Rhinoceros beetle horn development reveals deep parallels with dung beetles.

Beetle horns are attractive models for studying the evolution of novel traits, as they display diverse shapes, sizes, and numbers among closely related species within the family Scarabaeidae. Horns radiated prolifically and independently in two distant subfamilies of scarabs, the dung beetles (Scarabaeinae), and the rhinoceros beetles (Dynastinae). However, current knowledge of the mechanisms underlying horn diversification remains limited to a single genus of dung beetles, Onthophagus. Here we unveil 11 horn formation genes in a rhinoceros beetle, Trypoxylus dichotomus. These 11 genes are mostly categorized as larval head- and appendage-patterning genes that also are involved in Onthophagus horn formation, suggesting the same suite of genes was recruited in each lineage during horn evolution. Although our RNAi analyses reveal interesting differences in the functions of a few of these genes, the overwhelming conclusion is that both head and thoracic horns develop similarly in Trypoxylus and Onthophagus, originating in the same developmental regions and deploying similar portions of appendage patterning networks during their growth. Our findings highlight deep parallels in the development of rhinoceros and dung beetle horns, suggesting either that both horn types arose in the common ancestor of all scarabs, a surprising reconstruction of horn evolution that would mean the majority of scarab species (~35,000) actively repress horn growth, or that parallel origins of these extravagant structures resulted from repeated co-option of the same underlying developmental processes.

Vortioxetine versus placebo in major depressive disorder comorbid with social anxiety disorder.

BACKGROUND: Major Depressive Disorder (MDD) and Social Anxiety Disorder (SAD) are highly comorbid, yet the combined condition has not been subject to any placebo-controlled treatment trials. This study reports a trial of vortioxetine, an antidepressant that has also shown benefit in Generalized Anxiety Disorder (GAD), in patients meeting DSM-5 criteria for both MDD and SAD. METHODS: The study was a 12-week double-blind, placebo-controlled comparison of vortioxetine 10-20 mg/day or placebo administered on a 1:1 ratio. The study was designed to include 40 male or female outpatients aged 18-70 years. The primary endpoint was the "composite" Clinical Global Impression of Improvement (CGI-I) responder rate, factoring in improvement in both MDD and SAD features. Major secondary outcome measures were changes on the Montgomery Asberg Depression Rating Scale (MADRS) and Liebowitz Social Anxiety Scale (LSAS). RESULTS: On the composite CGI-I, 10 of 20 (50%) vortioxetine and six of 20 (30%) placebo-treated patients were rated as responders, a non-significant difference. However, vortioxetine-treated patients did show significantly greater improvement than those on placebo on both the MADRS (effect size 0.672) and LSAS (effect size 0.714). Efficacy in depression was seen before improvement in SAD. Adverse effects were similar to those previously reported. CONCLUSIONS: In this preliminary trial vortioxetine appears safe and effective for patients with MDD comorbid with SAD, with robust effect sizes on dimensional measures of both depression and social anxiety, but failure to separate from placebo on the primary outcome measure of composite responder rate. More studies of patients with comorbid conditions are needed, as this mirrors what is often seen in clinical practice.

Crystal structure of the plant receptor-like kinase TDR in complex with the TDIF peptide.

In plants, leucine-rich repeat receptor-like kinases (LRR-RKs) perceive ligands, including peptides and small molecules, to regulate various physiological processes. TDIF, a member of the CLE peptide family, specifically interacts with the LRR-RK TDR to inhibit meristem differentiation into tracheary elements, and promotes cell proliferation. Here we report the crystal structure of the extracellular domain of TDR in complex with the TDIF peptide. The extracellular domain of TDR adopts a superhelical structure comprising 22 LRRs, and specifically recognizes TDIF by its inner concave surface. Together with our biochemical and sequence analyses, our structure reveals a conserved TDIF-recognition mechanism of TDR among plant species. Furthermore, a structural comparison of TDR with other plant LRR-RKs suggested the activation mechanism of TDR by TDIF. The structure of this CLE peptide receptor provides insights into the recognition mechanism of the CLE family peptides.

