FGF4 retrogene on CFA 12 have opened up new areas of investigation to further define the pathophysiology of premature IVDD. While preliminary data from studies investigating FGF4 retrogenes in IVDD implicate FGF4 overexpression as a major disease factor, they have also highlighted knowledge gaps in our understanding of intervertebral disc herniation which is a complex and multifactorial disease process.Premature degeneration of the intervertebral disc and its association with specific chondrodystrophic dog breeds has been recognized for over a century. Several lines of evidence including disease breed predisposition, studies suggesting heritability of premature intervertebral disc degeneration (IVDD) and association of a dog chromosome 12 (CFA 12) locus with intervertebral disc calcification have strongly supported a genetic component in IVDD in dogs. Recent studies documenting association of IVDD with an overexpressing  The list of inherited neurological disorders in companion and production animals is ever expanding. There are over 120 known genetic variants for neurological disorders in dogs alone . Beyond FGF4 associated with insertion of FGF4 retrogenes on CFA12 and CFA18 appear to have broader influences on limb length across many breeds and are the only genes to have been implicated in body size in across-breed association studies  and has been used to describe extreme differences in limb length in several dog breeds, such as the Basset Hound, Dachshund, and Pekingese . Historiral disc .Fibroblast Growth Factor 4 is most highly expressed in the embryonic ectoderm, axial, paraxial, and lateral plate mesoderm, and tail bud   for Hansen Type I intervertebral disc disease  and polyA tracts (class 1 templated sequence insertion polymorphism) , 3534, 3orphism) .FGF4 retrogenes have been associated with clinical phenotypes in dogs.Retrocopies have historically been considered to be inactive in most cases due to lack of appropriate regulatory elements as well as genetic alterations that remove open reading frames, and documented association with disease states is relatively uncommon. However, retrotransposition is believed to play an important role in genome evolution  and as tFGF4 retrogene is 3,209 bp long (Gen Bank accession no. MF040221) and consists of the parental chromosome 18 FGF4 exons  with common 5′ UTR and exons but a shorter 3′ UTR and is inserted within a LINE element on the parental chromosome 18]. The CFA12 FGF4 retrogene is transcriptionally active resulting in a 20-fold increase in FGF expression in neonatal intervertebral disc from dogs homozygous for the CFA12 FGF4 retrogene insertion compared to disc from neonatal dogs with just the parental FGF4 genes  appeared and produced an obvious phenotype, strong selection was likely applied to retain them.Although we have little ancient historical data documenting IVDD prior to the late nineteenth century , we do kFGF4 retrogenes generally reflects the morphological phenotype and susceptibility to IVDD we recognize in clinical practice (FGF4 retrogene copy (CFA12 or CFA18) tend to have skeletal dysplasia with moderately short limbs, while those carrying both copies, such as Dachshunds, Corgis and Bassett Hounds, have the most severe form of disproportionate dwarfism. The breeds with a higher frequency of the CFA12 FGF4 insertion are the same breeds identified in the last 50 years as being predisposed to IVDD , it does not appear to be directly associated with development of IVDD since several of the highly susceptible short legged breeds carrying the CFA12 FGF4 retrogene such as Beagles, French Bulldogs and Cocker Spaniels do not carry the CFA18 FGF4 retrogene and interestingly contrast with the short legged breeds such as Cairn Terriers and West Highland White Terriers that are rarely reported to have IVDD and carry only the CFA18 FGF4 retrogene . Sunsertion , 42.FGF4 retrogene was present in 40 breeds, the CFA18 FGF4 retrogene in 32 breeds and both retrogenes in 23 breeds  in the general population of breeds such as Beagles, Dachshunds, French Bulldogs, and most spaniel breeds, and not surprisingly, this frequency increases when looking at the population of dogs that present with clinical IVDD . Consistent with the skeletal dysplasia phenotype common to both retrogenes, presence of 1 or 2 copies of either the CFA12 or CFA18 FGF4 retrogenes was also associated with “smaller” dogs (based on body weight).Genotyping of over 3,000 dogs from 75 breeds showed that the CFA12 3 breeds  42). Th. ThFGF4 FGF4 retrogene is associated with a younger age of presentation for decompressive surgery (mean 6.1 vs. 8.5 years) (FGF4 retrogene had a significantly younger age at surgery (median 3.7 years) compared to other breeds homozygous for the CFA12 FGF4 retrogene such as Dachshunds (median age 6.5 years)   42). In. InFGF4 5 years) , 52 thatFGF4 retrogene frequency also described dogs with calcified (Type I) IVD to be significantly smaller (median 8.1 vs. 25 kg) and to have a significantly younger age at presentation for surgery (5.5 vs. 9 years) compared to non-calcified IVD (Type II) dogs  compared to surgically treated animals with fibrous/non-calcified disc herniation/protrusion   dogs . Supporty 0.149) .FGF4 retrogene (84.8%) compared to 1 copy of the CFA12 FGF4 retrogene (63.8%) compared to zero copies (18.5%)  . Multivao 1 copy . Presencfication .FGF4 retrogene location on chromosome 12 was done using high vs. low radiographic calcification scores in Dachshunds  Dachshunds. This population would be necessary for a successful genome mapping study and is consistent with the CFA12 FGF4 retrogene dosage effects on radiographically apparent calcification subsequently demonstrated following identification of the CFA12 retrogene , relative risk for IVDD  associated with the CFA12 FGF4 retrogene ranged from 5.5 in Chihuahuas to 15.1 in mixed breed dogs.Prospective data determining the risk for IVDD associated with presence of the CFA12 erformed . LookingFGF4 retrogene. Clarification of these pathophysiological and clinical variables in the presentation of IVDD is important if genotyping is to be used as a tool for reduction in disease incidence. A more comprehensive picture will also increase breeder and owner confidence for eradication strategies that may potentially require major alterations in breed standards for those breeds with high allele frequencies.Several aspects of the clinical presentation of IVDD in dogs are not easily explained by a simple presence or absence of the CFA12 FGF4 retrogene allele frequency  as the major determinant of ratio changes. However, several genetic factors are likely to be contributing to conformation including CFA12 and 18 FGF4 retrogenes. Clinically affected Dachshunds were reported to have longer epaxial muscle fascicle lengths in one study, which could be a secondary effect of clinical IVDD; however, it is interesting to note that FGF4 is also a key signaling molecule in the development of myofibers and tendon of epaxial muscles arising from somitic mesoderm  length were associated with increased risk in one study  while a mesoderm , 68.FGF4 retrogene status of dogs within the Standard Wire Haired population which can be inferred from the successful GWAS mapping using this Dachshund subtype (FGF4 retrogene (FGF5 gene (likely loss of function based on recessive inheritance)  , 70. Altng limbs , 71. Redng limbs  and it iPO2 gene . RSPO2 sPO2 gene , an impoPO2 gene  and downPO2 gene .FGF4 retrogene presence and other factors) based on breed has been reported  gene associated with screw tail and brachycephaly  would provide further insight into the relative effects of the two retrogenes.Co-expression of the CFA12 and CFA18 e modest , potentie modest . The CFA the IVD  althoughFGF4 retrogenes documented to date may facilitate its expression in other chromosomal locations as well. This provokes the question of how many other times the FGF4 gene has been retrotransposed and then eliminated by selective breeding, or if additional retrocopies remain in some breeds and are responsible for other morphological phenotypes or contribute to IVDD.The location of a large CpG island in the two FGF4 retrogene were found in 12% (46/378) of dogs presenting for IVDD related surgery and with documented presence of calcified intervertebral discs  are defined, may have specific molecular mechanistic similarities to FGF4 overexpression and IVDD  in dogs. However, less definitive data are available regarding genetics of IVDD and associated low back pain and sciatica in the general human population. Heritability of risk factors for IVDD in humans was initially established using twin studies  may sometimes conflict with dog breeding phenotypic goals.The most critical unanswered question relating to the CFA12 FGF4 retrogene has been seen in almost all IVDD affected breeds, even those with high allele frequencies. In breeds with a high degree of segregation it should be possible to reduce or eliminate the retrogene from the population.Positively, some degree of segregation of the CFA12 FGF4 retrogenes. In high IVDD risk breeds such as Dachshunds, Bassets, Corgis and Pekingese that have both the CFA12 and CFA18 FGF4 retrogene, breeding away from the CFA12 FGF4 retrogene, while still maintaining the aesthetically desirable shortness in stature contributed by the CFA18 FGF4 retrogene is possible.Even in high allele frequency breeds, such as the Dachshund, there are frequency differences between populations [0.98 in USA/UK samples and 0.94 in Swiss samples ], indicaFGF4 retrogene, so that the allele frequency can be reduced.The dominant nature of the CFA12 FGF4 retrogene on IVDD and the very high allele frequency in some breeds means that eradication may be challenging. The Wire Haired Dachshund was developed through crossbreeding with Schnauzer and Terrier breeds which may explain the lower frequency of the CFA12 susceptibility locus and lower incidence of IVDD in some wire haired populations. Long term strategies may require a combination of testing and selection of heterozygous dogs, outbreeding, cross breeding, and alteration in breed standards, maybe with inclusion of additional CFA12 FGF4 retrogene-negative phenotypes within the breed standards. Whatever the future holds, from a veterinary perspective, current data suggest that breeding priorities should be for dogs with fewer copies of the CFA12 All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


