The caudal region of the rodent striatum, called the tail of the striatum (TS), is a relatively small area but might have a distinct function from other striatal subregions. Recent primate studies showed that this part of the striatum has a unique function in encoding long-term value memory of visual objects for habitual behavior. This function might be due to its specific connectivity. We identified inputs to the rat TS and compared those with inputs to the dorsomedial striatum (DMS) in the same animals. The TS directly received anatomical inputs from both sensory structures and value-coding regions, but the DMS did not. First, inputs from the sensory cortex and sensory thalamus to the TS were found; visual, auditory, somatosensory and gustatory cortex and thalamus projected to the TS but not to the DMS. Second, two value systems innervated the TS; dopamine and serotonin neurons in the lateral part of the substantia nigra pars compacta (SNc) and dorsal raphe nucleus projected to the TS, respectively. The DMS received inputs from the separate group of dopamine neurons in the medial part of the SNc. In addition, learning-related regions of the limbic system innervated the TS; the temporal areas and the basolateral amygdala selectively innervated the TS, but not the DMS. Our data showed that both sensory and value-processing structures innervated the TS, suggesting its plausible role in value-guided sensory-motor association for habitual behavior. The striatum receives inputs from various cortical and subcortical areas, and its subregions have different functions  and compared these with the inputs to the DMS which guides the goal-directed behavior in the same rats. Inputs from the sensory and reward systems in the cortical and subcortical structures were mainly examined. Furthermore, we investigated how the TS- and DMS-projecting neurons in each brain structure were topographically organized in the rostral-caudal axis.Male Sprague-Dawley rats aged 9–13 weeks (Orient Bio Inc.) were used. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University (SKKU).Rats were initially anesthetized with 5% isoflurane in oxygen for 3 min. After deep anesthetization, the heads were fixed to a stereotaxic frame (David Kopf Instruments) for incision and injection. The percentage of isoflurane was gradually decreased for the incision (3%) and injection (2%). After tracer injection, the injection needle and injection tower were removed, and the incision was sutured with surgical silk. Animals were then returned to their home cages. All procedures were performed under clean surgical condition.Anesthetized rats were placed in a stereotaxic frame, and the scalp was incised approximately 3 cm in length to expose the skull. We then drilled holes with 2-mm diameter in the skull to lower the injection needle (drill bit diameter: 1.35 mm). Red and green fluorescent retrobeads (Lumafluor) were used to compare TS-projecting neurons with DMS-projecting neurons in brain structures. Different fluorescent retrobeads were injected in the same rats to examine co-labeled neurons in the same brain slices. The injection sites and amounts are summarized in Table Seven to 10 days after injection, the rats were anesthetized with 5% isoflurane in oxygen for 3 min. For deep anesthesia, urethane  was intraperitoneally injected. To fix the brains, 250 ml of saline was perfused transcardially followed by the same volume of 4% paraformaldehyde. The brains were post-fixed overnight at 4°C and cryoprotected with 30% sucrose in phosphate-buffered saline . The processed brains were frozen in OCT compound at −80°C and keep in the freezer overnight. The next day, the brains were sectioned with a cryostat (Leica CM1950) at 50-μm thickness, and every 200 μm (rat #2 and #5) and 400 μm  slices were used for cell counting. Images of brain slices were obtained with a fluorescent microscope (Leica DMi8). Adjacent slices were used for Nissl staining and immunohistochemistry to define brain structures and cell types, respectively.To examine whether the TS-projecting neurons were dopaminergic or serotonergic, the SN slices of rat #2, #3 and #4 were labeled with tyrosine hydroxylase (TH) antibody, and the dorsal raphe nucleus (DRN) slices of rat #2, #3 and #4 were labeled with serotonin (5-hydroxytryptamine) or TH antibodies. After 30 min permeabilization by 0.5% Triton X-100 in PBS, the slices were blocked with 3% normal goat serum and 2% bovine serum albumin in PBS (pH 7.4). After blocking, the SN slices were incubated with mouse anti-TH antibody , and the adjacent slices  containing the DRN were separately incubated with rabbit anti-serotonin antibody  or mouse anti-TH antibody  overnight at room temperature. On the second day, the slices were washed three times with PBS and incubated with secondary antibodies for 2 h at room temperature. Goat anti-mouse IgG antibody conjugated with Alexa 647  was used as secondary antibody for TH staining in the SN. The donkey anti-rabbit IgG antibody conjugated with Alexa 488  and goat anti-mouse IgG antibody conjugated with Alexa 488  were used for serotonin and TH staining in the adjacent slices containing the DRN, respectively. After three washes with PBS, the slices were air-dried overnight at room temperature and mounted with VECTASHIELD (Vector). Cell images of the SN and DRN slices were scanned with a super resolution confocal microscope (Leica DLS).To reconstruct the striatum of rats injected with retrograde tracers Figures , 2A, theTo identify the brain structures that innervate the TS and DMS, we confirmed that the TS- and DMS-projecting neurons in each brain structure were consistently found in all six and four hemispheres, respectively. These labeled neurons in the confirmed brain structures were used for further analysis described below. To examine the rostral-caudal distributions of labeled neurons in the whole brain and brain structures, the number of neurons in each coronal slice was divided by the number of labeled cells in the whole brain  and two hemispheres , respectively, in Supplementary Figures. To analyze the labeled cell distribution in the rostral-caudal axis of each structure, we compared the percentage data in chosen series of the brain slices to the percentages in rest of the brain slices using Wilcoxon rank-sum test with MATLAB software .To compare the proportions of TS- and DMS-projecting neurons in a structure, the number of retrogradely labeled neurons was divided by the total number of labeled neurons in the cortex, subcortex and brain stem  of retrobead-injected rats were examined at 400-μm and 200-μm intervals Figure . We obseP < 0.001, Wilcoxon rank-sum test; Figures P < 0.001, Wilcoxon rank-sum test), agreeing with previous research . In V1 and V2L, we found more TS-projecting neurons in the central-caudal part than the rostral part .We found that the TS received inputs from the SN and DRN, two brain stem regions known to encode reward information .TS-projecting neurons were found in the midline and ipsilateral regions of the DRN Figure . These TThe DRN is also known to contain TH-positive neurons , to the TS Figures . In the P < 0.001, Wilcoxon rank-sum test; Figures P < 0.01 and P < 0.01, respectively, Wilcoxon rank-sum test; Figure In the thalamus, TS-projecting neurons were found in the MGB and were more numerous in the rostral-central region  and most DMS-projecting neurons in the rostral part , although some were found in layer 2 Figure , left. IP < 0.01, Wilcoxon rank-sum test; Figures P < 0.01, Wilcoxon rank-sum test; Figure TS-projecting neurons were found in the endopiriform area, an area involved in processing olfactory and gustatory information , while the DMS-projecting neurons tended to be located in the rostral-central part , while the DMS-projecting neurons were mostly located in the rostral region , and TS-projecting neurons were more located in the central part  and cingulate cortex , might process various sensory inputs  is an important structure in encoding reward value in primates and rodents  that innervate the primary sensory cortices directly projected to the TS (Alitto and Usrey, Dopaminergic input is thought to be involved in synaptic plasticity of the basal ganglia system Wickens, . DopaminIn addition to the SN dopaminergic input, serotonergic input to the TS was found in the DRN Figures . DiffereWe also found a few neurons projecting to the TS in the OFC. This result is consistent with the previous studies mostly using anterograde tracers; neurons in the OFC mainly innervate the rostral part of striatum (Berendse et al., We found that both layer 5 and 6 in some cortical regions projected to the TS. The cortical layer 5 is known to be a major input to the striatum, but some studies have shown that the cortical layer 6 also innervates the primate CDt and the rodent striatum Gerfen,  These diThe caudate tail of macaque monkeys mainly receives sensory inputs from the visual cortex and no or weak inputs from the somatosensory and olfactory regions (Saint-Cyr et al., As a general consideration in all retrograde tracer studies, some of the retrograde labeling may occur via damaged axons of passage near the injection site. In our tracer study, it is unlikely that this erroneous labeling heavily affected our results for two reasons. First, five rats had similar results, which is unlikely if there was substantial uptake by axons of passage. Second, all of the injection sites had minimal leakage. However, we still cannot exclude the possibility of en passage labeling completely. Addressing this issue will require further experiments, such as anatomical and functional tracings with genetic methods in future.One interesting question remains because the DLS is also known to guide habitual behavior; what are the anatomical and functional differences and similarities between the TS and the DLS? Based on previous reports (Fuccillo, HJ and HFK designed the research, performed the research and wrote the article. HJ analyzed the data with input from HFK.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.


