Overcoming challenges on an international project to advance systems engineering

The Body of Knowledge and Curriculum to Advance Systems Engineering (BKCASE) project's dual product development cycle spanned a three‐year period from the September 2009 to December, 2012. During this timeframe, BKCASE authors met quarterly at various locations, primarily in various regions of the United States, but also in Stockholm, Sweden; Toulouse, France; London, England; and Rome, Italy (BKCASE, 2009–2019). The team successfully worked through challenges and differences to produce The Guide to the Systems Engineering Body of Knowledge (SEBoK) wiki and a Graduate Reference Curriculum for Systems Engineering (GRCSE) publication. This article is a collection of personal stories from the team members that focus on overcoming obstacles to successfully produce the final published products

Neutrophil Extracellular Traps in Breast Cancer and Beyond: Current Perspectives on NET Stimuli, Thrombosis and Metastasis, and Clinical Utility for Diagnosis and Treatment

Abstract The formation of neutrophil extracellular traps (NETs), known as NETosis, was first observed as a novel immune response to bacterial infection, but has since been found to occur abnormally in a variety of other inflammatory disease states including cancer. Breast cancer is the most commonly diagnosed malignancy in women. In breast cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET-targeted therapies have shown success in preclinical cancer models and may prove valuable clinical targets in slowing or halting tumor progression in breast cancer patients. We will briefly outline the mechanisms by which NETs may form in the tumor microenvironment and circulation, including the crosstalk between neutrophils, tumor cells, endothelial cells, and platelets as well as the role of cancer-associated extracellular vesicles in modulating neutrophil behavior and NET extrusion. The prognostic implications of cancer-associated NETosis will be explored in addition to development of novel therapeutics aimed at targeting NET interactions to improve outcomes in patients with breast cancer

Regulation of Parkinson’s Disease-Associated Genes by Pumilio Proteins and MicroRNAs in the Human Neuroblastoma Cell Line SH-SY5Y

Parkinson’s disease (PD) is the second most common age-related, neurodegenerative disease. A small collection of genes has been linked to PD, including SNCA encoding the protein alpha-synuclein that aggregates in Lewy bodies, a hallmark of PD. Overexpression of these genes can lead to pathogenesis, yet the regulatory mechanisms that control protein production of the genes are not fully understood. The SH-SY5Y neuronal cell line is a common model for the study of neurodegenerative diseases, including PD. However, SH-SY5Y cells are difficult to transfect, a process necessary for genetic manipulations and downstream analysis of RNA and protein. Herein, I describe the assessment, successful optimization, and protocols for several techniques for use with SH-SY5Y cells. Research into the etiology of PD is a critical area of need. The research presented here provides evidence for the regulation of PD-associated genes by human Pumilio (PUM) proteins. PUM proteins belong to the PUF family of proteins, which are RNA-binding proteins that post-transcriptionally regulate gene expression through RNA binding motifs in the 3’ untranslated region (UTR) known as PUF Recognition Elements (PREs). The 3’UTRs of LRRK2, SNCA and SAT1 each contain multiple putative PREs. Knockdown (KD) of the two human PUF homologs, PUM1 and PUM2, in the SH-SY5Y neurodegenerative model cell line resulted in increased SNCA and LRRK2 mRNA, as well as alpha-synuclein levels, suggesting these genes are normally repressed by the PUM proteins. Some studies have indicated a relationship between PUM and microRNA activities on the same target, especially when their binding sites are close together. LRRK2, SNCA, and SAT1 3’UTRs each contain putative microRNA-binding sites, many near PREs. Small RNA-seq and microRNA qPCR assays were performed in wild type and PUM KD SH-SY5Y cells to analyze global and differential microRNA expression. Of the 1404 miRs determined to be expressed in SH-SY5Y, 21 microRNAs were differentially expressed, six of which were previously established to be altered in PD patient samples or research models. Collectively, these results demonstrate that PUMs and microRNAs play a multi-faceted role in regulating PD-associated genes

Known and novel miR raw read counts.

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Primers for RT-qPCR and site-directed mutagenesis.

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PREs in host genes of differentially expressed miRs.

PREs in host genes of differentially expressed miRs.</p

Known mature and novel miRNAs predicted by miRanda to target <i>PUM1</i>, <i>PUM2</i>, <i>SNCA</i>, <i>LRRK2</i>, and <i>SAT1</i>.

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Directed acyclic graphs for GO top 20.

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PRE and microRNA binding sites in the 3’UTRs of <i>LRRK2</i>, <i>SNCA</i>, and <i>SAT1</i>.

The black lines are a linear representation of the 3’UTRs of SAT1, SNCA, and LRRK2 transcripts. The number 1 indicates the nt immediately following the stop codon (5’ end of the 3’UTR). 3’UTR lengths for LRRK2 and SAT1 were based on transcript data from NCBI and 3’UTR length for SNCA was based on NCBI data as well as empirical data from expressed sequence tags [22]. Motifs with the sequence UGUA(A/U/C)AUA are considered canonical PREs and their locations on the 3’UTRs are represented by black boxes. Motifs that do not match the canonical sequence but contain a UGUA with downstream AU-rich region are considered non-canonical and are represented by gray boxes. The relative locations of predicted miR-binding sites are represented by red arrows. MiRs confirmed to be expressed in SH-SY5Y cells using sRNA-seq are bolded. MiRs with an asterisk (*) were determined to be differentially expressed based on DEseq2 analysis. MiR family conservation is based on TargetScanHuman 8.0 categorizations [71]. Broadly conserved miR families, those conserved among vertebrates, are represented by red text. All other levels of conservation are represented by black text. 8mer sites are highlighted in blue and 7mer-m8 sites are highlighted in green. 3’UTRs and predicted sites are not drawn to scale.</p

Differential expression analysis.

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