Corneal diseases constitute the second leading cause of vision loss and affect more than 10 million people globally. As there is a severe shortage of fresh donated corneas and an unknown risk of immune rejection with traditional heterografts, it is very important and urgent to construct a corneal equivalent to replace pathologic corneal tissue. Corneal tissue engineering has emerged as a practical strategy to develop corneal tissue substitutes, and the design of a scaffold with mechanical properties and transparency similar to that of natural cornea is paramount for the regeneration of corneal tissues. Nanofibrous scaffolds produced by electrospinning have high surface area–to-volume ratios and porosity that simulate the structure of protein fibers in native extra cellular matrix (ECM). The versatilities of electrospinning of polymer components, fiber structures, and functionalization have made the fabrication of nanofibrous scaffolds with suitable mechanical strength, transparency and biological properties for corneal tissue engineering feasible. In this paper, we review the recent developments of electrospun scaffolds for engineering corneal tissues, mainly including electrospun materials (single and blended polymers), fiber structures (isotropic or anisotropic), functionalization , applications  and future development perspectives. The cornea is a transparent, avascular, multi-laminar structure that forms a barrier to protect the intraocular structure and microenvironment while refracting light onto the retina ,2,3 and l-lactic acid (PLLA), poly(lactide-co-glycolide) (PLGA), polyethylene oxide (PEO), etc.) [Electrospinning is a versatile fabrication process that uses a high voltage between a syringe and a deposition target (or collector) to draw nano- or micro-scale fibers from the material dispensed by the syringe . Electro), etc.) ,32,33,34), etc.) . Further), etc.) ,37. The ), etc.) ,39,40,41Recently, electrospinning has attracted increased interest for fabricating biomimetic engineering functional corneal tissue due to the close structural resemblance of the constructs to native ECM and its high surface area–to-volume ratio and good porosity, which provide support for cell adhesion and movement, proliferation and differentiation, as well as excellent mechanical properties, easy manipulation of fiber properties, great material handling, suturability for implantation, and scalable production ,42,43. IAs mentioned above, over 200 polymers have been successfully electrospun to nano-scale or micro-scale fibers. However, only those with excellent biocompatibility have been widely utilized for tissue engineering applications. In addition to biocompatibility, electrospun scaffolds also need to offer additional properties required for corneal tissue engineering, including a degradation rate that is comparable to the regeneration rate of native ECM to provide sustained support for corneal tissue regeneration, mechanical properties that match those of the human cornea , and improved scaffold transparency for inducing visual function of the engineered corneal tissues. According to N-ethyl-N-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide-cross-linked collagen–chondroitin sulfate foam and seeded it with primary human corneal keratocytes. Retinal pigment epithelium (RPE) cells served as the epithelial layer after seeding on a dehydrothermally cross-linked collagen type I fibrous mat deposited directly on top of the foams by electrospinning. Physical characterizations and in vitro studies showed that the designed cornea replacement was suitable for cell attachment and growth, and co-culture of the two cell types induced more ECM deposition than single cell–seeded constructs [Natural materials, such as collagen, silk and gelatin, which have excellent biocompatibility, biodegradability and low immunogenicity, have been extensively utilized for corneal tissue engineering. Donna Phu, B.S. et al. electrospun type I collagen scaffolds for culturing corneal fibroblasts ex vivo that mimicked the microenvironment in the native cornea, and they recently investigated the effect of the scaffold nanostructure and composition on the phenotype of corneal stromal cells . Lindsaynstructs . An silknstructs .Although natural materials have many excellent biological properties, their poor mechanical properties will limit their clinical applications to a large extent. Synthetic polymers, especially FDA-approved polymers, such as PLGA, PCL, and PLA (poly lactic acid), have been used in corneal tissue engineering due to their superb mechanical properties. PCL has been electrospun into nanofibrous scaffolds C, allowiA blended electrospun scaffold can be fabricated using a polymer blend , aiming In addition to adjusting different polymer combinations, the ratio of polymer blends can also be changed to regulate the mechanical properties and the degradation rate to match natural ECM. For example, poly(glycerol sebacate) (PGS)/PCL nanofibrous scaffolds were fabricated by electrospinning different weight ratios of PGS and PCL  and the Young’s moduli decreased with the increasing PGS content. The Young's modulus of the 4:1 blended scaffold was determined to be 1–1.2 MPa, fitting nicely in the range of the mechanical properties of the native stroma . SF/P  has been demonstrated as an important factor in improving their performance for tissue engineering applications. By utilizing special collectors, aligned scaffold fibers can be obtained. It is well known that cells react differently to micro-topography and nano-topography, and mimicking natural tissues has been attempted by cell guidance using physical cues ,63. ThesChaotic fibers can be easily obtained using a traditional collector, such as a cylinder or a metal mesh. Pallavi Deshpande et al. electrospun chaotic PLGA mats to provide a biodegradable cell carrier