Search

Significance Paneth cells provide intestinal host defense against pathogens and control the healthy microbiota by secreting antimicrobial peptides. We show that the most abundant secreted Paneth cell products, human defensin HD-5 and HD-6, show a distinct susceptibility to proteolytic digestion by human duodenal fluid. While HD-5 is digested in many fragments, HD-6 is stable and still able to form nanonets. The occurring fragments of HD-5 were antimicrobially active against microorganisms. We provide proof of concept about microbiome modulating capacities in vivo, which includes an increase of Akkermansia sp. Our results indicate that fragmentation of defensins increases antimicrobial diversity and further adds to the complexity of host microbial interaction at interfaces. Fragmentation could lead to new antimicrobial peptides with possible therapeutic usage., Antimicrobial peptides, in particular α-defensins expressed by Paneth cells, control microbiota composition and play a key role in intestinal barrier function and homeostasis. Dynamic conditions in the local microenvironment, such as pH and redox potential, significantly affect the antimicrobial spectrum. In contrast to oxidized peptides, some reduced defensins exhibit increased vulnerability to proteolytic degradation. In this report, we investigated the susceptibility of Paneth-cell–specific human α-defensin 5 (HD-5) and -6 (HD-6) to intestinal proteases using natural human duodenal fluid. We systematically assessed proteolytic degradation using liquid chromatography–mass spectrometry and identified several active defensin fragments capable of impacting bacterial growth of both commensal and pathogenic origins. Of note, incubation of mucus with HD-5 resulted in 255–8,000 new antimicrobial combinations. In contrast, HD-6 remained stable with consistent preserved nanonet formation. In vivo studies demonstrated proof of concept that a HD-5 fragment shifted microbiota composition (e.g., increases of Akkermansia sp.) without decreasing diversity. Our data support the concept that secretion of host peptides results in an environmentally dependent increase of antimicrobial defense by clustering in active peptide fragments. This complex clustering mechanism dramatically increases the host’s ability to control pathogens and commensals. These findings broaden our understanding of host modulation of the microbiome as well as the complexity of human mucosal defense mechanisms, thus providing promising avenues to explore for drug development.

Klippel–Feil syndrome is a congenital vertebral anomaly, which is characterised by the fusion of at least two cervical vertebrae and a clinically broad set of symptoms, including congenital scoliosis and elevated scapula (Sprengel's deformity). Klippel–Feil syndrome is associated with mutations in MEOX1. The zebrafish mutant choker (cho) carries a mutation in its orthologue, meox1. Although zebrafish is being increasingly employed as fidelitous models of human spinal disease, the vertebral column of Meox1‐deficient fish has not been assessed for defects. Here, we describe the skeletal defects of meox1 (cho) mutant zebrafish utilising alizarin red to stain bones and chemical maceration of soft tissue to detect fusions in an unbiased manner. Obtained data reveal that meox1 (cho) mutants feature aspects of a number of described symptoms of patients who suffer from Klippel–Feil syndrome and have mutations in MEOX1. These include vertebral fusion, congenital scoliosis and an asymmetry of the pectoral girdle, which resembles Sprengel's deformity. Thus, the meox1 (cho) mutant zebrafish may serve as a useful tool to study the pathogenesis of the symptoms associated with Klippel–Feil syndrome.

The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish adamts5 during embryogenesis. This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated.

