Graham Greene Human Factor Epub To Mobi
Sandrine Willert
Salwei, M.E., Hoonakker, P.L.T., Wiegmann, D., Pulia, M., Patterson, B.W., Carayon, P. (2023). Post-implementation usability evaluation of a human factors-based clinical decision support for pulmonary embolism (PE) diagnosis (Dx): PE Dx Study Part 1. Human Factors in Healthcare.
We utilize novel mouse models of SARS-CoV and MERS-CoV to identify the host response to infection while identifying pathways and proteins that regulate the response. In the past we have identified STAT1, EGFR and other wound healing factors that regulate disease severity for SARS-CoV. After the emergence of MERS-CoV in 2012 we rapidly developed a mouse model for the virus and have been identifying the host factors and immune response to MERS-CoV infection. Wildtype mice are not permissive to MERS-CoV infection due to the an amino acid difference in the binding site of the MERS-CoV Spike protein with its cell surface receptor Dipeptidyl Peptidase IV (DPP4). We found that the mouse DPP4 does not bind MERS-CoV Spike but the human DPP4 does. We quickly realized that to study MERS-CoV pathogenesis in mice we would have to create a mouse that expressed human DPP4 instead of mouse DPP4. We rapidly produced and characterized a mouse that had human DPP4 knocked-in to the mouse DPP4 loci, replacing the gene but retaining the mouse DPP4 promoter. This allowed for the correct expression kinetics of the human DPP4 in the mice. We found that MERS-CoV infected these mice, produced significant lung pathology and induced a unique inflammatory response in the mice, different from that of SARS-CoV. Using this model, we have further focused on the effects of diabetic comorbidity on disease severity. The reason for focusing on diabetes is because of the patients that have lethal MERS-CoV infection, the vast majority have preexisting comorbidities, the largest of which is Type 2 diabetes (T2D). We have modeled T2D in our MERS-CoV mouse model by feeding the mice a high fat diet for 12 weeks and find that MERS-CoV infection induces a more severe disease in these mice compared to normal mice. The T2D mice have slower inflammatory cell infiltration, enhanced epithelial cell hyperplasia at sites of infection and a skewed immune response. We are currently investigating the role of the MERS-CoV receptor, DPP4, in the T2D response and how its activity effects the immune response to the virus. One striking difference is that Type 2 alveolar cells are infected immediately after inoculation in the diabetic mice, whereas they take 4 days after inoculation to become infected in normal mice. We believe that the Type 2 alveolar cells are more permissive in the diabetic mice to MERS-CoV due to a difference in accessibility of the virus to the cell surface and of a reduced innate immune response of these cells in diabetic mice. Mucus and surfactant levels are reduced on the surface of the alveoli in diabetic mice, allowing for increased virus deposition deep into the lungs, altering their microenvironment and enhancing infection. The same phenomenon will affect not just MERS-CoV but also other respiratory viruses including Influenza virus, bacterial infections including S. aureus and fungal infections including Aspergillus sp. We believe this model will be critical in the future for determining the role of diabetes and other comorbidities in the host response to MERS-CoV and potentially other pathogens.
One or more alternative mechanisms for the lengthening of telomeres other than telomerase were identified spanning from yeast to human normal cells and tumors. Two types of ALT mechanisms are known in yeast [22], a Rad51-dependent mechanism, mediated by homology recombination [23], and a Rad51-independent mechanism mediated by break-induced replication (BIR) [24]. In human, ALT activity has been detected also in non neoplastic somatic cells [13] and in embryonic cells [14], and recently also in canine sarcomas [25], indicating that this mechanism is conserved among mammalians. Cancers that have a mesenchymal origin are reported to activate ALT more frequently, while epithelial cancers rely on telomerase reactivation/re-expression [26, 27]. As mesenchymal stem cells are known to express minimal or no detectable amounts of telomerase [28], and harbor less frequent TERT mutations [29], this may predispose them to depend on ALT activation more frequently. ALT are characterized by telomere associated PML bodies (called APBs) containing HR proteins, sheltering factors and heterochromatin associated proteins such as HP1 [30, 31]. Moreover, a specific phosphorylated isoform of TRF1 has been found associated with and required for APBs formation [32, 33]. Therefore, chromatin modification appears to be one of the key factors determining the choice between TA and ALT. In this regard, the presence of one or more epigenetic repressors determining the TA to ALT switch has been known from years [34]. One of the best candidate for this function has been recently identified in the alpha thalassemia/mental retardation syndrome X-linked protein (ATRX), death-domain associated protein (DAXX) and