Generation of 3D lacrimal gland organoids from human pluripotent stem cells.

Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.

CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling.

Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.

Natural Selection Signatures in the Hondo and Ryukyu Japanese Subpopulations.

Natural selection signatures across Japanese subpopulations are under-explored. Here we conducted genome-wide selection scans with 622,926 single nucleotide polymorphisms for 20,366 Japanese individuals, who were recruited from the main-islands of Japanese Archipelago (Hondo) and the Ryukyu Archipelago (Ryukyu), representing two major Japanese subpopulations. The integrated haplotype score (iHS) analysis identified several signals in one or both subpopulations. We found a novel candidate locus at IKZF2, especially in Ryukyu. Significant signals were observed in the major histocompatibility complex region in both subpopulations. The lead variants differed and demonstrated substantial allele frequency differences between Hondo and Ryukyu. The lead variant in Hondo tags HLA-A*33:03-C*14:03-B*44:03-DRB1*13:02-DQB1*06:04-DPB1*04:01, a haplotype specific to Japanese and Korean. While in Ryukyu, the lead variant tags DRB1*15:01-DQB1*06:02, which had been recognized as a genetic risk factor for narcolepsy. In contrast, it is reported to confer protective effects against type 1 diabetes and human T lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis. The FastSMC analysis identified 8 loci potentially affected by selection within the past 20-150 generations, including 2 novel candidate loci. The analysis also showed differences in selection patterns of ALDH2 between Hondo and Ryukyu, a gene recognized to be specifically targeted by selection in East Asian. In summary, our study provided insights into the selection signatures within the Japanese and nominated potential sources of selection pressure.

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome presenting as recurrent aseptic peritonitis in a patient receiving peritoneal dialysis: a case report.

BACKGROUND: Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is caused by mutations in the ubiquitin-activating enzyme1 (UBA1) gene and characterised by an overlap between autoinflammatory and haematologic disorders. CASE PRESENTATION: We reported a case of a 67-year-Japanese man receiving peritoneal dialysis (PD) who had recurrent aseptic peritonitis caused by the VEXAS syndrome. He presented with unexplained fevers, headache, abdominal pain, conjunctival hyperaemia, ocular pain, auricular pain, arthralgia, and inflammatory skin lesions. Laboratory investigations showed high serum C-reactive protein concentration and increased cell count in PD effluent. He was treated with antibiotics for PD-related peritonitis, but this was unsuccessful. Fluorine-18-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography images demonstrated intense FDG uptake in his left superficial temporal artery, nasal septum, and bilateral auricles. The working diagnosis was giant cell arteritis, and he was treated with oral prednisolone (PSL) 15 mg daily with good response. However, he was unable to taper the dose to less than 10 mg daily because his symptoms flared up. Since Tocilizumab was initiated, he could taper PSL dose to 2 mg daily. Sanger sequencing of his peripheral blood sample showed a mutation of the UBA1 gene (c.122 T > C; p.Met41Thr). We made a final diagnosis of VEXAS syndrome. He suffered from flare of VEXAS syndrome at PSL of 1 mg daily with his cloudy PD effluent. PSL dose of 11 mg daily relieved the symptom within a few days. CONCLUSIONS: It is crucial to recognise aseptic peritonitis as one of the symptoms of VEXAS syndrome and pay attention to the systemic findings in the patients.

Morphological features of lower lumbar degenerative kyphosis.

PURPOSE: Kyphosis in the lower lumbar spine (L4-S1) significantly affects sagittal alignment. However, the characteristics of the spinopelvic parameters and compensatory mechanisms in patients with lower lumbar degenerative kyphosis (LLDK) have not been described in detail. The objective of this retrospective study was to analyze the morphological characteristics in patients with sagittal imbalance due to LLDK. METHODS: In this retrospective study, we reviewed the clinical records of consecutive patients who underwent corrective surgery for adult spinal deformity (ASD) at a single institution. We defined LLDK as (i) kyphotic deformity in lower lumbar spine (L4-S1) or (ii) inappropriate distribution of lordosis (lordosis distribution index < 40%) in the lower lumbar spine. Global spine parameters of ASD patients and MRI findings were compared between those with LLDK (LLDK group) and without LLDK (control group). RESULTS: A total of 95 patients were enrolled in this study, of which the LLDK group included 14 patients (14.7%). Compared to the control, LLDK presented significantly higher pelvic incidence (62.1° vs 52.6°) and pelvic tilt (40.0° vs 33.4°), larger lordosis at the thoracolumbar junction (12.0° vs -19.6°), and smaller thoracic kyphosis (9.3° vs 26.0°). In LLDK, there was significantly less disc degeneration at L2/3 and L3/4. CONCLUSION: LLDK patients had high pelvic incidence, large pelvic tilt, and a long compensatory curve at the thoracolumbar junction and thoracic spine region.

