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Supplementary Figure and Table Legends from GROα Is Highly Expressed in Adenocarcinoma of the Colon and Down-Regulates Fibulin-1

Supplementary Table 1 from GROα Is Highly Expressed in Adenocarcinoma of the Colon and Down-Regulates Fibulin-1

Supplementary Figure B from GROα Is Highly Expressed in Adenocarcinoma of the Colon and Down-Regulates Fibulin-1

Purpose: The growth-related oncogene α (GROα) is a secreted interleukin-like molecule that interacts with the CXCR2 G-protein–coupled receptor. We found that the mRNA and protein products of GROα are more highly expressed in neoplastic than normal colon epithelium, and we studied potential mechanisms by which GROα may contribute to tumor initiation or growth.Experimental Design: Cell lines that constitutively overexpress GROα were tested for growth rate, focus formation, and tumor formation in a xenograft model. GROα expression was determined by Affymetrix GeneChip (241 microdissected colon samples), real-time PCR (n = 32), and immunohistochemistry. Primary colon cancer samples were also employed to determine copy number changes and loss of heterozygosity related to the GROα and fibulin-1 genes.Results: In cell cultures, GROα transfection transformed NIH 3T3 cells, whereas inhibition of GROα by inhibitory RNA was associated with apoptosis, decreased growth rate, and marked up-regulation of the matrix protein fibulin-1. Forced expression of GROα was associated with decreased expression of fibulin-1. Expression of GROα mRNA was higher in primary adenocarcinomas (n = 132), adenomas (n = 32), and metastases (n = 52) than in normal colon epithelium (P < 0.001). These results were confirmed by real-time PCR and by immunohistochemistry. Samples of primary and metastatic colon cancer showed underexpression of fibulin-1 when compared with normal samples. There were no consistent changes in gene copy number of GROα or fibulin-1, implying a transcriptional basis for these findings.Conclusion: Elevated expression of GROα is frequent in adenocarcinoma of the colon and is associated with down-regulation of the matrix protein fibulin-1 in experimental models and in clinical samples. GROα overexpression abrogates contact inhibition in cell culture models, whereas inhibition of GROα expression is associated with apoptosis. Importantly, coexpression of fibulin-1 with GROα abrogates key aspects of the transformed phenotype, including tumor formation in a murine xenograft model. Targeting GRO proteins may provide new opportunities for treatment of colon cancer.

Purpose: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers.Experimental Design: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays.Results: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively).Conclusion: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.

CCR Translation on this Article from CpG Island Methylator Phenotype Associates with Low-Degree Chromosomal Abnormalities in Colorectal Cancer

Supplementary Information from The Signatures of Autozygosity among Patients with Colorectal Cancer

Previous studies have shown that among populations with a high rate of consanguinity, there is a significant increase in the prevalence of cancer. Single nucleotide polymorphism (SNP) array data (Affymetrix, 50K XbaI) analysis revealed long regions of homozygosity in genomic DNAs taken from tumor and matched normal tissues of colorectal cancer (CRC) patients. The presence of these regions in the genome may indicate levels of consanguinity in the individual's family lineage. We refer to these autozygous regions as identity-by-descent (IBD) segments. In this study, we compared IBD segments in 74 mostly Caucasian CRC patients (mean age of 66 years) to two control data sets: (a) 146 Caucasian individuals (mean age of 80 years) who participated in an age-related macular degeneration (AMD) study and (b) 118 cancer-free Caucasian individuals from the Framingham Heart Study (mean age of 67 years). Our results show that the percentage of CRC patients with IBD segments (≥4 Mb length and 50 SNPs probed) in the genome is at least twice as high as the AMD or Framingham control groups. Also, the average length of these IBD regions in the CRC patients is more than twice the length of the two control data sets. Compared with control groups, IBD segments are found to be more common among individuals of Jewish background. We believe that these IBD segments within CRC patients are likely to harbor important CRC-related genes with low-penetrance SNPs and/or mutations, and, indeed, two recently identified CRC predisposition SNPs in the 8q24 region were confirmed to be homozygous in one particular patient carrying an IBD segment covering the region. [Cancer Res 2008;68(8):2610–21]

Epigenetic modulation of gene expression is essential for tissue-specific development and maintenance in mammalian cells. Disruption of epigenetic processes, and the subsequent alteration of gene functions, can result in inappropriate activation or inhibition of various cellular signaling pathways, leading to cancer. Recent advancements in the understanding of the role of epigenetics in cancer initiation and progression have uncovered functions for DNA methylation, histone modifications, nucleosome positioning, and non-coding RNAs. Epigenetic therapies have shown some promise for hematological malignancies, and a wide range of epigenetic-based drugs are undergoing clinical trials. However, in a dynamic survival strategy, cancer cells exploit their heterogeneous population which frequently results in the rapid acquisition of therapy resistance. Here, we describe novel approaches in drug discovery targeting the epigenome, highlighting recent advances the selective degradation of target proteins using Proteolysis Targeting Chimera (PROTAC) to address drug resistance.

Detection of low-abundance mutations in cell-free DNA (cfDNA) is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR-LDR-qPCR assay capable of detecting point mutations at a single-molecule resolution in the presence of an excess of wild-type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer-binding regions as well as wild-type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single-well version of the assay detected 2 to 5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild-type human genomic DNA (hgDNA) from buffy coat. A 12-well (pixel) version of the assay was capable of single-molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000–2,250 GE of wild-type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.

Objective: Salivary gland epithelial cells (SGECs) are key cellular drivers in the pathogenesis of primary Sjögren's syndrome (SS); however, the mechanisms sustaining SGEC activation in primary SS remain unclear. We undertook this study to determine the role of autophagy in the survival and activation of SGECs in primary SS. Methods: Primary SGECs isolated from the minor SGs of patients with primary SS or sicca syndrome were evaluated by flow cytometry, immunoblotting, and immunofluorescence to assess autophagy (autophagic flux, light chain 3 IIB [LC3-IIB], p62, LC3-IIB+/lysosome-associated membrane protein 1 [LAMP-1] staining), apoptosis (annexin V/propidium iodide [PI], caspase 3), and activation (intercellular adhesion molecule, vascular cell adhesion molecule). Focus score and germinal center presence were assessed in the SGs from the same patients to assess correlation with histologic severity. Human SG (HSG) cells were stimulated in vitro with peripheral blood mononuclear cells (PBMCs) and serum from primary SS patients in the presence or absence of autophagy inhibitors to determine changes in autophagy and epithelial cell activation. Results: SGECs from primary SS patients (n=24) exhibited increased autophagy (autophagic flux [P=0.001]; LC3-IIB [P=0.02]; p62 [P=0.064]; and as indicated by LC3-IIB/LAMP-1+ staining), increased expression of antiapoptotic molecules (Bcl-2 [P=0.006]), and reduced apoptosis (annexin V/PI [P=0.002]; caspase 3 [P=0.057]), compared to samples from patients with sicca syndrome (n=16). Autophagy correlated with histologic disease severity. In vitro experiments on HSG cells stimulated with serum and PBMCs from primary SS patients confirmed activation of autophagy and expression of adhesion molecules, which was reverted upon pharmacologic inhibition of autophagy. Conclusion: In primary SS SGECs, inflammation induces autophagy and prosurvival mechanisms, which promote SGEC activation and mirror histologic severity. These findings indicate that autophagy is a central contributor to the pathogenesis of primary SS and a new therapeutic target.

