Tunneling nanotube-driven complete regeneration of murine fetal skin.

This study investigated the three-dimensional (3D) cellular interactions and tunneling nanotubes (TNTs) during fetal mouse skin regeneration on embryonic days 13 (E13) and 15 (E15). We aimed to understand spatial relationships among cell types involved in skin regeneration and assess the potential role of TNTs. Full-thickness skin incisions were performed in E13 and E15 embryos. Wound sites were collected, embedded in epoxy resin, processed for 3D reconstruction (1 µm thickness sections), and subjected to whole-mount immunostaining. We conducted in vitro co-culture experiments with fetal macrophages and fibroblasts to observe TNT formation. To assess the effect of TNTs on skin regeneration, an inhibiting agent (cytochalasin B) was administered to amniotic fluid. Results revealed that E13 epidermal keratinocytes interacted with dermal fibroblasts and macrophages, facilitating skin regrowth. TNT structures were observed at the E13-cell wound sites, among macrophages, and between macrophages and fibroblasts, confirmed through in vitro co-culture experiments. In vitro and utero cytochalasin B administration hindered those formation and inefficient skin texture regeneration at E13 wound sites. This emphasizes the necessity of 3D cellular interactions between epidermal and dermal cells during skin regeneration in mouse embryos at E13. The prevalence of TNT structures indicated their involvement in achieving complete skin texture restoration.

Factors Related to Satisfaction with Decision-making Regarding Human Papillomavirus Vaccination Behavior among Female University Students in Japan.

Introduction: Cervical cancer is the fourth most common cancer among women worldwide. Most cervical cancers are caused by persistent infection with human papillomavirus (HPV) acquired through sexual contact. Decision-making is the process of choosing among several options, and a better decision is one that the people engaged in the decision-making process express satisfaction with. Despite that HPV infection is associated with sexual behavior, no studies in Japan on HPV vaccination decision-making that include perspectives on sexuality exist. This study aimed to determine the factors that influence satisfaction with decision-making concerning HPV vaccination among female university students in Japan. Methods: The cross-sectional study was carried out by an anonymous self-administered questionnaire mail survey of 1988 female university students in Japan between April and July 2021. Of them, 301 agreed to participate in the survey. After the exclusion of those with missing data, the analysis included 252 (12.7%) students. We summarized descriptive statistics in terms of characteristics, satisfaction with decision-making regarding HPV vaccination, HPV vaccination behavior, knowledge, attitude about HPV vaccination, influencing factors, and perceptions and behaviors related to sexuality. Furthermore, we conducted multivariate analyses to investigate factors that influence satisfaction with decision-making regarding HPV vaccination. Results: Of the 252 participants, 102 (40.5%) were satisfied with their decisions regarding HPV vaccination. After adjustment for confounding factors, the multivariable-adjusted odds ratios (95% confidence intervals) for factors associated with satisfaction in decision-making regarding HPV vaccination were as follows: being vaccinated (vs. non-vaccinated) 5.46 (2.51-11.89), having high knowledge scores (vs. per 1 point) 1.09 (1.01-1.17), and having awareness about the risk of contracting sexually transmitted infections (STIs) via sexual intercourse (vs. per 1 point) 0.83 (0.72-0.96). Conclusions: Being vaccinated, having higher knowledge scores, and having lower awareness regarding the risk of STIs were associated with satisfied decision-making concerning HPV vaccination. Providing younger people with correct information about cervical cancer, HPV vaccines, and STI prevention contributes to increased satisfaction with their HPV vaccination decisions.

Secondary arteriovenous malformation due to subclavian vein occlusion.

An 80-year-old man underwent rectal resection and insertion of a central venous catheter through the left subclavian vein 16 years earlier. Following surgery, he developed edema of his left upper limb that became exacerbated and infected. Computed tomography showed occlusion of the subclavian vein and multiple arteriovenous shunts from the branches of the axillary artery to the venous sac of the axillary vein. Angiography confirmed numerous shunts between the branches of the axillary artery and vein and dilated collateral veins. Embolization of the venous sac was performed using coils, alcohol, and glue. Postprocedural angiography showed complete eradication of the nidus.