Genetic architecture underlying IgG-RF production is distinct from that of IgM-RF.

OBJECTIVE: HLA-DRB1 alleles, particularly the shared epitope (SE) alleles, are strongly associated with RA. Different genetic structures underlie the production of the various autoantibodies in RA. While extensive genetic analyses have been conducted to generate a detailed profile of ACPA, a representative autoantibody in RA, the genetic architecture underlying subfractions of RF other than IgM-RF, namely IgG-RF, known to be associated with rheumatoid vasculitis, is not well understood. METHODS: We enrolled a total of 743 RA subjects whose detailed autoantibody (IgG-RF, IgM-RF, and ACPA) data were available. We evaluated co-presence and correlations of the levels of these autoantibodies. We analysed associations between the presence or levels of the autoantibodies and HLA-DRB1 alleles for the 743 RA patients and 2008 healthy controls. RESULTS: We found both IgG-RF(+) and IgG-RF(-) RA subjects showed comparable associations with SE alleles, which was not observed for the other autoantibodies. Furthermore, there was a clear difference in SE allele associations between IgG-RF(+) and (-) subsets: the association with the IgG-RF(+) subsets was solely driven by HLA-DRB1*04:05, the most frequent SE allele in the Japanese population, while not only HLA-DRB1*04:05 but also HLA-DRB1*04:01, less frequent in the Japanese population but the most frequent SE allele in Europeans, were main drivers of the association in the IgG-RF(-) subset. We confirmed that these associations were irrespective of ACPA presence. CONCLUSION: We found a unique genetic architecture for IgG-RF(-) RA, which showed a strong association with a SE allele not frequent in the Japanese population but the most frequent SE allele in Europeans. The findings could shed light on uncovered RA pathology.

Living-Donor Liver Transplantation for Erythropoietic Protoporphyria: A Case Report and Literature Review.

Erythropoietic protoporphyria (EPP) is a very rare disease with an estimated prevalence of 1 in 200,000 individuals. Decreased ferrochelatase activity causes the accumulation of protoporphyrin in the body, and light exposure results in the generation of active oxygen, causing photosensitivity. Liver damage has the greatest influence on the prognosis, and liver transplantation is the only treatment option for patients with decompensated liver cirrhosis. We report a case of living-donor liver transplantation for decompensated liver cirrhosis associated with EPP. The patient was a 52-year-old male who led a normal life except for mild photosensitivity. When the patient was 37-year-old, hepatic dysfunction was noticed. At 48-year-old, high erythrocyte protoporphyrin levels, skin biopsy, and genetic tests resulted in a diagnosis of EPP. The patient underwent living- donor liver transplantation because of decompensated liver cirrhosis. In the operating room and intensive care unit, a special light-shielding film was applied to all light sources to block light with harmful wavelengths during treatment. Due to the need for special measures, a lecture on patients with EPP was given before surgery to deepen understanding among all medical professionals involved in the treatment. As a result, no adverse events occurred during the perioperative period, and the patient was discharged on the 46th post-operative day. Currently, the transplanted liver is functioning extremely well, and the patient is alive 3 years post-transplant. Herein, we describe a case of living donor liver transplantation for EPP with a brief literature review.

Small Intestinal Adenocarcinoma Arising at the Anastomotic Site after Kasai Operation for Biliary Atresia: A Case Report and Literature Review.

