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The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the extraembryonic domain, which is a hallmark of insect development but which is not yet well understood.

Unlike passive rupture of the human chorioamnion at birth, the insect extraembryonic (EE) tissues – the amnion and serosa – actively rupture and withdraw in late embryogenesis. Withdrawal is essential for development and has been a morphogenetic puzzle. Here, we use new fluorescent transgenic lines in the beetle Tribolium castaneum to show that the EE tissues dynamically form a basal-basal epithelial bilayer, contradicting the previous hypothesis of EE intercalation. We find that the EE tissues repeatedly detach and reattach throughout development and have distinct roles. Quantitative live imaging analyses show that the amnion initiates EE rupture in a specialized anterior-ventral cap. RNAi phenotypes demonstrate that the serosa contracts autonomously. Thus, apposition in a bilayer enables the amnion as 'initiator' to coordinate with the serosa as 'driver' to achieve withdrawal. This EE strategy may reflect evolutionary changes within the holometabolous insects and serves as a model to study interactions between developing epithelia. Early in development a protective fluid-filled sac forms around an embryo. In humans, this sac bursts during birth, but the sac surrounding insect embryos ruptures long before these animals begin to emerge from their eggs. This early rupture is important for insects to develop normally: if an insect embryo’s sac remains intact too long, the animal’s back will not close properly. The sac that surrounds insect embryos has two layers: an inner layer called the amnion, and a tough outer layer called the serosa. However, it has been difficult to study what happens to the amnion as the insect embryo develops because it is hard to distinguish it from the serosa. Now, Hilbrant et al. have used genetically engineered red flour beetles in which the cells of the amnion produce a fluorescent protein that can be viewed under a microscope. This allowed the amnion to be observed in living specimens during beetle embryo development, and revealed that the amnion attaches and detaches from the serosa more than once as the embryo develops. Furthermore, the amnion and serosa remain as distinct tissues as they withdraw from the embryo. Hilbrant et al. also found that the cells in part of the amnion near the head of the beetle embryo have a special shape before the sac ruptures. This region of the amnion breaks apart first, and the serosa breaks open a few minutes afterwards. Once the two layers have broken, they pull back from the embryo like a pillowcase being turned inside out as it is removed from a pillow. In normal beetles, this process is quite rapid and squeezes the embryo’s abdomen. But in beetles genetically manipulated to lack a serosa the process is slower because the amnion is not strong enough by itself to squeeze the embryo. Overall, the experiments show that the amnion starts the rupture of the red flour beetle embryo’s protective sac and the serosa drives the process of the sac being peeled back. Further research will now investigate the mechanics behind the two tissues’ roles and whether the amnion and serosa display similar behaviors in other related insect species.

The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. Contexte : les hémiptères (pucerons, cigales et punaises) constituent un ordre d'insectes clé, caractérisé par une grande diversité alimentaire et une excellente faisabilité expérimentale en génétique moléculaire. En nous appuyant sur le séquençage récent d'hémiptères nuisibles tels que les pucerons phloémifuges et les punaises de lit hématophages, nous présentons la séquence du génome et des analyses comparatives centrées sur la punaise de l'asclépiade Oncopeltus fasciatus, une espèce granivore de la famille des Lygaeidae.Résultats : le génome d'Oncopeltus (926 Mb) est bien représenté par l'assemblage actuel et le jeu de gènes officiel. Nous utilisons nos données génomiques et de séquençage d'ARN non seulement pour caractériser le répertoire de gènes codant pour les protéines et réaliser une interférence ARN spécifique à l'isoforme, mais aussi pour élucider les schémas d'évolution moléculaire et de physiologie. Nous observons une expansion et une diversification continues, spécifiques à la lignée, des protéines répressives à doigts de zinc C2H2. La découverte d'un gain et d'un renouvellement d'introns spécifiques aux hémiptères a également conduit à évaluer la lignée et la taille du génome comme prédicteurs de l'évolution de la structure des gènes. De plus, nous identifions des gains et des pertes enzymatiques corrélés à la biologie de l'alimentation, en particulier pour les réductions associées à une alimentation dérivée et fluide.Conclusions : grâce à la punaise de l’asclépiade, nous disposons désormais d’une masse critique d’espèces séquencées pour un ordre d’insectes hémimétaboles et un groupe proche des Holométaboles, améliorant considérablement la diversité génomique des insectes. Nous définissons ainsi les points communs entre les hémiptères et étudions comment les génomes des hémiptères reflètent des écologies alimentaires distinctes. Compte tenu de la force d’Oncopeltus comme modèle expérimental, ces nouvelles ressources de séquences renforcent les bases de la recherche moléculaire et mettent en lumière des considérations techniques pour l’analyse des génomes d’invertébrés de taille moyenne.

