MicroRNA-142-5p is Up-regulated on Allogeneic Immune Responses and Up-regulates MHC Class II Expression in Human Umbilical Vein Endothelial Cells.

PURPOSE: MicroRNA could be biomarker and therapeutic target for rejection. The aim of this study was to investigate the role of miR-142-5p in allogeneic immune responses using in vitro and in vivo models. MATERIALS AND METHODS: Primary and immortalized human umbilical vein endothelial cells (HUVECs) were cultured with unrelated blood mononuclear cells to induce allogeneic immune responses. Syngeneic and allogeneic skin graft was performed in mice. Flow cytometry, quantitative reverse transcription-polymerase chain reaction, and Western blotting was performed to understand the underlying mechanisms. RESULTS: miR-142-5p was up-regulated in primary HUVEC and a HUVEC line when allogeneic immune responses were elicited. miR-142-5p was also up-regulated in the murine allogeneic skin graft. Overexpression of miR-142-5p in HUVEC increased the expression of HLA-ABC and HLA-DR additively to allogeneic immune responses, suggesting a possible increase in alloantigen presentation. Inhibition of miR-142-5p reduced the expression of HLA-DR. ZEB1, a putative target gene of miR-142-5p, was down-regulated in HUVEC on allogeneic immune response as well as in murine allogeneic skin graft. CONCLUSION: These results suggest that the up-regulation of miR-142-5p on allogeneic immune response might facilitate endothelial activation to exacerbate rejection.

Usefulness of Rapiplug in nipple reconstruction to improve nipple projection.

BACKGROUND: The final stage of breast reconstruction after mastectomy for breast cancer is nipple reconstruction. However, a consistent and reliable method resulting in the most ideal aesthetic results has yet to be clarified. This study analysed the long-term outcomes of nipple reconstruction using Rapiplug. METHODS: Forty-one patients who underwent immediate breast reconstruction after mastectomy between January 2014 and February 2017 were enrolled. Nipple reconstruction was performed with C-V flap and Hammond flap, and hat-shaped Rapiplug was implanted at the flap core. Nipple projection and width were measured and nipple reduction rates were calculated immediately after and at 3, 6, and 12 months after surgery. Patient satisfaction was surveyed at the 12-month follow-up and compared with patient characteristics and other variables. RESULTS: Forty-one nipple reconstructions were performed in 41 patients. Most post-operative adverse events were resolved with conservative management, and revision was performed in only one case. The mean nipple projections were 8.9 ± 1.8, 7 ± 1.8, 5.6 ± 1.6 and 4.9 ± 1.6 mm immediately, and 3, 6 and 12 months after surgery, respectively, and the mean reduction rate of nipple size with reference to the size immediately after surgery was 22.2%, 37.2% and 44.7% at 3, 6 and 12 months after surgery, respectively. Patient satisfaction was 82.9% in overall outcome, and 85.3% of projection was good or excellent. CONCLUSION: Rapiplug can improve the long-term preservation of nipple projection after nipple reconstruction and is considered to be consistent and reliable with good aesthetic outcomes and no severe complications.

Publisher Correction: Magnetization Manipulation of a Flexible Magnetic Sensor by Controlled Stress Application.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Magnetization Manipulation of a Flexible Magnetic Sensor by Controlled Stress Application.

Spin-based electronic devices on polymer substrates have been intensively investigated because of several advantages in terms of weight, thickness, and flexibility, compared to rigid substrates. So far, most studies have focused on maintaining the functionality of devices with minimum degradation against mechanical deformation, as induced by stretching and bending of flexible devices. Here, we applied repetitive bending stress on a flexible magnetic layer and a spin-valve structure composed of Ta/NiFe/CoFe/Cu/Ni/IrMn/Ta on a polyimide (PI) substrate. It is found that the anisotropy can be enhanced or weakened depending upon the magnetostrictive properties under stress. In the flat state after bending, due to residual compressive stress, the magnetic anisotropy of the positive magnetostrictive free layer is weakened while that of the pinned layer with negative magnetostriction is enhanced. Thus, the magnetic configuration of the spin-valve is appropriate for use as a sensor. Through the bending process, we design a prototype magnetic sensor cell array and successfully show a sensing capability by detecting magnetic microbeads. This attempt demonstrates that appropriate control of stress, induced by repetitive bending of flexible magnetic layers, can be effectively used to modify the magnetic configurations for the magnetic sensor.

