Expressing exogenous genes in newts by transgenesis

The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (～20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 4-5 months.     

Controlling gene loss of function in newts with emphasis on lens regeneration

Here we describe a protocol for gene loss of function during regeneration in newts, specifically applied to lens regeneration. Knockdown with the use of morpholinos can be achieved both in vitro and in vivo, depending on the experimental design. These methods achieve desirable levels of gene knockdown, and thus can be compared with methods developed for use in other animals, such as zebrafish. The technology has been applied to study molecular mechanisms during the process of lens regeneration by knocking down genes at specific stages and examining their effects on other genes and lens differentiation. The protocol can take a few days or up to 20 d to complete, depending on the duration of the experiment.     

Genetic heterogeneity of hepatitis C virus in association with antiviral therapy determined by ultra-deep sequencing

Background and Aims

The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail.

Methods

Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy.

Results

Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents.

Conclusion

Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.     

Kyphectomy for severe kyphosis with pyogenic spondylitis associated with myelomeningocele: a case report

A 32-year-old woman was referred to our hospital for a refractory ulcer on her back. She had a history of myelomeningocele with spina bifida that was treated surgically at birth. The ulcer was located at the apex of the kyphosis. An X-ray film revealed a kyphosis of 154° between L1 and 3 and a scoliosis of 60° between T11 and L5. Computed tomography, magnetic resonance imaging and laboratory data indicated the presence of a pyogenic spondylitis at L2/3. To correct the kyphosis and remove the infected vertebrae together with the skin ulcer, kyphectomy was performed. Pedicle screws were inserted from T8 to T12 and from L4 to S1. The dural sac was transected and ligated at L2, followed by total kyphectomy of the L1-L3 vertebrae. The spinal column was reconstructed by approximating the ventral wall of the T12 vertebral body and the cranial endplate of the L4 vertebra. Postoperatively, the kyphosis was corrected to 61° and the scoliosis was corrected to 22°. In the present case, we treated the skin ulcer and pyogenic spondylitis successfully by kyphectomy, thereby, preventing recurrence of the ulcer and infection, and simultaneously obtaining sufficient correction of the spinal deformity.     

Use of a Modified α-N-Acetylgalactosaminidase in the Development of Enzyme Replacement Therapy for Fabry Disease

A modified α-N-acetylgalactosaminidase (NAGA) with α-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-α-D-galactopyranoside. It retained the original NAGA''s stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.     

Embryonic and posthatching treatments with sex steroids demasculinize the motivational aspects of crowing behavior in male Japanese quail

Demasculinizing action of embryonic estrogen on crowing behavior in male Japanese quails was examined. Eggs were treated with either 20 μg of estradiol benzoate (EB) or vehicle on the 10th day of incubation. Chicks hatched from both groups of eggs were injected daily with either testosterone propionate (TP; 10 μg/g b.w.), 5α-dihydrotestosterone (DHT, a non-aromatizable androgen; 10 μg/g b.w.), or vehicle from 11 to 50 days after hatching, and during this period their calling behaviors were observed. Irrespective of embryonic treatments, all birds received posthatching treatment with either TP or DHT, but not with vehicle, emitted crows in place of distress calls in a stress (non-sexual) context of being isolated in a recording chamber. The posthatching TP, but not posthatching DHT, induced crowing in a sexual context (crowing in their home-cages) from much earlier age than posthatching vehicle in the birds received control embryonic treatment with vehicle. The same TP treatment, however, completely eliminated the crowing in a sexual context in the birds received EB during their embryonic life. In the birds treated with either posthatching DHT or posthatching vehicle, the crowing in a sexual context was only slightly decreased by embryonic EB treatment. These data suggest that posthatching estrogen, derived from testosterone aromatization, enhances the demasculinizing action of embryonic estrogen, and thus strongly reduces the sexual motivation for crowing behavior. This demasculinizing action, however, would not influence vocal control system which generates acoustic pattern of crowing in the presence of androgens allowing the birds to crow in a non-sexual context.     

Structure–activity relationship study on α1 adrenergic receptor antagonists from beer

Hordatine A and aperidine have been previously isolated from beer as active ingredients, which bind to muscarinic M₃ receptor. In addition, these compounds have exhibited antagonist activity against the α_1A adrenoceptor. Although the relative structures of these two molecules have previously been determined, the absolute stereochemistry was unclear. Hence, to elucidate the absolute stereochemistry of natural hordatine A, we synthesized each enantiomer of hordatine A and aperidine from optically pure dehydrodi-p-coumaric acid. Several additional related compounds were also synthesized for structure–activity relationship studies. Chiral column HPLC analysis demonstrated that the absolute stereochemistry of natural hordatine A is (2S,3S), while based on the isomerization mechanism, the stereochemistry of aperidine is (2R,3S). The α_1A adrenoceptor binding activity of (2R,3R)-hordatine A is the most potent among the enantiomeric pairs of hordatines and aperidines. Furthermore, the related, synthetic compound, (2R,3R)-methyl benzofurancarboxylate exhibits antagonist activity against the α_1A adrenoceptor at a lower concentration than that of hordatine A.     

Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

Introduction

Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis.

Methods

Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb).

Results

Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss.

Conclusions

The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients.