One-step Cre-loxP organism creation by TAx9.

The creation of organisms with Cre-loxP conditional gene recombination systems often faces challenges, particularly when creating the initial (F0) generation with both a Cre recombinase and a DNA site flanked by loxP elements (floxed site). The primary reason is that it is difficult to synthesize a single plasmid with both the Cre gene and the floxed site, since Cre-mediated recombination spontaneously occurs when the plasmid is amplified in Escherichia coli bacterial cells. Here, we introduce an artificial nucleic acid sequence TATATATATATATATATA, named TAx9, that enables the integration of both the Cre gene and the floxed site into a single plasmid.

An adult myogenic cell line of the Japanese fire-bellied newt Cynops pyrrhogaster.

Adult myogenic cell lines are useful to study muscle development, repair and regeneration. In newts, which are known for their high regenerative capacity, myogenic cell lines have not been established in species other than the Eastern newt Notophthalmus viridescens. In this study, we established another myogenic cell line, named CpM01, from the skeletal muscle of the forearm of the adult Japanese fire-bellied newt Cynops pyrrhogaster.

Development of a ZRS Reporter System for the Newt () During Terrestrial Limb Regeneration.

Background: Newts, a type of urodele amphibian, offer remarkable insights into regenerative medicine due to their extraordinary tissue regeneration capabilities-a challenging feat in humans. During limb regeneration of adult newts, fascinating cellular and molecular processes are revealed, including scarless healing, de-differentiation of mature cells, and regeneration of limbs and digits. Sonic hedgehog (Shh), crucial for vertebrate limb development, is regulated by the zone of polarizing activity regulatory sequence (ZRS) in the limb bud zone of polarizing activity (ZPA).

Structural and functional analysis of the newt lymphatic system.

Regeneration competent vertebrates such as newts and salamanders possess a weakened adaptive immune system characterized by multiple connections between the lymphatic system and the blood vascular system called lymphatic hearts. The role of lymphatic vasculature and these lymphaticovenous connections in regeneration is unknown. We used in-vivo near-infrared lymphangiography, ultra-high frequency ultrasonography, micro-CT lymphangiography, and histological serial section 3-dimentional computer reconstruction to evaluate the lymphatic territories of Cynops pyrrhogaster.

Newtic1 Is a Component of Globular Structures That Accumulate along the Marginal Band of Erythrocytes in the Limb Blastema of Adult Newt, .

In adult newts, when a limb is amputated, a mesenchymal cell mass called the blastema is formed on the stump, where blood vessels filled with premature erythrocytes, named polychromatic normoblasts (PcNobs), elongate. We previously demonstrated that PcNobs in the blastema express an orphan gene, , and that they secrete growth factors such as BMP2 and TGFβ1 into the surrounding tissues. However, the relationship between Newtic1 expression and growth factor secretion was not clear since was thought to encode a membrane protein.

The latent dedifferentiation capacity of newt limb muscles is unleashed by a combination of metamorphosis and body growth.

Newts can regenerate their limbs throughout their life-span. Focusing on muscle, certain species of newts such as Cynops pyrrhogaster dedifferentiate muscle fibers in the limb stump and mobilize them for muscle creation in the regenerating limb, as they grow beyond metamorphosis. However, which developmental process is essential for muscle dedifferentiation, metamorphosis or body growth, is unknown.

Skin Wound Healing of the Adult Newt, : A Unique Re-Epithelialization and Scarless Model.

In surgical and cosmetic studies, scarless regeneration is an ideal method to heal skin wounds. To study the technologies that enable scarless skin wound healing in medicine, animal models are useful. However, four-limbed vertebrates, including humans, generally lose their competency of scarless regeneration as they transit to their terrestrial life-stages through metamorphosis, hatching or birth.

Reviewing the Effects of Skin Manipulations on Adult Newt Limb Regeneration: Implications for the Subcutaneous Origin of Axial Pattern Formation.

Newts are unique salamanders that can regenerate their limbs as postmetamorphic adults. In order to regenerate human limbs as newts do, it is necessary to determine whether the cells homologous to those contributing to the limb regeneration of adult newts also exist in humans. Previous skin manipulation studies in larval amphibians have suggested that stump skin plays a pivotal role in the axial patterning of regenerating limbs.

Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products.

