R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation.

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin ß1 Interactomes.

Importin ß1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin ß1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin ß1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.

B-cell antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Safety of intranasal human insulin: A review.

AIMS: To conduct a review in order to assess the safety of intranasal human insulin in clinical studies as well as the temporal stability of nasal insulin sprays. MATERIAL AND METHODS: An electronic search was performed using MEDLINE. We selected original research on intranasal human insulin without further additives in humans. The studies included could be of any design as long as they used human intranasal insulin as their study product. All outcomes and adverse side effects were extracted. RESULTS: A total of 38 studies in 1092 individuals receiving acute human intranasal insulin treatment and 18 studies in 832 individuals receiving human intranasal insulin treatment lasting between 21 days and 9.7 years were identified. No cases of symptomatic hypoglycaemia or severe adverse events (AEs) were reported. Transient local side effects in the nasal area were frequently experienced after intranasal insulin and placebo spray, while other AEs were less commonly reported. There were no reports of participants being excluded as a result of AEs. No instances of temporal stability of nasal insulin were reported in the literature. Tests on insulin that had been repacked into spray flasks showed that it had a chemical stability of up to 57 days. CONCLUSIONS: Our retrospective review of published studies on intranasal insulin did not reveal any safety concerns; however, there were insufficient data to ensure the long-term safety of this method of chronic insulin administration. Improved insulin preparations that cause less nasal irritation would be desirable for future treatment.

Non-alcoholic fatty liver disease and impaired proinsulin conversion as newly identified predictors of the long-term non-response to a lifestyle intervention for diabetes prevention: results from the TULIP study.

AIMS/HYPOTHESIS: Lifestyle intervention is effective to prevent type 2 diabetes. However, a considerable long-term non-response occurs to a standard lifestyle intervention. We investigated which risk phenotypes at baseline and their changes during the lifestyle intervention predict long-term glycaemic non-response to the intervention. METHODS: Of 300 participants at high risk for type 2 diabetes who participated in a 24 month lifestyle intervention with diet modification and increased physical activity, 190 participants could be re-examined after 8.7 ± 1.6 years. All individuals underwent a five-point 75 g OGTT and measurements of body fat compartments and liver fat content with MRI and spectroscopy at baseline, 9 and 24 months during the lifestyle intervention, and at long-term follow-up. Fasting proinsulin to insulin conversion (PI/I ratio) and insulin sensitivity and secretion were calculated from the OGTT. Non-response to lifestyle intervention was defined as no decrease in glycaemia, i.e. no decrease in AUC for glucose at 0-120 min during OGTT (AUCglucose0-120 min). RESULTS: Before the lifestyle intervention, 56% of participants had normal glucose regulation and 44% individuals had impaired fasting glucose and/or impaired glucose tolerance. At long-term follow-up, 11% had developed diabetes. Multivariable regression analysis with adjustment for age, sex, BMI and change in BMI during the lifestyle intervention revealed that baseline insulin secretion and insulin sensitivity, as well as change in insulin sensitivity during the lifestyle intervention, predicted long-term glycaemic control after 9 years. In addition, increased hepatic lipid content as well as impaired fasting proinsulin conversion at baseline were newly detected phenotypes that independently predicted long-term glycaemic control. CONCLUSIONS/INTERPRETATION: Increased hepatic lipid content and impaired proinsulin conversion are new predictors, independent of change in body weight, for non-response to lifestyle intervention in addition to the confirmed factors, impaired insulin secretion and insulin sensitivity.

B cell receptor signaling and associated pathways in the pathogenesis of chronic lymphocytic leukemia.

B cell antigen receptor (BCR) signaling is a key driver of growth and survival in both normal and malignant B cells. Several lines of evidence support an important pathogenic role of the BCR in chronic lymphocytic leukemia (CLL). The significant improvement of CLL patients' survival with the use of various BCR pathway targeting inhibitors, supports a crucial involvement of BCR signaling in the pathogenesis of CLL. Although the treatment landscape of CLL has significantly evolved in recent years, no agent has clearly demonstrated efficacy in patients with treatment-refractory CLL in the long run. To identify new drug targets and mechanisms of drug action in neoplastic B cells, a detailed understanding of the molecular mechanisms of leukemic transformation as well as CLL cell survival is required. In the last decades, studies of genetically modified CLL mouse models in line with CLL patient studies provided a variety of exciting data about BCR and BCR-associated kinases in their role in CLL pathogenesis as well as disease progression. BCR surface expression was identified as a particularly important factor regulating CLL cell survival. Also, BCR-associated kinases were shown to provide a crosstalk of the CLL cells with their tumor microenvironment, which highlights the significance of the cells' milieu in the assessment of disease progression and treatment. In this review, we summarize the major findings of recent CLL mouse as well as patient studies in regard to the BCR signalosome and discuss its relevance in the clinics.

