TDP-43 regulates cholesterol biosynthesis by inhibiting sterol regulatory element-binding protein 2.

Dyslipidemia is considered an essential component of the pathological process of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease. Although TAR DNA Binding Protein 43 kDa (TDP-43) links both familial and sporadic forms of ALS and cytoplasmic aggregates are a hallmark of most cases of ALS, the molecular mechanism and the in vivo relation of ALS dyslipidemia with TDP-43 have been unclear. To analyze the dyslipidemia-related gene expression by TDP-43, we performed expression microarray and RNA deep sequencing (RNA-Seq) using cell lines expressing high levels of TDP-43 and identified 434 significantly altered genes including sterol regulatory element-binding protein 2 (SREBP2), a master regulator of cholesterol homeostasis and its downstream genes. Elevated TDP-43 impaired SREBP2 transcriptional activity, leading to inhibition of cholesterol biosynthesis. The amount of cholesterol was significantly decreased in the spinal cords of TDP-43-overexpressed ALS model mice and in the cerebrospinal fluids of ALS patients. These results suggested that TDP-43 could play an essential role in cholesterol biosynthesis in relation to ALS dyslipidemia.

Preoperative vascular mapping for anterolateral thigh flap surgeries: A clinical trial of photoacoustic tomography imaging.

BACKGROUND: Photoacoustic tomography (PAT) is a noninvasive vascular imaging modality that uses near-infrared pulse laser beams and ultrasound (US) to visualize vessels. We previously demonstrated the utility of PAT for visualizing anterolateral thigh (ALT) perforators in a clinical study of 10 thighs in 5 healthy adults. Evaluation of the correlation between PAT and US findings showed that PAT had comparable diagnostic potential but was superior in visualizing subcutaneous microvessels; however, there was no comparison with intraoperative findings. In this study, we used a newly developed technique to transfer a PAT image to a body-attachable transparent sheet to compare PAT and intraoperative findings. METHODS: Eight patients were recruited in this prospective study. Patient age ranged from 32 to 79 years (average 60). Seven ALT flaps were applied in head and neck reconstruction. One flap was elevated in chest wall reconstruction. Each PAT scan of an 18 cm × 13.5 cm region took approximately 5 min. Acquired data were processed three-dimensionally using a novel imaging software program. Perforator vessel data from PAT imaging were traced and corrected for projection onto medical film sheets. The correlation between the perforator stem portions predicted by PAT and the intraoperative findings at the level of the fascia-penetrating points was evaluated, and distal branching patterns were analyzed. RESULTS: PAT imaging showed 16 perforators in 8 thighs. Intraoperative surgical findings revealed that all the perforator penetrating points at the deep fascia level matched the PAT findings within 10 mm. None of the eight ALT flaps demonstrated postoperative complications. The perforator complexes were classified as type I in three cases (19%), type II in eight cases (50%), and type III in five cases (31%). CONCLUSIONS: PAT imaging matched the intraoperative findings within 10 mm. Preoperative vascular evaluation allows for the creation of a vascular map for facilitating ALT flap surgeries.

Combined multiphoton imaging and biaxial tissue extension for quantitative analysis of geometric fiber organization in human reticular dermis.

The geometric organization of collagen fibers in human reticular dermis and its relationship to that of elastic fibers remain unclear. The tight packing and complex intertwining of dermal collagen fibers hinder accurate analysis of fiber orientation. We hypothesized that combined multiphoton microscopy and biaxial extension could overcome this issue. Continuous observation of fresh dermal sheets under biaxial extension revealed that the geometry of the elastic fiber network is maintained during expansion. Full-thickness human thigh skin samples were biaxially extended and cleared to visualize the entire reticular dermis. Throughout the dermis, collagen fibers straightened with increased inter-fiber spaces, making them more clearly identifiable after extension. The distribution of collagen fibers was evaluated with compilation of local orientation data. Two or three modes were confirmed in all superficial reticular layer samples. A high degree of local similarities in the direction of collagen and elastic fibers was observed. More than 80% of fibers had directional differences of ≤15°, regardless of layer. Understanding the geometric organization of fibers in the reticular dermis improves the understanding of mechanisms underlying the pliability of human skin. Combined multiphoton imaging and biaxial extension provides a research tool for studying the fibrous microarchitecture of the skin.

Critical Growth Processes for the Midfacial Morphogenesis in the Early Prenatal Period.

