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Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the β-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR2 interaction may suggest a possible immunoregulatory role of HSV glycoprotein C. [ABSTRACT FROM AUTHOR]

Purpose: Patients with MYC-amplified Group 3 medulloblastoma (MB) (subtype II) show poor progression-free survival rates. Class I histone deacetylase inhibitors (HDACi) are highly effective for the treatment of MYC-amplified MB in vitro and in vivo. Drug combination regimens including class I HDACi may represent an urgently needed novel treatment approach for this high risk disease., Methods: A medium-throughput in vitro combination drug screen was performed in three MYC-amplified and one non-MYC-amplified MB cell line testing 75 clinically relevant drugs alone and in combination with entinostat. The drug sensitivity score (DSS) was calculated based on metabolic inhibition quantified by CellTiter-Glo. The six top synergistic combination hits were evaluated in a 5 × 5 combination matrix and a seven-ray design. Synergy was validated and characterized by cell counts, caspase-3-like-activity and poly-(ADP-ribose)-polymerase-(PARP)-cleavage. On-target activity of drugs was validated by immunoprecipitation and western blot. BCL-XL dependency of the observed effect was explored with siRNA mediated knockdown of BCL2L1, and selective inhibition with targeted compounds (A-1331852, A-1155463)., Results: 20/75 drugs effectively reduced metabolic activity in combination with entinostat in all three MYC-amplified cell lines (DSS ≥ 10). The combination entinostat and navitoclax showed the strongest synergistic interaction across all MYC-amplified cell lines. siRNA mediated knockdown of BCL2L1, as well as targeted inhibition with selective inhibitors showed BCL-XL dependency of the observed effect. Increased cell death was associated with increased caspase-3-like-activity., Conclusion: Our study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells., (© 2024. The Author(s).)

Niemann-Pick type C1 disease (NPC1 [OMIM 257220]) is a rare and severe autosomal recessive disorder, characterized by a multitude of neurovisceral clinical manifestations and a fatal outcome with no effective treatment to date. Aiming to gain insights into the genetic aspects of the disease, clinical, genetic, and biomarker PPCS data from 602 patients referred from 47 countries and diagnosed with NPC1 in our laboratory were analyzed. Patients' clinical data were dissected using Human Phenotype Ontology (HPO) terms, and genotype-phenotype analysis was performed. The median age at diagnosis was 10.6 years (range 0-64.5 years), with 287 unique pathogenic/likely pathogenic (P/LP) variants identified, expanding NPC1 allelic heterogeneity. Importantly, 73 P/LP variants were previously unpublished. The most frequent variants detected were: c.3019C > G, p.(P1007A), c.3104C > T, p.(A1035V), and c.2861C > T, p.(S954L). Loss of function (LoF) variants were significantly associated with earlier age at diagnosis, highly increased biomarker levels, and a visceral phenotype (abnormal abdomen and liver morphology). On the other hand, the variants p.(P1007A) and p.(S954L) were significantly associated with later age at diagnosis (p < 0.001) and mildly elevated biomarker levels (p ≤ 0.002), consistent with the juvenile/adult form of NPC1. In addition, p.(I1061T), p.(S954L), and p.(A1035V) were associated with abnormality of eye movements (vertical supranuclear gaze palsy, p ≤ 0.05). We describe the largest and most heterogenous cohort of NPC1 patients published to date. Our results suggest that besides its utility in variant classification, the biomarker PPCS might serve to indicate disease severity/progression. In addition, we establish new genotype-phenotype relationships for "frequent" NPC1 variants., (© 2023. The Author(s).)

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75-78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs., (© 2022. The Author(s).)