Surgical indication of Furlow palatoplasty to treat submucous cleft palate.

This study investigated the effects of Furlow palatoplasty on children with submucous cleft palate (SMCP) and identified surgical indications by comparing SMCP and control patients. Twenty-three SMCP children (average age 28.9 months) who were nonsyndromic and underwent surgery between April 2010 and December 2016 were included. Facial computed tomography (CT) was performed preoperatively and at least 1 year postoperatively after a language test. Facial CT measurements were taken for 140 children aged 0-6 years without deformities (control group). Later surgery was associated with more severe nasality. In the coronal view, the difference in the maxillary tuberosity before and after surgery was 3.8 mm (p < 0.05). The height and width of the palatal arch (HNP and WNP) were well maintained (p > 0.05), whereas the angle of the levator veli palatini muscle (ALM) increased (p < 0.05). The nasopharynx was close to normal postoperatively. The distance between the medial pterygoid plates, the HNP, and the WNP were larger in SMCP patients preoperatively (p < 0.05), but these differences disappeared after surgery (p > 0.05). The ALM in SMCP patients was narrower preoperatively, but became flatter postoperatively (p < 0.05), indicating the repositioning of the levator muscle, with improvement of the velopharyngeal function. Furlow palatoplasty is indicated if the HNP and WNP values are larger, and the ALM value is less, in patients with SMCP than in those without.

A giant trichoblastic carcinoma.

Trichoblastic carcinoma usually occurs as a malignant transformation of the trichoblastoma, but is very rare. A 25-year-old man was admitted with trichoblastoma in the nuchal area with frequent recurrences since birth. The preoperative neck magnetic resonance image revealed lobulated soft tissue lesions involving superficial fascia and infiltrating into both proximal trapezius muscles. In our department, wide excision and reconstruction with a free anterolateral thigh flap were performed. Histological examination revealed skin adnexal carcinoma, originating from the hair follicles, consistent with trichoblastic carcinoma. There was no palpable mass 5 years postoperatively, and there was no recurrence on follow-up positron emission tomography-computed tomography. Trichoblastic carcinomas are rare and difficult to diagnose, but histopathological findings include atypical basaloid keratinocytes with crowded, hyperchromatic nuclei, and increased mitotic activity. The presence of hypercellular stroma is a criterion for distinguishing trichoblastic carcinoma from basal cell carcinoma. A rare giant trichoblastic carcinoma was reported, which was the biggest one in the literature.

Liver-directed gene therapy for murine glycogen storage disease type Ib.

Glycogen storage disease type-Ib (GSD-Ib), deficient in the glucose-6-phosphate transporter (G6PT), is characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenoma (HCA). We examined the efficacy of G6PT gene therapy in G6pt-/- mice using recombinant adeno-associated virus (rAAV) vectors, directed by either the G6PC or the G6PT promoter/enhancer. Both vectors corrected hepatic G6PT deficiency in murine GSD-Ib but the G6PC promoter/enhancer was more efficacious. Over a 78-week study, using dose titration of the rAAV vectors, we showed that G6pt-/- mice expressing 3-62% of normal hepatic G6PT activity exhibited a normalized liver phenotype. Two of the 12 mice expressing < 6% of normal hepatic G6PT activity developed HCA. All treated mice were leaner and more sensitive to insulin than wild-type mice. Mice expressing 3-22% of normal hepatic G6PT activity exhibited higher insulin sensitivity than mice expressing 44-62%. The levels of insulin sensitivity correlated with the magnitudes of hepatic carbohydrate response element binding protein signaling activation. In summary, we established the threshold of hepatic G6PT activity required to prevent tumor formation and showed that mice expressing 3-62% of normal hepatic G6PT activity maintained glucose homeostasis and were protected against age-related obesity and insulin resistance.

A new approach to nasomaxillary complex type of nasal bone fracture: Clip operation.