In Vivo Label-Free Observation of Tumor-Related Blood Vessels in Small Animals Using a Newly Designed Photoacoustic 3D Imaging System.

Photoacoustic (PA) technology can be used for non-invasive imaging of blood vessels. In this paper, we report on our prototype PA imaging system with a newly designed ultrasound sensor and its visualization performance of microvascular in animal. We fabricated an experimental system for animals using a high-frequency sensor. The system has two modes: still image mode by wide scanning and moving image mode by small rotation of sensor array. Optical test target, euthanized mice and rats, and live mice were used as objects. The results of optical test target showed that the spatial resolution was about two times higher than that of our conventional prototype. The image performance in vivo was evaluated in euthanized healthy mice and rats, allowing visualization of detailed blood vessels in the liver and kidneys. In tumor-bearing mice, different results of vascular induction were shown depending on the type of tumor and the method of transplantation. By utilizing the video imaging function, we were able to observe the movement of blood vessels around the tumor. We have demonstrated the feasibility of the system as a less invasive animal experimental device, as it can acquire vascular images in animals in a non-contrast and non-invasive manner.

Topical estrogen application promotes cutaneous wound healing in db/db female mice with type 2 diabetes.

Female sex hormones are beneficial effects for wound healing. However, till date, whether topical estrogen application can promote cutaneous wound healing in diabetes remains unclear. Therefore, the present study aimed to validate the effect of topical estrogen application on cutaneous wound healing in a type 2 diabetes db/db mice model. In total, 22 db/db female mice with type 2 diabetes and eight C57BL/6J female mice were subjected to two full-thickness wound injuries. The mice were divided into the db/db, db/db + estrogen, db/db + vehicle, and wild type (WT) groups. Wound healing was assessed until day 14. The db/db group had a significantly high wound area ratio (wound area/initial wound area) on days 3-14 and a significantly low re-epithelialization ratio on days 7 and 14. Moreover, their angiogenesis ratio was significantly low on day 7 and high on day 14. In contrast, compared with the db/db group, the db/db + estrogen group had a significantly lower wound area ratio on days 1-14 and angiogenesis ratio on day 14, thereby indicating early withdrawal of new blood vessels, as well as a significantly higher re-epithelialization ratio on days 7 and 14 and Ym1+ M2 macrophage/macrophage ratio on day 7. Moreover, microarray analysis showed that the top 10 upregulated or downregulated genes in the db/db group were reversed by estrogen treatment, particularly on day 14, in comparison with the WT group. Thus, topical estrogen application reduced the wound area, promoted re-epithelialization and angiogenesis, and increased the number of M2 macrophages in mice with type 2 diabetes. Furthermore, it improved the differential regulation of genes in db/db mice. Therefore, such treatment can enhance cutaneous wound healing in female mice with type 2 diabetes.

Complications of craniofacial surgery using the ultrasonic-assisted pinned resorbable system: A prospective report with a minimum follow-up of 30 months.

Several resorbable fixation systems are used for osteosynthesis in craniofacial surgery. Recently, ultrasonic-assisted pinned resorbable systems have been introduced; however, few studies have described the associated complications during the long-term follow-up until complete resorption. In this study, we investigated the complications of craniofacial surgery using the ultrasonic-assisted pinned resorbable system with a follow-up of at least 30 months. Among patients who underwent craniofacial surgery using a commercially available ultrasonic-assisted pinned resorbable system between 2014 and 2016, those with follow-up visits for at least 30 months were included in this study. We investigated the development of complications such as local infection, exposure of the device, and reoperation related to the implant. Twenty-four patients aged 6 months to 69.4 years (median: 3.5 years) were followed up for more than 30 months. None of the patients required reoperation regardless of implants. Further, no infection or device exposure was seen among all patients. However, two patients aged 6 and 22 months who underwent cranioplasty for craniosynostosis and another patient aged 148 months who underwent cranioplasty for cranial defect exhibited plate-related bulging in the scalp during the course of resorption between 7 and 12 months of follow-up. The bulges were characterized by swelling without pain or redness and resolved spontaneously within 18 months of follow-up, which was considered to occur after complete absorption of the plate. In conclusion, subcutaneous swelling is related to resorbable plates and has a benign clinical course. We recommend that patients be informed of this phenomenon preoperatively to relieve their anxiety.