The Small GTPase RHOA Links SLP65 Activation to PTEN Function in Pre B Cells and Is Essential for the Generation and Survival of Normal and Malignant B Cells.

The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA-deficient mice. The RhoA-deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA-deficient cells with RhoA or Foxo1, a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function.

Functionalization of Enzymatically Treated Apple Pomace from Juice Production by Extrusion Processing.

Food by-products can be used as natural and sustainable food ingredients. However, a modification is needed to improve the technofunctional properties according to the specific needs of designated applications. A lab-scale twin-screw extruder was used to process enzymatically treated apple pomace from commercial fruit juice production. To vary the range of the thermomechanical treatment, various screw speeds (200, 600, 1000 min-1), and screw configurations were applied to the raw material. Detailed chemical and functional analyses were performed to develop a comprehensive understanding of the impact of the extrusion processing on apple pomace composition and technofunctional properties as well as structures of individual polymers. Extrusion at moderate thermomechanical conditions increased the water absorption, swelling, and viscosity of the material. An increase in thermomechanical stress resulted in a higher water solubility index, but negatively affected the water absorption index, viscosity, and swelling. Scanning electron microscopy showed an extrusion-processing-related disruption of the cell wall. Dietary fiber analysis revealed an increase of soluble dietary fiber from 12.6 to 17.2 g/100 g dry matter at maximum thermo-mechanical treatment. Dietary fiber polysaccharide analysis demonstrated compositional changes, mainly in the insoluble dietary fiber fraction. In short, pectin polysaccharides seem to be susceptible to thermo-mechanical stress, especially arabinans as neutral side chains of rhamnogalacturonan I.

Extrusion Processing of Pure Chokeberry (Aronia melanocarpa) Pomace: Impact on Dietary Fiber Profile and Bioactive Compounds.

The partial substitution of starch with dietary fiber (DF) in extruded ready-to-eat texturized (RTE) cereals has been suggested as a strategy to reduce the high glycemic index of these food products. Here, we study the impact of extrusion processing on pure chokeberry (Aronia melanocarpa) pomace powder (CPP) rich in DF and polyphenols (PP) focusing on the content and profile of the DF fractions, stability of PP, and techno-functional properties of the extrudates. Using a co-rotating twin-screw extruder, different screw speeds were applied to CPP with different water contents (cw), which resulted in specific mechanical energies (SME) in the range of 145-222 Whkg-1 and material temperatures (TM) in the range of 123-155 °C. High molecular weight soluble DF contents slightly increase with increasing thermomechanical stress up to 16.1 ± 0.8 g/100 g dm as compared to CPP (11.5 ± 1.2 g/100 g dm), but total DF (TDF) contents (58.6 ± 0.8 g/100 g dm) did not change. DF structural analysis revealed extrusion-based changes in the portions of pectic polysaccharides (type I rhamnogalacturonan) in the soluble and insoluble DF fractions. Contents of thermolabile anthocyanins decrease linearly with SME and temperature from 1.80 ± 0.09 g/100 g dm in CPP to 0.24 ± 0.06 g/100 g dm (222 Whkg-1, 155 °C), but phenolic acids and flavonoids appear to be largely unaffected. Resulting techno-functional (water absorption and water solubility) and physical properties related to the sensory characteristics (expansion, hardness, and color) of pure CPP extrudates support the expectation that granulated CPP extrudates may be a suitable food ingredient rich in DF and PP.

The Influence of Extrusion Processing on the Gelation Properties of Apple Pomace Dispersions: Involved Cell Wall Components and Their Gelation Kinetics.

By-products of fruits and vegetables like apple pomace can serve as techno-functional ingredients in foods. Due to their physicochemical properties, e.g., viscosity, water absorption, or oil-binding, food by-products can modify the texture and sensory perception of products like yogurts and baked goods. It is known that, by extrusion processing, the properties of by-products can be altered. For example, by thermo-mechanical treatment, the capacity of food by-products to increase viscosity is improved. However, the mechanism and involved components leading to the viscosity increase are unknown. Therefore, the complex viscosity of apple pomace dispersions and the involved fractions as pectin (a major part of the water-soluble fraction), water-soluble and water-insoluble fraction, were measured. In the investigated range, an increase in the pectin yield and water solubility was observed with increasing thermo-mechanical treatment by extrusion processing. However, pectin and water-soluble cell wall components had only a limited effect on the complex viscosity of apple pomace dispersions. The insoluble fraction (particles) were investigated regarding their swelling behavior and influence on the complex viscosity. An intensification of thermo-mechanical treatment resulted in increasing swelling behavior.