Biopharmaceutical products are of great importance in the treatment or prevention of many diseases and represent a growing share of the global pharmaceutical market. The usual technology for protein synthesis (cell-based expression) faces certain obstacles, especially with 'difficult-to-express' proteins. Cell-free protein synthesis (CFPS) can overcome the main bottlenecks of cell-based expression.

Inter-professional and inter-departmental alcoholism rehabilitation program.

A 3-month alcoholism rehabilitation program at psychiatric hospitals is common in Japan for patients with alcohol use disorder (AUD). However, many AUD patients are often hospitalized for the treatment of digestive disorders due to alcohol-related liver diseases and pancreatitis. In this sense, AUD patients need to be better supported by professionals and departments in general hospitals.

Novel erythrocyte clumps revealed by an orphan gene Newtic1 in circulating blood and regenerating limbs of the adult newt.

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known.

Dietary administration of tetrodotoxin and its putative biosynthetic intermediates to the captive-reared non-toxic Japanese fire-bellied newt, Cynops pyrrhogaster.

The origin of tetrodotoxin (TTX) in amphibians has long been disputed. In this study, TTX and its putative biosynthetic intermediates or shunt compounds (4,9-anhydro-10-hemiketal-5-deoxyTTX and Cep-242) were dietary administered to the captive-reared, non-toxic Japanese fire-bellied newt, Cynops pyrrhogaster. After 4 weeks, the ingested compounds were detected mainly in the newt body using liquid chromatography-mass spectrometry (LC-MS), while these compounds were not converted into other TTX analogues in newts.

Implications of a Multi-Step Trigger of Retinal Regeneration in the Adult Newt.

The newt is an amazing four-limbed vertebrate that can regenerate various body parts including the retina. In this animal, when the neural retina (NR) is removed from the eye by surgery (retinectomy), both the NR and the retinal pigment epithelium (RPE) eventually regenerate through the process of reprogramming and proliferation of RPE cells. Thus far, we have pursued the onset mechanism of adult newt retinal regeneration.

[Differences in the mechanism of muscle regeneration between the larval and metamorphosed newts.].

The newt switches the cellular mechanism for limb regeneration from a stem/progenitor-based mechanism(larval mode)to a dedifferentiation-based one(adult mode)as it grows beyond metamorphosis. When it comes to muscle, larval newts regenerate muscle from muscle stem/progenitor cells such as satellite cells, but after metamorphosis the animals recruit muscle fibers for the same purpose by means of dedifferentiation in which muscle fibers(multinucleated cell)at the stump are fragmented to produce mononucleated cells, the primary cell source for new muscle. Dedifferentiation of muscle fibers may have been advantageous for the newt, during its evolution, to quickly regenerate the limbs by supplementing satellite cells whose number and potency become restricted by metamorphosis(transformation to adapt to terrestrial environments)or aging.

Turning the fate of reprogramming cells from retinal disorder to regeneration by Pax6 in newts.

The newt, a urodele amphibian, has an outstanding ability- even as an adult -to regenerate a functional retina through reprogramming and proliferation of the retinal pigment epithelium (RPE) cells, even though the neural retina is completely removed from the eye by surgery. It remains unknown how the newt invented such a superior mechanism. Here we show that disability of RPE cells to regenerate the retina brings about a symptom of proliferative vitreoretinopathy (PVR), even in the newt.

A developmentally regulated switch from stem cells to dedifferentiation for limb muscle regeneration in newts.

The newt, a urodele amphibian, is able to repeatedly regenerate its limbs throughout its lifespan, whereas other amphibians deteriorate or lose their ability to regenerate limbs after metamorphosis. It remains to be determined whether such an exceptional ability of the newt is either attributed to a strategy, which controls regeneration in larvae, or on a novel one invented by the newt after metamorphosis. Here we report that the newt switches the cellular mechanism for limb regeneration from a stem/progenitor-based mechanism (larval mode) to a dedifferentiation-based one (adult mode) as it transits beyond metamorphosis.

Expression of Two Classes of Pax6 Transcripts in Reprogramming Retinal Pigment Epithelium Cells of the Adult Newt.

The adult newt has the remarkable ability to regenerate a functional retina from retinal pigment epithelium (RPE) cells, even when the neural retina (NR) is completely lost from the eye. In this system, RPE cells are reprogrammed into a unique state of multipotent cells, named RPESCs, in an early phase of retinal regeneration. However, the signals that trigger reprogramming remain unknown.

