Investigating the functional significance of the upregulation of Cyclin D2 and p21 following Apc loss in vivo

The Apc gene encodes the Adenomatous polyposis coli tumour suppressor protein, the germ line mutation of which characterizes Familial Adenomatous Polyposis (FAP), an autosomal syndrome characterized by multiple colorectal lesions. Inactivation of the Apc gene is recognized as a key early event in the development of colorectal cancers and leads to the deregulation of the Wnt pathway and the activation of TCF/LEF target genes. This project focuses on the proto-oncogene c-Myc as it is a key Wnt target gene which is activated following loss of Apc in vivo. This upregulation is noteworthy as c-Myc is implicated in stem cell survival, proliferation, apoptosis and tumourigenesis. Previous studies have shown c-Myc dependency for both apoptosis and proliferation following activation of the Wnt pathway, however little is known about the role c-Myc plays in inducing apoptosis following DNA damage in vivo. To study this I have conditionally deleted c-Myc from the intestinal epithelium and examined the response of intestinal enterocytes following DNA damage. Remarkably, following DNA damage, c-Myc deficient enterocytes were unable to upregulate p53 and induce apoptosis, which was mechanistically due to an upregulation of MDM2. Taken together, results from this study showed for the first time in vivo, a key role for c-Myc in inducing apoptosis following DNA damage through control of p53. Previous studies from this lab have shown that within the intestinal epithelium, c-Myc is absolutely required for the hyper-proliferative phenotype that is observed following loss of Apc. Therefore one of the key aims of this thesis is to look downstream of c-Myc in order to delineate how c-Myc induces and controls this proliferation. Given that one of the key postulated functions of c-Myc is the transcriptional repression of p21, this thesis examines this hypothesis by investigating the significance of the upregulation of p21 following c-Myc deletion in Apc deficient intestinal enterocytes. To do this, I have generated triple knockout (TKO) intestines by intercrossing p21 knockout mice to mice where we can conditionally delete both Apc and c-Myc within the murine intestinal epithelium. Surprisingly, the levels of proliferation were the same between double knockout Apc Myc and TKO intestines, which had markedly less proliferation than Apc deficient intestines. However, unlike double knockout enterocytes, TKO intestinal enterocytes no longer moved up the crypt-villus axis and failed to generate villus. To examine which of these phenomena were key to tumourigenesis (differentiation or proliferation), we investigated whether TKO intestines could form intestinal adenomas and found that even in the absence of p21, c-Myc deficient cells were unable to form tumours. Taken together we have identified a novel role for p21 in driving differentiation following Apc and Myc deletion. This is consistent with the expression of p21 in the normal crypt at the crypt villus junction. Remarkably this function of p21 is independent of its key role as a cell cycle inhibitor. Moreover, this study also examined the importance of the upregulation of the Cyclin D/CDK4 complexes following Apc loss and their role in c-Myc dependent proliferation. Results from these studies showed that Cyclin D2 is required for efficient proliferation immediately following loss of Apc as well as for tumourigeneis in the Apc Min/+ mouse. Taken together, results from these studies showed that the upregulation of Cyclin D2 and CDK4 are c-Myc dependent and that the upregulation of these complexes are key for Wnt driven proliferation and tumourigenesis. Lastly, in this study I have examined whether Apc loss within the intestinal epithelium, where it is a bona fide tumour suppressor gene, can provoke senescence, and compared this to the ability of Apc gene deletion to trigger senescence in the renal epithelium, where it is not mutated in human cancer. This study showed that deletion of Apc within the renal epithelium invoked a p21 dependent senescence response, and Apc deficient renal epithelial cells were cleared and very rarely initiated tumourigenesis. However, combined Apc and p21 gene deletion rapidly initiated tumourigenesis, with all mice developing renal carcinoma by 2 months of age. In contrast to Apc deficient intestinal epithelium, this process was unaffected by loss of c-Myc. However within the intestinal epithelium, deletion of Apc did not invoke senescence, but lead to a highly proliferative, p21 independent response. Combined Apc and p21 gene loss had no impact on either the short term phenotypes of Apc loss or upon tumourigenesis. Taken together these results show for the first time that Apc loss in vivo can invoke a senescence program but in a context dependent fashion. This implies escape from senescence is not a crucial pathway in colorectal cancers that are initiated by Apc loss, and goes to explain why renal carcinoma is not observed in FAP patients who are germline heterozygous for APC. Therefore the aims for this thesis are: • To investigate the role of c-Myc in inducing apoptosis within the intestinal crypt, and whether this is p21 dependent? • To investigate the role of p21 in causing senescence of Apc deficient cells, and whether this is c-Myc dependent? • To determine the functional importance of repression of p21 by c-Myc in Apc deficient cells. • To determine the significance of Cyclin D2 upregulation within Apc deficient cells