The results showed that excess Na addition allowed us to compensate evaporation of sodium during the synthesis and sintering. Er2O3 was added as sintering aid to limit abnormal grain growth caused by h–BN addition. The modification of amount of Na and Er2O3 addition resulted in high purity KNN samples with tetragonal structure and apparent density higher than 97%. Finally, piezoelectric features of prepared dense samples were measured and presented.Analysis of dense Potassium Sodium Niobate (KNN) ceramic obtained by hot pressing (HP) method at 1100 °C are presented in this paper. The synthesis of KNN-based piezoelectrics meets the following challenges—low density of material, uncontrolled K/Na ratio, multiphase composition and formation of different KNN structures. The classical hot pressing approach results in contamination by carbon originating from graphite molds. The proposed hexagonal Boron Carbide (h-BN) layer between green sample and graphite mold could protect samples from carbon contamination. Additionally, the presence of h-BN may decrease the formation of oxygen vacancies, which allows us to maintain the semiconductor features of the KNN structure. Remaining issues were addressed with the addition of excess Na and Er T3. Its specific positioning combined with proper compositions stands for many functional properties, including ferromagnetism or thermoelectricity. KNN materials consist of two phases: ferroelectric potassium niobate (K1−xNbO3) and antiferroelectric sodium niobate (NaxNbO3) which differ in transition but crystallize in the almost the same perovskite structure. The basic transition dependency of K/Na ratio results in possibility of obtaining functional perovskite structure with outstanding piezoelectric properties, given that mentioned stoichiometric proportions are preserved, because of morphotropic phase boundary (MPB) that it creates in the tetragonal-rhombohedral transition [Ferroelectric materials, such as described above (except for Polyvinylidene Fluoride-PVDF), used as piezoelectrics have the crystalline structure of perovskite, which is commonly known as a structure similar to BaTiOansition .The main challenge of implementing KNN lead-free piezoelectric materials is the proper synthesis. While material can be relatively simply obtained by high temperature processing of uniaxially pressed samples, its expected properties could be decreased due to the small density of samples. Amongst possible reasons of this situation, most possible are: evaporation of sodium, alkali vacancies created during sintering and lack of liquid phase in the microstructure of pure KNN ,8,9.2 and Ar atmospheres respectively showed, that similar improvements of properties can be obtained by changing the environment [In face of those problems, a proper solutions were pursued by many researching teams experimenting with both environment and composition. Shimizu et al. showed that pure alkali niobate ferroelectric and dielectric properties can be significantly improved using only low oxygen partial pressure processes . Those sironment . Moreoveironment .0.5Nb0.5)O3 (CSN), it was shown, that sealed ceramic samples were denser that their unsealed counterparts. The overall improvement of electrical properties was also noted [Yang et al., while obtaining transparent lead-free ferroelectric ceramics, went one step further, employing sealed sintering process and comparing it to sintering in air. Although used KNN materials were differing due to the doping of Ca(Scso noted . Additioso noted .0.44Na0.52Li0.04)(Nb0.86Ta0.10Sb0.04)O3 in which lithium subtracts either potassium or sodium atoms, while alkaline earth metals are in niobium positions [33) with relatively high conversion ratios. Due to those findings, later iterations of solution were to follow [In addition to process changes, various dopants were reported to improve KNN synthesis processes and were considered as a possible solution to this problem. The simplest idea of doping with alkaline earth metals was proposed by Malič et al. but was generally lacking significant, if any, positive effect on densification of samples . Previouositions  and resuo follow ,18, showo follow .3+ substitution in perovskite structure could reduce coupling factor of materials but greatly improve its other piezoelectric properties [2O3 which was investigated by Zhao et al. and shown that it could result in creation of highly desired liquid phase during sintering [Another chance of improvement came with the involvement of rare earth metals oxides. Those materials were expected to improve samples, based on previous research, performed to improve PZT material systems. Those studies showed that Yboperties . In caseoperties  alongsidintering .2O3, were employed. In comparison to previous research [The aim of this study was to obtain a dense KNN-based material, using industrially applicable process, using the listed findings and preliminary studies performed prior to this paper, which would be tested in stress sensor. To achieve this task, dopants of rare-earth elements, namely Erresearch , processresearch . The tex0.5Na0.5NbO3), the following commercial reactants were used—Nb2O5 powder of CHANGSHA EASCHEM CO. Ltd.  and K2CO3, Na2CO3 carbonate powders produced by Lach-Ner . In the first step of KNN synthesis, the carbonate reactants were annealed at 150 °C for 24 h in order to remove residual water. Then K2CO3, Na2CO3 and Nb2O5 powders with 5 wt.% and 10 wt.% excess of sodium carbonate were mixed in ceramic mortar. The extra amount of Na2CO3 was added to compensate sodium evaporation process during synthesis. Thus prepared powders were dry homogenized in ball mill using 10.0 mm diameter silicon nitride milling media for 24 h. The reactants mixture were placed into alumina crucible with lid and heated up in air atmosphere to temperature of 950 °C, in which they were annealed for 2 h. The crushed and grinded KNN reaction bed was analyzed by X-ray diffraction method. The qualitative and quantitative X-ray Diffraction (XRD)/Rietveld phase composition analysis was made using PANalytical (model) diffractometer , equipped with Cu anode supported by X-pert HighScore software  for quantitative analysis of the samples.To synthesize KNN . having 0.65 m2/g specific surface area, 99.5% purity and 8.69 g/cm3 helium density. Due to company information average grain size of powder was estimated to d50 = 1.4 µm. The powder batches were dry homogenized in rotary mill using 5.0 mm Si3N4 grinding media for 24 h. Then mixtures were screened by 0.5 mm mesh sieve and placed in 25.0 mm diameter hot pressing graphite molds. To prevent excessive contamination of green samples by carbon, the graphite parts of the mold were covered by graphite foil and next it was sprayed by hexagonal boron nitride (h-BN) powder, as schematically showed in The obtained KNN powders were then mixed with 1 wt.% and 2 wt.% of Er2CO3 extra addition was manufactured in order to check: density, microstructure formation and phase composition. Cooling rate of HP process for those materials was very low of 3 °C/min. to reduce internal stresses caused by phase transformation and polycrystal formation during heat treatment.Prepared powders were hot pressed (HP) under 25 MPa of pressure for 2 h at 1100 °C in argon flow with heating/cooling rate of 10 °C/min and 8 °C/min. respectively. The applied pressure accelerated sintering, polycrystal densification and reduced evaporation of sodium. The obtained dense KNN sinters had diameter of 25.0 mm and thickness of 5.0 mm. Additionally, polycrystalline KNN reference sample without sintering activator and Na2O3 for O Kα line, Er2O3 for Er Mα and Er Lα lines. During calculations pressure variation method based on gas compensated technique was used. In order to confirm influence of HP applied pressure on material texturing, the reference sample was taken for ultrasonic measurement of longitudinal wave propagation in parallel and perpendicular directions to pressing axis. The ultrasonic examination was made by UZP-1  apparatus equipped with 1 MHz heads. In order to measure piezoelectric signal generated by samples in simulated real conditions, selected materials were subjected to poling process in custom built apparatus for 1 h in 2 kV/mm in silicon oil environment. It was followed by measurement of electric signal generated by piezoelectric properties of previously poled samples subjected to repetitive cyclic compression by custom built apparatus with force 150 N and 200 N for 2000 cycles. This equipment allows us to apply repetitive cyclic compression to rectangular or circular samples with set load between 100 to 1000 N. The analysis of these signals was performed by DasyLab 2016 software  in order to minimize distortions during measurements.The water contact with the sample is harmful due to the hygroscopic feature of KNN, for this reason, all samples handling was carefully proceeded in dry and no moisture conditions. The density measurements of KNN polycrystals was conducted in helium pycnometer AccuPyc II 1340 produced by Micrometrics Company . An average density value was calculated based on 30 measurement points. For microscopic observation purposes the sintered samples were included in resin and then polished by Struers polisher TegraPol-21 equipped with TegraForce-5 grinding discs under set load of 35 N and rotation speed of 300 rpm. The process was carried out in isopropanol environment. Surface preparation was done by diamond discs followed by final polishing with 0.25 um colloidal silica, which enabled mechanical etching of polycrystal to reveal microstructure details. Scanning electron microscope (SEM) FEI NOVA NANO SEM 200  was used for preliminary observations. Fracture morphology was recorded by use of secondary electrons (SE), while polished samples were observed in backscatter electron (BSE) mode. Detailed microstructural examinations were performed by use of scanning electron microscope FEI QUANTA 3D FEG SEM  equipped with energy dispersive spectrometer (EDS) . The applied energy of the electron beam was 15 keV (sufficient enough to excite the maximum intensity of Er Lα line) and beam current of 11 nA. Microstructure observations and microbeam analysis was performed in low vacuum conditions. Carbon dioxide gas was used to eliminate any reaction of the KNN with water vapor, which is the default gas in Variable Pressure SEM measurements. ZAF correction procedure was used for calculations. The following standards have been used for quantitative analysis: NaCl crystal for Na Kα line, KBr for K Kα line, pure niobium for Nb Lα line, Al4Nb10O30 in quantity of 83.5 wt.% and NaNbO3 in quantity of 11.5 wt.%, while planned stoichiometric KxNa1−xNbO3 existed as minor phase in quantity of approximately 5.0 wt.%. That may be caused by deficiency of sodium in the system, due to evaporation occurred at both synthesis and hot-pressing steps. The X-ray diffraction patterns obtained for reference sample revealed presence of two major phases: K3 measured by helium method on the polycrystalline grinded sample. Then, the material was examined by SEM. Morphological features of KNN fracture are presented in Relative density of manufactured reference material was estimated as 98.4% based on the theoretical one of 4.55 g/cmThe ultrasonic analysis of longitudinal wave propagation examined in perpendicular directions of the reference sinter confirms texturing influence of HP process, what is visible in the wave propagation anisotropy reaching 25%. The measurement directions and wave velocity are collected in 0.5Na0.5NbO3, Er2O3 sintering activator was added because of its positive effect in formation of both orthorhombic and tetragonal structures which enhance piezoelectric properties of obtained sinters [2CO3 and addition of 1 wt.% or 2 wt.% of Er2O3 were subjected to density measurements with results collected in 3, which confirmed that all of samples have more than 97% of relative density which is enough for piezoelectric generator manufacturing. This suggests that material may be strengthened because of low concentration of microstructure defects. Complete density data is presented in In order to obtain materials containing K sinters . The hot2CO3 and Er2O3 sintering aid allowed us to obtain almost pure KNN material in the tetragonal crystallographic structure. The increase of Er2O3 from 1 wt.% to 2 wt.% content and excess of Na2CO3 addition from 5 wt.% to 10 wt.% led to orthorhombic structure of K0.5Na0.5NbO3 and small quantities of KNN doped by erbium. However, the same mixture with both additions of 10 wt.% of Na2CO3 and 2% Er2O3 resulted in shift to orthorhombic structure. Lattice parameters of detected samples are gathered in The results of quantitative and qualitative XRD analysis presented in 2O3 is presented in The obtained KNN sinters were subjected to microstructural and chemical analysis by SEM-EDS method. The material fracture with addition of 1 wt.% of Er2CO3 resulted in non-stoichiometric composition of KNN in some parts of the material. The nucleation rate of fine grains was previously confirmed to be enhanced by the excess amount of Na2CO3 introduced to the system [Results of average elemental composition measured by EDS technique for KNN taken from two randomly chosen flat areas of the polished N5E1 sample are collected in e system .2CO3 excess from 5 wt.% to 10 wt.% resulted in a much more uniform fractures morphology as shown in 2O3 from 1 wt.% to 2 wt.%. Based on the results of our previous work [xNa1−xNbO3 were formed due to higher sodium content, which was enhanced by the excess amount of Na2CO3 introduced to the system.The N5E1 polycrystal is characterized by some elongated and geometrically oriented grains as marked in ous work , it can 2CO3 exhibited much lower values. This proves that small overabundance of Na in materials can be beneficial for piezoelectric properties but must be carefully optimized.Microstructure of sample N5E1 should have the best piezoelectric properties from all prepared samples thanks to its tetragonal crystallographic structure as shown in 2CO3 added to powder mixtures have a positive influence on maintaining the desired tetragonal KxNa1−xNbO3 stoichiometry. Material with 5 wt.% excess of Na2CO3 and 1 wt.% of Er2O3 (N5E1 sample) which possess domain microstructure, generated highest electric signal of all samples for both 150 N and 200 N load cyclic compression. Combination of 10 wt.% Na2CO3 and 2 wt.% Er2O3 (N10E2 sample) additions led to extensive formation of monoclinic KNN with low piezoelectric properties. The hot pressing process applied in KNN manufacturing stimulated texturing of microstructure, which was confirmed by ultrasonic measurements indicating anisotropy of properties. In case of reference sample, an addition of hexagonal boron nitride leads to sintering intensification and extensive grain growth up to 50 µm. The experiment proved that it is possible to obtain KNN polycrystalline samples characterized by large dimensions. The diameter of obtained sinters was of 25 mm with thickness of 5 mm. The main phases detected were either orthorhombic or tetragonal KNN. Obtained sinters generated relatively high piezoelectric signal under cyclic compression.The layer of h-BN on green samples was initially introduced as protective layer to prevent the excessive carbon contamination during hot pressing. Morphology of the samples in the areas close to sample surface showed a formation of much larger grains comparing to the grains in the central area of samples, which could result from limited diffusion of boron inside the samples during hot pressing. The excess amount of Na


