The impact of high-density lipoprotein (HDL) cholesterol on the development of atherosclerosis and diseases of systemic circulation has been well documented both in experimental and registry studies. Recent discoveries in pulmonary arterial hypertension (PAH) revealed a significant impact of HDL on pulmonary artery vasoreactivity and patients’ prognosis. The vasoprotective activity of HDL primarily involves vascular endothelium that also plays a central role in pulmonary arterial hypertension (PAH) pathobiology. However, the exact mechanism in which this lipoprotein fraction exerts its effect in pulmonary circulation is still under investigation. This paper reviews potential vasoprotective mechanisms of HDL in pulmonary circulation and presents current clinical reports on the role of HDL in PAH patients. The significance of plasma high-density lipoprotein (HDL) cholesterol concentration in the development and progression of diseases has been documented by reports from both experimental and observational studies. The strong inverse relationship between HDL and cardiovascular disease was first established by the Framingham Heart Study . This obPulmonary hypertension (PH) is defined as an increase in mean pulmonary arterial pressure ≥25 mmHg at rest as measured in right heart catheterization. Pulmonary arterial hypertension (PAH) describes a subject with pre-capillary PH (pulmonary artery wedge pressure ≤15 mmHg and a pulmonary vascular resistance >3 Wood units) in the absence of conditions such as PH due to lung diseases, chronic thromboembolic pulmonary hypertension, or other rare diseases . PAH is Recently, PAH has been considered as a systemic disease due to the involvement of numerous organs and tissues and metabolic and inflammatory abnormalities ,32,33. IThe molecular mechanism explaining the role of HDL in maintaining optimal pulmonary vascular resistance was identified as vasodilation via endothelial nitric oxide (NO) and prostacyclin release. Worth mentioning is the fact that most of the currently used PAH-specific medications, including phosphodiesterase 5 inhibitors, direct stimulators of soluble guanylate cyclase, and prostacyclin analogues, act on vascular endothelium by enhancing NO and prostacyclin signaling ,42. In tHDL also displays potent anti-inflammatory properties. It is worth noticing that, in animal models of PAH, both inflammation and altered immunity induce vascular remodeling. Importantly, it was shown that both pulmonary vascular cells and inflammatory cells can be an important source of inflammatory cytokines that lead to vascular contractility, proliferation, and remodeling typical for PAH. As recently shown, interleukin(IL)-1 can induce fibroblast growth factor 2 and, together with IL-6, plays a central role in promoting the proliferation of fibroblasts and smooth muscle cells of the pulmonary vasculature in PAH . HDL par1–S1P5) resulting in contradictory effects on vascular remodeling. Some studies documented that S1P can induce vasoconstriction and development of pulmonary vascular disease via S1P2 receptors [1 receptor with HDL-bound S1P was able to mitigate inflammation in both in-vitro and in-vivo models by downregulation of adhesion molecules. Interestingly, albumin-bound S1P failed to exert a similar anti-inflammatory effect [This represents a potent mechanism in which HDL can modulate both innate and adaptive immunity and therefore prevent pulmonary vascular remodeling. What is more, HDL can bind circulating lipopolysaccharides (LPS), facilitate its hepatic clearance, and prevent LPS-induced endotoxemia. HDL also exhibits a potent impact on monocyte differentiation and IL-1β secretion in response to LPS in metabolic syndrome patients . The vaseceptors . The othy effect .HDL-mediated anti-inflammatory properties are also closely associated with HDL’s ability to protect LDL and other lipoproteins from free radical-induced oxidative damage. Selective increase of HDL particles displaying decreased surface rigidity and rich in apoA-I were able to attenuate oxidative damage and mitigate inflammation. The antioxidant pathway includes a transfer of altered lipids from LDL to HDL particles and subsequent reduction of oxidized lipids by apoA-I and other HDL components . These pIn the last years, impaired glucose and insulin metabolism was proposed as a novel risk factor in pathobiology of PAH ,37. RecePlatelet activation is another phenomenon associated with numerous mechanisms observed in the development of pulmonary vascular disease. Thrombotic lesions have been indicated as common pathological findings in patients with PAH . ArterioRecent studies indicate that HDL particles can regulate gene expression by binding and transporting endogenous microRNA (miRNAs). miRNAs are involved in multiple cellular processes, including cell differentiation, proliferation, and apoptosis. An emerging body of evidence indicates a substantial role of miRNA signaling in maintaining homeostasis of the pulmonary circulation. Altered signaling of miRNA has been associated with the development of numerous PAH vascular hallmarks. These include endothelial dysfunction, smooth muscle cells proliferation, activation of fibroblasts, and formation of plexiform lesions . The bonThe vasoprotective properties of HDL have been recently shown to play a significant role in patients with different forms of PAH. Heresi et al. showed that PAH subjects are characterized with lower HDL level than controls after adjusting for demographic factors and cardiovascular medical history. Patients with higher HDL were also characterized by longer time to clinical worsening than subjects with lower HDL values. What is more, in PAH patients, serum HDL concentration was able to differentiate between survivors and non-survivors . In anotRecent experimental and clinical data indicate protective properties of HDL in PAH, translating into survival benefits in long-term observations. However, the exact mechanism in which this lipoprotein fraction exerts its effect in pulmonary circulation is still under investigation and needs to be fully explored before it can be translated to novel diagnostic and treatment methods ,88.


Functional coronary circulation is essential for a healthy heart in warm-blooded vertebrates, and coronary diseases can have a fatal consequence. Despite the growing interest, the knowledge about the coronary vessel development and the roles of new coronary vessel formation during heart regeneration is still limited. It is demonstrated that early revascularization is required for efficient heart regeneration. In this comprehensive review, we first describe the coronary vessel formation from an evolutionary perspective. We further discuss the cell origins of coronary endothelial cells and perivascular cells and summarize the critical signaling pathways regulating coronary vessel development. Lastly, we focus on the current knowledge about the molecular mechanisms regulating heart regeneration in zebrafish, a genetically tractable vertebrate model with a regenerative adult heart and well-developed coronary system. The heart pumps blood relentlessly during an animal’s lifespan and is critical for survival. Such functional load demands a continuous supply of nutrients and oxygen in heart muscle cells, cardiomyocytes. The coronary circulatory system made of coronary arteries, veins and capillaries meets this demand and provides a route for immune cells and other regulatory cells to constantly maintain heart function while protecting the heart from physiological and mechanical challenges. Thus, it is not surprising that coronary artery disease is the leading cause of human mortality . In adulThe coronary circulatory system is dispensable in many non-mammalian vertebrates . In all Scorpaena), and the Type-Ib hearts have non-penetrating superficial vessels on epicardium . The subclass Type-Ic hearts have the epicardial vessels, which enter subepicardial space and emptied in intratrabecular luminal space  [Conger). Both of Type-III and Type-IV have circulatory vessels in both compact and trabeculated myocardial layers, but they differ in the amount of cardiomyocyte compaction. The compact cardiomyocyte form <30% of the total heart mass in Type-III heart. Most elasmobranchs have a Type-III heart. The Type-IV heart has compact myocardium >30% of the total heart mass  . In all icefish) ,14 or aricefish)  have theWt1), epicardin/capsulin (Tcf21), T-box factor 18 (Tbx18) supporting such developmental and evolutionary link between PE and kidney [It is hypothesized that in the early vertebrates, coronary vasculature evolved from pronephric external glomeruli (PEG). Highly vasculogenic glomerular cells transferred to the heart and supplied it with blood vessel progenitors. Hypothetically, this could be the first step in evolution for myocardial vascularization. PEG is found in the representatives of the most primitive vertebrates . During evolution, the anterior-most part of pronephros disappeared, and the cardiac inflow tract (sinus venosus) and liver expanded. With time, a temporary embryonic structure, proepicardium (PE) emerged. Such evolutionary origin may be reflected in the developmental association between PE/PEG and liver/sinus venosus in the gnathostomes . PE is aFrom the early tubular structure, the heart gradually increases the myocardial layers to match the increased blood circulation demand in a rapidly growing embryo. At this stage, the myocardial growth occurs by cardiomyocyte proliferation (hyperplasia). In endothermic vertebrates , the heart initially becomes a Type-I like heart, with avascular trabecular myocardium and very thin mantle of compact cardiomyocytes. The emergence of the coronary circulatory system is a well-regulated combination of several processes, which include angiogenesis vasculogenesis, arterial development and maturation and correlates with the timing of myocardial growth . The devFrom the regenerative perspective, the vertebrate heart categorization based on myocardial and coronary vasculature  is allurThe principal cellular components of a blood vessel include the endothelial cells and the mural/perivascular cells. Endothelial cells form the vessel wall, and the mural cells encircle the endothelial wall on its abluminal surface and regulate blood vessel development/maturation, stability, and the contractile activity .The origin of the coronary endothelial cells has been the subject of interest for a long time. The findings in this aspect evolved and contradicted previous findings as the employed technologies improved over time, and different model organisms showed different origins. Based on the morphological studies, initially, the coronary arteries and veins were believed to sprout from the aorta and sinus venosus respectively ,26 by enSema3D+ and Scx+ PE cells have lesser contribution to coronary endothelial cells than the subepicardial endothelial precursor from sinus venosus and the endocardium. Furthermore, Sema3D+ and Scx+ PE cells can contribute to early sinus venosus and endocardium [In mice, advanced genetic lineage-tracing studies by utilizing the Cre-LoxP based method demonstrated that the sinus venosus and endocardium mostly contribute to forming coronary vasculature in a complementary fashion in different regions of the heart ,36,37,38ocardium . There aocardium ,40,41,42ocardium .In fish, there are a limited number of studies on coronary endothelial cell development. In sharks, coronary vasculature develops at the embryonic stage. In a dogfish study, the coronary veins first appeared as a