Incentivization Towards Advancement in Professionalism:Underlining the Impact and Importance of BSN Completion

Over 1000 Registered Nurses from across the United States will be surveyed in regards to their RN to BSN journey. These RNs have enrolled in Fort Hays State University’s (FHSU) RN to BSN program in the time since the IOM and RJWF report was released, and are either currently active in the degree completion process or have graduated. This study will allow us to determine if as a profession and a program, we are making an impact on the goals set forth by the IOM and RJWF report. Key concepts included in the study survey include motivation, incentivization, understanding, education, and professionalism

Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid

Objectives A number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells. Methods Fresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 h. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations and single-cell RNA-seq, and ELISA and LEGENDplex analysis of PsA SF were also performed. Results We observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be amongst those most highly upregulated by PsA monocytes/macrophages; and both proteins were elevated in PsA SF. Conclusions Using multiomic analyses we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance. Competing Interest Statement The authors have declared no competing interest

Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid

Objectives: A number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells. Methods: Fresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 hours. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations, single-cell RNA-seq, ELISA and LEGENDplex analysis of PsA SF were also performed. Results: We observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be among those most highly upregulated by PsA monocytes/macrophages in SF; and both proteins were elevated in PsA SF. Conclusions: Using multiomic analyses, we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance

Adversity in early life and pregnancy are immunologically distinct from total life adversity: macrophage-associated phenotypes in women exposed to interpersonal violence

Early childhood and pregnancy are two sensitive periods of heightened immune plasticity, when exposure to adversity may disproportionately increase health risks. However, we need deeper phenotyping to disentangle the impact of adversity during sensitive periods from that across the total lifespan. This study examined whether retrospective reports of adversity during childhood or pregnancy were associated with inflammatory imbalance, in an ethnically diverse cohort of 53 low-income women seeking family-based trauma treatment following exposure to interpersonal violence. Structured interviews assessed early life adversity (trauma exposure ≤ age 5), pregnancy adversity, and total lifetime adversity. Blood serum was assayed for pro-inflammatory (TNF-a, IL-1ß, IL-6, and CRP) and anti-inflammatory (IL-1RA, IL-4, and IL-10) cytokines. CD14+ monocytes were isolated in a subsample (n = 42) and gene expression assayed by RNA sequencing (Illumina HiSeq 4000; TruSeq cDNA library). The primary outcome was a macrophage-associated M1/M2 gene expression phenotype. To evaluate sensitivity and specificity, we contrasted M1/M2 gene expression with a second, clinically-validated macrophage-associated immunosuppressive phenotype (endotoxin tolerance) and with pro-inflammatory and anti-inflammatory cytokine levels. Adjusting for demographics, socioeconomic status, and psychopathology, higher adversity in early life (ß = .337, p = 0.029) and pregnancy (ß = .332, p = 0.032) were each associated with higher M1/M2 gene expression, whereas higher lifetime adversity (ß = −.341, p = 0.031) was associated with lower immunosuppressive gene expression. Adversity during sensitive periods was uniquely associated with M1/M2 imbalance, among low-income women with interpersonal violence exposure. Given that M1/M2 imbalance is found in sepsis, severe COVID-19 and myriad chronic diseases, these findings implicate novel immune mechanisms underlying the impact of adversity on health.publishedVersio

Inpatient Teleneurology Follow-up Has Comparable Outcomes to In-Person Neurology Follow-up

Background and Objectives Community emergency departments often transfer patients for lack of neurology coverage, potentially burdening patients and accepting facilities. Telestroke improves access to acute stroke care, but there is a lack of data on inpatient teleneurology and telestroke care. Methods From our prospective telestroke registry, we retrospectively reviewed 3702 consecutive patients who were seen via telestroke between September 2015 and December 2018. Patients who required transfer after initial telestroke evaluation or who were kept at hospitals without consistent neurology coverage were excluded from analysis. We compared baseline demographics, clinical characteristics, and hospital outcomes in patients who were subsequently followed remotely by a teleneurology neurohospitalist and those followed in person by a neurohospitalist. Results There were 447 (23%) patients followed by a teleneurology neurohospitalist and 1459 (77%) patients followed in person by a neurohospitalist. Both groups presented with similar stroke severity. In multivariate analysis, there were no significant differences in discharge disposition, stroke readmission rates, or 90-day modified Rankin Scale (mRS) scores. Length of stay was shorter with teleneurology follow-up. In the subgroup of patients who received tissue plasminogen activator, patients showed no differences in outcomes and had similar complication rates. Teleneurology follow-up resulted in a 3% transfer rate for higher level of care after admission. There remained no difference in outcomes in a subanalysis without Comprehensive Stroke Centers. A higher proportion of non-Hispanic Black patients and a lower proportion of Hispanic patients in the teleneurology follow-up group were possibly due to spoke location demographics. Discussion Teleneurology follow-up resulted in comparable outcomes to in-person neurology follow-up, with few transfers after admission. For select neurology and ischemic stroke patients, teleneurology follow-up provides an alternative to transfer for hospitals lacking neurology coverage