BackgroundThe quality of sexual life (QSL) is a complex and multimodal experience influenced by endogenous and external factors, including age, gender, and cultural environment. Rheumatic diseases, with their burden of pain, fatigue, organ damage, and disability, can severely impair sexual life and this is true also for Sjögren’s Syndrome, where simple tools to assess the QSL in everyday clinical practice are needed.ObjectivesTo translate ad adapt into Italian the Qualisex, a new brief questionnaire originally created for Rheumatoid Arthritis patients, for women with primary Sjögren’s Syndrome (pSS) and evaluate the impact of the disease on their sexuality.MethodsConsecutive sexually active pSS (according to ACR/EULAR 2016 criteria) patients aged >18 were asked to participate in this study approved by the local bioethics committee. With the permission of the developer, the French original version of the Qualisex questionnaire (consisting of 10 items, the higher the score, the greater the negative impact of the disease on the QSL) was translated and adapted into Italian according to current guidelines. In the absence of a gold standard assessment for sexuality in pSS, face and content validity was assessed cross-sectionally by correlations with other disease aspects such as anxiety and depression measured by Hospital Anxiety and Depression Scale (HADS), EULAR SS patient-reported index (ESSPRI), and quality of the relationship. As a measure of reliability, internal consistency was assessed through Cronbach’s Alpha coefficient. A Cronbach’s value >0.7 is generally regarded as satisfactory. The feasibility of the scale was indirectly assessed through missing data. To assess the factorial structure of the Italian version of the questionnaire an Exploratory Factor Analysis (EFA) was carried out. Moreover we also assessed the level of redundancy by means of intra-item correlation of the Qualisex questionnaire. ESSDAI (EULAR Sjögren’s Syndrome Disease Activity Index), and SSDDI (Sjogren’s Syndrome Disease Damage Index) were assessed as well. Analyses were carried out with IBM SPSS Statistics for Macintosh, version 22.0 (IBM Corp., Armonk, NY, USA).Results40 sexually active women with pSS and a median age of 53 (IQR 45.25-57.25) were enrolled. The EFA showed that the model with a single factor appeared to be highly significant (Chi235= 2943.10; pConclusionThe Italian version of the Qualisex questionnaire is a valid, reliable and useful tool to assess the quality of sexual life in pSS. QSL in pSS women has an inverse relationship with age, menopause, drug use, ESSPRI, mood disorders, and dissatisfaction with the partner, while, as previously reported, no correlation was found with disease activity, damage, and educational status. This further highlights the impact of subjective symptoms such as dryness, pain, fatigue, and the overall psychological well-being on patients’ life. Thus, it is critical for the physician to consider patients’ perspective.Disclosure of InterestsNone declared

BackgroundAlthough salivary gland epithelial cells (SGECs) are key players in the pathogenesis of autoimmune epithelitis that characterizes primary Sjӧgren’s Syndrome (pSS), the mechanisms sustaining SGECs activation in pSS remain largely undetermined. Therefore, therapeutic strategies to counteract SGECs activation in pSS are also lacking. In previous studies, we revealed that maladaptive autophagy sustains survival and pro-inflammatory activation of SGECs in pSS (1).ObjectivesTo determine the therapeutic potential of JAK/STAT inhibition with baricitinib to restore homeostatic regulation of SGECs in pSS, by reducing autophagy, survival, and expression of adhesion molecules.MethodsPrimary SGECs were isolated from minor salivary glands (SG) of large cohort of patients with pSS or sicca syndrome and subjected to mechanistic and functional studies including flow-cytometry, immunoblotting, and immunofluorescence to assess autophagy (autophagic-flux, LC3IIB, p62, LC3B+/LAMP1+ staining), apoptosis (annexin V/PI, Caspase-3) and activation (ICAM, VCAM). Focus score and germinal centers were determined in homologous SG biopsies to assess correlations of findings with histological disease severity. Primary SGECs of patients with pSS were treated with Baricitinib (1 mM) for 24 prior to assessment of autophagy, apoptosis and activation.ResultsSGECs from pSS patients (n=29) exhibited increased autophagy (as determined by autophagic-flux p=0.001; LC3IIB p=0.02; p62 p=0.064; LC3IIB/LAMP1+ staining), increased expression of anti-apoptotic molecules (Bcl2 p=0.006), and reduced apoptosis (Annexin-V/PI p=0.002, Caspase-3 p=0.057) compared to sicca (n=16). Induction of autophagy in pSS SGECs correlated with histologic disease severity. Treatment of pSS SGECs with baricitinib ex vivo suppressed autophagy, increased apoptosis, and reduced expression of adhesion molecules.ConclusionSGECs in the inflammatory milieu of pSS are characterized by induction of autophagy and pro-survival mechanisms, and by expression of adhesion molecules. These changes correlate with SG infiltration with immune cells and with histologic disease severity. Among clinically available therapies, the JAK/STAT inhibitor baricitinib effectively reduced autophagy, countered the state of maladaptive activation of SGECs, and restored epithelial cell homeostasis. Transcriptomics and metabolomics studies are ongoing to dissect the specific mechanisms responsible for these beneficial effects.References[1]Colafrancesco S, et al. Maladaptive autophagy in the pathogenesis of autoimmune epithelitis in Sjӧgren’s Syndrome. Arthritis Rheumatol 2021.Disclosure of InterestsSerena Colafrancesco Speakers bureau: NovartisSobi, Grant/research support from: Eli Lilly, cristiana barbati: None declared, Roberta Priori: None declared, Federico Giardina: None declared, angelica gattamelata: None declared, raffaella izzo: None declared, Bruna Cerbelli: None declared, Carla Giordano: None declared, Susanna Scarpa: None declared, Massimo Fusconi: None declared, Francesca Romana Spinelli Speakers bureau: Eli LillyPfizerAbbvie, Giulio Cavalli: None declared, cristiano alessandri: None declared, Fabrizio Conti: None declared