Topical estrogen application to wounds promotes delayed cutaneous wound healing in 80-week-old female mice.

Topical estrogen application to wounds is effective in promoting cutaneous wound healing. However, whether it promotes cutaneous wound healing in delayed cutaneous wound healing associated with advanced age remains to be elucidated. This study aimed to evaluate the effect of topical estrogen application to wounds in cutaneous wound healing in 80-week-old female mice. C57BL/6J female mice aged 82-85 and 12 weeks old were submitted to two full-thickness wounds. Mice were divided into four groups: aged group, topical estrogen wound treatment aged group (aged-E), vehicle wound treatment aged group (aged-vehicle), and young group. Wound healing was observed until day 14. In the aged group, wound area ratio (wound area / initial wound area) was significantly higher on days 3-14, ratio of re-epithelialization was significantly lower on day 3 and tended to be lower on day 14, and neutrophil number was significantly higher on day 7 compared with the young group. In contrast, in the aged-E group, wound area ratio was significantly smaller on days 1-14, re-epithelialization ratio was significantly higher on days 3-14, and neutrophil and macrophage number was significantly lower on days 3 and 7 compared with the aged group. These results demonstrate that topical estrogen application to wounds in 80-week-old female mice promoted cutaneous wound healing by reducing wound area and inflammatory response and promoting re-epithelialization.

The Incidence of Sleep Disordered Breathing One Week After Primary Palatoplasty: Evaluation With Overnight Pulse Oximetry.

BACKGROUND: Sleep disordered breathing (SDB) is defined as a series of disorders including snoring, obstructive sleep apnea, and hypopnea. Few studies investigated the incidence of SDB following primary palatoplasty with objective testing. The aims of this study were to elucidate the prevalence and degree of SDB approximately 1 week following primary palatoplasty with objective testing and to clarify the risk factors. METHOD: A retrospective review was performed on children who underwent primary palatoplasty between April 2013 and July 2017 at National Center for Child Health and Development, Tokyo, Japan. As a national center, the authors accept many syndromic patients. The authors keep all patients after palatoplasty intubated and observe them overnight in intensive care unit to reduce the risks of respiratory events. Patients were evaluated with overnight pulse oximetry on 5 to 7 days postoperatively. RESULTS: Forty-four patients were included, and 30% of the patients were associated with congenital anomaly. Thirteen patients (30%) were diagnosed with SDB. None of the patients required additional treatment after the evaluation. Laryngomalacia and postoperative oxygen requirement significantly correlated with postoperative SDB. CONCLUSION: Approximately one-third of the patients may be at the risk of SDB 1 week after primary palatoplasty. Patients with history of laryngomalacia or those who required oxygen support for prolonged time after primary palatoplasty should be cared for significantly high risk of postoperative SDB.

Analysis of Cranial Morphology of Healthy Infants Using Homologous Modeling.

OBJECTIVE: Data on cranial morphology of healthy individuals can be used as the guide in the treatment of cranial deformity. There are many reports analyzing the cranial morphology of healthy children in the past. But most of them focus on 2-dimensional values, and there are only a few reports, which analyzed the cranial morphology of Japanese healthy infants. We report a novel method that enables the comprehensive analysis of cranial morphology of Japanese healthy infants in 3D. METHODS: Craniofacial CT data of 20 healthy infants (9 males, 11 females) ranging in age from 1 to 11 months were collected. Based on the CT data, we created 20 homologous models of cranium using software specifically designed to support homologous modeling. We averaged vertex coordinates of the homologous models to create average model. We further performed principal component analysis, and created virtual models based on each principal component. The contribution rate was calculated, and the features described by each principal component were interpreted. RESULTS: We created the average cranial model of Japanese healthy infants. Seven principal components (cumulative contribution rate: 89.218%) were interpreted as to which part of the cranial shape each component was related to. The elements were extracted that may characterize the cranial morphology of some of the clinical conditions such as dolico/brachycephaly and deformational plagiocephaly. Some of these elements have not been mentioned in the past literature. CONCLUSION: Homologous modeling was considered to be valid and strong tool for comprehensive analysis of cranial morphology.