There has been growing interest in the therapeutic potential of Cannabidiol (CBD) stemming from independent evidence that CBD has antipsychotic and anxiolytic properties in patients with mental health disorders. CBD has been found to be non-inferior to antipsychotic medication in a 4-week clinical trial in acute schizophrenia  and also has been found to reduce anxiety symptoms in social phobia and following public speaking. Human data also suggest that it attenuates the cognitive impairments associated with use of the main psychoactive ingredient in cannabis. However, whether CBD may be useful in relieving symptoms and distress in patients at clinical high-risk of psychosis (CHR) has never been tested. Furthermore, how the beneficial effect of CBD on psychotic and anxiety symptoms may be mediated in the brain remains unclear.The aim of the present study was to investigate whether short-term treatment with CBD was associated with preliminary evidence of therapeutic benefit and understand the neurocognitive mechanisms.We investigated the effects of short-term (21 days) treatment with CBD on psychotic and anxiety symptoms in 33 CHR patients, using a placebo-controlled, double-blind, parallel-arm design .In the subjects who received 21 days of treatment, we used fMRI in conjunction with a verbal memory task to assess the effect of CBD relative to placebo treatment on medial temporal and striatal function.Of the 33 CHR patients recruited into the trial, 31 completed treatment for 21 days. Following 21-day treatment , CBD-treated (n=16) CHR patients showed a significantly greater reduction in anxiety (p=0.02) and in distress associated with psychotic symptoms (p=0.03) and a trend (p=0.14) toward greater reduction in the severity of psychotic symptoms compared to those treated with placebo (n=17) CHR patients . In addition, CBD was tolerated as well as placebo.Consistent with our predictions, treatment with CBD (n=15) attenuated the engagement during verbal encoding of the parahippocampal cortex, but increased activation in the putamen in CHR patients. A similar pattern of activation was evident during verbal recall, with CBD treatment associated with increased engagement in the putamen.Results from our proof-of-concept study suggest that 3-week treatment with CBD has beneficial effects on anxiety, attenuated psychotic symptoms and the distress associated with psychotic symptoms. They also suggest that short-term treatment with CBD modulates both medial temporal and striatal function in CHR patients, regions that are critically implicated in the CHR state. Coupled with the absence of significant adverse effects associated with CBD, a particularly important issue in relation to the treatment of CHR individuals, not all of whom develop a full-blown psychotic disorder, these data indicate that long-term treatment with CBD is likely to be efficacious in CHR patients.


Many cellular processes are associated with membrane remodeling. The BAR domainprotein family plays a key role in the formation and detection of localmembrane curvatures and in attracting other proteins, including the regulatorsof actin dynamics. Based on their structural and phylogenetic properties, BARdomains are divided into several groups which affect membrane in various waysand perform different functions in cells. However, recent studies haveuncovered evidence of functional differences even within the same group. Thisreview discusses the principles underlying the interactions of different groupsof BAR domains, and their individual representatives ,with membranes. Fig. 1).The preferential binding of BAR domains to curved membrane regions makes itpossible to attract target proteins to membrane rearrangement sites.During cell movement, the coordinated processes of actin polymerization and theinteraction between actin filaments and the cellular membrane push the activecell edge forward and result in filopodia formation. These processes arecoordinated by actin-binding proteins. Disruptions in the function ofactin-binding proteins that infringe on cell motility are a distinct feature ofneoblasts. BAR family proteins act as connecting links between actin dynamicsand membrane rearrangements in all eukaryotes. BAR domains were originallydefined as the conserved regions of the animal proteins BIN and amphiphysin, aswell as the yeast Rvs161 and Rvs167 proteins There are different ways through which BAR domain proteins can affect actinpolymerization. In some cases, they activate the actin nucleation factors WASP(Wiskott-Aldrich Syndrome Protein) and WAVE (Wiskott-Aldrich Verprolin Protein), while o2) required for the binding[In addition to attracting partner proteins, SH3 domains often function asregulators of the BAR domain activity ,9. Bind binding.The PICK protein, whose functions have to do with the internalization andexposure of AMPA receptors to the cell surface, provides an interesting exampleof BAR domain activity regulation .PICK isFig. 1)[According to the Uniprot database, the BAR family currently includes more than220 proteins ; crystalFig. 1).Classical BAR domains and N-BAR domains A classical BAR domain is a dimer where each monomer consists of three bentantiparallel α-helices . TheclaAmphiphysins are among the best-studied BAR domain proteins; their functionsare associated with neuronal endocytosis .MammalsThe crystal structure of the BAR domain of drosophila amphiphysin was obtainedin 2004, and a prediction was made that similar domains can be found in manyprotein groups . Based oFig. 2)reside at the ends of the BAR domain, between the α-helices 2 and 3 and on its concavesurface. Mutations in them reduce the ability of the BAR domain to bind to the membraneand modify liposomes in vitro. It has also been shown that the26 a.a.r. N-terminal sequence of amphiphysin has an unordered structure in thesolution but folds into an amphipathic helix (AH) when interacting with lipids.The insertion of an AH helps the BAR domain generate the membrane curvature[According to X-ray diffraction analysis data, clusters of positively charged amino acid residues. Later, in vitro experiments[Fig. 1).Clusters of positively charged amino acids that are responsiblefor the binding to negatively charged lipids in the membrane reside on theirconvex, rather than on the concave, surfaceand cause membrane curvature in the opposite direction as compared to BAR’s action[Similarly to the classical BAR domains, I-BAR domains consist of threeα-helices and form dimers; many of these dimers bind to liposomes andmodify their curvature in eriments-32.The s action, 33.The gene encoding IRSp53 is actively expressed in various mammalian cells andtissues, especially in neurons. IRSp53 knockout mice showed impaired learningskills and memory . IRSp53 2 clusters at the micron scale[The atomic structure of the I-BAR domain of the Pinkbar  protein was deciphered in 2011: the structure ischaracterized by an almost zero curvature .This pron scale.F-BAR domains Another broad group of BAR domain proteins contains the F-BAR domain (Fes/CIP4homology-BAR). F-BAR proteins were found in most eukaryotes except for plants;they are considered to be the key regulators of cellular membrane curvature. Most ofFig. 1).They consist of five α-helices: the short N-terminal helix, three long and oneshort C-terminal helices, followed by a short sequence responsible for homodimerization. Thesurfaces with which monomers interact with each other mostly containhydrophobic amino acid residues and several charged ones.Mutations in the conserved positively charged amino acid residueson the concave side of F-BAR dimers reduce the ability of proteins to bind tothe membrane and modify liposomes invitro [The F-BAR domain was first discovered in the CIP4 protein (CDC42-InteractingProtein 4) . The coninvitro , 52.In vitro experiments have demonstratedthat Rgd1p preferentially binds to liposomes containing PIP2. Decipheringof the crystal structure of the complex between Rgd1p and myo-inositol-1,2,3,4,5,6-hexakisphosphate (Ins P6), which acts as an analogue of thephosphoinositide lipid head, made it possible to identify which amino acidresidues the phosphoinositide-binding site consists of.Recent studies demonstrate that some F-BAR domains selectively bind tophosphoinositides , 54.ThuThe CIP4, FBP17, and FCHo2 proteins also exhibit specificity tophosphoinositides and contain a corresponding binding site, 52,57.The interaction between BAR domain proteins and the membrane generation ofmembrane curvature, its propagation, stabilization,and/or sensing, followed by the