diverticulum of the sinus venosus . Later, Vegfa expression was found in the ventricular and OFT myocardium, IVS, and at the AV junction [vegfaa normally express in the epicardium [−/−vegfaa mutant embryos show severe angiogenic defects. In the −/−vegfaa mutant fish rescued (by injecting vegfaa 121 or vegfaa 165 mRNA at the one-cell stage) to adulthood, the coronary vessels are irregularly distributed and thinner compared to wild-type fish [Origin and development of the coronary endothelial cells are regulated by the interaction between the cardiomyocyte layer, myocardium, and the flanking epithelial layers, epicardium, and endocardium. During development as the expanding myocardial layer become hypoxic it activates Hypoxia-inducible factor, HIF-1α in avian and mammalian embryos . In the junction ,58 whichjunction  indicatijunction . In anotjunction . This injunction . In zebricardium . The vegype fish .Ptc1) expression in the myocardium. HH signaling in the myocardium is required for myocardial expression of Vegfa, Vegfb, and angiopoietin-2 (Ang-2) and likely the expression of Vegfc in perivascular cells and these factors regulate coronary vascularization [Ang-1) promotes coronary vein formation. The subepicardial venous structures originate from the sinus venosus in mouse embryos. ANG-1 regulates the migration of APJ -negative immature endothelial cells from sinus venosus into the atrium and ventricular myocardium. These immature endothelial cells from the sinus venosus express TIE-2 receptor. When these endothelial cells enter myocardium, TIE-2 is activated by ANG-1 and promotes the endothelial cell migration, proliferation and differentiation into coronary veins [Fibroblast growth factor (FGF) is another signaling pathway that acts along VEGF in regulating coronary vasculature development. In a chick-quail chimera study when the transplant was preincubated with FGF and VEGF, the local vascularization of the compact myocardium increased significantly . Retinoirization . Myocardry veins .In juvenile zebrafish, the Cxcr4-Cxcl12 chemokine signaling regulates coronary angiogenesis. During zebrafish heart development, cardiomyocytes express the ligand Cxcl12b and guide Cxcr4a expressing endothelial cells to undergo angiogenesis to cover the ventricular myocardium . ConsistCoronary vessel formation is a versatile process that can adapt to the need of the myocardium. Different signaling pathways have been discovered to regulate coronary endothelial cells originating from different sources in mice. ELABELA (ELA)-APJ signaling axis is mainly required for the sinus venosus derived coronary endothelial cells in mouse embryos. When this developmental process is compromised, endocardium derived endothelial cells expand to compensate the lost sinus venosus derived cells to ensure normal heart development .Xenopus development, PE emerges asymmetrically and develops only from the right side around Hamburger-Hamilton stages 14, and stage 41 respectively [Xenopus) embryo. In zebrafish, pericardial flow generated by heartbeat promotes the release of PE cells from the lining of the pericardial cavity and directs the motion of the PE cells and their colonization of the myocardium [Mural cells/Perivascular cells (vascular smooth muscle cells and pericytes) in the coronary vasculatures have a unique developmental origin. Epicardium, the outermost epithelial layer of the heart, plays essential roles in the coronary vessel development. In early development, epicardium is formed from the PE cells that migrate to the surface of the heart. PE is highly conserved during the vertebrate heart development ,72,73,74ectively ,75. The ocardium . In chicocardium ,77. Afteocardium ,77.In an effort of fate mapping the proepicardial descendent cells, in the chicken embryo, the vital dye Dil or replication defective retrovirus  tagged PE cells were found to form coronary smooth muscles, perivascular connective tissue and endothelial cells. The study led to the conclusion that the coronary smooth muscle fate was predetermined in PE before their migration to the developing heart . Later, tcf21 positive epicardial cells contribute to the perivascular cells [The growth factor FGF, EGF and VEGF treatment to the cultured quail epicardial cells induces them to take the mesenchymal fate . Based oar cells . In anotar cells .Several cell-signaling mechanisms have been identified regulating mural cell recruitment on endothelial cells and their differentiation. Platelet-derived growth factor signaling is a major pathway, which regulates mural cell recruitments. Endothelial cells of the angiogenic vessel secrete the ligand PDGFB that binds with the receptor PDGFRβ on the pericyte surface ,86. PDGFflox/floxWt1-Cre: Notch) coronary artery development and perivascular cell differentiation were impaired [Rbpj, the notch signaling transcriptional regulator, abrogated smooth muscle cell differentiation from EPDCs and conditional Notch over-activation caused premature differentiation of smooth muscle cell [Notch signaling regulates mural cell differentiation depending on mural cell-endothelial cell contact. In the epicardial Notch loss of function mouse  fish, it was shown that in uninjured heart vegfaa normally express in the epicardium. After ventricular resection, vegfaa expression increased in the endocardium around the wound area. The global genetic cardiomyocyte ablation induces endocardial vegfaa expression throughout the heart [VEGF signaling regulates the fast revascularization of the wound area. In the cryoinjured heart, e injury . The exphe heart  indicati−/−vegfaa mutant heart showed delayed revascularization with a disorganized plexus formation. The ubiquitous overexpression of the dominant negative, dn-Vegfaa isoform under heat-shock 70-like (hsp70l) promoter severely affected revascularization of the cryoinjured area. In Tg(hsp70l:dn-vegfaa) fish, the vascular density at the injured area reduced ~75%, and this caused almost 60% decrease in cardiomyocyte proliferation at 7 days after the cryoinjury. Inhibiting early revascularization in the dn-vegfaa fish, changed the scar matrix composition (more collagen deposition instead of fibrin-collagen mixture) in the damaged area which eventually caused permanent collagen scar formation and incomplete regeneration [The cryoinjured neration .fgf17b ligand expression around day seven post-injury. This expression pattern is maintained until day 30, many cells in the regenerated myocardial wall express fgf17b. Epicardium around the injured area upregulates FGF receptors fgfr2 and fgfr4 expression following a similar timeline. Inhibiting the signaling by ubiquitous expression of the dominant-negative form of the Fgfr1 driven by the heat shock responsive hsp70 promoter, impairs cardiac regeneration and leaves a scar at day 30 after the injury. In the hsp70:dn-fgfr1 fish, epicardial cells fail to integrate into the regenerating tissue and the coronary neovascularization is severely impaired [FGF signaling is activated in epicardium during zebrafish heart regeneration. FGF activity promotes EMT of the EPDCs which drive coronary vascularization and resulting cardiac regeneration. In the ventricular amputation injury model, cardiomyocytes along with other cells in the injured area upregulate impaired .pdgfb. The receptor pdgfrβ expression was observed in the epicardium, subepicardium and the fibrin clot at the injury site at day six after amputation. The pdgfrβ expression is maintained at day 10 and day 14. In an in vitro heart explant culture system, recombinant Pdgfbb treatment induced the epicardium-derived epithelial cells to lose the cell-cell contact and undergo EMT like transition. Pdgfrβ inhibitor blocks epicardial cell proliferation, EMT, and new coronary vessel formation in the wound area. Together these findings suggest that after the cardiac injury, Pdgfrβ signaling is activated in the epicardium. The activated epicardial cells then proliferate and undergo EMT to generate mural cells and promote coronary vasculature formation [PDGF signaling is activated in the injured area of the amputated heart. After amputation, circulating thrombocyte in the wound area expresses the ligand ormation ,94.2O2). H2O2 is required for efficient heart regeneration after ventricular resection [NADPH-oxidase (Nox) and dual oxidase (Duox) enzymes in the plasma membrane catalyze electron transfer from NADPH to the molecular oxygen to generate superoxide anions. Superoxide dismutase readily converts superoxide anions into hydrogen peroxide  and promotes their degradation by ubiquitin/proteasome [2O2 derepresses pro-regenerative pErk1/2 MAP kinase signal which acts downstream of the FGF and PDGF signaling in the epicardium [At day three after the injury, oteasome . Dusp6 dicardium ,96.Tg(kdrl:EGFP) zebrafish with diphenyleneiodonium (DPI), a Duox/Nox enzyme inhibitor and BCI, an inhibitor of Dusp6 phosphatase activity, it was shown that H2O2 and Dusp6 regulate coronary vessel growth in the injured area. Inhibiting H2O2 generation by DPI treatment reduced +kdrl coronary vessel formation during heart regeneration. However, BCI co-treatment with DPI maintained +kdrl vascularization [dusp6 promoter Tg(dusp6:memGFP), dusp6 expression was observed in endothelial cells along with mild expression in the cardiomyocytes. During regeneration, nascent coronary vessels showed dusp6 expression in the regenerates. More importantly, dusp6 mutant hearts showed similar accelerated regeneration as the chemical inhibitor treated fish, where many fibrosis genes were downregulated. The uninjured dusp6 mutant heart showed thicker compact myocardium with more vessels showing pErk expression. In the dusp6 mutant fish, heart regeneration is only mildly affected Pdgfrβ signaling inhibition [dusp6 mutant fish is impacted by Pdgfrβ and Fgf signaling pathways.By treating rization . Recentlrization . Treatinhibition . It remaigf2b is upregulated in the endocardium and epicardium in the wound area after ventricular resection [aldh1a2 expression is strongly upregulated in the endocardium and sequentially in the atrial and the ventricular epicardium [Despite the characterization of the signaling pathways involved in new coronary vessel formation in zebrafish heart regeneration, it remains to be further investigated whether the coronary arteries regenerate the same architecture as in the uninjured hearts. Furthermore, the epicardium seems to play a significant role by interacting with other cell types  in coronary vascularization of the damaged area ,94,95. Aesection . Interesesection . It willesection . Interesicardium ,104. It Myocardial regeneration requires the formation of the new vasculature that is essential for the survival of the cardiac tissue and to adapt to the changing environment after traumatic damage. The research utilizing different model organisms have shed light on how the natural regeneration of the heart can be achieved. Many developmental processes of the hearts are recapitulated during cardiac regeneration. The studies in non-mammalian animal models have revealed molecular mechanisms of coronary vessel development and perhaps also in revascularization. Several important questions remain unanswered. The heterogeneity of different vascular cells needs to be further characterized in the non-mammalian models. It also remains unclear how blood flow and hemodynamics regulate the coronary vessel formation. With the recent advancement in molecular, genetic and imaging technologies in zebrafish and other models, these critical issues can be addressed in the near future.