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination plays a crucial role as pivotal strategies to curb the coronavirus disease-19 (COVID-19) pandemic. Despite the mass-scale vaccination, literature data about the incidence of disease flares in IIM patients are still not reported as well as the immunological condition.ObjectivesThe present study aimed to describe the clinical status of patients affected by IIM after vaccination against COVID19 in order to assess the number of relapses or immune-mediated reactions in a cohort of Italian patients with such disease.MethodsWe included all patients affected by IIM and followed by Myositis Clinic, Rheumatology and Respiratory Diseases Units, Siena University Hospital, Bari University Hospital, Policlinico Umberto I, Sapienza University, Rome, and Policlinico Paolo Giaccone, Palermo. Inclusion criteria were a recent (ResultsA total of 119 IIM patients (median, IQR 58 (47-66) years; 32 males) were consecutively enrolled. Fifty had a diagnosis of DM, 39 had PM and 30 had ASS. The median months of disease duration was 79.62±83.98. According to number of organs involvement, forty-two had only one, 45 had two organs involvement, 20 had three, 11 had four and one had five. The majority of them received two doses of COVID-19 vaccine, except four patients who refused the vaccination: 94 (78.9%) Cominarty, 16 (13.4%) Moderna, 5 (0.04%) AZ. Seven (0.06%) patients had flare after vaccination, the majority of them were mild except one major with three organs involved and one life-threatening with systemic involvement. In order to understand or predict the effect of demographic and clinical features on the flare development after vaccination, a logistic regression analysis was performed. The goodness-of-fit statistics showed a Chi2 associated with the Log ratio (L.R.) of 0.045. From the probability associated with the Chi-square tests, the Type II analysis showed the variable that most influences the development of flare was the number of organs involved (p=0.047).Sixty-eight patients received the third dose of COVID-19 vaccination: 51 (75%) Cominarty and 17 (25%) Moderna. Only one (0.01%) patient (the same who had life-threatening flare with systemic involvement after two doses) had flare after third dose and eventually died.ConclusionVaccines against SARS-CoV2 have provided, both in registratory studies and in preliminary real-life evidence, an overall good efficacy and safety. Nevertheless, only scanty data are available for rheumatic patients in general and the ones affected by IIM in particular. To the best of our knowledge, ours represent the largest cohort of IIM patients in which immunogenicity of anti-SARS-CoV2 vaccine was assessed. In line with real-life data from other diseases, we found a non-statistically significant risk of relapse in our patients, which occurred seldom, usually mild and in patients with a more severe and aggressive course of disease.ParametersFlare after two doses (n=7)No-flare after two doses (n=108)P valueAge (years)55 (51-68)59 (47-67)NSGender (M/F)2/530/82NSDiagnosis (DM/PM/ASS)2/2/348/36/28NSAntibodiesJo1225PL7-3PL12-1Ku-2Mi217PM/Scl15Ro5217TIG1g-5MDA5-6SRP-1SAE-2cN1a--NPX-1SSA-12Ds-DNA-1ANA (only positivity)-3negative227Length of disease (months)50 (19-200)60 (24-108)NSNumber of organs involved:One0360.0004Two243Three319Four110Five10Type of vaccination:Cominarty688NSModerna115AZ05Disease activity (PhGA≥2/PhGA3/427/81NSMDI3 (1-6.5)2 (1-4)NSCRP (mg/dL)0.1 (0.01-0.3)0.99 (0.3-2.9)0.0041ESR32 (14-39)15.5 (8-27.5)NSCPK111 (63-905)97.5 (63-158)NSTreatment at time of vaccination: GCs010NS Immunosuppressive319 Biologic12 Combination365 no-treatment-12Disclosure of InterestsNone declared

BackgroundVaccinations against SARS-CoV-2 represent a fundamental tool in controlling the pandemic. To date, data on the safety of anti-SARS-CoV-2 vaccines in patients with rare rheumatic diseases, such as systemic vasculitis, are limited.ObjectivesIn this study we aimed at evaluating the safety of anti-SARS-CoV-2 vaccines in a multicentric cohort of patients with systemic vasculitis.MethodsPatients with systemic vasculitis from two Rheumatology centres who had received anti-SARS-CoV-2 vaccine were retrospectively examined. The primary outcome was to evaluate, in this multi-centric cohort, the occurence of a disease flare after the administration of the vaccine, defined as development of clinical manifestations related to vasculitis with a concomitant increase in serum inflammatory markers. As a secondary outcome we aimed at evaluating, in a monocentric cohort of patients with vasculitis, the occurrence of adverse events (AEs) following vaccine administration compared to healthy controls (HC).ResultsWe examined 111 patients with systemic vasculitis (n=69 female, n=42 male), with a mean age of 64.3 (± 13) years. Sixty had ANCA-associated vasculitis (AAV), fourty-two had Giant-Cell Arterities (GCA), five had Periarteritis Nodosa, four had Takayasu’s arteritis. One-hundred and five patients received a mRNA vaccine and six a viral vector one. A disease flare occurred in only 2 patients (1.8%) after the first dose of a mRNA vaccine: both had AAV (microscopic poliangioitis) and developed a pulmunary disease flare (respiratory failure requiring hospitalization and treatment with high-dose glucocorticoids). Of note, one of these patients had multiple previous comorbidities, including a severe COPD. Multivaried analysis, adjusted for age and sex, performed in a single monocentric cohort of patients with systemic vasculitis [n=60 (39 AAV, 21 GCA), 37 female, 23 male, mean age 71 (± 12.5) years] demonstrated a statistically significant higher frequency of AEs in vasculitis patients compared to HC (p=0.015) after the first dose of vaccination. No significant differences in the frequency of AEs in vasculitis patients compared to HC after the second dose were detected. All the AEs were mild in both groups (malaise was the most frequently reported); no serious AEs were reported.ConclusionOur data show a very low incidence of disease flares after the administration of anti-SARS-CoV-2 vaccines in patients with systemic vasculitis. Patients with systemic vasculitis seem more prone to develop mild AEs after the first dose of the vaccine. Taken together, this data suggest a good risk profile for anti-SARS-CoV-2 vaccine in patients with systemic vasculitis.Disclosure of InterestsNone declared

The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCR–ligase detection reaction–real-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patient–derived cell-free DNAs. This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.

β-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.

BackgroundInterrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR.MethodsIn this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood.ResultsOur results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions ofNR5A2andPRKCB, and a non-coding region of chromosome 1 (upstream ofEFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27).ConclusionThis report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC50 (0.19 ± 0.08 μM) over controls (5.50 ± 0.09 μM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