Summary: The TCP-1 ring complex (TRiC) is a multi-subunit group II chaperonin that assists nascent or misfolded proteins to attain their native conformation in an ATP-dependent manner. Functional studies in yeast have suggested that TRiC is an essential and generalized component of the protein-folding machinery of eukaryotic cells. However, TRiC’s involvement in specific cellular processes within multicellular organisms is largely unknown because little validation of TRiC function exists in animals. Our in vivo analysis reveals a surprisingly specific role of TRiC in the biogenesis of skeletal muscle α-actin during sarcomere assembly in myofibers. TRiC acts at the sarcomere’s Z-disk, where it is required for efficient assembly of actin thin filaments. Binding of ATP specifically by the TRiC subunit Cct5 is required for efficient actin folding in vivo. Furthermore, mutant α-actin isoforms that result in nemaline myopathy in patients obtain their pathogenic conformation via this function of TRiC. : TRiC-deficient zebrafish feature specific defects in sarcomere and neurite formation. Berger et al. demonstrate a role for TRiC as a multiprotein scaffold positioned at the sarcomere’s Z-disk, where it enhances the processing of skeletal muscle α-actin. Accordingly, TRiC causes aggregation of myopathic α-actin variants in nemaline myopathy. Keywords: CCT, TRiC, chaperonin, folding, actin, zebrafish, muscle, myofibril, tubulin, nemaline myopathy

Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, 5 encode for components of striated muscle sarcomeres. In a genetic zebrafish screen the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganized and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerise to form thin filaments in the skeletal muscle of tmod4trg mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion, and early death. Myofibrils of tmod4trg mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones, and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods together with the reactivity of rods with phalloidin and an antibody against actinin is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4trg mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicate TMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4trg mutant will be a valuable tool to study human muscle disorders.

The muscle-specific UNC-45b assists in the folding of sarcomeric myosin. Analysis of the zebrafish unc-45b upstream region revealed that unc-45b promoter fragments reliably drive GFP expression after germline transmission. The muscle-specific 503-bp minimal promoter 503unc was identified to drive gene expression in the zebrafish musculature. In transgenic Tg(-503unc:GFP) zebrafish, GFP fluorescence was detected in the adaxial cells, their slow fiber descendants, and the fast muscle. At later stages, robust GFP fluorescence is eminent in the cardiac, cranial, fin, and trunk muscle, thereby recapitulating the unc-45b expression pattern. We propose that the 503unc promoter is a small and muscle-specific promoter that drives robust gene expression throughout the zebrafish musculature, making it a valuable tool for the exploration of zebrafish muscle.

Congenital myopathies are muscle degenerative disorders with a broad clinical spectrum. A number of myopathies have been associated with molecular defects within sarcomeres, the force-generating component of the muscle cell. Whereas the highly regular organization of the myofibril has been studied in detail, in vivo assembly of sarcomeres remains a poorly understood process. Therefore, a more detailed knowledge of sarcomere assembly is crucial to better understand the pathogenic mechanisms within myopathies. Recently, mutations in myosin XVIIIB (MYO18B) have been associated with cases of myopathies, although the underlying mechanism for the resulting pathology remains to be defined. To analyze the role of myosin XVIIIB in skeletal muscle disease, zebrafish mutants for myo18b were generated. Full loss of myo18b function results in a complete lack of sarcomeric structure, revealing a highly surprising and essential role for myo18b in sarcomere assembly. Importantly, scattered thin and thick filaments assemble throughout the sarcoplasm; but fail to organize into recognizable sarcomeric structures in myo18b null mutants. In myo18b partial loss-of-function mutants sarcomeric structures are assembled, but thin and thick filaments remain misaligned within these structures. These observations suggest a novel model of sarcomere assembly where Myo18b coordinates the integration of preformed thick and thin filaments into the sarcomere. Disruption of this highly coordinated process results in a block in sarcomere biogenesis and the onset of myopathic pathology.

Muscular dystrophies are a group of genetic disorders that specifically affect skeletal muscle and are characterized by progressive muscle degeneration and weakening. To develop therapies and treatments for these diseases, a better understanding of the molecular basis of muscular dystrophies is required. Thus, identification of causative genes mutated in specific disorders and the study of relevant animal models are imperative. Zebrafish genetic models of human muscle disorders often closely resemble disease pathogenesis, and the optical clarity of zebrafish embryos and larvae enables visualization of dynamic molecular processes in vivo. As an adjunct tool, morpholino studies provide insight into the molecular function of genes and allow rapid assessment of candidate genes for human muscular dystrophies. This unique set of attributes makes the zebrafish model system particularly valuable for the study of muscle diseases. This review discusses how recent research using zebrafish has shed light on the pathological basis of muscular dystrophies, with particular focus on the muscle cell membrane and the linkage between the myofibre cytoskeleton and the extracellular matrix.