BACKGROUND: Congenital midfacial hypoplasia often requires intensive treatments and is a typical condition for the Binder phenotype and syndromic craniosynostosis. The growth trait of the midfacial skeleton during the early fetal period has been assumed to be critical for such an anomaly. However, previous embryological studies using 2-dimensional analyses and specimens during the late fetal period have not been sufficient to reveal it. OBJECTIVE: To understand the morphogenesis of the midfacial skeleton in the early fetal period via 3-dimensional quantification of the growth trait and investigation of the developmental association between the growth centers and midface. METHODS: Magnetic resonance images were obtained from 60 human fetuses during the early fetal period. Three-dimensional shape changes in the craniofacial skeleton along growth were quantified and visualized using geometric morphometrics. Subsequently, the degree of development was computed. Furthermore, the developmental association between the growth centers and the midfacial skeleton was statistically investigated and visualized. RESULTS: The zygoma expanded drastically in the anterolateral dimension, and the lateral part of the maxilla developed forward until approximately 13 weeks of gestation. The growth centers such as the nasal septum and anterior portion of the sphenoid were highly associated with the forward growth of the midfacial skeleton (RV = 0.589; P < .001). CONCLUSIONS: The development of the midface, especially of the zygoma, before 13 weeks of gestation played an essential role in the midfacial development. Moreover, the growth centers had a strong association with midfacial forward growth before birth.

MiR-33a is a therapeutic target in SPG4-related hereditary spastic paraplegia human neurons.

Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3'-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.

Glucocorticoids suppress fibroblast apoptosis in an in vitro thermal injury model.

The wounds of full- and deep partial-thickness burns result in hypertrophic scars and lead to skin contracture more severely than those of superficial partial-thickness burns. Therefore, preventing burn progression may help improve the aesthetic and functional outcomes after healing. Although a number of studies have focused on elucidating the underlying mechanisms of and preventing burn wound progression, it is still difficult to rescue burned dermis unless early tangential excision is performed. To investigate the underlying mechanisms of and prevent cell death of heat-injured fibroblasts, we developed an in vitro experimental model of heat-injured fibroblasts. We confirmed that heating at 55°C for 30s caused fibroblast necrosis immediately after heating, whereas heating at 46°C for 30s induced apoptosis 24h after heating. We also found that the supplementation of 100ng/ml betamethasone to the culture medium after heating decreased the number of apoptotic cells and increased that of live cells. Our studies suggest that glucocorticoids suppress apoptosis of heat-injured fibroblasts and may be useful for preventing burn wound progression.

Visualising peripheral arterioles and venules through high-resolution and large-area photoacoustic imaging.

Photoacoustic (PA) imaging (PAI) has been shown to be a promising tool for non-invasive blood vessel imaging. A PAI system comprising a hemispherical detector array (HDA) has been reported previously as a method providing high morphological reproducibility. However, further improvements in diagnostic capability will require improving the image quality of PAI and fusing functional and morphological imaging. Our newly developed PAI system prototype not only enhances the PA image resolution but also acquires ultrasonic (US) B-mode images at continuous positions in the same coordinate axes. In addition, the pulse-to-pulse alternating laser irradiation shortens the measurement time difference between two wavelengths. We scanned extremities and breasts in an imaging region 140 mm in diameter and obtained 3D-PA images of fine blood vessels, including arterioles and venules. We could estimate whether a vessel was an artery or a vein by using the S-factor obtained from the PA images at two wavelengths, which corresponds approximately to the haemoglobin oxygen saturation. Furthermore, we observed tumour-related blood vessels around breast tumours with unprecedented resolution. In the future, clinical studies with our new PAI system will help to elucidate various mechanisms of vascular-associated diseases and events.

Tissue-engineered submillimeter-diameter vascular grafts for free flap survival in rat model.

Vascular grafts for free flap transfers should be of very small diameter and remain patent for approximately three weeks to supply blood until the revascularization from the surrounding tissue is established, with the autologous vein grafts acting as the gold standard. Artificial submillimeter-diameter vascular grafts with clinically useful size of 0.6 mm inner diameter and 5 cm length were prepared and evaluated by replacing the axial artery of free flap in rats. The rat tail artery, selected as a novel bioscaffold material, was decellularized using ultrahigh-hydrostatic pressure (UHP) method and compared with the detergent-based conventional method. To induce rapid endothelialization, the graft lumen was modified with synthesized peptides, having high affinity to the endothelial progenitor cells. The UHP method and peptide modification at 37 °C were found to preserve the extracellular matrix structure well, leading to the stable immobilization of the peptide at the luminal surface. These grafts showed the neointima formation, even at the center position far from the native vessels, remained patent for three weeks, and resulted in the flap survival in the rat free-flap model. The tissue-engineered vascular grafts with functionalized lumen have great future potential as an alternative to autologous vein grafts in free flap transfers.

