Local versus systemic control of bone and skeletal muscle mass by components of the transforming growth factor-ß signaling pathway.

Skeletal muscle and bone homeostasis are regulated by members of the myostatin/GDF-11/activin branch of the transforming growth factor-ß superfamily, which share many regulatory components, including inhibitory extracellular binding proteins and receptors that mediate signaling. Here, we present the results of genetic studies demonstrating a critical role for the binding protein follistatin (FST) in regulating both skeletal muscle and bone. Using an allelic series corresponding to varying expression levels of endogenous Fst, we show that FST acts in an exquisitely dose-dependent manner to regulate both muscle mass and bone density. Moreover, by employing a genetic strategy to target Fst expression only in the posterior (caudal) region of the animal, we show that the effects of Fst loss are mostly restricted to the posterior region, implying that locally produced FST plays a much more important role than circulating FST with respect to regulation of muscle and bone. Finally, we show that targeting receptors for these ligands specifically in osteoblasts leads to dramatic increases in bone mass, with trabecular bone volume fraction being increased by 12- to 13-fold and bone mineral density being increased by 8- to 9-fold in humeri, femurs, and lumbar vertebrae. These findings demonstrate that bone, like muscle, has an enormous inherent capacity for growth that is normally kept in check by this signaling system and suggest that the extent to which this regulatory mechanism may be used throughout the body to regulate tissue mass may be more significant than previously appreciated.

Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A.

Myostatin (MSTN) is a transforming growth factor-ß (TGF-ß) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-ß family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Targeting myostatin/activin A protects against skeletal muscle and bone loss during spaceflight.

Among the physiological consequences of extended spaceflight are loss of skeletal muscle and bone mass. One signaling pathway that plays an important role in maintaining muscle and bone homeostasis is that regulated by the secreted signaling proteins, myostatin (MSTN) and activin A. Here, we used both genetic and pharmacological approaches to investigate the effect of targeting MSTN/activin A signaling in mice that were sent to the International Space Station. Wild type mice lost significant muscle and bone mass during the 33 d spent in microgravity. Muscle weights of Mstn-/- mice, which are about twice those of wild type mice, were largely maintained during spaceflight. Systemic inhibition of MSTN/activin A signaling using a soluble form of the activin type IIB receptor (ACVR2B), which can bind each of these ligands, led to dramatic increases in both muscle and bone mass, with effects being comparable in ground and flight mice. Exposure to microgravity and treatment with the soluble receptor each led to alterations in numerous signaling pathways, which were reflected in changes in levels of key signaling components in the blood as well as their RNA expression levels in muscle and bone. These findings have implications for therapeutic strategies to combat the concomitant muscle and bone loss occurring in people afflicted with disuse atrophy on Earth as well as in astronauts in space, especially during prolonged missions.

Sensory evolution in a cavefish radiation: patterns of neuromast distribution and associated behaviour in Sinocyclocheilus (Cypriniformes: Cyprinidae).

The genus Sinocyclocheilus, comprising a large radiation of freshwater cavefishes, are well known for their presence of regressive features (e.g. variable eye reduction). Fewer constructive features are known, such as the expansion of the lateral line system (LLS), which is involved in detecting water movements. The precise relationship between LLS expansion and cave adaptation is not well understood. Here, we examine morphology and LLS-mediated behaviour in Sinocyclocheilus species characterized by broad variation in eye size, habitat and geographical distribution. Using live-staining techniques and automated behavioural analyses, we examined 26 Sinocyclocheilus species and quantified neuromast organ number, density and asymmetry within a phylogenetic context. We then examined how these morphological features may relate to wall-following, an established cave-associated behaviour mediated by the lateral line. We show that most species demonstrated laterality (i.e. asymmetry) in neuromast organs on the head, often biased to the right. We also found that wall-following behaviour was distinctive, particularly among eyeless species. Patterns of variation in LLS appear to correlate with the degree of eye loss, as well as geographical distribution. This work reveals that constructive LLS evolution is convergent across distant cavefish taxa and may mediate asymmetric behavioural features that enable survival in stark subterranean microenvironments.

