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OBJECTIVE: To compare two intensified insulin therapy regimens - continuous subcutaneous insulin infusion (CSII) against multiple daily insulin injection (MDI) - in Danish adolescents examined in a prospective, matched controlled study design. RESEARCH DESIGN AND METHODS: Thirty type 1 diabetic adolescents at CSII and 26 matched MDI controls were included in this open intention-to-treat study. Actrapid was used in both groups. Before study entry, all participants followed a brush-up course in order to minimize study effect. At each visit, the following parameters were recorded: hemoglobin A1c (HbA1c), insulin dose, weight, number of hypoglycemic and diabetic ketoacidosis (DKA) events, and the time resources used. At entry and exit of the study, diet registration and validated quality-of-life (QoL) questionnaires were filled by the participants. RESULTS: A non-significant decline in HbA1c was seen in both groups (p = 0.468); HbA1c decreased from 9.5 to 8.9% and from 9.7 to 9.5% in the CSII and MDI group, respectively. The insulin dose and the number of severe hypoglycemic events per patient were lower (non-significant) in the CSII group. Both groups showed increased body mass index - highest in the CSII group - and mild to moderate DKA episodes were only seen among CSII users. No differences could be demonstrated within the QoL or diet registrations. CONCLUSIONS: CSII treatment is beneficial as an intensified insulin therapy for selected type 1 diabetic patients and both MDI and CSII can be offered by the professional diabetes team to better tailor therapy. In future, there is a strong need to identify the characteristics of responders to CSII treatment in order to increase the efficacy and safety of CSII treatment. [ABSTRACT FROM AUTHOR]

Hepatoblastoma, the most prevalent pediatric liver cancer, almost always carries a WNT-activating CTNNB1 mutation, yet exhibits notable molecular heterogeneity. To characterize this heterogeneity and identify novel targeted therapies, we perform comprehensive analysis of hepatoblastomas and tumor-derived organoids using single-cell RNA-seq/ATAC-seq, spatial transcriptomics, and high-throughput drug profiling. We identify two distinct tumor epithelial signatures: hepatic 'fetal' and WNT-high 'embryonal', displaying divergent WNT signaling patterns. The fetal group is enriched for liver-specific WNT targets, while the embryonal group is enriched in canonical WNT target genes. Gene regulatory network analysis reveals enrichment of regulons related to hepatic functions such as bile acid, lipid and xenobiotic metabolism in the fetal subtype but not in the embryonal subtype. In addition, the dichotomous expression pattern of the transcription factors HNF4A and LEF1 allows for a clear distinction between the fetal and embryonal tumor cells. We also perform high-throughput drug screening using patient-derived tumor organoids and identify sensitivity to HDAC inhibitors. Intriguingly, embryonal and fetal tumor organoids are sensitive to FGFR and EGFR inhibitors, respectively, indicating a dependency on EGF/FGF signaling in hepatoblastoma tumorigenesis. In summary, our data uncover the molecular and drug sensitivity landscapes of hepatoblastoma and pave the way for the development of targeted therapies., Competing Interests: Competing interests: H.C. is currently head of pharma Research Early Development (pRED) at Roche and is an inventor on several patents related to organoid technology. His full disclosure is given at www.uu.nl/staff/JCClevers . The remaining authors declare no competing interests., (© 2024. The Author(s).)

Dictyostelium discoideum is a professional phagocyte frequently used to study cellular processes underlying the recognition, engulfment, and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control membrane properties, participate in signal transmission, and serve as adhesion molecules in recognition processes relevant to immunity and infection. By combining lipidomics with a bioinformatics-based cloning strategy, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum enabled identification of an enzyme that selectively catalyzes the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The IPC synthase, Dd IPCS1, shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum . These findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

ObjectivesFlavoured products are especially appealing to youth and contribute to the onset of waterpipe smoking and continued use of waterpipe tobacco. The goal of database and chemical analysis was to provide a clear overview of commonly used flavours and flavourings in tobacco and related waterpipe products, that is, herbal molasses and steam stones., Methods: In 2019, 249 waterpipe tobacco products were registered in the European Common Entry Gate by manufacturers to be marketed in The Netherlands. Flavour categories were assigned to the registered products based on their brand names and product descriptions. Nicotine and eleven 1111 flavourings were identified and quantified in waterpipe tobacco (n=8), herbal molasses (n=7) and steam stones (n=4) by extraction and gas chromatography-mass spectrometry (GC-MS) analysis., Results: Flavour categories could be assigned to 237 of 249 registered waterpipe tobacco products. Eight flavour main categories and 48 unique subcategories were identified and presented in a flavour wheel. All registered waterpipe tobacco products were flavoured, and the majority (78%) was fruit flavoured. Herbal molasses contained similar median flavouring levels, and steam stones contained lower median levels compared with waterpipe tobacco. Flavourings in waterpipe products were almost exclusively fruity and sweet, often in combination with menthol/mint flavourings., Conclusions: This study is the first to present a waterpipe tobacco flavour wheel, providing a quick overview of waterpipe tobacco flavours and thereby aiding communication among experts around the globe. GC-MS analysis revealed that the most prevalent flavourings are present in similar levels in herbal and tobacco waterpipe products. Banning flavourings in all waterpipe products would be a good strategy to reduce waterpipe smoking among youth., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Pediatric cancer is the leading cause of disease-related death in children and the need for better therapeutic options remains urgent. Due to the limited number of patients, target and drug development for pediatrics is often supplemented by data from studies focused on adult cancers. Recent evidence shows that pediatric cancers possess different vulnerabilities that should be explored independently from adult cancers., Methods: Using the publicly available Genomics of Drug Sensitivity in Cancer database, we explore therapeutic targets and biomarkers specific to the pediatric solid malignancies Ewing sarcoma, medulloblastoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma. Results are validated using cell viability assays and high-throughput drug screens are used to identify synergistic combinations., Results: Using published drug screening data, PARP is identified as a drug target of interest across multiple different pediatric malignancies. We validate these findings, and we show that efficacy can be improved when combined with conventional chemotherapeutics, namely topoisomerase inhibitors. Additionally, using gene set enrichment analysis, we identify ribosome biogenesis as a potential biomarker for PARP inhibition in pediatric cancer cell lines., Conclusion: Collectively, our results provide evidence to support the further development of PARP inhibition and the combination with TOP1 inhibition as a therapeutic approach in solid pediatric malignancies. Additionally, we propose ribosome biogenesis as a component to PARP inhibitor sensitivity that should be further investigated to help maximize the potential utility of PARP inhibition and combinations across pediatric solid malignancies., (© 2023. The Author(s).)

Introduction: Mutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma tumors and are associated with response to MEK inhibition in vitro . However, these inhibitors alone do not lead to tumor regression in vivo , indicating the need for combination therapy., Methods and Results: Via high-throughput combination screening, we identified that the MEK inhibitor trametinib can be combined with BCL-2 family member inhibitors, to efficiently inhibit growth of neuroblastoma cell lines with RAS-MAPK mutations. Suppressing the RAS-MAPK pathway with trametinib led to an increase in pro-apoptotic BIM, resulting in more BIM binding to anti-apoptotic BCL-2 family members. By favoring the formation of these complexes, trametinib treatment enhances sensitivity to compounds targeting anti-apoptotic BCL-2 family members. In vitro validation studies confirmed that this sensitizing effect is dependent on an active RAS-MAPK pathway. In vivo combination of trametinib with BCL-2 inhibitors led to tumor inhibition in NRAS -mutant and NF1 -deleted xenografts., Conclusion: Together, these results show that combining MEK inhibition with BCL-2 family member inhibition could potentially improve therapeutic outcomes for RAS-MAPK-mutated neuroblastoma patients., Competing Interests: HC is employed by Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Eleveld, Vernooij, Schild, Koopmans, Alles, Ebus, Dandis, van Tinteren, Caron, Koster, van Noesel, Tytgat, Eising, Versteeg, Dolman and Molenaar.)

