Coronary artery anomalies are rare congenital variants of coronary artery anatomy accounting second most common cause of sudden cardiac death in young competitive athletes. A single ostium coronary artery anomalous is an extremely rare variant with an incidence of less than 0.004%. They may present as chest pain, arrhythmia, or sudden death. Recently, advanced imaging techniques such as computed tomography and magnetic resonance imaging coronary angiography are becoming the alternatives investigation for diagnosis. We reported a rare case of 50 years old lady who presented with acute chest pain with normal electrocardiography, echocardiography, and cardiac markers. Coronary Computed tomography angiography revealed anomalous coronary artery anatomy with both right and left coronary artery arising from the large common trunk of the right coronary cusp, left main coronary artery having trans-septal course, there was no flow-limiting coronary artery disease. She was medically managed with a single antiplatelet, beta-blocker, and statin therapy. Recently, advanced imaging techniques such as computed tomography (CT) coronary angiography and magnetic resonance imaging coronary angiography are becoming the alternatives investigation for diagnosis.3Coronary artery anomalies and variants are rare congenital disorders of coronary artery anatomy. It accounts for the second most common cause of sudden cardiac death in young competitive athletes.We report a rare case of 50 years old female diagnosed as an anomalous coronary artery with left and right coronary artery arising from a single common trunk-off the right coronary cusp.We report a case of 50 years old female who visited the emergency department with a chief complaint of acute chest pain. She is a known case of hypothyroidism under thyroxine. However, she had no history of diabetes and hypertension. She is a non-smoker and non-alcoholic. On general physical examination, her vitals were stable with pulse rate 82 beats/ minute, blood pressure 100/80mmHg and systemic examination and laboratory investigations were within normal limit. Her ECG finding was normal; echo finding was normal left ventricular systolic and diastolic function with a normal left ventricular ejection fraction of 60%. Then, she was planned for CT coronary angiography in our center. It revealed anomalous coronary anatomy with both right and left coronary artery arising from large common trunk-off the right coronary cusp, left main coronary artery has a trans-septal course, there was no flow-limiting coronary artery disease , 2 and 3She was medically managed and discharged with single antiplatelet therapy, beta-blockers, and statin per-oral medications.4 Single ostium coronary artery is an extremely rare anomaly with a reported incidence of 0.04% of case of coronary angiography.5 Among different variants of anomalous coronary artery origins from the contralateral sinus of Valsalva, right-sided origin of left main coronary and left-sided RCA are the most common anomalies.6 For most variants of aberrant coronary origin, clinical significance and potential complications depend on its course. Depending upon the course of AOCA, the coronary arteries have 5 potential paths: pre-pulmonic, retro-aortic, intra-arterial, retro-cardiac, and trans-septal. In a trans-septal variant, the anomalous vessel passes through the septal muscle inferior to the plane of pulmonic valve giving off septal branches along the intra-myocardial course, whereas the anomalous vessel courses at the level of the pulmonic valve in the inter-arterial variant.7 Among various described courses of the coronary arteries, as they relate to the ascending aorta and pulmonary artery, the inter-arterial and trans-septal courses are clinically significant.Anomalous aortic origin of a coronary artery (AOCA) is a congenital abnormality of the origin arising from the sinus of Valsalva.6According to the coronary angiography done in 1,26,595 cases, the trans-septal variant was the most common in patients with the ectopic right-sided origin of the left coronary artery and right-sided single coronary types as reported in our case.7 CT angiography revealed single coronary artery arising from the right coronary sinus and giving rise to right coronary artery (RCA) and left main artery (LMA), LMA passes through septal myocardium and giving rise to left anterior descending and left circumflex arteries, RCA had a typical course within the right atrioventricular groove. Nevertheless, there was no evidence of atherosclerotic or calcific plaque despite angina symptoms. The average age of symptomatic presentation was 35 years, the oldest reported of 83years old, in our case the patient became symptomatic at the age of 50 years old. Additionally, a stress test may assist in stratifying the risk of future cardiac ischemic events in anomaly patients.7It has been stated that the abnormal origin and course of anomalous coronary arteries could make them more prone to atherosclerosis. Occasionally, they may be associated with sudden death due to compression by the aorta and pulmonary artery. According to Villa et al, approximately 15% of such cases can develop myocardial ischemia without atherosclerosis as reported in our case,The use of CT angiography to define this anomaly gives scope for clinicians to perform long term follow up and possibilities to test anomalies functionally to judge hemodynamic significance. In this case, CT angiography not only diagnosed the anomaly of the coronary artery it also prevented the invasive method .In conclusion, we report a rare anomaly of the common origin of right and left coronary from a single coronary ostium at the right sinus of Valsalva with a trans-septal course  course of the left coronary artery. These patients might be asymptomatic or may have events of sudden cardiac death. Patients are advised to avoid strenuous physical exertion.JNMA Case Report Consent Form was signed by the patient and the original article is attached with the patient's chart.


Kinases are an intensively studied drug target class in current pharmacological research as evidenced by the large number of kinase inhibitors being assessed in clinical trials. Kinase-targeted therapies have potential for treatment of a broad array of indications including central nervous system (CNS) disorders. In addition to the many variables which contribute to identification of a successful therapeutic molecule, drug discovery for CNS-related disorders also requires significant consideration of access to the target organ and specifically crossing the blood-brain barrier (BBB). To date, only a small number of kinase inhibitors have been reported that are specifically designed to be BBB permeable, which nonetheless demonstrates the potential for success. This review considers the potential for kinase inhibitors in the context of unmet medical need for neurodegenerative disease. A subset of kinases that have been the focus of clinical investigations over a 10-year period have been identified and discussed individually. For each kinase target, the data underpinning the validity of each in the context of neurodegenerative disease is critically evaluated. Selected molecules for each kinase are identified with information on modality, binding site and CNS penetrance, if known. Current clinical development in neurodegenerative disease are summarized. Collectively, the review indicates that kinase targets with sufficient rationale warrant careful design approaches with an emphasis on improving brain penetrance and selectivity. The protein kinase gene family comprises over 500 members and constitutes approximately 2% of all human genes. Protein kinases catalyze the transfer of the terminal phosphate group of adenosine triphosphate (ATP) onto an amino acid residue  within a polypeptide chain. Up to 20% of all human proteins may be modified by kinase activity which results in a functional change of the target protein by altering cellular activity, location or association with other proteins. Consequently, protein kinases regulate a range of cellular functions through the orchestrated propagation and amplification of cellular stimuli into distinct biological responses through coordinated signal transduction cascades . AberranThe catalytic subunits of protein kinases are highly conserved and over 200 structures have been solved. Protein kinases share a common bilobal-fold comprising a predominantly beta-sheet N-terminal and a helical C-terminal domain linked by a hinge polypeptide with a canonical catalytic (and ATP binding) site residing in the cleft between these two domains . InhibitIt can thus be appreciated that kinases are eminently “druggable” using small molecule approaches but still present several challenges including ATP competition (for type 1 inhibitors), selectivity, physicochemical properties, and intellectual property. Most kinase-targeted drugs developed have been for non-CNS indications such as cancer and inflammation and have not been optimized with respect to CNS exposure. Small molecule drugs have a proven track record with respect to achieving CNS penetration, but they need to be designed with certain properties to be permeable to traverse the endothelial membranes while avoiding the efflux transporters . ReducedThis review focuses on the challenges of targeting protein kinases for CNS disease indications with in-depth analyses on selected case studies for the underlying biological rationale. Here, we discuss in depth eleven protein kinase targets that have been progressed into clinical assessment for neurodegenerative disease indications from 2008 to 2018. These comprise ABL1, CSNK1D, CSF1R, MAP3K12, GSK3B, MAPK10, LRRK2, MAPK14, MTOR, RIPK1 and ROCK, discussed in the following sections and summarised in ABL1 gene reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits and protects against MPTP  challenges . Bosutinib is also in the clinic for “degenerative dementias” (NCT02921477), but no brain or plasma quantification data have been reported. Arguably, bafetinib (INNO-406) may be a better molecule to test the hypothesis that c-Abl mediates PD pathogenesis given the potentially improved CNS exposure  . Recentl of <0.1 . Recentlexposure . K-0706 The biological rationale for ABL1 is reasonable, particularly in PD with multiple biological substrates of the kinase being genetically linked and mechanistically connected to pathways of relevance to the disease. However, the molecules developed to date have suboptimal levels of CNS penetration and limited selectivity which may ultimately impact efficacy and tolerability since systemic exposures are likely to be in excess of those needed for CNS target engagement. Thus, more careful evaluation with suitably brain penetrant, potent, selective, and potentially better tolerated molecules may be warranted before the long-term therapeutic utility of selective ABL1 inhibitors in neurodegenerative patient populations can be fully ascertained.in vitro . Pfizer has reported no further development since October 2018 but Biogen has recently acquired the molecule with a $75 million upfront payment presumably to pursue this further in AD populations (January 2020)in vivo models; particularly in TDP43-related disorders which has seen the most effort to date. Pfizer has developed molecules rationally for selectivity, potency and brain penetrance which appear to be well tolerated in the clinic as far as we can currently ascertain. Thus, it would seem further assessment of these molecules in ND populations may be warranted.The biological rationale for this kinase in ND disorders would be significantly augmented via additional investigation in relevant Microglial cells have been described as the master regulators of the neuroinflammatory response associated with neurodegenerative disease. In numerous preclinical models of neurodegenerative disease, and in post-mortem brain samples from AD patients, an increase in microglia number and their proliferation rate has been observed . This prColony stimulating factor 1 receptor has two known ligands, CSF1 (colony stimulating factor 1) which is also expressed in microglia and IL-34 predominantly expressed in neurons . Ligand Structure-based drug development has been leveraged in some cases to rationally design CSF1R inhibitors including pexidartinib (PLX3397), GW2580, KI20227, AZD7507; JNJ-40346527, BLZ945/buparlisib or a set of DFG-out inhibitors . These mThe broad-spectrum class III RTK inhibitor, masitinib, is currently in phase III for ALS   and has While multiple data streams have indicated neuroinflammation and microglial activation as pathophysiological features of neurodegenerative disorders, it is not clear at what stage in the disease neuroinflammatory process would be the most appropriate point for intervention to lead to clinical benefit. There remains a question of when this detrimental effect of microglia may become relevant and consequently when would be an appropriate time to treat with this mechanism. The molecules developed and tested to date have suboptimal levels of CNS penetration and/or limited selectivity which again may impact the interpretation of the preclinical efficacy and ultimately clinical tolerability. That said there are preclinical data demonstrating a clear effect of CSF1R inhibitors . The strDual leucine zipper kinase, a mitogen-activated protein kinase kinase kinase, functions as an injury sensor that initiates the c-Jun N-terminal kinase (JNK)-dependent stress response in neurons to mediate context-dependent axon re- and degeneration . DLK actSmall molecule DLK inhibitors that reduced c-Jun phosphorylation in mouse models were initially identified following high throughput screening approaches and refined using a shape-based scaffold hopping approach , b. StruGenentech’s DLK inhibitor molecule is currently in PhI for ALS (NCT02655614).This case study represents a clear example of how structural and medicinal chemistry know how has been applied to actively develop a kinase inhibitor with CNS penetrance. It remains to be seen whether the preclinical assessment was sufficient to mitigate the risks associated with apparent context-dependent effects of DLK inhibition as the published data may suggest a narrow therapeutic index with potential toxicology concerns.GSK3B codes for two isoforms of a constitutively active serine/threonine kinase which phosphorylates over 40 substrates with key roles in Wnt signaling, insulin signaling, glycogen metabolism, development, mitotic regulation, inflammation (particularly the innate immune response), neurotransmitter and neurotrophic factor signaling and microtubule dynamics  in question. Analysis of the GSK3β structure as an unphosphorylated apo-enzyme together with Y216 monophosphorylated enzyme complexed with a peptide provided insight into both its regulation of the kinase activity and its preference for pre-phosphorylated substrates . DiverseDespite extensive promising preclinical data, GSK3β inhibitors have rarely reached clinical trials . MoleculPublished data suggests GSK3β inhibition may have therapeutic benefit across a range of neurodegenerative diseases. Many small molecule inhibitors have been generated, of which some appear to have a reasonable degree of CNS penetration. Toxicity may be one potential issue, likely due in part to the essential role of the enzyme in many fundamental physiological pathways, which has limited or prevented further exploration. Nonetheless, two molecules have been identified as being sufficiently safe for clinical investigation but have not yielded positive data for CNS indications.in vivo model phenotypes  family and regulate serine/threonine phosphorylation of several transcription factors in response to various stimuli, e.g., endoplasmic reticulum stress / unfolded protein response signaling, oxidative and excitotoxic stress, DNA damage, neurotoxins, cytokines, and fatty acids . Activatenotypes . Increasenotypes . JNK3, tenotypes . Kuan etenotypes , 2013. Cenotypes . Finallyenotypes .in vivo models (The JNK-interacting protein 1 (JIP1) derived peptide inhibitor  has the advantage of comparative specificity for JNK and is fused to the Tat peptide sequence to enable cellular delivery across the BBB . Peptideo models . More reo models . In addio models .Several ATP-competitive JNK inhibitors have been developed with a range of characteristics including good CNS penetration; only three are mentioned here as illustrations of their class. AS602801 (bentamapimod) is a pan-JNK inhibitor with good bioavailability and able to cross the BBB . SP60012apo and in complex with ligands or peptides has suggested the possibility for allosteric interactions between the catalytic and peptide binding sites and the A-loop  has been proposed to mediate synaptic function, proteostasis and endolysosomal function in response to stress signals and indeed, it is likely that there is cross-regulation between the p38 and JNK pathways [reviewed in in vivo .While potency, selectivity and SAR data is available for several individual p38α inhibitors; Astolfi and colleagues were the first to systematically assess these from a structural perspective [reviewed in Neflamapimod (VX745) was discovered through structure-based design at Vertex and previously assessed in rheumatoid arthritis in 2001. The molecule appears to be tolerated up to 125 mg twice daily for 12 weeks . As a reThe notion of MAPK pathway involvement in neurodegenerative disease is broadly supported in the literature. However, targeting these pathways are challenging due to (1) “crossover” between parallel pathways and (2) achieving selectivity and sufficient CNS exposure without notable systemic levels for a small molecule against any kinase in this pathway, including p38α. No drugs targeting this kinase are on the market yet for any indication, including chronic obstructive pulmonary disease, rheumatoid arthritis and corticosteroid-resistant asthma. Indeed, more than a dozen distinct molecules have failed in the clinic at least partly due to hepatotoxicity and development of tolerance. However, the reported favorable profile of neflamapimod and multiple ongoing clinical studies will hopefully inform whether this mechanism has therapeutic benefit.The serine/threonine kinase MTOR is a regulatory nexus for cell growth and metabolism in response to nutrients, growth factors and cellular energy conditions modulating autophagy, glucose metabolism, protein synthesis, and mitochondrial functions. MTOR is the catalytic subunit of two structurally and functionally distinct complexes with respect to protein composition: the rapamycin sensitive mTORC1 which functions as a nutrient sensor activated by insulin, growth factors and amino acids; and mTORC2 which has a role in cytoskeleton organization. Under nutrient-replete conditions, mTORC1 as an autophagy repressor via phosphorylation of ULK1, ATG13, ATG14L, and TFEB, key positive regulators of autophagy; and a positive regulator of protein synthesis via phosphorylation of S6K and eIF4E BP1-4 thus promoting global and cap-end translation . More reA consistent observation across neurodegenerative disease pathophysiology is the occurrence of specific misfolded proteins. These proteins ultimately appear as aggregates/inclusions that are proposed to infringe on normal cellular function including impaired protein clearance either by the ubiquitin-proteasome pathway or the lysosomal degradation pathway (including autophagy) [reviewed in The allosteric mTORC1 inhibitor rapamycin  was the first drug to be identified as an autophagy inducer through its interaction with FKBP12 which then binds to and inhibits the kinase activity of mTORC1 . CrystalRapamycin and its analogs have been used for many years in organ transplant patients , inflammation (via p65 and cFos) and cell death pathways (apoptosis and necroptosis but not necrosis) . For necin vivo . Similarin vivo .Multiple small molecule inhibitors for RIPK1 inhibition have been developed with a range of binding modes. Allosteric binders such as Nec-1s are precedented in addition to ATP competitive molecules such as GSK2982772 . The strDenali has announced that they have completed recruitment for assessment of DNL747, a claimed potent, oral dosed brain penetrant RIPK1 inhibitor, in ALS (NCT03757351) and AD (NCT03757325) populations. These studies should read out in mid-2020. Another Denali molecule, DNL104, a BBB-penetrant RIPK1 inhibitor has been reported to modulate pharmacodynamic markers in a healthy volunteer trial . GSK2982Although the rationale for this mechanism in necroptosis pathways seems reasonable, the clear weakness is the lack of clarity around the role this process plays in ongoing neurodegeneration; and whether blocking what is presumably quite a late stage cell death process will have real beneficial therapeutic consequences to the patient.Actin is one of three key components of the neuronal cytoskeleton and contributes to maintaining the axonal and somatodendritic compartments: the others being intermediate filaments and microtubules. A clear connection between cytoskeleton damage and neurodegeneration has emerged over the past few years such as actin cytoskeleton dysregulation and axonal transport defects [microtubule dependent and independent; reviewed in Several ROCK inhibitors have been developed in the past few decades that are predominantly ATP-competitive type 1 inhibitors exemplified by fasudil, the closely related ripasudil/K-115 and the chemically divergent Y-27632 . HoweverFasudil and ripasudil are already in use or in clinical trials for pathological conditions including glaucoma, cerebral vasospasm, hypertension, atherosclerosis, and aortic stiffness . DespiteAs for many other kinases, systemic inhibition of ROCK is expected to result in significant side effects. A molecule that avoids the blood pressure liabilities would be of great interest for clinical assessment in neurodegenerative disorders, however, to date this has proved very challenging.The rationale behind protein kinases as relevant targets for CNS drug discovery is aligned with the appreciation that many cellular processes are tightly regulated by reversible phosphorylation processes; and that at least a number of these may become aberrant in disease . Interespu,u) data together with confirmation of target engagement with clinical relevance such as PET (The greatest challenge facing any CNS-targeted drug is effective penetration of the BBB and this is certainly not restricted to kinase inhibitors . At leash as PET . Indeed,Another consistent theme in this review is the cross-reactivity seen with a range of kinase inhibitors although this is improving with kinase panel screening activities to assess selectivity . CautionStructurally guided approaches and/or fragment-based drug discovery are key approaches that can be leveraged which address many of these challenges. Kinases are eminently amenable to x-ray crystallography to generated detailed structural data that, together with biophysical and bioinformatic approaches, can be effectively leveraged to understand the three-dimensional binding mode of ligands and inform the rational optimization into potent and selective lead compounds with CNS drug-like properties . StructuCB and LD collected and reviewed the literature, wrote, and critically reviewed. Both authors contributed to the article and approved the submitted version.CB is a paid employee of Dementia Discovery Fund Ltd., as the CSO of LoQus23 Therapeutics. LD is a paid employee of Cerevance Ltd.