The Utility of Silk-elastin Hydrogel as a New Material for Wound Healing.

Cutaneous ulcers are treated with dressing materials and/or ointments to keep the wound in an appropriately moist environment. However, chronic cutaneous ulcers commonly have bacterial colonization that can cause local infection in such an environment. Therefore, the dressing materials and/or ointments should have antibacterial potency to treat chronic ulcers. Acute cutaneous wounds, by contrast, heal rapidly without local infection. The aim of treating acute cutaneous wounds is therefore not only wound closure but also preventing scar contracture after wound healing. However, no dressing materials or ointments available at present are simultaneously effective for preventing infection in chronic ulcers and reducing wound contracture in acute ulcers. Silk-elastin is a recombinant protein polymer with repeating units of silk-like and elastin-like blocks. Silk-elastin solution can self-assemble from a liquid to a hydrogel. We preliminarily reported that silk-elastin hydrogels have the potential to accelerate wound healing in decubitus ulcers of diabetic mice, which are animal models of severe, intractable cutaneous ulcers. In the present study, we examined the effects of silk-elastin hydrogels in chronic and acute ulcer models in comparison with conventional products (carboxymethyl cellulose gel). Silk-elastin hydrogels resulted in significantly higher epithelialization rates than conventional hydrogels in both the chronic and acute ulcer models and significantly larger areas of granulation tissue in acute ulcer models. These results show that silk-elastin hydrogel is a promising material for promoting the healing of cutaneous wounds, including decubitus ulcers, chronic ulcers, and acute ulcers.

Photoacoustic Tomography Shows the Branching Pattern of Anterolateral Thigh Perforators In Vivo.

The distal branching pattern of perforators is associated with thin anterolateral thigh flap failure. The purpose of this study was to investigate the feasibility of using photoacoustic tomography as a diagnostic imaging modality to identify anterolateral thigh perforators and their branching patterns in the subcutaneous layer. Ten thighs in five healthy men were studied. The anterolateral aspect of the midthigh was examined using photoacoustic tomography. The correlation between photoacoustic tomography and ultrasound findings was evaluated. To determine the detectability of photoacoustic tomography by depth, the depth of vessels in the stem portion was compared to the depth of the deep fascia measured by ultrasound. Branching patterns of vessels in the adipose and suprafascial layers were evaluated by three-dimensional observation. A total of 18 perforators were visualized by photoacoustic tomography. Photoacoustic tomography and ultrasound had comparable diagnostic potential for the detection of perforators. Photoacoustic tomography visualized microvessels in the subcutaneous layer, especially those in oblique or horizontal orientations. The estimated mean depth of visualized vessels was 9 mm; the maximum depth was 13 mm. There was a strong correlation between the depth of visualized vessels in the stem portion and the depth of the deep fascia. Three-dimensional observation of photoacoustic tomographic images showed the branching morphology of perforators. This study showed the applicability of photoacoustic tomography to identification of the branching patterns of anterolateral thigh perforators in vivo, although limited visualization of subfascial vessels is a technical issue. The authors believe that photoacoustic tomography has the potential to be a new imaging modality for thin anterolateral thigh flap surgery. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, IV.

HtrA1 Is Specifically Up-Regulated in Active Keloid Lesions and Stimulates Keloid Development.

Keloids occur after failure of the wound healing process; inflammation persists, and various treatments are ineffective. Keloid pathogenesis is still unclear. We have previously analysed the gene expression profiles in keloid tissue and found that HtrA1 was markedly up-regulated in the keloid lesions. HtrA1 is a serine protease suggested to play a role in the pathogenesis of various diseases, including age-related macular degeneration and osteoarthritis, by modulating extracellular matrix or cell surface proteins. We analysed HtrA1 localization and its role in keloid pathogenesis. Thirty keloid patients and twelve unrelated patients were enrolled for in situ hybridization, immunohistochemical, western blot, and cell proliferation analyses. Fibroblast-like cells expressed more HtrA1 in active keloid lesions than in surrounding lesions. The proportion of HtrA1-positive cells in keloids was significantly higher than that in normal skin, and HtrA1 protein was up-regulated relative to normal skin. Silencing HtrA1 gene expression significantly suppressed cell proliferation. HtrA1 was highly expressed in keloid tissues, and the suppression of the HtrA1 gene inhibited the proliferation of keloid-derived fibroblasts. HtrA1 may promote keloid development by accelerating cell proliferation and remodelling keloid-specific extracellular matrix or cell surface molecules. HtrA1 is suggested to have an important role in keloid pathogenesis.