system for limbal epithelial cells. The study showed that the limbal cells formed a continuous multilayer of cells on either side of the scaffold. Scaffolds of cells showed signs of the onset of degradation within two weeks in culture media at 37 °C. They suggested that this chaotic electrospun mat could be used as a replacement for the HAM in the treatment of limbal stem cell deficiency, lowering the risk of disease transmission to patients  Figure A,B. JingFor certain applications in tissue engineering, scaffolds with aligned fibers possess unique electrical, optical, and mechanical properties and are often more desirable to guide cell growth with the desired anisotropy ,66,67,68The aligned fibers mimicking the parallel orientation of native tissues have demonstrated favorable cell adhesion, migration and proliferation for corneal, cardiac, neural, and skeletal muscular tissues ,76. For In addition to the microstructure of electrospun scaffolds, transparency and mechanical properties are essential characteristics when constructing corneal substitutes in vitro.Optical transparency is an important property that should be considered while developing a bioengineered corneal construct. A healthy cornea is required for clear vision and it contributes two-thirds of the total refractive power of the eye, which is the most remarkable property of the cornea. For this reason, a corneal equivalent fabricated by tissue engineering should be able to transmit most of the visible light to mimic the natural behavior of the native cornea.2) plasma treatment. The results indicated that a plasma-treated stromal equivalent can transmit 37% more light than the untreated plasma stromal equivalent [Many polymers can be electrospun into nanofibrous scaffolds, including natural and synthetic materials. However, when applied in corneal tissue engineering, not all these materials are suitable because of their opacity or low transparency. There are three primary methods to improve the transparency of electrospun mats. One is post-modification of the scaffold via plasma discharge treatment. Additionally, plasma treatment further enhances the cell adhesion properties of these scaffolds. Surface modification by plasma treatment is a well-established method for modifying surface chemistry without changing morphology in an eco-friendly way . Plasma uivalent . Haleh Buivalent .The second method is through the blending of different materials, generally natural and synthetic polymers. Juan Ye et al. produced collagen/HA/PEO electrospun mat coatings with chitosan on the surface. The result showed that the scaffold had an excellent transparency . Chen, eNotably, keratocytes usually stay in the quiescent state and maintain non-crystalline structures to make the corneal transparent and optimal refraction. Recent studies have identified the importance of the intracellular protein expression of keratocytes in maintaining corneal transparency . The kerNot only do the scaffolds need to provide high transparency and suitable refractive power, but they must be able to carry the tension induced from high intraocular pressure and eye movements . The scaHaleh Bakhshandeh et al. electrospun PCL nanofibrous scaffolds post-treated with plasma to improve their mechanical properties. The result demonstrated that the Young’s modulus value of the electrospun PCL skirt was 7.5 MPa, which is in line with the elasticity range of natural human corneas (0.3–7 MPa) . ElectroScaffolds for corneal tissue engineering should support cell survival and promote cell differentiation toward the corneal phenotype, which can be achieved by electrospun scaffolds due their bio-mimicking nanofibrous structure and excellent biocompatibility ,81.N-acetylglucosamine-6-O-sulfotransferase (CHST6) and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), indicating that the stem cells on the substrate differentiated into keratocytes [Some electrospun scaffolds have also shown the ability to promote the differentiation of corneal stem cells into corneal epithelial cells or keratocytes. For example, Shweta Sharma at al. electrospun PCL nanofibrous scaffolds and inoculated limbal epithelial stem cells on their surfaces. The cells on the scaffold exhibited high bio-viability, and the expression of differentiation markers K3/12 suggests that cultivated limbal epithelial stem cells had the potential to differentiate into mature corneal epithelial cells  Figure A. A subsatocytes  (Figure Control and maintenance of the corneal cell phenotype is vital for developing in vitro tissue-engineered strategies for corneal repair. Much research work has been performed to study how to maintain the phenotype of corneal cells, such as limbus stem cells, corneal epithelial cells and keratocytes. Limbus stem cells were demonstrated to retain a normal corneal stem cell phenotype on the electrospun silk scaffolds , PCL scaAs the cells cultured on electrospun scaffolds are capable of survival, differentiation and maintaining their phenotype, further attention should be paid to inducing the formation of corneal tissues. Because it is difficult to construct full-thickness corneal equivalents that include all five layers, most researchers have focused on the fabrication of one layer or two layers, such as the corneal epithelial layer or the stromal layer.Shweta Sharma et al. electrospun PCL nanofibrous scaffold and inoculated limbus epithelial cells on the surface of the scaffold. The results showed that cells infiltrated the nanofibers and successfully formed a 3D corneal epithelium, which was viable for two weeks . AlirezaRecently, because of the increasing population with corneal disease and the short-comings of donated fresh cornea, many researchers have devoted themselves to the field of corneal tissue engineering and have already achieved extensive advancements. Although there are still many