Fibrous dysplasia (FD) is a congenital disorder arising from sporadic mutation of the α-subunit of the Gs stimulatory protein. Osseous changes are characterised by the replacement and distortion of normal bone with poorly organised, structurally unsound, fibrous tissue. The disease process may be localised to a single or multiple bones. In McCune-Albright syndrome (MAS), fibrous dysplasia is associated with hyperfunction of endocrine organs and overproduction of melanin in the skin, while Mazabraud syndrome FD is associated with intramuscular myxomas. In radiology, FD is very often automatically associated with the term “ground glass matrix”. However, FD is a complex disease, and knowledge of its unique pathogenesis and course are crucial to understanding imaging findings and potential complications. This article aims to not only summarise the spectrum of radiological findings of osseous and extra-osseous abnormalities associated with FD but also to highlight the pathological base of the disease evolution, corresponding imaging changes and complications based on the disease distribution. We also have provided current recommendations for clinical management and follow-up of patients with FD.FD is often a part of complex disease, involving not only bone but also multiple other organs.• FD lesions are characterised by age-related histological, radiographical and clinical transformations.• Radiologists play a crucial role in the identification of osseous complications associated with FD.• The craniofacial form of the disease is the most common type of FD and the most difficult form to manage.• Patients with McCune-Albright syndrome may have different extra-skeletal abnormalities, which often require follow-up.•  Fibrous dysplasia (FD) is a disorder caused by sporadic mutation of the α-subunit of the Gs stimulatory protein, in which bone is replaced and distorted by poorly organised, structurally unsound, fibrous tissue. In McCune-Albright and Mazabraud syndromes, FD is associated with a range of extra-skeletal abnormalities. For many radiologists, FD is often automatically associated with the term “ground glass bone matrix”; however, FD is often a part of complex disease, and knowledge of its unique pathogenesis and disease course is crucial to understanding imaging findings, their changes over time, and potential complications.This article summarises more than 25 years of observation of patients with FD under the protocol “Screening and natural history study of fibrous dysplasia” at the National Institutes of Health , . We describe the spectrum of osseous and extra-osseous radiological findings related to FD and highlight the pathological base of disease evolution, corresponding imaging changes, complications based on disease distribution, and important surveillance and management techniques for radiologists.The disease process may be localised to a single bone (monostotic FD) or multiple bones (polyostotic FD). The current convention is to preserve the eponym Jaffe-Lichtenstein disease for isolated FD. The name McCune-Albright syndrome is used when FD is associated with extra-skeletal abnormalities. Mazabraud syndrome is reserved for cases of FD with associated intramuscular myxomas. Cherubism has historically been considered a variant of FD, but it is a genetically distinct disease. This format has been codified in the Paris Nomenclature of Constitutional Diseases of the Bone  Table 11.Table 1s stimulatory protein (GNAS mutations) leading to activation and inappropriate cyclic adenosine monophosphate (cAMP) overproduction  as might be expected . Signal Technetium 99m-methyldiphosphonate (99m-Tc-MDP) bone scan is used to detect metabolically active lesions and assess the extent of disease, especially in young patients . Small aGNAS mutations undergo apoptosis, leading to a decreased number of FD cells and, thus, changing the classic radiographical appearance of ground glass to a more dense and sclerotic pattern , leading to deformity, fracture and pain . The risFD is the most common benign rib lesion. In adults, FD of the ribs is often discovered incidentally and is usually asymptomatic; however, it may present with obvious deformity or pain.Approximately 2–3% of patients with FD have extra-skeletal disease, known as McCune-Albright syndrome (MAS). Bone changes in MAS are often more severe than in polyostotic FD without extra-skeletal manifestations. MAS patients have the most extensive disease and the most complicated course of the disease, regularly experience multiple fractures, and require adequate surgical treatment .Eighty-five percent of female patients have functionally active ovarian cysts, resulting in gonadotropin-independent precocious puberty. A typical ultrasound finding in these patients is a large unilateral ovarian cyst, which can sometimes be haemorrhagic and appear to have mixed cystic and solid elements Fig. . RecurreAbout 80% of male patients have testicular abnormalities that typically manifest as unilateral or bilateral testicular enlargement. Ultrasound may demonstrate hyperechoic lesions (49%), hypoechoic lesions (30%), microlithiasis (30%), heterogeneous lesions (47%) and focal calcifications (11%). These radiological findings often correspond to areas of Leydig and/or Sertoli cell hyperplasia. Imaging cannot differentiate benign from malignant pathology Fig. . The malHyperthyroidism is common (38%); evidence of thyroid involvement without frank hyperthyroidism is even more common (63%). Ultrasound typically demonstrates diffuse benign changes such as multinodular goitre, and mixed cystic and solid nodules. Malignant transformation of affected thyroid tissue has rarely been reported Fig.  25].Fig.Fig25].FGNAS mutations in the anterior pituitary that can lead to autonomous growth hormone production, typically accompanied by hyperprolactinaemia. The abnormality is usually diagnosed during young adulthood and is almost always associated with skull base FD. Among patients with growth hormone excess, adenoma can be seen in about 54% and macroadenoma in more than two-thirds of those cases . IPMN can progress to invasive adenocarcinoma, suggesting that IPMN is an important precursor to pancreatic malignancy. IPMNs occur at an early age, and optimal care is evolving. Recently, liver adenomas and choledochal cysts were also described in patients with MAS. Screening symptomatic patients with abdominal MRI or magnetic resonance cholangiopancreatography (MRCP) is recommended , which often require follow-up.Many patients with FD undergo repeated imaging with radiation; therefore, high radiation exposure is a concern. Efforts should be made to reduce cumulative radiation risks for these patients.