Evolving in the darkness: Phylogenomics of Sinocyclocheilus cavefishes highlights recent diversification and cryptic diversity.

Troglomorphism-any morphological adaptation enabling life to the constant darkness of caves, such as loss of pigment, reduced eyesight or blindness, over-developed tactile and olfactory organs-has long intrigued biologists. However, inferring the proximate and ultimate mechanisms driving the evolution of troglomorphism (stygomorphism) in freshwater fish requires a sound understanding of the evolutionary relationships between surface and stygomorphic lineages. We use Restriction Site Associated DNA Sequencing (RADseq) to better understand the evolution of the Sinocyclocheilus fishes of China. With a remarkable array of derived stygomorphic traits, they comprise the largest cavefish diversification in the world, emerging as a multi-species model system to study evolutionary novelty. We sequenced a total of 120 individuals throughout the Sinocyclocheilus distribution. The data comprised a total of 646,497 bp per individual, including 4378 loci and 67,983 SNPs shared across a minimum of 114 individuals at a given locus. Phylogenetic analyses using either the concatenated RAD loci (RAxML) or the SNPs under a coalescent model (SVDquartets, SNAPP) showed a high degree of congruence with similar topologies and high node support (>95 for most nodes in the phylogeny). The major clades recovered conform to a pattern previously established using Sanger-based mt-DNA sequences, with a few notable exceptions. We now recognize six major clades in this group, elevating the blind cavefish S. tianlinensis and the micro-eyed S. microphthalmus as two new distinct clades due to their deep divergence from other clades. PCA plots of the SNP data also support the recognition of six major clusters of species congruent with the identified clades in ordination space. A Bayes factor delimitation (BFD) analysis showed support for 21 species, recognizing 19 previously described species and two putative new cryptic ones. Two species whose identities were previously disputed, S. furcodorsalis and S. tianeensis, are supported here as distinct species. In addition, our multi-species calibrated tree in SNAPP suggests that the genus Sinocyclocheilus originated around 10.16 Mya, with most speciation events occurring in the last 2 Mya, likely favored by the uplift of the Qinghai-Tibetan Plateau and cave occupation induced by climate-driven aridification during this period. These results provide a firm basis for future comparative studies on the evolution of Sinocyclocheilus and its adaptations to cave life.

Pictorial Imaging-Histopathology Correlation in a Rabbit with Hepatic VX2 Tumor Treated by Transarterial Vascular Disrupting Agent Administration.

Cancer vasculature is immature, disorganized and hyperpermeable and can serve as a target for anti-cancer therapies. Vascular disrupting agents (VDAs) are tubulin protein binding and depolymerizing agents that induce rapid tumoral vascular shutdown and subsequent cancer necrosis. However, two clinical problems exist with all VDAs, i.e. 1) incomplete anticancer effect and 2) dose-dependent toxicity. To tackle these problems, in our ongoing research, a novel VDA C118P is applied by transarterial administration of half the intravenous dose in rabbits with implanted VX2 liver tumor to assess its therapeutic efficacy. Nearly complete tumor necrosis was achieved by only a single arterial dose of C118P at 5 mg/kg, which was documented in a representative case by in vivo digital subtraction arteriogram (DSA) and magnetic resonance imaging (MRI), and further confirmed by ex vivo microangiogram and histopathology. This convincing and promising preliminary outcome would warrant further comprehensive studies to explore the potentials of VDAs by transarterial administration either in mono-drug or in combination for management of solid cancers.

Aqueous two-phase systems based on deep eutectic solvents and their application in green separation processes.