β-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

Background:The Mediterranean Diet (MD) has anti-inflammatory and immunomodulatory effects1,2 suggesting a protective role in rheumatic diseases. There is limited knowledge about the eating habits and the nutritional state in patients with isolated Sjögren Syndrome (SS) living within the Mediterranean area.Objectives:assessment of adherence to the MD and analysis of the nutritional state in women with SS and their correlations with the clinical, laboratory and histological data of the disease.Methods:patients classified as isolated SS according to AECG criteria 20023 who had undergone to minor salivary gland biopsy during the previous twelve months were consecutively enrolled during follow-up visits. The adherence to the MD was assessed by the Med Diet (MDiet)4 which includes eleven groups of foods; to each group is assigned a value ranging between 0 and 5 based on the frequency of monthly intake. The total score spans from 0 (poor adherence) to 55 (maximum adherence). The level of physical activity was measured by the 6-minutes walking test (6MWT) and by the International Physical Activity Questionnaire (IPAQ). Systemic disease activity was evaluated with the EULAR SS disease activity index (ESSDAI); EULAR SS patient-reported index (ESSPRI) was calculated as well. A subgroup of patients was asked to fill a daily food diary processed with FOOD CONS software which allows to study in detail their eating habits. Nutritional state, muscle strength and basal metabolic rate were assessed. Alcoholism or drug abuse, diabetes mellitus, specific dietary models, treatment with drugs and/or food supplements with anti-inflammatory and/or antioxidant activity were considered exclusion criteria. Multivariate linear regression was performed with R project for Statistical Computing.Results:N= 40 N= 26Age, median (range) 53 (25-80) 33 (25-71)BMI, median (range) 21 (19-29.3) 25.1 (19-33.7)MedDiet score, median (range) 33 (26-43) 33 (23-40)ESSDAI, median (range) 2 (0-16) 1 (0-16)ESSPRI, median (range) 6 (0-8.6) 5.3 (1.6-9)ESSPRI dryness, median (range) 6 (0-10) 6 (2-10)Focus score, median (range) 2.5 (0-9.6) 1.7 (0.8-6.24)ASM kg, median (range) - 16.8 (13.3-21.7)IPAQ meters, median (range) - 1386 (99-11865)6MWT meters, median (range) - 595 (536-680)BMI, body mass index; ASM appendicular skeletal mass; IPAQ International Physical Activity Questionnaire; 6MWT, six minute walking test.MDiet was administered to 40 female SS outpatients. Even if not reaching significativity, patients with a higher focus score in their MSG have a lower value of MDiet score (p = 0.058, r = -1.00). The MDiet score is not associated with ESSDAI (p = 0.85, r 0.02), but only with lower serum levels of C3 (p = 0.004, r = - 0.08).In 26 patients, daily food questionnaire shows that their diet consists of 43% of carbohydrates while fats represent 40% of total energy intake, the remaining 17% daily energy comes from proteins. Fat consumption is higher compared to the levels of energy and nutrient intake for the Italian population5. Six patients had a reduction in muscle mass; sarcopenia is not associated to ESSDAI (p = 0.610).The MDiet score and the amount eaten of polyunsaturated fatty acids (PUFA) were reduced in patients with high value of subscale dryness of ESSPRI (p = 0.057, r -1.21; p =0.610, r -1.00).Conclusion:This study highlights a lower degree of glandular lymphocytic infiltration (expressed as focus score) in minor salivary glands in patients following MD, so its anti-inflammatory role of seems to be confirmed. SS patients have an unbalanced diet because of a higher intake of fat foods, likely for their lubricating effect. Despite the absence of correlation with objective parameters, the increased dryness in patients with a reduced intake of PUFA arouses our interest in a future study including omega-3 supplementation.References:[1]Schwingshackl L et al., Nutr Metab Cardiovasc Dis 2014[2]Mena MP et al., Am J Clin Nutr. 2009[3]Vitali C et al, ARD 2002[4]Panagiotakos D et al., J Med Food 2007[5]LARN 2014Disclosure of Interests:None declared

Background:The severe measures of lockdown imposed in Italy to limit the SARS coronavirus 2 disease (COVID-19) spread caused an increase of reported anxiety, depression and suicidal rate among general population. Patients affected by rheumatic disorders feature an increased risk of mood disorders for the chronic course of the disease itself and for the related disability.Objectives:Aim of this study was to investigate the impact of COVID-19 lockdown on emotional well-being of a large cohort of rheumatic patients through a telemedicine approach.Methods:Patients in follow-up in rheumatologic out-patient clinics of our hospital were invited to participate to an online survey. They were asked also to invite their best friend, matched for age and sex, to participate the survey, as control group. The online survey included demographic questions and validated, psychometric scales for stress vulnerability (Stress Vulnerability Scale-SVS), resilience (Resilience Scale-RS), depression (Zung’s depression questionnaire-Zung-D) and anxiety (Zung’s anxiety questionnaire-Zung-A) evaluation.Results:The cohort was composed by 484 subjects (84,1% F, 15,9% M). The number of subjects and the frequency of various diagnosis are shown in Table 1. According to the psychometric scales, 55,5% and 43,3% of subject showed respectively an increased stress vulnerability and a reduced resiliency. Moreover, 64% and 40,5% of the enrolled subjects reported respectively anxiety and depressive symptoms worthy of psychiatric attention. There was a significant different distribution of scores for SVS (pConclusion:Rheumatic patients developed a high frequency of anxiety and depressive symptoms following COVID-19 lockdown, of which a large part should be referred for specialist attention according to their severity. There was a large variability of the symptoms reported among the various diagnosis. CTD, SSJ and FM patients were the most susceptible to the development of anxiety, depression and stress vulnerability. The application of psycometric scales through a telemedicine approach represents a useful tool to identify patients with higher levels of anxiety and depression.Table 1.DIAGNOSISFrequencyPercentControls459,3RA8216,9PSA214,3UA40,8SPA71,4CTD7014,5FM7916,3Myositis81,7Behcet’s112,3Vasculitis163,3APS61,2Other AID132,7SSJ12225,2Total484100RA: Rheumatoid Arthritis, PSA: Psoriatic Arthritis; UA: Undifferentiated Arthritis; SPA: Spondyloarthritis; CTD: Connective tissues diseases (including Systemic Lupus Erythematosus, Scleroderma, Undifferentiated Connettivitis, Mixed Connettivitis); FM: Fibromyalgia; APS: Anti-phospholipid syndrome; Other AID: Other autoimmune/inflammatory disorders (including Adult-onset Still disease, IgG4 related disease); SSJ: Sjogren SyndromeFigure 1.Disclosure of Interests:None declared

Background:Abatacept (ABA) is a biological drug approved for the treatment of rheumatoid arthritis (RA) patients that, by working on CTLA4, can inhibit T-cell activation. Randomized controlled trials have demonstrated both the efficacy and a good safety profile, characterized by a lower infectious risk in comparison with other biological DMARDs, in RA patients. In a real-life setting, the drug retention rate could be considered as a surrogate of drug effectiveness. Data from the literature reported a retention rate of ABA ranging from 55 to 76% at 12 months and from 54 to 64% at 24 months (1-3).Objectives:In the present longitudinal analysis, we evaluated the retention rate of ABA in a large monocentric RA cohort.Methods:We enrolled consecutive RA patients starting treatment with intravenous (IV) or subcutaneous (SC) ABA according to the standard of care. All the patients fulfilled the 2010 ACR/EULAR classification criteria for RA. For each patient, we collected demographic parameters, serological status, previous and concomitant treatments, and disease activity by DAS28 with C reactive protein (DAS28-CRP). All the patients were assessed at baseline, and after 4 and 12 months (T4 and T12, respectively). The reasons for withdrawal of treatment were registered and classified as primary or secondary inefficacy or adverse events (AEs). Kaplan-Meier statistical analysis has been done to evaluate the survival of the treatment in patients with at least 12 months follow-up.Results:We evaluated 161 patients [M/F 21/140; median age 67 years (IQR 21.7), median disease duration 180 months (IQR 161)]. RF was positive in 70.3% of patients, ACPA in 66.4%. ABA was the first biological DMARD in 66 patients (41%). At baseline, the median DAS28-CRP was 4.3 (IQR 1.6) and ABA was administered in association with MTX in 96 patients (59.6%). One hundred-eleven patients (68.9%) started SC ABA [M/F 16/95; median age 64.5 years (IQR 21.5), median disease duration 156 months (IQR 132)], the remaining 50 IV ABA [M/F 5/45, median age 71 years (IQR 60.2), median disease duration 187 months (IQR 157)]. Median age and disease duration were significantly higher in patients receiving IV in comparison with SC ABA (p=0.008 and p=0.03, respectively). We found a significant reduction of DAS28-CRP values during the follow-up in comparison with baseline [4 months: median 3.5 (IQR 1.9), pConclusion:In our monocentric RA cohort, we have observed a high retention rate of ABA at both 12 and 24 months, confirming the good profile of this drug in terms of effectiveness and safety, irrespective of the route of administration and association with MTX.References:[1]Cagnotto, Arthritis Res Ther 2020; (2) Salmon, J Clin Med 2020; Westhovens, Rheumatol Int 2020.Acknowledgements:I would like to acknowledge Dr. F. Ceccarelli, for her patience.Disclosure of Interests:None declared

