Gli1+-PDL Cells Contribute to Alveolar Bone Homeostasis and Regeneration.

The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) that can differentiate into osteoblasts, cementoblasts, and fibroblasts. Nevertheless, the distribution and characteristics of these cells remain uncertain. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Therefore, in the present study, the differentiation ability of Gli1+ cells was examined using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. In 4-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were only slightly detected in the PDL, around endomucin-expressing blood vessels. These cells had proliferated over time, localizing in the PDL as well as on the bone and cementum surfaces at day 28. However, in 8-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were quiescent, as most cells were not immunoreactive for Ki-67. These cells in 8-wk-old mice exhibited high colony-forming unit fibroblast activity and were capable of osteogenic, chondrogenic, and adipogenic differentiation in vitro. In addition, after transplantation of teeth of iGli1/Tomato mice into the hypodermis of wild-type mice, Tomato fluorescence indicating the progeny of Gli1+ cells was detected in the osteoblasts and osteocytes of the regenerated bone. These results demonstrate that Gli1+ cells in the PDL were MSCs and could contribute to the alveolar bone regeneration.

Successful trachelectomy and re-vaginoplasty for cervico-vaginal stenosis following unsuccessful uterovaginal anastomosis and vaginoplasty in congenital cervical and vaginal aplasia: description of technique and a case report.

We describe a successful surgical technique of abdominal trachelectomy and re-vaginoplasty for cervico-vaginal stenosis following unsuccessful uterovaginal anastomosis and vaginoplasty in a patient with congenital cervical and vaginal aplasia. After the surgical procedure, cervico-vaginal stenosis was resolved and periodic menstruation without dysmenorrhoea resumed. While long-term follow-up is essential to ensure successful pregnancy and delivery, we conclude that this novel surgical procedure is a promising alternative for improvement of the quality of life and normal sexual function, and for preservation of fertility in patients with cervical and vaginal aplasia.

The poly(A) polymerase beta gene may not be associated with azoospermia caused by Sertoli-cell-only syndrome in Japanese patients by comparing patients and normal controls.

Approximately 15% of couples are infertile, with half of these cases being due to a male factor. Testis-specific cytoplasmic poly(A) polymerase beta (PAPOLB) is known to be critical for spermatogenesis. In mice, the loss of function of the Papolb gene results in the arrest of spermiogenesis and in male infertility. To analyse the role of the PAPOLB gene in human male infertility, this study investigated the relevance of this gene to human Sertoli-cell-only syndrome (SCOS) with azoospermia. Mutation analysis of the PAPOLB coding region was performed on 139 Japanese patients by PCR and direct sequence analysis. No critical mutations directly causing SCOS were detected, but three single-nucleotide polymorphisms (SNPs; SNP1 (c1101C > T), SNP2 (c1347T > C) and SNP3 (c1903C > A)) were found in the coding region. However, there were no significant associations in the allelic and genotypic distributions of these three SNPs between the SCOS and control groups (p>.05). This study suggests a lack of association of PAPOLB with azoospermia due to SCOS in humans.

Changes in penile length after radical prostatectomy: effect of neoadjuvant androgen deprivation therapy.

Although reports have shown evidence for penile length (PL) shortening after radical prostatectomy (RP), the association between neoadjuvant androgen deprivation therapy (NADT) and PL after RP has yet to be determined. This study evaluates chronological changes in PL after NADT and RP. Stretched PLs (SPLs) of 143 patients, 41 of whom had undergone NADT, were measured before, 10 days after, and 1, 3, 6, 9, 12, 18, and 24 months after RP. Chronological erectile function and testosterone levels were then evaluated. SPL was shortest 10 days after RP in both the NADT (-) and NADT (+) groups and gradually recovered in length thereafter. SPL in the NADT (-) group was significantly longer than that in the NADT (+) group before RP. However, no significant differences in SPLs were found between both groups 6 months after RP. Although all subjects in the NADT (+) group had testosterone levels of <50 ng/dL before RP, such levels increased after RP. Before RP, the NADT (-) group was found to have significantly better erectile function than the NADT (+) group. However, differences in erectile function between the NADT (-) and NADT (+) groups after RP were not significant. This report is the first to show that among patients with prostate cancer, those who underwent NADT had greater PL recovery after RP than those who did not. Data regarding PL recovery after NADT and RP obtained in this study could be useful for patients with prostate cancer who plan to undergo such procedures.

Cerebrospinal fluid orexin-A levels in systemic lupus erythematosus patients presenting with excessive daytime sleepiness.