Structural and functional insights into IZUMO1 recognition by JUNO in mammalian fertilization.

Sperm-egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO (also known as folate receptor (FR) 4 or IZUMO1R) on the egg surface. Whereas other FRs bind and uptake folates, JUNO binds IZUMO1 and establishes the cell-cell adhesion. However, the mechanism of IZUMO1 recognition by JUNO has remained elusive. Here we report the crystal structure of mouse JUNO, at 2.3 Å resolution. A structural comparison of JUNO with the FRs revealed that JUNO and the FRs have similar overall structures, but JUNO lacks the folate-binding pocket, thereby explaining the inability of JUNO to bind folate. Further complementation of Juno knockout eggs with mutant Juno messenger RNAs revealed that the conserved, surface-exposed tryptophan residue of JUNO is required for sperm binding and fertilization. Our structure-based in vivo functional analyses provide a framework towards a mechanistic understanding of mammalian gamete recognition.

Structural basis for specific inhibition of Autotaxin by a DNA aptamer.

ATX is a plasma lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) and produces lysophosphatidic acid. To date, no ATX-inhibition-mediated treatment strategies for human diseases have been established. Here, we report anti-ATX DNA aptamers that inhibit ATX with high specificity and efficacy. We solved the crystal structure of ATX in complex with the anti-ATX aptamer RB011, at 2.0-Å resolution. RB011 binds in the vicinity of the active site through base-specific interactions, thus preventing the access of the choline moiety of LPC substrates. Using the structural information, we developed the modified anti-ATX DNA aptamer RB014, which exhibited in vivo efficacy in a bleomycin-induced pulmonary fibrosis mouse model. Our findings reveal the structural basis for the specific inhibition of ATX by the anti-ATX aptamer and highlight the therapeutic potential of anti-ATX aptamers for the treatment of human diseases, such as pulmonary fibrosis.

Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase.

Choline is an essential nutrient for all living cells and is produced extracellularly by sequential degradation of phosphatidylcholine (PC). However, little is known about how choline is produced extracellularly. Here, we report that ENPP6, a choline-specific phosphodiesterase, hydrolyzes glycerophosphocholine (GPC), a degradation product of PC, as a physiological substrate and participates in choline metabolism. ENPP6 is highly expressed in liver sinusoidal endothelial cells and developing oligodendrocytes, which actively incorporate choline and synthesize PC. ENPP6-deficient mice exhibited fatty liver and hypomyelination, well known choline-deficient phenotypes. The choline moiety of GPC was incorporated into PC in an ENPP6-dependent manner both in vivo and in vitro. The crystal structure of ENPP6 in complex with phosphocholine revealed that the choline moiety of the phosphocholine is recognized by a choline-binding pocket formed by conserved aromatic and acidic residues. The present study provides the molecular basis for ENPP6-mediated choline metabolism at atomic, cellular and tissue levels.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6.

Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Šresolution. The crystal belonged to space group P1, with unit-cell parameters a=63.7, b=68.8, c=69.7 Å, α=60.6, ß=87.0, γ=68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

[Awareness about medical expenses and certifications of eligibility for limited health insurance payments for chemotherapy among clinicians at the Ehime Cancer Care Network Priority Hospitals (Ehime Cancer Kyoten Hospitals)].

The "Cancer Chemotherapy and its Management" subcommittee at the Ehime Cancer Care Network Priority Hospitals (Ehime Cancer Kyoten Hospitals)with a focus on medical expenses associated with chemotherapy, surveyed awareness among 98 clinicians regarding certifications of eligibility for Limited Health Insurance Payments during cancer treatment. This committee also lists social and clinical problems encountered at the Ehime Cancer Care Network Priority Hospitals. In our survey, 78% of clinicians were consulted about medical expenses associated with chemotherapy and were actively involved in resolving medical expense problems and resulting correspondences. However, only 38% of clinicians could explain the details of the Japanese guideline on the catastrophic cap and the certifications of eligibility for Limited Health Insurance Payments. This knowledge deficit was more pronounced in younger residents. From our analyses of the awareness about medical expenses among clinicians, we recommend the establishment of the following systems for the management of cancer patients. First, establish a reporting system and early consultation on the catastrophic cap and the certifications of eligibility before initiating cancer treatment. Second, education regarding medical expenses should be mandatory for clinicians, especially for young residents. Third, patients with cancer suffering in the interval of the medical expense and the social system should be relieved with new systems.