Background:Sjögren’s syndrome (SS) is a systemic autoimmune disease mainly affecting exocrine glands and characterized by a progressive lymphocytic infiltration of salivary and lacrimal glands with consequent loss of function and development of sicca symptoms. Autoimmune thyroiditis (AT) is the most frequent autoimmune disease associated with SS and detectable in about 10 - 30% of cases1. Interestingly, patients with concomitant SS and AT seem to display a more attenuated phenotype compared to patients with solely SS. It is also noteworthy that up to 30% of patients with AT experience sicca symptoms without a clear diagnosis SS2. At the light of these evidences, it is unclear whether SS and AT represent two distinct nosological entities or different expressions of the same pathology.Objectives:Aim of this study is to evaluate the prevalence of AT in a large monocentric cohort of patients with SS and to define its clinical and laboratory characteristics compared to isolated SS.Methods:Consecutive patients with SS (AECG criteria) referring to our “Sjögren Clinic” (Sapienza University of Rome) were enrolled and divided in two groups: SS with AT (group 1) and SS without AT (group 2). Group 1 was further divided in two subgroups depending on the presence (1a) or absence (1b) of anti Ro/SSA antibodies. The following clinical and laboratory data were retrospectively collected for all patients: concomitant celiac disease, arthralgia, lung involvement, purpura, lymphoma, presence of ANA, anti-Ro/SSA, anti-La/SSB, rheumatoid factor, cryoglobulins, leukopenia and hypergammaglobulinaemia. These characteristics were compared between the following groups: group 1, group 2, group 1a and 1b. For statistic Chi Square and Fisher’s test analysis were performed.Results:Six-hundred and three SS patients were enrolled (group 1 n=135; group 2 n=381; group 1a n=96; group 1b n=39). The prevalence of AT was 135/603 (22.3%). When comparing SS patients with or without AT (group 1 vs group 2) the frequency of rheumatoid factor was significantly higher in group 2 compared to group 1 (p=0.006). No case of lymphoma was recorded in group 1 while 14 cases of lymphoma were ascertained in group 2 (p=0,08). Conversely, celiac disease was higher in group 1 compared to group 2 (p=0.01). No other differences between these groups were identified. Stratifying SS patients with AT according to the presence (group 1a) or not (group 1b) of anti Ro/SSA antibodies, ANA, rheumatoid factor and hypergammaglobulinemia were significantly more positive in group 1a compared to group 1b (p=0.0002, p=0.002, p=0.02, respectively); no clinical differences were identified.Conclusion:In this study, we confirm the presence of a less aggressive disease in patients with SS and AT compared to solely SS. The higher prevalence of rheumatoid factor and lymphoma occurrence in SS without AT, strictly suggest a more severe phenotype in this subset. Although is known that in SS patients with anti Ro/SSA+ antibodies and RF there is a more aggressive disease, in SS with AT the presence or absence of such autoantibodies do not seems to associate with any difference in clinical severity. Follow up studies are presently being carried out in order to provide conformation of a less sever phenotype and a better disease outcome in patients with associated SS and AT.References:[1]Jara, L.J., Navarro, C., Brito-Zerón, M.P. et al. Thyroid disease in Sjögren’s syndrome. Clin Rheumatol 26, 1601–1606 (2007)[2]Vera D. Milic, Goran Radunovic, Ivan Boricic, Sanja Ognjanovic, Radmila Petrovic, Marija Radak-Perovic, Nada Vujasinovic-Stupar, Nemanja Damjanov, High prevalence of autoimmune thyroid disease in subjects with sicca symptoms without Sjögren’s syndrome, Rheumatology, Volume 52, Issue 4, April 2013, Pages 754–755.Disclosure of Interests:None declared

Background:Resilience is the ability to react positively to stressful life events, a multidimensional feature that varies in relation to context, time, age, sex, culture and personal experience, appearing among the most important traits in patients suffering from rheumatic diseases1. Several studies focus on patients with RA and SLE and the role of resilience in the respective clinical manifestations, as well as in the development of anxiety and depression2,3. Conversely, the data available regarding patients with primary Sjögren’s Syndrome (pSS) are limited.Objectives:To assess, in women with pSS (classified according to the criteria of Vitali et al.4), the relationship between resilience and anxiety, depression, health, fatigue, physical activity and quality of life in relation to disease activity and duration and in consideration of demographic, job and cultural characteristics.Methods:74 female patients with pSS afferent to the dedicated clinic of the University Hospital Policlinico Umberto I of Rome were recruited. Resilience was assessed by administering the Italian validated version of the Resilience Scale (RS-14)5consisting of 14 items, each of which is assigned a score from 1 to 7, with a range from 14 to 98. Higher scores relate to greater resilience. ESSDAI (EULAR Sjögren’s syndrome disease activity index), ESSPRI (EULAR Sjogren’s Syndrome Patient Reported Index), SSDDI (Sjogren’s Syndrome Disease Damage Index) were assessed and EuroQol / GH EQ VAS (visual analogue scale), HADS (Hospital Anxiety and Depression Scale), SF-12 (Short-form 12 health survey), FAS (Fatigue Assesment Scale), IPAQ (International Physical Activity Questionnaire), FACIT-F (Functional Assessment of Chronic Illness Therapy – Fatigue) questionnaires were submitted. Educational qualifications and job were also considered. The statistical analysis was carried out by means of Spearman’s correlation.Results:No relationship was found between resilience, systemic disease activity, disease duration, patient-reported symptoms and damage. Furthermore, no apparent link was found between socio-demographic characteristics, employment and resilience. Conversely, an inverse relationship was found between resilience and mood disorders (p=0.0379), with greater resilience associated with a better perception of quality of life (p=0.0232) and general health (p=0.0002), mainly mental (p=0.0001) than physical (p=0.0035), as well as less fatigue (p=0.0079) and more phyisically active lifestyle (p=0.0012)Conclusion:For the first time, the role of resilience in women with pSS in relation to their disease and other individual parameters was assessed. The most resilient patients are less depressed and show better perception of their health. Greater resilience tends to correlate with less anxiety, physical and mental fatigue and a more active lyfestile, while there was no relation between resilience value, active disease and socio-demographic features.References:[1]Rojas M. et al., Resilience in women with autoimmune rheumatic diseases, Joint Bone Spine (2017).[2]Sílvia Fernanda Cal et al., Resilience in systemic lupus erythematosus, Psychology, Health & Medicine (2013), 18:5, 558-563.[3]NeiLi Xu et al., Associations of perceived social support and positive psychological resources with fatigue symptom in patients with rheumatoid arthritis.PLoS One. 2017; 12(3).[4]Vitali C, et al. Classification criteria for Sjogren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002; 61:554-558.[5]Camilla Callegari et al., Reliability and validity of the Italian version of the 14-item Resilience Scale, Psychology Research and Behavior Management 2016:9 277–284.Disclosure of Interests:None declared