Biliary atresia is an obliterative cholangiopathy of unknown etiology. Hepatic portoenterostomy, in which obliterated extrahepatic bile ducts are resected and bile flow is restored, known as Kasai operation, is performed within 3 months after birth. While this operation enhances long-term survival of patients, the occurrence of primary malignant hepatic tumors has been increasing. We report a case of small intestinal adenocarcinoma arising at the anastomotic site after Kasai operation. A 49-year-old man, who underwent Kasai operation for biliary atresia when he was 2 months old, experienced rapidly progressive jaundice and liver dysfunction. Deceased-donor liver transplantation was performed for liver failure. Macroscopically, there was a white-yellow tumor located at the anastomotic site of hepatic portoenterostomy of the resected liver. Pathological examination revealed a well-differentiated adenocarcinoma with some Paneth cells in the neoplastic lesion. Immunohistochemically, the tumor cells were negative for cytokeratin 7 (CK7) but positive for cytokeratin 20 (CK20) and a homeobox domain-containing transcription factor (CDX2). Mucin expression in tumor cells was negative for mucin 1 (MUC1) and mucin 6 (MUC6) and positive for mucin 2 (MUC2) and mucin 5AC (MUC5AC). The pathological diagnosis was small intestinal adenocarcinoma originating from the jejunum. The patient was discharged 48 days after the operation. The patient had not experienced recurrence at 10 months after the operation. This is the first report of small intestinal adenocarcinoma arising at the anastomotic site after Kasai operation for biliary atresia. Special care should be taken for the patients after Kasai operation with acute progressive jaundice and liver dysfunction because there is a possibility of malignancy in their native liver.

Co-ordinated ocular development from human iPS cells and recovery of corneal function.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

Cholesterol Granuloma of the Liver Mimicking Malignant Tumor: A Case Report.

Cholesterol granuloma is a benign, tumor-like lesion with an accumulation of cholesterol crystals in the tissue and is a consequence of a chronic inflammatory reaction. It commonly occurs in the middle ear but rarely in the liver. There is only one previous case report of cholesterol granuloma of the liver, which was caused by cholesterol hepatolithiasis. We report a case of cholesterol granuloma of the liver in a patient with no intrahepatic cholesterol stones; it was difficult to rule out malignant liver tumor preoperatively. The patient was a 79-year-old woman in whom a lesion in the liver was detected on abdominal ultrasonography. She was referred to our hospital for detailed examination and treatment. Abdominal contrast-enhanced computed tomography showed a 20 mm lesion with ring enhancement in the lateral segment of the liver during the arterial and delayed phases. Since a malignant tumor could not be ruled out radiologically, laparoscopic lateral segment hepatectomy was performed for definitive diagnosis and treatment. The resection specimen showed a yellowish-white lesion measuring 15 mm in diameter. Pathological examination showed a granulomatous lesion with cholesterol crystals surrounded by foreign body giant cells. The lesion was diagnosed as cholesterol granuloma of the liver. The postoperative course was good, and the patient was discharged on postoperative day 5. She was healthy, and no recurrence of the cholesterol granuloma was detected at the 5-month follow-up. This is the first case report of cholesterol granuloma of the liver mimicking a malignant liver tumor in a patient with no intrahepatic cholesterol stones.

Impact of surgical masks on fraction of inspired oxygen during oxygen therapy depends on the type of oxygen masks and respiratory conditions: volunteer- and simulation-based studies.

PURPOSE: We investigated the impact of surgical masks (SM) during oxygen therapy using oxygen masks in volunteer- and simulation-based studies. METHODS: Fifteen volunteers wore the Hudson RCI® or Open-Face Mask® with/without an SM. The fraction of inspired oxygen concentration (FIO2), end-tidal CO2 (EtCO2), partial pressure of inspired CO2 (PICO2), and respiratory rate (RR) were measured. The oxygen flow rate increased from 0 to 10 L/min. In the simulation-based study, FIO2 was measured using a simulator that reproduced spontaneous breathing. RR was 12 or 24 bpm, and the tidal volume (Tv) was 300, 500, or 700 mL. The effect of oxygen mask fitting conditions was also examined. The primary outcome measure was FIO2 at 6 L/min. RESULTS: In the volunteer-based study, FIO2 was reduced when the SM was used with the Hudson RCI® or Open-Face Mask®. The FIO2 drop was larger with the Open-Face Mask® than with the Hudson RCI®. The RR, EtCO2, and PICO2 significantly changed with the SM, but the differences were not clinically meaningful. In the simulation-based study, the SM with the Hudson RCI® did not reduce FIO2, but the SM with the Open-Face Mask® significantly decreased FIO2 under several conditions. However, the SM with the Hudson Mask® reduced FIO2 when the fit of the mask was inadequate. With the Open-Face Mask®, lower RR and Tv resulted in larger differences in FIO2. CONCLUSIONS: The SM decreased FIO2 during oxygen therapy with oxygen masks. The impact of SM depended on the type of the oxygen mask, mask fitting, and respiratory condition.