recruitment of cytosolicprotein factors to a specific site in the cell[The main functions of BAR domains include the the cell. Generatgeneration of the membrane curvature takeplace due to the electrostatic binding of the BAR domain to the membrane and,in some cases, due to the incorporation of an N-terminal AH into the membrane[The initial stages of membrane. Bindingmembrane. Theincmembrane. It was membrane. Howevermembrane, 66do npropagation requires an interaction between many BARdomains. The structure formed by them on the membrane surface is known as ascaffold. All BAR domain proteins are believed to be capable of scaffoldformation; the scaffold structure largely determines the result of the effecton the membrane. In its turn, the scaffold structure depends on the proteinconcentration and membrane tension. It was shown by coarse-grained moleculardynamics simulation that when present at low concentrations, N-BAR domainsaggregate on flat membranes and liposomes to form a filamentary structure andnetworks; after a 20% surface density is achieved, they start forming amembrane protrusion [Curvature otrusion . N-BAR potrusion .The sensitivity of BAR domains to membrane curvature The investigation of the fluorescence intensity of the proteins on the membranetubules formed by giant liposomes has shown that BAR domains can act asdetectors of membrane curvature: the density of arrangement of the BAR domainsbound to membrane tubules is several dozen or even hundreds of times higherthan that of the BAR domains residing on a flat membrane. All the tested BARdomain proteins amphiphysin ,endophiFig. 3).Since X-ray diffraction analysis does not provide any idea on how proteinsinteract with a full-size membrane, reconstructions of the oligomers of BARdomains bound to membrane tubules were obtained by cryo-electron microscopy, 72, 73Fig. 3A).The large regions of the unoccupied membrane between theneighboring bundles (~50 Å) can be attributed to the need to provideaccess for GTPases, with which endophilin interacts during endocytosis[Studies of the arrangement of the F-BAR domains of endophilin on the membranetubule have demonstrated that they are oriented at a 10° angle withrespect to each other . The BARdomains of amphiphysin are more densely packed than those in the endophilinstructure and in such a way that one end of the BAR domain is oriented inwardinto the membrane, while the other one is oriented outwards. Unlike inendophilin, they are stably connected with one another by AHs, which presumablypartake in curvature initiation. As a result, the tubules formed by amphiphysinare much more rigid. This agrees well with the biological functions of theseproteins: amphiphysin forms stable T-tubules, while endophilin is involved inthe formation of the dynamic structures that quickly assemble and disassembleduring endocytosis [Another oligomeric structure that was studied using cryo-electron microscopyand helical reconstruction was the structure of the BAR domains of theamphiphysin II isoform involved in the organization of T-tubules.This assumption has been confirmed by cryo-electron microscopy[X-ray crystallography was used to determine the structures of individual F-BARdomains and to propose a scheme of their interaction with membranes. Incrystals, F-BAR domains form flat scaffolds where the BAR domains have alateral orientation. The BAR domains interact with each other through theterminal and lateral regions. When interacting with the membrane, the BARdomains turn so that the curved side carrying the positively charged amino acidresidues faces the membrane; the flat scaffold acquires a ring shape, and thenit becomes helical and twists around the tubule being formedand be actively involved in curvature propagation[Fig. 3E).The isolated I-BAR domains can actively form the membrane curvature. Howeverpagation.On the Stabilization of the membrane curvature in vitroexperiments, the absence of AHs made endophilin unable to modifyliposomes and form tubules. This has also been demonstrated by moleculardynamics simulation [The significance of N-terminal AHs in stabilizing interaction with lipids wasestablished using various methods ,79. In mulation .A hypotThe endophilin structure determined by cryo-electron microscopy indicated thatthe insertions of neighboring  dimersdo not interact with each other and are oriented towards the membrane. Thisdifferentiated them from the arrangement in the crystal structure and in theliposome-bound state. The difference was later attributed to two conformationalstates: at high protein concentrations sufficient for oligomer formation, theN-BAR domain resides closer to the membrane, thus contributing to a deeperincorporation of AH , impedinIn addition to attracting partner proteins, the SH3 domain of endophilinregulates the activity of the N-BAR domain. It was demonstrated by a moleculardynamics simulation that the SH3 domain in solution binds to the N-terminal AHdue to hydrophobic interactions and the formation of salt bridges betweencharged residues . The negRecently there has been evidence that not all BAR domains exhibit activity inthe formation of membrane tubules or membrane invagination. The yeast proteinCdc15p involved in cytokinesis oligomerizes into filaments and does not causemembrane modification .OligomeNontraditional orientation: The F-BAR domain protein Nervous wreck Drosophilamelanogaster is a convenient model for studying synaptic growthregulation, since the muscle area increases more than one hundredfold withinfour days, which is accompanied by a significant increase in the number ofneuronal contacts. Neuronal growth regulation includes both the retrogradesignals from the muscle and anterograde signals from the neuron to the musclecell [Neuronal growth and the formation of new connections, the processes underlyinglearning and memory, are controlled by growth factors. Receptors bound togrowth factors are moved inside the cell by endocytosis and sent to specialcellular compartments, where they can undergo modification or degradation, orinteract with other proteins .Determiclecell . Mutatioclecell -90.In vitro studies have shown that, unlike other F-BARproteins, Nwk causes the formation of cellular protrusions rather thaninvaginations [The F-BAR protein Nervous wreck (Nwk) exhibits limited homology to other F-BARproteins. Fig. 4A).2, which has a specific S-shape[Fig. 4B).Electron microscopy was used to study the ways in which the F-BAR domains of the Cip4and Nwk proteins were organized on lipids. As expected, the Cip4 F-BAR domainswere found to aggregate into linear filaments, while the Nwk F-BAR domains werefound to form higher order V-, N-shaped, and zigzag structures[Fig. 4C)[In order to study the interaction between the Nwk F-BAR domain and themembrane, its model was built based on the known crystal structure of thehomologous F-BAR domain FCHO S-shape.The end regions of the dimer that are responsible for oligomerization andelectrostatic interactions between the membrane and the concave side of F-BARplay a crucial role in this process .ProtrusThe functioning of Nwk in neurons is apparently ensured not only by its F-BARdomain, but also by two SH3 domains, each of them binding to certain proteins.Nwk in recycling endosomes interacts with the SNX16 protein belonging to thesorting nexin family, which, in its turn, is bound to the presynaptic growthfactor Tkv . The int2 concentration in the membrane leads to a rise in thedegree of order of lipids, which is characterized by an alignment ofhydrocarbon tails, increased bilayer thickness, decreased lateral diffusion coefficient, etc.[2 concentrationsuppresses the membrane-deforming activity of the F-BAR domain FBP17 inin vivo experiments [Another important function of Nwk SH3 domains is the regulation of F-BARactivity. The SH3b domain was shown to bind to F-BAR; however, this does notresult in a loss of its membrane-binding ability but only increases the amountof the negatively charged lipids needed for binding .Both thnt, etc., 99.Thent, etc..The memeriments .Bindingeriments  or byineriments .Howeverin vitro studies and electron microscopy haveallowed researchers to explain how the schemes of oligomerization of BARdomains result in the formation of various membrane structures. It has beendemonstrated how the activity of some BAR domain proteins can be regulated byintra-protein and protein–protein interactions, as well as what themechanism for achieving a specificity of partner protein recruitment is.However, despite the significant progress in understanding the role of BARdomain proteins in cell activity, many questions still remain to be answered.Taking into account that changes in the expression level and mutations in thegenes encoding BAR domain proteins are related to many serious diseases, thisfield of research is of interest both for biology and medicine.Deciphering the crystal structures of BAR domains has made it possible todescribe the mechanisms of changes in the membrane shape at the molecularlevel, while