WOS: 000423194500015, Science Citation Index Expanded, The "New Mediterranean Biodiversity Records" series includes new records of marine species found in the Mediterranean basin and/or information on the spatial distribution of already established species of particular interest. The current article presents information on 21 marine taxa classified per country according to their geographic position in the Mediterranean, from west to east. The new records per ecoregion are as follows: Algeria: the first record of the fish Etrumeus golanii is reported along the Algerian coast. Tunisia: the alien jellyfish Phyllorhiza punctata is reported for the first time in the Gulf of Gabes. Italy: the first record of Siganus rivulatus in the Strait of Sicily and a new record of Katsuwonus pelamis from the central Tyrrhenian Sea are reported. The establishment of the isopod of the genus Mesanthura in the northern Tyrrhenian with some notes on its ecology are also included. Croatia: signs of establishment of the Lessepsian Siganus luridus and the occurrence of the alien mollusc Rapana venosa are reported. Albania: the first record of the elasmobranch Alopias superciliosus and a recent sighting of the rare monk seal Monachus monachus in Albanian waters are given. Greece: signs of the establishment of the fish Parupeneus forsskali and of the ascidian Hermania momus in Hellenic Aegean waters are reported. Turkey: a new record of the fish P. forsskali and of the halacarid mites Acaromantis monnioti and Simognathus adriaticus are given, while the first case of Remora australis in association with delphinids and the occurrence of the sea star Coscinasterias tenuispina are reported. Also, the establishment of the two alien species Isognomon legumen and Viriola sp. [cf. corrugata] are presented. Egypt: the fish Bathygobius cyclopterus is reported for the first time in Mediterranean Sea waters. Also, a new record of Pagellus bogaraveo and a first record of Seriola fasciata in Egyptian Mediterranean waters are reported. Lebanon: the first record of Dondice banyulensis is presented., Interreg MED Project Mpa-Adapt Guiding Mediterranean MPAs through the climate change era: building resilience and adaptation; Croatian Science Foundation (HRZZ) [IP-2016-06-5251]; Rufford Foundation [19938-1]; Rufford Foundation; Marine Mammals Research Association; Interreg MED Project Mpa-Adapt Guiding Mediterranean MPAs through the climate change era: building resilience and adaptation; Croatian Science Foundation (HRZZ) [IP-2016-06-5251]; Rufford Foundation [19938-1]; Rufford Foundation; Marine Mammals Research Association; [R 2000 Eb F 42 / L43], Azzurro E. and Giardina F. are indebted to Giuseppe Maggiore and Peppino Sorrentino for providing all the information regarding the capture of Siganus rivulatus in Lampedusa Island; their study was supported by the Interreg MED Project Mpa-Adapt Guiding Mediterranean MPAs through the climate change era: building resilience and adaptation. Rabaoui L. and El Zrelli R. would like to thank all fishermen who provided information on the recent occurrence of Phyllorhiza punctata in the Gulf of Gabes. Dulcic J. and Dragicevic B. acknowledge the Croatian Science Foundation (HRZZ) that partly supported their work under project IP-2016-06-5251. Giovos I. and Cakalli M. would like to warmly thank the fisherman Arsen Jupe for helping and assisting them in taking all the measurements. Bakiu R. and Cakalli M. would like to thank the Rufford Foundation (Project ref. 19938-1) for financially supporting the Mediterranean monk seal monitoring program along the Ionian coast of Albania; furthermore, they wish to warmly thank Eng. Agron Bakiu for the power supply system that he built for the camera traps.. Bas A.A. and Gonulal O. would like to thank Prof. Dr. Ivan Sazima and Isil Danyer for their help on species identification and the Rufford Foundation and Marine Mammals Research Association for their financial support. Durucan F. and Boyaci Y.O. wish to express special thanks to Dr. Matteo Dal Zotto (Universita degli Studi di Modena e Reggio Emilia, Department of Life Sciences, Modena, Italy) who kindly sent them a copy of Dr. Morselli's article that was lacking from the first author's library. Durucan F. would like to thank the anonymous reviewers for their useful comments and also Suleyman Demirel University, Fisheries Faculty, Ecology and Limnology laboratory (Isparta, Turkey) for providing laboratory facilities. Crocetta F. and Bitar G. thank Jean Vacelet (Marseille, France) for the photo of Dondice banyulensis taken during the Franco-Lebanese cooperation program - CEDRE (1999-2002) (R 2000 Eb F 42 / L43). Kassar A. and Hemida F. are deeply grateful to the fisherman Benssada Djamel for providing the specimen of Etrumeus golanii.

On July 2017 during a scientific survey, a single individual of Ophioblennius atlanticus (Valenciennes, 1836) was caught at the entrance of Lampedusa harbour, in proximity to the Marine Protected Area of the Pelagie Islands. This record confirms the introduction of this fish into Mediterranean waters, adding a new species to the list of Italian NIS. Possible pathways of introduction and the relevance of this finding for Mediterranean MPAs are briefly discussed.

Purpose: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. Experimental Design: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. Results: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). Conclusion: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.

The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase 3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.

T-cadherin is a 95kDa glycoprotein member of the cadherin family of adhesion molecules attached to the extracellular surface of the cell membrane through a glycosyl-phosphatidylinositol (GPI)-anchor. Whether a T-cadherin ectodomain apical targeting signal or the GPI-anchor itself targets this protein to the apical membrane is not known. Chimeras of the reporter EGFP and T-cadherin have demonstrated that a minimal construct consisting of the C-terminal 25 amino acids including the N690 (omega-site) of T-cadherin was sufficient to GPI-anchor the EGFP protein. However, efficient GPI-anchor with minimal secretion of the protein required an additional 5 residues (omega-1 to omega-5). The GPI-anchored chimeras fractionated to the Triton X-100 detergent insoluble fraction and were released to the cell culture supernatant by phosphoinositide-specific phospho-lipase C digestion. When expressed in MDCK cells, all GPI-anchored chimeras targeted to the basolateral membrane, while the T/N-chimera and the wild-type T-cadherin targeted to the apical membrane. Therefore, T-cadherin is an example of another rare GPI-anchored protein where the anchor itself is not sufficient for apical targeting in MDCK cells.