[Incidence of dysgeusia associated with chemotherapy for cancer].

Chemotherapy for cancer has been reported to have many side effects. Dysgeusia or taste disorder is a common complaint among cancer patients undergoing ambulatory chemotherapy. The present study was undertaken to establish the importance of dysgeusia as a side effect of chemotherapy in these patients. The study included 356 patients who visted Shikoku Cancer Center to undergo outpatient cancer chemotherapy. Of these patients, 156(43.8%)experienced dysgeusia. Of the 156 patients, 34 were male and 122 were female. The incidence of dysgeusia was higher in patients receiving FOLFOX6(oxaliplatin+ 5-FU), docetaxel(DTX), paclitaxel(PTX), docetaxel+cyclophosphamide(TC)or epirubicin+cyclophosphamide(EC) than in those receiving other regimens. When the occurrence of dysgeusia was difficult to define, the changes in taste sensations were subtle for salty and umami taste. This disorder affected appetite: 87.2%of patients experienced loss of appetite. In addition, 66.7% of patients were distressed by this disorder. Dysgeusia may significantly reduce the quality of life of patients undergoing chemotherapy for cancer. Therefore, patient support is important for patients who experience dysgeusia.

[Current problems for outpatients undergoing cancer chemotherapy in Ehime Priority Hospitals of Cancer Care Network (Ehime Cancer Kyoten Hospitals)].

Ehime Priority Hospitals of Cancer Care Network(Ehime Cancer Kyoten Hospitals)regularly have meetings to discus the current problems in cancer care in Ehime Prefecture. We established three subcommittees:"Registration of Cancer Incident," "Critical Paths for the Management of Patients with Cancer,"and"Palliative Care for Patients with Advanced Cancer"to exchange our opinions. We recently set up a new subcommittee related to the physical and spiritual care of patients undergoing chemotherapy treatment,"A Subcommittee dealing with Cancer Chemotherapy and its Management"."This subcommittee has tried to identify current problems with chemotherapy for outpatients in each institution through questionnaire and analysis. As a result of this survey, it was found that Ehime Priority Hospitals have total of seventy-three beds for outpatients undergoing chemotherapy, and that they performed chemotherapy 19, 671 times in 2008. A total of eight oncology physicians and sixteen oncology nurses were engaged in performing chemotherapy in this system. The questions patients most frequently asked during chemotherapy concerned the management of therapy-related complications, dealing with problems at night and during holidays after chemotherapy, and financial problems related to the costs of treatment. In this study we found three issues that need to be managed in Ehime Priority Hospitals. First, for the nursing of outpatients undergoing chemotherapy, more staff engaged in different types of care is required. Second, a new system to deal with emergencies at night and during holidays after chemotherapy is necessary, because Ehime Priority Hospitals use the same system to deal with chemotherapy patients as for other patients. Third, cooperation between pharmacies and out-clinics is important for patient compliance during chemotherapy, especially for the administration of oral anti-tumor agents. Ehime Priority Hospitals of Cancer Care Network is trying to improve each institution while dealing with these problems.

Autoimmune thyroid diseases in 65 Japanese women with Turner syndrome.