Generation and validation of a PITX2-EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells.

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator-like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2-IRES2-EGFP sequence downstream of the stop codon in exon 8 of PITX2 Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2-EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

Brønsted Acid-Catalyzed Aza-Ferrier Reaction of N,O-Allenyl Acetals: Synthesis of ß-Amino-α-methylene Aldehydes.

A Tf2NH-catalyzed aza-Ferrier reaction of N,O-allenyl acetals was reported. This protocol provided various types of ß-amino-α-methylene aldehydes as the products. The N,O-allenyl acetal substrates were easily prepared by base-induced isomerization of N,O-propargyl acetals with Triton B. The N,O-propargyl acetals were prepared from the corresponding aldehydes or lactams. Further synthetic applications of the products were also described.

Leptin promotes the fat preference associated with low-temperature acclimation in mice.

Although fluctuations in energy metabolism are known to influence intake as well as nutrient selection, there are no definitive reports on how food preferences change with changes in habitat temperature. We investigated the effects of habitat temperature on appetite and food preference and elucidated the underlying mechanism by conducting a feeding experiment and a leptin administration test on mice reared at low temperatures. Our results showed that the increased food intake and HFD preference observed in the 10°C group were induced by decrease in plasma leptin concentration. Then, a leptin administration experiment was conducted to clarify the relationship between leptin and food preference with low-temperature acclimation. The control group reared in 10°C significantly preferred the HFD, but the leptin-administered group did not. These results show that the peripheral system appetite-regulating hormone leptin not only acts to suppress appetite but also might inhibit preference for lipids in low-temperature acclimation.

The effect of 1% glucose loading on metabolism in the elderly patients during remifentanil-induced anesthesia: a randomized controlled trial.

BACKGROUND: Previous studies showed that remifentanil-induced anesthesia can inhibit surgical stress response in non-diabetic adult patients and that low-dose glucose loading during anesthesia may attenuate fat catabolism. However, little is known about the influence of glucose loading on metabolism in elderly patients, whose condition may be influenced by decreased basal metabolism and increased insulin resistance. We hypothesized that, in elderly patients, intraoperative low glucose infusion may attenuate the catabolism of fat without causing harmful hyperglycemia during remifentanil-induced anesthesia. METHODS: Elderly, non-diabetic patients scheduled to undergo elective surgery were enrolled and randomized to receive no glucose (0G group) or low-dose glucose infusion (0.1 g/kg/hr. for 1 h followed by 0.05 g/kg/hr. for 1 h; LG group) during surgery. Glucose, adrenocorticotropic hormone (ACTH), 3-methylhistidine (3-MH), insulin, cortisol, free fatty acid (FFA), creatinine (Cr), and ketone body levels were measured pre-anesthesia, 1 h post-glucose infusion, at the end of surgery, and on the following morning. RESULTS: A total of 31 patients (aged 75-85) were included (0G, n = 16; LG, n = 15). ACTH levels during anesthesia decreased significantly in both groups. In the LG group, glucose levels increased significantly after glucose loading but hyperglycemia was not observed. During surgery, ketone bodies and FFA were significantly lower in the LG group than the 0G group. There were no significant differences in insulin, Cr, 3-MH, and 3-MH/Cr between the two groups. CONCLUSION: Remifentanil-induced anesthesia inhibited surgical stress response in elderly patients. Intraoperative low-dose glucose infusion attenuated catabolism of fat without inducing hyperglycemia. TRIAL REGISTRATION: This study has been registered with the University hospital Medical Information Network Center (http://www.umin.ac.jp/english/). TRIAL REGISTRATION NUMBER: UMIN000016189. The initial registration date: January 12th 2015.

Genetic analysis of IgG4-related disease.