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene. As in humans, zebrafish dystrophin is initially expressed at the peripheral ends of the myofibres adjacent to the myotendinous junction and gradually shifts to non-junctional sites. Dystrophin-deficient zebrafish larvae are characterised by abundant necrotic fibres being replaced by mono-nucleated infiltrates, extensive fibrosis accompanied by inflammation, and a broader variation in muscle fibre cross-sectional areas. Muscle progenitor proliferation cannot compensate for the extensive skeletal muscle loss. Live imaging of dystrophin-deficient zebrafish larvae documents detaching myofibres elicited by muscle contraction. Correspondingly, the progressive phenotype of dystrophin-deficient zebrafish resembles many aspects of the human disease, suggesting that specific advantages of the zebrafish model system, such as the ability to undertake in vivo drug screens and real time analysis of muscle fibre loss, could be used to make novel insights relevant to understanding and treating the pathological basis of dystrophin-deficient muscular dystrophy.

In skeletal muscle, the dystrophin-associated glycoprotein complex forms a link between the actin cytoskeleton and the extracellular matrix that is critical for muscle integrity. Within this complex resides the sarcoglycan subcomplex, which consists of four transmembrane glycoproteins (alpha-, beta-, gamma-, and delta-sarcoglycan). During assembly, beta-sarcoglycan tightly associates with delta-sarcoglycan to form a functional core that then recruits gamma- and alpha-sarcoglycan to form the sarcoglycan complex. Together with sarcospan, the sarcoglycan complex binds other components of the dystrophin-associated glycoprotein complex and integrates into the myofibre's membrane. Once integrated, the sarcoglycan complex plays a pivotal role in mechanically stabilising the sarcolemma as well as the dystrophin-associated glycoprotein complex. Additionally, the sarcoglycan complex undergoes chemical modifications in response to muscle contractions, thereby transducing mechanical information into a cellular signal. Mutations in the sarcoglycans induce limb girdle muscular dystrophy, and several animal models have been established to study the molecular biology and function of the sarcoglycan complex. This review discusses the role of the sarcoglycan complex in skeletal muscle and describes the functional deficiencies that lead to muscular dystrophies.

Dividing asymmetrically to fix muscle Resident tissue stem cells called satellite cells repair muscle after injury. However, how satellite cells operate inside living tissue is unclear. Gurevich et al. exploited the optical clarity of zebrafish larvae and used a series of genetic approaches to study muscle injury. After injury, satellite cells divide asymmetrically to generate a progenitor pool for muscle replacement and at the same time “self-renew” the satellite stem cell. This results in regeneration that is highly clonal in nature, validating many decades of in vitro analyses examining the regenerative capacity of skeletal muscle. Science , this issue p. 136

BACKGROUND AND OBJECTIVE Symptomatic patients with multivessel coronary disease (MVD) benefit from both coronary artery bypass grafting (CABG) and percutaneous coronary angioplasty (PTCA). The >>German Angioplasty Bypass Investigation<< (GABI-I) trial randomized patients to one of these treatment strategies between 1986 and 1991. In order to evaluate the impact of current technology, in particular coronary stents, the GABI-II trial was initiated, which in 1996 and 1997 prospectively enrolled patients according to the initial GABI-I criteria. PATIENTS AND METHODS Into the study 136 consecutive patients (108 men, 28 women; 63 +/- 12 years) were included. Patients from GABI-I served as controls. RESULTS A mean of 2.1 +/- 0.5 vessels were treated per patient (vs. 1.9 +/- 0.5 vessels in the PTCA arm of GABI-I) and 63 % of the lesions were covered with stents. With respect to the primary endpoint less patients remained with a CCS class III or IV in GABI-II after 12 months (1,5 % vs. 8 % in the PTCA arm of GABI-I, p