The vertebrate clade diverged into Chondrichthyes  and Osteichthyes fishes (bony fishes) approximately 420 mya, with each group accumulating vast anatomical and physiological differences, including skin properties. The skin of Chondrichthyes fishes is covered in dermal denticles, whereas Osteichthyes fishes are covered in scales and are mucous rich. The divergence time among these two fish groups is hypothesized to result in predictable variation among symbionts. Here, using shotgun metagenomics, we test if patterns of diversity in the skin surface microbiome across the two fish clades match predictions made by phylosymbiosis theory. We hypothesize (1) the skin microbiome will be host and clade-specific, (2) evolutionary difference in elasmobranch and teleost will correspond with a concomitant increase in host-microbiome dissimilarity, and (3) the skin structure of the two groups will affect the taxonomic and functional composition of the microbiomes.We show that the taxonomic and functional composition of the microbiomes is host-specific. Teleost fish had lower average microbiome within clade similarity compared to among clade comparison, but their composition is not different among clade in a null based model. Elasmobranch’s average similarity within clade was not different than across clade and not different in a null based model of comparison. In the comparison of host distance with microbiome distance, we found that the taxonomic composition of the microbiome was related to host distance for the elasmobranchs, but not the teleost fishes. In comparison, the gene function composition was not related to the host-organism distance for elasmobranchs but was negatively correlated with host distance for teleost fishes.Our results show the patterns of phylosymbiosis are not consistent across both fish clades, with the elasmobranchs showing phylosymbiosis, while the teleost fish are not. The discrepancy may be linked to alternative processes underpinning microbiome assemblage, including possible historical host-microbiome evolution of the elasmobranchs and convergent evolution in the teleost which filter specific microbial groups. Our comparison of the microbiomes among fishes represents an investigation into the microbial relationships of the oldest divergence of extant vertebrate hosts and reveals that microbial relationships are not consistent across evolutionary timescales.Video abstract Phylogenetically diverse microorganisms  colonize living surfaces. These organisms collectively form the microbiome, which is involved in processes such as host development , host nupCO2 [Phylosymbiosis is reported more often from studies investigating internal compartments of the organism, such as the gut of mammals and the plant root endosphere, than from external surfaces, such as leaf and skin surfaces . ExternapCO2 , 25.Gadus morhua), the sampling location affected microbiome structure [Alopias vulpinus) was species-specific. There was higher microbiome similarity among individual thresher sharks, compared to individuals of another host and the surrounding seawater [Carcharhinus melanopterus) [The most abundant group of marine vertebrates is the fishes, including Chondrichthyes (cartilaginous) and Osteichthyes (bony or ray-finned) fishes. These fishes diverged approximately 420 mya , and thetructure . Within seawater . Similaropterus) . These ropterus) . This reThe skin of marine organisms is a dynamic interface with constant exposure to the surrounding environment, and therefore, predicting processes that govern microbiome assemblage in this space is complex. We developed a sampling framework to identify whether the skin microbiome from elasmobranch and teleost fishes exhibit phylosymbiosis, amidst the web of possible alternative drivers of microbiome structure, in both the taxonomic and gene function dimensions. In the marine environment, processes that influence functional gene composition in the microbiomes vary from processes that influence microbial taxonomic composition , 36. WitAlopias vulpinus), whale shark (Rhincodon typus), leopard shark (Triakis semifasciata), and round ray . Teleost fishes included the bay blennies (Hypsoblennius gentilis), California flounder , California killifish (Fundulus parvipinnis), shiner perch (Cymatogaster aggregata), and bay pipefish (Syngnathus leptorhynchus). First, we asked whether the skin microbiome of these marine fishes is host-specific. A central tenet of phylosymbiosis is that the variability of within-species microbiome similarity will be lower than that across host species; thus, we expect little variation among samples from the same species relative to samples among species. We extend the central tenet of phylosymbiosis to the elasmobranch and teleost clades. We determine whether the host evolutionary divergence extended to the accumulation of microbiome differences or if processes that result in patterns of phylosymbiosis appear to erode. Our results, from 38 total individuals from within 9 species, demonstrate that patterns of phylosymbiosis vary across clades and that convergent evolution of host traits may influence host microbiomes in teleost fish.Elasmobranchs investigated include the common thresher shark (n = 21) nested within the group elasmobranch and 5 bony fish species .We hypothesized that the taxonomic and functional compositions of the microbiomes would be host-specific, i.e., microbiomes sampled from individuals of the same host species will be more similar than microbiomes sampled from individuals of a difference host species. To address this question, we first compared the similarity of microbiome composition within and across host species within their respective clades . Taxonomic similarity within host species was higher than compared with the microbiomes among host species . For functional gene comparisons between clades, we find no difference in mean similarity scores between intra-clade and inter-clade pairwise comparisons  is predicted to be greater than pairwise species comparisons among clades (inter-clade). The phylogenetic similarity of the microbiome shows that intra-elasmobranch similarity is not different than inter-clade comparisons; however, teleost fishes . For instance, the killifish microbiome was phylogenetically more similar to the thresher shark than to the founder . Blennie microbiomes were more similar to leopard shark and stingray microbiomes than to pipefish . However, the elasmobranch and teleost clades were not significantly different , supporting phylosymbiosis. For teleost, there was no significant relationship between host distance and microbial phylogenetic distance. In contrast, the gene function comparison showed the reverse trend. There was a lack of relationship between host evolutionary distances for gene function of the microbiomes for elasmobranchs . The decreasing slope indicates that functional similarity is increasing with increasing host evolutionary distance.Phylosymbiosis argues that increasing host evolutionary distance results in accumulated microbiome divergence (increasing dissimilarity or distance). To test this hypothesis, we compared the host’s evolutionary distance (based on the phylogenetic assessment of the COX1 gene) to the divergence of the microbiome for each clade (note that the clades were compared separately because of variations in the evolutionary clock). In elasmobranchs, we found a significant increase in microbiome distance with increasing host evolutionary distance . The phylogenetic assessment of the elasmobranch species based on the COX1 gene suggests they have a more recent common ancestor relative to the teleost fish species. However, we note caution in the interpretation of the results based on the COX1 gene, as the mitochondrial DNA among Chondrichthyes fish species accumulates nucleotide differences at rates much slower than their Osteichthyes fish counterparts . TherefoTeleost fish, however, lack a consistent phylosymbiosis relationship, which may be the result of convergent evolution of traits in skin features selecting for specific microbiome inhabitants. The increasing microbiome similarity  with increasing host distance suggests convergent evolution for traits that fish use to select and maintain a microbiome , 41. SimThe lack of a pattern for phylosymbiosis in the genes required to live on elasmobranch fishes may occur because mucus is not a selective mechanism. There are low amounts of mucus excreted onto the skin surface , except Microbiome patterns observed in this study could also be the result of conserved symbiosis from a distant shared ancestor of elasmobranch and teleost fishes, whereby some interactions have been maintained and others have been lost. Such processes have been hypothesized for the convergence observed in some teleost fish and mammal gut microbiome , in whicCarcharhinus melanopterus) accounted for high variation in microbiome composition [The lack of consistency in the relationship of microbiomes across clades could also be the result of eco-environmental effects, such as biogeography. Capture sites of blacktip reefs sharks  collection and processing are described in detail in Doane et al. [n = 7) samples were collected from La Jolla, CA, USA, in September 2013 using a hook and line methods, with the shark brought into the boat for microbial sampling. Whale shark (n = 4) microbial samples were collected from La Paz, Mexico, in February 2014. The skin microbiota was obtained via a two-way modified syringe in which expelled water is recollected in the backside of the syringe [n = 4) were put into 5-L shallow bins filled with bay seawater until ready to sample. Rays were lifted just out of the water, and the 2-way syringe was used to collect microbes from the dorsal surface just posterior of the eyes. The 2-way syringe was preloaded with a sterile PBST solution [Common thresher shark Table ; n = 6 csolution . The othsolution . The watDNA was extracted from the microbes captured on the filter using a modified column purification protocol from Macherey-Nagel Nucleospin Tissue kit as described in Doane et al. . In brieAll libraries (including common thresher shark), were cleaned using PRINSEQ software  to filtehttp://erick.matsen.org/pplacer/generated_rst/guppy.html). Tree visualization was conducted using iTOL v3 [A total of 38 metagenomic libraries were used in the analysis. Function genes and phylogenetic microbiome diversity were annotated in the following manner. Functional genes were assigned using SUPERFOCUS , which f iTOL v3 . Query r iTOL v3 p value correction. We tested for differences in β-diversity among clade (elasmobranch versus teleost fishes) and species  using a two-factor nested PERMANOVA. A permutational t test was used to determine which species were causing the differences when the main effect test was significant. Nonmetric multidimensional scaling (nMDS) was used to visualize the microbiome (gene function and phylogenetic) dissimilarity or distance in ordination space. To test if skin microbiome composition was linked with host phylogeny, we calculated host distance by aligning the cytochrome c oxidase I (COX1) gene of each species using Clustal Omega [Gene function matrices were standardized to the summed total of each sample. The phylogenetic matrix was not standardized, as PhyloSift provides a weighted normalization output. The similarity in microbiomes was calculated for gene functions using Bray-Curtis dissimilarity matrices and for phylogenetic distance using Kantorovich-Rubinstein metric (KR-distances from here on) . For aimal Omega  on the EAll statistics were run using R (v3.5.1) and Primer package 6 (v6.1.15) with PERMANOVA+ (v1.0.5). All figures were generated ggplot2 package in R (v3.5.1).Additional file 1 Supplemental Table 1. All pairwise comparisons among species across all community dimensions. P (perm) is the calculated p-value based on permuted values. BC similarity is Bray-Curtis similarity. Phylogenetic distance is the KR distance method.