For example, isobutyramido- stilbene undergoes FeCl3 promoted cyclization to produce only indoline, while n-butyramidostilbene, under the same conditions, produces both indoline and bisindoline.The  Previously undiscovered steric, conformational and stereoelectronic effects have been considered in this study involving eight stilbenes.Although previous studies by our group have demonstrated the synthetic utility of benzophenone  as an effective additive for dramatically improving the yield of indolines (and suppressing bisindoline formation), the present study demonstrated that even in the absence of benzophenone, the amide structure can also under certain conditions discriminate between the indoline and bisindoline forms (enhancing the former and suppressing the latter). Stilbene 9 with an ortho-methoxy substituent dramatically shifts the electron density in the olefinic bond  as the single electron transfer reagent [We would like to draw the reader’s attention to some intriguing electronic effects. For example, stilbene bond see . The C(7 reagent ,20,21. F


Recent policy initiatives, including Bundled Payments for Care Improvement (BPCI) Initiative by the Centers for Medicare and Medicaid Health Services (CMS), encourage healthcare providers to manage the total episode of care, rather than just the surgical episode. Surgical site infections (SSI) following total joint replacement result in preventable morbidity and suffering for patients and excess healthcare utilization for healthcare providers. This study sought to estimate the additional resources associated with SSIs within the 90-day episode of care following hip and knee joint replacement.Using the 2013 Nationwide Readmissions Database (NRD), healthcare resource utilization was compared between propensity score matched patient groups with and without SSI-related readmissions within the 90-day episode of care following total joint replacement.Surgical site infections were associated with significantly longer hospital length of stay and increased costs following hip and knee joint replacement procedures. Generalized estimating equation regression results confirmed that additional costs associated with SSIs following both cohorts were significant, with additional hospital length of stay and costs following total hip and knee replacement procedures ranging from 4.9 to 5.2 days and $12,689 to $12,890, respectively.Surgical site infections following total joint replacement account for significant additional healthcare resource use within the 90-day episode of care. As life expectancy continues to improve, healthcare administrators anticipate a corresponding rise in the incidence of total joint replacement, particularly primary hip and knee procedures –3. The dRecent policy initiatives, including the Comprehensive Care for Joint Replacement (CJR) program from the Centers for Medicare and Medicaid Health Services (CMS), are intended to encourage healthcare providers to consider best practices in managing patients over a 90-day episode of care, rather than just the acute care period , 12. Bunhttps://www.hcup-us.ahrq.gov/db/nation/nrd/nrddbdocumentation.jsp) national database that captures all-payer hospital inpatient-related events, and its flexibility allows various readmission-related analyses. Capturing more than 14 million discharges in 2013, 21 US states are accounted for in the database, representing over 2000 hospitals.The current study sought to estimate the incidence and additional economic burden associated with surgical site infections within the 90-day episode of care period using the National Readmissions Database (NRD) of the Healthcare Cost and Utilization Project (HCUP). This unique database captures information on discharges with and without repeat hospital visits per year, allowing for the longitudinal study of patient populations through a 12-month period, such that readmissions and associated resource use following an event of interest can be captured and followed over time. With an increased emphasis on improved healthcare delivery while containing costs, this database has the capability of identifying causes of readmissions, making it possible to critically assess relevant patient populations and proactively develop strategies to successfully mitigate inefficiencies, while improving outcomes. The HCUP NRD is a publicly available  primary procedure code for an elective total hip or knee joint replacement procedure were included into the study. Patients who presented with bilateral joint procedures were excluded from the study.The date of admission for the joint replacement was defined as the index date, and each patient was followed forward in time to capture any repeat visits associated with surgical site infections within 90 days of the primary joint replacement procedure. Readmissions due to a primary diagnosis of surgical site infections identified based on the presence of any of the ICD-9 CM diagnostic codes which include 99666, 99667, 99669, 99851, and 99859 were identified  through the 90-day post-discharge period.t tests and generalized estimating equations which accounted for the matched nature of the final study sample were used to assess the relationship between hospital resource use and the presence/absence of SSIs following TJA. All analyses were carried out using the statistical analytical software, SAS 9.4. Descriptive statistics  were used to summarize baseline sociodemographic, clinical characteristics, and health care utilization cost patterns. McNemar’s and generalizing estimating equation (GEE) regression analyses were also conducted as deemed appropriate.Frequencies, means, and standard deviations were used in univariate analyses to describe the study population; thereafter, using a caliper-propensity score without replacement matching technique, patients identified to present with SSI within the 90-day post-index event discharge period were matched with patients without SSIs based on age, gender, and primary payer status . Other baseline variables included were Zip income quartile, hospital bed size, hospital teaching status, and Charlson comorbidity scores  and LOS  between patients with SSIs  and patients without SSIs  within 90 days post-discharge was statistically significant.A total of 48,143 adult ≥ 45-year-old patients were identified to have met inclusion criteria within the study period; of this, 403 (0.84%) were identified to have surgical site infection-related readmissions. Table p < 0.0001 associated with patients with SSIs  compared to their peers without SSIs ; by LOS, patients readmitted due to SSIs (8.02 days) spent an additional 4.9 days; p < 0.0001 in the hospital compared to their peers without SSI-related readmissions with an LOS of 3.12 days  met the inclusion criteria for readmission due to surgical site infection within the 90-day episode of care period. Table 2% met thAlthough 400,000 hip and knee joint replacement procedures were completed in 2014, accounting for over $7 billion in hospitalizations , qualityThe findings of the current study confirm that the additional costs associated with SSIs within the 90-day episode of care following total joint replacement are significant. However, patient suffering due to additional treatments of these preventable infections are unquantifiable and so is the pressure that these events can put on providers that are subject to working based on bundled payments. With projections suggesting that joint replacement procedures are on the rise , there iAlthough there are treatment protocols and guidelines designed to improve perioperative efficiencies, curtail and/or prevent the incidence and severity of preventable surgical site infections following joint replacement –16, a moWith the surgical site infection-related readmissions observed to be primarily due to the internal device used, it would also be important to consider how outcomes differ by the specific internal joint prosthesis used in joint replacement. While device costs remain a significant determining factor in the choice of the prosthesis used, there is a need to assess how long-term outcomes impact overall treatment costs. The extended period of antibiotic treatment and revisions as a result of infections from surgical implants are considered significant and in most cases avoidable. This is of crucial financial importance as complication of device, implant, or graft is considered one of the five most expensive conditions by payer, accounting for over $12.4 million and 632,000 hospital stays .Our study adds to the existing literature estimating the economic burden of SSIs over the 90-day episode of care period following hip and knee joint replacement, utilizing the Nationwide Readmissions Database, a nationally representative database with the capability of capturing readmissions following an event of interest –26. We eSurgical site infections following total joint replacement are associated with significant healthcare costs within the 90-day episode of care. Healthcare providers, particularly those subject to quality improvement initiatives and bundled payments, are encouraged to consider evidence-based protocols to avoid excess morbidity, patient suffering, and the financial costs associated with these events.


Many secreted peptides used for cell–cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies. Chlamydomonas [Cilia are membrane-bound cellular projections containing a microtubule-based scaffold, the axoneme, that is templated directly from the triplet microtubules of a modified centriole termed the basal body. Cilia are sensory organelles that can be characterized as motile (capable of cell and/or fluid propulsion) or immotile   Fig. 1aa1a, b. Tydomonas –5.Fig. 1Nearly all cells in the human body (except those of lymphoid and myeloid origin) build a cilium at some point in their life cycle . In addiCilia are highly complex: proteomic, transcriptomic and comparative genomics approaches in various organisms have identified many hundreds of proteins associated with these organelles , 9; indeChlamydomonas, enhanced ciliary power output during viscous loading is mediated by increased trafficking of the dynein regulatory factor Lis1 into cilia [Chlamydomonas, are confined to its motile cilia [The cilium is a discrete cellular compartment; entry into both motile and immotile cilia is controlled in part by a multi-subunit gate termed the transition zone , 19  in building cilia [Chlamydomonas and metazoans  suggesting that it dates to the last eukaryotic common ancestor and represents an important aspect of ciliogenesis. Here we review the evidence supporting a role for the PAM protein and its amidating activity in ciliary assembly, suggest models for PAM function in this process, and describe how interactions of PAM with the actin cytoskeleton might alter both cilia and microvilli, leading to broad and generalized effects on cytoskeleton-based cellular protrusions. Furthermore, we address the intriguing phylogenetic question of how some organisms that lack PAM can still build cilia and briefly discuss the more clinical implications of merging the fields of ciliogenesis and peptidergic signaling.Recent studies have uncovered an unanticipated role for a secretory pathway enzyme involved in bioactive peptide synthesis  first dChlamydomonas, the IFT-A complex associates with a specific “cytoplasmic” dynein isoform to return IFT particles to the ciliary base and ultimately to export proteins out of the cilium [Since the ciliary