Scientific Reports 10.1038/srep23220, published online 21 March 2016Correction to: This Article contains an error in Figure 2. During the final preparation of the figures prior to publication, the wrong version of Figure 2 containing an incorrect graph legend was used. The correct Figure 2 appears below as Figure


Facioscapulohumeral muscular dystrophy (FSHD) is a myopathy with prevalence of 1 in 20,000. Almost all patients affected by FSHD carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Assessment of size of D4Z4 alleles is commonly used for FSHD diagnosis. However, the extended molecular testing has expanded the spectrum of clinical phenotypes. In particular, D4Z4 alleles with 9–10 repeat have been found in healthy individuals, in subjects with FSHD or affected by other myopathies. These findings weakened the strict relationship between observed phenotypes and their underlying genotypes, complicating the interpretation of molecular findings for diagnosis and genetic counseling. In light of the wide clinical variability detected in carriers of D4Z4 alleles with 9–10 repeats, we applied a standardized methodology, the Comprehensive Clinical Evaluation Form (CCEF), to describe and characterize the phenotype of 244 individuals carrying D4Z4 alleles with 9–10 repeats (134 index cases and 110 relatives). The study shows that 54.5% of index cases display a classical FSHD phenotype with typical facial and scapular muscle weakness, whereas 20.1% present incomplete phenotype with facial weakness or scapular girdle weakness, 6.7% display minor signs such as winged scapula or hyperCKemia, without functional motor impairment, and 18.7% of index cases show more complex phenotypes with atypical clinical features. Family studies revealed that 70.9% of relatives carrying 9–10 D4Z4 reduced alleles has no motor impairment, whereas a few relatives (10.0%) display a classical FSHD phenotype. Importantly all relatives of index cases with no FSHD phenotype were healthy carriers. These data establish the low penetrance of D4Z4 alleles with 9–10 repeats. We recommend the use of CCEF for the standardized clinical assessment integrated by family studies and further molecular investigation for appropriate diagnosis and genetic counseling. Especially in presence of atypical phenotypes and/or sporadic cases with all healthy relatives is not possible to perform conclusive diagnosis of FSHD, but all these cases need further studies for a proper diagnosis, to search novel causative genetic defects or investigate environmental factors or co-morbidities that may trigger the pathogenic process. These evidences are also fundamental for the stratification of patients eligible for clinical trials. Our work reinforces the value of large genotype–phenotype studies to define criteria for clinical practice and genetic counseling in rare diseases. The disease is characterized by a peculiar distribution of muscle weakness affecting facial and shoulder girdle muscles. Abdominal muscles also become affected, leading to a characteristic hyperlordotic posture. The selective and precocious weakness of tibialis anterior muscle is typical at lower limb, eventually followed by proximal leg muscles involvement4.Facioscapulohumeral muscular dystrophy  has prevalence of one in 8–20,0005. Two genetically distinct disease subtypes, FSHD1 and FSHD2, have been described up to now6. FSHD1 is associated with contractions of a polymorphic macrosatellite repeat on chromosome 4q35.27. This region consists of tandemly arrayed 3.3 kb D4Z4 repeat elements ranging from 11 to > 100 repeat units in healthy subjects. Individuals displaying FSHD symptoms and carrying D4Z4 alleles with 10 or fewer repeat units are genetically defined as FSHD18. They represent 95% of people with FSHD.Presently, FSHD diagnosis is based on molecular findingshttp://www.musclegenetable.fr). FSHD1 and FSHD2, considered clinically undistinguishable, are characterized by DNA hypomethylation of the 4q35 D4Z4 array9.FSHD2 defines 5–10% of affected individuals carrying two D4Z4 arrays in the healthy range (> 10 repeat units). FSHD2 is defined as the cause of SMCHD1 mutation in OMIM and Gene Table  have been recently added to the list of possible contributors to disease onset and modifiers of disease severity, and also to explain a digenic inheritance for FSHD25; even though more extended analysis suggest the limited contribution of these factors27. This clinical and molecular complexity has complicated FSHD diagnosis, clinical practice and genetic counseling. Based on the above considerations, how can we interpret and use the result of the FSHD molecular test?FSHD is characterized by reduced penetrance and wide variability in the clinical expression among patients and within families28, making the definition of a clear cut-off point problematic.Here, we investigate the occurrence of the FSHD classical phenotype in 134 index cases and 110 relatives carrying alleles with 9–10 D4Z4 repeat units from the Italian National Registry for FSHD (INRF). Alleles of this size, named borderline D4Z4 reduced alleles (bDRA), are considered the upper size of the diagnostic range and pose the major diagnostic challenges. In fact, a wide phenotypic spectrum including atypical or incomplete phenotypes with no affected relatives is frequently observed in subjects carrying a bDRAwww.fshd.it)29. Out of 1340 index cases bearing a DRA with 1–10 repeats, we identified 166 subjects (14.6%) carrying a bDRA . Clinical and molecular analysis was extended to all available and willing to participate relatives. Phenotypic characterization was performed on 134 index cases and 110 relatives carrying a bDRA from 58 unrelated families. Seventy-six index cases did not have available relatives carrying a bDRA .In this study, we enrolled subjects carrying a bDRA. Index cases were identified through the INRF considering a 9-year time-window (2008–2016). The INRF database contains clinical and molecular data of subjects examined by the Italian Clinical Network for FSHD 31. Subjects were further classified on the basis of the clinical signs considering typical and atypical features, as listed in the CCEF : (1) subjects presenting facial and scapular girdle muscle weakness typical of FSHD , (2) subjects with muscle weakness limited to scapular girdle or facial muscles , (3) asymptomatic without any functional motor impairment (FSHD score 0) or healthy subjects , (4) subjects with myopathic phenotype presenting clinical features not consistent with FSHD canonical phenotype . Age at onset was estimated as self-reported anamnestic record32.We used the Comprehensive Clinical Evaluation Form (CCEF) for the clinical characterization of each subjectEcoRI, EcoRI/BlnI. Digested DNA was separated by pulsed field gel electrophoresis (PFGE) in 1% agarose gels, as previously described18. Allele sizes were estimated by Southern hybridization with probe p13E-11 of 7 μg of EcoRI-digested, EcoRI/BlnI-digested genomic DNA extracted from peripheral blood lymphocytes, electrophoresed in a 0.4% agarose gel, for 62–64 h at 35 V, alongside an 8–48 kb marker (Bio-Rad). Participants carrying alleles of 36–41 kb (9–10 D4Z4 units) in size were included in the study. Within the European population, pathological D4Z4 contractions usually are associated with the permissive 4qA haplotype in the subtelomeric region of chromosome 4q. The qA polymorphism was assessed by HindIII digestion and hybridization with qA probe. Restriction fragments were detected by autoradiography or by using the Typhoon Trio system . To verify that the obtained shortened D4Z4 fragment on chromosome 4 has a causative 4qA haplotype, an additional HindIII Southern blot was performed as suggested for the molecular diagnosis of FSHD133.DNA was prepared from isolated lymphocytes according to standard procedures. In brief, restriction endonuclease digestion of DNA was performed in agarose plugs with the appropriate restriction enzyme: t test or ANOVA.We used descriptive statistics for quantitative variables (mean and standard deviation) and qualitative variables (relative frequencies). Associations between qualitative variables were assessed using chi-square test or Fisher exact test. Associations between clinical parameters were described and tested using Pearson r correlation coefficient. Associations between quantitative variables and qualitative variables were evaluated using The INRF database was approved by the Provincial Ethics Committee of Modena (2712/CE). Informed written consent was obtained from all study participants, in accordance with the ethical standards of the 1964 Declaration of Helsinki.This manuscript does not contain any individual person’s data in any form. Each patient was identified by a unique alphanumeric identification code and all data were made anonymous.The clinical characterization of 134 index cases carrying a bDRA  in all patients falling in categories A, B, or D. We therefore evaluated whether clinical categories may represent different stages of disease progression that is pre-symptomatic carriers are included in category C, mildly affected individuals are in category B and fully manifesting subjects are in category A. If this is the case one would expect category C subjects to be the youngest and category A the oldest. Figure Among the 73 index cases classified as category A, 2 presented severe facial weakness and were classified as subcategory A1, 25 showed the classical pattern of facial weakness (subcategory A2), 46 presented mild facial involvement (subcategory A3) . Thus, subcategory B1 patients have a milder clinical phenotype.Facial-sparing scapular myopathy is often detected in clinical practice and it must be distinguished from other forms of myopathy including scapular peroneal syndromeThree index cases, aged 31, 52 and 70 years, were classified as subcategory B2 showing facial weakness without scapular girdle involvement. They presented a mild motor impairment, FSHD score 1, 1, 4 respectively. Two of them had isolated weakness of facial muscles.Out of 134 index cases, 25 index cases (18.7%) were classified as category D. Sixteen patients were identified as subcategory D1 for the presence of additional atypical features, more frequently including prevalent pelvic girdle weakness and axial involvement with bent spine and dropped head. Nine index cases (6.7%) presented phenotypes inconsistent with FSHD, such as isolated axial weakness (i.e. bent spine syndrome), or other clinical conditions described in Table There was also the case of a woman  from 58 unrelated families  as reported in Supplementary Table 1.We then analyzed whether the clinical category of the index case can predict the phenotype observed in the relatives carrying the same bDRA. We subdivided the probands in two groups on the basis of the CCEF phenotype observed in their relatives. One group included 19 probands whose relatives had classical FSHD or incomplete phenotype (categories A and B); the other group included 39 probands whose relatives were healthy or had a complex phenotype (categories C and D). This analysis shows that the distribution of clinical categories among the 58 probands subdivided on the basis of the phenotypic categories of their relatives does not significantly differ . Cases are distributed on the basis of the size of D4Z4 alleles reported as number of Repeat Units and the correspondent molecular weight as kilobases(kb).Supplementary Figure 2. Selection of index cases and their relatives carrying a bDRA for genotype–phenotype correlation analysis.Supplementary Figure 3. Clinical categories of CCEF (from reference ).Supplementary Table 1. Distribution of probands on the basis of the clinical phenotype of relatives.