Dependoparvovirus. These EVE sequences show characteristics previously unseen in modern, mammalian AAVs, but instead appear more similar to the more primitive, autonomously replicating and pathogenic waterfowl dependoparvoviruses. Phylogeny reconstruction suggests that the whippomorph EVE orthologue derives from exogenous ancestors of autonomous and highly pathogenic dependoparvovirus lineages, believed to have uniquely co-evolved with waterfowl birds to present date. In contrast, ancestors of the two other mammalian orthologues (Lagomorpha and Vespertilionidae) likely shared the same lineage as all other known mammalian exogenous AAVs. Comparative in silico analysis of the EVE genomes revealed remarkable overall conservation of AAV rep and cap genes, despite millions of years of integration within the host germline. Modelling these proteins identified unexpected variety, even between orthologues, in previously defined capsid viral protein (VP) variable regions, especially in those related to the three- and fivefold symmetry axes of the capsid. Moreover, the normally well-conserved phospholipase A2 domain of the predicted minor VP1 also exhibited a high degree of sequence variance. These findings may indicate unique biological properties for these virus ‘fossils’ relative to extant dependoparvoviruses and suggest key regions to explore within capsid sequences that may confer novel properties for engineered gene therapy vectors based on paleovirology data.Endogenous viral elements (EVEs) are genetic remnants of viruses that have integrated into host genomes millions of years ago and retained as heritable elements passed on to offspring until present-day. As a result, EVEs provide an opportunity to analyse the genomes of extinct viruses utilizing these genomic viral fossils to study evolution of viruses over large timescales. Analysis of sequences from near full-length EVEs of dependoparvoviral origin identified within three mammalian taxa, Whippomorpha , Vespertilionidae (smooth-nosed bats), and Lagomorpha , indicates that distinct ancestral dependoparvovirus species integrated into these host genomes approximately 77 to 23 million years ago. These ancestral viruses are unique relative to modern adeno-associated viruses (AAVs), and distinct from extant species of genus  Parvoviridae is classified into two subfamilies; Densovirinae and Parvovirinae, based on distinct host ranges infecting either invertebrates or vertebrates, respectively  was originally identified as a contaminant in adenovirus stocks, as indicated by their name , which encode four non-structural Rep proteins and three structural capsid viral proteins (VPs), via alternative splicing and alternative translation initiation. The mRNA populations of AAVs are transcribed in temporal order from three promoters, located at map units 5, 19, and 40, in the AAV2 genome and utilize a common polyadenylation site, directly upstream of the right ITR . A thirdassembly .Capsid structures of AAV serotypes are known to be highly conserved, possessing icosahedral symmetry assembled from 60 monomers composed of the minor VP1 and VP2, and major VP3, in a 1:1:10 ratio, respectively. The VPs are overlapping with VP1 containing a unique N-terminal sequence (VP1u) that encodes a phospholipase A2 (PLA2) enzyme required for infection. The ordered structure of the overlapping VP3 region contains conserved β-strands, forming a β-barrel ‘jelly roll’ core linked by surface-exposed variable loops that confer the virion surface morphology . The surAAVs have proven utility as gene therapy vectors that are safe and efficacious for delivery and expression of therapeutic genes due to non-pathogenicity and ability to infect both dividing and non-dividing cells. However, utility of AAV-based vectors can be compromised when patients have previously been exposed to wild-type AAV serotypes that are closely related to therapeutic capsids used in gene therapy. As a result, these individuals are ineligible for therapy utilizing conventional AAV vectors. Estimates indicate that nearly 70 per cent of the population have pre-existing antibodies to common AAV strains and this prevalence of pre-existing immunity remains an obstacle for widespread therapeutic use . TherefoEVEs are DNA sequences derived from viruses that once circulated in a host population, but integrated into cells of the host germline, thus they to become endogenous sequences within host genomes . Due to Parvoviridae, which is composed of various genera including Ambidensovirus, Protoparvovirus, Amdoparvovirus, and Dependoparvovirus  in a broad range of eukaryotic species  was confirmed in all cetacean specimens analysed (Hippopotamus amphibius reference genome (ASM299558v1). The Dp.1-Whippo element is integrated into an intron of the Pax5 gene of the Whippomorpha suborder (Cetacea and hippopotamuses)  reference genome. Integration of this EPV into the ancestral whippomorph germline therefore must have occurred after the ruminantia and whippomorpha lineages split, but before divergence of cetaceans and hippos. Based on time-calibrated phylogenies for speciation of cetacea and hippos suggests a window of 60 to 55 million years ago (MYA), in which the ancestral dependoparvovirus represented by the Dp.1 sequence would have circulated and infected progenitors of the Whippomorpha suborder.Presence of the cetacean EPV (Dp.1-Whippo) first identified in the analysed  and the tamuses)  of the Crep and cap genes have undergone varying degrees of disruption among the species analysed. The most intact copy is found in the minke whale  and the most degraded within the oceanic dolphins (family Delphinidae). The rep sequences in hippo and minke whale EVEs contained only four frameshifts. Terminal deletion of the rep gene was observed in all species, with 267 nt being deleted from the 5′-end, and 312 nt of the 3′-end (large Rep-coding sequence) relative to the most closely related dependoparvovirus, namely goose parvovirus, with which the minke whale element Rep displays 57 per cent amino acid (aa) identity. The least intact rep was found in the Dp.1 sequence of the killer whale (Orcinus orca), with six frameshifts and five stop codons. A broader number of post-integration mutations were observed in the whippomorph cap gene. Despite these indels, the entire aa sequence of VP1 could be reconstructed, with the exception of the hippo EPV where the C-terminus past VR-IX is missing  of the CHRS-2 family were identified within cetacean EPVs. Members of the Delphinidae clade had two copies of the CHRS-2 SINE, present in both the rep and cap genes (Mesoplodon stejnegeri and Ziphius cavirostris) possessed a single CHRS-2 SINE integrated into the cap region of their EPV while baleen whales, represented by the minke whale (B.bonaerensis) and basal toothed whales, represented by the sperm whale (Physeter catodon), and hippos had no copies of the SINE within their respective EPV sequences   were chosen as representatives for cetacean species lacking any SINE integrations in their EPV  and cetaceans with SINE integrations in both rep and cap regions (T.truncatus). Following transcriptome sequencing of cell lines, only two sequencing reads were mapped to the EPV locus of T.truncatus but both reads corresponded to the CHR-2 SINE sequence nested within the EPV’s VP region and not the EVE sequence itself. CHR-2 sequences are known to occur at numerous loci within cetacean genomes and share incredibly high levels of sequence identity, often differing only by a few nucleotides. These two mapped reads are not suggestive for expression of the EVEs, but likely result from sequencing errors leading to altered mapping of other CHR-2 SINE elements from elsewhere in the genome to the EVE loci’s SINE  appear to retain a level of EVE expression, unlike the dolphin EVE containing SINE integrations within their EPV.Two cetacean cell lines  for fourteen species, revealed eight chiroptera species, all members of Vespertilionidae, carry orthologous copies of this EPV, which is fixed within an intron of the KIF6 gene  of vesper bats based on radiation of these families to no greater than 49 to 38 MYA: the divergence dates for Miniopteridae and Vespertilionidae, but earlier than 30 to 19 MYA, during the speciation of the Eptesicus and Myotis genera of bats  to six (genus Myotis) and the in-frame stop codons from one to as many as five when comparing Myotis versus Plecotus, respectively . The presence of an additional, small ORF encoding a 10-aa-long C-terminal extension, analogous to the AAV Rep68/40 C-terminal tails, could be identified as well as both splice sites with high confidence (>0.95 on the donor and 0.78–0.98 on the acceptor site), thus suggesting that the ancestral virus harboured a similar transcription pattern to that of modern primate AAVs   . A heaviOchotona princeps), although in this species only a portion of the rep gene remains. These results indicate that the EPV integration occurred in the germline of the MRCA of all lagomorphs. Based on evolutionary timescales of lagomorphs, the MRCA would have been infected with a circulating AAV prior to divergence of Ochotonidae and Leporidae families ∼50 MYA (Mus musculus) show that this element is exclusive to the order Lagomorpha, indicating that this EPV derives from an ancient dependoparvovirus that was incorporated into the germline of the common ancestor of extant lagomorphs after the Rodentia/Lagomorpha split, estimated to have occurred between 66 and 47 MYA , and splice donor and acceptor sites of the Rep68/Rep40 homologues  cell line RK13 (ATCC® CCL-37) mapped to both rep and cap regions of the rabbit EPV. Thirty-one sequencing reads mapped to the rep region and were distributed across the entire length of rep,whereas four sequencing reads mapped to the 5′ end of cap  and B. Uus genus . In the us genus . The PLAus genus  In the celements . Within sequence . In the Sequences and models for VP showed the entire VRII of Dp.3-Lagomorpha was absent, probably due to a deletion . This exDependoparvovirus to be at least 50 million years old (18).EVEs represent genomic fossils that function as surrogates for ancestral viral genomes able to provide insight into ancient, presumed extinct, viruses. These EPVs derived from ancestral dependoparvoviruses represent distinct viral genotypes, including the three distinct orthologues of this study, confirmed by their independent clustering relative to extant AAVs in phylogenetic trees . We wereAnseriform dependoparvovirus 1 possess characteristics distinguishing them from mammalian members of genus Dependoparvovirus, namely their pathogenicity in waterfowl and autonomous replication capabilities  origin, based on both the possibility of autonomous replication in waterfowl and squamate reptiles, as well as via phylogenetic inference .While there are no known exogenous dependoparvoviruses for Lagomorpha and Whippomorpha species, Chiropteran AAVs have been identified from a variety of bat species, including a fully sequenced genome from ricketti . Despiteoviruses . This stLoxodonta africana) that also showed expression of EPVs and harboured intact NS- and Rep-encoding genes (Ellobius lutescens) genome, an intact NS gene fused with a bZIP transcription factor’s ORF . Regardless of translation, the illustrated transcription of Cetacean and Leporidae EPV sequences may provide a direct benefit for the hosts themselves, particularly as non-coding RNA (ncRNA). Specifically, ncRNAs have been identified that are important components for regulating innate immune responses and stimulating antiviral responses , which lack this conserved domain , or tissue samples provided through museum collection archives  (Loan authorization request No. 2016.020 Mamm), the University of Alaska Museum (UAM) Museum of the North (Loan authorization request No. 2013.13 Mamm), National Oceanography and Atmospheric Administration (NOAA), and the National Marine Mammals Fisheries Service (NMFS) Regional Stranding Network Associates ).in silico for the EVE sequence and, for EVE positive species, their sequence data included with sequences derived from the tissue samples to survey a total of 19 Cetacean and one Hippo species, all members of the whippomorph suborder (DNA was extracted using Qiagen DNeasy Blood & Tissue Kit (Cat No./ID: 69506), followed by PCR amplification with Phusion High-fidelity polymerase (New England Biolabs) using primers indicated in suborder , eight lsuborder , and twesuborder . DNA seqsuborder , then insuborder . Maximumsuborder . PhylogeTime-calibrated phylogenies for evolution and speciation of host species previously published for cetacean , lagomorEVE sequences previously identified within marsupial species  were anarep, cap, and AAP were reconstructed. Annotation was carried out using the Artemis Genome Browser . RNA-seq reads from species lacking a complete reference genome , were mapped to their respective EVE sequences determined via Sanger sequencing.Cell lines from four, EVE-positive species  were culSylvilagus audobonni displayed the most intact capsid, therefore this sequence was selected for protein modelling building for Leporidae, whereas one representative of all capsid variations was subjected to model building from the other two taxa.As homology modelling does not tolerate unknown or missing residues, it was necessary to reconstruct the presumed original residues altered by in-frame stop codons prior to further analysis. Alignments were done, with or without the incorporation of structural data as described above, to evaluate stop codons based on a parsimonic evolutionary approach to reconstruct the most-probable original codon. Briefly, we utilized a substitution matrix, in which transition and transversion events of aa codons received weighted scores based on the expected probabilities for the different types of substitutions, assuming transitions to happen at a higher frequency. For each stop codon position all potential residues observed in exogenous AAV data were considered as candidates. Potential residues in which the least number of nucleotide substitutions required to restore a coding residue, according to the scoring matrix, were selected to replace the stop codons in question. The EVE from http://viperdb.scripps.edu/)  . The UCSps.edu/) .MN782247–MN782272.Newly described EVE sequence data available at NCBI with GenBank accession numbers veaa043_Supplementary_DataClick here for additional data file.


We report a patient with symptomatic low-flow high-grade aortic valve stenosis and myelodysplastic syndrome. Preinterventional imaging revealed an anomalous origin of the right coronary artery only defined by CT. The patient was classified as high risk in regard to conventional cardiac surgery by our heart team and therefore scheduled for transcatheter aortic valve repair (TAVR). The case presentation describes the potential effect of this anatomical coronary variant with regard to the peri- and postinterventional outcome: anomalous origin of the right coronary artery may lead to severe ischemia during TAVR due to occlusion of the coronary vessel. Conversion to open surgery with immediate coronary bypass surgery may rapidly restore myocardial perfusion and enhance clinical outcome of the patient. Transcatheter aortic valve repair (TAVR) has become the preferred therapy in patients with symptomatic high-grade aortic valve (AV) stenosis and for whom open heart surgery is contraindicated. Excellent peri- and postoperative outcomes for TAVR patients at high or intermediate surgical risk have led to a more frequent use of this therapy at many centers -2. NeverA 76-year-old man presented to our cardiology department with atypical chest pain and shortness of breath. Physical examination revealed a loud 4/6 systolic murmur related to the AV. Transthoracic echocardiography showed low-flow high-grade AV stenosis . Former clinical history included MDS that was first diagnosed in 2018 with leukopenia, thrombopenia, and anemia but no actual need for chemotherapy or transfusion of blood cells or plasma. Invasive coronary angiography and multislice CT following a specific TAVR protocol, as well as a transesophageal echocardiography (TEE), were performed. Coronary angiography revealed diffuse coronary sclerosis without significant stenoses in the left anterior descending artery and circumflex vessels. Despite the use of different diagnostic coronary catheter types and selective intubation, visualization of the RCA could not be achieved due to a suspected anomalous origin of the vessel Figures . Due to the comorbidities related to MDS and the high probability of infectiologic complications occurring after conventional open valve surgery, the heart team at our institution decided to perform a transfemoral TAVR procedure. The intervention was achieved using a balloon-expandable 26-mm Edwards Sapien 3 transcatheter valve . The choice of the bioprosthetic valve type was based on local expertise and suitable anatomical features such as degree and extent of valve calcification.After the release of the transcatheter valve, unspecific electrocardiogram (ECG) ST-segment depression alterations occurred with hemodynamic worsening and a decrease in systolic blood pressure to 70 mm Hg. Severe right ventricular dysfunction with unchanged left ventricular ejection fraction (EF) was revealed on TEE. The right ventricular wall motion was reduced, and right ventricle dilatation was recorded. There were no signs of pericardial effusion or peripheral bleeding. The TAVR valve was judged to be in the correct position, and no AV regurgitation was present.Fluid substitution and high-dosage catecholamine infusions were administered, although no clinical benefit was observed. Due to persistent ECG ST-segment changes, we suspected an ischemic right ventricular infarction and occlusion of the RCA. Following an acute worsening of hemodynamics, we opted for intervention consisting of a conversion to sternotomy and a single venous bypass graft surgery of the RCA. The patient’s hemodynamic situation rapidly stabilized, and postoperative weaning was unproblematic. A subsequent echocardiography revealed minimal reduction of global left ventricular EF 47%), minimal AV regurgitation, and no remaining AV stenosis  of Valsalva may lead to hemodynamic and ischemic complications, especially in TAVR patients. The CT diagnosis and incidence of ACAOS anatomy have been previously described and are related to potential clinical outcomes. There is poor evidence considering the influence of ACAOS anatomy in TAVR procedures -6. In thIn patients with anomalous RCA origin, TAVR may lead to myocardial ischemia with poor interventional therapeutic options. Careful consideration of TAVR should be mandatory for these patients, especially in the presence of dominant RCA anatomy. When patients develop severe hemodynamic complications during the TAVR procedure, conversion to emergency bypass surgery may be the only therapeutic alternative.