As a new environmentally friendly separation technology, deep eutectic solvent based aqueous two-phase systems are extensively applied in various fields. Herein, we review recent advances in this field and highlight the possible directions of future developments. This article focuses on the effects of deep eutectic solvent and inorganic salts on the phase equilibrium, the microstructure of deep eutectic solvent based aqueous two-phase systems, the applications of deep eutectic solvent based aqueous two-phase systems in separation (proteins, biopolymers, saponins, and organic acids), and removal and recovery technologies for deep eutectic solvent from aqueous two-phase systems.

Foxo1 nucleo-cytoplasmic distribution and unidirectional nuclear influx are the same in nuclei in a single skeletal muscle fiber but vary between fibers.

Foxo transcription factors promote protein breakdown and atrophy of skeletal muscle fibers. Foxo transcriptional effectiveness is largely determined by phosphorylation-dependent nucleo-cytoplasmic shuttling. Imaging Foxo1-green fluorescent protein (GFP) over time in 124 nuclei in 68 multinucleated adult skeletal muscle fibers under control culture conditions reveals large variability between fibers in Foxo1-GFP nucleo-cytoplasmic concentration ratio (N/C) and in the apparent rate coefficient ( kI') for Foxo1-GFP unidirectional nuclear influx (measured with efflux blocked by leptomycin B). Pairs of values of N/C or of kI' from different nuclei in the same fiber were essentially the same, but only weakly correlated in nuclei from different fibers in the same culture well. Thus, fiber to fiber variability of cellular factors, but not extracellular factors, determines Foxo1 distribution. Over all nuclei, N/C and kI' were closely proportional, indicating that kI' is the major determinant of Foxo1 distribution. IGF-I activation of Foxo kinase Akt reduces variability by decreasing kI' and N/C in all fibers. However, inhibiting Akt did not drive kI' uniformly high, indicating other pathways in Foxo1 regulation.

The first study on therapeutic efficacies of a vascular disrupting agent CA4P among primary hepatocellular carcinomas with a full spectrum of differentiation and vascularity: Correlation of MRI-microangiography-histopathology in rats.

To better inform the next clinical trials of vascular disrupting agent combretastatin-A4-phosphate (CA4P) in patients with hepatic malignancies, this preclinical study aimed at evaluating CA4P therapeutic efficacy in rats with primary hepatocellular carcinomas (HCCs) of a full spectrum of differentiation and vascularity by magnetic resonance imaging (MRI), microangiography and histopathology. Ninety-six HCCs were raised in 25 rats by diethylnitrosamine gavage. Tumor growth was monitored by T2-/T1-weighted-MRI (T2WI, T1WI) using a 3.0 T scanner. Early vascular response and later intratumoral necrosis were detected by dynamic-contrast-enhanced (DCE) MRI and diffusion-weighted-imaging (DWI) before, 1 and 12 hr after CA4P iv-administration. In vivo MRI-findings were validated by postmortem-techniques. Multi-parametric MRI revealed rapid CA4P-induced tumor vascular shutdown within 1 hr, followed by variable intratumoral necrosis at 12 hr. Tumor volumes decreased by 10% at 1 hr (p < 0.05), but resumed at 12 hr. Correlations of semi-quantitative DCE parameter initial-area-under-the-gadolinium-curve (IAUGC30) with histopathology proved partial vascular closure and compensational reopening (p < 0.05). The higher grades of vascularity prevented those residual tumor tissues from CA4P-caused ischemic necrosis. By histopathology using a 4-scale cellular-differentiation criteria and a 4-grade tumor-vascularity classification, percentage of CA4P-induced necrosis negatively correlated with HCC differentiation (r = -0.404, p < 0.001) and tumor vascularity (r = -0.370, p < 0.001). Ordinal-logistic-regression helped to predict early tumor responses to CA4P in terms of tumoral differentiation and vascularity. Our study demonstrated that CA4P could induce vascular shutdown in primary HCCs within 1 hr, resulting in various degrees of tumor necrosis at 12 hr. MRI as a real-time imaging biomarker may help to define tumor vascularity and differentiation and further to predict CA4P therapeutic outcomes.