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Neuroblastoma is the most common extracranial solid tumor found in children and despite intense multi-modal therapeutic approaches, low overall survival rates of high-risk patients persist. Tumors with heterozygous loss of chromosome 11q and MYCN amplification are two genetically distinct subsets of neuroblastoma that are associated with poor patient outcome. Using an isogenic 11q deleted model system and high-throughput drug screening, we identify checkpoint kinase 1 (CHK1) as a potential therapeutic target for 11q deleted neuroblastoma. Further investigation reveals MYCN amplification as a possible additional biomarker for CHK1 inhibition, independent of 11q loss. Overall, our study highlights the potential power of studying chromosomal aberrations to guide preclinical development of novel drug targets and combinations. Additionally, our study builds on the growing evidence that DNA damage repair and replication stress response pathways offer therapeutic vulnerabilities for the treatment of neuroblastoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Keller, Eleveld, Schild, van den Handel, van den Boogaard, Amo-Addae, Eising, Ober, Koopmans, Looijenga, Tytgat, Ylstra, Molenaar, Dolman and van Hooff.)

Background: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations., Methods: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled., Results: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRAS Q61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition., Conclusions: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells., (© 2022. The Author(s).)

The lysosome is the major catabolic organelle in the cell that has been established as a key metabolic signaling center. Mutations in many lysosomal proteins have catastrophic effects and cause neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many evolutionary conserved proteins. Proteins reach vacuoles via the Vps10-dependent endosomal vacuolar protein sorting pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. A systematic understanding of the cargo spectrum of each pathway is completely lacking. Here, we use quantitative proteomics of purified vacuoles to generate the yeast lysosomal biogenesis map. This dataset harbors information on the cargo-receptor relationship of almost all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data show how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis., (© 2022 Eising et al.)

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast ( Saccharomyces cerevisiae ) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Schmidt et al.)

Bioorthogonal reactions can be performed selectively in the presence of any biological functional group and are widely used to achieve site-selective chemical modifications of biomolecules. The click-to-release reaction is a bioorthogonal bond-cleavage variant that has gained much interest over the last few years. The bioorthogonal reaction between tetrazines and trans-cyclooctenes or vinyl ethers, for example, initiates the release of a small molecule immediately after the cycloaddition with tetrazines. Recently, our group reported that vinylboronic acids (VBAs) give exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder reaction with tetrazines that are substituted with boron-coordinating ligands. In the present study, we show that VBAs can be used in a click-to-release variant and demonstrate its bioorthogonality with a VBA-protected doxorubicin prodrug. We show that the cytotoxicity of doxorubicin is silenced by the attachment of the VBA, and activity can be largely restored upon the reaction with a tetrazine, inducing cell death.

Cellular homeostasis requires the ubiquitin-dependent degradation of membrane proteins. This was assumed to be mediated exclusively either by endoplasmic reticulum-associated degradation (ERAD) or by endosomal sorting complexes required for transport (ESCRT)-dependent lysosomal degradation. We identified in Saccharomyces cerevisiae an additional pathway that selectively extracts membrane proteins at Golgi and endosomes for degradation by cytosolic proteasomes. One endogenous substrate of this endosome and Golgi-associated degradation pathway (EGAD) is the ER-resident membrane protein Orm2, a negative regulator of sphingolipid biosynthesis. Orm2 degradation is initiated by phosphorylation, which triggers its ER export. Once on Golgi and endosomes, Orm2 is poly-ubiquitinated by the membrane-embedded "Defective in SREBP cleavage" (Dsc) ubiquitin ligase complex. Cdc48/VCP then extracts ubiquitinated Orm2 from membranes, which is tightly coupled to the proteasomal degradation of Orm2. Thereby, EGAD prevents the accumulation of Orm2 at the ER and in post-ER compartments and promotes the controlled de-repression of sphingolipid biosynthesis. Thus, the selective degradation of membrane proteins by EGAD contributes to proteostasis and lipid homeostasis in eukaryotic cells., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Proteins and lipids of the plasma membrane underlie constant remodeling via a combination of the secretory- and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted for recycling to the plasma membrane or degradation in vacuoles. Previously we have shown a role for the GARP complex in sphingolipid sorting and homeostasis (Fröhlich et al. 2015). However, the majority of cargo sorted in a GARP dependent process remain largely unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry based vacuolar proteomics and lipidomics to show that recycling of two specific groups of proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of the lipid composition are the first occurring defects in GARP mutants. Our assay can be adapted to systematically map cargo of the entire endocytic pathway., Competing Interests: SE, LT, FF No competing interests declared, (© 2019, Eising et al.)