Objective Involvement of the hypothalamus is rare in patients with systemic lupus erythematosus (SLE). In this study, we measured cerebrospinal fluid (CSF) orexin-A levels in SLE patients with hypothalamic lesions to investigate whether the orexin system plays a role in SLE patients with hypothalamic lesions who present with excessive daytime sleepiness (EDS). Methods Orexin-A levels were measured in CSF from four patients with SLE who presented with hypothalamic lesions detected by MRI. Three patients underwent repeated CSF testing. All patients met the updated American College of Rheumatology revised criteria for SLE. Results Tests for serum anti-aquaporin-4 antibodies, CSF myelin basic protein and CSF oligoclonal bands were negative in all patients. All patients presented with EDS. Low to intermediate CSF orexin-A levels (92-180 pg/ml) were observed in three patients in the acute stage, two of whom (patients 1 and 2) underwent repeated testing and showed increased CSF orexin-A levels, reduced abnormal hypothalamic lesion intensities detected by MRI and EDS dissipation at follow-up. In contrast, CSF orexin-A levels were normal in one patient (patient 4) while in the acute stage and at follow-up, despite improvements in EDS and MRI findings. Patient 4 showed markedly increased CSF interleukin-6 levels (1130 pg/ml) and a slightly involved hypothalamus than the other patients. Conclusions Our findings suggest that the orexinergic system has a role in EDS in SLE patients with hypothalamic lesions. Furthermore, cytokine-mediated tissue damage might cause EDS without orexinergic involvement.

Characterization of PTEN mutations in brain cancer reveals that pten mono-ubiquitination promotes protein stability and nuclear localization.

PTEN is a PIP3 phosphatase that antagonizes oncogenic PI3-kinase signalling. Due to its critical role in suppressing the potent signalling pathway, it is one of the most mutated tumour suppressors, especially in brain tumours. It is generally thought that PTEN deficiencies predominantly result from either loss of expression or enzymatic activity. By analysing PTEN in malignant glioblastoma primary cells derived from 16 of our patients, we report mutations that block localization of PTEN at the plasma membrane and nucleus without affecting lipid phosphatase activity. Cellular and biochemical analyses as well as structural modelling revealed that two mutations disrupt intramolecular interaction of PTEN and open its conformation, enhancing polyubiquitination of PTEN and decreasing protein stability. Moreover, promoting mono-ubiquitination increases protein stability and nuclear localization of mutant PTEN. Thus, our findings provide a molecular mechanism for cancer-associated PTEN defects and may lead to a brain cancer treatment that targets PTEN mono-ubiquitination.

Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro.

While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid-gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.

High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.

BACKGROUND: The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment. RESULTS: We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration. CONCLUSIONS: Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.

Prognostic significance of GRP78/BiP expression in patients with Stage III/IV hypopharyngeal squamous cell carcinoma.

The immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein 78 (GRP78) plays an essential role in the endoplasmic reticulum (ER) stress, and GRP78/BiP is known to be highly expressed in various human neoplasms. The clinicopathological features of GRP78/BiP expression in patients with advanced hypopharyngeal squamous cell carcinoma (HSCC) remain unclear. The aim of this study is to elucidate the prognostic significance of GRP78/BiP for HSCC.A total of 68 patients with advanced HSCC (stage III/IV) were analyzed, and tumor specimens were stained with immunohistochemistry for GRP78/BiP, Ki-67, and microvessel density (MVD), as determined through CD34 and p53 levels. GRP78/BiP was highly expressed in 80.8% (55/68) of all patients. The expression level of GRP78/BiP disclosed no significant relationship with any variables. Multivariate analysis confirmed that low expression of GRP78/BiP was an independent prognostic factor for predicting poor overall survival and progression-free survival in patients with advanced HSCC. The decreasing expression of GRP78/BiP was identified as a significant predictor related to shorter survival duration after surgery for advanced HSCC. Our study suggests that the reduced expression of GRP78/BiP contributes to worse survival for patients with advanced head and neck cancer.

A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome.

About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia.

A new class of cancer-associated PTEN mutations defined by membrane translocation defects.

Phosphatase and tensin homolog (PTEN), which negatively regulates tumorigenic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signaling, is a commonly mutated tumor suppressor. The majority of cancer-associated PTEN mutations block its essential PIP3 phosphatase activity. However, there is a group of clinically identified PTEN mutations that maintain enzymatic activity, and it is unknown how these mutations contribute to tumor pathogenesis. Here, we show that these enzymatically competent PTEN mutants fail to translocate to the plasma membrane where PTEN converts PIP3 to PI(4,5)P2. Artificial membrane tethering of the PTEN mutants effectively restores tumor suppressor activity and represses excess PIP3 signaling in cells. Thus, our findings reveal a novel mechanism of tumorigenic PTEN deficiency.