Establishment and application of a method for screening the therapeutic drugs of ethanol-induced liver injury based on cellular metabonomics.

A drug-screening method to test the capacity of drugs to protect against ethanol-induced liver injury based on cellular metabonomics was established and applied in this study. It screens for the ability to protect against ethanol-induced liver injury by considering changes in the cellular metabolites of human normal liver L-02 cells subjected to ethanol treatment. This method considers cellular metabolites as the main analytical index, principal component analysis and orthogonal partial least squares discriminant analysis as the main multi- and megavariate data analysis methods, and vitamin C as the standard substance to determine the ability to protect against ethanol-induced liver injury. Ability to protect against ethanol-induced liver injury unit = [190 - 50× (14.318 - 10 × Y predictive value)1/2 ] × ability 1 µg/mL vitamin C. Olive leaf extract, Lycium barbarum L extract and fish roe peptide were screened using the established methods. Olive leaf OP phase had the strongest ability to protect against ethanol-induced liver injury, at 81.88. The value for L. barbarum L was 37.56. The fish roe peptide water phase was 63.07. All three have the ability to protect against ethanol-induced liver injury. The drug-screening method for ability to protect against ethanol-induced liver injury based on cell metabonomics is a fast, accurate and effective method for quantitative detection of ability to protect against ethanol-induced liver injury.

The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP.

Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.

Vascular disrupting agent in pancreatic and hepatic tumour allografts: observations of location-dependent efficacy by MRI, microangiography and histomorphology.

BACKGROUND: Tumours growing in organs of different vascular environment could exhibit diverse responses to vascular disrupting agent (VDA). This study was aimed to identify in vivo imaging biomarkers for evaluation of pancreatic and hepatic tumours and comparison of their responses to a VDA Combretastatin A4 Phosphate (CA4P) using multiparametric MRI. METHODS: Male WAG/Rij rats were used for orthotopic pancreatic head tumour and hepatic tumour implantation; tumour growth was monitored by 3D isotropic MRI using a 3.0-T clinic scanner. Therapeutic intervention using CA4P was investigated by in vivo quantitative MRI measurements including T2/T1 relaxation mapping, diffusion kurtosis imaging and dynamic contrast-enhancement (DCE) imaging. Animals were scarified 10 h after CA4P treatment for ex vivo validation using microangiography and histomorphology. RESULTS: State-of-the-art clinical MRI protocols were successfully adapted for imaging small animal tumour with high reliability. One hour after CA4P injection, marked vascular shutdown was detected with DCE MRI in both pancreatic and hepatic tumours. However, 10 h later, therapeutic necrosis was limited in pancreatic tumours compared with that in hepatic tumours (P<0.01). Heterogeneous therapeutic changes were depicted in tumour lesions using pixel-wise Tofts model, which was generated from dynamic T1 mapping. In addition, tumour responses including haemorrhage, oedema and necrosis were detected using quantitative T2/T1 relaxation maps and diffusion kurtosis images, and were validated using histomorphology. CONCLUSIONS: Using multiparametric imaging biomarkers, hepatic tumours were found to be significantly more responsive to CA4P than pancreatic tumours, which could be of reference for designing future clinical trials on this agent.

Characterization of a rat orthotopic pancreatic head tumor model using three-dimensional and quantitative multi-parametric MRI.

The purpose of this study was to investigate the reliability of 3D isotropic MRI and quantitative multi-parametric MRI characterization on an orthotopic pancreatic head tumor model in rats. 3D isotropic T2 -weighted MRI was performed as a routine for tumor longitudinal follow-up and volume estimation. Common bile duct diameter was measured from 3D multiplanar reconstruction. Quantitative multi-parametric measurements including pixel-wise T2 , T1 relaxivity, apparent diffusion coefficient (ADC) and apparent diffusion kurtosis mapping were performed twice throughout tumor growth. Semi-quantitative and quantitative analyses based on an extended Tofts model were applied to region-of-interest-based dynamic contrast-enhanced imaging, followed by contrast ratio measurement on standard contrast-enhanced imaging. Moreover, low-level texture-based analysis was inspected for T2 , T1 , ADC and contrast ratio measurements. Results indicated that multi-parametric MRI showed good reproducibility for tumor characterization; the measurements were not affected by tumor growth. Tumor growth was further confirmed with histology examinations. To conclude, state-of-the-art clinical MRI techniques were translated to this preclinical tumor model with high reliability, and have paved the way for translational oncology studies on this tumor model.