challenges for the regeneration of full-thickness corneal tissues, the problems that we need to focus on are much clearer than they were a decade ago. The primary work is the fabrication of scaffolds that can mimic the ECM found in native cornea. For this purpose, various methods and materials have been tested for their performance in corneal tissue engineering. Among these methods, the technique of electrospinning has been proven as an effective approach and has been widely studied in recent years. The scaffold fabricated by electrospinning has a high volume-to–surface area ratio and porosity, which simulates natural ECM and provides an atmosphere for corneal cells to attach, move, proliferate and differentiate. By integrating the advanced electrospun scaffold design with fast-growing biotechnologies, it is possible to partially or fully overcome the existing challenges of corneal tissue engineering and to make the translation of electrospun-based engineered corneal tissues for clinical applications more realistic.The electrospun scaffolds have already shown their abilities for engineering corneal tissues in vitro and many studies have developed optimal scaffolds with similar morphology, mechanical properties and transparency to natural corneal engineering. Previous studies have inoculated corneal cells on scaffolds and studied the interaction of cells and scaffolds, and further had in vivo transplantation. However, few studies have studied the corneal tissue repair mechanism after transplanting scaffolds into injured cornea. This is very important and if this issue is addressed, it can serve as a model of the repair mechanism for other tissues in the body.


Inability to efficiently implement high-throughput field phenotyping is increasingly perceived as a key component that limits genetic gain in breeding programs. Field phenotyping must be integrated into a wider context than just choosing the correct selection traits, deployment tools, evaluation platforms, or basic data-management methods. Phenotyping means more than conducting such activities in a resource-efficient manner; it also requires appropriate trial management and spatial variability handling, definition of key constraining conditions prevalent in the target population of environments, and the development of more comprehensive data management, including crop modeling. This review will provide a wide perspective on how field phenotyping is best implemented. It will also outline how to bridge the gap between breeders and ‘phenotypers’ in an effective manner. Although the development of effective field-based high-throughput phenotyping remains a bottleneck for future breeding advances, particularly those related to yield potential and stress resilience, progress in sensors, aeronautics, and high-performance computing is paving the way.The growing interest in affordable solutions for remote-sensing approaches as well as data management may facilitate technological adoption by breeders.However, phenotyping is more than just choosing the correct selection traits, deployment tools, evaluation platforms, or basic data-management methods.Trial management and spatial variability handling, definition of the key environmental conditions that are prevalent in the targeted regions, and the development of more comprehensive data management, including crop modeling, are components of the phenotyping pipeline that need to be taken into consideration. Genetic gain is the amount of increase in performance achieved per unit time through artificial selection In summary, genetic gain within a breeding program can be accelerated in a number of ways Box 1Vegetation indices derived from RGB images  have shoet al., unpublished results). In the case of Miller et al.In terms of sensors, the near future promises a widening of RGB image utilization. Several diverse factors support this prediction: (i) this is a really affordable way of phenotyping, given the low cost of high-resolution RGB cameras and the existence of open source software to formulate different categories of vegetation indices; (ii) the much higher resolution (at least four times) of RGB cameras compared with multispectral images makes the former more suitable for installation in aerial platforms; (iii) even from nanosatellites and microsatellites RGB images are amenable for important applications other than vegetation indices. To date, RGB imaging has proven its value not only in formulating vegetation indices that report on plant cover senescence, or the impact of foliar diseases, but also in a wide range of other applications see  in Box 3Nanosatellites and microsatellites are emerging as an effective low-cost option for collecting data like sow date and yields on small farms across the developing world Alt-text: Box 1Much of what is currently considered high-throughput phenotyping is based on remote sensing . The mosRemote-sensing tools allow assessment of physiological yield components that are clearly and conceptually related to crop productivity and stress adaption in terms of resource acquisition , resource use efficiency, or downstream biomass partitioning However, equivocating remote sensing with high-throughput phenotyping is an oversimplification, because there are different categories of traits of an analytical nature, particularly grain/fruit quality traits, as well as analytical indicators such as stable (carbon) isotope, which have all proven their value light detection and ranging (LiDAR) and radio detection and ranging (radar), which use active sensors, will be also addressed (Box 2Radar via synthetic aperture radar (SAR) systems is able to provide a wide range of plant physiological measurements relevant to high-throughput phenotyping in crop breeding: soil humidity, root characteristics, plant architecture, and counting fruits beneath the canopy LiDAR is another