Substance misuse is a critical social and health care issue, and learning how to effectively screen for misuse and perform a brief intervention is useful for all health care professions. As an intercollegiate, interprofessional group, we developed a mechanism for delivering interprofessional education (IPE) using SBIRT  as a tool to identify potential substance misuse.A total of 1,255 students from nursing, pharmacy, medicine, physician assistant, social work, dietetics, and occupational therapy programs participated in the training and evaluation of this IPE experience over 2 academic years. The training incorporated asynchronous SBIRT training, in-person student role-plays, and a standardized patient (SP) interaction.A significant majority of participants indicated that this IPE experience enhanced their interprofessional skills (91%), was useful for interprofessional development (79%), was relevant to their career (92%), and would benefit their clients (93%). Faculty debrief sessions supported the efficacy of SBIRT as a platform for IPE.Students believed that utilizing SBIRT as an interprofessional learning experience enhanced their overall educational experience and assisted with developing interprofessional relationships and that team-based care would lead to improved patient outcomes. Faculty found this learning activity to be effective in developing student insight regarding future professional peers and patient interview skill development through role-plays with peers and SPs. The screening, brief intervention, and referral to treatment (SBIRT) technique is an evidence-based tool to identify potential misuse and prevent abuse of alcohol and/or illicit drugs. SBIRT was selected for this interprofessional activity because it incorporates common curricular content, including team-based patient-centered care, motivational interviewing skills, and population health and prevention.1 The goal of IPE is to prepare workforce-ready health care team members who practice collaboratively in order to improve health outcomes.2 In response to the growing complexity of care, as well as accreditation requirements, health professions programs now integrate teamwork and communication content in their curricula. In order to create meaningful IPE learning opportunities, educators strive to design, implement, and evaluate learning opportunities that simulate patient care scenarios students will likely encounter in clinical settings.Interprofessional education (IPE) programs require strong faculty commitment and a dedication to ongoing collaboration.3 Using SBIRT as the vector for IPE, the goal of this program was to provide a meaningful interprofessional learning activity for students  in medicine, nursing (BSN and DNP), pharmacy, physician assistant, social work, dietetics, and occupational therapy programs.The resource described here drew from lessons learned through a previous interprofessional Health Service and Research Administration–funded program that included faculty development, implementation of an interprofessional team-based program to teach collaborative practice skills using standardized patients (SPs), and multiple assessment and evaluation strategies to provide formative feedback to students.MedEdPORTAL publications have shared SBIRT cases for medical resident training using SPs4 or using SBIRT to facilitate interprofessional instruction of postgraduate health care professionals.6 In this module, we offer a guide for presession independent learning followed by SBIRT cases for a single in-person session of peer role-play practice and SP interactions, with students from seven different health professions as the target learners.Other This project was reviewed by the Washington State University Institutional Review Board (the host university), which determined the project satisfied the criteria for exempt research.We designed this SBIRT educational module for teaching interprofessional student learners from concept to skills application. We assembled an interprofessional team comprising medicine, nursing (BSN and DNP), pharmacy, physician assistant, social work, dietetics, and occupational therapy faculty to design, implement, and evaluate the educational module and learning objectives . StudentFor student participants, the educational module consisted of a required 2-hour online informational component (completed at any point prior to the live session) and a 2-hour in-person application session. Participation was required as a class assignment in each profession-specific program.7 These two trainings were similar in defining SBIRT, introducing screening tools for identifying drug and alcohol misuse, demonstrating a brief intervention, and discussing impacts and benefits of using SBIRT. Prior to the in-person session, all students were required to complete the online preclass component and watch a video8  and SBIRT Oregon were selected.a video8  that proa video8 .9  over the course of three cases. Case roles were color coded by rounds so students could more easily follow the format  core competencies  were addressed by the SPICE tool, so we added two questions regarding the remaining two IPEC core competencies .17 One question asked students to rate SBIRT as an effective interprofessional learning activity, and one question addressed how useful the activity was to their interprofessional development.All students were asked to complete a survey at the conclusion of the training . The sur1.Based on your experience, what do you believe are the benefits of an interprofessional team approach to caring for patients with substance abuse behaviors?2.What about the training was most useful in supporting your work responsibilities?3.How can Center for Substance Abuse Training improve its training?Finally, students completed three open-ended qualitative questions:18 The lead qualitative researcher monitored the process for quality and rigor by employing reflexivity and triangulation. Reflexivity acknowledges the presence of bias and seeks to mitigate its influence during the data-analysis process.20 Triangulation is a methodological check on the validity of the data analysis and was accomplished by triangulating iterative coding phases among four qualitative researchers. Conceptual codes and subcodes21 were developed as an outcome of this process.We examined these responses qualitatively using principles of inductive reasoning—a common approach in qualitative analysis that allows researchers to gain insights beyond those possible through quantitative methods alone.Qualtrics Research Suite software was used to deploy the anonymous, web-based electronic survey. Quantitative data were summarized as frequencies (percentages) for each item.In addition to the feedback from SPs , students provided peer-to-peer verbal feedback immediately following each role-play and SP interaction using the observational rubric . Observe•The training enhanced my skills in this topic area .•I expect to use the information gained from this training .•I expect this training to benefit my clients .Over 2 academic years from 2016 to 2018, 1,255 students participated in the interprofessional activity and completed any survey questions. Of these, 985 (79%) found the activity very useful/useful to their interprofessional development (learning objectives 1 and 4). Students were very satisfied/satisfied with the training regarding the following:Students' perceived value of working with interprofessional teams was high following the training activity .Qualitative data analysis resulted in seven themes across the three categories of usefulness (learning objectives 1 and 3), evaluation (learning objective 1), and benefit (learning objectives 2 and 4). 22A discipline-specific evaluation of student performance and perceptions of the potential impact of the training using a smaller cohort of students has been described elsewhere.23 as well as accreditation requirements and the increasing recognition that health professional students benefit from engaging in cooperative work early in their training. We successfully used SBIRT as a model for SP simulation and realistic interprofessional collaboration.Growing interest in IPE activities stems from demonstrated improvements in patient safety7 materials) and had varying levels of prior experience with SPs. Students highly valued this SBIRT IPE learning experience as indicated by postexperience results.We developed our cases with input and review from professionals in all seven participating disciplines; thus, all student participants were able to fully engage as equal members of the team in the SP interaction. The students learned about substance use and SBIRT from preclass preparation materials , that they expected to use the information (93%), and that it would benefit their clients (93%). This activity gave students an opportunity to gain skills they found useful to their practice with current and future patients.With three universities and seven disciplines involved, scheduling the interactive sessions was complex, but all disciplines successfully embedded the activity into an existing course. Interprofessional faculty collaboration was critical; this included early communication regarding logistics so each faculty could plan their courses accordingly, as well as regular meetings to coordinate the experience and then engage in ongoing assessment for program improvements. Numerous small-group sessions at varying times of day were needed to accommodate all disciplines because of highly diverse student schedules.Strengths of this module include the focus on previsit interprofessional collaborative dialogue and planning, which allowed students to discuss and decide what each member's roles and responsibilities would be. Taking turns interviewing and counseling the SP helped develop each student's communication skills as well as the ability to observe other team members and provide real-time feedback using a rubric.Potential limitations to this activity include the need for multiple health professions to be present, which may be a challenge, especially in more rural educational settings. SP simulations were done in triads, so this activity could be done with as few as two to three different health professions. In low-resource areas where programs may not have availability or funding for SP actors, faculty or students can use the scripts to play these roles. We included a videoconferencing option for students in rural educational sites; however, both students and faculty agreed that the technological frustrations outweighed the benefits of this component. A further limitation was that due to the small number of students in some professions, we did not identify themed perceptions by profession in order to protect participants' anonymity.In future iterations of this activity, we would incorporate students describing their roles and those of other professions in screening for substance misuse during the icebreaker activity , slide 2Educational Objectives.docxAdministrative Instructions Prior to Session.docxStudent Overview of SBIRT Components - Email Prior.docxStudent Prep - ADEPT Video.mp4AUDIT Screening Tool - Email and Print.docxDemonstration - SBIRT Colorado.mp4Faculty Overview and Agenda.docxSBIRT Slides for Live Session.pptxFaculty Script for Slide Presentation.docxSBIRT Pocket Card - Print.pdfStudent Agenda - Print.docxPeer Role-Play Case 1-Print ORANGE-Observer.docxPeer Role-Play Case 1-Print ORANGE-Patient.docxPeer Role-Play Case 1-Print ORANGE-Provider.docxPeer Role-Play Case 2-Print BLUE-Observer.docxPeer Role-Play Case 2-Print BLUE-Patient.docxPeer Role-Play Case 2-Print BLUE-Provider.docxPeer Role-Play Case 3-Print GREEN-Observer.docxPeer Role-Play Case 3-Print GREEN-Patient.docxPeer Role-Play Case 3-Print GREEN-Provider.docxSP Case Jamie Quimby.docxSP AUDIT Screen Jamie Quimby.pdfSP Case Pat Stewart.docxSP AUDIT Screen Pat Stewart.pdfEvaluation Tool.docxAll appendices are peer reviewed as integral parts of the Original Publication.


