Pgc suppresses the zygotically acting RNA decay pathway to protect germ plasm RNAs in the Drosophila embryo.

Specification of germ cells is pivotal to ensure continuation of animal species. In many animal embryos, germ cell specification depends on maternally supplied determinants in the germ plasm. Drosophila polar granule component (pgc) mRNA is a component of the germ plasm. pgc encodes a small protein that is transiently expressed in newly formed pole cells, the germline progenitors, where it globally represses mRNA transcription. pgc is also required for pole cell survival, but the mechanism linking transcriptional repression to pole cell survival remains elusive. We report that pole cells lacking pgc show premature loss of germ plasm mRNAs, including the germ cell survival factor nanos, and undergo apoptosis. We found that pgc- pole cells misexpress multiple miRNA genes. Reduction of miRNA pathway activity in pgc- embryos partially suppressed germ plasm mRNA degradation and pole cell death, suggesting that Pgc represses zygotic miRNA transcription in pole cells to protect germ plasm mRNAs. Interestingly, germ plasm mRNAs are protected from miRNA-mediated degradation in vertebrates, albeit by a different mechanism. Thus, independently evolved mechanisms are used to silence miRNAs during germ cell specification.

Maternal Nanos inhibits Importin-α2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline.

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. Here, we show that Nos represses somatic gene expression by inhibiting translation of maternal importin-α2 (impα2) mRNA. Mis-expression of Impα2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, we propose that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a 'double-lock' mechanism to inhibit somatic gene expression in germline progenitors.

Mortality and risk factors on admission in toxic epidermal necrolysis: A cohort study of 59 patients.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but life-threatening disorders characterized by widespread epidermal necrosis of the skin and mucosa. The severity-of-illness scoring system for TEN (SCORTEN) was widely used since 2000 as a standard prognostic tool consisting of seven clinical values. METHODS: To evaluate the prognosis using current treatments and risk factors for mortality, we retrospectively analyzed 59 cases of TEN, including SJS/TEN overlap treated in two university hospitals from January 2000 to March 2020. RESULTS: The mortality rate of TEN was 13.6% (8/59). All patients treated with high-dose steroid administration in combination with plasma exchange and/or immunoglobulin therapy recovered. Logistic regression analysis showed nine clinical composite scores, namely: heart rate (≧120 bpm), malignancy present, percentage of body surface area with epidermal detachment (>10%), blood urea nitrogen (>28 mg/dL), serum bicarbonate level (<20 mEq/L), serum glucose level (>252 mg/dL), age (≧71 years), the interval between disease onset and treatment initiation at the specialty hospital (≧8 days), and respiratory disorder within 48 h after admission. The receiver operating characteristic curves confirmed a high potential for predicting the prognosis of TEN. CONCLUSIONS: Recent developments in treatment strategies have contributed to the improved prognosis of TEN patients. A modified severity scoring model composed of nine scores may be helpful in the prediction of TEN prognosis in recent patients. Further large-scale studies are needed to confirm mortality findings to improve prognostication in patients with TEN.

[A LONG PROSPECTIVE OBSERVATIONAL STUDY ON PREVALENCE OF ATOPIC DERMATITIS IN EARLY CHILDHOOD TO ADOLESCENCE].

BACKGROUND: There are few prospective observational studies on the prevalence of atopic dermatitis (AD) in children. We aimed to prospectively investigate the dynamics of change in the prevalence of AD from early childhood to adolescence. METHODS: We conducted a survey with a modified questionnaire to diagnose AD based on The International Study of Asthma and Allergies in Childhood questionnaire with 1230 13-year-old children who were born in the Minami ward, Yokohama City between May 2004 and June 2005 and had undergone physical examinations by dermatologists at 3 years of age. Among the 422 children who answered the questionnaire, 210 had undergone periodic physical examinations by dermatologists from 4 months to 3 years of age (Cohort 1), whereas 212 had undergone physical examinations only at 3 years of age (Cohort 2). RESULTS: The prevalence of AD was 16.9% in 422 children at 13 years of age, with 22.9%, 16.6%, 20.0% and 18.3% prevalence at 4 months, 18 months, 3 years and 13 years of age, respectively, in children who were followed up long-term. The frequency of AD occurrence per year decreased after the age of 3 years; a history of AD at this age was significantly related to AD at 13 years of age (Fisher's exact test, p<0.001). CONCLUSION: In conclusion, it was suggested that AD in 3-year-old children is one of the risk factors for the development of AD in 13-year-old children.

Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing.

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.

Retrospective analysis of Stevens-Johnson syndrome and toxic epidermal necrolysis in 87 Japanese patients--Treatment and outcome.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but severe adverse drug reactions with high mortality. METHODS: To present the clinical characteristics of SJS and TEN in Japan and evaluate the efficacy of treatments, we retrospectively analyzed cases of SJS and TEN treated in 2 university hospitals during 2000-2013. RESULTS: Fifty-two cases of SJS (21 males and 31 females; average age, 55.1 years) and 35 cases of TEN (17 males and 18 females; average age, 56.6 years) were included in this study. Twenty-eight cases of SJS (53.8%) and all cases of TEN were caused by drugs. Hepatitis was the most common organ involvement in both SJS and TEN. Renal dysfunction, intestinal disorder, and respiratory disorder were also involved in some cases. The major complication was pneumonia and sepsis. All cases except for 3 cases were treated systemically with corticosteroids. Steroid pulse therapy was performed in 88.6% of TEN. Plasmapheresis and/or immunoglobulin therapy was combined with steroid therapy mainly in TEN after 2007. The mortality rate was 6.9% and the rates for SJS and TEN were 1.9% and 14.3%, respectively. These were much lower than predicted mortality according to a severity-of-illness scoring system for TEN prognosis (SCORTEN) score. When comparing the mortality rate between 2000-2006 and 2007-2013, it was decreased from 4.5% to 0.0% in SJS and from 22.2% to 5.3% in TEN. CONCLUSIONS: Treatment with steroid pulse therapy in combination with plasmapheresis and/or immunoglobulin therapy seems to have contributed to prognostic improvement in SJS/TEN.

Drosophila Mon2 couples Oskar-induced endocytosis with actin remodeling for cortical anchorage of the germ plasm.

Drosophila pole (germ) plasm contains germline and abdominal determinants. Its assembly begins with the localization and translation of oskar (osk) RNA at the oocyte posterior, to which the pole plasm must be restricted for proper embryonic development. Osk stimulates endocytosis, which in turn promotes actin remodeling to form long F-actin projections at the oocyte posterior pole. Although the endocytosis-coupled actin remodeling appears to be crucial for the pole plasm anchoring, the mechanism linking Osk-induced endocytic activity and actin remodeling is unknown. Here, we report that a Golgi-endosomal protein, Mon2, acts downstream of Osk to remodel cortical actin and to anchor the pole plasm. Mon2 interacts with two actin nucleators known to be involved in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. We also found that these actin regulators are required for Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. We propose that, in response to the Osk-mediated endocytic activation, vesicle-localized Mon2 acts as a scaffold that instructs the actin-remodeling complex to form long F-actin projections. This Mon2-mediated coupling event is crucial to restrict the pole plasm to the oocyte posterior cortex.

Drosophila Pgc protein inhibits P-TEFb recruitment to chromatin in primordial germ cells.

Germ cells are the only cells that transmit genetic information to the next generation, and they therefore must be prevented from differentiating inappropriately into somatic cells. A common mechanism by which germline progenitors are protected from differentiation-inducing signals is a transient and global repression of RNA polymerase II (RNAPII)-dependent transcription. In both Drosophila and Caenorhabditis elegans embryos, the repression of messenger RNA transcription during germ cell specification correlates with an absence of phosphorylation of Ser 2 residues in the carboxy-terminal domain of RNAPII (hereafter called CTD), a critical modification for transcriptional elongation. Here we show that, in Drosophila embryos, a small protein encoded by polar granule component (pgc) is essential for repressing CTD Ser 2 phosphorylation in newly formed pole cells, the germline progenitors. Ectopic Pgc expression in somatic cells is sufficient to repress CTD Ser 2 phosphorylation. Furthermore, Pgc interacts, physically and genetically, with positive transcription elongation factor b (P-TEFb), the CTD Ser 2 kinase complex, and prevents its recruitment to transcription sites. These results indicate that Pgc is a cell-type-specific P-TEFb inhibitor that has a fundamental role in Drosophila germ cell specification. In C. elegans embryos, PIE-1 protein segregates to germline blastomeres, and is thought to repress mRNA transcription through interaction with P-TEFb. Thus, inhibition of P-TEFb is probably a common mechanism during germ cell specification in the disparate organisms C. elegans and Drosophila.