Turner syndrome (TS) is associated with a number of complications including thyroid disease. In this study, the prevalence of thyroid disease was evaluated in Japanese women with TS. The medical charts of 65 TS women (age 30+/-9 years old, range: 15-61), treated with estrogen replacement therapy or with antiosteoporotic pharmaceuticals at our outpatient clinic, were reviewed. History of thyroid disease, titer of thyroid autoantibodies, and thyroid function were recorded. Thyroid autoantibodies were undetectable in 28 of 65 women (43%), and thyroid function was normal in all these women. Of the 37 women with thyroid autoantibodies (57%), 3 had Graves' disease and 20 women were hypothyroidism and diagnosed as Hashimoto' s thyroiditis. The resting 14 women with euthyroidism were also considered to be so-called probable cases of Hashimoto' s thyroiditis. In 20 women with hypothyroidism, 14 (70%) received replacement therapy with levothyroxine. The replacement with levothyroxine started between age 17 and 60 (median: 31 years old). These data showed that more than half of Japanese women with TS in adulthood had thyroid autoantibodies. In women with TS, monitoring of thyroid hormone is important to detect hypothyroidism earlier and start adequate replacement therapy.

Growth hormone (GH) or insulin-like growth factor (IGF)-I represses 11beta-hydroxysteroid dehydrogenase type 1 (HSD1) mRNA expression in 3T3-L1 cells and its activity in their homogenates.

Patients with growth hormone (GH) deficiency (GHD) have a clinical feature of visceral adiposity and it has been reported that these patients have an increased active cortisol (F)/inactive cortisone (E) metabolite ratio. 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an enzyme expressed in liver and adipose tissue that acts principally as a reductase converting E to F. In the present study, we investigated the effects of GH or IGF-I on the activity of 11beta- HSD1 in 3T3-L1 cell homogenates and its mRNA expression. First, we showed that 11beta-HSD1 activity and mRNA levels were low in preadipocytes and increased throughout the process of adipogenesis. When fully differentiated adipocytes were treated with GH for various times, the activity of 11beta-HSD1 was significantly decreased after 4 h and 8 h but was restored to basal levels after 24 h. After 8 h of GH stimulation, 11beta-HSD1 mRNA levels were decreased compared with basal levels. IGF-I treatment of adipocytes resulted in rapid decreases in 11beta-HSD1 activity as well as mRNA levels; however, IGF-I treatment for 24 h increased 11beta-HSD1 activity. In long-term cultured adipocytes, GH or IGF-I showed only inhibitory effects on 11beta-HSD1 activity. In conclusion, 11beta-HSD1 activity was suppressed by GH or IGF-I in differentiated adipocytes, probably due to a reduction of 11beta-HSD1 mRNA levels. These data suggest that under the conditions of low GH or IGF-I concentrations, 11beta-HSD1 activity in adipose tissue is maintained at high levels, leading to an increase in active cortisol that induces adipogenesis and/or lipogenesis. Thus, visceral adiposity in patients with GHD might be related to increased 11beta-HSD1 activity.

Metabolic co-morbidities revealed in patients with childhood-onset adult GH deficiency after cessation of GH replacement therapy for short stature.

GH therapy was approved in 2006 for treatment of adult growth hormone deficiency (GHD) in Japan. Until then, GH was used only to treat short stature in children with GHD and the treatment was stopped when the final height was reached. In the present study, we investigated metabolic co-morbidities experienced by adults with childhood-onset (CO) GHD after the cessation of GH. Forty-two patients with COGHD (M/F 22/20, age at follow up when the retrospective analysis was carried out: 18-52 yr) treated with GH in childhood were studied. We reviewed the medical records of these patients to determine the metabolic co-morbidities that developed after cessation of GH. The median age was 19 yrs (range: 14-38) at cessation of GH, and the following co-morbidities were observed: hypertriglyceridemia in 15 (41%) patients, non-alcoholic fatty liver disease (NAFLD) in 11 (29%) patients, hypercholesterolemia in 10 (26%) patients, diabetes mellitus (DM) in 4 (10%) patients, and hypertension in 1 (2.4%) patient. The median BMI when these complications became overt was 23.5 kg/m(2) for those with hypertriglyceridemia, 26.0 kg/m(2) for those with NAFLD, 20.9 kg/m(2) for those with hypercholesterolemia, and 27.2 kg/m(2 ) for those with DM. More than two co-morbidities were experienced by 32% of men and 30% of women. In conclusion, adults with COGHD after the cessation of GH have multiple metabolic co-morbidities. Lifelong GH replacement might be important for improving the overall metabolic profiles in these patients.