A robust transcriptional program in newts undergoing multiple events of lens regeneration throughout their lifespan.

Newts have the ability to repeatedly regenerate their lens even during ageing. However, it is unclear whether this regeneration reflects an undisturbed genetic activity. To answer this question, we compared the transcriptomes of lenses, irises and tails from aged newts that had undergone lens regeneration 19 times with the equivalent tissues from young newts that had never experienced lens regeneration.

Confirmation of the absence of tetrodotoxin and its analogues in the juveniles of the Japanese fire-bellied newt, Cynops pyrrhogaster, captive-reared from eggs in the laboratory using HILIC-LC-MS.

The tetrodotoxin (TTX) contents of the Japanese fire-bellied newt, Cynops pyrrhogaster, captive-reared from eggs to metamorphosed juveniles with a non-toxic diet for 70 weeks, as well as wild-caught juvenile newts, were investigated using a high-resolution hydrophilic interaction chromatography-LC-MS. TTX was detected in 0- to 22-week-old captive-reared juvenile newts but was not detected (<15 ng/g) in the 36- to 70-week-old newts, while significant levels of TTX (1.3-14 μg/g) were detected in the wild-caught juveniles.

The newt (Cynops pyrrhogaster) RPE65 promoter: molecular cloning, characterization and functional analysis.

The adult newt has the ability to regenerate the neural retina following injury, a process achieved primarily by the retinal pigment epithelium (RPE). To deliver exogenous genes to the RPE for genetic manipulation of regenerative events, we isolated the newt RPE65 promoter region by genome walking. First, we cloned the 2.

A transcriptome for the study of early processes of retinal regeneration in the adult newt, Cynops pyrrhogaster.

Retinal regeneration in the adult newt is a useful system to uncover essential mechanisms underlying the regeneration of body parts of this animal as well as to find clues to treat retinal disorders such as proliferative vitreoretinopathy. Here, to facilitate the study of early processes of retinal regeneration, we provide a de novo assembly transcriptome and inferred proteome of the Japanese fire bellied newt (Cynops pyrrhogaster), which was obtained from eyeball samples of day 0-14 after surgical removal of the lens and neural retina. This transcriptome (237,120 in silico transcripts) contains most information of cDNAs/ESTs which has been reported in newts (C.

The newt reprograms mature RPE cells into a unique multipotent state for retinal regeneration.

The reprogramming of retinal pigment epithelium (RPE) cells in the adult newt immediately after retinal injury is an area of active research for the study of retinal disorders and regeneration. We demonstrate here that unlike embryonic/larval retinal regeneration, adult newt RPE cells are not directly reprogrammed into retinal stem/progenitor cells; instead, they are programmed into a unique state of multipotency that is similar to the early optic vesicle (embryo) but preserves certain adult characteristics. These cells then differentiate into two populations from which the prospective-neural retina and -RPE layers are formed with the correct polarity.

The retinal pigment epithelium: an important player of retinal disorders and regeneration.

The retinal pigment epithelium (RPE) is a partner of the neural retina and is indispensable for vision. In humans, proliferation and transformation (cell-type switching) of RPE cells after a traumatic injury of the neural retina causes a retinal disorder leading to loss of vision. In contrast, in certain adult amphibians such as Xenopus laevis and the newt, a similar process in RPE cells leads to regeneration of the entire retina.

MEK-ERK signaling in adult newt retinal pigment epithelium cells is strengthened immediately after surgical induction of retinal regeneration.

Adult newt retinal pigment epithelium (RPE) cells are mitotically quiescent in the physiological condition, but upon a traumatic injury of the neural retina (NR) they re-enter the cell-cycle and eventually regenerate the missing NR. Here, to understand the mechanism underlying the cell-cycle re-entry of RPE cells following NR injury, we first investigated changes in MEK-ERK signaling activity in RPE cells upon removal of the NR (retinectomy) from the eye of living animals, and found that ERK-mediated signaling activity is elevated quickly (in 30 min) upon retinectomy. In addition, we found, in in vitro analyses, that immediate early activation of MEK-ERK signaling may occur in RPE cells upon NR injury, intensifying the MEK-ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway, and that 1-h blockade of such early MEK-ERK signaling interferes with the cell-cycle re-entry, which occurs 5-10 days later.

Metamorphosis inhibition: an alternative rearing protocol for the newt, Cynops pyrrhogaster.

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently, a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the laboratory.