To evaluate the different freeze-thaw variables which modify the mechanical properties of treated coals, the effects of freezing time, number of freeze-thaw cycles, and the moisture content of coal were studied using combined uniaxial compression and acoustic emission testing systems. Freezing the samples with LN2 for increasing amounts of time degraded the strength of coal within a certain limit. Comparison to freezing time, freeze-thaw cycling caused much more damage to the coal strength. The third variable studied, freeze-thaw damage resulting from high moisture content, was restricted by the coal’s moisture saturation limit. Based on the experimental results, equations describing the amount of damage caused by each of the different freeze-thaw variables were empirically regressed. Additionally, by using the ultrasonic wave detection method and fractal dimension analyses, how freeze-thaw induced fractures in the coal was quantitatively analyzed. The results also showed that the velocity of ultrasonic waves had a negative correlation with coal permeability, and the freeze-thaw cycles significantly augment the permeability of frozen-thawed coal masses.Freeze-thaw induced fracturing coal by liquid nitrogen (LN The feasibility of this fracturing technology has been studied for the exploitation of unconventional oil and gas6. An unconventional gas, the worldwide reserves of coal-bed methane (CBM) are very large8. High-efficiency extraction and utilization of CBM not only can decrease greenhouse gas emission and avoid gas disasters in coal mines but can also augment the world’s energy supply12.For fracturing coal using an anhydrous fluid, liquid nitrogen . The relationships between axial strain and the freeze-thaw variables are shown in Figs. 22. By collecting the acoustic emission signals from the frozen-thawed coal during the compression, the extension, connection, and failure of the freeze-thaw induced fractures in the coal can be inferred25.Uniaxial compression causes stress concentrations and slippage friction around freeze-thaw induced fractures. Some of the strain energy in the coal is released in the form of elastic waves and these waves can be detected as acoustic emissions (AE)2 freeze-thaw treatments. Then the freeze-thaw induced fractures were compacted during uniaxial compression and the compression generates even more fractures when the original freeze-thaw induced fractures reached their ultimate strengths. Subsequently, fractures connected and degrade the coal samples to even more extent. The frozen-thawed coal produced a large number of acoustic emissions at different stages of compression including friction and compression signals, cracking signals, and splitting signals.A few micro-fractures existed in the original coal samples. Additional fractures at many scales were generated in the coal samples by the LNIn this study, the compression failure of the frozen-thawed coal was divided into three stages: compaction, elastic deformation and yield failure stages. Figure 2 freezing time, number of freeze-thaw cycles and moisture content in the coal) is shown in Fig. 2 freezing time and freeze-thaw cycles while a positive correlation with moisture contents in coal. Equations fit to the relationship between the three freeze-thaw variables and the ultimate uniaxial compressive strength for these coal samples are listed in Table Uniaxial compressive strength refers to the ultimate stress on a sample at failure under uniaxial compression. The smaller the uniaxial compressive strength, the easier the coal can be damaged under the load. The relationship between uniaxial compressive strength of the frozen-thawed coal and freeze-thaw variables  and freeze-thaw damage variable D. The relationship between freeze-thaw damage variable D and freezing time T is exponential as is the relationship between D and the number of freeze-thaw cycles C. The relationship between D and the coal’s moisture content, w, is linear. The equations for these relationships are shown below.Figure D reaches a value of about 0.12. This appears to be the limit for coal damage from freezing time alone. However, D continues to rise with an increase in the number of freeze-thaw cycles and the rate of damage suffered by the coal increases after 20 freeze-thaw cycles. The variable D shows a negative correlation with moisture contents and this suggests that the lower the moisture content, the more seriously the coal has been damaged in the elastic stage of deformation, and the result is consistent with the results from the acoustic emissions measurements. Because the maximum saturated moisture content for coal is with the 10–15% range, the influence of moisture contents on freeze-thaw damage is restricted by the fully saturation condition.Figure 31. Among these methods, box dimension based on grid coverage is most widely used for quantitatively analyzing fracture surfaces30.Fractal dimension is an important parameter for characterizing fracture distribution and connectivity of coal and rocks. Surface geometries of coal fractures can be quantitatively described by fractal dimension. The surfaces of fractures generated in the coal during freeze-thaw cycles were studied using fractal geometry. Fractal dimension can be calculated by several methods including correlation, similarity, capacity, information dimension, and box dimensionδ so as to calculate the number, N(δ), of fractures in the grids. Different N(δ) can be obtained by changing the side length of the grids. The methods for grid partitioning on the fracture surfaces of frozen-thawed coal and fracture calculation are provided in Supplementary Fig. δ) of fractures and side length δ of grids using the least square method. The slope of the regression line represents the fractal dimension of the fracture surfaces30. The regression equation is shown as equation  and water-ice (H2O) phase transitions after pumping LN2 into a coal seam causes three fracturing effects through expansion, frost heaving, and low-temperature fracturing. Because a few water-bearing initial fractures existed in the original coal, water in the fractures froze and expanded . Th. ThT, nuK increased exponentially with T and C and linearly with w as expressed by equations (Permeability quations –14). Th. ThK inc38. As expressed in equation .2) freeze-thaw treatments results in dissimilar damage to the coal samples. When the coal is frozen, the absolute values of axial and hoop strains in the frozen-thawed coal decrease as freezing time increase, the number of freeze-thaw cycles, and moisture contents in the coal increase. The uniaxial compressive strengths and elastic moduli of the coal samples are negatively correlated with freezing time and the number of freeze-thaw cycles and positively correlated with the coal’s moisture content. However, the coal’s Poisson’s ratio shows contrary results. The uniaxial test results are consistent with the acoustic emission data. The results allowed equations quantifying the changes caused by the three different freeze-thaw variables to the mechanical properties of the coal to be written.Modifying samples using different liquid nitrogen (LND increases to about 0.12 but does not rise above that value. Variable D is negatively correlated with the coal’s moisture contents which shows that the lower the moisture content is, the larger the amount of damage done in the elastic stage of deformation. However, the upper limit of freeze-thaw damage related to moisture content that the coal can sustain is controlled by the coal’s water saturation limit.With increasing freezing time, the value of the freeze-thaw damage variable 2 shows numerous advantages for modifying coal reservoirs and therefore is expected to become one of the important technologies for developing CBM resources efficiently.Using the box counting method on fractures in the coal shows that fractal dimension increases from 1.06 for samples frozen-thawed through five freeze-thaw cycles to 1.23 for samples subject to 25 cycles. The direction in which the fractal dimension changed is always positive. The ultrasonic wave velocities exhibit a