IgG4-related disease (IgG4-RD) is an emerging concept of a novel clinical entity, characterized by the swelling of the affected organs, increase in serum total IgG and IgG4 levels, infiltration of plasmacyte and eosinophil, fibrosis of the affected lesions and good response to corticosteroid. IgG4-RD includes diseases with organ-specific fibrosis and infiltration of IgG4-positive plasmacyte, previously known as type 1 autoimmune pancreatitis (AIP), Mikulicz's disease and others. Although the precise mechanisms of the pathogenesis of IgG4-RD are not yet understood, some studies have suggested genetic components contributing to the onset of IgG4-RD or its subgroup. The recent emergence of the concept of IgG4-RD has made it difficult to conduct genetic analyses of IgG4-RD as a whole. When the analyses are restricted to the various subgroups of IgG4-RD, they require a large number of DNA samples from patients satisfying IgG4-RD diagnostic criteria not completely overlapping the criteria in type 1 AIP, Mikulicz's disease and others. Not only HLA but also non-HLA genes have been described as IgG4-RD risk genes, particularly in type 1 AIP. In this mini-review article, we will explore previous studies analyzing genetic associations with IgG4-RD and its subgroups, and discuss the future direction of the research addressing the existing problems.

GABAergic Local Interneurons Shape Female Fruit Fly Response to Mating Songs.

Many animals use acoustic signals to attract a potential mating partner. In fruit flies (Drosophila melanogaster), the courtship pulse song has a species-specific interpulse interval (IPI) that activates mating. Although a series of auditory neurons in the fly brain exhibit different tuning patterns to IPIs, it is unclear how the response of each neuron is tuned. Here, we studied the neural circuitry regulating the activity of antennal mechanosensory and motor center (AMMC)-B1 neurons, key secondary auditory neurons in the excitatory neural pathway that relay song information. By performing Ca2+ imaging in female flies, we found that the IPI selectivity observed in AMMC-B1 neurons differs from that of upstream auditory sensory neurons [Johnston's organ (JO)-B]. Selective knock-down of a GABAA receptor subunit in AMMC-B1 neurons increased their response to short IPIs, suggesting that GABA suppresses AMMC-B1 activity at these IPIs. Connection mapping identified two GABAergic local interneurons that synapse with AMMC-B1 and JO-B. Ca2+ imaging combined with neuronal silencing revealed that these local interneurons, AMMC-LN and AMMC-B2, shape the response pattern of AMMC-B1 neurons at a 15 ms IPI. Neuronal silencing studies further suggested that both GABAergic local interneurons suppress the behavioral response to artificial pulse songs in flies, particularly those with a 15 ms IPI. Altogether, we identified a circuit containing two GABAergic local interneurons that affects the temporal tuning of AMMC-B1 neurons in the song relay pathway and the behavioral response to the courtship song. Our findings suggest that feedforward inhibitory pathways adjust the behavioral response to courtship pulse songs in female flies.SIGNIFICANCE STATEMENT To understand how the brain detects time intervals between sound elements, we studied the neural pathway that relays species-specific courtship song information in female Drosophila melanogaster We demonstrate that the signal transmission from auditory sensory neurons to key secondary auditory neurons antennal mechanosensory and motor center (AMMC)-B1 is the first-step to generate time interval selectivity of neurons in the song relay pathway. Two GABAergic local interneurons are suggested to shape the interval selectivity of AMMC-B1 neurons by receiving auditory inputs and in turn providing feedforward inhibition onto AMMC-B1 neurons. Furthermore, these GABAergic local interneurons suppress the song response behavior in an interval-dependent manner. Our results provide new insights into the neural circuit basis to adjust neuronal and behavioral responses to a species-specific communication sound.

Shared epitope defines distinct associations of cigarette smoking with levels of anticitrullinated protein antibody and rheumatoid factor.