matrix lacks ribosomes, proteins are not synthesized in this cellular compartment. Cargoes destined for this organelle must be recognized, sorted and trafficked from the cytoplasm or endomembrane system. This process involves the intraflagellar transport (IFT) machinery that mediates bi-directional transport along ciliary doublet microtubules Fig. . The mule cilium . In nemae cilium . In addie cilium , includie cilium .Fig. 3MeThe trafficking of membrane proteins such as PAM into and out of the cilium poses further challenges , and thePAM (EC 1.14.17.3) is best known for its role in the neuroendocrine system, where it catalyzes a late step (α-amidation) in the synthesis of many secreted bioactive peptides such as oxytocin, vasopressin, gonadotropin-releasing hormone and neuropeptide Y . As peptpeptidylglycine α-hydroxylating monooxygenase  14.5 with ventricular hypertrophy, massive edema and a poorly formed vasculature [pam/−− zebrafish embryos exhibit small eyes, cyst-like protrusions associated with the pronephros, hydrocephalus and edema, dying at ~ 10 days post-fertilization , strongly suggesting that this enzyme was present in the last eukaryotic common ancestor which is thought to have had a motile cilium that was also used for signaling [It is now clear that bifunctional, integral membrane PAM predates evolution of the nervous system . A PAM-lignaling , 66.In mammalian cells, PAM is subject to complex, cell type-specific trafficking through the biosynthetic and endocytic pathways ; the traChlamydomonas, ~ 7% of the total PHM activity is present in cilia [Chlamydomonas cilia. Similarly, Chlamydomonas PAM lacking most of its CD exhibits impaired trafficking; although the mutant protein is still found in the cilia, it tends to accumulate in the secretory pathway [Chlamydomonas PAM is distributed appropriately when expressed in murine cells  that traffics to the nucleus and alters the expression of a subset of genes , 71. Golin cilia . Disrupt pathway . The tralls Fig. b.Chlamydomonas cilia using antibodies against both the PAM luminal domain and the CD; the latter only yielded a signal after the ciliary membrane had been permeabilized [Chlamydomonas cilium, and biochemical fractionation demonstrated that PAM associates with the microtubular axoneme as does another ciliary membrane protein—the non-selective cation channel, polycystin 2 [Given PAM’s topology in the secretory pathway, the catalytic domains are predicted to be on the outside of the cilium Fig. a where tabilized . Intrigucystin 2 ; whetherChlamydomonas, lack of PAM results in upregulation of an actin paralogue that can functionally compensate for the loss of canonical actin in essential cellular systems; upregulation of this paralogue also occurs in the ida5 actin-null mutant [Chlamydomonas mimics loss of actin. Furthermore, in Chlamydomonas cells lacking PAM, cellular actin is reorganized from a diffuse array spread throughout the cytoplasm into a few phalloidin-stained foci [The actin cytoskeleton is intimately involved in the formation of cilia , has effl mutant , so in tned foci .Fig. 6PAPam mRNA is one of the most abundant [pam-null mutant embryos as PAM protein can be made at very early developmental stages from the large maternally derived mRNA stores. However, after several days of development, there is no detectable amidating activity and a dramatic loss of microvilli and motile cilia is observed along almost the entire length of the pronephros, suggesting that these structures either cannot form or be maintained in the absence of PAM. As PAM is absent from the microvilli themselves , and exhibited a normal contractile vacuole cycle, which is another process heavily dependent on membrane trafficking. Thus, the ciliary and Golgi defects observed following the loss of Schmidtea mediterranea and two vertebrates (zebrafish and mice) [pam-null zebrafish [Demonstration that PAM plays a role in metazoan ciliogenesis came from studies in the planarian nd mice) , 38. PlaPam/−− embryos were much shorter than controls (0.5 versus 0.9 μm), suggesting that they represent immature or aberrant forms  in the cell body to build two approximately half-length cilia without additional protein synthesis [Chlamydomonas cells expressing CrPAMΔCD, a mutant with functional catalytic cores that lacks ~ 75% of the CD, have enhanced enzyme activity as the truncated protein accumulates in the secretory pathway, and make full-length cilia that are completely motile [Drosophila) the PAL protein also has an associated TMD/CD, recent database searches suggest that one insect order—the lepidoptera—express only soluble PAL lacking the TMD/CD again indicating the catalytic domains may be important. Although insects lack multiciliated epithelial cells, they still build ciliated motile sperm and use modified cilia for mechano- and chemo-sensation and in the chordotonal organ [A key question raised by these observations is whether the ciliary phenotypes are caused by the lack of PAM-mediated protein–protein interactions, by the loss of amidating activity per se, or both. Currently, several lines of evidence provide support for a role for amidating activity. First, planaria exhibited the strongest ciliogenesis phenotype only when both membrane-PAM and soluble PHM were targeted together, thereby reducing amidating activity to very low levels. Furthermore, knockdown of soluble PHM alone resulted in ciliary loss and the slow muscle-driven motility characteristic of animals with defective motile cilia , again synthesis . Third, y motile . Furthery motile , and whial organ –93. Thesal organ ), selectal organ , 96 and Chlamydomonas, loss of PAM alters Golgi structure as the membrane stacks become more highly curved [Chlamydomonas affects formation of the TZ and disrupts or modifies the ciliary gate such that IFT components accumulate in the post-TZ stubs [Chlamydomonas responds to the loss of PAM with a large increase in the level of two TZ proteins (CEP290 and NPHP4) and an IFT-A protein (IFT139) [The available evidence supports a species-specific role for amidating activity in promoting ciliogenesis. However, the identity of the substrate(s), product(s) and downstream target(s) important for this process remain unknown. In addition to a general lack of amidated products in the secretory pathway, PAM deficiency leads to several other distinct effects, each of which might also impact a different aspect of the ciliogenic pathway Fig. . In Chlay curved . This may curved , 80. Secy curved . ClathriTZ stubs . Potenti(IFT139) ; these pPlasmodium to extrude a membrane-less axoneme assembled in the cytoplasm [In multiciliated epithelial cells of both planaria Fig. b and zebytoplasm . In the ytoplasm ), cytosoChlamydomonas, it becomes stably associated with the microtubular axoneme [Chlamydomonas, ciliary PAM might function in trans, amidating substrates present on the surface of the interacting cilium. In Chlamydomonas, the ciliary membrane is the only membrane normally directly exposed to the environment . Local generation of amidated factors might play a key role in providing a sufficiently high concentration of modified products for productive downstream signaling. As PAM CD binds actin directly with sub-μM affinity [In addition to its role in cilia formation, PAM is also trafficked into these organelles; in  axoneme . The topaffinity , it is aaffinity , 99.Intriguingly, PAM is tightly associated with the axoneme and in vitro, unlike most ciliary membrane proteins, PAM is only solubilized when demembranated cilia are treated with increased salt concentrations, as observed for several other axonemal substructures such as the dynein arms. This interaction might occur directly, through association of the intraciliary PAM CD with the axoneme, or indirectly, through an axoneme-associated component (which might even extend across the membrane into the extracellular space). Why is this unusual transmembrane protein tethering necessary? One possible explanation is that it provides a mechanism to avoid the general, non-specific or unregulated loss of membrane-associated PAM from the cilium—either in ectosomes budded from the ciliary membrane or from BBSome/IFT-mediated recycling back to the cell body. A further puzzle is the apparent 250–300 nm spacing for PAM observed by immunogold EM ; this spMonosiga brevicollis (XP_001746677) and both PHM- (GGOY01024199) and PAL-like (GGOY01026207) sequences in Salpingoeca urceolata. Similarly, membrane-PAM is present in the filasterean Capsaspora owczarzaki (XP_004363913). A second clear instance of PAM loss occurs in the green plant lineage. Although PAM is found in ciliated unicellular and colonial chlorophyte algae , it is missing in all land plants, including mosses and Ginkgo, which both have flagellated sperm cells.PAM and/or separate PHM and PAL modules are present throughout metazoans and the chlorophyte green algae; in addition to key roles in peptidergic signaling, in both these lineages PAM is involved in ciliogenesis. However, the pattern of PAM-like gene expression in other eukaryotic groups is more varied, suggesting that this enzyme has undergone multiple independent loss events during evolution and that in some organisms, ciliary assembly in the absence of PAM occurs Fig. . For exaTrypanosoma), stramenopiles , and fornicates , also completely lack PAM. Intriguingly, a more complex pattern of loss is apparent in the ciliated alveolates. Some, such as Tetrahymena and Paramecium, do not encode PAM-like sequences. However, PHM- (EX872387) and PAL-like (EX872386) partial sequences were previously reported in another alveolate—the dinoflagellate Karenia brevis [Emiliania huxleyi and Chrysochromulina sp. express a soluble PHM + PAL . Haptophytes have calcite-based coccoliths forming an exoskeleton and two motile cilia. Furthermore, they assemble an additional thin microtubule-based cellular protrusion (the haptonema), and use bidirectional transport along this structure for food/prey retrieval [Chlamydomonas cilia [Genomic studies reveal that numerous other ciliated groups, including excavates  lack PAM. However, that PAM likely plays non-cilia related roles in at least some unicellular organisms is evidenced by its retention in the filasterean C. owczarzaki, which appears to lack all cilia-specific genes [Ostreococcus lucimarinus, which is entirely missing any IFT machinery, also has PAM; this organism does not build cilia and retains only a single axonemal inner arm dynein that may have been repurposed for a non-ciliary role [Coccomyxa sp. and Chlorella variabilis [A broad and diverse array of non-ciliated unicellular organisms , a highly conserved copper-, molecular oxygen- and ascorbate-dependent secretory pathway enzyme, in building cilia. This connection has been observed in both vertebrates and algae, suggesting that it dates to the last common ancestor of eukaryotes and represents a fundamental feature of ciliogenesis. Current data support a role for amidating activity in forming the ciliary gate and regulation of the IFT system. Numerous studies have also revealed a connection between PAM and control of the actin cytoskeleton, and lead to the suggestion that PAM might play a central role in trafficking of membrane or other components to these cytoskeleton-based cellular extensions. Key issues for the future include identifying the PAM substrate(s) involved in ciliogenesis and determining the role and fate of ciliary-localized PAM and its potential amidated products.