negative correlation with coal permeability and show that the freeze-thaw cycles have a significant effect on increasing the frozen-thawed coal’s permeability. Freeze-thaw fracturing with LNThe coal samples used for these experiments were lignite collected from the Shengli coalfield in Inner Mongolia, China. The maceral contents and proximate analyses for the coal samples are listed in Supplementary Table There are three main factors that control how freeze-thawing modifies and fractures coal. They are freezing time, number of freeze-thaw cycles, and the moisture content of coal. After being numbered, the coal samples were treated in a vacuum saturation device until they were fully saturated with water. Coal samples with different moisture contents were then produced by drying them for different lengths of time in a vacuum drying oven. Then samples with different moisture contents, including some completely saturated samples, were frozen separately for 1, 5, 10, 20, 30, 40, 50, and 60 min. Another set of samples was used for freeze-thaw experiments. The freeze-thaw experiments were conducted for 1, 5, 10, 15, 20, 25 and 30 cycles, each cycle involving 5 min of freezing and 5 min of thawing at room temperature. A third set of coal samples with different water contents were frozen for 90 min. These samples had moisture contents of 0%, 5.13%, 7.9%, 11.9%, and 13.9%. Following these freezing procedures, uniaxial compression tests were conducted on the samples. These compression tests included strain monitoring and the collection of acoustic emission signals during the compression tests and were conducted with the equipment shown in Supplementary Fig. Factors controlling the mechanical properties degradation and permeability of coal subjected to liquid nitrogen freeze-thaw


Novel silica nanowires and interconnected nanorings were firstly synthesized on a graphite paper by typical thermal catalytic chemical vapor deposition method, using silicon and carbon black powders as raw materials. The field emission scanning electron microscopy, energy dispersive x-ray spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and transmission electron microscopy were used to investigate the composition and structure characterization, which indicates that the silica nanowires and interconnected nanorings were amorphous. The growth of the as-prepared silica nanowires and interconnected nanorings was related to the vapor-liquid-solid mechanism, but the nanowire-ring structure may be due to the polycentric nucleation and periodic stable growth with gradual direction changes. The room temperature photoluminescence emission spectrum showed that the silica nanostructures emitted strong blue light at 460 nm, resulting from the combination of neutral oxygen vacancy (≡Si-Si≡) and selftrapped excitons. The as-synthesized novel silica nanowires and interconnected nanorings could be a potential candidate for applications in future light-emitting diodes and optoelectronic nanodevices. As known to all, the properties and potential applications for nanomaterials were always affected by their morphology and structure. Therefore, most studies recently, on one side, were focused on the development of nanostructures  with multifarious morphologies, such as nanowires, nanobelts, nanorods, nanotubes, nanocables, nanorings, and nanochains12. And on the other hand, various methods also have been applied to assemble the as-prepared nanostructures into desired architectures for the practical needs of nanophotonics and nanoelectronics, and reported many exciting nano-micro-scale structures for function oxides , like fish bones, combs, dendrite spines, bridges, and windmills17.One dimensional (1D) nanostructured materials have attracted worldwide intense attention due to their novel optical and electrical properties, and owned potential applications in the integrated energy storage, sensors, photonics, and electronics devices19. Similarly, the observed luminescence properties of silica nanostructures are significantly different in various morphologies and also different processing methods20, such as the laser-ablated silica nanowires with two strong blue bands at 420 and 470 nm21, the thermal evaporated silica nanowires with strong green emission at 540 nm22, the chemical vapor deposition (CVD) jellyfish-like nanowires with a blue emission band at 415 nm18, as well as the spring-shaped, fishbone-shaped, frog-egg-shaped, and pearl-shaped silica nanowires with different properties and corresponding applications27. For the development of classical optical waveguides, the different cross sections would result in different transverse optical modes28. Thus, various structures and shapes for waveguides were studied and reported recently, such as Y-branching29, star type30, and ring structure31. Therefore, the preparation of silica nanowires with novel structures is now considered as an interesting and meaningful research direction.Among these functional nanostructures, silica nanowires are important optical materials for potential application in low dimensional wave-guides for future integrated optical devices and high-resolution optical heads of scanning near-field optical microscopes, due to their intense and stable emissionIn this paper, the novel silica interconnected nanorings were prepared and reported for the first time. Silica powder and coke powder were used as raw materials, and aluminum nitrate acted as a catalyst. The growth mechanism for the silica nanowire (NWs) and interconnected nanorings (ICNRs) was discussed in detail. The photoluminescence (PL) spectrum was investigated to clarify the optical properties of as-prepared samples. We think this study could provide a new way to synthesize novel morphology nanostructures of silica and other oxide or non-oxide materials for the application of new generation nano-devices with tunable photoelectric performance.3)3 as catalyst, and argon as flowing gas. The morphology results indicated that of these silica nanostructures were mainly composed of NWs and ICNRs. For here, the amount of the ICNRs nanostructures in the products is about 50%, and this study is the first time to report the novel structure so far. Both the wire size of nanostructures is uniform and its diameter is about 200 nm. According to the separated ICNRs images shown in Fig. Figure −1 and 812 cm−1 are attributed to the Si-O-Si stretching vibration, and the peak at about 1135 cm−1 is correspond to the Si-O stretching vibration of amorphous SiOx. Moreover, XPS data was also used to confirm the elemental makeup and the oxidation state of the as-prepared nanostructures. As is shown in Fig. 233. No any other obvious Si XPS peaks are observed here. The atom ratio of Si (30.31%) and O (61.24%) is calculated to be 1:2.02, which is just a little higher than the ideal proportion of Si and O elements for SiO2 due to some atomic oxygen adsorbed on the surface. Therefore, it can be concluded the composition as-prepared samples are silica products.Figure Further investigation on the internal microstructure of the as-fabricated nanostructures was initiated by TEM equipped with EDS, and the details were presented in Fig. 34. Herein, Al(NO3)3 serves as the catalyst precursor and Al plays an important role in the growth and formation of the novel silica nanostructures, which was observed at the end of the nanowires by EDS spectra. A growth model for the silica nanostructures was proposed and shown in Fig. 35. The Al atoms coalesce to nanoparticles, then deposit on the graphite paper and finally combine into micro-particles with high catalytic activity for the growth of silica nanostructures. The second step is the formation of gaseous silicon species. It is apparent that the silica is reduced to gas phase silicon monoxide and silica by carbon black, which also enables the formation of silica nanostructures by the VLS mechanism. The third step is the formation of silica nanostructures, including the nucleation step and growth step. Theoretically, the nucleation process could be further divided into monocentric and polycentric nucleation. When the gaseous silicon and silicon monoxide transport to the surface of the graphite paper and react with Al to form Al-Si-O droplets, the monocentric nucleation sites obtain firstly around the catalyst droplets. Simultaneously, both the silicon and silicon monoxide are oxidized to amorphous silica by the oxygen remained in the furnace or released from the refractory lining. As a result, the silica NWs begin to form, while the catalyst particles are lifted up from