An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.Cytoplasmic calcium ([Ca Because Ca2+ acts as a stress signal, it is one of the first responses plants have to environmental stimuli. This fast response is achieved through an influx of extracellular Ca2+ and an efflux of stored Ca2+ from the vacuole, endoplasmic reticulum (ER), and mitochondria that is achieved via a concentration gradient between the intracellular Ca2+ stores and the cytoplasm ..2+-bindiM domain .2+ sensors in plants are driven by constitutive promoters such as Cauliflower Mosaic Virus 35S (35S) and Ubiquitin 10 (UBQ10) [2+ sensors enabled the functional study of [Ca2+]cyt changes across entire plant organs [2+]cyt signature. While [Ca2+]cyt changes typically observed in the entire plant organ likely originate from more than one cell type, we lack tools to monitor these changes within specific tissues or cells cyt changes in roots treated with chemicals previously shown to induce [Ca2+]cyt increases in plants.To date, expression of CGaMP and other genetically-encoded Ca (UBQ10) ,13. Altht organs , it is lor cells . To addrromoters . We demo2+]cyt sensors that are available to the scientific community are driven by constitutive promoters. A. thaliana lines expressing the UBQ10:GCaMP3 construct, which we generated and used previously to study [Ca2+]cyt oscillations in root hairs   solution without ATP to roots. We found that applying MS solution triggered a rise in [Ca2+]cyt that was delayed and lower in amplitude compared to the [Ca2+]cyt change caused by ATP  . In mamm2+]cyt . While p [2+]cyt ,26,27. C decline A 5,25].,25.2+]cyd by ATP A. This oresponse ,20,27,282+]cyt signatures. For example, the PIN2:GCaMP3 lines, which expressed GCaMP3 in the epidermis and cortex , NaCl treatment resulted in cell-specific [Ca2+]cyt transients in the early elongation zone [UBQ10:GCaMP3 exhibited late-onset [Ca2+]cyt transients  in cells of the elongation zone  using freshly prepared MS plates for planting seeds and (2) pretreating roots two times with MS solution prior to application of the actual chemical solution. These modifications in our treatment protocols were tested using ATP which typically generated peaks with the highest amplitude.As shown in the preceding sections, the 0.5X MS control solution induced [Cad cortex . Althoug2+]cyt transients triggered by the MS solution were significantly dampened when fresh plates were used for the experiments . Under these modified growth and pretreatment conditions, the ATP-induced [Ca2+]cyt increases were lower in amplitude when compared to the [Ca2+]cyt increases shown in 2+]cyt transients, these [Ca2+]cyt responses were better separated from the [Ca2+]cyt responses induced by MS solution, particularly in lines expressing UBQ10:GCaMP3 and those in which GCaMP3 was expressed in root surface cell types and the cortex , and analyzed the relationship between chemicals using Analysis of Variance (ANOVA) with Tukey Post-Hoc test (3+ showed similar responses (except ATHB8:GCaMP3 to Al3+) while adapted ATP:MS showed significantly slower reaction times to both ATP and glutamate  [2+]cyt transients when compared to UBQ10:GCaMP3 lines , is required for root growth recovery after encountering high salinity conditions cyt transients in the cortex revealed by the PEP:GCaMP3-expressing lines could be a mechanism to maintain root growth integrity. The cell type GCaMP3 lines described here should be useful in testing this hypothesis.The line that was most similar to . (2010)  . The [Ca2+]cyt transients observed after MS treatment were reminiscent of touch-induced [Ca2+]cyt responses [2+]cyt signaling [2+]cyt imaging involved planting seed of the reporter lines on a thin layer of agarose to secure the root while at the same time allowing for the root cells to be accessible to the various chemical treatments  (http://neb.com) and cloned into a modified pCAMBIA1390 vector cyt-dependent GCaMP3 fluorescence, images of growing roots were acquired every 1 s for 10 to 15 min using the Leica SP8-X confocal microscope. Images were captured at a scanning speed of 600 MHz and pixel resolution of 512 × 300. Baseline GCaMP3 fluorescence of the roots was first acquired for 1 min after placing seedlings on the stage of the microscope. Twenty µL of the treatment solution was then added on top of the agarose medium where the root was growing using an adjustable volume pipette while imaging continued. From the collected images, the average fluorescence intensity was acquired by marking a rectangular region of interest corresponding to the specific cell type using the rectangular selection tool of the SPX-8 LAS software. GCaMP3 fluorescence (F) values were normalized using the formula To measure [Caonstruct . Another


S1 TableMeasurements were done on 3, 4 and 5 days-old seedlings. The presented values are the mean of twelve replicates per accession and treatment.(XLS)Click here for additional data file.S2 TableValues were obtained by dividing the RGR presented in (XLS)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableThe threshold used to declare these SNPs significant is 5% Bonferroni. If the gene was found in the respective analysis, it is denoted with a 1 in the table, where a value of 0 indicates no significant association.(XLSX)Click here for additional data file.


Figure 3 as published. Parts A and B were labeled in the incorrect order, the order of these panels within the figure was altered during reviewer responses and the figure legend was not updated correctly in the process. The correct legend appears below.In the original article, there was a mistake in the legend for Figure 3. (A) MCAv was determined continuously by transcranial doppler ultrasound (TCD) and presented as change in MCAv from rest in each protocol for each participant. (B) PETCO2 was determined by measurement of breath-by-breath respiratory gas exchange and ventilation. Data were averaged for 1 min during seated rest (Rest); over 30 s during the final minute of four 4 min high intensity (85% HRmax) interval bouts ; across the duration of four 30 s supramaximal (200% Wmax) sprint intervals , and over a 30 s period, 15 s into the recovery following each bout in both interval protocols (Reco 1 – 4). Data are presented as mean ± SD (n = 24). Significance  between time points is denoted by * for differences from resting values;€ for differences between interval (ex) and recovery (reco). Significant differences between protocols are denoted by Ω.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


The authors issue this Correction to address the following issues that were noted after this article  was publIn Questions were raised about the lack of loading control data reported in  for the As demonstrated in Fig 2A and For Figs 2B-2D, different amounts of purified iNOSfl proteins were loaded in the plus versus minus CaM lanes so as to obtain approximately equivalent iNOSfl levels (130 KD bands) across lanes within each gel or blot. Loading volumes needed to achieve equivalent iNOSfl levels and were determined through standardization experiments for which data were not reported in the article. The iNOS blots in Fig 2B and 2D served as the controls for the CaM blot in Fig 2B and the heme gel in Fig 2C, respectively. For each of these experiments, duplicate gels were prepared for the control and experimental assays by loading the same purified iNOSfl proteins on parallel gels.In The article’s Data Availability statement says, “All relevant data are within the paper.” The underlying data were not included with  but are PLOS ONE’s Editorial Board reviewed the updated figures, supporting data files, and information provided above regarding western blot controls and advised that they address the previous concerns and errors in the original manuscript. The Academic Editor commented that the corrections do not change the article’s conclusions.A member of The authors apologize for the errors in the published article.S1 File(PPT)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 FileThe blot image provided for Fig 2D is a digital image of the original blot reported in the published figure; levels were adjusted in the image file so that band intensities would align approximately with bands observed by the Ponceau S staining. The original film from the Fig 2D blot experiment is no longer available.(ZIP)Click here for additional data file.S4 File(TIF)Click here for additional data file.S5 File(JPG)Click here for additional data file.S6 File(ZIP)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(ZIP)Click here for additional data file.S9 File(ZIP)Click here for additional data file.


Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity. Published 12, October 2018We recently discovered an error in the original Figure 3A, in which the RIP quantification figure in Suz12 was accidentally duplicated from Ezh2. This occurred inadvertently during the conversion of histogram plot to dot format with mislabeled file name of raw data. The figure has been corrected so that the corrected Figure 3A now reflects the correct quantification. The correction does not affect or change any of the conclusions of the manuscript. We apologize for the mistake and any inconvenience or confusion this error may have caused:Corrected Figure 3Original Figure 3The article has been corrected accordingly.