Modification of Apple Pomace by Extrusion Processing: Studies on the Composition, Polymer Structures, and Functional Properties.

By-products of fruit and vegetable processing are an inexpensive and sustainable source of dietary fiber, potentially offering valuable functional properties such as water binding and thickening. Due to these favorable properties, they can be utilized to reformulate widely-consumed foods, e.g., bakery products or beverages. In this study, apple pomace was used as a model system to study whether extrusion technology affects food by-product functionality and thus has the potential to broaden the application of by-products in foods. The effect of the process parameters and the extent of thermo-mechanical treatment on the structural and functional properties of apple pomace were analyzed after extrusion trials using various screw speeds, water contents, and barrel temperatures. Compared to the raw material, apple pomace extruded at Tbarrel = 100 °C, n = 700 min-1 and mH2O = 17% showed an increased water solubility up to 33%. The water absorption increased from 5 to 19 Pa·s and the paste viscosity from 5 to 339 Pa·s by extrusion processing. Analyses of dietary fiber contents and fiber polysaccharide structures revealed that thermo-mechanical stress (n = 700 min-1, mH2O = 22%) increased the content of soluble dietary fiber from 12.5 to 16.7 g/100 g dry matter, and that the harshest conditions even enabled the formation of low-molecular-weight dietary fiber. Arabinans (as neutral rhamnogalacturonan I side chains) appeared to be most sensitive to thermo-mechanical stress, whereas xylans (i.e., a group of minor polysaccharides) were an example of a more stable fiber polysaccharide. Also, the degree of methylation of the pectic polysaccharides was strongly reduced from 50% to 15% when thermo-mechanical stress was applied. Imaging and pore size analysis showed that extrusion processing could disrupt the rigid cell wall macromolecular structure.

Impact of defined thermomechanical treatment on the structure and content of dietary fiber and the stability and bioaccessibility of polyphenols of chokeberry (Aronia melanocarpa) pomace.

Dietary fiber is a potential replacement for other ingredients such as starch in reformulated extruded breakfast cereals. Analysis of chokeberry pomace powder revealed a total dietary fiber content of 57.8 ± 2 g/100 g with 76% being insoluble, 20% high molecular soluble and 4% low molecular soluble dietary fiber. The fiber polysaccharide composition was analyzed in detail by using a variety of analytical approaches. Extrusion-like processing conditions were studies in a Closed Cavity Rheometer enabling the application of defined thermal (temperature range 100-160 °C) and mechanical treatments (shear rates between 0.1 s-1 and 50 s-1) to chokeberry pomace powder. Application of temperatures up to 140 °C irrespective of the mechanical treatment does not remarkably alter dietary fiber structure or content, but reduces the initial content of total polyphenols by about 40% to a final content of 3.3 ± 0.5 g/100 g including 0.63 ± 0.1 g/100 g of anthocyanins, 0.18 ± 0.02 g/100 g of phenolic acids and 0.090 ± 0.007 g/100 g of flavonols, respectively. The retained polyphenols are fully bioaccessible after in vitro digestion, and antioxidant capacity remains unchanged as compared to the untreated pomace powder. Glucose bioaccessibility remains unaffected, whereas glucose content is reduced. It is concluded that chokeberry pomace powder is a good source of dietary fiber with the potential to partially substitute starch in extruded breakfast cereals.

Synergism between IL7R and CXCR4 drives BCR-ABL induced transformation in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Ph+ acute lymphoblastic leukemia (ALL) is characterized by the expression of an oncogenic fusion kinase termed BCR-ABL1. Here, we show that interleukin 7 receptor (IL7R) interacts with the chemokine receptor CXCR4 to recruit BCR-ABL1 and JAK kinases in close proximity. Treatment with BCR-ABL1 kinase inhibitors results in elevated expression of IL7R which enables the survival of transformed cells when IL7 was added together with the kinase inhibitors. Importantly, treatment with anti-IL7R antibodies prevents leukemia development in xenotransplantation models using patient-derived Ph+ ALL cells. Our results suggest that the association between IL7R and CXCR4 serves as molecular platform for BCR-ABL1-induced transformation and development of Ph+ ALL. Targeting this platform with anti-IL7R antibody eliminates Ph+ ALL cells including those with resistance to commonly used ABL1 kinase inhibitors. Thus, anti-IL7R antibodies may provide alternative treatment options for ALL in general and may suppress incurable drug-resistant leukemia forms.