the graphite paper exactly as the normal VLS mechanism said. If the silica trunks periodically grow stably during the growth process, the silica nanowires would obtain on the graphite paper. But on the other hand, some Al-Si-O nanoparticles may also deposit on the top surface of the nanowire trunks  during the periodic stable growth, which would form two or more new growth centers (namely polycentric nucleation) and result in the branch-Y-shaped nanowires. The branch of Y-shaped nanowires can also grow comparable stably, but their growth directions may change and tend to from a ring. The change of growth direction may originate from the dislocation inside the nanostructures, but the growth mechanism of such a nanowire-ring formation process is still not clear. A possible explanation may be the effects of Van der Waals interactions or the surface energy minimization37. When the top of the branched nanowires meet together, the Al-Si-O catalytic droplets combine to become one new growth center. Then the next immediate procedure is another stable growth of silica nanowires. If the polycentric droplets were formed in the further reaction procedure, the nanowire-ring structure would be acquired again due to the polycentric nucleation and periodic stable growth with the gradual direction changes. Finally, the silica NWs and ICNRs products were obtained.These results indicate that the growth process of the amorphous silica nanostructures in our study could be explained by vapor-liquid-solid (VLS) mechanism, which has been put forward by Wagner and Ellis38. Thus the PL process could be discribed as follow: silica nanostructures firstly absorb the energy of 4.88 eV photons by ground-to-singlet transition or energy transfer from excited states , then then the energy is tranferred from the excited singlet to the triplet states by intersystem crossing, and finally emit the 2.76 eV emission by forbidden transition from the triplet state to the singlet state39. However, the emission at 2.30 eV observed in this study is scarely reported and the exact mechanism of the green emission for silica nanostructures also remains unclear22. The possible reason may be the rediative recombination of the selftrapped excitons, such as e-h pairs or excitons formed by the two-photon cross band-gap excitation40. Another possibility might be ascribed the surplus oxygen in local context of the amorphous silica products38. According to the recent literature, the PL properties of silica nanowires are different due to their various structures and stoichiometry41. The novel amorphous silica NWs and ICNRs here could be potential optical materials for visible light region.In order to investigate the optical properties of the novel silica nanostructures, the PL spectrum was recorded at room temperature using a Xe lamp as the excitation source. Figure 2 powders and coke powders as raw materials. The as-prepared silica nanostructures mainly present as the morphologies of NWs and ICNRs, which are both about 200 nm in diameter. The structure investigations indicate that these nanostructures are amorphous. The growth of the silica nanowires are related to the normal vapor-liquid-solid (VLS) mechanism with monocentric nucleation and periodic stable growth process. While the growth process of silica interconnected nanorings are in accordance with the polycentric nucleation and periodic stable growth with the gradual direction changes. The PL measurement indicates that the strong blue emission spectrum for the as-synthesized silica nanostructures may be resulted from the combination of neutral oxygen vacancy (≡Si-Si≡) and selftrapped excitons. This study provides a simple method to gain new morphology for silica nanostructures, and the obtained novel silica NWs and ICNRs with outstanding optical properties could be considered as candidates for the application in optoelectronic nanodevices and light-emitting diode (LED) materials.Novel silica nanowire (NWs) and interconnected nanorings (ICNRs) have been successfully prepared via a typical thermal catalytic chemical vapor deposition (CVD) route using SiO2 powders and coke powders, were well-mixed in an agate mortar for 30 min. About 2 g of the milled mixtures was loaded in one side of a corundum crucible, and a graphite paper was placed on the opposite side dispersed with several drops of Al(NO3)3 aqueous solutions on both the top and bottom surfaces and dried in air. The aluminum crucible was put into the center of the long corundum tube in a tube furnace. The mixed powders were placed close to the argon-intake end. The experimental tube was vacuumized firstly by a rotary pump and then argon was introduced and kept flowing with a furnace pressure of 0.15 MPa during the whole heat treatment process. Meanwhile, the setup was heat from room temperature to 1350 °C at 3 °C/min, held for 3 h, and then cooled to room temperature naturally. Two white layers of products were finally obtained on both two sides of the graphite paper.The typical synthesis reaction was conducted by a simple CVD procedure in a corundum tube furnace, using argon (purity 99.99%) as a carrier gas. The experimental setup is shown in Fig. 2-F30, USA) equipped with energy dispersive X-ray spectroscopy (EDS). Fourier transform infrared spectroscopy (FT-IR) data were collected by a Nicolet IR100/200 spectrophotometer from 450 cm−1 to 1500 cm−1, using the standard KBr pellet technique. A PHI 5300 X-ray photoelectron spectroscopy system  was used to acquire the chemical states of Si 2p, and O 1s with a chamber base pressure of approximately 10−7 torr using Mg Kα X-rays at a take-off angle of 45°, and the total power was of 400 W. The PL spectra of as-prepared samples were recorded by fluorescence spectrophotometer  using a 150 W Xe lamp as excitation source. All the measurements were determined at room temperature.The as-prepared samples were characterized by field emission scanning electron microscopy  and transmission electron microscopy (TEM/HRTEM, FEI-Tecnai-G


MicroRNAs (miRNAs) regulate gene expression through interactions with target sites within mRNAs, leading to enhanced degradation of the mRNA or inhibition of translation. Skeletal muscle expresses many different miRNAs with important roles in adulthood myogenesis (regeneration) and myofibre hypertrophy and atrophy, processes associated with muscle ageing. However, the large number of miRNAs and their targets mean that a complex network of pathways exists, making it difficult to predict the effect of selected miRNAs on age-related muscle wasting. Computational modelling has the potential to aid this process as it is possible to combine models of individual miRNA:target interactions to form an integrated network. As yet, no models of these interactions in muscle exist. We created the first model of miRNA:target interactions in myogenesis based on experimental evidence of individual miRNAs which were next validated and used to make testable predictions. Our model confirms that miRNAs regulate key interactions during myogenesis and can act by promoting the switch between quiescent/proliferating/differentiating myoblasts and by maintaining the differentiation process. We propose that a threshold level of miR-1 acts in the initial switch to differentiation, with miR-181 keeping the switch on and miR-378 maintaining the differentiation and miR-143 inhibiting myogenesis. The mechanisms leading to this loss in homeostasis are not fully understood but it is hypothesised that changes in gene expression and dysregulation of cellular signalling pathways are key contributors. Recently, miRNAs have been shown to play important roles in muscle maintenance during adulthood and ageing5. Muscles express their own set of muscle-enriched miRNAs known as myomiRs: miR-1, -206, -208, -133, -486 and -4996. Satellite cells, adult muscle stem cells contributing to muscle regeneration, express different sets of miRNAs during quiescence and activation1 suggesting the role of miRNAs in regulating satellite cell homeostasis. Moreover, the expression profile of miRNAs in satellite cells and muscle is altered during exercise and ageing8. The formation of new muscle fibres and repair of injured myofibres (myogenesis) requires the differentiation of myogenic progenitors or satellite cells, respectively9 and several miRNAs have been shown to regulate myogenesis in adulthood, including miR-1, miR-206, miR-27, miR-378 and miR-18112.Muscle homeostasis is perturbed in ageing leading to loss of muscle mass and function - sarcopenia. Muscle homeostasis depends on the