Image-based phenotypic drug profiling is receiving increasing attention in drug discovery and precision medicine. Compared to classical end-point measurements quantifying drug response, image-based profiling enables both the quantification of drug response and characterization of disease entities and drug-induced cell-death phenotypes. Here, we aim to quantify image-based drug responses in patient-derived 3D spheroid tumor cell cultures, tackling the challenges of a lack of single-cell-segmentation methods and limited patient-derived material. Therefore, we investigate deep transfer learning with patient-by-patient fine-tuning for cell-viability quantification. We fine-tune a convolutional neural network (pre-trained on ImageNet) with 210 control images specific to a single training cell line and 54 additional screen -specific assay control images. This method of image-based drug profiling is validated on 6 cell lines with known drug sensitivities, and further tested with primary patient-derived samples in a medium-throughput setting. Network outputs at different drug concentrations are used for drug-sensitivity scoring, and dense-layer activations are used in t-distributed stochastic neighbor embeddings of drugs to visualize groups of drugs with similar cell-death phenotypes. Image-based cell-line experiments show strong correlation to metabolic results ( R ≈ 0.7 ) and confirm expected hits, indicating the predictive power of deep learning to identify drug-hit candidates for individual patients. In patient-derived samples, combining drug sensitivity scoring with phenotypic analysis may provide opportunities for complementary combination treatments. Deep transfer learning with patient-by-patient fine-tuning is a promising, segmentation-free image-analysis approach for precision medicine and drug discovery.

To present our experience using a multiomic approach, which integrates genetic and biochemical testing as a first-line diagnostic tool for patients with inherited metabolic disorders (IMDs). A cohort of 3720 patients from 62 countries was tested using a panel including 206 genes with single nucleotide and copy number variant (SNV/CNV) detection, followed by semi-automatic variant filtering and reflex biochemical testing (25 assays). In 1389 patients (37%), a genetic diagnosis was achieved. Within this cohort, the highest diagnostic yield was obtained for patients from Asia (57.5%, mainly from Pakistan). Overall, 701 pathogenic/likely pathogenic unique SNVs and 40 CNVs were identified. In 620 patients, the result of the biochemical tests guided variant classification and reporting. Top five diagnosed diseases were: Gaucher disease, Niemann-Pick disease type A/B, phenylketonuria, mucopolysaccharidosis type I, and Wilson disease. We show that integrated genetic and biochemical testing facilitated the decision on clinical relevance of the variants and led to a high diagnostic yield (37%), which is comparable to exome/genome sequencing. More importantly, up to 43% of these patients (n = 610) could benefit from medical treatments (e.g., enzyme replacement therapy). This multiomic approach constitutes a unique and highly effective tool for the genetic diagnosis of IMDs., (© 2022. The Author(s).)

The importance of acetogens for H 2 turnover and overall anaerobic degradation in peatlands remains elusive. In the well-studied minerotrophic peatland fen Schlöppnerbrunnen, H 2 -consuming acetogens are conceptualized to be largely outcompeted by iron reducers, sulfate reducers, and hydrogenotrophic methanogens in bulk peat soil. However, in root zones of graminoids, fermenters thriving on rhizodeposits and root litter might temporarily provide sufficient H 2 for acetogens. In the present study, root-free peat soils from around the roots of Molinia caerulea and Carex rostrata (i.e., two graminoids common in fen Schlöpnnerbrunnen) were anoxically incubated with or without supplemental H 2 to simulate conditions of high and low H 2 availability in the fen. In unsupplemented soil treatments, H 2 concentrations were largely below the detection limit (∼10 ppmV) and possibly too low for acetogens and methanogens, an assumption supported by the finding that neither acetate nor methane substantially accumulated. In the presence of supplemental H 2 , acetate accumulation exceeded CH 4 accumulation in Molinia soil whereas acetate and methane accumulated equally in Carex soil. However, reductant recoveries indicated that initially, additional unknown processes were involved either in H 2 consumption or the consumption of acetate produced by H 2 -consuming acetogens. 16S rRNA and 16S rRNA gene analyses revealed that potential acetogens ( Clostridium, Holophagaceae ), methanogens ( Methanocellales, Methanobacterium ), iron reducers ( Geobacter ), and physiologically uncharacterized phylotypes ( Acidobacteria, Actinobacteria, Bacteroidetes ) were stimulated by supplemental H 2 in soil treatments. Phylotypes closely related to clostridial acetogens were also active in soil-free Molinia and Carex root treatments with or without supplemental H 2 . Due to pronounced fermentation activities, H 2 consumption was less obvious in root treatments, and acetogens likely thrived on root organic carbon and fermentation products (e.g., ethanol) in addition to H 2 . Collectively, the data highlighted that in fen Schlöppnerbrunnen, acetogens are associated to graminoid roots and inhabit the peat soil around the roots, where they have to compete for H 2 with methanogens and iron reducers. Furthermore, the study underscored that the metabolically flexible acetogens do not rely on H 2 , potentially a key advantage over other H 2 consumers under the highly dynamic conditions characteristic for the root-zones of graminoids in peatlands., 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 Meier, Oppermann, Drake and Schmidt.)

