B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

Optimal entropic properties of SARS-CoV-2 RNA sequences.

The reaction of the scientific community against the COVID-19 pandemic has generated a huge (approx. 106 entries) dataset of genome sequences collected worldwide and spanning a relatively short time window. These unprecedented conditions together with the certain identification of the reference viral genome sequence allow for an original statistical study of mutations in the virus genome. In this paper, we compute the Shannon entropy of every sequence in the dataset as well as the relative entropy and the mutual information between the reference sequence and the mutated ones. These functions, originally developed in information theory, measure the information content of a sequence and allows us to study the random character of mutation mechanism in terms of its entropy and information gain or loss. We show that this approach allows us to set in new format known features of the SARS-CoV-2 mutation mechanism like the CT bias, but also to discover new optimal entropic properties of the mutation process in the sense that the virus mutation mechanism track closely theoretically computable lower bounds for the entropy decrease and the information transfer.

Elemental Fingerprinting Combined with Machine Learning Techniques as a Powerful Tool for Geographical Discrimination of Honeys from Nearby Regions.

Discrimination of honey based on geographical origin is a common fraudulent practice and is one of the most investigated topics in honey authentication. This research aims to discriminate honeys according to their geographical origin by combining elemental fingerprinting with machine-learning techniques. In particular, the main objective of this study is to distinguish the origin of unifloral and multifloral honeys produced in neighboring regions, such as Sardinia (Italy) and Spain. The elemental compositions of 247 honeys were determined using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The origins of honey were differentiated using Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA), and Random Forest (RF). Compared to LDA, RF demonstrated greater stability and better classification performance. The best classification was based on geographical origin, achieving 90% accuracy using Na, Mg, Mn, Sr, Zn, Ce, Nd, Eu, and Tb as predictors.

Thermal degradation of red cabbage (Brassica oleracea L. var. Capitata f. rubra) anthocyanins in a water model extract under accelerated shelf-life testing.

Red cabbage (RC) represents a source of anthocyanins acylated with hydroxycinnamic acids (HCA) that are described to enhance their stability. Nevertheless, data about their thermal degradation are still controversial. Our aim was to comprehensively analyse the degradation kinetics of individual RC anthocyanins in a model aqueous extract treated at 40 °C × 30 days to simulate severe but realistic storage conditions. Free anthocyanins and radical-scavenging capacity showed different kinetics. The results confirm the high stability of RC anthocyanins (t1/2: 16.4-18.4 days), although HPLC analyses of each molecule displayed distinct kinetics with t1/2 from 12.6 to 35.1 days. In particular, the sinapoyl acylation negatively affected the stability of the anthocyanins, while the forms monoacylated with glycosyl p-coumaric and ferulic acids exhibited higher stability. In conclusion, our results indicate that acylation is not a prerogative of stability, as this is instead more dependent on specific acylation patterns and the glycosylation of HCA.

Nanoencapsulation of Anthocyanins from Red Cabbage (Brassica oleracea L. var. Capitata f. rubra) through Coacervation of Whey Protein Isolate and Apple High Methoxyl Pectin.

Encapsulation is a valuable strategy to protect and deliver anthocyanins (ACNs), phenolic compounds with outstanding antioxidant capacity but limited stability. In this study, coacervation was used to encapsulate an ACN-rich red cabbage extract (RCE). Two agri-food by-product polymers, whey protein isolate (WPI) and apple high-methoxyl pectin (HMP), were blended at pH 4.0 in a specific ratio to induce the formation of nanoparticles (NPs). The process optimisation yielded a monodispersed population (PDI < 0.200) of negatively charged (-17 mV) NPs with an average diameter of 380 nm. RCE concentration influenced size, charge, and antioxidant capacity in a dose-dependent manner. NPs were also sensitive to pH increases from 4 to 7, showing a progressive breakdown. The encapsulation efficiency was 30%, with the retention of ACNs within the polymeric matrix being influenced by their chemical structure: diacylated and/or C3-triglucoside forms were more efficiently encapsulated than monoacylated C3-diglucosides. In conclusion, we report a promising, simple, and sustainable method to produce monodispersed NPs for ACN encapsulation and delivery. Evidence of differential binding of ACNs to NPs, dependent on specific acylation/glycosylation patterns, indicates that care must be taken in the choice of the appropriate NP formulation for the encapsulation of phenolic compounds.