Summit

The UAS Writing Center dedicates this journal to Dr. Michael Collins and Dr. Andrea Lunsford, who created and inspired this project.It is with great enthusiasm that the UAS Writing Center launches our inaugural edition of Summit: The UAS Writing Center’s Collection of Exceptional Academic Works for 2019/2020. In the Writing Center, we have the privilege of seeing the wide range of talents and interests of our UAS students and the exceptional way that they communicate their research, analysis, and philosophical ruminations through essays, speeches, and written reports. The tutors in the Writing Center engage with their peers in fascinating discussions of historical events, political debates, scientific discoveries, teaching pedagogy, and business theory (to name a few). We celebrate when students receive stellar grades on their assignments, but we also recognize that good writing should be shared beyond the classroom. Summit emerged from a desire to highlight and publish the extraordinary writing that we get the honor to see in the Writing Center every day. This journal, however, is more than just a showcase of undergraduate writing. It has also been an opportunity for the undergraduate student tutors to engage in a collaborative project that required time management, careful communication, attention to detail and leadership skills. The editors and contributors have persevered through unimaginable (until now) challenges including unprecedented budget cuts at the university and a global pandemic that forced us to move our work online. At a time when the present feels chaotic and worrying, the undergraduate students at UAS remind me that our future is in good hands. I am confident in our new generation of leaders as I’ve watched our students think critically, engage in the community, and work tirelessly for projects they believe in. It is joy to present our first edition of Summit! Allison Neeland UAS Writing Center SpecialistSpecial thanks to: OUR FUNDERS UAS Student Government, Fall 2019 OUR JUDGES Thank you to our team of faculty and student judges OUR SUPPORTERS Provost Karen Carey Kelly Jensen Alison Krein Karen Mitchell Allison Neeland Elise Tomlinson And, of course, a huge thank you to the students of UAS for supporting our journal through their submissions. We look forward to the future of this journal.Contents Forward ........................................................................................................................... 1 Ernestine Hayes Award for Excellence in Academic Writing ........................................ 2 Veterans, Veracity, and Depictions of Violence: An Examination of All Quiet on the Western Front, Germany’s Interwar Period, and the Interplay Between the Two .......... 4 The Influence of German Societal Changes and Remarque’s Im Westen nichts Neues During the Interwar Era................................................................................................. 13 Estrogenic Endocrine Disrupting Chemicals as a Water Pollutant: Sources, Effects and Degradation ................................................................................................................... 21 Presence and Effects of Per- and Poly- Fluoroalkyl Substances in the Environment ... 32 A Study in Milk ............................................................................................................. 44 Acknowledgements Page .............................................................................................. 55Ye

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity

Perspective and priorities for improvement of parathyroid hormone (PTH) measurement – A view from the IFCC Working Group for PTH

Parathyroid hormone (PTH) measurement in serum or plasma is a necessary tool for the exploration of calcium/phosphate disorders, and is widely used as a surrogate marker to assess skeletal and mineral disorders associated with chronic kidney disease (CKD), referred to as CKD-bone mineral disorders (CKD-MBD). CKD currently affects >10% of the adult population in the United States and represents a major health issue worldwide. Disturbances in mineral metabolism and fractures in CKD patients are associated with increased morbidity and mortality. Appropriate identification and management of CKD-MBD is therefore critical to improving clinical outcome. Recent increases in understanding of the complex pathophysiology of CKD, which involves calcium, phosphate and magnesium balance, and is also influenced by vitamin D status and fibroblast growth factor (FGF)-23 production, should facilitate such improvement. Development of evidence-based recommendations about how best to use PTH is limited by considerable method-related variation in results, of up to 5-fold, as well as by lack of clarity about which PTH metabolites these methods recognise. This makes it difficult to compare PTH results from different studies and to develop common reference intervals and/or decision levels for treatment. The implications of these method-related differences for current clinical practice are reviewed here. Work being undertaken by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to improve the comparability of PTH measurements worldwide is also described