The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms.

Background: An increased prevalence of Parkinson's disease (PD) disease has been previously reported in subjects with Fabry disease (FD) carrying alpha-galactosidase (GLA) mutations and their first-line relatives. Moreover, decreased alpha-galactosidase A (AGLA) enzymatic activity has been reported among cases with PD compared to controls., Objective: The aim of our study was to determine the prevalence of FD among patients with PD., Methods: We recruited 236 consecutive patients with PD from February 2018 to December 2020. Clinical and sociodemographic data, including the MDS-UPDRS-III scores and HY stage (the Hoehn and Yahr scale), were collected, and in-depth phenotyping was performed in subjects with identified GLA variants. A multistep approach, including standard determination of AGLA activity and LysoGb3 in males, and next-generation based GLA sequencing in all females and males with abnormal AGLA levels was performed in a routine diagnostic setting., Results: The mean age of our patients was 68.9 ± 8.9 years, 130 were men (55.1%), and the mean disease duration was 7.77 ± 5.35 years. Among 130 men, AGLA levels were low in 20 patients (15%), and subsequent Lyso-Gb3 testing showed values within the reference range for all tested subjects. In 126 subsequently genetically tested patients, four heterozygous p.(Asp313Tyr) GLA variants (3.2%, MAF 0.016) were identified; all were females. None of the 4 GLA variant carriers identified had any clinical manifestation suggestive of FD., Conclusions: The results of this study suggest a possible relationship between FD and PD in a small proportion of cases. Nevertheless, the GLA variant found in our cohort is classified as a variant of unknown significance. Therefore, its pathogenic causative role in the context of PD needs further elucidation, and these findings should be interpreted with caution., Competing Interests: The authors have no conflicts of interest., (Copyright © 2022 Alexandra Lackova et al.)

Respiratory complex I drives proton translocation across energy-transducing membranes by NADH oxidation coupled with (ubi)quinone reduction. In humans, its dysfunction is associated with neurodegenerative diseases. The Escherichia coli complex represents the structural minimal form of an energy-converting NADH:ubiquinone oxidoreductase. Here, we report the structure of the peripheral arm of the E. coli complex I consisting of six subunits, the FMN cofactor, and nine iron-sulfur clusters at 2.7 Å resolution obtained by cryo electron microscopy. While the cofactors are in equivalent positions as in the complex from other species, individual subunits are adapted to the absence of supernumerary proteins to guarantee structural stability. The catalytically important subunits NuoC and D are fused resulting in a specific architecture of functional importance. Striking features of the E. coli complex are scrutinized by mutagenesis and biochemical characterization of the variants. Moreover, the arrangement of the subunits sheds light on the unknown assembly of the complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