The rs1001179 SNP and CpG methylation regulate catalase expression in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an extremely variable clinical course. We have recently shown that high catalase (CAT) expression identifies patients with an aggressive clinical course. Elucidating mechanisms regulating CAT expression in CLL is preeminent to understand disease mechanisms and develop strategies for improving its clinical management. In this study, we investigated the role of the CAT promoter rs1001179 single nucleotide polymorphism (SNP) and of the CpG Island II methylation encompassing this SNP in the regulation of CAT expression in CLL. Leukemic cells harboring the rs1001179 SNP T allele exhibited a significantly higher CAT expression compared with cells bearing the CC genotype. CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-ß transcription factors. Moreover, CLL cells exhibited lower methylation levels than normal B cells, in line with the higher CAT mRNA and protein expressed by CLL in comparison with normal B cells. Methylation levels at specific CpG sites negatively correlated with CAT levels in CLL cells. Inhibition of methyltransferase activity induced a significant increase in CAT levels, thus functionally validating the role of CpG methylation in regulating CAT expression in CLL. Finally, the CT/TT genotypes were associated with lower methylation and higher CAT levels, suggesting that the rs1001179 T allele and CpG methylation may interact in regulating CAT expression in CLL. This study identifies genetic and epigenetic mechanisms underlying differential expression of CAT, which could be of crucial relevance for the development of therapies targeting redox regulatory pathways in CLL.

Spray-drying Microencapsulation of an Extract from Tilia tomentosa Moench Flowers: Physicochemical Characterization and in Vitro Intestinal Activity.

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36-47% compared to MD12 and + 18-24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products.

Phospho-Specific Flow Cytometry Reveals Signaling Heterogeneity in T-Cell Acute Lymphoblastic Leukemia Cell Lines.

Several signaling pathways are aberrantly activated in T-ALL due to genetic alterations of their components and in response to external microenvironmental cues. To functionally characterize elements of the signaling network in T-ALL, here we analyzed ten signaling proteins that are frequently altered in T-ALL -namely Akt, Erk1/2, JNK, Lck, NF-κB p65, p38, STAT3, STAT5, ZAP70, Rb- in Jurkat, CEM and MOLT4 cell lines, using phospho-specific flow cytometry. Phosphorylation statuses of signaling proteins were measured in the basal condition or under modulation with H2O2, PMA, CXCL12 or IL7. Signaling profiles are characterized by a high variability across the analyzed T-ALL cell lines. Hierarchical clustering analysis documents that higher intrinsic phosphorylation of Erk1/2, Lck, ZAP70, and Akt, together with ZAP70 phosphorylation induced by H2O2, identifies Jurkat cells. In contrast, CEM are characterized by higher intrinsic phosphorylation of JNK and Rb and higher responsiveness of Akt to external stimuli. MOLT4 cells are characterized by higher basal STAT3 phosphorylation. These data document that phospho-specific flow cytometry reveals a high variability in intrinsic as well as modulated signaling networks across different T-ALL cell lines. Characterizing signaling network profiles across individual leukemia could provide the basis to identify molecular targets for personalized T-ALL therapy.

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01.

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite ( SeO 3 2 - ) into the less bioavailable elemental selenium (Se0) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of SeO 3 2 - exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with SeO 3 2 - . The results show substantial biochemical changes in SeITE01 cells when exposed to SeO 3 2 - . The initial uptake of SeO 3 2 - by SeITE01 cells (3h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against SeO 3 2 - are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of SeO 3 2 - to Se0. In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from SeO 3 2 - toxicity while it is converting into SeNPs.