J. L. Cook, J. F. Giardina, Z. Zhang and R. N. Re. Intracellular Angiotensin II Increases the Long Isoform of PDGF mRNA in Rat Hepatoma Cells. Journal of Molecular and Cellular Cardiology (2002) 34, 1525–1537. Our recent published studies suggest that angiotensin II (AII), generated and retained intracellularly, enhances growth of H4-II-E-C3 rat hepatoma cells, an average of 33%. Proliferation conferred by introduction of a plasmid [ Ang(-S)Exp/pSVL ] encoding a signal sequence-depleted angiotensinogen [Ang(-S)Exp] into these cells (which we have shown possess ACE and renin mRNAs) is mediated, at least in part, by enhanced PDGF-A chain mRNA production and protein secretion. The mitogenic effect is inhibited by losartan suggesting that it involves AII interaction with an AT1-like receptor. Introduction of anti-AII antibodies into the medium of these transfected cells has no effect upon growth of the cells, suggesting that AII is retained by the cells and that intracellular AII is growth stimulatory. In the present study, we sought to further characterize the intracellular localization and mode of action of Ang(-S)Exp. Consistent with our expectations, we now show that a fusion product of Ang(-S)Exp with green fluorescent protein [Ang(-S)Exp/EGFP], generated from an expression plasmid, is abundant and primarily cytoplasmic. Wild-type angiotensinogen/EGFP, in contrast, is only detectable following a cold-block (which acts to enhance folding-kinetics and slow secretion) and is largely restricted to the secretory pathway. We further show, using semi-quantitative RT/PCR that the long isoform of PDGF mRNA is elevated in Ang(-S)Exp transfected cells and in AII-treated naive cells but not in losartan-treated Ang(-S)Exp transfected cells. We identify C-terminal amidation recognition sites within the long-form protein (that are not present in the short-form) and show that these cells possess PAM (amidating enzyme precursor) and carboxypeptidase E mRNAs (the corresponding proteins of which are sufficient for amidation). Inhibitors of amidation inhibit growth of naive and Ang(-S)Cntr/ pSVL -transfected cells (2.6-fold for phenylbutenoic acid and 3.5-fold for disulfiram treatment) but more profoundly inhibit growth of Ang(-S)Exp/pSVL -transfected cells (6.7-fold for phenylbutenoic acid and 13-fold for disulfiram). In conclusion, these data confirm that signal sequence-depleted Ang(-S)Exp is retained within cells and is largely cytoplasmic. Because C-terminal amidation is absolutely required for full biological potency of a number of peptide hormones (including oxytocin, gastrin and calcitonin), we postulate that growth effects of both intracellular AII and exogenous AII can be conferred by PDGF long-form, possibly through an amidation-dependent mechanism.

Previous studies have suggested that neuronal apoptosis is the result of an abortive attempt to re-enter the cell cycle, and more recently the cyclin-dependent (CDKs) and the mitogen-activated protein (MAP) kinases, two superfamilies of kinases that influence and control cell cycle progression, have been implicated in neuronal apoptosis. Here, to examine whether CDK/MAPK related pathways are involved in excitotoxicity, we studied the actions of various kinase inhibitors on apoptosis induced by the ionotropic glutamate (Glu) receptor agonist, kainate (KA), in primary cultures of murine cerebellar granule cells (CGCs). KA-mediated neurotoxicity was concentration-dependent, as determined by a cell viability assay monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and largely apoptotic in nature, as shown by morphological examination and labelling of DNA fragmentation in situ using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick-end labelling (TUNEL). KA-mediated neurotoxicity and apoptosis was completely attenuated by the mixed CDK and MAP kinase inhibitor, olomoucine, in a concentration-dependent manner (50 – 600 μM), and partially by roscovitine (1 – 100 μM), a more selective CDK inihibitor. The p38 MAP kinase inhibitor, SB203580 (1 – 100 μM), partially attenuated KA receptor-mediated apoptosis, as did the MAP kinase kinase inhibitors PD98509 (1 – 100 μM) and U0126 (1 – 100 μM). These findings provide new evidence for a complex network of interacting pathways involving CDK/MAPK that control apoptosis downstream of KA receptor activation in excitotoxic neuronal cell death. British Journal of Pharmacology (2002) 135, 1733–1742; doi:10.1038/sj.bjp.0704636

The involvement of low-affinity kainate (KA) receptors in neuronal injury was investigated by employing a variety of agonists active at GluR5-7. Their excitotoxic profiles were determined in primary cultures of cerebellar granule cells, which abundantly expressed low-affinity KA receptors, and in the absence of any AMPA receptor-mediated neurotoxicity. Neurotoxicity induced by these compounds was analysed by phase contrast microscopy, a cell viability assay, the TUNEL technique (apoptosis), and by employing propidium iodide (PI; necrosis). All agonists induced concentration-dependent neurotoxicity, with rank order (EC(50) values; microM): (S)-iodowillardiine (IW) 0.2(2S,4R)-4-methylglutamate (4-MG) 36(2S,4R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) 46KA 74(RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4yl)propanoic acid (ATPA) 88. IW exposure resulted in apoptosis at lower concentrations (30 microM) and necrosis at higher concentrations, both of which were attenuated by CNQX (50 microM), but not MK-801 (10 microM). ATPA-mediated neurotoxicity was purely apoptotic and was attenuated by the non-NMDA receptor antagonists. Both IW and ATPA induced injury with the morphological characteristics of apoptosis shown by the presence of TUNEL-positive neurones. LY339434-mediated neuronal injury was only attenuated by MK-801 and was necrotic in nature. Similarly, 4-MG (30 microM) exposure caused necrosis that was partially attenuated by MK-801 (10 microM) and CNQX (50 microM). The patterns of neurotoxicity possessed a complex pharmacological profile, demonstrated an apoptotic-necrotic continuum and were inconsistent with past findings, further outlining the importance of characterizing novel compounds at native receptors. ATPA and to a lesser extent IW appear to be suitable drugs for low-affinity KA receptors. Since toxicity-mediated by low-affinity KA receptors seem likely to contribute to neurodegenerative conditions, our study importantly examines the excitotoxic profile of these novel agonists.

AM-36 is a novel neuroprotective agent incorporating both antioxidant and Na(+) channel blocking actions. In cerebral ischaemia, loss of cellular ion homeostasis due to Na(+) channel activation, together with increased reactive oxygen species (ROS) production, are thought to contribute to neuronal death. Since neuronal death in the penumbra of the ischaemic lesion is suggested to occur by apoptosis, we investigated the ability of AM-36, antioxidants and Na(+) channel antagonists to inhibit toxicity induced by the neurotoxin, veratridine in cultured cerebellar granule cells (CGC's). Veratridine (10 - 300 microM) concentration-dependently reduced cell viability of cultured CGC's. Under the experimental conditions employed, cell death induced by veratridine (100 microM) possessed the characteristics of apoptosis as assessed by morphology, TUNEL staining and DNA laddering on agarose gels. Neurotoxicity and apoptosis induced by veratridine (100 microM) were inhibited to a maximum of 50% by the antioxidants, U74500A (0.1 - 10 microM) and U83836E (0.03 - 10 microM), and to a maximum of 30% by the Na(+) channel blocker, dibucaine (0.1 - 100 microM). In contrast, AM-36 (0.01 - 10 microM) completely inhibited veratridine-induced toxicity ( IC(50) 1.7 (1.5 - 1.9) microM, 95% confidence intervals (CI) in parentheses) and concentration-dependently inhibited apoptosis. These findings suggest veratridine-induced toxicity and apoptosis are partially mediated by generation of ROS. AM-36, which combines both Na(+) channel blocking and antioxidant activity, provided superior neuroprotection compared with agents possessing only one of these actions. This bifunctional profile of activity may underlie the potent neuroprotective effects of AM-36 recently found in a stroke model in conscious rats.

A low-cost tide gauge was developed and field tested to demonstrate a technology that would enable more cost-effective and greater sampling of spatially variable water levels and ocean surface waves. The gauge was designed to be adaptable to expendable and, possibly, air-deployed use for applications such as support of naval operations. The gauge incorporates a single printed circuit board that includes a very low power 3.3-Vdc microprocessor and 1 Mbyte of nonvolatile flash memory. A low-cost solid-state pressure sensor provides pressure data that are corrected automatically as a function of measured pressure and temperature and are processed within the gauge to provide low-frequency water levels and nondirectional surface wave information. Gauge-operating lifetimes range from more than four months to more than two years, depending on the data collection mode (tide or tide–wave) and the data collection interval (half-hourly or hourly). Gauge measurements are compared to measurements from a Sea-B...