Histone 3.3 complex [35,36,37]. Nevertheless, the role of ATRX/DAXX and H3.3 is not completely clarified. ATRX is a chromatin remodeling protein that presents a SWI/SNF2-type ATPase/helicase and a plant homeodomain-like zinc finger. ATRX/DAXX complex localizes mainly in the nucleus and is associated with PML nuclear bodies and other subnuclear domains [38]. Functional studies shows that loss of ATRX function is necessary but not sufficient for activation of ALT [39]. Mechanicistically, it has been demonstrated that ATRX can bind and suppress R-loops at transcribed telomeres, which are more frequent in ALT [40], bind to MRN complex and contribute to the replication fork restart [41]. Recent evidence shows that ATRX knock down suppresses the NHEJ in favor of HR, contributing to the increase of replication defects and genomic instability [42,43,44], thus suggesting a possible mechanism of induction of ALT activity by ATRX loss of function. The Homology Recombination dependent ALT pathway in human cancer is a RAD51 mediated processes, which is similar to the yeast Type I ALT and requires the integrity of the MRN (MRE11-RAD50-NBS1) recombination complex [45, 46] (Scheme 1). In agreement with an epigenetic control in the predisposition to acquire a TA or ALT phenotype, ALT cells are characterized by overall less H3K9 and H4K20 trimethylation as well as more H3 and H4 acetylation at subtelomeric and telomeric regions. The mechanisms leading to chromatin decompaction in ALT involve the regulation of the DNMT and HDAC enzymes, the CHK1 kinase, as well as other chromatin remodelling factors reviewed in [47]. Several HR proteins were already known to be targeted by miRNA (acknowledged in [47]), although only recently, a direct role of miRNA in the TA/ALT switch has been demonstrated [48]. The different chromatin organization at subtelomeric regions lead ALT telomeres to be hyper-transcribed into long ncRNA transcripts called telomeric repeat-containing RNA (TERRA) [49]. TERRA have been implicated in the regulation of telomerase, in the formation of heterochromatin at telomeres, and in telomere stability [50]. Recently, Graf and coauthors revealed differential regulation of TERRA according to the cell cycle and to telomere length, uncovering an elegant feedback loop for telomere length maintenance [51]. Moreover, TERRA was found to bind to extra-telomeric chromatin and to influences the transcription of nearby genes; additionally, TERRA was found associated with a proteome involved in diverse processes, including chromatin remodeling and transcription [52]. TERRA R-loops forming at telomeres in yeast and human cells predispose telomeres to double-strand breaks and homology-directed repair (HDR) [53]. In some cases, HDR can drive telomere elongation and allow cells to escape senescence [54, 55]. This has led to speculation that TERRA can trigger the initiating events leading to alternative lengthening of telomeres (ALT).
CYP2B6 has been much studied as a classical CAR target gene, but the CAR target gene profile appears to be fairly overlapping with PXR (Kobayashi et al. 2015). No ChIP-Seq analysis revealing the CAR binding to human CYP genes has been published so far, although the human CAR interactome has been studied in a mouse model (Niu et al. 2018). Interestingly, this investigation showed that CAR targets several genes coding for other transcription factors including PXR and AHR introducing additional level of complexity to the induction mechanisms (Niu et al. 2018).
CYP2A6 is induced in humans in vivo by CAR, PXR, ERα, and NRF2 agonists (Tables 12, 13, 14). The regulation of CYP2A6 by ERα and NRF2 sets it apart as no other CYP enzyme is known to be regulated in vivo by these transcription factors. CYP2A6 is induced through ERα by phytoestrogens such as genistein (in legumes such as soybeans)(Y. Chen et al. 2011; Mazur 1998) and quercetin (in tea, vegetables, fruits, and berries) (Chen et al. 2009; Chun et al. 2012) as well as ethinyl estradiol of oral contraceptives (Benowitz et al. 2006; Berlin et al. 2007; Sinues et al. 2008). Exposure to cadmium measured as urine cadmium excretion is associated with CYP2A6 activity probed with coumarin 7-hydroxylation but only in non-smokers (Satarug et al. 2004a, b). In smokers, CYP2A6 activity is known to be reduced (inhibition) by an unknown mechanism (Hukkanen et al. 2005) and as smoking is also an important source of cadmium (induction), it is not surprising that smoking can confound the association between cadmium exposure and CYP2A6 activity. The effect of cadmium on CYP2A6 is most likely mediated by NRF2 as is the induction caused by sulforaphane present in cruciferous vegetables (Abu-Bakar et al. 2004; Yokota et al. 2011). All medications known to induce CYP2A6 are combined CAR/PXR activators and it is not known which nuclear receptor is more important for CYP2A6 induction in vivo as there is some evidence for the involvement of both (Itoh et al. 2006). Rifampicin treatment for 6 days had no effect on CYP2A6 activity measured as coumarin hydroxylation (Rautio et al. 1994) arguing against the role of PXR in the in vivo regulation.