Discovery and identification of potential biomarkers for alcohol-induced oxidative stress based on cellular metabolomics.

Biomarkers involved in alcohol-induced oxidative stress play an important role in alcoholic liver disease prevention and diagnosis. Alcohol-induced oxidative stress in human liver L-02 cells was used to discover the potential biomarkers. Metabolites from L-02 cells induced by alcohol were measured by high-performance liquid chromatography and mass spectrometry. Fourteen metabolites that allowed discrimination between control and model groups were discovered by multivariate statistical data analysis (i.e. principal components analysis, orthogonal partial least-squares discriminate analysis). Based on the retention time, UV spectrum and LC-MS findings of the samples and compared with the authentic standards, eight biomarkers involved in alcohol-induced oxidative stress, namely, malic acid, oxidized glutathione, γ-glutamyl-cysteinyl-glycine, adenosine triphosphate, phenylalanine, adenosine monophosphate, nitrotyrosine and tryptophan, were identified. These biomarkers offered important targets for disease diagnosis and other researches.

Discovery of biomarkers for oxidative stress based on cellular metabolomics.

Oxidative stress has a close relationship with various pathologic physiology phenomena and the potential biomarkers of oxidative stress may provide evidence for clinical diagnosis or disease prevention. Metabolomics was employed to identify the potential biomarkers of oxidative stress. High-performance liquid chromatography-diode array detector, mass spectrometry and partial least squares discriminate analysis were used in this study. The 10, 15 and 13 metabolites were considered to discriminate the model group, vitamin E-treated group and l-glutathione-treated group, respectively. Some of them have been identified, namely, malic acid, vitamin C, reduced glutathione and tryptophan. Identification of other potential biomarkers should be conducted and their physiological significance also needs to be elaborated.

[Research progress on biological activities of Olea europaea leaf extract].

Olea europaea oil is one of the most important part of the "Mediterranean dietary pattern", and a lot of epidemiological evidences showed that people with the Mediterranean diet having a lower morbidity of the cardiovascular system diseases, skin cancer and colon cancer. The health benefits of a Mediterranean diet not only attributed to monounsaturated fatty acids and vitamins and other nutrients in O. europaea oil, but also the phenolic compounds named as antioxidant effect. Studies have shown that O. europaea leaf contains much more antioxidant activity composition than the fruit, and oleuropein, flavonoids such polyphenols are the main active ingredients in O. europaea leaf. A small amount of O. europaea was introduced into China in 1956, after widely cultivated in Fujian, Guangdong, Taiwan, Sichuan, Shaanxi, Yunnan, and Longnan in Gansu province is the biggest O. europaea planting area in the country. In every winter pruning O. europaea will produce a large number of the leaves, which could be a high added value products (phenolic compounds) of rich source. This article through consulting the literature at home and abroad, classified and summarized the biological activity research status of O. europaea leaf extract and the possible mechanisms, including antimicrobial, antitumor, antioxidation, and on the function of brain, cardiovascular system, anti-diabetes, anti-inflammatory and analgesia and so on. At the same time looked ahead to its development prospects of O. europaea leaf extract, it has variety and high content of active ingredients, and antioxidant synergy, which provide a theoretical basis for the further development and utilization of O. europaea leaf. And O. europaea leaf extract has a rich cheap source and good bioavailability, which provided a broad space in the application of medical and health care.