KCNE1B blurs the genetic basis of KCNE1-LQTS patients” [KCNE1B, absent from the GRCh37 version of the human genome, is an artifact introduced in GRCh38:KCNE1 (T) is different from the reference nucleotide for the paralogue position of the KCNE1B pseudogene (C), according to GRCh38  capture panel [KCNE1 SNV, while the mother is wild type. PCR was performed using primers that, according to GRCh38, should not be able to distinguish between KCNE1 and KCNE1B , the mother is wild type and, therefore, the daughter could not possibly inherit two variant alleles from her parents  and homozygous KCNE1 reference (C/C) genotypes for rs1805128, with no sign of the reference KCNE1B (T) allele (Supplementary Table We have PCR amplified the regions containing the positions for rs1805127 and rs1805128 SNPs on DNA from lymphoblastoid cell lines from ten individuals representing ten different ethnic origins from four continents in the same conditions unable to distinguish In regard to the Viewpoint by Pantou et al. “The potential presence of the highly similar paralogue gene KCNE1B is an artifact that should be reviewed and curated. This would reduce the probabilities of false negative results caused by misalignment of variant reads originating from the true and clinically relevant KCNE1 locus to the KCNE1B region of the GRCh38 reference genome.In our opinion, these data, together with those presented by Pantou et al., strongly suggest that Supplementary Materials and MethodsSupplementary Table 1Supplementary Table 2Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3


Scanning tunnelling microscopy is used to compare the properties of PTCDA molecules adsorbed on a partly CaF1-covered Si(111) surface with deposition on thicker CaF2/CaF1/Si(111) films. The identification of mostly single molecules on the CaF1/Si(111) interface layer is explained by the presence of atomic-size defects within this layer. Geometry-optimisation calculations using density functional theory reveal a geometry on CaF2(111) of nearly flat-lying PTCDA molecules with two oxygen atoms displaced towards calcium surface ions. This geometry is in agreement with the experimental observations.Thin insulating films are commonly employed for the electronic decoupling of molecules as they enable a preservation of the intrinsic molecular electronic functionality. Here, the molecular properties of 3,4,9,10-perylene tetracarboxylic dianhydride (PTCDA) adsorbed on insulating CaF The study of molecular adsorption on thin insulating films is motivated by the possibility to investigate and utilise molecular properties in their largely undisturbed state . MoleculA particularly well-studied case of surface-specific molecular properties is the adsorption of 3,4,9,10-perylene tetracarboxylic dianhydride (PTCDA) on metal –12, semi2 thin films grown on Si(111) surfaces, with PTCDA as the probe molecule. In particular, the adsorption properties of PTCDA on CaF2/CaF1/Si(111), CaF1/Si(111), as well as Si(111)-(7 × 7) surfaces are experimentally investigated by high-resolution scanning tunnelling microscopy (STM) and the adsorption geometry on a CaF2(111) slab is theoretically modelled using density functional theory (DFT). A prominent difference of the molecular properties on the different surface areas is the presence of mostly single molecules in CaF1/Si(111) regions, while ultrasmall molecular assemblies are experimentally observed on thicker CaF2 films. A rather flat-lying geometry is found from geometry-optimisation calculations of a single PTCDA molecule on a CaF2 slab using DFT, whereby an interaction between two carbonyl oxygen atoms and two surface calcium ions leads to a slight deformation of the PTCDA molecule.Here, the understanding of molecular adsorption on insulating thin films is extended by studying an insulator-on-semiconductor system, namely CaFSample preparation and STM experiments were performed under ultrahigh-vacuum conditions. Highly B-doped p-type Si(111) samples  were used as substrates. The (7 × 7) reconstruction was formed by flash cycles and the (7 × 7) surface quality was checked by STM imaging.2 material  was deposited from an e-beam evaporator  on silicon samples held at about 600 °C by direct-current heating. Further details on the sample preparation and CaF2/Si(111) surface properties can be found in [Ub. Gwyddion [CaFfound in –29. PTCDfound in . Image dGwyddion  was used2 triple layers. The lowest triple layer was held fixed. A tolerance of 10−4 Ha/Bohr was used.Density functional theory calculations were performed using cp2k (http://www.cp2k.org)  and para1-covered Si(111)-(7 × 7) and CaF2/CaF1/Si(111) surfaces. Representative overview images acquired at 77 K on the three different surfaces areas are reproduced in PTCDA adsorption was studied by STM after deposition on partly CaF2 films on Si(111) requires the formation of a CaF1 interface layer as the first step. This interface layer is generated by an interface reaction between CaF2 and the silicon surface [2 to CaF1 and F. The dissociated fluorine atoms mostly desorb from the surface, likely in the form of SixF molecules [2 layers can then be grown stoichiometrically on the interface layer by successive CaF2 deposition. The CaF1/Si(111) surface has a (1 × 1) termination after etching the Si(111)-(7 × 7) reconstruction.The growth of ordered CaF surface –29, wherolecules –29. Thic1/Si(111) areas  thick film is shown in On the CaFsee also  is the psee also  was only1 and CaF2 areas. A detailed statistical analysis is shown in 2 direction from more than 1000 molecules in a total of 10 images. The CaF2 directions were determined from filled-state imaging of the (7 × 7) reconstruction [2 on Si(111) at the chosen growth parameters, the CaF2 direction is identical to the Si  direction that was determined from STM images. Three equivalent CaF2 directions are depicted at the right of the exemplary image in 2 direction, especially after deposition on samples held at room temperature. The width of the peaks is attributed to uncertainties of the angle measurement and the asymmetry to a statistical artefact. However, it is also noteworthy that the intermediate minimum in the distribution is less pronounced in measurements performed after PTCDA deposition on cooled samples, which suggests an increased barrier at the estimated sample temperature of 140 K to arrive in the optimum adsorption geometry.The STM data in truction . Due to 1/Si(111) and CaF2/CaF1/Si(111) areas from measuring the pairwise molecular separations of a total of about 400 (1600) molecules on the CaF1 (CaF2) area in the STM image in 2/CaF1/Si(111) areas, while no clear preference is apparent from the distribution on the CaF1/Si(111) interface layer areas. As the surface lattice periodicities are identical for the interface and subsequent layers due to the extremely small mismatch between the silicon and CaF2 lattice constants [1 areas can rather be explained by nucleation at defects present in the interface layer. This is substantiated by data in 2 surfaces are mostly defect-free.A second statistical analysis was performed for the nearest-neighbour distances between nearby PTCDA molecules on CaFonstants , the abs2(111) slab (only the top layer is shown in 2(111) surface. In this geometry, the PTCDA molecule is aligned with the long axis along the  direction, which corresponds to an alignment with an angle of 30° off a CaF2 direction  CaF2(111) [2(111) is likely the result of an interplay between the carbonyl oxygen–calcium attraction and the repulsion between the other oxygen atoms and the surface fluorine atoms, as well as the resulting deformation of the PTCDA molecule.The PTCDA molecule is in a nearly flat-lying geometry, with the exception that two of the four carbonyl oxygen atoms are displaced out of the PTCDA plane towards a surface calcium ion. An attraction between the molecular oxygen and surface calcium atoms is suggested from an oxygen–calcium distance of about 2.8 Å. Note that this distance is larger than 2.4 Å recently found for the oxygen–calcium distance of a carboxylic acid moiety of a ferrocene derivative that binds vertically to CaF2(111) . The oxy3 of the electron density difference Δρ = ρfull − ρslab − ρPTCDA. This difference was calculated as a difference between the electron densities of the full (ρfull), CaF2 slab (ρslab), and PTCDA gas phase (ρPTCDA) systems. The main finding is electron accumulation below the carbonyl oxygen atoms, in agreement with the attractive interaction with the surface calcium atom already identified before from the oxygen displacement.2(111) system are depicted in 1 thin film is of strong LUMO character, whereas the filled-state image  as well as the three lowest unoccupied molecular orbitals  as calculated with cp2k for the PTCDA/CaFte image  on the Chin film  reveals A slightly deformed adsorption geometry including a small tilt of the molecule with respect to the surface plane is furthermore in agreement with the experimental observation of a slight asymmetry in the imaged lobe height, see for example 1/Si(111), and CaF2/CaF1/Si(111) surface areas were studied by STM at low temperatures. Single molecules were identified on the CaF1/Si(111) interface layer while ultrasmall molecular assemblies were found on the CaF2/CaF1/Si(111) areas. The presence of mostly single PTCDA molecules in CaF1 regions is rationalised by nucleation at defects present within the CaF1 interface layer. In contrast, the CaF2/CaF1 layer is mostly defect-free. A statistical analysis revealed a preferred molecular orientation of the long molecular axis along a CaF2 direction, in full agreement with the DFT-calculated optimum adsorption geometry. The DFT-based analysis furthermore revealed a nearly flat-lying molecular adsorption geometry with a downward displacement of two carbonyl oxygen atoms. Based on an analysis of the electron density difference, these atoms are attracted towards the surface calcium ions. A comparison of calculated molecular orbital shapes with the experimental STM data suggests a strong influence of the LUMO in filled-state STM imaging on the CaF1 interface layer. Instead, the absence of long-range order on the CaF2 films is explained by a mismatch of the common PTCDA motifs with the CaF2 surface structure.Adsorption properties of PTCDA molecules on Si(111)-(7 × 7), CaF