Isolated left ventricular noncompaction (LVNC) is a rare form of cardiomyopathy that is characterized by deep intertrabecular recesses and abnormal trabeculations that can be observed on transthoracic echocardiogram (TTE) or cardiac MRI (CMR) studies. Our case describes a 41-year-old male who presented with exertional chest pain and was discovered to have significantly reduced left ventricular ejection fraction (LVEF) which was nonischemic in etiology as confirmed by cardiac catheterization. Subsequent evaluation with CMR imaging revealed noncompaction of the left ventricle. The patient received defibrillation and lifelong anticoagulation given his elevated risk of sudden cardiac death (SCD). This case highlights the importance of considering unconventional etiologies of cardiomyopathy when investigating new-onset heart failure as well as the necessity of life-saving measures such as anticoagulation and defibrillator implantation in view of arrhythmogenic structural heart diseases. Left ventricular noncompaction (LVNC) is an uncommon cardiomyopathy estimated to occur between 0.014 and 1.3 percent in the general population. It is characterized by deep intertrabecular recesses and abnormal trabeculations popularly believed to result from a failure of a phase of cardiac development during which the normal myocardium compacts inward. However, it can also involve abnormalities during ventricular wall formation or remodeling which can be transient or permanent .Clinical manifestations vary widely and can range from asymptomatic disease to symptoms of heart failure, left ventricular systolic dysfunction, thromboembolism, Wolff-Parkinson-White (WPW) syndrome, atrial and ventricular arrhythmias, bundle branch blocks, and sudden cardiac death. The most common manifestations are heart failure and ventricular systolic dysfunction.There is currently no universally-accepted criteria for classifying and diagnosing LVNC. Depending on the organization, it may be classified as a genetic cardiomyopathy or as an unclassified cardiomyopathy , 4. DiagThe patient was a 41-year-old male with a past medical history of unspecified congenital heart disease, hypothyroidism, and hypogonadism (taking intramuscular testosterone injections twice weekly), who presented with a chief complaint of chest pain during exertion of 1-month duration. The pain was described as 10 out of 10 in intensity, burning with a band-like tightness in nature, and located in the epigastric area with radiation toward the substernal region. His initial cardiac enzymes were significant for a troponin level of 0.62 ng/mL. His B-type natriuretic peptide (BNP) level was 1,190. An electrocardiogram (ECG) revealed a sinus rhythm with right superior axis deviation, biatrial enlargement, and right ventricular hypertrophy. Further evaluation with a TTE revealed that the patient had a LVEF of 20-29% with a markedly dilated left atrium and left ventricle. Subsequently, a cardiac catheterization did not reveal evidence of obstructive coronary artery disease. Given the trivial history of congenital heart disease and nonischemic cardiomyopathy, a cardiac MRI was then conducted and revealed a noncompacted to compacted myocardium ratio of greater than 2.3 in the left ventricle, global hypokinesis with severely depressed left and right ventricle ejection fractions of 27% and 26%, biatrial enlargement, and no evidence of a left ventricular thrombus . Some literature supports the use of anticoagulation in patients with LVEF less than 40%, a history of atrial fibrillation, or a prior cardioembolic event . All pat


Burn patients have a high risk of sepsis-related mortality even after surviving the initial injury. Immunosuppression increases the risk of sepsis after burn injury, as does the disruption of the intestinal epithelial barrier, which allows the translocation of bacteria and bacterial products into the circulation. The integrity of the intestinal epithelial barrier is largely maintained by the intestinal microbiota. Burn injury has been reported to result in significant changes in the intestinal microbiome composition. In this mouse study, we confirm these taxonomic differences in a full-thickness scald injury model using CF-1 mice. For the first time, we also address alterations in functional gene expression of the intestinal microbiota after burn injury to assess the microbiome’s physiological capabilities for overgrowth and pathogenic invasion: 38 pathways were differentially abundant between the sham and burn injury mice, including bacterial invasion of epithelial cells and gap- and adherens junction pathways. Burn patients, who survive the initial trauma, develop a state of relative immune compromise, making them highly susceptible to infections , 2. Burnde novo synthesis of nutrients and assistance in immune development and function [Firmicute, Proteobacteria and Bacterioidete , 9. Changes to the microbiome result in a dysbiosis, that disrupts this regulatory environment, enabling low abundant and pathogenic populations (i.e. Clostridium difficile) to expand [In healthy individuals, the gut microbiome protects against pathogenic microbes and helps maintain the intestinal epithelial barrier . This cofunction . The guto expand , 11. Dyso expand . Thus, tSeveral factors have been reported to affect the composition and biodiversity of the microbiome, including diet, environment, medication, infection/inflammation and burn injury itself –15. On dad libitum access to pellet diet and water. All experiments are approved by the Institution Animal Care and Use Committee (IACUC number 08-09-19-01) of the University of Cincinnati.Male CF-1 mice were obtained from Charles River Laboratories  at five weeks of age and allowed to acclimate for one to two weeks prior to conducting experiments. All mice were housed in standard environmental conditions and had We used a scald burn injury model as previously described  Briefly,2 at a 30% fill rate and cecal fecal samples (100–200mg) were collected. Nucleic acid isolation was performed with the MoBio PowerMag Microbiome kit  according to the manufacturer’s guidelines and optimized for high-throughput processing. All samples were quantified via the Qubit Quant-iT dsDNA High Sensitivity Kit  to ensure that they met minimum concentration and mass of DNA.On post burn day 1 (PBD1), mice were euthanized with COSamples were prepared for sequencing with the Illumina Nextera XT kit and quantified with Quant-iT dsDNA High Sensitivity assays. Libraries were pooled and run with 100 bp paired-end sequencing protocols on the Illumina NextSeq 500 platform.Host sequences were removed with Kraken , which uMetaPhIAn2  was useFiltered DNA sequences were mapped against a reference database of all proteins within the KEGG databases (version 75.0). The database is composed of 8,973,418 protein sequences extracted from 2,854 genomes. The search of translated DNA sequences was executed using Diamond  and hitsRichness is the sum of unique species found in each sample. Shannon diversity utilizes the richness of a sample along with the relative abundance of the present OTUs to calculate a diversity index.All profiles are inter-compared in pair-wise fashion to determine a dissimilarity score and store it in a distance dissimilarity matrix. Distance functions produce low dissimilarity scores when comparing similar samples. Abundance-weighted sample pair-wise differences were calculated using the Bray-Curtis dissimilarity. Bray-Curtis dissimilarity is calculated by the ratio of the summed absolute differences in counts to the sum of abundances in the two samples .Two-dimensional ordinations and hierarchical clustering maps of the samples in the form of dendrograms were created to graphically summarize the inter-sample relationships. Principal Coordinate Analysis (PCoA) is a method of two-dimensional ordination plotting that is used to help visualize complex relationships between samples. PCoA uses the sample-to-sample dissimilarity values to position the points relative to each other by maximizing the linear correlation between the dissimilarity values and the plot distances.Permutational Analysis of Variance (PERMANOVA) is utilized for finding significant differences among discrete categorical or continuous variables. In this randomization/Monte Carlo permutation test, the samples are randomly reassigned to the various sample categories, and the between-category differences are compared to the true between-category differences. PERMANOVA utilizes the sample-to-sample distance matrix directly, not a derived ordination or clustering outcome.The Wilcoxon Rank Sum test was employed to identify differentially abundant taxa, genes or pathways. Where samples could be paired across categories, a paired Wilcoxon Signed Rank test was employed. P-values were adjusted by Benjamini-Hochberg procedure to control for false discovery rates from multiple testing.Mice were subjected to a 28% full-thickness scald injury. To assess their overall health status after burn injury, we first assessed changes in body weight. On post burn day 1 (PBD1) mice had lost 3.3 g body weight on average, corresponding to approx. 10% of their total body mass . We alsoIn order to analyze potential changes to the microbiome upon burn injury, cecal feces samples were collected on PBD1. The taxonomic richness  was statistically similar between both groups: Similar sums of unique species were found in each sample  and the Firmicutes  and Spleen focus forming virus (genus Gammaretrovirus) were enriched in the burn injury mice samples . A reduced abundance of this pathway corresponds to the reduced abundance of Actinobacteriae in burn injured mice and constitutes a loss of a self-regulation of the microbiome. The second downregulated pathway, N-glycan biosynthesis, can result in immune evasion, as non-glycosylated bacterial proteins are often no longer recognized by the host immune system [Tetracycline biosynthesis was one of the suppressed pathways. Tetracyclines are antibiotics produced by e system .Many of the metabolic pathways that were more abundant in the microbiome of burn injury mice have potential immunomodulatory consequences: Increased production of prostaglandin E2 –either through increased biosynthesis of its precursor alpha-linoleic acid, through increased arachidonic-acid biosynthesis or through increased ether lipid metabolism—suppresses the type 1 immune response .Bacteriodetes found in burn injury microbiome samples. Bacteriodetes are major producers of LPS. Bacteriodetes-derived LPS has been reported to be immunoinhibitory, which may contribute to disease susceptibility upon burn injury [LPS biosynthesis was also more abundant upon burn injury. This matches the expansion of n injury . Addition injury . LPS bion injury .A large number of significantly affected pathways upon burn injury are linked to infection and pathogenic invasion. Particularly the pathways regulating the actin cytoskeleton, gap- and adherens junctions and bacterial invasion of epithelial cells would contribute to the disruption of the intestinal barrier and enable the translocation of bacteria and subsequent sepsis.Enterobacteriaceae. We also identify functional gene expression changes upon burn injury, linking particular pathways to dysbiosis, lack of barrier integrity and pathogenic invasion for the first time. The disruption of the intestinal barrier following dysbiosis and the resulting translocation of bacteria and their products from the gut lumen to the circulation can lead to (Gram-negative) bacteremia, resulting in sepsis, multi-organ failure and even death of burn patients.Our data confirms taxonomic differences to the intestinal microbiome upon burn injury, particularly the overgrowth of S1 TableTable 1 includes individual data points for body weight before burn and on PBD1, spleen weight, spleen-to-body weight ratio, serum G-CSF and serum IL-6 levels for each sample.(XLS)Click here for additional data file.S2 TableTable 2 includes individual data points on richness, Shannon index and dissimilarity matrix for beta diversity measures.(XLS)Click here for additional data file.S3 TableTable 3 includes all count tables and the mean abundance values for the different phyla, families and strains.(XLS)Click here for additional data file.S4 TableTable 4 includes individual data points on pathway alpha diversity and count tables for pathways and individual genes, as well as the mean log2 fold change values for each gene and pathway.(XLS)Click here for additional data file.


