Subgroup B Adenovirus Type 35 Early Region 3 mRNAs Differ from Those of the Subgroup C Adenoviruses

AbstractAdenovirus type 35 (Ad35) is a member of Ad subgroup B, DNA homology cluster B2. The B2 Ads are unique in that they are isolated most frequently from immunosupressed individuals such as AIDS patients and bone marrow transplant recipients and in that they have a tropism for the urinary tract. One region of the Ad genome which may influence serotype specific pathology is early region 3 (E3). E3 of subgroup C Ad2 and Ad5 has been shown to encode proteins which counteract the immune response to Ad infection. While a great deal is known about gene expression of the subgroup C Ad E3s, little is known about the E3 gene expression from the subgroup B Ads. Although some E3 open reading frames (ORFs) are shared between subgroups B and C, there are additional ORFs that appear in subgroup B. This paper demonstrates the results of an analysis of gene expression from the Ad35 E3 and describes differences in splicing and polyadenylation between the Ad35 and Ad2 E3s. RT–PCR, cDNA sequencing, RNase protection, 3′ RACE, and Northern blotting techniques were utilized to identify, quantify, and determine the structure of six Ad35 E3 mRNAs predicted to encode at least seven proteins. A common intron that is removed during splicing of the subgroup C E3 mRNAs is not removed from Ad35 E3 mRNAs, and only one E3 polyadenylation signal is present in the Ad35 E3 while two polyadenylation signals are used in the formation of subgroup C E3 mRNAs. The quantity of individual mRNAs encoding homologous proteins for Ad35 and Ad2 also differ substantially, presumably because of the absence in Ad35 ofcis-acting signals which have been shown to be important for regulation of Ad2 E3 pre-mRNA processing. Such information should contribute to an understanding of the role the E3 plays in determining subgroup B Ad pathogenesis in general and Ad35 pathogenesis in particular

Assessment of Hypoxia Inducible Factor Levels in Cancer Cell Lines upon Hypoxic Induction Using a Novel Reporter Construct

Hypoxia Inducible Factor (HIF) signaling pathway is important for tumor cells with limited oxygen supplies, as it is shown to be involved in the process of proliferation and angiogenesis. Given its pivotal role in cancer biology, robust assays for tracking changes in HIF expression are necessary for understanding its regulation in cancer as well as developing therapies that target HIF signaling. Here we report a novel HIF reporter construct containing tandem repeats of minimum HIF binding sites upstream of eYFP coding sequence. We show that the reporter construct has an excellent signal to background ratio and the reporter activity is HIF dependent and directly correlates with HIF protein levels. By utilizing this new construct, we assayed HIF activity levels in different cancer cell lines cultured in various degrees of hypoxia. This analysis reveals a surprising cancer cell line specific variation of HIF activity in the same level of hypoxia. We further show that in two cervical cancer cell lines, ME180 and HeLa, the different HIF activity levels observed correlate with the levels of hsp90, a cofactor that protects HIF against VHL-independent degradation. This novel HIF reporter construct serves as a tool to rapidly define HIF activity levels and therefore the therapeutic capacity of potential HIF repressors in individual cancers

Breast Cancer DNA Methylation Profiles Are Associated with Tumor Size and Alcohol and Folate Intake

Although tumor size and lymph node involvement are the current cornerstones of breast cancer prognosis, they have not been extensively explored in relation to tumor methylation attributes in conjunction with other tumor and patient dietary and hormonal characteristics. Using primary breast tumors from 162 (AJCC stage I–IV) women from the Kaiser Division of Research Pathways Study and the Illumina GoldenGate methylation bead-array platform, we measured 1,413 autosomal CpG loci associated with 773 cancer-related genes and validated select CpG loci with Sequenom EpiTYPER. Tumor grade, size, estrogen and progesterone receptor status, and triple negative status were significantly (Q-values <0.05) associated with altered methylation of 209, 74, 183, 69, and 130 loci, respectively. Unsupervised clustering, using a recursively partitioned mixture model (RPMM), of all autosomal CpG loci revealed eight distinct methylation classes. Methylation class membership was significantly associated with patient race (P<0.02) and tumor size (P<0.001) in univariate tests. Using multinomial logistic regression to adjust for potential confounders, patient age and tumor size, as well as known disease risk factors of alcohol intake and total dietary folate, were all significantly (P<0.0001) associated with methylation class membership. Breast cancer prognostic characteristics and risk-related exposures appear to be associated with gene-specific tumor methylation, as well as overall methylation patterns

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

An essential function for the ATR-Activation-Domain (AAD) of TopBP1 in mouse development and cellular senescence

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

Am I getting an accurate picture: a tool to assess clinical handover in remote settings?

BACKGROUND: Good clinical handover is critical to safe medical care. Little research has investigated handover in rural settings. In a remote setting where nurses and medical students give telephone handover to an aeromedical retrieval service, we developed a tool by which the receiving clinician might assess the handover; and investigated factors impacting on the reliability and validity of that assessment. METHODS: Researchers consulted with clinicians to develop an assessment tool, based on the ISBAR handover framework, combining validity evidence and the existing literature. The tool was applied 'live' by receiving clinicians and from recorded handovers by academic assessors. The tool's performance was analysed using generalisability theory. Receiving clinicians and assessors provided feedback. RESULTS: Reliability for assessing a call was good (G = 0.73 with 4 assessments). The scale had a single factor structure with good internal consistency (Cronbach's alpha = 0.8). The group mean for the global score for nurses and students was 2.30 (SD 0.85) out of a maximum 3.0, with no difference between these sub-groups. CONCLUSIONS: We have developed and evaluated a tool to assess high-stakes handover in a remote setting. It showed good reliability and was easy for working clinicians to use. Further investigation and use is warranted beyond this setting

Integrative analysis of RUNX1 downstream pathways and target genes

Background: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBF[Beta], and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBF[Beta]. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications

American Association of Plastic Surgeons Consensus on Breast Implant-Associated Anaplastic Large-Cell Lymphoma

BACKGROUND: In the absence of high-quality evidence, there is a need for guidelines and multidisciplinary consensus recommendations on breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL). The purpose of this expert consensus conference was to evaluate the existing evidence regarding the diagnosis and management of BIA-ALCL caused by textured implants. This article aims to provide evidence-based recommendations regarding the management and prevention of BIA-ALCL. METHODS: A comprehensive search was conducted in the MEDLINE, Cochrane Library, and Embase databases, and supplemented by manual searches of relevant English-language articles and related articles sections. Studies focusing on breast surgery and lymphoma associated with breast implants were included for analysis. Meta-analyses were performed and reviewed by experts selected by the American Association of Plastic Surgeons using a Delphi consensus method. RESULTS: A total of 840 articles published between January of 2011 and January of 2023 were initially identified and screened. The full text of 188 articles was assessed. An additional 43 articles were excluded for focus, and 145 articles were included in the synthesis of results, with 105 of them being case reports or case series. The analysis encompassed a comprehensive examination of the selected articles to determine the incidence, risk factors, clinical presentation, diagnostic approaches, and treatment modalities related to BIA-ALCL. CONCLUSIONS: Plastic surgeons should be aware of the elevated risks by implant surface type, implement appropriate patient surveillance, and follow the recommendations outlined in this statement to ensure patient safety and optimize outcomes. Ongoing research on the pathogenesis, genetic drivers, and preventative and prophylactic measures for BIA-ALCL is crucial for improving patient care