Clinical features of insulin-like growth factor-II producing non-islet-cell tumor hypoglycemia.

In some patients with non-islet-cell tumor hypoglycemia (NICTH), a high molecular weight form of IGF-II (big IGF-II) derived from tumors is present in the circulation and might be associated with recurrent hypoglycemia. In this study, in order to survey the clinical characteristics of patients with IGF-II producing NICTH, we analyzed the medical records of 78 patients with NICTH (M/F 44/34, age 62+/-1.8, range; 9-86 years.) whose serum contained a large amount of big IGF-II. Hepatocellular carcinoma and gastric carcinoma were the most common causes of NICTH. The diameters of the tumors were more than 10 cm in 70% of the patients. Basal immunoreactive insulin (IRI) levels were less than 3 microU/dl in 79% of the patients. Hypoglycemic attack was the onset of disease in 31 of 65 cases (48%), but the tumor was revealed prior to the occurrence of hypoglycemia in 34 cases (52%). Twenty-five of 47 (53%) patients had decreased serum potassium levels. These data suggested that hypoinsulinemic hypoglycemia associated with the presence of a large tumor supports the diagnosis of IGF-II producing NICTH. Hypokalemia was associated with hypoglycemia in some patients. The BMI (21.4+/-0.6 kg/m2) and serum total protein levels (6.6+/-0.1g/dl) were preserved at the occurrence of first hypoglycemic attack suggesting that malnutrition might not be the main cause of hypoglycemia in most patients.

Dynamics of microtubules and positioning of female pronucleus during bovine parthenogenesis.

The zygote centrosome, consisting of both paternal and maternal centrosomal components, is the microtubule-organizing center necessary for pronuclear migration and positioning in fertilization. Maternal centrosomal function in microtubule organization and pronuclear positioning, however, remains unclear. In the present study, we sought to elucidate the function of maternal centrosomes during bovine parthenotes in the microtubule organizational processes required to move the pronucleus to the cell center without sperm centrosomal components. Microtubule organization, pronuclear position, and distribution of gamma-tubulin, which is thought to be the major component of maternal centrosomal material, were imaged by immunocytochemistry and conventional epifluorescence microscopy. In bovine parthenotes treated with paclitaxel, a microtubule-stabilizing drug, the cytoplasmic microtubule asters became organized after chemical activation, and the microtubules radiated dynamically toward the female pronucleus. The microtubule patterns correlated well with pronuclear movement to the cell center. Microtubules aggregated at regions of gamma-tubulin concentration, but gamma-tubulin did not localize to a spot until the first interphase of bovine parthenogenesis. These findings indicate that gamma-tubulin is responsible for microtubule organization as the maternal centrosome. In bovine parthenogenesis, the maternal centrosome then organizes cytoplasmic microtubules to move the female pronucleus into the cell center. We propose that the maternal centrosome plays a role as a functional centrosome despite the lack of a sperm contribution, making this structure less competent for microtubule organization in comparison with centrosomes containing sperm centrosomal components.

Microtubule organization during rabbit fertilization by intracytoplasmic sperm injection with and without sperm centrosome.