In meiosis, crossovers between homologous chromosomes link them together. This enables them to attach to microtubules of the meiotic spindle as a unit, such that the homologs will be pulled away from one another at anaphase I. Homologous pairs can sometimes fail to become linked by crossovers. In some organisms, these non-exchange partners are still able to segregate properly. In several organisms, associations between the centromeres of non-exchange partners occur in meiotic prophase. These associations have been proposed to promote segregation in meiosis I. But it is unclear how centromere pairing could promote subsequent proper segregation. Here we report that meiotic centromere pairing of chromosomes in mouse spermatocytes allows the formation of an association between chromosome pairs. We find that heterochromatin regions of homologous centromeres remain associated even after centromere-pairing dissolves. Our results suggest the model that, in mouse spermatocytes, heterochromatin maintains the association of homologous centromeres in the absence crossing-over.The online version of this article (10.1007/s00412-019-00708-6) contains supplementary material, which is available to authorized users. Drosophila and yeast, a high proportion of non-exchange chromosomes (those without crossovers) partition correctly in meiosis I  partners  that runs along their aligned axes. In budding yeast, the SC disassembles from the chromosome arms in late prophase except at the centromeres where it mediates their pairing , with a block of pericentromeric heterochromatin adjacent to the end that harbors the centromere   Fig. . This doPrevious studies have suggested that in early prophase centromeres are in heterologous clusters that appear as large chromocenters and that by zygotene they have organized as homologous pairs , consistent with the model that associations of the homologous pericentromeric regions keep the centromeres together.If associations between blocks of pericentromeric heterochromatin help to keep homologous centromeres together, then those with more abundant heterochromatin might remain together more efficiently. To test this idea, we used the levels of methylated histone 3 lysine 9 (H3K9Me3) as an indicator of the amount of heterochromatin. H3K9Me3 is a post-translational modification of constitutive pericentromeric heterochromatin  chromosomes scored)  Fig.. We concers Fig.  C. Thus,ers Fig. .Fig. 5Th− CENs). Thus, cantharidin treatment in diplotene causes the loss of centromeric SYCP1 after the centromeres have been paired. This allowed us to ask whether centromeres that are no longer directly tethered by SC continued to share a common chromocenter. The loss of SYCP1 did not result in a separation of the centromere pairs; instead, they remained joined by a shared cloud of heterochromatin , before homologous pairing occurs, centromeres associate in pairs or clusters independent of sequence homology  disrupted the close juxtaposition of homologous centromeres (centromere pairing) but did not affect heterochromatin interactions between homologous pairs. Thus, there must be a mechanism that stabilizes heterochromatin connections between homolog pairs independently of the SC.When and how are homologous heterochromatin connections established? Our results define a period of prophase I in which the SC promotes the stable homologous pericentromeric heterochromatin interactions observed between diplotene chromosomes. First, prior to synapsis, spermatocytes display high numbers of non-homologous centromeres connected by heterochromatin. Second, diplotene Drosophila oocytes during early meiosis suggested the possibility that heterochromatin connections may be established during DNA replication  that recognize heterochromatin methylation marks  were used in this study.The Oklahoma Medical Research Foundation Animal Care and Use Committee (IACUC) approved all animal protocols. Wild-type (C57BL/6) and We employed established experimental approaches for the visualization of chromosomes in chromosome surface spreads


Plasmodium falciparum is needed to explore pathogen biology and evaluate antimalarial targets. It is, however, aggravated by a low transfection efficiency, a paucity of selectable markers and a biased A/T-rich genome. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. We recently devised a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest. Although this attB is silent and without effect on intron splicing or protein translation and function, it allows efficient gene modification with minimal risk of unwanted changes at other genomic sites. We describe the range of applications for this new method as well as specific cases where it is preferred over CRISPR-Cas9 and other technologies. The advantages and limitations of various strategies for endogenous gene editing are also discussed.Genetic manipulation of the human malaria parasite  Plasmodium falciparum, a virulent human malaria parasite, acquires resistance to most antimalarial drugs quickly, new drug targets and a better understanding of resistance mechanisms are needed to sustain advances in global health. These goals depend on transfection studies in cultured P. falciparum parasites [Despite advances from combination therapies and public health measures such as bednets, more than 400,000 people still die of malaria annually. Since arasites –3. BecauGene manipulation in this system requires single or double crossover homologous recombination into the haploid genome as the parasite lacks the enzymes to carryout non-homologous end joining. Historically, these experiments relied on infrequent, coincidental genome breaks at or near the target site, often necessitating many months of continuous cultivation and drug-cycling to obtain detectable levels of integration . FurtherpiggyBac transposase and mycobacteriophage Bxb1 integrase facilitated stable integration of transgenes and reporters [P. falciparum, with zinc finger nucleases (ZFN) and clustered regulatory interspaced short palindromic repeats (CRISPRs) successfully used to study parasite biology [Subsequent implementation of eporters , 9, but  biology –15. Gene biology .P. falciparum, we recently implemented a novel application of the Bxb1 integrase transposition technology to permit targeted gene replacements [attB element into an intron of the target gene; we found that this 40 bp insertion is well-tolerated as it does not adversely affect either intron splicing or subsequent translation and function of the encoded protein. Once integrated, this attB element permits rapid recombination with an attP site on subsequent transfection plasmids to replace the downstream sequence with desired modifications. Although our strategy requires an additional transfection to first introduce the attB site, it then enables an unlimited array of distal modifications including site-directed mutations, insertions, deletions, epitope tagging, and conditional knockdown through the introduction of ddFKBP or EcDHFR-based destabilization domains, the TetR aptamer module, or the glmS riboswitch [To address these concerns and to add another option for DNA transfection in acements . Our appboswitch –21. AfteattP sequence in the phage and an attB sequence in the Mycobacterium smegmatis groEL1 gene with minimal fully effective sequences of 48 and 38 bp, respectively. This recombination enables phage integration into the bacterial genome [. Recombination begins when Bxb1 dimers bind to each of these sites with high affinity (Kd estimated at 70 nM); these dimers then interact with each other to form a synaptic tetramer, a process known as synapsis [attB and attP  can facilitate the reverse reaction and excision of the phage from the host genome [att sequences that confer specificity for attB-attP recombination and prevent the reverse attL-attR recombination reaction [att sites, controlled cleavage and rotation, hybridization and religation [Bxb1 integrase is a mycobacteriophage serine integrase that catalyses recombination between an l genome –24. Recosynapsis . All fouttP Fig. ; cleavagt genome . Structureaction . Kineticligation –31. WhenattP-containing plasmid with an engineered attB in the parasite genome; Bxb1 integrase was expressed from a co-transfected helper plasmid. The attB sequence, otherwise not present in P. falciparum, was introduced into the nonessential cg6 gene; several such cg6-attB lines have been generated in distinct genetic backgrounds [attP plasmid is a modified version of a parasite gene, the endogenous copy is not affected by transfection and is expected to remain functional, yielding a merodiploid whose phenotype may be more difficult to study. Expression levels for the transgene may also confound interpretation: genomic site effects due to expression from a heterologous cg6 locus or concatemerization of the attP plasmid prior to integration may lead to unanticipated transcript levels. Another concern is that the phenotype under study may be affected by disruption of the cg6 gene. Production of lines that carry attB at nonessential loci other than cg6 could address some of these concerns, but this has not been undertaken to our knowledge.Nkrumah et al.  demonstrkgrounds . These skgrounds . NeverthPlasmodium spp.While Cre recombinase, a distinct phage recombinase from the tyrosine integrase family , has beeattB into an intron of the gene of interest  offers excellent prospects for introduction of a silent attB without requiring modifying the encoded protein’s sequence; our clag3 attB intron has been validated and should work in most cases based on studies confirming faithful recognition and splicing of heterologous introns [We envision that this approach will be broadly applicable to study of , 179 bp , simplif introns , these sboundary . For gen introns .attB-loaded intron into clag3 via single crossover recombination, but this may be simplified by CRISPR-Cas9 transfection with a donor plasmid that contains homology arms of 200–300 bp on either side of the intron; if CRISPR-Cas9 is used, it is critical to select the single guide RNA (sgRNA) and cleavage site carefully with proximity to the intron and on-target efficiency score the most critical factors in P. falciparum transfections [attB sequence. Although some workers have argued that limiting dilution cloning is not required because subpopulations without this integration event will be removed by cloning after the second transfection, we caution against this because genome-level modifications at non-target sites may grow preferentially in the second transfection, aggravating attempts to identify the desired final clone.We originally introduced the fections . After pattB site has been successfully introduced, the full range of target gene modifications can be generated with a second transfection with two plasmids . The pLN-attP plasmid carries the attP element followed by the distal intronic sequence with a 3’ splice site for faithful splicing after integration. Any sequence following this attP-intron will be inserted into the genome upon attB-attP recombination as facilitated by the Bxb1 integrase expressed from the pINT helper plasmid.After the silent attB in a target gene’s intron distinguishes our approach from previous use of this technology in malaria research and has two important consequences. It disrupts the target gene by inserting the entire pLN-attP plasmid in the gene after the modified intron. It also achieves