balance between muscle hypertrophy and atrophy and successful muscle regeneration following injury. During ageing, muscle regeneration is defective and this is attributed, at least to some degree, to changes in satellite cell properties13. On the other hand, MyoD has been shown to upregulate the expression of many miRNAs, including miR-114. Pax3/Pax7 are expressed in embryonic myogenic progenitor cells and in quiescent satellite cells, whereas MyoD expression is low in the undifferentiated progenitors. Upon activation of satellite cells/myogenic progenitors, the expression of Pax3 is downregulated, whereas MyoD expression is upregulated following satellite cell activation. The downregulation of Pax3/Pax7 expression is partly mediated by miR-1 and miR-206, which ensure complete silencing of the Pax genes to confer robustness to the timing of myogenesis in development13. In addition, miR-206 and miR-486 have been shown to induce myoblast differentiation by downregulating Pax7 expression15, and miR-27 inhibits Pax3 expression in satellite cells16. It has also been shown that Pax3 protein is regulated by monoubiquitination and degradation by the proteasome17. On the other hand, MyoD expression has been shown to be inhibited by Musculin (Msc) , expressed during early stages of myogenesis and down-regulated at later stages. Therefore, the balance between these two, as well as other myogenic factors such as Myf-518, contributes to the regulation of early stages of myogenesis: commitment and differentiation. Moreover, the expression of miR-1 and miR-133 has been shown to decrease during skeletal muscle hypertrophy and ageing19.We have shown that miR-1 and miR-206 down-regulate the expression of the transcription factor Pax3, which inhibits MyoD, a myogenic regulatory factor (MRF) that is essential for myogenesis to occur12. miR-181 inhibits Hox-A11 at the protein level, which suggests that it inhibits translation of Hox-A11 mRNA12. miR-181 has also been shown to inhibit Sirt-1 expression at the protein level, which leads to a decrease in myotube size20. This may be partly mediated by Sirt-1 inhibition of FoxO3, a transcription factor that upregulates the muscle-specific E3 ligase Mafbx which in turn leads to an increase in protein degradation and muscle atrophy. Interestingly, MyoD has been shown to be one of the targets of Mafbx21. Sirt-1 has also been shown to promote cell proliferation by decreasing the expression of the cyclin-dependent kinase inhibitor, p2122 and to block the induction of autophagy-related genes leading to a decrease in proteolysis23. Therefore, miR-181 may inhibit Sirt-1-induced hypertrophy. Moreover, upregulation of miR-181 may also lead to inhibition of myoblast proliferation. miR-181a has been shown to be down-regulated in mice and human skeletal tissue with age20, therefore it may contribute to dysregulation of myogenesis with age.Another miRNA, miR-181, is strongly upregulated during myogenesis and positively regulates myogenic differentiation through inhibiting Hox-A11, another antagonist of MyoD, expression11. miR-378 may provide robustness to myogenesis through inhibition of Msc expression during later stages of the differentiation process. During C2C12 differentiation, miR-378 expression is increased more than four-fold, with a slight increase on day 1, a larger increase on day 2 and no further increase up to day 4, suggesting its key role during early steps of myogenesis11. Interestingly, miR-378 is encoded within an intron of the Ppargc1b gene which encodes PGC-1β, a regulator of energy metabolism. As miR-378 is co-expressed with PGC-1β, it has been suggested to play a role in metabolism and known interactions with target genes has confirmed this (reviewed in24). miR-378a-5p has been shown to be down-regulated with age in men7. This study also showed that after exercise miR-378a-5p is downregulated in young but not in older people, suggesting a blunting effect of age on miR-378 expression in muscle following exercise. miR-378 and several other miRNAs also target the components of the insulin-like growth factor-1 (IGF-1) signalling pathway, resulting in loss of muscle homeostasis7. However, there have been no studies to date to show that miR-378a-3p, the strand investigated in other studies changes with age3.During myogenesis, MyoD activation leads to upregulation of the expression of several miRNAs, including miR-378, an inhibitor of Msc25. miR-143-3p expression is downregulated in satellite cells and muscle progenitors during ageing, and changes in miR-143:Igfbp5 interactions have been proposed to be a part of a compensatory mechanism aimed at improving the efficiency of myogenesis during ageing. Interestingly, treatment of myoblasts with the cytokine IL-6, which has been shown to be elevated in circulation during ageing26, led to lower expression of miR-143 and an increase in Igfbp525. However, it is not yet clear how IL-6 leads to the reduction in miR-143 expression.A recent study identified miR-143–3p as a regulator of the IGF-binding protein-5 (Igfbp5), that regulates IGF signalling through binding IGF-1 and IGF-2, in primary myoblastsWe have previously demonstrated the utility of computer simulation models to examine the effect of miRNAs in gene regulatory circuits (in submission). These models show how miRNAs affect target gene expression by different mechanisms such as positive feedback, negative feedback and positive feedforward loops. Each type of mechanism leads to different effects on the target genes and includes pulse-like responses, extended repression, delays in responses and fine-tuning. The aim of this study was to use computational modelling to increase our understanding of the mechanisms by which individual miRNA:target interactions affect the decline in muscle regeneration during ageing. In order to do this, we constructed network models of a subset of miRNAs that are known to be important in the maintenance of muscle homeostasis and are up- or down-regulated during ageing. To start modelling ageing-associated changes in the muscle, we chose the networks involving miR-1, miR-143, miR-181, and miR-378, miRNAs with roles in muscle homeostasis and expression of which is dysregulated in muscle and satellite cells during ageing.In order to create a model of the effects of selected miRNAs on myogenesis, we first overexpressed and inhibited these miRNAs in undifferentiated C2C12 cells. Overexpressing miR-1, miR-181, or miR-378 improved the efficiency of myogenesis, whereas increased levels of miR-143 decreased myogenesis efficiency as compared to scrambled-transfected controls Fig. . Convers14 and that miR-1 enhances the degradation of Pax3 mRNA13. In addition, as Pax3 expression was found to be negatively correlated with MyoD13 we assumed that Pax3 inhibits transcription of MyoD resulting in a negative feedback loop  in the total absence of miR-1 and compared this to our results in which MyoD induces miR-1 expression. In order to fit our model to this data, it was necessary to assume that miR-1 enhances degradation of Pax3 transcript but does not inhibit Pax3 translation. Therefore, we needed to include a reaction in which Pax3 mRNA bound by miR-1 could be translated into protein. The model output shows that the presence of miR-1 leads to about a 30–50% reduction in Pax3 transcript and protein at 48 h, which is in good agreement with the experimental data13 , miR-143 levels return to normal  modelling standard37), and also established as a tool in modelling congenital heart disease (reviewed in38). Despite the success of modelling in these fields, there is still a paucity of computational models in the study of human diseases. In particular, to our knowledge, there is currently a lack of computational models of the underlying molecular mechanisms leading to muscle dysfunction in ageing and disease. Our method has the potential to be used to help increase our understanding of other diseases related to muscle such as Duchenne Muscular Dystrophy (DMD) and myositis. DMD is a severe type of muscular dystrophy for which there is currently no cure. It is caused by a mutation in the dystrophin gene, leading to dysregulation in calcium and other signalling pathways, mitochondrial dysfunction and necrosis of muscle fibres. However, the underlying pathways are complex and not fully understood, suggesting the need for computational modelling. Considering the limited success of current therapies undergoing clinical trials, additional computational approaches could possibly be used to enhance the success of exon skipping or