Nuclear Punctate Distribution of ALL-1 is Conferred by Distinct Elements at the N-terminus of the Protein

The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which plays a critical role in maintaining proper spatial and temporal expression of the Antennapedia-bithorax homeotic genes determining the fruit fly's body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its -1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

MTOR and STAT3 pathway hyper-activation is associated with elevated interleukin-6 levels in patients with shwachman-diamond syndrome: Further evidence of lymphoid lineage impairment

Shwachman–Diamond syndrome (SDS) is a rare inherited bone marrow failure syndrome, resulting in neutropenia and a risk of myeloid neoplasia. A mutation in a ribosome maturation factor accounts for almost all of the cases. Lymphoid involvement in SDS has not been well characterized. We recently reported that lymphocyte subpopulations are reduced in SDS patients. We have also shown that the mTOR-STAT3 pathway is hyper-activated in SDS myeloid cell populations. Here we show that mTOR-STAT3 signaling is markedly upregulated in the lymphoid compartment of SDS patients. Furthermore, our data reveal elevated IL-6 levels in cellular supernatants obtained from lymphoblasts, bone marrow mononuclear and mesenchymal stromal cells, and plasma samples obtained from a cohort of 10 patients. Of note, everolimus-mediated inhibition of mTOR signaling is associated with basal state of phosphorylated STAT3. Finally, inhibition of mTOR-STAT3 pathway activation leads to normalization of IL-6 expression in SDS cells. Altogether, our data strengthen the hypothesis that SDS affects both lymphoid and myeloid blood compartment and suggest everolimus as a potential therapeutic agent to reduce excessive mTOR-STAT3 activation in SDS

Prev Chronic Dis

IntroductionObesity is highly prevalent among American Indians, and effective prevention efforts require caregiver involvement. We examined American Indian (AI) parents' assessment of and level of concern about their kindergarten child's weight status.MethodsWe collected baseline data (fall of 2005 and fall of 2006) on children and their parents or caregivers for a school-based obesity prevention trial (Bright Start) on an AI reservation in South Dakota. The current study uses 413 parent-child pairs. Age- and sex-adjusted body mass index percentiles were categorized as very underweight (<5th percentile), slightly underweight (5th to <15th percentile), normal weight (15th to <85th percentile), overweight (85th to <95th percentile), and obese ( 6595th percentile). Parents or caregivers reported their assessment of and concerns about their child's weight status as well as sociodemographic characteristics. We used mixed-model multivariable analysis to examine associations between sociodemographic characteristics and the probability of parents underclassifying or overclassifying their child's weight status; analyses were adjusted for school as a random effect.ResultsChildren were evenly divided by sex and had a mean age of 5.8 years. Twenty-nine percent of children and 86% of parents were overweight or obese. Approximately 33% (n = 138) of parents underclassified and 7% (n = 29) of parents overclassified their child's weight status. Higher parental weight status and higher concern about their child's weight status increased the probability of underclassification (P for trend = .02 for both).ConclusionIn this sample of at-risk children, one-third of parents underclassified their child's weight status. Childhood obesity prevention programs need to increase awareness and recognition of childhood obesity and address parental weight issues.2012649

Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

BACKGROUND: Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION: Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE: CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials

Activity of chemotherapy in mucinous ovarian cancer with a recurrence free interval of more than 6 months: results from the SOCRATES retrospective study