Pharmacologic Effects of Cannabidiol on Acute Reperfused Myocardial Infarction in Rabbits: Evaluated With 3.0T Cardiac Magnetic Resonance Imaging and Histopathology.

Cannabidiol (CBD) has anti-inflammatory effects. We explored its therapeutic effects on cardiac ischemia-reperfusion injury with an experimental imaging platform. Reperfused acute myocardial infarction (AMI) was induced in rabbits with a 90-minute coronary artery occlusion followed by 24-hour reperfusion. Before reperfusion, rabbits received 2 intravenous doses of 100 µg/kg CBD (n = 10) or vehicle (control, n = 10). Evans blue was intravenously injected for later detection of the AMI core. Cardiac magnetic resonance imaging was performed to evaluate cardiac morphology and function. After euthanasia, blood troponin I (cTnI) was assessed, and the heart was excised and infused with multifunctional red iodized oil dye. The heart was sliced for digital radiography to quantify the perfusion density rate, area at risk (AAR), and myocardial salvage index, followed by histomorphologic staining. Compared with controls, CBD treatment improved systolic wall thickening (P < 0.05), significantly increased blood flow in the AAR (P < 0.05), significantly decreased microvascular obstruction (P < 0.05), increased the perfusion density rate by 1.7-fold, lowered the AMI core/AAR ratio (P < 0.05), and increased the myocardial salvage index (P < 0.05). These improvements were associated with reductions in serum cTnI, cardiac leukocyte infiltration, and myocellular apoptosis (P < 0.05). Thus, CBD therapy reduced AMI size and facilitated restoration of left ventricular function. We demonstrated that this experimental platform has potential theragnostic utility.

Mathematical modeling reveals modulation of both nuclear influx and efflux of Foxo1 by the IGF-I/PI3K/Akt pathway in skeletal muscle fibers.

Foxo family transcription factors contribute to muscle atrophy by promoting transcription of the ubiquitin ligases muscle-specific RING finger protein and muscle atrophy F-box/atrogin-1. Foxo transcriptional effectiveness is largely determined by its nuclear-cytoplasmic distribution, with unphosphorylated Foxo1 transported into nuclei and phosphorylated Foxo1 transported out of nuclei. We expressed the fluorescent fusion protein Foxo1-green fluorescent protein (GFP) in cultured adult mouse flexor digitorum brevis muscle fibers and tracked the time course of the nuclear-to-cytoplasmic Foxo1-GFP mean pixel fluorescence ratio (N/C) in living fibers by confocal imaging. We previously showed that IGF-I, which activates the Foxo kinase Akt/PKB, caused a rapid marked decline in N/C, whereas inhibition of Akt caused a modest increase in N/C. Here we develop a two-state mathematical model for Foxo1 nuclear-cytoplasmic redistribution, where Foxo phosphorylation/dephosphorylation is assumed to be fast compared with nuclear influx and efflux. Cytoplasmic Foxo1-GFP mean pixel fluorescence is constant due to the much larger cytoplasmic than nuclear volume. Analysis of N/C time courses reveals that IGF-I strongly increased unidirectional nuclear efflux, indicating similarly increased fractional phosphorylation of Foxo1 within nuclei, and decreased unidirectional nuclear influx, indicating increased cytoplasmic fractional phosphorylation of Foxo1. Inhibition of Akt increased Foxo1 unidirectional nuclear influx, consistent with block of Foxo1 cytoplasmic phosphorylation, but did not decrease Foxo1 unidirectional nuclear efflux, indicating that Akt may not be involved in Foxo1 nuclear efflux under control conditions. New media change experiments show that cultured fibers release IGF-I-like factors, which maintain low nuclear Foxo1 in the medium. This study demonstrates the power of quantitative modeling of observed nuclear fluxes.

Wall-following - Phylogenetic context of an enhanced behaviour in stygomorphic Sinocyclocheilus (Cypriniformes: Cyprinidae) cavefishes.