Effects of Combination Treatments with Astaxanthin-Loaded Microparticles and Pentoxifylline on Intracellular ROS and Radiosensitivity of J774A.1 Macrophages.

Radiation-induced fibrosis (RIF) is a serious, yet incurable, complication of external beam radiation therapy for the treatment of cancer. Macrophages are key cellular actors in RIF because of their ability to produce reactive oxidants, such as reactive oxygen species (ROS) and inflammatory cytokines that, in turn, are the drivers of pro-fibrotic pathways. In a previous work, we showed that phagocytosis could be exploited to deliver the potent natural antioxidant astaxanthin specifically to macrophages. For this purpose, astaxanthin encapsulated into µm-sized protein particles could specifically target macrophages that can uptake the particles by phagocytosis. In these cells, astaxanthin microparticles significantly reduced intracellular ROS levels and the secretion of bioactive TGFß and increased cell survival after radiation treatments. Here we show that pentoxifylline, a drug currently used for the treatment of muscle pain resulting from peripheral artery disease, amplifies the effects of astaxanthin microparticles on J774A.1 macrophages. Combination treatments with pentoxifylline and encapsulated astaxanthin might reduce the risk of RIF in cancer patients.

Phagocytosis of Astaxanthin-Loaded Microparticles Modulates TGFß Production and Intracellular ROS Levels in J774A.1 Macrophages.

Radiation-induced fibrosis is a serious long-lasting side effect of radiation therapy. Central to this condition is the role of macrophages that, activated by radiation-induced reactive oxygen species and tissue cell damage, produce pro-inflammatory cytokines, such as transforming growth factor beta (TGFß). This, in turn, recruits fibroblasts at the site of the lesion that initiates fibrosis. We investigated whether astaxanthin, an antioxidant molecule extracted from marine and freshwater organisms, could help control macrophage activation. To this purpose, we encapsulated food-grade astaxanthin from Haematococcus pluvialis into micrometer-sized whey protein particles to specifically target macrophages that can uptake material within this size range by phagocytosis. The data show that astaxanthin-loaded microparticles are resistant to radiation, are well-tolerated by J774A.1 macrophages, induce in these cells a significant reduction of intracellular reactive oxygen species and inhibit the release of active TGFß as evaluated in a bioassay with transformed MFB-F11 fibroblasts. Micro-encapsulation of bioactive molecules is a promising strategy to specifically target phagocytic cells and modulate their own functions.

Effects of CD20 antibodies and kinase inhibitors on B-cell receptor signalling and survival of chronic lymphocytic leukaemia cells.

Recently, clinical trial results have established inhibitors of B-cell receptor (BCR)-associated kinase (BAKi), with or without CD20 moniclonal antibodies (mAbs), as the preferred first-line treatment for most chronic lymphocytic leukaemia (CLL) patients. Using phosphospecific flow cytometry, we showed that in leukaemic cells from CLL patients the CD20 therapeutic antibodies - rituximab, ofatumumab, and obinutuzumab - inhibited BCR signalling pathways targeting preferentially pBTKY551 - but not BTKY223 - and pAKT. On the contrary, ibrutinib and idelalisib reduced pBTKY223 to a higher extent than pBTKY551 . The strong reduction of pAKT induced by idelalisib was enhanced by its combination with rituximab or ofatumumab. Moreover, CD20 mAbs and BAKi induced the death of leukaemia cells that was significantly potentiated by their combination. Analysis of the enhancement of cell death in these combinations revealed an approximately additive enhancement induced by rituximab or obinutuzumab combined with ibrutinib or idelalisib. Taken together, our data identified negative regulatory effects of CD20 mAbs and their combinations with BAKi on BCR signalling and cell survival in CLL. In conclusion, this study advances our understanding of mechanisms of action of CD20 mAbs as single agents or in combination with BAKi and could inform on the potential of combined therapies in ongoing and future clinical trials in patients with CLL.

The control of acidity in tumor cells: a biophysical model.

Acidosis of the tumor microenvironment leads to cancer invasion, progression and resistance to therapies. We present a biophysical model that describes how tumor cells regulate intracellular and extracellular acidity while they grow in a microenvironment characterized by increasing acidity and hypoxia. The model takes into account the dynamic interplay between glucose and [Formula: see text] consumption with lactate and [Formula: see text] production and connects these processes to [Formula: see text] and [Formula: see text] fluxes inside and outside cells. We have validated the model with independent experimental data and used it to investigate how and to which extent tumor cells can survive in adverse micro-environments characterized by acidity and hypoxia. The simulations show a dominance of the [Formula: see text] exchanges in well-oxygenated regions, and of [Formula: see text] exchanges in the inner hypoxic regions where tumor cells are known to acquire malignant phenotypes. The model also includes the activity of the enzyme Carbonic Anhydrase 9 (CA9), a known marker of tumor aggressiveness, and the simulations demonstrate that CA9 acts as a nonlinear [Formula: see text] equalizer at any [Formula: see text] level in cells that grow in acidic extracellular environments.

Oxygen in the Tumor Microenvironment: Mathematical and Numerical Modeling.

There are many reasons to try to achieve a good grasp of the distribution of oxygen in the tumor microenvironment. The lack of oxygen - hypoxia - is a main actor in the evolution of tumors and in their growth and appears to be just as important in tumor invasion and metastasis. Mathematical models of the distribution of oxygen in tumors which are based on reaction-diffusion equations provide partial but qualitatively significant descriptions of the measured oxygen concentrations in the tumor microenvironment, especially when they incorporate important elements of the blood vessel network such as the blood vessel size and spatial distribution and the pulsation of local pressure due to blood circulation. Here, we review our mathematical and numerical approaches to the distribution of oxygen that yield insights both on the role of the distribution of blood vessel density and size and on the fluctuations of blood pressure.

Fine-grained simulations of the microenvironment of vascularized tumours.

One of many important features of the tumour microenvironment is that it is a place of active Darwinian selection where different tumour clones become adapted to the variety of ecological niches that make up the microenvironment. These evolutionary processes turn the microenvironment into a powerful source of tumour heterogeneity and contribute to the development of drug resistance in cancer. Here, we describe a computational tool to study the ecology of the microenvironment and report results about the ecology of the tumour microenvironment and its evolutionary dynamics.

Planar AFM macro-probes to study the biomechanical properties of large cells and 3D cell spheroids.

The ability to measure mechanical response of cells under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Living cells have been mechanically investigated by several approaches. Among them, atomic force microscopy (AFM) is widely used thanks to its high versatility and sensitivity. In the case of large cells or 3D multicellular aggregates, standard AFM probes may not be appropriate to investigate the mechanical properties of the whole biological system. Owing to their size, standard AFM probes can compress only a single somatic cell or part of it. To fill this gap, we have designed and fabricated planar AFM macro-probes compatible with commercial AFM instruments. The probes are constituted of a large flat compression plate, connected to the chip by two flexible arms, whose mechanical characteristics are tuned for specific biological applications. As proof of concept, we have used the macro-probes to measure the viscoelasticity of large spherical biological systems, which have a diameter above 100 µm: human oocytes and 3D cell spheroids. Compression experiments are combined with visual inspection, using a side-view configuration imaging, which allows us to monitor the sample morphology during the compression and to correlate it with the viscoelastic parameters. Our measurements provide a quantitative estimate of the relaxation times of such biological systems, which are discussed in relation to data present in literature. The broad applicability of the AFM macro-probes can be relevant to study the biomechanical features in any biological process involving large soft materials. STATEMENT OF SIGNIFICANCE: The understanding of the role of physical factors in defining cell and tissue functions requires to develop new methods or improve the existing ones to accurately measure the biomechanical properties. AFM is a sensitive and versatile tool to measure the mechanical features from single proteins to single cells. When cells or cell aggregates exceed few tens of microns, AFM suffers from limitations. On these biological systems the control of the contact area and the application of a precise uniform compression becomes crucial. A modification of the standard cantilevers fabrication allowed us obtaining AFM macro-probes, having large planar contact area and spring constant suitable for biological investigations. They were demonstrated valuable to characterize the mechanical properties of large hierarchical biological systems.

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein.

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.

Dynamical Detection of Boundaries and Cavities in Biophysical Cell-Based Simulations of Growing Tumor Tissues.

Cell-based lattice-free simulations of the growth of tumor tissues require the definition of geometrical and topological relations among cells and the other basic elements of the simulation (most notably the local and the global environments). This is necessary for the correct description of the biochemistry of tumor tissues, and to implement the biomechanical interactions among cells. Weak cell-cell forces and the necrosis of tumor tissues due to poor vascularization can lead to the formation of cavities - i.e., regions without viable cells and filled with cellular debris and fluids. It is important to give an accurate geometrical/topological description of the resulting microenvironment that plays an important role in the pathology of cancer. In this paper, we concentrate on simulations of the growth of avascular solid tumors and we describe the STAR (Shape of Tumors from Algorithmic Reconstruction) algorithm that defines the shape of clusters of cells and searches for the boundary and cavities in a 3D environment. The algorithm is GPU-based and exploits the high degree of parallelism of GPUs. The final implementation achieves a 30-fold speedup with respect to a previous CPU-based version.

Effects of microencapsulation by ionic gelation on the oxidative stability of flaxseed oil.

Flaxseed oil is a major source of omega-3 polyunsaturated fatty acids (PUFAs), as it contains nearly 50% of alpha-linolenic acid. For this reason it is highly susceptible to auto-oxidation. The aim of the work was to increase the stability of flaxseed oil by a microencapsulation process based on ionic gelation through vibrating-nozzle extrusion technology, using pectin as shell material. Two different drying systems, passive air drying (AD) and fluid bed (FB), were compared. The results show that the encapsulation efficiency is very high (up to 98%). Besides being approximately 20-fold faster, FB gives beads showing on average higher payload (76% vs 68%) and lower peroxide value (9.64 vs 21.33) than the AD. An accelerated test carried out on FB-dried beads shows that the oxidative stability of encapsulated oil is 13-fold higher than bulk oil (PV FB: 20 vs PV oil: 260), demonstrating the protecting effect of microencapsulation.

Low catalase expression confers redox hypersensitivity and identifies an indolent clinical behavior in CLL.

B-cell receptor (BCR) signaling is a key determinant of variable clinical behavior and a target for therapeutic interventions in chronic lymphocytic leukemia (CLL). Endogenously produced H2O2 is thought to fine-tune the BCR signaling by reversibly inhibiting phosphatases. However, little is known about how CLL cells sense and respond to such redox cues and what effect they have on CLL. We characterized the response of BCR signaling proteins to exogenous H2O2 in cells from patients with CLL, using phosphospecific flow cytometry. Exogenous H2O2 in the absence of BCR engagement induced a signaling response of BCR proteins that was higher in CLL with favorable prognostic parameters and an indolent clinical course. We identified low catalase expression as a possible mechanism accounting for redox signaling hypersensitivity. Decreased catalase could cause an escalated accumulation of exogenous H2O2 in leukemic cells with a consequent greater inhibition of phosphatases and an increase of redox signaling sensitivity. Moreover, lower levels of catalase were significantly associated with a slower progression of the disease. In leukemic cells characterized by redox hypersensitivity, we also documented an elevated accumulation of ROS and an increased mitochondrial amount. Taken together, our data identified redox sensitivity and metabolic profiles that are linked to differential clinical behavior in CLL. This study advances our understanding of the redox and signaling heterogeneity of CLL and provides the rationale for the development of therapies targeting redox pathways in CLL.