Cell-penetrating peptides are able to transport a wide variety of cargo across cell membranes. Although promising, they are not often considered for therapeutic purposes as they lack controllable activity and cell selectivity. We have developed an activation strategy based on a split octa-arginine cell-penetrating peptide (CPP) that can be activated by means of bioorthogonal ligation. To this end we prepared two non-penetrating tetra-arginine halves, functionalized either with a tetrazine or with a complementary bicyclo[6.1.0]nonyne (BCN) group. We demonstrate that an active octa-arginine can be reconstituted in situ upon mixing the complementary split peptides. The resulting activated peptide is taken up as efficiently as the well-established cell-penetrating peptide octa-arginine. The activation of the oligo-arginines can also be achieved using trans -cyclooctene (TCO) as a ligation partner, while norbornene appears too kinetically slow for use in situ . We further show that this strategy can be applied successfully to transport a large protein into living cells. Our results validate a promising first step in achieving control over cell penetration and to use CPPs for therapeutic approaches.

Bioorthogonal reactions are selective transformations that are not affected by any biological functional group and are widely used for chemical modification of biomolecules. Recently, we reported that vinylboronic acids (VBAs) gave exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction with tetrazines bearing a boron-coordinating pyridyl moiety compared to tetrazines lacking such a substituent. In this integrated experimental and theoretical study, we show how the reaction rate of the VBA-tetrazine ligation can be accelerated by shifting the equilibrium from boronic acid to the boronate anion in the reaction mixture. Quantum chemical activation strain analyses reveal that this rate enhancement is a direct consequence of the excellent electron-donating capability of the boronate anion in which the π HOMO is pushed to a higher energy due to the net negative potential of this species. We have explored the second-order rate constants of several tetrazines containing potential VBA-coordinating hydroxyl substituents. We observed an increase in rate constants of several orders of magnitude compared to the tetrazines lacking a hydroxyl substituent. Furthermore, we find the hydroxyl-substituted tetrazines to be more selective toward VBAs than toward the commonly used bioorthogonal reactant norbornene, and more stable in aqueous environment than the previously studied tetrazines containing a pyridyl substituent.

Mapping proteins at a specific subcellular location is essential to gaining detailed insight on local protein dynamics. We have developed an enzymatic strategy to label proteins on a subcellular level using arylamine N-acetyltransferase (NAT). The NAT enzyme activates an arylhydroxamic acid functionality into a nitrenium ion that reacts fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. The electron density on the aromatic ring proved important for probe activation as strong labeling was only observed with an arylhydroxamic acid bearing an electron donating substituent. We further demonstrate that, using this electron rich arylhydroxamic acid, clear labeling was achieved on a subcellular level in living cells that were transfected with a genetically targeted NAT to the nucleus or the cytosol.

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Virus capsids, i.e., viruses devoid of their genetic material, are suitable nanocarriers for biomedical applications such as drug delivery and diagnostic imaging. For this purpose, the reliable encapsulation of cargo in such a protein nanocage is crucial, which can be accomplished by the covalent attachment of the compounds of interest to the protein domains positioned at the interior of the cage. This approach is particularly valid for the capsid proteins of the cowpea chlorotic mottle virus (CCMV), which have their N-termini located at the inside of the capsid structure. Here, we examined several site-selective modification methods for covalent attachment and encapsulation of cargo at the N-terminus of the CCMV protein. Initially, we explored approaches to introduce an N-terminal azide functionality, which would allow the subsequent bioorthogonal modification with a strained alkyne to attach the desired cargo. As these methods showed compatibility issues with the CCMV capsid proteins, a strategy based on 2-pyridinecarboxaldehydes for site-specific N-terminal protein modification was employed. This method allowed the successful modification of the proteins, and was applied for the introduction of a bioorthogonal vinylboronic acid moiety. In a subsequent reaction, the proteins could be modified further with a fluorophore using the tetrazine ligation. The application of capsid assembly conditions on the functionalized proteins led to successful particle formation, showing the potential of this covalent encapsulation strategy.