Togaviridae family that are mainly transmitted by arthropods such as mosquitoes. In the last decades, several alphaviruses have re-emerged, causing outbreaks worldwide. One example is the re-emergence of chikungunya virus (CHIKV) in 2004, which caused massive epidemics in the Indian Ocean region after which the virus dramatically spread to the Americas in late 2013. Besides CHIKV, other alphaviruses, such as the Ross River virus (RRV), Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV), have emerged and have become a serious public health concern in recent years. Infections with the Old World alphaviruses  are primarily associated with polyarthritis and myalgia that can persist for months to years. On the other hand, New World alphaviruses such as VEEV cause mainly neurological disease. Despite the worldwide (re-)emergence of these viruses, there are no antivirals or vaccines available for the treatment or prevention of infections with alphaviruses. It is therefore of utmost importance to develop antiviral strategies against these viruses. We here provided an overview of the reported antiviral strategies against arthritogenic alphaviruses. In addition, we highlighted the future perspectives for the development and the proper use of such antivirals.Alphaviruses are members of the  Alphaviruses are a group of human and animal viruses that belong to the family Togaviridae. Alphaviruses are mainly transmitted by the bite of hematophagous arthropods  . ClassicSeveral arthritogenic alphaviruses have (re-)emerged worldwide and have become a major public health threat. However, there is no approved antiviral drug or vaccine for the treatment or prevention of these viral infections. The current treatment depends on symptomatic relief using analgesics, antipyretics, nonsteroidal anti-inflammatory drugs, and, in severe cases, methotrexate .Arthritogenic alphaviruses are generally maintained in the wild via enzootic/epizootic transmission cycles between an arthropod vector (mostly mosquitoes) and animal reservoirs such as primates, forest birds, and macropods ,5. On ceAedes mosquitoes . Co. Co12]. In theory, all steps in the alphavirus replication cycle can be potential targets for antiviral drug development. These steps involve an interplay between viral proteins and host factors. The major advantage of host-targeting antivirals is the potential broad-spectrum activity against more than one alphavirus and the lower possibility that the virus will develop resistance. However, caution must be taken to avoid (serious) side effects when targeting a host factor. We here reviewed the reported antiviral strategies against arthritogenic alphaviruses replication.To date, the entry receptors for arthritogenic alphaviruses are not fully elucidated. Interestingly, the adhesion molecule Mxra8 has been recently identified as an entry receptor for several arthritogenic alphaviruses including CHIKV, ONNV, RRV, and MAYV . TreatmeAnother possible target for blocking viral entry is prohibitin. Prohibitin-1 is a signaling protein that was previously identified as a receptor for CHIKV in mammalian cells . TargetiThe broad-spectrum antiviral drug arbidol  has alsoViral mRNA capping plays an essential role in subsequent downstream processing, translation, nuclear export, and stability of mRNA. For alphaviruses, the viral RNA capping involves the RNA methyltransferase (MTase) and guanylyltransferase (GTase) activities of nsP1, as well as the RNA triphosphatase function of nsP2. Interestingly, the alphavirus capping mechanism is unique and proceeds in a sequence that is distinct from the capping mechanism in the host cell . TherefoThe first class of small molecules reported to target nsP1 is the MADTP series, which has a triazolopyrimidinone scaffold ,23 Figu. This clIn another study, a fluorescence polarization-based GTP competition screen led to the identification of several hit compounds that were able to compete with GTP for the CHIKV nsP1-GTP binding site . One of The viral protease has been shown to be an excellent target to inhibit replication of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) . Similar50 values in the low µM range. Based on molecular docking studies, it was proposed that these compounds may interact with the nsP2 protease domain [The crystal structures or homology models of the nsP2 protease of different arthritogenic alphaviruses were extensively explored in molecular docking and molecular dynamics studies in order to identify potential alphavirus inhibitors ,35,36,37® compounds library (Sigma Aldrich) against the crystal structure of CHIKV CP protease [In contrast to the multiple studies targeting the nsP2 protease, studies exploring the capsid protease are scarce. The first study confirming that the alphavirus CP protease could be a potential druggable target was published very recently . Three iprotease . The antprotease .Antiviral drugs that target the viral polymerase are currently available for treatment of the infections with several viruses including HIV, hepatitis B virus (HBV), HCV, and herpes. For alphaviruses, the viral nsP4 protein possesses the viral RNA-dependent RNA polymerase (RdRp) activity and could be a potential target for development of broad-alphavirus inhibitors . FavipirThe approved HCV polymerase inhibitor sofosbuvir, a nucleotide analog , has beeThe alphavirus 6K protein is considered a viroporin due to its ability to form ion channels that facilitate viral assembly and release through the cellular membranes . RecentlCrotalus durissus terrificus) venom have been reported to have a virucidal effect against several enveloped viruses, including CHIKV [Ginkgo biloba plant, which has been recently shown to inhibit the in vitro replication of CHIKV and MAYV through a direct virucidal effect [Canistrocarpus cervicornis) also had potent virucidal activity against CHIKV [Several natural products have been reported to exert virucidal activity against arthritogenic alphaviruses ,45,46. Sng CHIKV . Anotherst CHIKV .As mentioned before, the fusion of the viral envelope glycoprotein E1 with the endosomal membrane is triggered by low pH in the endosome . ConsequThe envelope of alphavirus particles is acquired during budding from the lipid plasma membrane and plays an important role during virus entry into the host cell. The lipid composition of the alphavirus envelope has been demonstrated to be critical for virus particle stability and infectivity . It has de novo synthesis of long-chain fatty acids and their early desaturation, respectively [The fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD1) are two key enzymes which are essential for the ectively . Both enectively ,55. Inhiectively ,55 or thectively ), as welectively , reducedectively .Other potential targets in the host lipid pathway are the liver X receptors (LXRα and LXRβ), host transcription factors essential for the intracellular cholesterol homeostasis through regulation of cholesterol exporters . The LXRHalofuginone  is a potThe ubiquitin-proteasome system plays a major role in the degradation of cellular protein and has been previously reported to regulate the intracellular levels of certain viral proteins including the alphavirus nsP4 protein . The clide novo biosynthesis of guanine nucleotides [de novo pyrimidine biosynthesis [The inosine monophosphate dehydrogenase enzyme (IMPDH) catalyzes an essential step in the leotides . Inhibitleotides ,68. Anotleotides . The antleotides . The obsynthesis .Targeting cellular kinases has been reported as a potential strategy to inhibit the replication of arthritogenic alphaviruses. The Src family kinases (SFKs) comprise a group of membrane-associated kinases that mediate signal transduction of certain cellular receptors and have been reported to promote the replication of several viruses, including HCV and dengue virus (DENV) . A kinomAnother example of involved cellular kinase pathways is the major mitogen-activated protein kinase (MAPK) signaling pathway. This pathway was found to be activated during CHIKV infection and to play a role in infectious alphavirus particles formation . ReducinA different example is the protein kinase C (PKC) family which comprises related serine/threonine kinases that regulate many cellular processes such as proliferation, differentiation and apoptosis . PKC actCellular ion channels have been shown be involved in the entry and genome replication of several viruses . Using sPost-translational modifications of alphavirus structural proteins are essential for the production of mature alphavirus virions . CellulaDigoxin is a cardiac glycoside that inhibits the sodium-potassium ATPase activity. In a high- throughput antiviral screening, digoxin has been identified as a potent CHIKV inhibitor in human cell lines . MoreoveSerotonin or 5-hydroxytryptamine (5-HT) receptors are mostly G-protein coupled receptors that play a key role in several physiological functions and signaling pathways . Interes−/− C57BL/6 mice infected with CHIKV showed more severe disease symptoms compared with the infected wild-type mice, which was attributed to an altered CD4 T-cell response [The host innate immune responses, mainly type I interferon (IFN) signaling, play an essential role in controlling acute alphavirus infections . Recombiresponse . Tiloronresponse . Recentlresponse . Polyinoresponse ,92. The response ,92. The response . Consequresponse ,95. Receresponse . This moresponse .Another promising class of immunomodulators are the heparan sulfate mimetics . Heparan sulfate mimetics inhibit the glycosidase heparinase which degrades degrade heparan sulfate, a key component of the extracellular matrix. Pentosan polysulfate is used for the treatment of interstitial cystitis and osteoarthritis, whereas pixatimod is currently developed as an anticancer drug. Both drugs have been reported to reduce the severity of alphavirus-induced disease in vivo through the modulation of inflammatory infiltrates and cytokine levels ,98. TreaThe worldwide re-emergence of arthritogenic alphaviruses and the high morbidity rate associated with their infections underline the need for potent and safe antiviral drugs against these viruses. Efficacious antiviral drugs, if administered in time, could reduce the severity of the disease symptoms during the acute infection by lowering the viral loads in the infected patient. Moreover, reducing viremia in infected patients using antivirals may indirectly limit virus transmission by mosquitoes and hence might reduce the chance for massive epidemics . Since tCurrently, approved antiviral drugs are available for the treatment of only a limited number of viruses such as HIV, HBV, HCV, influenza, and herpes viruses. By investing sufficient time and efforts, it could also be possible to develop safe and potent antivirals for the treatment and/or prophylaxis of arthritogenic alphavirus infections. As discussed in this review, several molecules with in vitro anti-alphavirus activity have been reported, but most of these are still in the early stages of preclinical development. A next important and indispensable step is the evaluation of in vivo infection models. Only few of the reported molecules have been tested in alphavirus infection models so far. Infection models in small animals are available for several alphaviruses , but theThe development of specific antiviral drugs against each arthritogenic alphavirus separately will not be economically viable. Therefore, pan-alphavirus inhibitors will be the prime strategy to cope with this challenge. Since the alphavirus nsP2 (protease) and nsP4  proteins have conserved catalytic domains, both are considered potential targets for development of broad-spectrum antivirals for arthritogenic alphaviruses. Another promising target is Mxra8, as it functions as an entry receptor for several arthritogenic alphaviruses . DesigniAnother beneficial strategy to control emerging viral infections is the repurposing of drugs that have been approved for other diseases. Because repurposed drugs have already been intensively studied in patients and thus the safety profile is well known, the clinical evaluation of such drugs could be fast-tracked during viral epidemics. Furthermore, production strategies for these drugs have been implemented. However, repurposed drugs cannot be expected to be highly potent inhibitors of alphaviruses, as these were not developed specifically against this particular virus. Examples of approved drugs that would be of interest to evaluate in clinical trials during epidemics of arthritogenic alphaviruses include sofosbuvir, favipiravir, orlistat, and tilorone, since these drugs showed promising antiviral activities, not only in cell culture, but also in vivo animal models.Another challenge for alphavirus antiviral drug development is how such an antiviral drug could be used practically. The viremia of alphaviruses is short (<1 week). Therefore, antiviral therapy that targets virus replication should be initiated soon after the start of the infection to be efficacious. This requires an early diagnosis, but this is currently difficult. The therapeutic use of antiviral drugs could therefore be complicated. Pre-exposure prophylaxis of household members of an infected patient has been proposed as a useful strategy, since the probability of arthritogenic alphavirus transmission such as CHIKV has been reported to be up to 12% between household members . Another