PURPOSE: Nasal bone fractures comprise almost 40% of all facial injuries. Most are initially reduced using closed reduction. This study introduces a newly developed method, the clip operation via endonasal approach. MATERIALS AND METHODS: The operation was performed in these patients by a single surgeon extensively experienced in facial bone fractures. An absorbable mesh plate made into a clip was used for fixation after open reduction via the endonasal approach. No screws were used for fixation. Nasal packing was removed the first day after surgery; aluminum splinting was removed the third week after surgery. Three-dimensional facial computed tomography and cephalolateral radiography were performed preoperatively and postoperatively. Plastic surgeon satisfaction and postoperative complications were assessed. RESULTS: Fracture relapse was not observed. Reduction status was well maintained. Postoperative complications occurred, with a low final incidence of 1.8% in the third postoperative month. Plastic surgeon satisfaction was very high at 4.58. This operation takes 5-10 min, and is simple to perform. It entails a short hospitalization, and the duration during which nasal packing and aluminum splint are maintained is comparable. Undesirable functional, aesthetic complications and secondary surgery resulting from inaccurate relapse were reduced. CONCLUSION: The clip operation is a useful technique for correcting nasal bone fractures, especially nasomaxillary complex type.

Downregulation of pathways implicated in liver inflammation and tumorigenesis of glycogen storage disease type Ia mice receiving gene therapy.

Glycogen storage disease type Ia (GSD-Ia) is characterized by impaired glucose homeostasis and long-term risks of hepatocellular adenoma (HCA) and carcinoma (HCC). We have shown that the non-tumor-bearing (NT), recombinant adeno-associated virus (rAAV) vector-treated GSD-Ia mice (AAV-NT mice) expressing a wide range (0.9-63%) of normal hepatic glucose-6-phosphatase-α activity maintain glucose homeostasis and display physiologic features mimicking animals living under calorie restriction (CR). We now show that in AAV-NT mice, the signaling pathways of the CR mediators, AMP-activated protein kinase (AMPK) and sirtuin-1 are activated. AMPK/sirtuin-1 inhibit the activity of STAT3 (signal transducer and activator of transcription 3) and NFκB (nuclear factor κB), the pro-inflammatory and cancer-promoting transcription factors. Sirtuin-1 also inhibits cancer metastasis via increasing the expression of E-cadherin, a tumor suppressor, and decreasing the expression of mesenchymal markers. Consistently, in AAV-NT mice, hepatic levels of active STAT3 and NFκB-p65 were reduced as were expression of mesenchymal markers, STAT3 targets, NFκB targets and ß-catenin targets, all of which were consistent with the promotion of tumorigenesis. AAV-NT mice also expressed increased levels of E-cadherin and fibroblast growth factor 21 (FGF21), targets of sirtuin-1, and ß-klotho, which can acts as a tumor suppressor. Importantly, treating AAV-NT mice with a sirtuin-1 inhibitor markedly reversed many of the observed anti-inflammatory/anti-tumorigenic signaling pathways. In summary, activation of hepatic AMPK/sirtuin-1 and FGF21/ß-klotho signaling pathways combined with down-regulation of STAT3/NFκB-mediated inflammatory and tumorigenic signaling pathways can explain the absence of hepatic tumors in AAV-NT mice.

Glycogen storage disease type Ia mice with less than 2% of normal hepatic glucose-6-phosphatase-α activity restored are at risk of developing hepatic tumors.

Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3-63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring <2% of normal hepatic G6Pase-α activity are at risk of tumor development.

Glycogen storage disease type Ib neutrophils exhibit impaired cell adhesion and migration.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is an inherited autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Neutrophils play an essential role in the defense against invading pathogens. The recruitment of neutrophils towards the inflammation sites in response to inflammatory stimuli is a tightly regulated process involving rolling, adhesion, and transmigration. In this study, we investigated the role of G6PT in neutrophil adhesion and migration using in vivo and in vitro models. We showed that the GSD-Ib (G6pt-/-) mice manifested severe neutropenia in both blood and bone marrow, and treating G6pt-/- mice with granulocyte colony-stimulating factor (G-CSF) corrected neutropenia. However, upon thioglycolate challenge, neutrophils from both untreated and G-CSF-treated G6pt-/-mice exhibited decreased ability to migrate to the peritoneal cavity. In vitro migration and cell adhesion of G6PT-deficient neutrophils were also significantly impaired. Defects in cell migration were not due to enhanced apoptosis or altered fMLP receptor expression. Remarkably, the expression of the ß2 integrins CD11a and CD11b, which are critical for cell adhesion, was greatly decreased in G6PT-deficient neutrophils. This study suggests that deficiencies in G6PT cause impairment in neutrophil adhesion and migration via aberrant expression of ß2 integrins, and our finding should facilitate the development of novel therapies for GSD-Ib.

Feasibility of the Use of RapiGraft and Skin Grafting in Reconstructive Surgery.

BACKGROUND: Skin grafting is a relatively simple and thus widely used procedure. However, the elastic and structural quality of grafted skin is poor. Recently, various dermal substitutes have been developed to overcome this disadvantage of split-thickness skin grafts. The present study aims to determine the feasibility of RapiGraft as a new dermal substitute. METHODS: This prospective study included 20 patients with partial- or full-thickness skin defects; the patients were enrolled between January 2013 and March 2014. After skin defect debridement, the wound was divided into two parts by an imaginary line. Split-thickness skin grafting alone was performed on one side (group A), and RapiGraft and split-thickness skin grafting were used on the other side (group B). All patients were evaluated using photographs and self-questionnaires. The Manchester scar scale (MSS), a chromameter, and a durometer were used for the scar evaluation. The average follow-up period was 6 months. RESULTS: The skin graft take rates were 93% in group A and 89% in group B, a non-significant difference (P=0.082). Statistically, group B had significantly lower MSS, vascularity, and pigmentation results than group A (P<0.05 for all). However, the groups did not differ significantly in pliability (P=0.155). CONCLUSIONS: The present study indicates that a simultaneous application of RapiGraft and a split-thickness skin graft is safe and yields improved results. Therefore, we conclude that the use of RapiGraft along with skin grafting will be beneficial for patients requiring reconstructive surgery.

Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation.

Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.

Pro178 and Pro183 of selenoprotein S are essential residues for interaction with p97(VCP) during endoplasmic reticulum-associated degradation.

During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro(178) and Pro(183) of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress.

Inhibition of 14-3-3 binding to Rictor of mTORC2 for Akt phosphorylation at Ser473 is regulated by selenoprotein W.

14-3-3 reduces cell proliferation by inhibiting the activity of proteins involved in the signaling pathway that includes Akt kinase. Activation of Akt is enhanced by activating the mammalian target of rapamycin complex 2 (mTORC2). 14-3-3 is also a negative regulator of the mTORC2/Akt pathway, by interacting with a component of mTORC2. Recently, we reported that selenoprotein W (SelW) regulated the interaction between 14-3-3 and its target protein, CDC25B. Here, we show that the binding of Rictor, a component of mTORC2, to 14-3-3, is regulated by the interaction of 14-3-3 with SelW. When SelW was down-regulated, mTORC2-dependent phosphorylation of Akt at Ser473 was decreased. However, the phosphorylation of Thr308 was not affected. The interaction of Rictor with 14-3-3 was increased in SelW-knockdown cells, as compared to control cells. SelW-knockdown cells were also more sensitive to DNA damage induced by etoposide, than control cells. This phenomenon was due to the decreased phosphorylation of Akt at Ser473. We also found that ectopic expression of SelW(U13C) reduced the interaction between Rictor and 14-3-3, leading to Akt phosphorylation at Ser473. Taken together, these findings demonstrate that SelW activates the mTORC2/Akt pathway for Akt phosphorylation at Ser473, by interrupting the binding of Rictor to 14-3-3.

Regulation of cytosolic phospholipase A2 phosphorylation by proteolytic cleavage of annexin A1 in activated mast cells.

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow-derived mast cells. This cleavage event was inhibited when intracellular Ca(2+) signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A(2) (cPLA(2)) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA(2) interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA(2) mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA(2) and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA(2) and production of eicosanoids during mast-cell activation.