Androgen receptor overexpression in prostate cancer in type 2 diabetes.

OBJECTIVE: While prostate cancer does not occur more often in men with diabetes, survival is markedly reduced in this patient group. Androgen signaling is a known and major driver for prostate cancer progression. Therefore, we analyzed major components of the androgen signaling chain and cell proliferation in relation to type 2 diabetes. METHODS: Tumor content of 70 prostate tissue samples of men with type 2 diabetes and 59 samples of patients without diabetes was quantified by an experienced pathologist, and a subset of 51 samples was immunohistochemically stained for androgen receptor (AR). mRNA expression of AR, insulin receptor isoform A (IR-A) and B (IR-B), IGF-1 receptor (IGF1R), Cyp27A1 and Cyp7B1, PSA gene KLK3, PSMA gene FOLH1, Ki-67 gene MKI67, and estrogen receptor beta (ESR2) were analyzed by RT-qPCR. RESULTS: AR mRNA and protein expression were associated with the tumor content only in men with diabetes. AR expression also correlated with downstream targets PSA (KLK3) and PSMA (FOLH1) and increased cell proliferation. Only in diabetes, AR expression was correlated to higher IR-A/IR-B ratio and lower IR-B/IGF1R ratio, thus, in favor of the mitogenic isoforms. Reduced Cyp27A1 and increased Cyp7B1 expressions in tumor suggest lower levels of protective estrogen receptor ligands in diabetes. CONCLUSIONS: We report elevated androgen receptor signaling and activity presumably due to altered insulin/IGF-1 receptors and decreased levels of protective estrogen receptor ligands in prostate cancer in men with diabetes. Our results reveal new insights why these patients have a worse prognosis. These findings provide the basis for future clinical trials to investigate treatment response in patients with prostate cancer and diabetes.

FTO Genotype Interacts with Improvement in Aerobic Fitness on Body Weight Loss During Lifestyle Intervention.

OBJECTIVE: Not every participant responds with a comparable body weight loss to lifestyle intervention, despite the same compliance. Genetic factors may explain parts of this difference. Variation in fat mass and obesity-associated gene (FTO) is the strongest common genetic determinant of body weight. The aim of the present study was to evaluate the impact of FTO genotype differences in the link between improvement of fitness and reduction of body weight during a lifestyle intervention. METHODS: We genotyped 292 healthy subjects for FTO rs8050136. Participants underwent a 9-month lifestyle intervention. Before and after intervention, aerobic fitness was tested by bicycle (VO2max) and treadmill spiroergometry (individual anaerobic threshold (IAT), subgroup of N = 192). RESULTS: Participants lost body weight (p < 0.0001) independent of FTO genotype (p = 0.5). There was a significant correlation between improvement in VO2max and decrease in body weight (p < 0.0001). FTO genotype interacted with this relationship (p = 0.0042 for VO2max, p = 0.0049 for IAT). When stratifying the cohort according to their improvement in VO2max, FTO obesity-risk A-allele carriers in the higher quartiles of improvement in fitness lost significantly less body weight. CONCLUSIONS: Our data reveal that genetic variation in FTO impacts on body weight reduction during lifestyle intervention only in subjects with marked improvement in aerobic fitness.

TGF-ß Contributes to Impaired Exercise Response by Suppression of Mitochondrial Key Regulators in Skeletal Muscle.

A substantial number of people at risk of developing type 2 diabetes could not improve insulin sensitivity by physical training intervention. We studied the mechanisms of this impaired exercise response in 20 middle-aged individuals at high risk of developing type 2 diabetes who performed 8 weeks of controlled cycling and walking training at 80% individual Vo2 peak. Participants identified as nonresponders in insulin sensitivity (based on the Matsuda index) did not differ in preintervention parameters compared with high responders. The failure to increase insulin sensitivity after training correlates with impaired upregulation of mitochondrial fuel oxidation genes in skeletal muscle, and with the suppression of the upstream regulators PGC1α and AMPKα2. The muscle transcriptomes of the nonresponders are further characterized by the activation of transforming growth factor (TGF)-ß and TGF-ß target genes, which is associated with increases in inflammatory and macrophage markers. TGF-ß1 as inhibitor of mitochondrial regulators and insulin signaling is validated in human skeletal muscle cells. Activated TGF-ß1 signaling downregulates the abundance of PGC1α, AMPKα2, the mitochondrial transcription factor TFAM, and mitochondrial enzymes. Thus, the data suggest that increased TGF-ß activity in skeletal muscle can attenuate the improvement of mitochondrial fuel oxidation after training and contribute to the failure to increase insulin sensitivity.

High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage.