Chronic lymphocytic leukemia (CLL) is a dynamic malignancy in which intraclonal subfractions differ in activation history and responsiveness to microenvironmental signals. Here, we investigated the expression and inducibility of IL-12 family receptor subunits (IL-23R, IL-12Rβ1, IL-12Rβ2) and the related receptor complexes in recirculating CLL cells, with a focus on CXCR4/CD5-defined fractions: the proliferative fraction (PF; CXCR4dim/CD5bright; most recently divided, tissue-emigrated cells) and the resting fraction (RF; CXCR4bright/CD5dim; older, quiescent cells). At baseline, IL-12Rβ1 was enriched in the PF and was associated with a higher proportion of cells expressing IL-23R and IL-12R receptor complexes. Concomitantly, RT-qPCR disclosed higher IL-12Rβ1 mRNA levels. Following antigen-independent activation with CpG or CpG + IL-15, there was a marked increase in IL-23R and IL-12Rβ1 but not in IL-12Rβ2 surface expression, resulting in preferential upregulation of the IL-23R complex over the IL-12R complex. Fraction-specific analyses showed stronger induction of IL-23R and IL-23R complex expression in PF compared with RF. These findings identify an intraclonal bias toward IL-23 responsiveness in the CLL cells with a phenotype of recently divided, tissue-emigrated cells and suggest the IL-23/IL-23R axis as a potential therapeutic target.

Background/Objectives: The immunoglobulin heavy-chain variable (IGHV) gene repertoire represents a characteristic feature of chronic lymphocytic leukemia (CLL), although its configuration is not well defined at the early disease stages. The IGHV repertoire of a cohort of early CLL patients was analyzed and compared to that of a "real-world" reference cohort. Methods: Patients from the O-CLL1 observational protocol, which enrolled only Binet stage A cases within twelve months from diagnosis, were studied. IGHV/IGHJ rearrangements were sequenced and annotated following ERIC recommendations, and stereotyped subsets were assigned using ARResT/AssignSubsets. The repertoire features were compared with the dataset of a real-world cohort of patients with heterogeneous staging (CTR cohort) and with published early-diagnosis series. Results: IGHV and IGHJ gene distributions and HCDR3-length profiles in O-CLL1 closely mirrored those of CTR, indicating that the BcR IG repertoire at diagnosis is already defined rather than being selected during disease progression. Mutated IGHV (M-CLL) predominated, with a frequency of stereotyped BcR IG comparable to that of other early-diagnosis cohorts. However, within this conserved framework, subset #4 was over-represented among M-CLL from O-CLL without an increased overall IGHV4-34 gene usage, suggestive of a selective expansion rather than a recombinational bias. Subset #4 cases retained canonical HCDR3 motifs and showed time-to-first-treatment like other M-CLL, likely reflecting the younger age structure of O-CLL1. Conclusions: Early-diagnosis CLL displays a biased IGHV repertoire with stereotyped configurations characteristic of CLL, including subsets that are rare in the normal B-cell repertoire. These findings support a central role for antigen-driven selection in shaping CLL evolution.

Although chronic lymphocytic leukemia (CLL) is diagnosed by identifying a circulating B-cell clone that exceeds 5x103/μL, additional distinct clones (ADC) have been identified in various studies. Notably, the numbers of ADC documented in these studies has increased as the various technologies evolved. To better define the frequency and the characteristics of ADC in CLL, we used a next-generation sequencing platform that affords high sequencing depth along with steps that limit overcounting to analyze IGHV-IGHD-IGHJ gene rearrangements in circulating CD5+ B cells from 57 patients. Notably, all patients had at least one ADC, in addition to the clinically relevant clone. In 46 patients for whom lymphocyte count data were available, 44 had at least one ADC above the threshold of 1 B cell/μL and, remarkably, the average number of ADC was 12 per patient. Notably, in two patients, the predominant ADC qualified clinically as a separate CLL clone and in the remaining cases as low/high-count monoclonal B-cell lymphocytosis clones. Moreover, in 11 patients studied longitudinally, predominant ADC were persistent and often increased in number. ADC in patients with CLL exhibited 4-fold more stereotyped IGHV-IGHD-IGH rearrangements than found in CD5+ B cells from healthy individuals, and IGHV use, somatic mutations, and Ig isotype distribution were similar between predominant ADC and clinically relevant clones. Thus, finding multiple expanded clones within the CD5+ B cells is the rule in patients with CLL, indicating that leukemogenesis is a multiclonal process that likely involves competition among B cells with special B-cell receptor features.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties . We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Abstract Although chronic lymphocytic leukemia (CLL) is diagnosed by identifying in a patient’s blood a B-cell clone that exceeds 5x106/µL, additional clones have on occasion been identified. To characterize such clones in patients with CLL, we analyzed by NGS the IGHV-IGHD-IGHJ gene rearrangements in circulating CD5+ B cells from 57 untreated patients. In every patient, we identified additional distinct clonotypes (ADCs) from the clinically relevant clone (CRC). Even when defining clonal expansion using a rigorous threshold, 68% of patients had at least one expanded ADC. The predominant ADC (pADC) reached numbers qualifying as a CLL clone in two patients and qualifying as low/high-count monoclonal B-cell lymphocytosis in each other case. Over time, pADCs enlarged or shrank, suggesting an ongoing selection process. Stereotyped IGHV-D-J rearrangements were enriched in ADCs and IGHV usage and mutations revealed similarities specifically between pADCs and CRCs, while minor ADCs more closely resembled normal CD5+ B-cells. Thus, finding multiple expanded clones within the CD5+ B-cell population of patients with CLL is more the rule rather than the exception. Thus, leukemogenesis involves a competitive selection for dominance of more than one B-cell clone, with the process being influenced to some degree by the structure of the B-cell receptor.

Chronic Lymphocytic Leukemia (CLL) is caused by the clonal expansion of CD5+ B lymphocytes in the circulation, peripheral lymphoid organs and bone marrow [...]

Recently, cases of fortuitous discovery of Chronic Lymphocytic Leukemia (CLL) during hospitalization for Coronavirus disease (COVID-19) have been reported. These patients did not show a monoclonal B cell expansion before COVID-19 but were diagnosed with CLL upon a sudden lymphocytosis that occurred during hospitalization. The (hyper)lymphocytosis during COVID-19 was also described in patients with overt CLL disease. Contextually, lymphocytosis is an unexpected phenomenon since it is an uncommon feature in the COVID-19 patient population, who rather tend to experience lymphopenia. Thus, lymphocytosis that arises during COVID-19 infection is a thought-provoking behavior, strikingly in contrast with that observed in non-CLL individuals. Herein, we speculate about the possible mechanisms involved with the observed phenomenon. Many of the plausible explanations might have an adverse impact on these CLL patients and further clinical and laboratory investigations might be desirable.

The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

Additional file 2. Identification details of the proteins examined in this study. In the second sheet, protein identification details are given, such as protein band, protein accession and description, protein theoretical mass and pI values, protein score, protein matches, protein sequence coverage (%) and protein emPAI value. In the third sheet, peptide identification details are given, among which peptide rank, peptide experimental m/z value, peptide experimental Mr. value, peptide charge, peptide delta score, peptide score and peptide sequence.

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10–30 μM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process.

SH3BGRL3 is a gene belonging to SH3BGR family, it is ubiquitously expressed and encodes for a 93 AA thiorerodoxin-like protein evolutionarily conserved. A proteomic study reported that SH3BGRL3 binds the cytoplasmatic domain of ERBB2 receptor. On this basis we performed immuno-staining experiments in FLAG-SH3BGRL3 transfected SKBR3 cell line that showed SH3BGRL3 and ERBB2 co-localization. Nonetheless, co-immunoprecipitation (Co-IP) of ERBB2 using FLAG-SH3BGRL3 as bait and vice versa was not achievable. Therefore, to investigate SH3BGRL3 potential interactors we performed Co-IP experiments from SKBR3 lysates transfected with FLAG-SH3BGRL3 followed by mass spectrometry analysis. The results revealed myosin 1c (Myo1c) as a candidate interactor. Subsequent Co-IP experiments followed by WB analysis validated the interaction between the two proteins. To map the interaction site we performed Co-IP experiments using SKBR3 cells co-transfected with FLAG-SH3BGRL3 and HA tagged deletion mutants of Myo1c that showed SH3BGRL3 binding to the neck region of Myo1c. Since Myo1c neck region binds calmodulin in a Ca2+ dependent way, we assessed if the binding was Ca2+ dependent also for SH3BGRL3. The experiments showed that SH3BGRL3 binds the Myo1c neck in presence of Ca2+, differently from calmodulin that binds it in absence of Ca2+. Myo1c is a motor protein involved, among its different functions, in cell membrane dynamics. Thus we investigated SH3BGRL3 involvement in cell migration using MDA-MB-231 cell line. We transfected MDA-MB-231 cells with FLAG-SH3BGRL3 and performed immuno-staining and Co-IP experiments that showed co-localization and interaction of Myo1c and SH3BGRL3. Accordingly, we performed migration assays using boyden chambers after silencing or not SH3BGRL3 expression by means siRNAs. The results showed a statistically significant decrease in migration capacity of silenced cells respect to controls. Our data show that SH3BGRL3 binds Myo1c neck region in a Ca2+ dependent way and that this interaction is involved in cell migration in our model.

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation in the blood, bone marrow and lymphoid tissues of monoclonal B lymphocytes with a distinctive phenotype characterized by the expression of CD5 and CD23 antigens and a low expression of surface Immunoglobulins (sIG). The course of the disease is heterogeneous with some patients show- ing an aggressive form and a second group with a more stable disease; these groups are well defined by the absence or presence of somatic mutation on the IGHV gene, respectively. In CLL clones the rearrangements of the B-cell Receptor (BCR) IGV regions exhibit distinc- tive features. In spite of the enormous diversity that can be created in the IGHV region up to 30% of CLL clones exhibit very similar (stereotyped) BCR which have been categorized in Sub- sets. Of these, 19 are most represented therefore are defined as “major Subsets”. The identifica- tion of stereotyped receptors is codified on the basis of HCDR3 features in the context of the use of certain IGHV genes. The above observations sustain the notion of an antigen drive role in CLL ontogeny. In addition, stimulation via BCR appears to be critical for leukemic cell survival/proliferation as suggested by the possibility to inhibit CLL growth by modern inhibitors of BCR-associat- ed kinases. Furthermore, has been reported existence of a BCR homotypic interaction between BCR epitopes leading to receptor de-clustering and allowing the activation of autonomous sign- aling. Thus, this homotypic BCR–BCR interaction provides a further mechanism of CLL cell stimulation. Whether CLL-like stereotyped BCR are present in the B-cell repertoire of healthy subjects remains largely unexplored. We addressed this issue in splenic B lymphocyte subpopulations and used a high throughput sequencing technique to collect IGV gene rearranged sequences. In particular, we separated and analyzed follicular mantle B cells (sIgDbright IgM+ CD38-CD27-), germinal center (GC) B cells (CD38+, sIgD-, CD24-), marginal zone B cells (sIgDlow, sIgM+, CD38-) and switched memory B cells (sIgD-sIgM-CD38-). In addition, we focus on IGV rear- rangements using IGHV1 family genes because these are the most represented among the major subsets identified in CLL. Our study shows that CLL stereotyped receptors can be traced in a sizeable proportion of most of splenic B-cell subpopulations indicating that these receptors are part of the normal B-cell repertoire.

Recent studies suggest that the commonly prescribed anti-osteoporosis drugs bisphosphonates (BPs) might also exhibit antitumor activity. We investigated a possible anticancer effect of BPs on B-chronic lymphocytic leukemia (CLL) cells obtained from peripheral blood of 26 CLL patients. Zoledronate, etidronate and clodronate were administered in vitro simultaneously to following activation stimuli: i) CD40L-expressing fibroblasts, ii) soluble recombinant CD40L produced in our laboratory +IL-4, iii) CpG ODN 2006+IL-15 with or without bone marrow stromal cells (BMSC). CLL cell viability, activation/proliferation were monitored by flow cytometry. We unexpectedly observed that BPs generated a protective effect from spontaneous apoptosis in 11/26 (42%) patients (viability + 18%-392%) and an augmentation in CLL cell activation/proliferation in 61% of the samples (S+G2M phase: +100%±25). Interestingly, protection from spontaneous apoptosis or increment of cell activation, required the presence of either fibroblasts, BMSC or autologous Nurse Like Cells (NLC). We thus hypothesized that supportive cells are involved in the BPs effects either through cell-cell interactions with leukemic cells or T cells, or through soluble factors release in the medium. Functional experiments with transwells suggest that stromal cells, in presence of Clodronate, release soluble factors in the medium that may probably concur to the unexpected Clodronate-mediated enhancement of CLL cell activation/proliferation. This work is in progress and several critical questions on the mechanisms are still unanswered. Nevertheless, the phenomenological data argue that caution should be taken when administering BPs against osteoporosis in elderly persons, who could have Monoclonal B Lymphocytosis or CLL.

Chronic lymphocytic leukemia (CLL) cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. We previously demonstrated that the antidiabetic drug metformin, known to also inhibit oxidative phosphorylation (OXPHOS), inhibits cell cycle entry of leukemic cells derived ex-vivo from the peripheral blood of CLL patients and stimulated in vitro by cell culture systems that recreate a microenvironment to drive their proliferation (Bruno et al, Oncotarget, 2015). However, overtly proliferating CLL cells were resistant to the cytostatic effects of metformin. Since metformin switched the energetic metabolism of activated, not yet proliferating, CLL cells from OXPHOS to accelerated glycolysis, in the present study we asked whether combining metformin with glycolysis impairment could inhibit also proliferating CLL cells. Still, CLL cells recovered from a transitory block and rescued in vitro proliferation. What kind of energetic reprogramming was involved in the resistance of proliferating CLL cells to glucose utilization? Recent studies highlight on the role of fatty acid utilization of CLL cells. We asked 1) whether inhibitors of lipid metabolism could impair proliferation of in vitro stimulated CLL cells; 2) whether impairing glucose energetic pathways could act synergistically with beta oxidation inhibitors.We found that inhibitors of critical steps of fatty acid metabolisms, such as carnitine-palmitoyl transferase 1A (CPT1A) -rate-limiting enzyme for fatty acid import into mitochondria- or Peroxisome Proliferator-Activated Receptor (PPAR)-alpha -regulator of beta-oxidation- administered at clinically achievable doses, were ineffective on quiescent CLL cells and on CLL cells stimulated by the microenvironment during the first stages of activation. Conversely, remarkable susceptibility to undergo apoptosis was observed at later stages of cell activation and during overt proliferation. Synergism with impairment of other energetic pathways occurred depending on the stage of activation of the in vitro stimulated CLL cells.The results suggest that energetic metabolic pathways could be relevant targets for CLL treatment, provided that the complex metabolic reprogramming network during the transition of leukemic cells from quiescence to proliferation, and back, are clearly elucidated. This work was supported by grants from AIRC IG15426.

Crucial for CLL development and progression are iterative cycles of cell re-activation and proliferation in lymphoid tissues. Since cellular fatty acid (FA) import and oxidation (FAO) were recently reported to be upregulated in CLL, we explored in-vitro effects of ST1326, a reversible inhibitor of carnitine-palmitoyl transferase 1A (CPT1A), on leukemic cells subject to activating microenvironment-mimicking stimuli. ST1326 induced dose-dependent mitochondrial dysfunction and cell death, which were remarkably higher in activated/proliferating than quiescent CLL cells. Drug sensitivity was observed irrespective of the presence of TP53 alterations or chromosomal abnormalities. ST1326 cytotoxicity was associated with decreased levels of intracellular acetylCoA and phosphorylated STAT3, known to favor CLL cell proliferation and upregulate antiapoptotic Bcl-2 family members. Indeed, rising of Mcl-1 and Bcl-xl expression in response to microenvironment stimulation was impaired by ST1326. Drug combination experiments with the BH3-mimetic ABT-199/Venetoclax, whose effects are counteracted by Mcl-1/Bcl-xl and cell proliferation, showed strong ST1326-mediated potentiation of ABT-199 cytotoxicity in activated/proliferating CLL cells. The data indicate that CLL cells turning to an activated/proliferating state become more dependent on FAO and more sensitive to FAO-antagonists, and pave the way for ST1326 as an adjuvant tool in anti-CLL drug-combination regimens with drugs that loose efficacy on proliferating leukemic cells.

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease.1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients.Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts.At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival-and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis.In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules.Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

SUMMARY B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in human adults of the western world and no definitive cure is yet available. One key factor in CLL pathogenesis and disease progression is misbalanced Bcl-2 cell death machinery that is shifted towards protection from apoptosis. Thus, strategies to counteract the antiapoptotic action of the Bcl-2 family in CLL cells are being explored. The Bcl-2 family is composed of a growing number of proteins related to Bcl-2 by sequence homology and their interactions regulate the cell’s decision to die. The features of one particular subclass, the BH3-only proteins, are being studied and exploited for the development of therapeutic anticancer approaches that specifically target antiapoptotic Bcl-2 proteins overexpressed in tumors, including CLL. Preclinical and clinical efficacy and toxicity of the most effective among these ‘BH3 mimetics’ are presented, together with a model that accounts for the differential sensitivity of CLL and normal cells to Bcl-2 neutralization.