a promoter-trap because the sequence inserted distal to the intron upon recombination will be spliced in frame behind the native promoter and gene sequence upstream of the intron. Thus, the outcome in most cases is an effective gene replacement. In one application, this distal sequence could be the remainder of the gene with desired modifications. These modifications may include site-directed mutations, internal insertions or deletions (indels), domain swaps, addition of C-terminal epitope tags on the encoded protein, modified codon usage, and/or altered transcriptional regulatory elements . To demonstrate the range of possible modifications, we reported the production of a merodiploid parasite that expresses two CLAG3 proteins with distinct functional properties and separate epitope tags using this approach; although these clag3 genes are large (5.3 kb each) and we desired distinct epitope tags on two proteins expressed under a single promoter, our strategy and use of the clag3-attB line permitted facile introduction with a single plasmid .attB-intron is disregarded, and the pLN-attP plasmid may carry an entire gene cassette with required 5’ and 3’ untranslated regions . This re-codonization would require custom synthesis of a 6.3 kb DNA, which is both prohibitively expensive and time-consuming for most research labs.Another important case where the on  is exemattB-intron method should be considered, especially for labs that have committed to specific genes and require a broad collection of gene modifications for biochemical and/or structural studies.DNA transfection technologies represent an important tool for basic and translational malaria research. We remain limited by a low transfection efficiency, relatively slow parasite replication, a paucity of selection markers, and other difficulties specific to this important pathogen. Ongoing improvements in the methods available for generating parasite lines with desired genome modifications make this is an exciting time for malaria research. While CRISPR-Cas9 is currently the method-of-choice for most gene editing experiments, other strategies such as our


Acute myocardial infarction (AMI) is one of the leading causes of mortality and morbidity worldwide. There has been an extensive search for cardioprotective therapies to reduce myocardial ischemia-reperfusion (I/R) injury. Remote ischemic preconditioning (RIPC) is a phenomenon that relies on the body's endogenous protective modalities against I/R injury. In RIPC, non-lethal brief I/R of one organ or tissue confers protection against subsequent lethal I/R injury in an organ remote to the briefly ischemic organ or tissue. Initially it was believed to be limited to direct myocardial protection, however it soon became apparent that RIPC applied to other organs such as kidney, liver, intestine, skeletal muscle can reduce myocardial infarct size. Intriguing discoveries have been made in extending the concept of RIPC to other organs than the heart. Over the years, the underlying mechanisms of RIPC have been widely sought and discussed. The involvement of blood-borne factors as mediators of RIPC has been suggested by a number of research groups. The main purpose of this review article is to summarize the possible circulating mediators of RIPC, and recent studies to establish the clinical efficacy of these mediators in cardioprotection from lethal I/R injury. Impaired coronary circulation during myocardial ischemia leads to lethal hypoxic injury. Rapid restoration of coronary blood flow through either thrombolytic therapy or percutaneous coronary intervention (PCI) is essential to limiting myocardial infarction (MI) size, preserving left ventricular (LV) ejection fraction and preventing LV remodeling. Revascularization via either thrombolytic therapy or PCI reduces the mortality rate of patients suffering a heart attack by almost 50% . Paradoxet al. used a canine model to provide the first demonstration that brief episodes of non-lethal I/R to the heart prior to sustained ischemia can dramatically reduce MI size, an endogenous cardioprotective phenomenon termed ischemic preconditioning (IPC) . T. T23]. Tfunction . ShimizuR injury . These a C (PKC) . Breivik pathway . Interes pathway . The hum pathway .et al. who demonstrated that transient occlusion of the anterior mesenteric artery can mediate cardioprotection, which can be abrogated by ganglionic blockers , , 215],  and PCI RIPC has provided an innovative non-invasive therapeutic strategy to prevent acute I/R injury in susceptible organs and tissues with some variability. Non-invasive procedures such as using a blood pressure measuring cuff around the arm to achieve protection against I/R injury has facilitated its translation from bench to bedside. Though there are several clinical trials that did not show beneficial effects of RIPC, further mechanistic studies will help us understand the underlying cause of the failure of these studies. Optimal modality, site and duration of RIPC remains unclear. RIPC may nonetheless benefit children and adults undergoing certain elective surgeries where there is potential to improve clinical outcomes. Future insights into the control of circulating mediators of RIPC, including transcriptional regulation and secretion into the bloodstream will assist the development of pharmacologic approaches stimulating protective signaling pathways in target organs.


MocR bacterial transcriptional regulators are a subfamily within the GntR family. The MocR proteins possess an N-terminal domaincontaining the winged Helix-Turn-Helix (wHTH) motif and a C-terminal domain whose architecture is homologous to the fold type-Ipyridoxal 5'-phosphate (PLP) dependent enzymes and whose archetypical protein is aspartate aminotransferase (AAT). The ancestor of thefold type-I PLP dependent super-family is considered one of the earliest enzymes. The members of this super-family are the product ofevolution which resulted in a diversified protein population able to catalyze a set of reactions on substrates often containing amino groups.The MocR regulators are activators or repressors of gene control within many metabolic pathways often involving PLP enzymes. Thisdiversity implies that MocR specifically responds to different classes of effector molecules. Therefore, it is of interest to compare the AATdomains of MocR from six bacteria phyla. Multi dimensional scaling and cluster analyses suggested that at least three subgroups existwithin the population that reflects functional specialization rather than taxonomic origin. The AAT-domains of the three clusters displayvariable degree of similarity to different fold type-I PLP enzyme families. The results support the hypothesis that independent fusionevents generated at least three different MocR subgroups. The vast GntR family ,2 was naIn general, the MocR regulators are involved as activators orrepressors in the control of many, important metabolic networksnot yet fully characterized but often involving PLP-dependentenzymes ,24. MoreMocRs represent an interesting case of evolution of chimericproteins . To veriBacterial protein sequences from complete proteomes have beenretrieved for each phylum from UniProt Data Bank  accessedMultidimensional scaling (MDS) and clustering techniques asimplemented in R modules "bios2mds"  and "cluHMMer suite  was emplDifferences discriminating the sequence clusters have beententatively identified through Seq2Logo web server(http://www.cbs.dtu.dk/biotools/Seq2Logo/). Seq2Logo displaysThe proteomes of bacteria from the most populated phyla wereconsidered to the purpose of this analysis: Actinobacteria,Firmicutes, Alpha proteobacteria, Beta proteobacteria, Gammaproteobacteria,and Bacteroidetes. The AAT-domain sequencesfrom the MocRs of the six phyla were merged into a single dataset,which was filtered at 50% sequence identity level by the cd-hit tool.The final taxonomical composition of the AAT-domain set,accounting for a total of 1331 sequences, is reported in To test for the existence of AAT-domain subgroups within thecollected MocR set, the multidimensional scaling analysis (MDS)implemented in the R package "bios2msd"  has beenThe R "Kmeans" function has been applied to the distance matrixof the AAT-like domains. To establish the optimal number ofclusters, silhouette score analysis  has beenAAT-domain sequences assigned by K-mean clustering to each ofthe three clusters have been separately aligned with clustal omega.A Profile Hidden Markov Model (HMM) has been calculated foreach multiple alignment with the program HMMbuild. Each HMMprofile has been utilized as a HMM search query over the Pfam-Adatabank. Only hits showing an E-value lower than 0.1 wereconsidered. The distribution of the Pfam hits obtained by eachHMM profile is displayed in To spot the sequence features discriminating the three clusters,corresponding Logos have been compared. To avoid noise due toinaccuracies in sequence alignment, Logo comparison has beenrestricted to blocks, namely to portions of the multiple sequencealignment made of at least three consecutive columns eachcontaining not more than 90% gaps . The posThe MocR regulators are chimeric proteins emerged from ancestralfusion events between a gene coding for an HTH domain and aneffector/dimerization domain belonging to a vast and diversifiedenzyme superfamily, the fold type-I PLP-dependent enzymes .AspartaThe results obtained by MDS and clustering analyses support thenotion that at least three MocR subgroups can be distinguished.The clustering does not reflect apparently the taxonomicclassification of the source species since MocR sequences belongingto the same bacterial phylum agglomerate into different clusters.Consequently, clustering seems to reflect functional rather thanevolutionary proximity. Cluster analysis attributes the referenceMocRs  to threeThe attempt to identify the positions characterizing the MocRsequences assigned to different clusters on the basis of the multiplesequence alignment was compounded by the high dissimilarity ofthe sequences: the final alignment contains many longinsertions/deletions and the average pairwise percentage identitywas around 20%. For this reason, attention has been focussed ontoconserved blocks. Interestingly, the blocks add up to a structural"core" surrounding the PLP binding site of the GabR AAT-domainstructure along with the helix connecting the large and smalldomain of the single subunit . This coHMMsearch suggests that the three clusters have variable degreesof similarity to the Pfam families corresponding to different foldtype-I families. All MocR profiles displays high similarity to thePfam Aminotran_1_2 family that collects most of the fold type-IPLP dependent aminotransferases and to the familyAminotran_MocR, collecting the major domain of triptofanases.However, GabR cluster profile retrieves very few sequencesbelonging to other fold type-I families while PtsJ and EnuR profilesdo . A sharpThe results reported here support the hypothesis that the MocRregulators emerged after independent ancestral fusion eventsbetween a HTH domain and at least three already catalyticallyspecialized PLP dependent enzymes of fold type-I. This hypothesisis also coherent with the conception that regulation machineryshould emerge after evolution of the metabolic pathway under itscontrol as, for example, in the case of prokaryotic BdzR regulatorinvolved in the anaerobic degradation of benzoate . Howevernone