De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome.

Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.

Near-Infrared Fluorescence Imaging Directly Visualizes Lymphatic Drainage Pathways and Connections between Superficial and Deep Lymphatic Systems in the Mouse Hindlimb.

Since lymphedema rarely develops in the mouse hindlimb, the underlying mechanisms remain unclear. We herein investigated the resolution of chronic hindlimb lymphedema in mice using a Near-Infrared Fluorescence (NIRF) imaging system. Nineteen 7-28-week-old BALB/c male and female mice were injected with two dyes for lymphography and dissection. Lymphadenectomy was performed on six male mice to completely obstruct lymph flow in the hindlimb. Edematous changes in both hindlimbs were compared until 60 days after surgery. The NIRF imaging system detected three lymphatic collecting systems in the mouse hindlimb: superficial lateral, superficial medial, and deep medial. It also showed connections between the superficial and deep lymphatic systems in the inguinal region. Lymphadenectomy of the iliac, inguinal, and popliteal lymph nodes caused edematous changes. However, lymph flow in these operated areas restarted within 60 days and the severity of lymphedema appeared to be low. NIRF imaging showed that the deep medial system and a connection between the superficial and deep lymphatic systems in the inguinal region drain lymph from the hindlimb. This is the one reasons why lymphedema does not develop in the mouse hindlimb. The stable obstruction of lymph flow in these three systems is desired to develop chronic lymphedema.

Cross talk between TP53 and c-Myc in the pathophysiology of Diamond-Blackfan anemia: Evidence from RPL11-deficient in vivo and in vitro models.

Mutations in genes encoding ribosomal proteins have been identified in Diamond-Blackfan anemia (DBA), a rare genetic disorder that presents with a prominent erythroid phenotype. TP53 has been implicated in the pathophysiology of DBA with ribosomal protein (RP) L11 playing a crucial role in the TP53 response. Interestingly, RPL11 also controls the transcriptional activity of c-Myc, an oncoprotein that positively regulates ribosome biogenesis. In the present study, we analyzed the consequences of rpl11 depletion on erythropoiesis and ribosome biogenesis in zebrafish. As expected, Rpl11-deficient zebrafish exhibited defects in ribosome biogenesis and an anemia phenotype. However, co-inhibition of Tp53 did not alleviate the erythroid aplasia in these fish. Next, we explored the role of c-Myc in RPL11-deficient cellular and animal models. c-Myc and its target nucleolar proteins showed upregulation and increased localization in the head region of Rpl11-deficient zebrafish, where the morphological abnormalities and tp53 expression were more pronounced. Interestingly, in blood cells derived from DBA patients with mutations in RPL11, the biogenesis of ribosomes was defective, but the expression level of c-Myc and its target nucleolar proteins was unchanged. The results suggest a model whereby RPL11 deficiency activates the synthesis of c-Myc target nucleolar proteins, which subsequently triggers a p53 response. These results further demonstrate that the induction of Tp53 mediates the morphological, but not erythroid, defects associated with RPL11 deficiency.

Protective effects of oral glutathione on fasting-induced intestinal atrophy through oxidative stress.