Unlike passive rupture of the human chorioamnion at birth, the insect extraembryonic (EE) tissues the amnion and serosa actively rupture and withdraw in late embryogenesis. Despite its importance for successful development, EE morphogenesis remains poorly understood. Contradicting the hypothesis of a single, fused EE membrane, we show that both tissues persist as discrete epithelia within a bilayer, using new tissue-specific EGFP transgenic lines in the beetle Tribolium castaneum. Quantitative live imaging analyses show that the amnion initiates EE rupture in a specialized anterior-ventral cap, while RNAi manipulation of EE tissue complement and function reveals that the serosa is autonomously contractile. Thus the bilayer efficiently coordinates the amnion as initiator and serosa as driver to achieve withdrawal. The novel bilayer architecture may reflect evolutionary changes in the EE tissues specific to holometabolous insects. More generally, tissue apposition in a bilayer exemplifies a high degree of functional interaction between developing epithelia.

Background Transforming Growth Factor (TGF) and Bone Morphogenetic Protein (BMP) signalling critically influence pancreatic ductal adenocarcinoma (PDAC) progression, with TGF-B paradoxically exerting both tumour-promoting and -suppressive effects. Parallel to this observation, the specific context-dependent, spatial dynamics of these pathways and their interaction with the tumour microenvironment (TME) remain poorly understood. Methods We performed a spatially resolved analysis of PDAC on a multi-region tissue microarray cohort of 117 curatively resected PDAC specimens consisting of tumour centre (TC), tumour front (TF), and stromal(-predominant) tissue cores each. Protein (ID1, pSMAD2) and mRNA (TGF-A, TGF-B1/2, BMP4, GREM1) expression were assessed in each tissue compartment by immunohistochemistry and in situ hybridization, respectively, quantified by digital image analysis, and correlated with clinicopathologic features. Results ID1 was significantly overexpressed in PDAC cells compared to associated stroma ( p  < 0.01), while pSMAD2 was largely absent in PDAC cells, but preserved among associated stroma compartments, particularly in TF cores ( p  = 0.04). Higher stromal GREM1 signal correlated with reduced overall tumoural ID1 protein expression ( p  = 0.02), and TGF-B2 high /TGF-A low stroma was significantly associated with worse survival ( p  < 0.01). Intratumoural TGF-B2 was inversely correlated with stromal pSMAD2 expression ( p  = 0.03) and was associated with lymph node involvement ( p  = 0.02). FOXP3 + regulatory T-cells were significantly reduced in TGF-B2 high tumours ( p  = 0.04), while higher tumoural TGF-B1 exhibited a trend towards increased FOXP3 + cells ( p  = 0.08). Conclusions Our spatial analysis reveals intratumoural heterogeneity of TGF/BMP signalling and its significance for PDAC progression. Notably, stromal TGF-B2 emerges as a prognostic biomarker, while TGF-B1 and ID1 are implicated in adverse clinical and pathologic features. These findings highlight the importance of TGF/BMP signalling niches in the TME with implications for PDAC biology and can inform the development of future therapeutic strategies.