In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the  jnk gene family is known to act within this pathway but its specific role in heart development has been difficult to establish using mouse mutants because genes within the family overlap in function and embryos mutant for more than one jnk gene die before the heart is fully formed. In these studies, we used zebrafish to determine the role of jnk1 in heart development and found that a specific gene transcript from zebrafish jnk1a is needed to produce the full complement of cardiomyocytes that form the first part of the ventricular heart chamber. We show that this happens because jnk1a is required for the normal expression of hand2, a gene required to make ventricular heart cells. Furthermore, we can fully reverse the abnormalities in jnk1a embryos by increasing hand2 gene activity. This part of the zebrafish ventricular chamber is equivalent to the left ventricle in humans and understanding how jnk genes work may help understand why some human heart malformations are associated with an underdeveloped left ventricle.The planar cell polarity pathway is a genetic cascade important for normal development of the heart. The  Vangl2, Dvl2 and Celsr1 have been shown to cause cardiac malformations  –5, but fiewed in ), specifiewed in . Inactiviewed in ,9 and Rhiewed in  also proSOS1 found in patients with Noonan syndrome, has been shown to directly activate Jnk  x35 cycles, 72°C (5min). These conditions ensured products obtained were within the linear phase of PCR (jnk1a) and NheI-HF  and gel densitometry performed using Fiji [ef1α value and with regard to the efficiency of the PCR reaction as established with plasmids containing full length transcripts  using a high-speed digital video camera (640x480 pixels at 127 frames/sec) . Data was analysed using Fiji  and simuEmbryos were incubated in 10mM BrdU  in 15% DMSO for 30 min on ice at the 10-somite stage (ss). Then, embryos were washed in E3 medium and allowed to develop until 12ss. After fixing in 4% PFA overnight at 4°C, embryos were stored in methanol at -20°C. Embryos were rehydrated in PBS, then permeabilized using Proteinase K and post-fixed in PFA 4%. After incubation in 2N HCl for 1 hour, standard immunohistochemistry using anti-BrdU and anti-GFP antibodies was carried out. The proportion of BrdU-positive cardiomyocytes was assessed as above.TUNEL staining  was performed as previously described  in wholemyl7:gfp embryos at the 14 ss were hand dechorionated and oriented in 1.5% low melting agarose. A 20-image stack , using a 20x objective at 3μm intervals, was acquired every 6 minutes between 15–16 ss to 19–20 ss. Manual tracking was performed using Fiji [An established protocol  with mining Fiji  and XY ci, yi) and the final  coordinatesMean speed calculated from each measurable displacement per unit time between the initial  and the final  coordinatesMean velocity calculated from the absolute displacement per unit time between initial  and, depending on the number of groups to compare, either Student t test or ANOVA with Tukey's post-hoc analysis was performed. Data is presented as mean and SEM.S1 Figjnk1a and jnk1b genes are capable of producing exon 7 and short C-terminus containing transcripts that fully match human JNK1 transcripts. However, whilst Ex8 derived from jnk1b matches the human peptide the Ex8 from jnk1a is divergent and contains a serine rather than threonine residue (*). In contrast whilst the long C-terminal extension provided by jnk1a matches the human, the jnk1b long terminal extension is highly divergent and differs by 9/39 amino acids including insertion of an additional threonine residue.Black text indicates identical amino acids, green text indicates favourable amino acid substitutions and red text divergent amino acid residues. Exons 2–6 and 9–12 are common to all transcripts and highly conserved. Both (TIF)Click here for additional data file.S2 FigA) specificity of primers used in RT-PCR splicing assay. Primers sets to identify Ex7 and Ex8 containing variants of both jnk1a and jnk1b transcripts were assessed against all 8 transcripts contained within plasmids, using primers for the plasmid backbone (M13) as loading control. Restriction enzyme digests showing original PCR product and complete digestion by enzyme to identify C-terminal extension in jnk1a and jnk1b transcripts. (B) Graphical representation of gel densitometry indicating the splicing assay was carried out during the linear phase of amplification for jnk1a and jnk1b Ex7 and Ex8 PCR (Arbitrary logarithmic Units). (C) PCR with plasmid containing transcripts were used as positive controls for PCR reactions and indicated efficiency of each reaction.((TIF)Click here for additional data file.S3 FigA) Prior to heart formation all transcripts are present throughout the embryo at the start of gastrulation (6hpf) and the end of gastrulation (10hpf). The most abundant transcripts by RT-PCR splicing assay are indicated by dashed lines. (B) WISH for all jnk1a and jnk1b alternatively spliced transcripts at 24hpf and 48hpf. Transcripts with obvious expression within heart—jnk1a Ex7Lg and jnk1b Ex8Sh—are indicated by arrows. (C) Close up of heart in all jnk1a transcripts at 24 and 48hpf. Outline of heart indicated by dashed lines. Absent expression indicated by arrow heads and expression in heart indicated by arrows in jnk1a Ex7Lg, jnk1a Ex8Lg and jnk1b Ex8Sh. (D) WISH demonstrated expression of jnk2 and jnk3 in the developing brain (arrows) but not heart (arrowheads). (E) RT-PCR of extracted hearts indicates that jnk2 is expressed at low level at 24 and 48 hpf, whilst there is late onset of jnk3 gene expression by 48hpf. ef1alpha is used as a loading control.Chromogenic WISH is not quantitative but indicates the extent of expression. ((TIF)Click here for additional data file.S4 Fign = 3 clutches in all cases.(TIF)Click here for additional data file.S5 FigA) Cardiac morphology at 28 hpf. All ventricular cardiomyocytes are labelled by MF20 antibody (red) and atrial cells by both MF20 and S46 antibody (appear yellow). Normal appearances seen in wildtype (wt) embryos. Ventricular hypoplasia (indicated by size of white bar) is seen in MZjnk1a null mutants but is not seen in MZjnk1b null mutants. There is no additional ventricular hypoplasia in MZjnk1a/MZjnk1b double mutants. (B) MF20 antibody staining to identify somites/skeletal muscle blocks in wild type (wt) and MZjnk1a/MZjnk1b mutants (C) Somite counting excludes global somatic developmental delay in MZjnk1a/MZjnk1b null mutants.  TUNEL labelling at 12ss stage in wild type (wt) and MZjnk1a/MZjnk1b mutants. Myocytes in the ALPM are identified by Mef2 antibody. The percentage TUNEL positive cells in Mef2 population is unchanged in MZjnk1a/MZjnk1b null mutants.  BrdU incorporation in cardiomyocytes from 10ss to 12ss. Quantification shows that there is no difference in BrdU incorporation index in MZjnk1a/MZjnk1b null mutants compared to wild type (wt) controls. ns = nonsignificant.((TIF)Click here for additional data file.S1 TableGuide RNA sequences of CRISPR Cas9 mutant production and Genotyping primers used for selection of mutants. Transcript cloning indicated are transcript sequences; add additional sequence for Gateway or restriction enzyme cloning. RT-PCR splicing assay primers, see RT-PCR primers for jnk2 and jnk3 and in-situ hybridization primer sequences used to produce sense and non-sense probes.(DOCX)Click here for additional data file.S2 TableNucleotide sequences for all jnk1a and jnk1b transcripts based on results of bacterial subcloning and translation into peptide sequences.(DOCX)Click here for additional data file.S1 Movie(MOV)Click here for additional data file.S2 Movie(MOV)Click here for additional data file.


Dear Editor,IDH1-mutated relapsed or refractory acute myeloid leukemia (AML) showed an overall response rate of 41.6% and a complete remission rate of 21.6% with a median duration of response of 8.2 months [IDH1 mutations do not show a clear prognostic effect in AML patients who are treated with standard induction and consolidation therapy [The first clinical IDH1 inhibitor ivosidenib as a single agent in  therapy . It is uWe evaluated the mIDH1 inhibitor BAY1436032 in sequential or simultaneous combination with cytarabine plus doxorubicin in a previously reported IDH1 mutant PDX mouse model , the per2The findings from our preclinical study show that simultaneously combining an IDH1mutant inhibitor with cytarabine plus doxorubicin substantially inhibits leukemia in vivo. These findings are in accordance with our previous study in which we showed a synergistic effect of simultaneous administration of an IDH1 inhibitor with the hypomethylating agent azacitidine compared with sequential administration . A phaseESM 1(DOCX 22 kb)