Aims Rotablation is a widely used technique for the treatment of complex coronary artery lesions but is so far only poorly supported by controlled studies. The Comparison of Balloon-Angioplasty versus Rotational Atherectomy study (COBRA) is a multicentre, prospective, randomized trial to compare short- and long-term effects of percutaneous transluminal coronary angioplasty (PTCA) and rotablation in patients with angiographically pre-defined complex coronary artery lesions. Methods and Results At seven clinical sites 502 patients with pre-defined complex coronary artery lesions were assigned to either PTCA (n=250) or rotablation (n=252). Primary end-points were procedural success, 6-month restenosis rates in the treated segments, and major cardiac events during follow-up. Procedural success was achieved in 78% (PTCA), and 85% (rotablation) ( P =0·038) of cases. Crossover from PTCA to rotablation was 4% and 10% vice versa ( P =0·019). There was no difference between PTCA and rotablation with respect to procedure-related complications such as Q wave infarctions (2·4% each), emergency bypass surgery (1·2% versus 2·4%), and death (1·6% versus 0·4%). However, more stents were required after PTCA (14·9% versus 6·4%, P

Spermine oxidase is a FAD-containing enzyme involved in polyamines catabolism, selectively oxidizing spermine to produce H2O2, spermidine, and 3-aminopropanal. Spermine oxidase is highly expressed in the mouse brain and plays a key role in regulating the levels of spermine, which is involved in protein synthesis, cell division and cell growth. Spermine is normally released by neurons at synaptic sites where it exerts a neuromodulatory function, by specifically interacting with different types of ion channels, and with ionotropic glutamate receptors. In order to get an insight into the neurobiological roles of spermine oxidase and spermine, we have deregulated spermine oxidase gene expression producing and characterizing the transgenic mouse model JoSMOrec, conditionally overexpressing the enzyme in the neocortex. We have investigated the effects of spermine oxidase overexpression in the mouse neocortex by transcript accumulation, immunohistochemical analysis, enzymatic assays and polyamine content in young and aged animals. Transgenic JoSMOrec mice showed in the neocortex a higher H2O2 production in respect to Wild-Type controls, indicating an increase of oxidative stress due to SMO overexpression. Moreover, the response of transgenic mice to excitotoxic brain injury, induced by kainic acid injection, was evaluated by analysing the behavioural phenotype, the immunodistribution of neural cell populations, and the ultrastructural features of neocortical neurons. Spermine oxidase overexpression and the consequently altered polyamine levels in the neocortex affects the cytoarchitecture in the adult and aging brain, as well as after neurotoxic insult. It resulted that the transgenic JoSMOrec mouse line is more sensitive to KA than Wild-Type mice, indicating an important role of spermine oxidase during excitotoxicity. These results provide novel evidences of the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain. © 2013 Cervelli et al.

The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene,identifying laminin β2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.

Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.

Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic alpha cell fate specification while simultaneously repressing the beta and delta cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the alpha cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of beta and delta cells. Concurrently, a remarkable increase in the number of cells displaying alpha cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult beta cells led to a loss of the beta cell phenotype and a concomitant increase in a number of cells with alpha or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of beta cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.

We generated a transgenic mouse line named E1-Ngn2/Cre that expresses Cre recombinase and GFP under the control of the E1 enhancer element of the gene Ngn2 (Scardigli et al.: Neuron 31:203-217, 2001). Cre-recombinase activity and GFP fluorescence are consistent with the reported expression pattern controlled by the E1-Ngn2 enhancer. Recombination was detected in the progenitor domains p1 and p2 in the ventricular zone of the neural tube and in distinct domains of the pretectum, the dorsal and ventral thalamus, the tegmentum of the mesencephalon, and the hindbrain. In the developing cortex, Cre-recombinase activity is confined to a subpopulation of progenitors predominantly in the region of the ventral and lateral pallium. The E1-Ngn2/Cre mouse line thus provides an excellent novel tool for a region-specific conditional mutagenesis in the developing CNS.