Food-dependent exercise-induced anaphylaxis caused by leek and several allergens of the allium family.

Exercise-induced anaphylaxis (EIA) is a relatively rare condition but can be a diagnostic pitfall in daily practice. Leek allergy is extremely rare, and there have been no reports, to our knowledge, of leek-dependent EIA. Here, we report the first case of exercise- and leek-induced anaphylaxis. An 18-year-old woman presented with symptoms of anaphylaxis after exercise in the morning. Prick-to-prick tests for leek was 1+ and challenge test for heated leek was negative, but leek-dependent physical exertion challenge test evoked anaphylaxis. We diagnosed food-dependent EIA by some additional tests including immunoblotting assay with patient's serum. Leek allergy is an extremely rare condition, so careful interview and investigation of allergens is important to eliminate causative substances of anaphylaxis.

Endocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi responses.

RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.

[Case of food-dependent exercise-induced anaphylaxis diagnosed by the provocation test with cuttlefish after the pretreatment with 1.5 g of aspirin].

A 29-year-old woman had an episode of urticaria at the age of 17 while exercising after eating fried cuttlefish. For years thereafter, she experienced several episodes of urticaria after eating seafood. At the age of 29, she ate grilled seafood, including cuttlefish for supper after taking loxoprofen for lumbago. One hour later, she developed generalized urticaria accompanied by nausea, abdominal pain, swelling of the lips, and dyspnea while walking; she was taken to a hospital. She was then referred to us for further examination of the etiology of her anaphylactic reactions. The level of specific IgE measured using Immuno CAP was negative for all kinds of foods, including cuttlefish. However, a skin prick test was positive for raw and cooked cuttlefish. Provocation tests were performed on admission by combining the intake of cuttlefish and aspirin under the suspicion of cuttlefish allergy enhanced by nonsteroidal anti-inflammatory drugs and exercise. As a result, she developed no symptoms except for slight itching of the oral mucosa after eating 20 g or 100 g of cuttlefish with or without concomitant administration of 0.5 g of aspirin. Finally, generalized urticaria appeared after challenge with cuttlefish and 1.5 g of aspirin. She was diagnosed with food-dependent exercise-induced anaphylaxis (FDEIA) caused by cuttlefish. She has not developed urticaria since she started to avoid eating cuttlefish. Our results indicated that in provocation tests for the diagnosis of FDEIA, allergic reactions could not only be induced by food intake but could also be enhanced by aspirin in a dose-dependent manner.

[A case of an allergic reaction due to Anisakis simplex after the ingestion of salted fish guts made of Sagittated calamari: allergen analysis with recombinant and purified Anisakis simplex allergens].

A 75-year-old man ingested salted fish guts made of Sagittated calamari which he caught in the daytime, with alcohol and then dozed. Five hours later, he woke up due to itching over his entire body and noticed generalized urticaria and a swollen tongue, which was too large for him to close his mouth. Serum total IgE was 456 IU/ml and ImmunoCAP was positive for anisakis, but negative for squid, shrimp, and ascaris. A skin prick test (SPT) was positive for anisakis extract (10 mg/ml) and house dust mites, but negative for squid and shrimp. He was diagnosed with IgE-mediated allergy due to Anisakis simplex after the ingestion of salted fish guts made of Sagittated calamari, which had been parasitized by Anisakis simplex. Furthermore, we performed SPT with six extracts of purified or recombinant allergens (Ani s 1, 3, 4, 5, 6, and 8) to identify the causative allergens in this case. Only Ani s 3 (tropomyosin) was positive, indicating that Ani s 3 was the causative allergen in this case. Third stage larvae of the nematode Anisakis simplex often parasitize not only marine fish but also invertebrates, including squid. It is necessary to consider Anisakis simplex allergy for urticarial reactions that develop after the ingestion of squid.

Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis.

Translational control is a critical process in the spatio-temporal restriction of protein production. In Drosophila oogenesis, translational repression of oskar (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation. This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive. Here, we report that an ovarian protein, Cup, is required to repress precocious osk translation. Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins. A mutant Cup protein lacking this sequence fails to repress osk translation in vivo. Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner. These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex.

[Enhancement of nonsteroidal, anti-inflammatory drugs and preventive effect of antihistamines and disodium cromoglycate on wheat allergy].

BACKGROUND: Aspirin has been known to be an enhancer to wheat allergy, including wheat-dependent, exercise-induced anaphylaxis. OBJECTIVE: To investigate whether nonsteroidal, anti-inflammatory drugs (NSAIDs) other than aspirin would enhance allergic reactions after wheat ingestion and whether antihistamines and disodium cromoglycate would prevent these reactions. METHODS: Seven cases, whose reactions after wheat ingestion were enhanced by aspirin on challenge tests, were enrolled. Skin prick tests (SPT) and CAP-RAST were undergone for wheat and gluten. We used challenge tests of wheat after pretreatment of NSAIDs and preventive drugs. RESULTS: Four cases were diagnosed with wheat allergy, 3 cases had wheat-dependent, salicylic acid-induced anaphylaxis. SPT and CAP-RAST were positive for wheat and gluten in 5 of 7 cases and 4 of 7 cases, respectively. Dicrofenac enhanced the allergic reactions after wheat ingestion in 1 of 2 cases, whereas etodolac failed to enhance the symptoms in all 5 cases performed. Furthermore, disodium cromoglycate could not completely prevent the allergic reaction in all 4 cases and even enhanced the reaction in 1 case of them. To see an inhibitory effect of antihistamines on the symptoms, fexofenadine (in 2, 1 and 1 case, respectively), olopatadine, and chlorpheniramine were administrated before the challenge test, and as a result these drugs were found to have inhibitory effects on the allergic reaction. CONCLUSION: In this study, it was suggested that etodolac might be a relatively safe anti-inflammatory drug on wheat allergy and antihistamines could prevent allergic reactions more than DSCG in patients with wheat allergy.

Repression of early zygotic transcription in the germline.

Germ cells, the progenitors of gametes, are often specified and segregated from somatic lineages early in embryogenesis. As germ cells are essential to create the next generation in sexually reproducing organisms, they must be prevented from differentiating inappropriately into somatic cells. In Drosophila and Caenorhabditis elegans embryos, this is governed by the transient and global repression of mRNA transcription. Furthermore, the inhibition of somatic transcriptional programs is also crucial for germ cell specification in the mouse. Therefore, the active repression of somatic transcriptional programs appears to be a common mechanism for launching the germline. In this review, we will discuss the mechanisms of transcriptional repression during germ cell specification and their interspecies similarities and differences.

Germ cell-autonomous Wunen2 is required for germline development in Drosophila embryos.

In many animals, primordial germ cells (PGCs) migrate through the embryo towards the future gonad, a process guided by attractive and repulsive cues provided from surrounding somatic cells. In Drosophila, the two related lipid phosphate phosphatases (LPPs), Wunen (Wun) and Wun2, are thought to degrade extracellular substrates and to act redundantly in somatic cells to provide a repulsive environment to steer the migration of PGCs, or pole cells. Wun and Wun2 also affect the viability of pole cells, because overexpression of either one in somatic cells causes pole cell death. However, the means by which they regulate pole cell migration and survival remains elusive. We report that Wun2 has a maternal function required for the survival of pole cells during their migration to the gonad. Maternal wun2 RNA was found to be concentrated in pole cells and pole cell-specific expression of wun2 rescued the pole cell death phenotype of the maternal wun2 mutant, suggesting that wun2 activity in pole cells is required for their survival. Furthermore, we obtained genetic evidence that pole cell survival requires a proper balance of LPP activity in pole cells and somatic cells. We propose that Wun2 in pole cells competes with somatic Wun and Wun2 for a common lipid phosphate substrate, which is required by pole cells to produce their survival signal. In somatic cells, Wun and Wun2 may provide a repulsive environment for pole cell migration by depleting this extracellular substrate.