High throughput screening methods, measuring the sensitivity and resistance of tumor cells to drug treatments have been rapidly evolving. Not only do these screens allow correlating response profiles to tumor genomic features for developing novel predictors of treatment response, but they can also add evidence for therapy decision making in precision oncology. Recent analysis methods developed for either assessing single agents or combination drug efficacies enable quantification of dose-response curves with restricted symmetric fit settings. Here, we introduce iTReX, a user-friendly and interactive Shiny/R application, for both the analysis of mono- and combination therapy responses. The application features an extended version of the drug sensitivity score (DSS) based on the integral of an advanced five-parameter dose-response curve model and a differential DSS for combination therapy profiling. Additionally, iTReX includes modules that visualize drug target interaction networks and support the detection of matches between top therapy hits and the sample omics features to enable the identification of druggable targets and biomarkers. iTReX enables the analysis of various quantitative drug or therapy response readouts (e.g. luminescence, fluorescence microscopy) and multiple treatment strategies (drug treatments, radiation). Using iTReX we validate a cost-effective drug combination screening approach and reveal the application's ability to identify potential sample-specific biomarkers based on drug target interaction networks. The iTReX web application is accessible at https://itrex.kitz-heidelberg.de., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fen Schlöppnerbrunnen is a moderately acidic methane-emitting peatland overgrown by Molinia caerulea and other wetland graminoids (e.g. Carex rostrata). Recently, the accumulation of H2, an indicator for fermentation, was observed with anoxically incubated C. rostrata roots but not with root-free fen soil. Based on this finding, we hypothesized that root-derived organic carbon has a higher capacity to promote fermentation processes than peat organic carbon from root-free fen soil. To address this hypothesis, C. rostrata and M. caerulea roots were anoxically incubated with or without fen soil and the product profiles of root treatments were compared with those of root-free soil treatments. Ethanol, acetate, propionate, butyrate, H2 and CO2 accumulated in root treatments and collective amounts of carbon in accumulating products were 20-200 times higher than those in root-free soil treatments, in which mainly CO2 accumulated. Analyses of 16S rRNA and 16S rRNA gene sequences revealed that Clostridium, Propionispira and Rahnella, representatives of butyrate, propionate and mixed acid fermenters, respectively, were relatively enriched in root treatments. In contrast, differences of the microbial community before and after incubation were marginal in root-free soil treatments. Collectively, these findings supported the assumed stimulatory effect of root-derived organic carbon on fen fermenters., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytochrome bd quinol:O 2 oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b 595 is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding., (© 2021. The Author(s).)

Ixodes ricinus is the main vector of tick-borne diseases in Europe. An immunization trial of calves with soluble extracts of I. ricinus salivary glands (SGE) or midgut (ME) previously showed a strong response against subsequent tick challenge, resulting in diminished tick feeding success. Immune sera from these trials were used for the co-immunoprecipitation of tick tissue extracts, followed by LC-MS/MS analyses. This resulted in the identification of 46 immunodominant proteins that were differentially recognized by the serum of immunized calves. Some of these proteins had previously also drawn attention as potential anti-tick vaccine candidates using other approaches. Selected proteins were studied in more detail by measuring their relative expression in tick tissues and RNA interference (RNAi) studies. The strongest RNAi phenotypes were observed for MG6 (A0A147BXB7), a protein containing eight fibronectin type III domains predominantly expressed in tick midgut and ovaries of feeding females, and SG2 (A0A0K8RKT7), a glutathione-S-transferase that was found to be upregulated in all investigated tissues upon feeding. The results demonstrated that co-immunoprecipitation of tick proteins with host immune sera followed by protein identification using LC-MS/MS is a valid approach to identify antigen-antibody interactions, and could be integrated into anti-tick vaccine discovery pipelines.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in Escherichia coli complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213G H variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed., 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 © 2021 Nuber, Mérono, Oppermann, Schimpf, Wohlwend and Friedrich.)

Energy converting NADH:ubiquinone oxidoreductase, complex I, is the first enzyme of respiratory chains in most eukaryotes and many bacteria. The complex comprises a peripheral arm catalyzing electron transfer and a membrane arm involved in proton-translocation. In Escherichia coli, the peripheral arm features a non-covalently bound flavin mononucleotide and nine iron-sulfur (Fe/S)-clusters. Very little is known about the incorporation of the Fe/S-clusters into the E. coli complex I. ErpA, an A-type carrier protein is discussed to act as a Fe/S-cluster carrier protein. To contribute to the understanding of ErpA for the assembly of E. coli complex I, we analyzed an erpA knock-out strain. Deletion of erpA decreased the complex I content in cytoplasmic membranes to approximately one third and the NADH oxidase activity to one fifth. EPR spectroscopy showed the presence of all Fe/S-clusters of the complex in the membrane but only in minor quantities. Sucrose gradient centrifugation and native PAGE revealed the presence of a marginal amount of a stable and fully assembled complex extractable from the membrane. Thus, ErpA is not essential for the assembly of complex I but its absence leads to a strong decrease of a functional complex in the cytoplasmic membrane due to a major lack of all EPR-detectable Fe/S-clusters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gaucher disease (GD) is a lysosomal storage disorder that responds well to enzyme replacement therapy (ERT). Certain laboratory parameters, including blood concentration of glucosylsphingosine (Lyso-Gb1), the lyso-derivate of the common glycolipid glucocerebroside, correlate with clinical improvement and are therefore considered candidate-monitoring biomarkers. Whether they can indicate a reduction or loss of treatment efficiency, however, has not been systematically addressed for obvious reasons. We established and validated measurement of Lyso-Gb1 from dried blood spots (DBSs) by mass spectrometry. We then characterized the assay's longitudinal performance in 19 stably ERT-treated GD patients by dense monitoring over a 3-year period. The observed level of fluctuation was accounted for in the subsequent development of a unifying data normalization concept. The resulting approach was eventually applied to data from Lyso-Gb1 measurements after an involuntary treatment break for all 19 patients. It enabled separation of the "under treatment" versus "not under treatment" conditions with high sensitivity and specificity. We conclude that Lyso-Gb1 determination from DBSs indicates treatment issues already at an early stage before clinical consequences arise. In addition to its previously shown diagnostic utility, Lyso-Gb1 thereby qualifies as a monitoring biomarker in GD patients.