Understanding the optical and electronic mechanisms that govern charge separation, transport and recombination in these devices will enhance their current conversion efficiencies under illumination to sunlight. In this work, density functional theory with Perdew-Burke Ernzerhof (PBE) functional approach was used to explore the optical and electronic properties of three modeled TiO2 brookite clusters, (TiO2)n=5,8,68. The simulated optical absorption spectra for (TiO2)5 and (TiO2)8 clusters show excitation around 200–400 nm, with (TiO2)8 cluster showing higher absorbance than the corresponding (TiO2)5 cluster. The density of states and the projected density of states of the clusters were computed using Grid-base Projector Augmented Wave (GPAW) and PBE exchange correlation functional in a bid to further understand their electronic structure. The density of states spectra reveal surface valence and conduction bands separated by a band gap of 1.10, 2.31, and 1.37 eV for (TiO2)5, (TiO2)8, and (TiO2)68 clusters, respectively. Adsorption of croconate dyes onto the cluster shifted the absorption peaks to higher wavelengths.A range of solution-processed organic and hybrid organic−inorganic solar cells, such as dye-sensitized and bulk heterojunction organic solar cells have been intensely developed recently. TiO In recent years, there has been alarming increase in energy demand across the globe ,2,3; fos2 has attracted a wide range of interests in the research community owing to its proven ability for many applications in the medical field .The absorption spectra of (TiOT method  in which2)5 and (TiO2)8 complex presented were computed using GPAW and TD-DFT [2)5 also shows a slight absorption peak at around 400 nm, suggesting the absorption peak at higher wavelength than (TiO2)8. Maximum peak height indicates that the higher absorbance is notable in (TiO2)8 brookite absorption spectra than in the (TiO2)5 absorption spectra. The results of the absorption spectra of (TiO2)5 and (TiO2)8 generally agrees with findings from literature in the sense that TiO2 is majorly sensitive in the UV region of the solar spectrum owing to its wide band gap (3.0–3.2 eV) [The absorption spectra of  ,65.2)5, (TiO2)8 and (TiO2)68 nanoclusters were computed using GPAW [2)5, (TiO2)8 and (TiO2)68 are presented in The density of states and the projected density of states of  state, whereas the lowest unoccupied state of the conduction band is mainly dominated by the contributions of titanium 3d atomic orbitals as illustrated in d and p orbitals, while contributions from oxygen atoms are minimal.The density of states (DOS) is composed of the surface valence and conduction bands separated by a wide band gap. The PDOS results for the clusters show that both the oxygen and titanium atomic orbitals contributes to the valence states, with the oxygen 22 investigated in this work is shown in the 2 brookite semiconductor material employed in this study was used to generate models of the three TiO2 clusters. Brookite TiO2 has an orthorhombic crystalline structure with eight formula units in the orthorhombic cell.The structural model for brookite polymorph of TiOThe optimized molecular geometries of the croconate dyes CR1 and CR2 considered for this study are presented in the 3), which is an alkyl derived from methane, with one carbon atom bonded to three hydrogen atoms while CR2 contains an electron withdrawing carboxyl group (–COOH), which is an organic compound situated in the carboxylic acid, one carbon atom bonded to two oxygen atoms and one hydrogen atom.CR1 contains electron donating methyl group (CH2)5, (TiO2)8 and (TiO2)68 brookite clusters through the diketo group as presented in 2)5, (TiO2)8 and (TiO2)68 brookite clusters are investigated in this section. A fascinating feature about croconate dyes is their good solvatochromic shift, they can absorb photons in the near infrared region [2 complexes were relaxed upon adsorption.The croconate dyes CR1 and CR2 are adsorbed onto the surface of 5 is 3.93 eV, CR2-(TiO2)5 is 5.53 eV, CR1-(TiO2)8 is 0.75 eV, CR2-(TiO2)8 is 0.68 eV, CR-(TiO2)68 is 4.74 eV, and CR2-(TiO2)68 is 4.95 eV.The computed adsorption energies are presented in 2 nanocluster [2)8 brookite cluster than the one with the electron withdrawing moiety (CR2). However, the dye with the electron withdrawing moiety (CR2) binds more strongly to (TiO2)5 and (TiO2)68 brookite cluster than the dye with the electron donating methyl (CR1). The results suggest that the dye molecules react differently to different surfaces and sizes of TiO2 brookite clusters.The positive adsorption energies denote the binding ability of the dye molecules to the surface of TiOocluster . The res2)5 are presented in 2)5 cluster. The absorption maxima of (TiO2)5 brookite cluster, which was located around 200 nm has now shifted to higher wavelength around 700–900 nm as shown in 2)8 are presented in 2)8 cluster and it is evident that the absorption maxima with the dyes have shifted to higher wavelength. The absorption maxima of (TiO2)8 brookite cluster that was located around 200 nm has now shifted to higher wavelength around 600–900 nm. In both cases, the CR1 and CR2 dye molecules show a bathochromic shift upon adsorption of the dyes on TiO2 cluster. The bathochromic shift observed after adsorption of CR1 and CR2 dye molecules on TiO2 brookite cluster suggests good optical properties of the dye molecules and corroborates with reports from literature that the adsorption of dye molecules on TiO2 improves its optical response and helps overcome its limited spectral sensitivity in the UV region, thereby improving its photocatalytic properties and overall DSSCs device efficiencies [The optical spectra of CR1 and CR2 dyes adsorbed on the (TiOciencies .2)5, (TiO2)8 and (TiO2)68 brookite cluster are presented in 2 clusters where the unoccupied electronic states are situated. This suggests good electronic coupling between the occupied excited state of the dye and the unoccupied acceptor levels of the semiconductor conduction band. The localization of the HOMO electronic level on the dye molecules and the LUMO electronic level on the TiO2 clusters implies efficient separation of charge upon adsorption and electron injection from the dye excited state into the TiO2 semiconductor conduction band.The isodensity surfaces of the molecular orbital involved in photoexcitation of CR1 and CR2 dye molecules adsorbed on 5, (TiO2)8 and (TiO2)68 were computed using GPAW and PBE exchange correction functional. The TDOS and PDOS spectra are presented in 2)5, dyes-(TiO2)8 and dyes-(TiO2)68, respectively. The DOS is composed of the surface valence and conduction bands separated by a wide band gap. The density of states for the clean (TiO2)5, (TiO2)8 and (TiO2)68 clusters before the dyes were adsorbed were presented previously in 2 cluster alone and the DOS spectrum of croconate dyes adsorbed on TiO2 clusters, it is observed that the adsorption of the CR1 and CR2 dye molecules results in new occupied molecular orbitals introduced to the band gap of the TiO2 clusters upon the absorption of the dye molecules as seen in 2. Comparing between the DOS spectra of all the clean (TiO2)5, (TiO2)8 and (TiO2)68 clusters and the DOS spectra of the dyes adsorbed on the clusters, it is clear that the adsorption of the dyes on TiO2 clusters results in a shift of the conduction band edge of TiO2 to higher energy levels, and consequently narrowing of the band gap between the occupied valence states and the unoccupied conduction band. Additionally, upon adsorption of the dye molecules on the TiO2 clusters, the DOS results reveal that the process introduces new occupied electronic orbitals between the two states where there was a broad band gap initially and in the conduction band of the TiO2 clusters.In order to understand further the electronic structure of CR1 and CR2 dye molecules adsorbed on TiO2)5, dyes-(TiO2)8 and dyes-(TiO2)68 show the contribution of the atomic orbitals in the occupied states and unoccupied states. The PDOS results for the clean clusters showed that both the oxygen and titanium atomic orbitals contributes to the valence states, the oxygen 2p atomic orbitals contributes mostly to the highest occupied valence band state, whereas the lowest unoccupied state of the conduction band is mainly dominated by the contributions of titanium 3d atomic orbitals. The valence band is dominated by the p atomic orbitals of oxygen with a little contribution from the p atomic orbitals of titanium. The major contribution in the conduction band comes from the titanium orbitals, especially the d and p orbitals. The PDOS spectra of all the dyes-(TiO2)5, dyes-(TiO2)8 and dyes-(TiO2)68 complexes show major contributions from the 3d orbitals of titanium, 2p orbitals of carbon, 2p orbitals oxygen, s orbitals of hydrogen to the valence states. Carbon p orbitals contribute majorly to the conduction band.The PDOS spectra of the dyes-n where n = 5, 8 and 68 were modeled from the optimized ground state bulk structure of TiO2 brookite. (TiO2)5 brookite cluster is a nanostructured form of brookite TiO2 modeled from the bulk structure with dimension repeated in x, 2)8 brookite cluster with the stoichiometry of (TiO2)8 is modeled from the bulk structure of brookite, while (TiO2)68 is a supercell which was created from the repetition of the optimized TiO2 brookite bulk structure unit cell in repeated by 2 × 2 × 2 Å in three dimensions. The trend for TiO2 is (TiO2)n with Ti and O in the ratio 1:2, respectively. The clusters were then exported to (GPAW) [The bulk structure of brookite TiOorted in . The thro (GPAW)  softwareo (GPAW)  for furt2 brookite clusters that were generated are presented in 2)5 brookite nanocluster with five titanium and 10 oxygen atoms cleaved from brookite bulk structure as described in the previous section. 2)8 comprising eight titanium and 16 oxygen atoms whereas 2)68 supercell 2 × 2 × 2 Å comprising 68 titanium atoms and 136 oxygen atoms.All DFT calculations were performed within an atomic simulation environment (ASE) with GPAW software while the structures were visualized with Avogadro. GPAW is a Python based program-package formulated with density-functional theory (DFT) combined with the grid space projector-augmented wave (GPAW). The three TiO2 as well as the main features of oxygen vacancies in rutile and anatase polymorphs. GPAW implements the projector-augmented wave method with the smooth wave-functions and electron density represented on real space grids. The structures were considered to have converged when the maximum forces that were acting on all the atoms were about 0.05 N. The periodic boundary conditions were implemented on the supercell during the relaxation and was set to none for the nonperiodic brookite cluster models. The atoms of the cluster were reoriented during the relaxation until the ground state structure was reached, where they became stable and the forces converged to a maximum value of 0.05 N.All the structures were relaxed in vacuum using GPAW with the PBE. The exchange correlation energy was approximated within the generalized gradient approximation PBE  whereby The UV/Vis, total density of states (TDOS) and partial density of states (PDOS) of the nanocluster structures were computed using the trajectory files obtained from the relaxed structures. The UV/Vis was calculated in vacuum and the TDOS and PDOS were computed using the PBE functional.2)5, (TiO2)8 and (TiO2)68 clusters. We also investigated the adsorption of visible and near infra-red light harvesting croconate dyes onto the three TiO2 brookite clusters, namely (TiO2)5, (TiO2)8 and (TiO2)68. Our findings reveal that (TiO2)5 cluster shows higher wavelength absorption than the corresponding (TiO2)8 and (TiO2)68 clusters owing to its miniaturized size. The band gap of (TiO2)5 cluster also shows a smaller band gap than the corresponding (TiO2)8 and (TiO2)68 clusters, which generally suggests that the nanostructure of TiO2 will enhance more charge transport for solar cell applications. The simulated optical absorption spectrum for (TiO2)5 and (TiO2)8 cluster shows excitation around 200–400 nm, with (TiO2)8 cluster showing higher absorbance than the corresponding (TiO2)5 cluster. The computed density of states and the projected density of states spectra for (TiO2)5, (TiO2)8 and (TiO2)68 clusters reveal surface valence and conduction bands separated by energy band gaps of 1.10, 2.31, and 1.37 eV for (TiO2)5, (TiO2)8 and (TiO2)68 clusters, respectively. The projected density of states spectrum reveals that 2p atomic orbitals contribute mostly to the highest occupied valence band state, whereas the lowest unoccupied state of the conduction band is mainly dominated by the contributions of titanium 3d atomic orbitals. Our findings showed that the adsorption of croconate dyes on the cluster shifted the absorption spectrum to higher wavelengths. The absorption maxima of the clusters alone located in the UV region was shifted to the visible and near infra-red region of the solar spectrum, making it possible to absorb in the whole spectral region. The isodensity surfaces show that HOMO were more localized on the dye while the LUMO were delocalized on the TiO2 semiconductor, depicting that electrons were transferred from the dye excited states to the semiconductor acceptor states. Our findings generally show that the optical and electronic properties of TiO2 cluster vary with the size of the cluster.First principle calculations based on density functional theory (DFT) and time dependent (TD-DFT) were used to investigate the optical and electronic properties of  less susceptible to UV degradation. The research also provides significant findings for the fabrication of brookite semiconductor based DSSCs.Findings from this research are of good significance as the theoretical knowledge and the results could be a guide to experiments on dye molecules adsorbed onto brookite polymorph for application as electron transporting material in nanostructured TiO