In neocortical neuronal cultures, (S)-AMPA caused neurotoxicity which was concentration-dependent, receptor-mediated, slow and apoptotic in nature. (S)-AMPA (3-600 microM) failed to produce rapid neuronal swelling, but morphological observations and monitoring of viability at 24-72 h revealed 50% cell death consistent with apoptosis. (S)-AMPA induced cell shrinkage, neurite blebbing and nuclear condensation. Cyclothiazide (50 and 100 microM), which blocks AMPA receptor desensitization potentiated excitotoxicity with 75% of neurones undergoing slow death. The AMPA-selective antagonist GYKI 52466 (10-50 microM), attenuated (S)-AMPA-mediated neurotoxicity. DNA condensation, a hallmark of apoptosis, was found by labelling neurones with the DNA binding dye 4,6-diamidino-2-phenylindole HCl (DAPI). Gel electrophoresis revealed DNA fragmentation, which was increased by cyclothiazide and reduced by GYKI 52466 and cycloheximide. Overstimulation of the AMPA receptor produces a novel form of neuronal death, which is apoptotic, very slow in nature, and which could contribute to various neuropathologies.

We have investigated the involvement of c-Jun in cell death induced by exposure of primary cultures of murine cerebellar granule cells to the glutamate receptor agonist kainate (KA) and evaluated its possible use as a marker for apoptosis. Using cerebellar granule cell neurones from postnatal day 7 mice, we found that 1 hr exposure to KA (1–1000 μM) induced a concentration-dependent neuronal cell death with characteristic apoptotic morphology, including cell shrinkage, neurite blebbing and DNA fragmentation. In addition KA-induced a concentration-dependent expression of c-Jun mRNA and protein as determined by in situ hybridization and immunocytochemistry respectively. DNA fragmentation was detected using terminal transferase-mediated nick-end (TUNEL) labelling and agarose gel electrophoresis. KA-induced cell death was significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM), which shifted the concentration-response curve significantly rightward. The number of apoptotic cell bodies, determined by TUNEL, was also reduced by CNQX (50 μM), with only 15–20% of neurones staining positive after exposure to 1mM KA. In addition, the number of positively stained cells for c-Jun protein and mRNA was substantially reduced by CNQX (50 μM) as determined by random and representative cell counts. These results show for the first time that KA induced apoptotic neuronal death in cultured murine cerebellar granule cells involves the induction of c-Jun mRNA and protein, suggesting the involvement of this immediate early gene in excitotoxic receptor-mediated apoptosis and its potential use as a marker for apoptotic cell death. J. Neurosci. Res. 52:69–82, 1998. © 1998 Wiley-Liss, Inc.

We have previously shown that oligonucleotides designed to bind in triplex fashion to a specific p53 binding site homology inhibit the proliferation of colon cancer cells in vitro. The present study was designed to extend these observations in an in vivo model. HCT 116 human colon carcinoma cells were injected subcutaneously into Ncr nude mice and tumors formed at one to two weeks. Tumors were injected daily for 14 days with either triplex forming oligonucleotide (Hoog 1), a scrambled Hoog 1 oligonucleotide (Hoog3) as control, or vehicle. Tumor size was measured twice weekly. Active triplex forming oligonucleotide (Hoog1) reduced tumor size in comparison to either control oligonucleotide (Hoog3) or vehicle. Tumor sizes in the three groups were significantly different (P < 0.001). Student Newman Keuls test shows statistically significant differences between the experimental group and each of the control and vehicle groups (P < 0.05). A triplex forming oligonucleotide directed at a p53 consensus binding site reduces tumor growth suggesting a novel method of tumor inhibition.

Cardiac myocytes (AT-1 cells) derived from heart tumors of mice transgenic for an atrial natriuretic factor promoter, SV40 large T-antigen DNA transgene, demonstrate properties consistent with normal cardiac myocytes but retain the capacity to proliferate in culture. We studied the renin-angiotensin system (RAS) and related growth regulation of these cells because AT-1 cells (or transgenically similar cells) may be useful to repair injured myocardium. This study reveals two separate and distinct findings: 1) AT-1 cells proliferate or hypertrophy in response to angiotensin II (ANG II), depending on their competence to proceed through the cell cycle; and 2) AT-1 cells possess components of a RAS, and angiotensinogen antisense experiments suggest that the RAS is functional in these cells. Specifically, AT-1 cells proliferate in response to ANG II in low-serum medium but hypertrophy in response to ANG II when first treated with mitomycin C (at a concentration that inhibits DNA replication but is not cytotoxic). The ANG II-mediated proliferative and hypertrophic responses are inhibited by DuP 753. In addition, there is a significant increase in the protein-to-DNA ratio of cells, which are proliferation-inhibited in the absence of ANG II treatment (20%, P < 0.05). DuP 753 also inhibits this hypertrophy, suggesting that these cells possess a functional RAS. AT-1 cells contain mRNAs for angiotensin-converting enzyme, renin, angiotensinogen, and the AT1 receptor as determined by sequence analysis of polymerase chain reaction amplification products. Antisense oligonucleotides complementary to the angiotensinogen mRNA specifically inhibit angiotensinogen mRNA accumulation and proliferation of AT-1 cells. In summary, these cells contain a growth-regulating RAS, suggesting that such a system may play a significant role in left ventricular hypertrophy.

The record of several individuals of the African hind Cephalopholis taeniops from Lampedusa Island (Italy) is here reported, with discussion on the biogeographical importance of the Sicily channel as a crossroad for exotic fish of Atlantic and Indo-pacific origin.

During disease progression the cells that comprise solid malignancies undergo significant changes in gene copy number and chromosome structure. Colorectal cancer provides an excellent model to study this process. To indentify and characterize chromosomal abnormalities in colorectal cancer, we performed a statistical analysis of 299 expression and 130 SNP arrays profiled at different stages of the disease, including normal tissue, adenoma, stages 1–4 adenocarcinoma, and metastasis. We identified broad (> 1/2 chromosomal arm) and focal (< 1/2 chromosomal arm) events. Broad amplifications were noted on chromosomes 7, 8q, 13q, 20, and X and broad deletions on chromosomes 4, 8p, 14q, 15q, 17p, 18, 20p, and 22q. Focal events (gains or losses) were identified in regions containing known cancer pathway genes, such as VEGFA , MYC , MET , FGF6 , FGF23 , LYN , MMP9 , MYBL2 , AURKA , UBE2C , and PTEN . Other focal events encompassed potential new candidate tumor suppressors (losses) and oncogenes (gains), including CCDC68 , CSMD1 , POLR1D , and PMEPA1 . From the expression data, we identified genes whose expression levels reflected their copy number changes and used this relationship to impute copy number changes to samples without accompanying SNP data. This analysis provided the statistical power to show that deletions of 8p, 4p, and 15q are associated with survival and disease progression, and that samples with simultaneous deletions in 18q, 8p, 4p, and 15q have a particularly poor prognosis. Annotation analysis reveals that the oxidative phosphorylation pathway shows a strong tendency for decreased expression in the samples characterized by poor prognosis.