Muscle atrophy in chronic inflammatory demyelinating polyneuropathy: a computed tomography assessment.

BACKGROUND AND PURPOSE: Muscle atrophy is generally mild in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) compared with the severity and duration of the muscle weakness. Muscle atrophy was evaluated using computed tomography (CT) in patients with CIDP. METHODS: Thirty-one patients with typical CIDP who satisfied the diagnostic criteria for the definite CIDP classification proposed by the European Federation of Neurological Societies and the Peripheral Nerve Society were assessed. The clinicopathological findings in patients with muscle atrophy were also compared with those in patients without atrophy. RESULTS: Computed tomography evidence was found of marked muscle atrophy with findings suggestive of fatty degeneration in 11 of the 31 patients with CIDP. CT-assessed muscle atrophy was in the lower extremities, particularly in the ankle plantarflexor muscles. Muscle weakness, which reflects the presence of muscle atrophy, tended to be more pronounced in the lower extremities than in the upper extremities in patients with muscle atrophy, whereas the upper and lower limbs tended to be equally affected in patients without muscle atrophy. Nerve conduction examinations revealed significantly greater reductions in compound muscle action potential amplitudes in the tibial nerves of patients with muscle atrophy. Sural nerve biopsy findings were similar in both groups. The functional prognoses after immunomodulatory therapies were significantly poorer amongst patients with muscle atrophy. CONCLUSIONS: Muscle atrophy was present in a subgroup of patients with CIDP, including patients with a typical form of the disease. These patients tended to demonstrate predominant motor impairments of the lower extremities and poorer functional prognoses.

Antidepressant and anxiolytic profiles of newly synthesized arginine vasopressin V1B receptor antagonists: TASP0233278 and TASP0390325.

BACKGROUND AND PURPOSE: Vasopressin V1B receptor antagonists may be effective for the treatment of depression and anxiety and the objective of this study was to characterize the pharmacological profiles of two newly synthesized arginine vasopressin receptor 1B (V1B receptor) antagonists, TASP0233278 and TASP0390325. EXPERIMENTAL APPROACH: We investigated the in vitro profiles of TASP0233278 and TASP0390325. In addition, the effect of TASP0390325 on the increase in plasma adrenocorticotropic hormone (ACTH) levels induced by corticotropin-releasing factor (CRF)/desmopressin (dDAVP) was investigated. We also investigated the antidepressant and anxiolytic profiles of TASP0233278 and TASP0390325 in animal models. KEY RESULTS: Both TASP0233278 and TASP0390325 showed a high affinity and potent antagonist activity for V1B receptors. Oral administration of TASP0390325 antagonized the increase in plasma ACTH levels induced by CRF/dDAVP in rats, indicating that TASP0390325 blocks the anterior pituitary V1B receptor in vivo. Oral administration of TASP0233278 or TASP0390325 also exerted antidepressant effects in two models of depression (a forced swimming test and an olfactory bulbectomy model). Moreover, TASP0233278 improved depressive-like behaviour induced by repeated treatment with corticosterone, a model that has been shown to be resistant to treatment with currently prescribed antidepressants. In addition to depression models, TASP0233278 or TASP0390325 exerted anxiolytic effects in several anxiety models (social interaction, elevated plus-maze, stress-induced hyperthermia, separation-induced ultrasonic vocalization and sodium lactate-induced panic-like responses in panic-prone rats). CONCLUSION: TASP0233278 and TASP0390325 are potent and orally active V1B receptor antagonists with antidepressant and anxiolytic activities in rodents.

Mechanism of human PTEN localization revealed by heterologous expression in Dictyostelium.

Phosphatase and tensin homolog (PTEN) is one of the most frequently mutated tumor suppressor genes in cancers. PTEN has a central role in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signaling and converts PIP3 to phosphatidylinositol (4,5)-bisphosphate at the plasma membrane. Despite its importance, the mechanism that mediates membrane localization of PTEN is poorly understood. Here, we generated a library that contains green fluorescent protein fused to randomly mutated human PTEN and expressed the library in Dictyostelium cells. Using live cell imaging, we identified mutations that enhance the association of PTEN with the plasma membrane. These mutations were located in four separate regions, including the phosphatase catalytic site, the calcium-binding region 3 (CBR3) loop, the Cα2 loop and the C-terminal tail phosphorylation site. The phosphatase catalytic site, the CBR3 loop and the Cα2 loop formed the membrane-binding regulatory interface and interacted with the inhibitory phosphorylated C-terminal tail. Furthermore, we showed that membrane recruitment of PTEN is required for PTEN function in cells. Thus, heterologous expression system in Dictyostelium cells provides mechanistic and functional insight into membrane localization of PTEN.