While most studies of the niche focus on those residing in the bone marrow (BM), a better understanding of the fetal liver niche during development is vital to design human pluripotent stem cell (PSC) culture and may provide valuable insights with regard to expanding HSCs ex vivo for transplantation. This review will discuss the importance of the fetal liver niche in HSC expansion, a feat that occurs during development and has great clinical potential. We will also discuss emerging approaches to generate expandable HSC in cell culture that attain more complexity in the form of cells or organoid models in combination with engineering and systems biology approaches. Overall, delivering HSC by charting developmental principles will help in the understanding of the molecular and biological interactions between HSCs and fetal liver cells for their controlled maturation and expansion.Clinical transplants of hematopoietic stem cells (HSC) can provide a lifesaving therapy for many hematological diseases; however, therapeutic applications are hampered by donor availability. In vivo Hematopoietic stem cells (HSCs) are a unique cell population that, in addition to self-renewal, is capable of giving rise to all the types of cells in the blood system. Clinically, this has translated into applications such as bone marrow (BM) and umbilical cord blood (UCB) transplants for patients with various hematological diseases , 2. HoweInduced pluripotent stem cell (iPSC)-derived HSCs would offer another alternative source for transplantation. To derive HSCs from iPSCs in vitro without transgene over-expression, it is essential to understand the developmental mechanisms through which the first HSCs are produced and expand during fetal organogenesis since iPSC differentiation recapitulates aspects of embryogenesis. Importantly, the biological and molecular characteristics of fetal HSCs are considerably different from the adult HSCs. In particular, fetal HSCs expand tremendously without developing leukemia due to unique resilience mechanisms with, for example, an increased expression of DNA repair and antioxidant genes . Thus, pDuring fetal development, hematopoiesis occurs in multiple waves throughout the developing embryo and fetus, including extraembryonic yolk sac (YS), the para-aortic region of the embryo, fetal liver, and placenta before eventually homing to the bone marrow where it occurs just before birth. The first hematopoiesis is observed in the YS as early as embryonic day (E) 8.0 in mouse and Carnegie Stage (CS) 8 in humans, often referred to as primitive hematopoiesis, producing embryonic type erythroblasts that have large nuclei and express embryonic globin genes, and primitive type macrophages. The following wave is consisting of YS and embryo-derived adult type hematopoiesis that produces erythro-myeloid progenitors (EMPs) and lymphoid precursors. Finally, the first BM-repopulating HSCs are produced in the aorta-gonad-mesonephros (AGM) region at E10–11/CS13–16. These de novo HSCs seed the fetal liver and the placenta around E11–12/CS16–17 and expand to support hematopoiesis after birth , 6. In t. This review will explore the stages of hematopoiesis around the time of fetal liver development while focusing on work done in mice except where noted, as well as discuss the current state of research into culturing HSCs and attempts at ex vivo expansion.While most studies of the HSC niche involve those residing in the BM, a better understanding of the fetal liver niche is essential to recapitulate it in vitro and may provide clinically significant information with regard to expanding HSCs ex vivo for transplantation. Organoid research, particularly liver organoids, is a rapidly growing field, in which cells are directly taken from patients or differentiated from iPSCs (reviewed elsewhere ). These −/− chimera mouse models where β1−/− HSCs failed to seed the fetal liver while the production of HSCs was not impaired [The first HSC emergence specifically takes place in the endothelial cells of the dorsal aorta as hemogenic endothelial cells (HECs) lining the vessel undergo endothelial to hematopoietic transition (EHT) , 10. Durimpaired , 17. It impaired . These rThe developing liver is made of a mixture of cell types including both hepatic and hematopoietic lineages Fig. . The liv+ stromal cells adjacent to portal vessels [+ cells secrete cytokines or have direct cross talk with HSCs.Understanding the specific location and cell types that form the HSC niche in the fetal liver has long been an area of research. Early hematopoiesis in the fetal liver occurs prior to the full maturation of the hepatic vascular system. Because of this, hematopoietic cells including HSCs are found in the liver parenchyma where they are able to directly interact with various cell types Fig. . It has  vessels . HoweverIn order to determine the factors and stromal cells that support HSC expansion, fetal liver stromal cells were isolated and tested their HSC supportive ability , 23. AFT+Dlk1+ fetal liver stromal cells express angptl3, in addition to alpha-fetoprotein , CXCL12, and essential hematopoietic cytokines including SCF, thrombopoietin (TPO), and insulin-like growth factor 2 (IGF2). SCF+Dlk1+ cells maintained E15 fetal liver HSC repopulating ability during 4 days of co-culture. Others have reported that Dlk1+ fetal hepatic progenitors secrete SCF and Epo, confirmed by ELISA assays and immunostaining [+ fetal hepatoblasts secrete essential hematopoietic cytokines and support HSC expansion. Since Tie-2 and Angiopoietin-1 signaling has a critical role in maintaining HSC quiescence in the BM niche, it will be interesting to examine the interaction of fetal liver HSCs with angptl3 during expansion in the fetal liver niche [From freshly isolated fetal liver stromal cells, angiopoietin-like 2 and 3  have been identified to expand HSCs . The folstaining . Thus, ier niche .Another main type of stromal cells in the liver is endothelial cells. As previously mentioned, HSCs are found in close proximity to endothelial cells in the developing fetal liver. More recently, it has been observed that endothelial cell-selective adhesion molecule (ESAM) expressed on endothelial cells and HSCs plays an essential role in this interaction in the fetal liver and is crucial to erythropoiesis . Endothe−/− mouse fetal liver was reduced at multiple stages due to upregulated SHH in non-hematopoietic cells [Each of the hepatic and hematopoietic lineages develops together during the process of fetal liver hematopoiesis. This co-differentiation system allows for a complex interaction network that is still not fully described. However, it is known that the niche environment promotes differentiation or maturation to both lineages through cellular cross talk. During fetal hematopoietic development, macrophages release Oncostatin M (OSM) which is important for hepatocyte differentiation . Similaric cells . This reBile acids have also been shown to play a key role in HSC expansion in the fetal liver . DespiteloCD133lo stromal cells have been reported to support CD34hiCD133hi fetal liver HSCs [loCD133lo cells were isolated from human fetal liver (15–23-week gestation), shown to express AFP, CK18, CK19, and EpCAM (CD326), like fetal hepatocyte progenitors, and differentiated into hepatocytes both in vitro and vivo, expressing albumin, HNF1A, and HNF1B [loCD133lo cells maintained SCID-repopulating ability of CD34hiCD133hi fetal liver HSCs after 7 days co-culture [loCD133lo cells are immature hepatocyte progenitors and support fetal liver hematopoiesis. In another report, human fetal liver cells have also been cultured for short term (6 days) and were able to maintain hepatic, endothelial, and hematopoietic lineages [The interaction between hepatoblasts and HSCs is also seen in human fetal liver. Human fetal liver CD34ver HSCs . CD34loCnd HNF1B . CD34loC-culture . These rlineages . These cThus, it is likely that the crosstalk between hepatoblasts and hematopoietic cells leads both hepatoblast maturation into hepatocytes as well as HSC expansion. Insights gained from these co-culture experiments may help design expansion systems without the need for fetal cells. Future experiments may focus on iPSC-derived hepatic/HSC cultures, which often remain fetal in nature and may be a useful source for modeling HSC in the fetal liver microenvironment.One of the more obvious and long attempted methods for in vitro production of HSC for transplant is directed differentiation from embryonic stem (ES) cell or iPSC sources (reviewed elsewhere ). In addAnother important factor is the selection of potential media for use in fetal liver HSC cell culture experiments. There are several commercially available media that focus on expansion culture of HSC, regardless of cell source; however, these provide limited expansion and are not yet in use for clinical expansion of HSC. Furthermore, culture of fetal liver HSC may have different media requirements, especially if liver cells are included in a co-culture system. Differentiation of hepatic cells from iPSC or embryonic stem cells (ESC)  requires a series of growth factors including BMP4, FGF2, HGF, and Oncostatin M –43. TablWhile fetal liver HSCs home to the bone marrow around birth and settle in the BM niche, there are differences in biological and molecular characteristics between fetal liver and adult BM HSCs, including cell surface markers, lineage differentiation bias upon transplantation, gene expression patterns, and cell cycle rates , 45. HSCThe gene signatures of HSC play a key role in their trafficking during development as well. The exact gene signature of HSC at various stages of development is still an area of current research, and recent technological advances in single cell transcriptomic analyses are revealing a huge amount of data to unravel this mystery. Recently, a combination of single cell RNA sequencing (scRNAseq) and ATACseq was used in fetal HSCs from AGM, fetal liver, and bone marrow to analyze gene signature differences including epigenetic changes in mice and humans. Single cell sequencing of the human AGM region has revealed two separate populations of early HSCs, similar to the separate waves of early hematopoiesis in model organisms, with the earlier one lacking arterial markers . SequencMany attempts at expanding HSC ex vivo utilize the cytokines present in the fetal liver, with SCF and TPO being the main two. Recently, a breakthrough in ex vivo HSC culture was reported with murine HSCs capable of being expanded between 236 and 899-fold . This stIn the field of bioengineering, a combination of engineering approaches has been utilized to create a human HSC niche for ex vivo culture Fig. . HoweverIt has been shown that the HSC in the fetal liver exist in close proximity to certain cells, specifically around the portal vessels . Future During embryogenesis, hemodynamic flow (shear stress) is critical for proper development of hematopoietic cells in vivo . MicroflAnother possibility for niche creation is through differentiation of iPSC or ESC  into a complex multilineage hepatic culture. Recently, iPSC were engineered to overexpress GATA6 to induce differentiation into the three germ layers . These iThis review has discussed the importance of the fetal liver niche in HSC expansion, a feat that occurs during development and has great clinical potential, yet has thus far proven difficult to recapitulate for ex vivo HSC expansion. Recent studies have increasingly honed in on specific cytokines cocktails for culturing HSC, and while some experiments are looking at modeling the HSC niche in culture with co-culture or ECM systems, many of these focus on the BM niche. This might not be ideal for the large-scale expansion that is desired and present in the fetal liver because iPSC-derived HSCs, if produced, would have similar characteristics of fetal HSCs. Current in vitro co-culture systems involving fetal liver HSCs largely involve with primary fetal cells which limits experiments to mouse cells , 37. As