Thenar Dysplasia in Radial Polydactyly Depends on the Level of Bifurcation.

BACKGROUND: Little is known about thenar dysplasia in radial polydactyly, other than that thenar hypoplasia occasionally occurs in radial polydactyly with triphalangism. In particular, the phenotype and level of duplication associated with thenar dysplasia remain unclear. METHODS: The abductor pollicis brevis and flexor pollicis brevis muscles were visualized using three-dimensional ultrasound, and their horizontal geometry was assessed using a biaxial level classification system. Subjects were categorized into three phenotypes according to the developmental condition of the radial thumb. The relationship between the level of distribution of the muscles and the level of the bifurcation of the radial thumb was investigated. RESULTS: Nineteen patients with radial polydactyly without triphalangism were included. There were 10 patients with the nonfloating type, three with the floating type, and six with the rudimentary type. All patients with bifurcation at or more distal to the metacarpophalangeal joint had normal thenar muscle distribution, but the muscles in patients with bifurcation at or more proximal to the level of the metacarpals were confined, regardless of phenotype. The level of muscle distribution was strongly correlated with the level of the bifurcation of the radial thumb. CONCLUSIONS: These findings suggest that formation of the thenar muscles in the longitudinal direction in radial polydactyly might depend on the level of bifurcation of the radial thumb. The presence of thenar dysplasia even in floating-type or rudimentary-type duplications is of clinical and etiologic importance.

Efficacy of the dual controlled release of HGF and bFGF impregnated with a collagen/gelatin scaffold.

BACKGROUND: We previously developed collagen/gelatin sponges (CGS) able to sustain and release basic fibroblast growth factor (bFGF) and reported that this CGS impregnated with bFGF promoted dermis-like tissue formation. We herein confirmed the single-sustained release of hepatocyte growth factor (HGF) and the dual sustained release of HGF and bFGF from CGSs, and explored its efficacy using a murine model of skin defects. MATERIALS AND METHODS: The sustained release of HGF alone and both HGF and bFGF from CGSs were evaluated in vitro. CGSs (8 mm in diameter) impregnated with normal saline solution (NSS) (NSS group), HGF solution (10 or 50 µg/cm2) (HGF-L or HGF-H group), bFGF solution (7 µg/cm2) (bFGF group), or HGF (10 µg/cm2) and bFGF (7 µg/cm2) solution (HGF + bFGF group) were implanted into full-thickness skin defects on the backs of mice. The wound area, neoepithelium length, dermis-like tissue formation and newly formed capillaries were evaluated. RESULTS: The single release of HGF and the dual release of HGF and bFGF from CGSs were confirmed. At week 1, the wound closure and neoepithelium length were promoted in the HGF-L group compared with the NSS group. At week 2, the wound closure, neoepithelium length, dermis-like tissue formation and newly formed capillary formation were promoted in the bFGF and HGF + bFGF groups compared with the NSS and HGF-H groups. Newly formed capillary formation was superior in the HGF + bFGF group compared with the bFGF group. CONCLUSIONS: The dual release of HGF and bFGF from CGS was a promising treatment for full-thickness skin defects.

Development of a novel bioabsorbable implant that is substituted by adipose tissue in vivo.

Recently, adipose tissue has been regenerated by combining scaffolds, growth factors, and/or adipose-tissue-derived stromal cells. However, the safety of growth factors and adipose-tissue-derived stromal cells has not been confirmed in cancer patients. We reported the regeneration of adipose tissue in the internal space of a polypropylene mesh containing a collagen sponge (CS), without using any growth factors or cells. We herein explored the formation of adipose tissue, using the bioabsorbable implant containing CS, in rats. We prepared the implants without and with CS, using threads of either poly-l-lactide-co-ε-caprolactone or poly-l-lactic acid (PLLA), and measured their strengths. The procedure was performed in the rat inguinal region. In the control group, no operative procedure was performed. In the sham-operation group, skin incision without implantation was performed. The other groups received CS alone and the 2 implants with and without CS. The areas of formed tissue and adipose tissue inside the implants and the remnants of CS were evaluated. All implants maintained the internal space before implantation. At 6 and 12 months after implantation, the internal space was maintained and the formation of adipose tissue was promoted in the 2 PLLA groups. At 6 months, the internal space was maintained, and more adipose tissue was formed in the PLLA-with-CS group than in the PLLA group. Porcine collagen was absorbed within 3 months. The PLLA implant with CS is a novel bioabsorbable implant that is replaced with autologous adipose tissue after implantation.

A Quick Evaluation of TRAM Flap Viability using Fingerstall-Type Tissue Oximetry.

Supplemental Digital Content is available in the text.

Melanin pigments in the melanocytic nevus regress spontaneously after inactivation by high hydrostatic pressure.

We report a novel treatment for giant congenital melanocytic nevi (GCMN) that involves the reuse of resected nevus tissue after high hydrostatic pressurization (HHP). However, the remaining melanin pigments in the inactivated nevus tissue pose a problem; therefore, we performed a long-term observation of the color change of inactivated nevus tissue after HHP. Pressurized nevus specimens (200 MPa group, n = 9) and non-pressurized nevus tissues (control group, n = 9) were subcutaneously implanted into nude mice (BALB/c-nu) and then harvested 3, 6, and 12 months later. Color changes of the nevus specimens were evaluated. In the 200 MPa group, the specimen color gradually regressed and turned white, and brightness values were significantly higher in the 200 MPa group than in the control group after 6 months. This indicated that melanin pigments in the pressurized nevus tissue had spontaneously degraded and regressed. Therefore, it is not necessary to remove melanin pigments in HHP-treated nevus tissue.

The development of a novel wound healing material, silk-elastin sponge.

Silk-elastin is a recombinant protein polymer with repeating units of silk and elastin blocks. This novel wound healing promoting material has the ability to self-assemble from a liquid to a gel. We have already reported that an aqueous solution of silk-elastin has the potential to accelerate wound healing; however, there are several problems in applying silk-elastin in the clinical setting. To solve these problems, we developed a silk-elastin sponge that is easy to use in the clinical setting. In the present study, we examined whether the wound healing effect of the silk-elastin sponge is equal to the aqueous solution of silk-elastin in vivo. The granulation tissue formation promoting effect of the silk-elastin sponge was equal to that of the aqueous solution the silk-elastin, as after application to the wound surface, the sponge was absorbed and dissolved by the exudate. At body temperature the silk-elastin then formed temperature gel. The silk-elastin gel that was obtained contained abundant cytokines from the exudate. We believe that silk-elastin sponge can be applied to various wounds that are difficult to treat with the aqueous solution.

Quantitation of nasal development in the early prenatal period using geometric morphometrics and MRI: a new insight into the critical period of Binder phenotype.

OBJECTIVES: Disturbance of the development of the nasal septum in the early prenatal period causes congenital facial anomalies characterized by a flat nose and defects of the anterior nasal spine (ANS), such as Binder phenotype. The present research aimed to assess the development of the nasal septum and the ANS with growth in the early prenatal period. METHODS: Magnetic resonance images were obtained from 56 specimens. Mid-sagittal images were analyzed by using geometric morphometrics for the development of the nasal septum, and angle analysis was performed for the development of the ANS. Additionally, we calculated and visualized the ontogenetic allometry of the nasal septum. RESULTS: Our results showed that the nasal septum changed shape in the anteroposterior direction in smaller specimens, while it maintained an almost isometric shape in larger specimens. Furthermore, mathematical evidence revealed that the maturation periods of the shapes of the ANS and the nasal septum were around 12 and 14 weeks of gestation, respectively. CONCLUSION: The anteroposterior development of the nasal septum is specific until 14 weeks of gestation, and it is important for nasal protrusion and the development of the ANS. Therefore, the disturbance of such development could induce low nasal deformity, including Binder phenotype. © 2017 John Wiley & Sons, Ltd.

Patient With Cleft Maxillary Hypoplasia Who Underwent Distraction Osteogenesis and Conservative Therapies to Postoperative Velopharyngeal Insufficiency.

The authors performed distraction osteogenesis using The Maxillary Distractor System (SYNTHES) to maxillary hypoplasia patient with cleft lip palate, and consequently improved the aesthetic complexion of the patient. Velopharyngeal insufficiency developed after bone elongation; the authors improved the insufficiency with conservative therapies such as articulatory training using the bulb attached palatal lift prosthesis. The authors were successful and accepted postoperative speech outcome.

DIEP flap breast reconstruction preserving a lumbar peritoneal shunt tube.

We herein report a case of immediate deep inferior epigastric perforator flap (DIEP flap) breast reconstruction surgery in a 50-year-old female patient with a lumbar peritoneal shunt tube. We performed DIEP flap reconstruction in the patient by withdrawing and subsequently reinserting the abdominal side of an implanted shunt tube.