•Investigation of endogenous formation of 1-propanol and methanol after consumption of an alcoholic beverage.•In a drinking experiment 24 volunteers consumed congener free vodka.•The kinetic parameters of ethanol, methanol and 1-propanol were evaluated.•Results confirm the formation of 1-propanol and methanol.•Evaluation of concentrations of 1-propanol must include the endogenous formation. In cases of drunk-driving, allegations that alcohol has been consumed after the incident, are proved by analyzing congener alcohols in the blood sample. 1-Propanol, one of the main congener compounds, was tested, whether it is also endogenously formed when a person has consumed alcoholic beverages. Eleven male and 13 female volunteers consumed congener-free vodka (37.5 vol% ethanol, individual doses: 0.15–0.32 l) within one hour. Blood samples were taken up to 10 h and analyzed for ethanol and congener alcohols by headspace gas chromatography-mass spectrometry. Ethanol concentrations reached in blood a maximum of 0.65–1.23 g/l and decreased by 0.18 g/l/h (median values). Of the congener alcohols analyzed, only methanol and 1-propanol were detected in the plasma samples of all subjects. The endogenous methanol concentration increased from 0.66 mg/l by 0.22 mg/l/h to 2.19 mg/l (medians). 1-Propanol was not detected prior to alcohol consumption. Maximum concentrations of 0.10–0.32 mg/L were measured after 1.0–4.5 h. A plateau of the 1-propanol concentration was observed in the plasma samples of the 18 subjects lasting for 0.5–4.0 h and this alcohol was completely eliminated at ethanol concentrations of 0.17 g/l (median, range 0.03–0.55 g/l). The results of the study confirm the formation of 1-propanol after consumption of 1-propanol-free beverages, which should be taken into account when evaluating its concentration.

The interplay of Dirac physics and induced superconductivity at the interface of a 3D topological insulator (TI) with an s-wave superconductor (S) provides a new platform for topologically protected quantum computation based on elusive Majorana modes. To employ such S–TI hybrid devices in future topological quantum computation architectures, a process is required that allows for device fabrication under ultrahigh vacuum conditions. Here, we report on the selective area growth of (Bi,Sb)2Te3 TI thin films and stencil lithography of superconductive Nb for a full in situ fabrication of S–TI hybrid devices via molecular-beam epitaxy. A dielectric capping layer was deposited as a final step to protect the delicate surfaces of the S–TI hybrids at ambient conditions. Transport experiments in as-prepared Josephson junctions show highly transparent S–TI interfaces and a missing first Shapiro step, which indicates the presence of Majorana bound states. To move from single junctions towards complex circuitry for future topological quantum computation architectures, we monolithically integrated two aligned hardmasks to the substrate prior to growth. The presented process provides new possibilities to deliberately combine delicate quantum materials in situ at the nanoscale.

Prognosis of metastatic melanoma improved with the development of checkpoint inhibitors. The role of tumor infiltrating lymphocytes (TILs) in lymph node metastases of stage III melanoma remains unclear. We retrospectively characterized TILs in primary melanomas and matched lymph node metastases (stage III melanoma) of patients treated with the checkpoint inhibitor ipilimumab. Tumor infiltrating lymphocytes were characterized for CD3, CD4, and CD8 expressions by immunohistochemistry. 4/9 patients (44%) responded to treatment with ipilimumab (1 complete and 2 partial remissions, 1 stable disease). All responders exhibited CD4 and CD8 T-cell infiltration in their lymph node metastases, whereas all non-responders did not show an infiltration of the lymph node metastasis with TILs. The correlation between the presence and absence of TILs in responders vs. non-responders was statistically significant (p = 0.008). Median distant metastases free survival, i.e., progression from stage III to stage IV melanoma, was similar in responders and non-responders (22.1 vs. 19.3 months; p = 0.462). Median progression free and overall survival show a trend in favor of the patients having TIL rich lymph node metastases (6.8 vs. 3.3 months, p = 0.09; and all alive at last follow-up vs. 8.2 months, respectively, p = 0.08). Our data suggest a correlation between the T-cell infiltration of the lymph node metastases in stage III melanoma and the response to ipilimumab once these patients progress to stage IV disease.