The introduction of disease-specific therapy for patients with type I Gaucher disease (GD1) was a revolution in the management of patients, but not without cost. Thus, the management of mildly affected patients is still debated. We herein report a long-term follow-up (median (range) of 20 (5-58) years) of 103 GD1 patients who have never received enzymatic or substrate reduction therapy. The median (range) platelet count and hemoglobin levels in last assessment of all but six patients who refused therapy (although recommended and approved) were 152 (56-408) × 10 3 /mL and 13.1 (7.6-16.8) g/dL, respectively. Most patients had mild hepatosplenomegaly. Nine patients were splenectomized. No patient developed clinical bone disease. The median (range) lyso-Gb1 levels at last visit was 108.5 (8.1-711) ng/mL; lowest for patients with R496H/other and highest for patients refusing therapy. This rather large cohort with long follow-up confirms that mildly affected patients may remain stable for many years without GD-specific therapy. The challenge for the future, when newborn screening may detect all patients, is to be able to predict which of the early diagnosed patients is at risk for disease-related complications and therefore for early treatment, and who may remain asymptomatic or minimally affected with no need for disease-specific therapy., Competing Interests: The SZMC Gaucher Unit receives support from Sanofi/Genzyme for participation in the ICGG Registry, from Shire/Takeda for the GOS Registry, and Pfizer for TALIAS. The Unit also receives research grants from Shire/Takeda and Pfizer. C.C., M.H., S.O., and L.D. are employees of Centogene AG the company that has done the lysoGb1 analysis. A.R. is the founder and CEO of Centogene AG. He has received honoraria and speakers’ fees from Shire/Takeda. A.Z. receives honoraria from Shire/Takeda and Pfizer and consultancy fee from Shire/Takeda and Prevail therapeutics. S.R.-V. receives speakers’ fees and travel support from Shire/Takeda, Pfizer, and Sanofi/Genzyme.

Stable flies (Stomoxys calcitrans Linnaeus, 1758) can have a considerable negative impact on animal well-being, health, and productivity. Since insecticides constitute the mainstay for their control, this study aimed at assessing the occurrence of insecticide resistance in S. calcitrans on dairy farms in Brandenburg, Germany. First, the susceptibility of stable flies from 40 dairy farms to a deltamethrin-impregnated fabric was evaluated using the FlyBox ® field test method. Then, S. calcitrans strains from 10 farms were reared in the laboratory, and the offspring was tested against the adulticides deltamethrin and azamethiphos and the larvicides cyromazine and pyriproxyfen. The FlyBox ® method indicated 100% resistance in stable flies against deltamethrin. Later, to the offspring of those 10 established laboratory strains previously caught on suspected dairy farms, these field findings could be confirmed with mortalities well below 90% 24 h following topical application of the calculated LD 95 of deltamethrin and azamethiphos. The ten strains could therefore be classified as resistant to the tested insecticides. In contrast, exposure to the insect growth regulators cyromazine and pyriproxyfen at their recommended concentrations demonstrated 100% efficacy. Both larvicides inhibited the moulting process of the stable fly larval stages completely, showing that the stable fly strains tested were susceptible to them. The intensive use of insecticides in recent decades has probably promoted the development of insecticide resistance. Systematic surveys in different livestock production systems and vigilance are therefore deemed necessary for estimating the risk of insecticide resistance development on a nationwide scale.