AIM: To determine whether oral glutathione (GSH) administration can alleviate the effects of fasting-induced intestinal atrophy in the small intestinal mucosa. METHODS: Rats were divided into eight groups. One group was fed ad libitum, another was fed ad libitum and received oral GSH, and six groups were administrated saline (SA) or GSH orally during fasting. Mucosal height, apoptosis, and cell proliferation in the jejunum were histologically evaluated. iNOS protein expression (by immunohistochemistry), nitrite levels (by high performance liquid chromatography, as a measure of NO production), 8-hydroxydeoxyguanosine formation (by ELISA, indicating ROS levels), glutathione/oxidized glutathione (GSH/GSSG) ratio (by enzymatic colorimetric detection), and γ-glutamyl transpeptidase (Ggt1) mRNA levels in the jejunum (by semi-quantitative RT-PCR) were also estimated. RESULTS: Oral GSH administration was demonstrated to drastically reduce fasting-induced intestinal atrophy in the jejunum. In particular, jejunal mucosal height was enhanced in GSH-treated animals compared to SA-treated animals [527.2 ± 6.9 for 50 mg/kg GSH, 567.6 ± 5.4 for 500 mg/kg GSH vs 483.1 ± 4.9 (µm), P < 0.01 at 72 h]. This effect was consistent with decreasing changes in GSH-treated animals compared to SA-treated animals for iNOS protein staining [0.337 ± 0.016 for 50 mg/kg GSH, 0.317 ± 0.017 for 500 mg/kg GSH vs 0.430 ± 0.023 (area of staining part/area of tissue), P < 0.01 at 72 h] and NO [2.99 ± 0.29 for 50 mg/kg GSH, 2.88 ± 0.19 for 500 mg/kg GSH vs 5.34 ± 0.35 (nmol/g tissue), P < 0.01 at 72 h] and ROS [3.92 ± 0.46 for 50 mg/kg GSH, 4.58 ± 0.29 for 500 mg/kg GSH vs 6.42 ± 0.52 (8-OHdG pg/µg DNA), P < 0.01, P < 0.05 at 72 h, respectively] levels as apoptosis mediators in the jejunum. Furthermore, oral GSH administration attenuated cell proliferation decreases in the fasting jejunum [182.5 ± 1.9 for 500 mg/kg GSH vs 155.8 ± 3.4 (5-BrdU positive cells/10 crypts), P < 0.01 at 72 h]. Notably, both GSH concentration and Ggt1 mRNA expression in the jejunum were also attenuated in rats following oral administration of GSH during fasting as compared with fasting alone [0.45 ± 0.12 vs 0.97 ± 0.06 (nmol/mg tissue), P < 0.01; 1.01 ± 0.11 vs 2.79 ± 0.39 (Ggt1 mRNA/Gapdh mRNA), P < 0.01 for 500 mg/kg GSH at 48 h, respectively]. CONCLUSION: Oral GSH administration during fasting enhances jejunal regenerative potential to minimize intestinal mucosal atrophy by diminishing fasting-mediated ROS generation and enterocyte apoptosis and enhancing cell proliferation.

Lymph Drainage During Wound Healing in a Hindlimb Lymphedema Mouse Model.

BACKGROUND: Although lymphedematous skin exhibits delayed wound healing, little is known about lymph drainage during wound healing. We investigated the wound healing process in the presence of lymphatic dysfunction. METHODS AND RESULTS: The right inguinal lymph nodes (iLNs) and the surrounding tissue were excised in each mouse (the operation side), and a sham operation was performed in the left hindlimb (the control side). The next day, full-thickness wounds were made on both hindlimbs. The right hindlimb exhibited acute edema until day 3; however, it started to improve after day 4, and the wound area and epithelialization ratio were similar on both sides. Indocyanine green (ICG) was injected into both hindlimbs to observe lymph flow. On the operation side, ICG leaked out of the surgical site or remained at the injection site until day 2. Some lymph flow toward the existing lymph vessels was seen on day 3, and on day 10, lymph flow toward the axial LNs was detected on the operation side in all mice. On the operation side, the number of dermal lymph vessels was significantly increased on days 3 and 15. The dermal lymph vessel area of the peripheral wound was significantly smaller on the operation side. CONCLUSIONS: In a hindlimb lymphedema mouse model, lymph transiently accumulated in subcutaneous tissue, and then was gradually absorbed by the existing lymph vessels. The increase in the number of lymph vessels contributes to lymph drainage during wound healing. Acute lymphedema because of transient lymphatic dysfunction has little effect on wound healing.