OBJECTS: Although the association of cigarette smoking (CS) with susceptibility to rheumatoid arthritis (RA) has been established, the impact of CS on anticitrullinated cyclic peptide/protein antibody (ACPA) and rheumatoid factor (RF) levels in RA has yet been clear, especially in relation to shared epitope (SE) alleles. METHODS: A total of 6239 subjects, the largest Asian study ever, from two independent Japanese cohorts were enrolled. Precise smoking histories, levels of ACPA and RF, and HLA-DRB1 allele status were withdrawn from databases. Associations between CS and high ACPA or RF levels, defined by the top quartiles, were evaluated. The effect of HLA-DRB1 alleles on the association was further investigated. RESULTS: CS at RA onset conferred the risks of high levels of both antibodies, especially RF (OR 2.06, p=7.4×10-14; ACPA, OR 1.29, p=0.012), suggesting that RF level is more sensitive to CS than ACPA level. The patients who had quitted CS before RA onset showed a trend of decreased risks of developing high levels of ACPA or RF, and the risks steadily decreased according to the cessation years. The association of CS with high ACPA level was observed only in subjects carrying SE alleles, while the association of high RF level was observed regardless of SE. CONCLUSIONS: CS confers the risks of high autoantibody levels in RA in different manners; CS interacts with SE alleles on ACPA level, while CS impacts on RF level despite SE allele. These data suggest novel distinct production mechanisms of RF and ACPA.

A single male auditory response test to quantify auditory behavioral responses in Drosophila melanogaster.

Many animals utilize auditory signals to communicate with conspecific individuals. During courtship, males of the fruit fly Drosophila melanogaster and related species produce a courtship song comprised of sine and pulse songs by vibrating their wings. The pulse song increases female receptivity and male courtship activity, indicating that it functions as a sexual signal. One song parameter, interpulse interval (IPI), varies among closely related species. In D. melanogaster, a song with a conspecific IPI induces a stronger behavioral response than heterospecific songs, indicating the ability of the flies to discriminate conspecific IPI. Traditionally, the fly's response to the song is measured under grouped conditions, in which the effect of sensory modalities other than audition cannot be excluded. Here, to quantify the individual ability to discriminate a conspecific song, we systematically analyzed the auditory response of single male flies to sound with various parameters. Moreover, we applied this method, termed SMART (Single Male Auditory Response Test), to two sister species for potential application in a comparative approach. By quantifying the locomotor activity of single D. melanogaster males during sound exposure, we detected increased locomotor activity in response to pulse songs, but not to white noise or pure tone. The conspecific song evoked stronger response than the heterospecific songs, and ablation of their antennal receivers severely suppressed the locomotor increase. A pulse song with a small IPI variation evoked a continuous response, while the response to songs with highly variable IPIs tends to be rapidly decayed. This provides the first evidence that fruit flies discriminate IPI variations, which possibly inform the age and social contexts of the singer. Sister species, D. sechellia, exhibited a locomotor response to pulse song, while D. simulans exhibited no behavioral response. This suggests that auditory and other stimuli that elicit this behavioral response are diversified among Drosophila species.

The function of appendage patterning genes in mandible development of the sexually dimorphic stag beetle.

One of the defining features of the evolutionary success of insects is the morphological diversification of their appendages, especially mouthparts. Although most insects share a common mouthpart ground plan, there is remarkable diversity in the relative size and shapes of these appendages among different insect lineages. One of the most prominent examples of mouthpart modification can be found in the enlargement of mandibles in stag beetles (Coleoptera, Insecta). In order to understand the proximate mechanisms of mouthpart modification, we investigated the function of appendage-patterning genes in mandibular enlargement during extreme growth of the sexually dimorphic mandibles of the stag beetle Cyclommatus metallifer. Based on knowledge from Drosophila and Tribolium studies, we focused on seven appendage patterning genes (Distal-less (Dll), aristaless (al), dachshund (dac), homothorax (hth), Epidermal growth factor receptor (Egfr), escargot (esg), and Keren (Krn). In order to characterize the developmental function of these genes, we performed functional analyses by using RNA interference (RNAi). Importantly, we found that RNAi knockdown of dac resulted in a significant mandible size reduction in males but not in female mandibles. In addition to reducing the size of mandibles, dac knockdown also resulted in a loss of the serrate teeth structures on the mandibles of males and females. We found that al and hth play a significant role during morphogenesis of the large male-specific inner mandibular tooth. On the other hand, knockdown of the distal selector gene Dll did not affect mandible development, supporting the hypothesis that mandibles likely do not contain the distal-most region of the ancestral appendage and therefore co-option of Dll expression is unlikely to be involved in mandible enlargement in stag beetles. In addition to mandible development, we explored possible roles of these genes in controlling the divergent antennal morphology of Coleoptera.