massive RC tears, because we believe that clinical and functional outcomes in patients with massive RC tears should be considered separately. Moreover, our selection and recruitment process, our assessment criteria and our follow up were extremely rigorous and performed in a strict scientific fashion. With the number of patients enrolled, the results of our study can be considered univocal.Major strengths of the present study are: (1) two fully trained surgeon (G.R. and F.F.), each with over 25 years of experience, performed all the operation using a well-defined established technique; (2) no patients experienced surgical complications; (3) the follow up evaluations were performed blindly by two independent examiners (U.G.L and S.P.); (4) the evaluation of ROM and strength was performed according to standard measurement guidelines; (5) the mean follow up is long enough to consider that, by then, the results of surgery had stabilized and recovery effected; (6) the number of patients enrolled in the study can guarantee adequate results; (7) patients with massive RC tears were excluded; (8) all the patients were followed by a full-trained physiotherapist during the rehabilitation exercises.However, several limitations of our study must be underlined: (1) we did not obtain a base-line evaluation and a base-line clinical score; (2) we included patients with an RC tear regardless of the presence of subacromial spurs and of the width of the subacromial space; (3) we did not address the issue of single versus double-row repair ,48,49; (In conclusion, within the limitations outlined above, better results were obtained by the patients who received a limited early passive motion rehabilitation protocol, in which passive external rotation ROM, as well as active pendulum exercise, were restricted during the first 2 weeks after surgery, compared to patients who underwent aggressive early passive motion rehabilitation.In this study, two different rehabilitation protocols after arthroscopic RCR for the management of full-thickness RC tears were compared. Better clinical and functional outcomes were obtained by the patients who underwent restricted rehabilitation, in which passive external rotation ROM, as well as active pendulum exercise, were excluded during the first 2 weeks after surgery, compared to patients who received an aggressive early motion rehabilitation protocol including passive external rotation up to 30° and active pendulum exercises. The difference between the two groups resulted in statistically significant concerning active forward flexion ROM. However, no statistically significant differences were found between the two groups concerning the other ROMs analyzed, clinical scores, and muscle strength.