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present in the cell. The tetrazine ligation is very suitable as a bioorthogonal reaction because of its selectivity and high reaction rates with several alkenes and alkynes. Recently, we described vinylboronic acids (VBAs) as novel hydrophilic bioorthogonal moieties that react efficiently with dipyridyl- s-tetrazines and used them for protein modification in cell lysate. It is not clear, however, whether VBAs are suitable for labeling experiments in living cells because of the possible coordination with, for example, vicinal carbohydrate diols. Here, we evaluated VBAs as bioorthogonal reactants for labeling of proteins in living cells using an irreversible inhibitor of the proteasome and compared the reactivity to that of an inhibitor containing norbornene, a widely used reactant for the tetrazine ligation. No large differences were observed between the VBA and norbornene probes in a two-step labeling approach with a cell-penetrable fluorescent tetrazine, indicating that the VBA gives little or no side reactions with diols and can be used efficiently for protein labeling in living cells.

Background: COPD is a heterogeneous chronic inflammatory disease of the airways and it is well accepted that the GOLD classification does not fully represent the complex clinical manifestations of COPD and this classification therefore is not well suited for phenotyping of individual patients with COPD. Besides the chronic inflammation in the lung compartment, there is also a systemic inflammation present in COPD patients. This systemic inflammation is associated with elevated levels of cytokines in the peripheral blood, but the precise composition is unknown. Therefore, differences in phenotype of peripheral blood neutrophils in vivo could be used as a read out for the overall systemic inflammation in COPD., Method: Our aim was to utilize an unsupervised method to assess the proteomic profile of peripheral neutrophils of stable COPD patients and healthy age matched controls to find potential differences in these profiles as read-out of inflammatory phenotypes. We performed fluorescence two-dimensional difference gel electrophoresis with the lysates of peripheral neutrophils of controls and stable COPD patients., Results: We identified two groups of COPD patients based on the differentially regulated proteins and hierarchical clustering whereas there was no difference in lung function between these two COPD groups. The neutrophils from one of the COPD groups were less responsive to bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF)., Conclusion: This illustrates that systemic inflammatory signals do not necessarily correlate with the GOLD classification and that inflammatory phenotyping can significantly add in an improved diagnosis of single COPD patients., Trial Registration: Clinicaltrials.gov: NCT00807469 registered December 11th 2008.

Background/aim: An in-depth understanding of the early phase of type 1 diabetes (T1D) pathogenesis is important for targeting primary prevention. We examined if 14 preselected mediators of immune responses differed in neonates that later developed T1D compared to control neonates., Methods: The study is a case-control study with a 1:2 matching. The individuals were born between 1981 through 2002. Cases were validated using the National Patient Register and the Danish Childhood Diabetes Register. Interleukin(IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, interferon gamma, tumor necrosis factor alpha, transforming growth factor beta 1 (active form), leptin, adiponectin, c-reactive protein, mannose-binding lectin and soluble triggering receptor expressed on myeloid cells-1 were measured by using a flowmetric Luminex xMAP® technology. We tested two models both including a number of possible confounders. In the first model (model 1) we also adjusted for HLA-DQB1 genotype. A total of 1930 groups of assay-matched cases and controls (4746 individuals) were included in the statistical analyses., Results: Adiponectin was negatively associated with later risk of T1D in both models (relative change (RC), model 1: 0.95, P=0.046 and model 2: 0.95, P=0.006). IL-10 and IL-12 were both positively associated with T1D risk in the model 2 (RC, 1.19, P=0.006 and 1.07, P=0.02, respectively)-these results were borderline significant in model 1, but showed the same direction as the results from model 2., Conclusions: Our results indicate that specific immunological signatures are already present at time of birth in children developing T1D before the age of 18years., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bioorthogonal reactions are widely used for the chemical modification of biomolecules. The application of vinylboronic acids (VBAs) as non-strained, synthetically accessible and water-soluble reaction partners in a bioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction with 3,6-dipyridyl-s-tetrazines is described. Depending on the substituents, VBA derivatives give second-order rate constants up to 27 m(-1)  s(-1) in aqueous environments at room temperature, which is suitable for biological labeling applications. The VBAs are shown to be biocompatible, non-toxic, and highly stable in aqueous media and cell lysate. Furthermore, VBAs can be used orthogonally to the strain-promoted alkyne-azide cycloaddition for protein modification, making them attractive complements to the bioorthogonal molecular toolbox., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations.

Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies.