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Alzheimer’s disease (AD) is a neurodegenerative disorder accounting for over 50% of all dementia patients and representing a leading cause of death worldwide for the global ageing population. The lack of effective treatments for overt AD urges the discovery of biomarkers for early diagnosis, i.e., in subjects with mild cognitive impairment (MCI) or prodromal AD. The brain is exposed to oxidative stress as levels of reactive oxygen species (ROS) are increased, whereas cellular antioxidant defenses are decreased. Increased ROS levels can damage cellular structures or molecules, leading to protein, lipid, DNA, or RNA oxidation. Oxidative damage is involved in the molecular mechanisms which link the accumulation of amyloid-β and neurofibrillary tangles, containing hyperphosphorylated tau, to microglia response. In this scenario, microglia are thought to play a crucial role not only in the early events of AD pathogenesis but also in the progression of the disease. This review will focus on oxidative damage products as possible peripheral biomarkers in AD and in the preclinical phases of the disease. Particular attention will be paid to biological fluids such as blood, CSF, urine, and saliva, and potential future use of molecules contained in such body fluids for early differential diagnosis and monitoring the disease course. We will also review the role of oxidative damage and microglia in the pathogenesis of AD and, more broadly, in neurodegeneration.

Although extracellular vesicles (EVs) were initially relegated to a waste disposal role, nowadays, they have gained multiple fundamental functions working as messengers in intercellular communication as well as exerting active roles in physiological and pathological processes. Accumulating evidence proves the involvement of EVs in many diseases, including those of the central nervous system (CNS), such as multiple sclerosis (MS). Indeed, these membrane-bound particles, produced in any type of cell, carry and release a vast range of bioactive molecules (nucleic acids, proteins, and lipids), conferring genotypic and phenotypic changes to the recipient cell. This means that not only EVs per se but their content, especially, could reveal new candidate disease biomarkers and/or therapeutic agents. This review is intended to provide an overview regarding current knowledge about EVs’ involvement in MS, analyzing the potential versatility of EVs as a new therapeutic tool and source of biomarkers.

Homozygous mutations in Triggering Receptor Expressed on Myeloid cells 2 gene (TREM2) are one of the major causes of Nasu Hakola Disease (NHD). We analysed Peripheral Blood Mononuclear Cells (PBMC) profile of 164 inflammatory factors in patients with NHD carrying the TREM2 Q33X mutation as compared with heterozygous and wild type individuals. Several molecules related to bone formation and angiogenesis were altered in NHD compared to non-carriers: Bone Morphogenetic Protein (BMP)-1 mRNA levels were significantly increased in PBMC (2.32 fold-increase; P = 0.01), as were Transforming Growth Factor Beta (TGFB)3 levels (1.51 fold-increase; P = 0.02). Conversely, CXCL5 and Pro Platelet Basic Protein (PPBP) were strongly downregulated (−28.26, −9.85 fold-decrease over non-carriers, respectively, P = 0.01), as well as Platelet Factor 4 Variant 1 (PF4V1; −41.44, P = 0.03). Among other inflammatory factors evaluated, Interleukin (IL)-15 and Tumor Necrosis Factor Superfamily Member (TNFSF)4 mRNA levels were decreased in NHD as compared with non-carriers (−2.25 and −3.87 fold-decrease, P = 0.01 and 0.001, respectively). In heterozygous individuals, no significant differences were observed, apart from IL-15 mRNA levels, that were decreased at the same extent as NHD (−2.05 fold-decrease over non-carriers, P = 0.002). We identified a signature in PBMC from patients with NHD consisting of strongly decreased mRNA levels of CXCL5, PPBP, PF4V1, mildly decreased IL-15 and TNFSF4 and mildly increased BMP-1 and TGFB3.

We report the case of two monozygotic twins with Thr272fs mutation in progranulin gene. Both patients developed frontotemporal dementia with 5 years difference in age at onset (Twin 1:73 years, Twin 2:68 years), with early behavioral, language, dysexecutive, and memory problems. They had the same formal education (5 years), but while Twin 1 dedicated more to social and leisure activity, Twin 2 worked all her life. At neuroimaging (MRI for Twin 1 and CT for Twin 2), they both showed asymmetric atrophy with left predominance. The two were discordant for total tau levels in cerebrospinal fluid, neuropsychological testing, and smoking habits. The description of the twins can help identify environmental factors that influence the onset and phenotype of frontotemporal dementia.

LncRNA PCR arrays containing 90 common LncRNAs were used to screen lncRNA expression levels in PBMC from a discovery population of patients with MS. Data from discovery and replications cohorts showed a generalized dysregulation of lncRNA levels in MS patients compared with controls. MALAT1, MEG9, NRON, ANRIL, TUG1, XIST, SOX2OT, GOMAFU, HULC, BACE-1AS were significantly downregulated in MS patients in comparison with controls. Therefore, we performed a validation analysis in an independent cohort of Belgian origin. In this study, NRON and TUG1 downregulations in MS patients compared with controls were confirmed (p ≤ .05 and p ≤ .0001 respectively), whereas considering the other lncRNAs, the statistical threshold was not reached. LncRNAs profiling could thus represent a new challenge in the research of easy detectable biomarkers of disease susceptibility and progression. ispartof: Journal Of Neuroimmunology vol:324 pages:129-135 ispartof: location:Netherlands status: Published online

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of the corticospinal motor neurons, which ultimately leads to death. The repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) represents the most common genetic cause of ALS and it is also involved in the pathogenesis of other neurodegenerative disorders. To offer insights into C9ORF72-mediated pathogenesis, we quantitatively analyzed the proteome of patient-derived primary skin fibroblasts from ALS patients carrying the C9ORF72 mutation compared with ALS patients who tested negative for it. Differentially expressed proteins were identified, used to generate a protein-protein interaction network and subjected to a functional enrichment analysis to unveil altered molecular pathways. ALS patients were also compared with patients affected by frontotemporal dementia carrying the C9ORF72 repeat expansion. As a result, we demonstrated that the molecular pathways mainly altered in fibroblasts (e.g., protein homeostasis) mirror the alterations observed in C9ORF72-mutated neurons. Moreover, we highlighted novel molecular pathways (nuclear and mitochondrial transports, vesicle trafficking, mitochondrial bioenergetics, glucose metabolism, ER-phagosome crosstalk and Slit/Robo signaling pathway) which might be further investigated as C9ORF72-specific pathogenetic mechanisms. Data are available via ProteomeXchange with the identifier PXD023866.

Aquaporin4 (AQP4) is a water channel protein located at astrocyte foot processes that plays a role in glymphatic system, a highly organized fluid transport pathway which seems to be involved in Alzheimer's disease (AD) and normal pressure hydrocephalus (NPH) pathophysiology. Cerebrospinal fluid (CSF) AQP4 levels were determined in 11 patients with AD, 10 patients with NPH, and 9 controls. We found significantly reduced AQP4 in AD patients, a trend in reduction in NPH patients, and a correlation between AQP4 and amyloid-β CSF levels. This study indicates the potential role of AQP4 and glymphatic system in neurodegenerative diseases pathophysiology.

Small extracellular vesicles (EVs) are able to pass from the central nervous system (CNS) into peripheral blood and contain molecule markers of their parental origin. The aim of our study was to isolate and characterize total and neural-derived small EVs (NDEVs) and their micro RNA (miRNA) cargo in Alzheimer&rsquo, s disease (AD) patients. Small NDEVs were isolated from plasma in a population consisting of 40 AD patients and 40 healthy subjects (CTRLs) using high throughput Advanced TaqMan miRNA OpenArrays&reg, which enables the simultaneous determination of 754 miRNAs. MiR-23a-3p, miR-223-3p, miR-100-3p and miR-190-5p showed a significant dysregulation in small NDEVs from AD patients as compared with controls (1.16 &plusmn, 0.49 versus 7.54 &plusmn, 2.5, p = 0.026, 9.32 &plusmn, 2.27 versus 0.66 &plusmn, 0.18, p &lt, 0.0001, 0.069 &plusmn, 0.01 versus 0.5 &plusmn, 0.1, p &lt, 0.0001 and 2.9 &plusmn, 1.2 versus 1.93 &plusmn, 0.9, p &lt, 0.05, respectively). A further validation analysis confirmed that miR-23a-3p, miR-223-3p and miR-190a-5p levels in small NDEVs from AD patients were significantly upregulated as compared with controls (p = 0.008, p = 0.016, p = 0.003, respectively) whereas miR-100-3p levels were significantly downregulated (p = 0.008). This is the first study that carries out the comparison between total plasma small EV population and NDEVs, demonstrating the presence of a specific AD NDEV miRNA signature.

SummaryMyelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34+ CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

Summary Amplification of 1q21 occurs in approximately 30% of de novo and 70% of relapsed multiple myeloma (MM) and is correlated with disease progression and drug resistance. Here, we provide evidence that the 1q21 amplification-driven overexpression of ILF2 in MM promotes tolerance of genomic instability and drives resistance to DNA-damaging agents. Mechanistically, elevated ILF2 expression exerts resistance to genotoxic agents by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65, which promotes mRNA processing and the stabilization of transcripts involved in homologous recombination in response to DNA damage. The intimate link between 1q21-amplified ILF2 and the regulation of RNA splicing of DNA repair genes may be exploited to optimize the use of DNA-damaging agents in patients with high-risk MM.

We explored the molecular mechanisms involved in the establishement of CMA-03/06, an IL-6-independent variant of the multiple myeloma cell line CMA-03 previously generated in our Institution. CMA-03/06 cells grow in the absence of IL-6 with a doubling time comparable with that of CMA-03 cells; neither the addition of IL6 (IL-6) to the culture medium nor co-culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL-6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL-6 independence of CMA-03/06 cells is not apparently due to the development of an autocrine IL-6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA-03/06 cells showed an activated pattern of the NF-κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA-03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA-03/06 cells display a higher sensibility to NF-κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL-6 independence of CMA-03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell-line CMA-03 and its variant CMA-03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL-6-independent growth of myeloma cells.

ZAP-70 is a marker of clinical outcome in chronic lymphocytic leukemia (CLL), however its assessment suffers from a lack of standardization consensus. To identify novel markers able to surrogate IGHV mutational status, CD19(+)CD5(+)-B-lymphocytes from 216 patients enrolled in a prospective study (ClinicalTrial.gov Identifier: NCT00917540 ), underwent gene expression profiling. Samples were split into CLL-Training (n = 102) and CLL-Validation (n = 114) sets, and an independent supervised analysis for IGHV mutational status was performed considering all genes with gene expression equal or above that of ZAP-70. Thirty-one genes (23 up- and 8 down-regulated) and 23 genes (18 up- and 5 down-regulated) satisfied these criteria in the CLL-Training and CLL-Validation sets, respectively, and 20 common genes (15 up and 5 down) were found to be differentially regulated in both sets. Two (SNORA70F, NRIP1) of the down-regulated and 6 (SEPT10, ZNF667, TGFBR3, MBOAT1, LPL, CRY1) of the up-regulated genes were significantly associated with a reduced risk of disease progression in both sets. Forcing the afore-mentioned genes in a Cox multivariate model together with IGHV mutational status, only CRY1 (HR = 2.3, 95% CI: 1.1–4.9, P = .027) and MBOAT1 (HR = 2.1, 95% CI: 1.1–3.7, P = .018) retained their independent prognostic impact, supporting the hypothesis that these genes may potentially act as surrogates for predicting IGHV mutational status.

We explored the molecular mechanisms involved in the establishement of CMA-03/06, an IL-6-independent variant of the multiple myeloma cell line CMA-03 previously generated in our Institution. CMA-03/06 cells grow in the absence of IL-6 with a doubling time comparable with that of CMA-03 cells; neither the addition of IL6 (IL-6) to the culture medium nor co-culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL-6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL-6 independence of CMA-03/06 cells is not apparently due to the development of an autocrine IL-6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA-03/06 cells showed an activated pattern of the NF-κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA-03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA-03/06 cells display a higher sensibility to NF-κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL-6 independence of CMA-03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell-line CMA-03 and its variant CMA-03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL-6-independent growth of myeloma cells.

The 1q21 amplification, which occurs in approximately 40% of de novo and 70% of relapsed MM, is among the most frequent chromosomal aberrations in multiple myeloma (MM) patients and is considered a very high-risk genetic feature that is especially correlated with disease progression and drug resistance. To uncover novel 1q21 MM-critical genes, we first identified a list of 78 potential 1q21 drivers, which were located in the minimal common region of amplification of 254 MM samples and showed copy number-driven expression. These 78 candidates were then subjected to an shRNA screen to identify those genes involved in selective death and/or growth inhibition of MM cells carrying the 1q21 amplification. Using this approach, we identified and functionally validated the Interleukin-2 enhancer binding factor 2 (ILF2) as one of key 1q21 amplification-specific genes. ILF2 downregulation in 1q21-amplified MM cells resulted in multinucleated phenotypes and abnormal nuclear morphologies, findings that are consistent with the DNA damage-induced genomic instability that is associated with DNA repair defects that occur during cellular replication. Correspondingly, ILF2 downregulation was associated with a significant increase in the activation of the ATM (but not ATR or DNA-PK) pathway and accumulation of gH2AX foci, which are indicative of double-strand DNA breaks, and resulted in caspase 3-mediated apoptosis. Therefore, we sought to determine whether ILF2 is involved in the genome damage repair that occurs during cellular replication. To this end, we evaluated whether ILF2 depletion could affect the efficiency of non-homologous end joining (NHEJ) or homologous recombination (HR), the two major repair pathways in mammalian cells. We observed a profound impairment of HR in ILF2-depleted cells (p=0.038), whereas NHEJ was unaltered after ILF2 downregulation. Conversely, enforced ILF2 expression significantly enhanced HR efficiency in MM cells (p=0.008). To further support the role of ILF2 in the regulation of the DNA repair pathway in MM cells, we evaluated whether ILF2 downregulation increased MM sensitivity to DNA-damaging agents routinely used in the treatment of MM. Employing the interstrand crosslinker melphalan as an instigator of double-strand DNA breaks, we found that ILF2-depleted MM cells subjected to continuous melphalan treatment showed increased accumulation of γH2AX and apoptosis. Consistent with these findings, elevated ILF2 expression significantly correlated with poor survival in MM patients treated with high-dose melphalan followed by tandem autologous transplantation (n=256, p=0.01). Mechanistically, mass spectrometry analysis showed that ILF2 interacted with numerous RNA binding proteins directly involved in the regulation of DNA damage response by modulating alternative splicing of specific pre-mRNAs. RNA-sequencing experiments confirmed that ILF2 depletion resulted in aberrant splicing of genes involved in the DNA repair pathway, including ERCC1, FANCD2, and EXO1. RNA immunoprecipitation sequencing experiments showed that ILF2 directly bound to transcripts involved in the regulation of the HR pathway, including components of BRCA1 protein complex. Furthermore, in an attempt to dissect the ILF2 protein interacting network involved in the DNA repair regulation in response to DNA damage activation, we found that ILF2 mediated drug resistance in a dose-dependent manner by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65 to promote mRNA processing and stabilization of DNA repair genes, including FANCD2 and EXO1, in response to DNA damage. In conclusion, our study reveals an intimate relationship among 1q21 amplification, mRNA splicing, and DNA repair in the control of DNA damage response in MM. Given that 1q21 amplification is one of the most frequent copy number alterations in cancer, synthetic lethality approaches based on targeting gain-of-functions associated with ILF2 may have a broad spectrum of applications to potentiate the sensitivity of cancer cells to chemotherapeutic agents. Disclosures Giuliani: Janssen: Research Funding; Celgene: Research Funding.

In the last decade, significant effort has been directed toward the stratification of multiple myeloma (MM) patients for targeted therapy, and many studies have shown that some genetic alterations, especially t(4;14) translocation, loss of the short arm of chromosome 17, and amplification of chromosome 1q21, are associated with a poor outcome. The 1q21 amplicon spans a region of 10-15 Mb and contains a large number of possible candidate genes; it is among the most frequent chromosomal aberrations in patients with MM and is associated with poor prognosis, disease progression, and drug resistance. Therefore, the identification of critical 1q21 genes may yield potential therapeutic targets for this high-risk MM subgroup and provide a rationale for patient stratification. In an effort to accomplish this goal, we first identified a high-priority list of 78 copy number-driven 1q21 MM-relevant genes by integrating high-resolution array comparative genomic hybridization (aCGH) and matched expression profiling of the 254 MM samples deposited in the Multiple Myeloma Research Consortium (MMRC) database. Then, we performed a high-throughput systematic shRNA screen in vitroto identify 1q21 genes whose loss of function resulted in the selective death and/or growth inhibition of MM cells carrying the 1q21 amplification. We used shRNA targeting (excluding shRNAs that displayed cytotoxic activity regardless of 1q21 amplification) and a GFP competitive growth assay to identify 1q21-resident targets whose downregulation significantly decreased the percentage of GFP-positive MM cells with 1q21 amplification over a time of 7 days. These assays identified UBAPL2, INTS3, LASS2, KRTCAP2, and ILF2 as key targets for further analysis. Secondary validation experiments in the MM cell lines JJN3 and H929 confirmed that the downregulation of all of our top five candidate genes induced significant levels of apoptosis, inhibition of proliferation, and cell cycle arrest. Integration of copy number analysis, expression profiling, and clinical outcome indicated that only UBAPL2 and ILF2 were highly significant prognostic genes, and target validation in NOD-SCID mice showed that ILF2, but not UBAPL2, downregulation had a significant impact on in vivo survival. Therefore, we sought to further characterize ILF2’s role in 1q21-amplified MM. ILF2 encodes NF45, the regulatory subunit of NF90/NF110 complexes, which are involved in mitotic control, DNA break repair, and RNA splicing regulation. Downregulation of ILF2 in MM cells with 1q21 amplification resulted in multinucleated phenotypes and abnormal nuclear morphologies (nucleoplasmic bridges and buds and micronuclei) that were associated with a significant accumulation of phospho-H2AX foci and DNA damage response activation, increased sensitivity to the DNA damaging agent melphalan, and impaired activation of DNA repair pathways. Experiments of immunoprecipitation combined with mass spectometry showed that ILF2 interacts with numerous RNA binding proteins directly implicated in DNA repair or regulation of DNA damage response by modulating alternative splicing and stability of specific pre-mRNAs. Accordingly, RNA-seq analysis of ILF2-depleted MM cells, when compared to cells carrying scrambled shRNAs, identified specific changes in RNA splicing patterns both before and after treatment with melphalan. In conclusion, our studies have revealed an unanticipated link between 1q21 amplification, DNA damage response, and RNA splicing. We identified ILF2 as a key driver of this interaction, and our findings support the development of strategies designed to modulate ILF2 expression in patients with high-risk MM carrying 1q21 amplification. Disclosures No relevant conflicts of interest to declare.

Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic neoplastic disorders that are characterized by ineffective myeloid differentiation and dysplasia as well as telomere shortening and accumulated DNA damage in progenitor cells. Less understood is whether DNA damage is the instigator of impaired progenitor cell differentiation and MDS development. Telomerase deficient mice have served as a model system to demonstrate the adverse effects of wide-spread endogenous DNA damage signaling on stem cell function in vivo. In recent studies, we sought to determine whether persistent physiological DNA damage can impair the function of specific hematopoietic lineages by employing the 4-hydroxytamoxifen (OHT)-inducible telomerase reverse transcriptase-estrogen receptor (TERTER) model. For the first time, we demonstrate that late generation TERTER/ER mice with dysfunctional telomeres exhibit hallmark features of MDS, including peripheral blood cytopenias, bone marrow (BM) hyper-cellularity, and an increased myeloid-to-erythroid progenitor ratio in the absence of increased apoptosis. Severe tri-lineage myelodysplasia, and an increase of immature, morphologically abnormal myeloid blasts frequently with pronounced monocytic differentiation were consistent with refractory anemia with excess of blasts (RAEB) or chronic myelo-monocytic leukemia (CMML), a specific sub-group of MDS that is characterized by a high propensity to develop acute myeloid leukemia (AML). Accordingly, approximately 5% of aged TERTER/ER mice progressed to AML, as demonstrated by a marked increase of BM myeloid blasts, and infiltration of myeloid precursors into the splenic white-red pulp architecture, resulting in myeloid sarcoma with the complete effacement of lymphoid follicles. Compared to control mice with intact telomeres, the progenitor compartment of telomere dysfunctional mice shows a significant increase in the number of granulocyte-macrophage progenitors (GMP) with a concomitant loss of the megakaryocyte-erythroid progenitors (MEP) and slight reduction in the number of common myeloid progenitors (CMP), which is consistent with the condition of skewed myeloid differentiation occurring in MDS patients with higher risk of leukemic transformation. Transplantation experiments of long-term hematopoietic stem cells isolated from telomere dysfunctional mice into wild type congenic recipients revealed that the level of donor-derived skewed myeloid differentiation was comparable to that observed at steady state in the same telomere dysfunctional mice before transplantation, suggesting that impaired progenitor differentiation occurred as a result of cell intrinsic defects of telomere dysfunctional hematopoietic cells. In the setting of telomere dysfunction, somatic in vivo and in vitro telomerase reactivation reduced DNA damage signaling and specifically reversed defective differentiation and MDS phenotypes. Unbiased transcriptomic network analyses of CMP with telomere dysfunction revealed profound down-regulation of genes in the mRNA splicing and processing pathways which was rescued by telomerase reactivation, indicating that telomere dysfunction-induced DNA damage response can impact on the expression of genes involved in splicing regulation. RNA-seq analysis of telomere dysfunctional CMP suggested altered splicing activity at the level of exon usage and identified aberrantly spliced variants of genes implicated in chromatin remodeling, and histone modifications. The prominence of aberrantly spliced epigenetic regulators prompted us to hypothesize that there was a link between impaired myeloid differentiation and aberrant splicing patterns as a result of telomere dysfunction-induced repression of splicing components. In conclusion, our studies have revealed an unanticipated link between telomere biology, RNA splicing, and MDS pathogenesis and support the development of strategies designed to modulate the downstream targets of splicing alterations in specific hematopoietic populations. Disclosures No relevant conflicts of interest to declare.