The role of glucosylsphingosine (lyso-Gb1), a downstream metabolic product of glucosylceramide, for monitoring treated and untreated children with Gaucher disease (GD) has not yet been studied. We reviewed the clinical charts of 81 children (<18 years), 35 with mild type 1 GD (GD1), 34 with severe GD1 and 12 with type 3 GD (GD3), followed at Shaare Zedek Medical Center between 2014-2018. Disease severity for GD1 was based on genotypes. Forty children (87%) with severe GD1 and GD3 received enzyme replacement therapy (ERT) compared to two children (6%) with mild GD1. Lyso-Gb1 measurements were conducted on dried blood spot samples taken at each clinic visit. Lyso-Gb1 levels were significantly lower in children with mild compared to severe GD1 ( p = 0.009). In untreated children, lyso-Gb1 levels were inversely correlated with platelet counts. During follow-up, lyso-Gb1 increased in almost 50% of untreated children, more commonly in younger children. In treated children, lyso-Gb1 levels were inversely correlated with hemoglobin levels. The increase of lyso-Gb1 while receiving ERT, seen in eight children, was partly associated with compliance and weight gain. Lyso-Gb1 seems to be a useful biomarker for monitoring children with GD and should be included in the routine follow-up. Progressive increase in lyso-Gb1 levels in untreated children suggests ERT initiation.

Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K 2 HPO 4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC-MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Glucorticoids (GCs) such as dexamethasone (DEX) remain important treatments for Chronic Lymphocytic Leukemia (CLL) but the mechanisms are poorly understood and resistance is inevitable. Proliferation centers (PC) in lymph nodes and bone marrow offer protection against many cytotoxic drugs and circulating CLL cells were found to acquire resistance to DEX-mediated killing in conditions encountered in PCs including stimulation by toll-like receptor agonists and interactions with stromal cells. The resistant state was associated with impaired glucocorticoid receptor-mediated gene expression, autocrine activation of STAT3 through Janus Kinases (JAKs), and increased glycolysis. The JAK1/2 inhibitor ruxolitinib blocked STAT3-phosphorylation and partially improved DEX-mediated killing of stimulated CLL cells in vitro but not in CLL patients in vivo. An automated microscopy-based screen of a kinase inhibitor library implicated an additional protective role for the PI3K/AKT/FOXO pathway. Blocking this pathway with the glycolysis inhibitor 2-deoxyglucose (2-DG) or the PI3K-inhibitors idelalisib and buparlisib increased DEX-mediated killing but did not block STAT3-phosphorylation. Combining idelalisib or buparlisib with ruxolitinib greatly increased killing by DEX. These observations suggest that glucocorticoid resistance in CLL cells may be overcome by combining JAK and PI3K inhibitors.

Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax., (© 2016 by The American Society of Hematology.)

The inhibition of apoptosis enables the survival and proliferation of tumors and contributes to resistance to conventional chemotherapy agents and is therefore a very promising avenue for the development of new agents that will enhance current cancer therapies. The BCL-2 family proteins orchestrate apoptosis at the mitochondria and endoplasmic reticulum and are involved in other processes such as autophagy and unfolded protein response (UPR) that lead to different types of cell death. Over the past decade, significant efforts have been made to restore apoptosis using small molecules that modulate the activity of BCL-2 family proteins. The small molecule ABT-199, which antagonizes the activity of BCL-2, is currently the furthest in clinical trials and shows promising activity in many lymphoid malignancies as a single agent and in combination with conventional chemotherapy agents. Here, we discuss strategies to improve the specificity of pharmacologically modulating various antiapoptotic BCL-2 family proteins, review additional BCL-2 family protein interactions that can be exploited for the improvement of conventional anticancer therapies, and highlight important points of consideration for assessing the activity of small-molecule BCL-2 family protein modulators., (©2015 American Association for Cancer Research.)