Effect of dentist's clinical experience on treatment satisfaction of a complete denture.

The relationship between the prosthodontic experience of dentists and satisfaction of complete denture wearers remains unknown. To investigate whether a prosthodontist's clinical experience affects treatment satisfaction of a complete denture wearer. From April 2004 to July 2006, we conducted a randomised controlled trial at two centres, including 74 edentulous patients; of these, 32 and 30 were randomly allocated to the ED or ID group, respectively. All the patients rated their satisfaction with dentures, including general satisfaction and satisfaction of chewing ability, speaking, cleaning, stability, retention, comfort and aesthetics. These satisfaction ratings were measured by a 100-mm visual analog scale (VAS). Perceived chewing ability to foods, divided into five grades, was measured using a questionnaire. The mastication index (MI) was calculated for each grade. General satisfaction and satisfaction of speaking, stability and retention were significantly higher in the ED than in the ID group (P = 0·049, 0·003, 0·019 and 0·041, respectively). No significant difference existed between the MI of the ED (71·3 ± 18·4) and ID group (64·1 ± 16·53). However, the perceived chewing ability of grade 5 food, whose texture was the hardest among all the grades, was significantly higher in the ED group than in the ID group. Within its limitations, this study showed that a clinician's prosthodontic experience affects a complete denture wearer's satisfaction ratings.

Possible involvement of CD14+ CD16+ monocyte lineage cells in the epidermal damage of Stevens-Johnson syndrome and toxic epidermal necrolysis.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are characterized by keratinocyte apoptosis and necrosis, resulting in epidermal detachment. Although monocytes abundantly infiltrate the epidermis in SJS/TEN skin lesions, the properties and functions of these cells have not been fully examined. OBJECTIVES: To determine the properties of monocytes infiltrating into the epidermis in SJS/TEN. METHODS: Immunostaining of skin sections was performed to examine the membrane markers of monocytes infiltrating into skin lesions. RESULTS: Immunostaining of cryosections from 11 SJS/TEN skin lesions revealed numerous CD14+ monocytes located along the dermoepidermal junction and throughout the epidermis. The cells coexpressed CD16, CD11c and HLA-DR. CD14+ CD16+ cells were identified in very early lesions without epidermal damage, suggesting that their infiltration is a cause, rather than a result, of epidermal damage. Moreover, these cells expressed CD80, CD86 and CD137 ligand, indicative of their ability to facilitate the proliferation and cytotoxicity of CD8+ T cells. CD16+ cells infiltrating the epidermis and detected at the dermoepidermal junction were immunostained and counted in paraffin-embedded skin sections obtained from 47 patients with drug rash manifested as TEN, SJS, maculopapular-type rash or erythema multiform-type rash. The number of CD16+ monocytes infiltrating the epidermis increased significantly, depending on the grade of epidermal damage. CONCLUSIONS: These findings suggest that the appearance of CD14+ CD16+ cells of monocyte lineage plays an important role in the epidermal damage associated with SJS/TEN, most probably by enhancing the cytotoxicity of CD8+ T cells.

Differences in odor identification among clinical subtypes of Parkinson's disease.

OBJECTIVE: Olfactory dysfunction is a non-motor symptom in idiopathic Parkinson's disease (PD). We investigated whether this dysfunction differs among clinical subtypes of PD. METHODS: Participants comprised of 90 patients with idiopathic PD and without dementia. Olfactory function was evaluated using the odor stick identification test for Japanese, which evaluated the detection of 12 odorants familiar to Japanese participants. Patients were divided into tremor-dominant type (TDT), akinetic-rigid type (ART), and mixed type (MXT) PD subgroups using part III of the Unified Parkinson's Disease Rating Scale. RESULTS: Fifty-five patients were classified as ART, 21 as MXT, and 14 as TDT. There were no differences in age, sex, or duration of illness among the subtypes. Subjective symptoms of impaired sense of smell were significantly higher (P<0.05) in the ART than in the TDT. Mean odor identification score was 4.3 in the ART, 5.2 in MXT, and 6.6 in TDT. It was significantly lower in the ART than in the TDT (P<0.01). CONCLUSION: Olfactory dysfunction differed among the clinical subtypes of PD. This suggests that olfactory function might relate to prognosis of patients with PD.