Effectiveness of Changing the Application of Japanese Honey to a Hydrocolloid Dressing in Between the Inflammatory and Proliferative Phases on Cutaneous Wound Healing in Male Mice.

INTRODUCTION: The aim of this study was to investigate the effects of changing the application of Japanese honey to a hydrocolloid dressing (HCD) in between the inflammatory and proliferative phases on cutaneous wound healing in 8-week-old, BALB/cCrSlc male mice. MATERIALS AND METHODS: Mice were divided into 4 groups: acacia honey followed by a HCD, buckwheat flour honey followed by a HCD, Chinese milk vetch honey followed by a HCD, and a HCD alone (control group). All mice received 2 full-thickness wounds on both sides of the dorsum using a Disposable Biopsy Punch. The wounds of the control group were covered with a HCD, whereas wounds in the other groups were treated with 0.1 mL of the relevant type of honey until day 3 post-wound and then were covered with a HCD from days 4 to 14. RESULTS: In the experimental groups, the wound area ratio was significantly smaller in the inflammatory phase but significantly larger in the proliferative phase. Reepithelialization, collagen deposition, and wound contraction were significantly delayed compared with those in the control group. DISCUSSION: The re-expansion of the wounds in the proliferative phase could not be prevented, and reepithelialization, collagen deposition, and wound contraction were delayed compared with those upon the use of a HCD. CONCLUSION: The study's authors concluded that these methods do not promote cutaneous wound healing better than the use of a HCD alone.

17ß-Estradiol administration promotes delayed cutaneous wound healing in 40-week ovariectomised female mice.

This study investigated the effect of 17ß-estradiol on wound healing in 40-week ovariectomised female mice. Thirty-six-week-old female mice were divided into three groups: medication with 17ß-estradiol after ovariectomy (OVX + 17ß-estradiol), ovariectomy (OVX) and sham (SHAM). The mice received two full-thickness wounds, and the OVX + 17ß-estradiol group was administered 17ß-estradiol at 0·01 g/day until healing. In the OVX + 17ß-estradiol group, the ratio of wound area was significantly smaller than those of the OVX and SHAM groups on days 1-3, 5, 6, 8-12 and 9-12, respectively, the numbers of neutrophils and macrophages were significantly smaller than those on days 3 and 7, the ratio of re-epithelialisation was significantly higher than those on days 3 and 11, the ratio of myofibroblasts was significantly higher than those on day 11 and smaller on day 14, and the ratio of collagen fibres was significantly larger than that of the OVX group on days 7-14. We found that 17ß-estradiol administration promotes cutaneous wound healing in 40-week female mice by reducing wound area, shortening inflammatory response, and promoting re-epithelialisation, collagen deposition and wound contraction. Our results suggest that cutaneous wound healing that is delayed because of ageing is promoted by exogenous and continuous 17ß-estradiol administration.

The effect of 17ß-estradiol on cutaneous wound healing in protein-malnourished ovariectomized female mouse model.

Cutaneous wound healing is delayed by protein malnutrition (PM). On the other hand, estrogen promotes cutaneous wound healing by its anti-inflammatory and cell proliferation effects. Therefore, we hypothesized that estrogen administration in protein-malnourished ovariectomized (OVX) female mice might improve the inflammatory response and promote cutaneous wound healing as well as normal nutrition. To test this hypothesis, we used full-thickness excisional wounds in Control SHAM, PM SHAM, PM OVX and PM OVX+17ß-estradiol mice. The Control diet included 200 g/kg protein and the PM diet included 30 g/kg protein. The ratio of wound area in the Control SHAM group was significantly smaller than those in the three PM groups. In addition, microscopic findings also showed that the ratio of collagen fibers, the ratio of myofibroblasts and the number of new blood vessels in the Control SHAM group were significantly greater than those in the three PM groups. However, the number of Ym1-positive cells as an anti-inflammatory M2-like macrophage marker in the PM OVX+17ß-estradiol group was significantly higher than those in the other three groups. These results indicate that the appearance of anti-inflammatory M2-like macrophages was promoted by estrogen administration; however, it could not promote cutaneous wound healing upon a low-protein diet. Therefore, it may be confirmed that nutrition is more important for promoting cutaneous wound healing than estrogen administration.

Identification of glycoproteins carrying a target glycan-motif by liquid chromatography/multiple-stage mass spectrometry: identification of Lewis x-conjugated glycoproteins in mouse kidney.

Certain glycan motifs in glycoproteins are involved in several biological events and diseases. To understand the roles of these motifs, a method is needed to identify the glycoproteins that carry them. We previously demonstrated that liquid chromatography-multiple-stage mass spectrometry (LC-MSn) allowed for differentiation of oligosaccharides attached to Lewis-motifs, such as Lewisx(Lex, Galbeta1-4(Fucalpha1-3)GlcNAc) from other glycans. We successfully discriminated Lex-conjugated oligosaccharides from other N-linked oligosaccharides derived from mouse kidney proteins by using Lewis-motif-distinctive ions, a deoxyhexose (dHex)+hexose (Hex)+N-acetylhexsosamine (HexNAc) fragment (m/z 512), and a Hex+HexNAc fragment (m/z 366). In the present study, we demonstrated that this method could be used to identify the Lex-conjugated glycoproteins. All proteins in the mouse kidney were digested into peptides, and the fucosylated glycopeptides were enriched by lectin-affinity chromatography. The resulting fucosylated glycopeptides were subjected to two different runs of LC-MSn using a Fourier- transform ion cyclotron resonance mass spectrometer (FTICR-MS) and an ion trap-type mass spectrometer. After the first run, we picked out product ion spectra of the expected Lex-conjugated glycopeptides based on the presence of Lewis-motif-distinctive ions and assigned a peptide+HexNAc or peptide+(dHex)HexNAc fragment in each spectrum. Then the fucosylated glycopeptides were subjected to a second run in which the peptide-related fragments were set as precursor ions. We successfully identified gamma-glutamyl transpeptidase 1 (gamma-GTP1), low-density lipoprotein receptor-related protein 2 (LRP2), and a cubilin precursor as Lex-conjugated glycoproteins by sequencing of 2-5 glycopeptides. In addition, it was deduced that cadherin 16, dipeptidase I, H-2 class I histocompatibility antigen, K-K alpha precursor (H2-Kk), and alanyl (membrane) aminopeptidase could be Lex-conjugated glycoproteins from the good agreement between the experimental and theoretical masses and fragment patterns. The results indicated that our method could be applicable for the identification and screening of glycoproteins carrying target glycan-motifs, such as Lewis epitopes.

Deficiency of ribosomal protein S19 during early embryogenesis leads to reduction of erythrocytes in a zebrafish model of Diamond-Blackfan anemia.

Ribosomes are responsible for protein synthesis in all cells. Ribosomal protein S19 (RPS19) is one of the 79 ribosomal proteins (RPs) in vertebrates. Heterozygous mutations in RPS19 have been identified in 25% of patients with Diamond-Blackfan anemia (DBA), but the relationship between RPS19 mutations and the pure red-cell aplasia of DBA is unclear. In this study, we developed an RPS19-deficient zebrafish by knocking down rps19 using a Morpholino antisense oligo. The RPS19-deficient animals showed a dramatic decrease in blood cells as well as deformities in the head and tail regions at early developmental stages. These phenotypes were rescued by injection of zebrafish rps19 mRNA, but not by injection of rps19 mRNAs with mutations that have been identified in DBA patients. Our results indicate that rps19 is essential for hematopoietic differentiation during early embryogenesis. The effects were specific to rps19, but knocking down the genes for three other RPs, rpl35, rpl35a and rplp2, produced similar phenotypes, suggesting that these genes might have a common function in zebrafish erythropoiesis. The RPS19-deficient zebrafish will provide a valuable tool for investigating the molecular mechanisms of DBA development in humans.