<p>Abstract</p> <p>Background</p> <p>Mucinous ovarian carcinoma have a poorer prognosis compared with other histological subtypes. The aim of this study was to evaluate, retrospectively, the activity of chemotherapy in patients with platinum sensitive recurrent mucinous ovarian cancer.</p> <p>Methods</p> <p>The SOCRATES study retrospectively assessed the pattern of care of a cohort of patients with recurrent platinum-sensitive ovarian cancer observed in the years 2000–2002 in 37 Italian centres. Data were collected between April and September 2005. Patients with recurrent ovarian cancer with > 6 months of platinum free interval were considered eligible.</p> <p>Results</p> <p>Twenty patients with mucinous histotype and 388 patients with other histotypes were analyzed. At baseline, mucinous tumours differed from the others for an higher number of patients with lower tumor grading (p = 0.0056) and less advanced FIGO stage (p = 0.025). At time of recurrence, a statistically significant difference was found in performance status (worse in mucinous, p = 0.024). About 20% of patients underwent secondary cytoreduction in both groups, but a lower number of patients were optimally debulked in the mucinous group (p = 0.03). Patients with mucinous cancer received more frequently single agent platinum than platinum based-combination therapy or other non-platinum schedules as second line therapy (p = 0.026), with a response rate lower than in non-mucinous group (36.4% vs 62.6%, respectively, p = 0.04). Median time to progression and overall survival were worse for mucinous ovarian cancer. Finally, mucinous cancer received a lower number of chemotherapy lines (p = 0.0023).</p> <p>Conclusion</p> <p>This analysis shows that platinum sensitive mucinous ovarian cancer has a poor response to chemotherapy. Studies dedicated to this histological subgroup are needed.</p

CDKN1B mutation and copy number variation are associated with tumor aggressiveness in luminal breast cancer

The CDKN1B gene, encoding for the CDK inhibitor p27kip1, is mutated in defined human cancer subtypes, including breast, prostate carcinomas and small intestine neuroendocrine tumors. Lessons learned from small intestine neuroendocrine tumors suggest that CDKN1B mutations could be subclonal, raising the question of whether a deeper sequencing approach could lead to the identification of higher numbers of patients with mutations. Here, we addressed this question and analyzed human cancer biopsies from breast (n = 396), ovarian (n = 110) and head and neck squamous carcinoma (n = 202) patients, using an ultra-deep sequencing approach. Notwithstanding this effort, the mutation rate of CDKN1B remained substantially aligned with values from the literature, showing that essentially only hormone receptor-positive breast cancer displayed CDKN1B mutations in a relevant number of cases (3%). However, the analysis of copy number variation showed that another fraction of luminal breast cancer displayed loss (8%) or gain (6%) of the CDKN1B gene, further reinforcing the idea that the function of p27kip1 is important in this type of tumor. Intriguingly, an enrichment for CDKN1B alterations was found in samples from premenopausal luminal breast cancer patients (n = 227, 4%) and in circulating cell-free DNA from metastatic luminal breast cancer patients (n = 59, 8.5%), suggesting that CDKN1B alterations could correlate with tumor aggressiveness and/or occur later during disease progression. Notably, many of the identified somatic mutations resulted in p27kip1 protein truncation, leading to loss of most of the protein or of its C-terminal domain. Using a gene-editing approach in a luminal breast cancer cell line, MCF-7, we observed that the expression of p27kip1 truncating mutants that lose the C-terminal domains failed to rescue most of the phenotypes induced by CDKN1B gene knockout, indicating that the functions retained by the C-terminal portion are critical for its role as an oncosuppressor, at least in luminal breast cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley &amp; Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

BACKGROUND: Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers. CONCLUSIONS/SIGNIFICANCE: Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers

Inflammasome-Mediated IL-1β Production in Humans with Cystic Fibrosis

Inflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF). Interleukin-1 beta (IL-1β) is a key inflammatory mediator. Secretion of biologically active IL-1β involves inflammasome-mediated processing. Little is known about the contribution of IL-1β and the inflammasomes in CF inflammatory disease. This study examines inflammasome-mediated IL-1β production in CF bronchial epithelial cell lines and human patients with CF.Bronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1β compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation. In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1β and strongly upregulated caspase-1 activity upon inflammasome stimulation. PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1β production when stimulated with inflammasome activators. This IL-1β production was dependent on NF-κB activity and could be enhanced by priming with LPS. Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1β or IL-8 production in response to P. aeruginosa.Hematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1β in CF. PBMCs derived from CF subjects display preserved inflammasome activation and IL-1β secretion in response to the major CF pathogen Pseudomonas aeruginosa. However, our data do not support the hypothesis that increased IL-1β production in CF subjects is due to an intrinsic increase in NF-κB activity through loss of CFTR function