to propose other therapies. Myositis is charactersied by the infiltration of immune cells into the muscle leading to chronic inflammation, however, the mechanisms by which damage to muscle occurs is poorly understood. Particularly, the mechanism by which changes in the immune cells infiltrating the muscle and changes within the muscle tissue itself contribute to the onset of myositis are not understood. We suggest that computational modelling could be useful in simulating the effects of an increase in pro-inflammatory cytokines on the function of muscle cells.Computational modelling has previously been extensively used in the field of cancer . Sirt-1 activity is also dysregulated in ageing leading to lower expression of antioxidants and increased oxidative stress42. Moreover, Sirt-1 has been shown to positively regulate metabolic state of satellite cells and autophagy, which is also associated with FoxO3 activity43. Therefore, including more detail of the pathways involving Sirt-1 would be important in the context of modelling age-related changes in muscle regeneration.The miR-181 model suggests that above a threshold level, miR-181 promotes myogenesis, however it may also lead to either muscle atrophy or inhibition of hypertrophy. This is due to the inclusion of two targets: Hox-A11 and Sirt-1, which both inhibit myogenesis via different pathways. In addition, Sirt-1 inhibits muscle atrophy and promotes hypertrophy. Therefore, the effect of miR-181 via Sirt-1 is complex and requires further investigation into what determines the different outcomes due to Sirt-1. External conditions such as mechanical signals and nutrient supply regulate Sirt-1 activity and so the effect of Sirt-1 may depend on the energy status of the cell.  in agreement with our experimental data Fig.  and a pr48, miR-128 downregulates genes involved in the insulin/IGF1 signalling pathway in skeletal muscle49, and miR-206, a miRNA in the same family as miR-1 also targets Pax313. For a comprehensive discussion of the role of miRNAs in skeletal muscle we refer the reader to recent reviews50. Since miRNAs are involved in many different gene regulatory circuits and affect multiple signalling pathways, it will be important to investigate the cross-talk between miRNAs, their regulators and their targets. Computational modelling can contribute to this by combining models of individual miRNAs into an integrated model and then simulate the effect of perturbing one or more components on the overall behaviour of the system. We illustrated this approach by combining each of the models described in this paper into an integrated model. The output from this model showed that crosstalk between the pathways affected levels of miRNAs themselves, especially those that are regulated by MyoD, due to different pathways having an inhibitory effect on MyoD activity. In addition, we examined the effects of overexpressing each miRNA individually and also in combination and showed that miR-1, miR-181 and miR-378 all have positive effects on myogenesis, whereas overexpression of miR-143 leads to detrimental effects due to lower levels of myogenin. This is consistent with our experimental data  or for making predictions. It should be pointed out that we do not claim that we have developed the “best models” as modelling is a dynamic process and future research may produce new data which is inconsistent with our models. In this case the model output may provide clues as to what is missing or incorrect in the models so that modifications can be made. SBML models are very amenable to this process and examples of adapting models for new data has been illustrated in the literature.in vivo studies, so that only targets that have been predicted by the model to be beneficial need to tested. The models can also take into account redundancy as has been observed for miR-1 and miR-206 in which single knock-outs in mice have hardly any phenotype52, highlighting the possible need for a multiple target approach. Finding the best combination of multiple targets may be infeasible and unethical in an experimental setting but well-suited to a computational approach.Finally, there are major advantages of using computational modelling from both financial and ethical perspectives. For example, computer simulation can be used to investigate the effects of single and multiple targets before 53 and a Python tool to convert the code into SBML31. Simulations were performed in COPASI 4.1854 using either the deterministic LSODA algorithm55 or the stochastic direct method which is based on the Gillespie algorithm56. The LSODA algorithm solves stiff and non-stiff systems of ordinary differential equations by automatic selection55. We used the COPASI default parameters for this solver . The stochastic Gillespie direct method simulates every reaction, updating the number of molecules of each species after a reaction occurs. At each time-step a random number is generated to determine the next reaction to occur and the time interval to that reaction. The probability of any particular reaction occurring is proportional to its associated parameter value multiplied by the number of substrate molecules. Each stochastic simulation produces a different trajectory. Therefore multiple stochastic simulations were also carried out on a cluster using software developed at Newcastle University57 which also used the Gillespie algorithm. This software is encoded in the C programming language and is available on request. Simulation data were analysed and plotted in R using ggplot258. Network diagrams of the models were created in CellDesigner59 which used the Systems Biology Graphical Notation60.All models were constructed using SBML shorthand61. This allowed for isolation of a pure population of primary myoblasts retaining the expression patterns of selected miRNAs.Primary myoblasts from adult (6 months old) and old (24 months old) mice were prepared from EDL muscles via collagenase and dispase digestion as described by us previously52. Images were taken using Zeiss fluorescent microscope and analysed using ImageJ as in25.Mouse primary myoblasts were maintained in DMEM media supplemented with 20% fetal bovine serum, 10% horse serum and 1% penicillin/streptomycin. To induce myogenic differentiation 90% confluent cells were cultured in DMEM supplemented with 5% horse serum and 1% penicillin/streptomycin (DM). Myoblast differentiation was assessed 10 days following the switch to DM by immunostaining for myosin heavy chain using the MF20 antibody13, as indicated in figures, using Lipofectamine 2000TM 52. AntimiR-scrambled-transfected cell served as controls. This provided a transfection efficiency of 40–70%, depending on the molecule transfected as described previously52.Myoblasts were transfected with 100 nM miRNA mimic or antimiR (Qiagen) or scrambled antagomiR not predicted to bind to any know miRNA62. cDNA synthesis (mRNA) was performed using 500ng RNA and SuperScript II according to the manufacturer’s protocol (Invitrogen). cDNA synthesis (miRNA) was performed using 100ng RNA and miRscript RT kit II according to the manufacturer’s protocol (Qiagen). qPCR analysis was performed using miRScript SybrGreen Mastermix or sso-Advanced SybrGreen Mastermix in a 20 µl reaction . Expression relative to β-2 microblobulin and/or 18S (mRNA) or Rnu-6 and/or Snord-61 (miRNA) was calculated using delta delta Ct method.RNA isolation and quantitative real time PCR were performed using standard methods as described previously64. These myoblasts retained the expression patterns of miRNAs.The study was performed using male wild type C57Bl/6 mice . Mice were obtained from Charles River (Margate). All mice were maintained under specific-pathogen free conditions and fed ad libitum a standard chow and maintained under barrier on a 12-h light-dark cycle. For tissue collection, mice were culled by cervical dislocation. The tissues were immediately excised, frozen and stored at −80 °C. Experiments were performed in accordance with UK Home Office guidelines under the UK Animals (Scientific Procedures) Act 1986 and received ethical approval from the University of Liverpool Animal Welfare and Ethical Review Body (AWERB). Primary myoblasts from adult (7 months old) and old (24 months old) mice were prepared from EDL muscles following single fibre isolation as previously described65 and assigned the identifiers MODEL1704110000-MODEL1704110004.The SBML code for each of the four individual models and the integrated model was deposited in the public repository, BiomodelsSupplementary Information