Aim: In most mammalian fertilization, the sperm introduces the centrosome, which acts as a microtubule organizing center (MTOC) and is essential for pronuclear movement. In rabbit fertilization, biparental centrosomal contribution in microtubule organization has been suggested. Methods: To reveal the function and inheritance of the centrosome during rabbit fertilization, we compared microtubule organization and early embryonal development following intracytoplasmic sperm injection (ICSI) with and without sperm centrosome. Sperm centrosome was removed by sonication, and the isolated sperm head was injected by a Piezo-driven micromanipulator. Samples were studied by light microscope after immunocytological stain. Results: The sperm aster formation was observed 2-3 h after ICSI with intact sperm. In contrast, microtubules were organized between the male and female pronucleus without a nucleation site in the eggs after ICSI with an isolated sperm head. In the late pronuclear stage following ICSI with an isolated sperm head, microtubule organization was the same as in late pronuclear stage eggs after intact sperm injection. The first mitotic spindle was organized in eggs following ICSI with an isolated sperm head, as observed in eggs following ICSI with an intact sperm. Conclusions: These results indicate that the MTOC is in oocyte cytoplasm during fertilization and fulfils the function when the sperm centrosome is absent. (Reprod Med Biol 2005; 4: 169-178).

Cytoskeletal dynamics during mammalian gametegenesis and fertilization: Implications for human reproduction.

From gamete to neonate, human fertilization is a series of cell motilities (motion and morphological changes). Cytoskeletons play a role in cell motility as they work as a field worker in the cell. The present study is a review of dynamic motility of cytoskeletons (microfilaments and microtubules) during mammalian gamategenesis and fertilization. Dynamic and proper organization of cytoskeletons is crucial for the completion of oocyte maturation and spermatogenesis. By intracytoplasmic sperm injection, some difficulties in fertilization by sperm entry into the egg cytoplasm are overcome. However, the goal of fertilization is the union of the male and female genome, and sperm incorporation into an oocyte is nothing but the beginning of fertilization. Sperm centrosomal function, which introduces microtubule organization and promotes pronuclear apposition and first mitotic spindle formation, plays the leading role in the 'motility' of post-intracytoplasmic sperm injection events in fertilization. The present review introduces novel challenges in functional assessment of the human sperm centrosome. Furthermore, microtubule organization during development without the sperm centrosome (e.g. parthenogenesis) is mentioned. (Reprod Med Biol 2005; 4: 179-187).

Use of Mammalian eggs for assessment of human sperm function: molecular and cellular analyses of fertilization by intracytoplasmic sperm injection.

PROBLEMS: Intracytoplasmic sperm injection (ICSI ) has been described as the 'cure' for male sterility because a single sperm can now be directly introduced into an egg with some chance of pregnancy. While ICSI has revolutionized the practice of assisted reproductive techniques (ART), there are few molecular and cellular studies about its safety and efficacy. Even by using ICSI, fertilization in humans succeeds only if the sperm effectively accomplishes a number of tasks including 'post-ICSI events' in fertilization. To assess the function of human sperm after ICSI, we used heterologous ICSI with human sperm into animal eggs. Egg activation, sperm decondensation and sperm centorosomal function were examined in sperm from fertile men and infertile patients. METHODS: Sperm from fertile men and infertile patients were injected into hamster, rabbit and bovine eggs by Piezo micromanipulator, and studied in decondensation of sperm nuclei, egg activation and microtubule organization. RESULTS: Decondensation human sperm head following ICSI into hamster eggs occurred initially form basal lesion, and apical portion of sperm nuclei which is surrounded by acrosome and perinuclear theca, still condensed in early pronuclear stage. Radial array of microtubules from sperm centrosome 'sperm aster' which is essential for pronuclear movement was observed in 30% rabbit eggs following ICSI with human sperm. By heterologous ICSI system with fertile human sperm and bovine eggs, 83.3% of eggs was activated and 60% eggs had sperm aster, indicating that bovine Piezo ICSI system is appropriate for assessing human sperm oocyte activation ability and human sperm centrosomal function. Oocyte activation and sperm centrosomal function were significantly low in sperm from men with globozoospermia and men with dysplasia of fibrous sheath. CONCLUSION: These assays indicate differences of the process of fertilization between in vitro fertilization and ICSI, and reflect the human sperm function especially for the 'post-ICSI events' in fertilization. More molecular and cellular analyses in fertilization by ICSI are needed for improvement of ART.