Immunohistochemistry (IHQ) is commonly used for the detection of mismatch repair proteins deficiency (MMRD). One very infrequent abnormal pattern of MMR protein expression is the loss of PMS2 and MSH6, with intact expression of MLH1 and MSH2.PMS2 mutations (R134* germline mutation in one tumour and M1R and c.1239delA somatic mutation in the other) as the primary event and somatic MSH6 mutation (c.3261dupC) as the secondary event in both tumours.We review the frequency of this MMRD IHC pattern among 108 colorectal (CRCs) and 35 endometrial cancers in our files with loss of expression of at least one protein, and present two CRCs showing loss of PMS2 and MSH6 protein expression (1.9% of CRCs). NGS analysis of these tumours identified This study suggests that Next Generation Sequencing (NGS) tumour analysis should be considered in the algorithm of Lynch syndrome screening to detect MMR gen somatic mutation in inconclusive cases. MLH1 promoter hypermetylation, the most common immunohistochemical pattern observed in MMRD tumours is MLH1/PMS2 loss [MLH1 germline mutation, whereas MSH2/MSH6 loss occurs in LS due to MSH2 germline mutations [PMS2 [MSH6 [Mismatch repair proteins deficiency (MMRD) can be studied by different methods in tumour tissue, but the most commonly used in the routine practice are immunohistochemistry (IHC) to analyze the expression of MMR proteins  and/or microsatellite instability (MSI) analysis . Since tMS2 loss . This pautations . In addiS2 [MSH6 , 6, respPMS2 mutations (germline in one tumour and somatic in the other) as the primary event and somatic MSH6 mutation as the secondary event in both tumours. These cases illustrated the utility of NGS on tumour tissue for LS screening in inconclusive cases.One very infrequent abnormal pattern of MMR protein expression is the loss of PMS2 and MSH6, with intact expression of MLH1 and MSH2 . In thisCRCs and ECs with loss of any MMR protein diagnosed between 2010 and 2019 were identified in our Laboratory Information System. Indications for the study of MMR protein expression have changed along this period of time from screening for LS in patients with CRC and Bethesda criteria to universal screening, not only for the identification of patients with LS, but also to select the more appropriate chemotherapy or immunotherapy treatment. In the same way, universal screening for LS by IHC has been implemented in our centre for all ECs. In two cases, IHQ of MMR protein and NGS were realized –9 [see AWe identified 108 CRCs and 35 ECs with loss of at least one MMR protein. The different patterns of expression and their relative frequencies are presented in Table Patient 1 was a 41-year old female without a personal history of cancer, including father and grandfather with bladder cancer and a brother with melanoma. She presented in 2007 with a pT3N0 tumour of the right colon  and received adjuvant Capecitabine. In 2016 a liver mass of 12 cm was identified and the patient received 3 cycles of neoadjuvant chemotherapy (FOLFOX). The pathologic examination showed the metastatic nature of the lesion and poor response to chemotherapy (approximately 10% of tumour regression). There was no evidence of disease in the last follow-up in 2020. Liver metastases showed loss of PMS2 and MSH6, but intact expression of MLH1 and MSH2 . The tumour showed loss of PMS2 and MSH6 staining with intact staining for MLH1 and MSH2 [see Additional file NGS was performed in the liver metastasis in patient 1 and in the primary tumour in patient 2. Poor DNA integrity precluded NGS analysis of the primary tumour in patient 1.PMS2, accompanied by a somatic missense mutation (G271S). Patient 2 portrayed two putative truncating somatic mutations (M1R and c.1239delA) in PMS2. Interestingly, tumour samples from both patients portrayed the same somatic frameshift mutation in MSH6 (c.3261dupC). In addition, a second missense somatic mutation located in a close genomic position to the previously one, was detected in both patients   located in a mononucleotide tract, typical of MMRD.Our data tend to support the hypothesis that in both tumours genetic alterations in PMS2 mutation, no PMS2 alterations were reported in the other patient. The authors also observed a somatic mutation in the same mononucleotide tract in MSH6 in three additional CRCs with MLH1/PMS2 loss, although the subjacent genetic or epigenetic alteration of these three cases was not reported.Although a complete review of all published series about MMRD is out of the scope of this study, we have only identified two similar cases included in the series reported by McCarthy et al. , who fouPMS2 (47%) or MLH1 (24%) germline mutation. These germline MLH1 mutations produced MLH1 proteins that retain antigenicity but are non-functional compromising stability of MLH1-PMS2 complexes and inducing PMS2 loss. Kato et al. [MLH1 promoter hypermetylation. Only 1 out of 5 patients tested for germline mutations carried a PMS2 germline mutation and any of them carried a MLH1 germline mutations. Finally, Pearlman et al. [PMS2.Previous studies have reported that isolated loss of PMS2 represents approximately 0.4% of CRCs and 2.2–7% of ECs with MMRD, respectively , 12. Then et al.  reportedPMS2 mutations than among LS patients carrying germline mutations in other MMR genes  [Tumours with isolated PMS2 loss seem to have specific clinicopathological features. The risk of developing CRC is lower among LS patients carrying germline In patient 1, MSH6 loss of expression occurred only in liver metastases and after the use of chemotherapy. MSH6 loss expression has been reported in some CRCs after the use of neoadjuvant therapy . AlthougPOLE. Whereas one of the mutations was reported as probably benign in previous reports, the somatic mutation E277G detected in the tumour of patient 1 has been reported as germline and pathogenic in one family with familial CRC [These two cases showed a somatic mutation in the exonuclease domain of lial CRC . The rolPMS2 germline and/or somatic mutations as the possible primary event and somatic mutations of MSH6 as a possible secondary event of these cases. NGS tumour analysis should be considered in the algorism of LS screening for inconclusive cases.Summarizing, loss of PMS2 and MSH6 expression with intact expression of MLH1 and MSH2 is very infrequent and was observed in 1.9% of CRC with IHC-MMRD, but not in ECs. NGS analysis of two cases demonstrated Additional file 1. Immunohistochemistry: Clones and criteria used for the interpretation. Massive parallel sequencing: Description of the NGS panel. Sanger sequencing: Primers used and variants obtained from Sanger sequencing.Additional file 2. Figure case 2.Additional file 3. 2013 CARE Checklist.


Within the eukaryotic nucleus the genomic DNA is organized into chromatin by stably interacting with the histone proteins as well as with several other nuclear components including non-histone proteins and non-coding RNAs. Together these interactions distribute the genetic material into chromatin subdomains which can exhibit higher and lower compaction levels. This organization contributes to differentially control the access to genomic sequences encoding key regulatory genetic information. In this context, epigenetic mechanisms play a critical role in the regulation of gene expression as they modify the degree of chromatin compaction to facilitate both activation and repression of transcription. Among the most studied epigenetic mechanisms we find the methylation of DNA, ATP-dependent chromatin remodeling, and enzyme-mediated deposition and elimination of post-translational modifications at histone and non-histone proteins. In this mini review, we discuss evidence that supports the role of these epigenetic mechanisms during transcriptional control of osteoblast-related genes. Special attention is dedicated to mechanisms of epigenetic control operating at the Runx2 and Sp7 genes coding for the two principal master regulators of the osteogenic lineage during mesenchymal stem cell commitment. Osteoblast lineage commitment is regulated by a coordinated set of extra cellular stimuli and developmentally-regulated signaling pathways, including those mediated by bone morphogenic proteins (BMPs), Wnt-ligands, steroid hormones, and growth factors, among others . FollowiWe discuss research demonstrating the role of epigenetic mechanisms in controlling gene transcription during mesenchymal cell commitment to the osteogenic lineage. We first overview key components of epigenetic mechanisms that control gene expression in mammals and then describe how these epigenetic processes contribute to regulate transcription of the Runx2 and Sp7 genes, two critical osteogenic master regulators.Nuclear DNA is organized as chromatin. Nucleosomes are the fundamental units of this chromatin and are composed of an approximately 147 bp DNA segment wrapped around an octamer of histone proteins  . A portiMolecular components that regulate chromatin organization and transcription are critical players during cell differentiation. Among them the large group of “histone post-translational modifications” (HPTMs), which are considered a principal “epigenetic” mechanism . HPTMs mGenetic studies allowed the identification of the Polycomb Group (PcG) and the Trithorax Group (TrxG) complexes, which mediate inhibition and activation of transcription, respectively, during embryogenesis . One sigSeveral mammalian TrxG complexes, including COMPASS (Complex of Proteins Associated with Set1a/b) and the Mixed Lineage Leukemia (MLL1–5)-containing COMPASS-like complexes have been reported . TrxG-meSet1-COMPASS mediates global genomic deposition of H3K4me3 in most mammalian cells and therefore its function is tightly associated with transcriptional activation of a large number of genes. MLL2-COMPASS-like has been shown responsible for H3K4me3 deposition at promoters in embryonic stem cells (ESCs), contributing to transcriptional upregulation of homeobox genes . InteresChromatin domains with decreased enrichment of H3K4me3 or H3K27me3 can also be maintained through the activity of lysine demethylases . In partMethylation at H3K9  is strongly associated with the formation of highly compacted and transcriptionally repressed chromatin (heterochromatin) . The metcis or by transferring histone octamers in an ATP-dependent manner. This changes the exposure of regulatory motifs thereby facilitating or preventing recognition by cognate factors  activities (Genomic DNA can be methylated at cytosines that are followed by guanosines (CpG dinucleotides). The DNA methyl-transferases that mediate this modification belong to a well-conserved family of proteins that include both maintenance (Dnmt1) and tivities . Most retivities . DNA demtivities . Importativities .Enrichment of H3K9me3/2 at transcriptionally-inactive chromatin can be associated with the presence of 5mCpG. This is due to the ability of the proteins that “write” and “read” these two epigenetic marks to form complexes  thereby Differentiation of mesenchymal stem cells (MSCs) to the osteogenic lineage requires the expression and function of two master transcription factors: Runx2 (Runt Related Transcription Factor 2) and Sp7 . These factors bind to and control the expression of numerous downstream target genes that code for proteins that are necessary for establishing the osteoblast phenotype . Runx2 aActive transcription of the Runx2 and Sp7 genes is accompanied by an epigenetic landscape that includes increased H3K4me3 enrichment at both promoter regions . Hence, It has been determined that an enrichment of H3K4me1 accompanies transcriptional silence at the Runx2 and Sp7 genes in non-osteoblastic and mesenchymal uncommitted cells ,b. As MLMaintenance of chromatin with low H3K4me3 enrichment (and hence elevated H3K4me1) at the Runx2 promoter can be also achieved by binding and activity of the H3K4 demethylase Jarid1b/Kdm5b , which cFindings from several teams revealed that regulating the repressive mark H3K27me3 represents an important step during the differentiation of MSCs toward osteoblasts. It was demonstrated that the activity of the H3K27 demethylases Utx/Kdm6a and Jmjd3/Kdm6b is critical for erasing this mark from the promoter of the Runx2 and Sp7 genes  and hencThe repressive mark H3K9me3 plays an important role during differentiation of MSCs to the osteoblastic lineage ,b. H3K9mAlthough most studies support the role of the H3K27me3 and H3K9me3 marks during repression of both master osteogenic genes, recent analyses indicate that for certain cells, this role may have to be re-evaluated. Thus, analysis of uncommitted human umbilical cord-derived Wharton Jelly MSCs (WJ-MSCs) showed that the Runx2-P1 promoter region does not exhibit significant enrichment of H3K9me3 and H3K27me3 . InteresHistone lysine acetylation also plays a relevant role during bone formation as it supports increased expression of critical genes for osteoblast differentiation . ChromatDNA methylation (5mCpG) can regulate osteogenic differentiation of MSCs. Addition of the DNA methyl-transferase inhibitor 5-Azacytidine (5-Aza) to human and murine mesenchymal cells enhances their ability to engage osteogenesis by reducing 5mCpG enrichment at regulatory regions of osseous genes . In partex vivo. Recent results demonstrate that the substrate stiffness at which hMSCs are grown in culture can induce rapid and stable changes in chromatin organization and the associated epigenetic landscapes that permanently up- or down-regulate genes (Controlling lineage commitment in MSCs represents a critical challenge to overcome skeletal deficiencies in patients. Extensive research during the last 20 years has shed light into potential targeting of specific epigenetic mechanisms that control bone-related gene expression. Future studies will need to consider the influence of the mechanical environments at which MSCs are maintained and differentiated te genes , hence mMM analyzed the data and wrote most parts of the manuscript. MC wrote parts of the text and prepared the figures. GN analyzed the data, wrote parts of the text and helped preparing the figures. 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.