With 75 known species, the freshwater fish genus Sinocyclocheilus is the largest cavefish radiation in the world and shows multiple adaptations for cave-dwelling (stygomorphic adaptations), which include a range of traits such as eye degeneration (normal-eyed, micro-eyed and eyeless), depigmentation of skin, and in some species, the presence of "horns". Their behavioural adaptations to subterranean environments, however, are poorly understood. Wall-following (WF) behaviour, where an organism remains in close contact with the boundary demarcating its habitat when in the dark, is a peculiar behaviour observed in a wide range of animals and is enhanced in cave dwellers. Hence, we hypothesise that wall-following is also present in Sinocyclocheilus, possibly enhanced in eyeless species compared to eye bearing (normal-/micro-eyed species). Using 13 species representative of Sinocyclocheilus radiation and eye morphs, we designed a series of assays, based on pre-existing methods for Astyanax mexicanus behavioural experiments, to examine wall-following behaviour under three conditions. Our results indicate that eyeless species exhibit significantly enhanced intensities of WF compared to normal-eyed species, with micro-eyed forms demonstrating intermediate intensities in the WF distance. Using a mtDNA based dated phylogeny (chronogram with four clades A-D), we traced the degree of WF of these forms to outline common patterns. We show that the intensity of WF behaviour is higher in the subterranean clades compared to clades dominated by normal-eyed free-living species. We also found that eyeless species are highly sensitive to vibrations, whereas normal-eyed species are the least sensitive. Since WF behaviour is presented to some degree in all Sinocyclocheilus species, and given that these fishes evolved in the late Miocene, we identify this behaviour as being ancestral with WF enhancement related to cave occupation. Results from this diversification-scale study of cavefish behaviour suggest that enhanced wall-following behaviour may be a convergent trait across all stygomorphic lineages.

Opposing HDAC4 nuclear fluxes due to phosphorylation by ß-adrenergic activated protein kinase A or by activity or Epac activated CaMKII in skeletal muscle fibres.

Class IIa histone deacetylases (HDACs) move between skeletal muscle fibre cytoplasm and nuclei in response to various stimuli, suppressing activity of the exclusively nuclear transcription factor Mef2. Protein kinase A (PKA) phosphorylates class IIa HDACs in cardiac muscle, resulting in HDAC nuclear accumulation, but this has not been examined in skeletal muscle. Using HDAC4-green fluorescent protein (HDAC4-GFP) expressed in isolated skeletal muscle fibres, we now show that activation of PKA by the beta-receptor agonist isoproterenol or dibutyryl (Db) cAMP causes a steady HDAC4-GFP nuclear influx. The beta-receptor blocker propranolol or PKA inhibitor Rp-cAMPS blocks the effects of isoproterenol on the nuclear influx of HDAC4-GFP, and Rp-cAMPS blocks the effects of Db cAMP. The HDAC4-GFP construct having serines 265 and 266 replaced with alanines, HDAC4 (S265/266A)-GFP, did not respond to beta-receptor or PKA activation. Immunoprecipitation results show that HDAC4-GFP is a substrate of PKA, but HDAC4 (S265/266A)-GFP is not, implicating HDAC4 serines 265/266 as the site(s) phosphorylated by PKA. During 10 Hz trains of muscle fibre electrical stimulation, the nuclear efflux rate of HDAC4-GFP, but not of HDAC4 (S265/266)-GFP, was decreased by PKA activation, directly demonstrating antagonism between the effects of fibre stimulation and beta-adrenergic activation of PKA on HDAC4 nuclear fluxes. 8-CPT, a specific activator of Epac, caused nuclear efflux of HDAC4-GFP, opposite to the effect of PKA. Db cAMP increased both phosphorylated PKA and GTP-bound Rap1. Our results demonstrate that the PKA and CaMKII pathways play important opposing roles in skeletal muscle gene expression by oppositely affecting the subcellular localization of HDAC4.