active sensor technique benefitting from technological advancements that have delivered more meaningful data at higher resolutions with less size and weight to the extent that it has become amenable to field plant phenotyping applications Alt-text: Box 2In the following sections, we highlight some remote-sensing techniques, which may be relevant in terms of special capacities and/or cost. In addition, ddressed , becauseBox 3The concept of the phenotypic platform is wide and ranges from platforms working under fully controlled conditions to field platforms or even platforms designed with a clear objective of ‘deep phenotyping’ of specific traits , or elusive plant parts . The choice of platform will depend on the scale of the intended work, including the sensors deployed, their resolution, and associated costs.Within the category of field phenotyping platforms, the alternatives are still very diverse Aerial platforms are a flexible alternative. Within this category and compared with the use of manned planes and even balloons, unmanned aerial platforms, also termed drones , are attracting increasing attention due to their growing reliability and decreasing cost https://www.planet.com/products/hi-res-monitoring/ and https://directory.eoportal.org/web/eoportal/satellite-missions/s/skysat).In addition, over the coming years nanosatellites and microsatellites mounted with high-resolution RGB cameras may become an alternative to aerial phenotyping platforms Alt-text: Box 3Remote-sensing tools are usually integrated into phenotyping platforms . Some ofWhile the early detection of plant stress  remains a challenge for phenotyping To date, most remote-sensing applications of multispectral/hyperspectral sensors and imagers have focused on using vegetative indices to infer overall plant status, with the normalized difference vegetation index (NDVI) being the most well-known option in situ  fast evaluation of these traits (particularly using leaf adaptors). In addition, hyperspectral imaging has been tested for its ability to detect biotic stresses Different studies have demonstrated that hyperspectral data 400–2500 nm) can be utilized to infer leaf chemical properties in various species 0–2500 nmThe category of hyperspectral assessments may also include near-infrared reflectance spectroscopy (NIRS), which is a basic tool in quality laboratories, for example, and NIRS imagers have been successfully mounted on harvesters to save time and sample handling costs. NIRS may be also deployed in the analysis of other traits with potential for phenotyping such as stable isotopes, and nitrogen and mineral content  within the concept of genetic gain will assist in monitoring success. High-throughput phenotyping can contribute both directly and indirectly to genetic gain .Obtaining accurate and inexpensive estimates of genetic value of individuals is central to breeding. Many routine traits such as stand establishment, phenology, abiotic stress severity, disease severity and progression, plant height, heading or flowering date, lodging, and yield components remain largely manual within breeding programs, particularly within the public sector https://www.google.com/patents/US9335313; https://www.google.com/patents/WO2017021285A1; https://www.google.com/patents/US20090046890]. New image analysis protocols are available to measure yield components using both a line scanner and conveyor belt and flatbed scanner Recently, there have been many advances in the development of high-throughput phenotyping tools for ‘breeder-preferred’ traits  has been estimated at 45 plots per hour, compared with 3000 plots per hour using a phenomobile equipped with an ultrasonic canopy height sensor Manual measurements of key traits are time consuming and the application of high-throughput phenotyping tools could significantly reduce labor requirements. For example, the rate of manual measurements of plant height in rice  application Crop models translate processes related to crop growth and development into mathematical equations However, mechanisms to capture the interaction of combined stress are often not properly fitted within models www.agmip.org), Global Future and Harvest Choice (www.harvestchoice.org), and the Climate Change, Agricultural and Food Security program (ccafs.cgiar.org). Significant progress has been recently made on ORYZA to improve the predictability of rice models under extreme conditions such as water- and nitrogen-stressed conditions Besides the aforementioned limitations, renewed interest in crop modeling has been associated with several international, multidisciplinary collaborations to update the ‘engines’ of existing models, including the Agricultural Model Intercomparison and Improvement Program  if it is demonstrated as something valuable in terms of genetic gains achieved with resources invested. Moreover, in least developed countries, the public sector, rather than the private sector, continues to have a predominant role in crop breeding. To ensure that advances in phenotyping can be translated into yield gains in these countries, it is essential that low-cost phenotyping tools are developed. In that context, affordable, easy-to-handle, and reliable tools and platforms for large-scale  field phenotyping may pave the way. Among such tools the wide range of applications for RGB images makes them good candidates.To capitalize on advances in phenotyping and molecular technologies, greater progress is needed in areas of environmental characterization and data collection and management. Aligning breeding programs to future demands requires increasingly strategic choices: identification of rigorously prioritized, market-informed product profiles; understanding current and future target population of environments; balancing farmer and consumer needs; and accounting for value chain participant concerns. The engagement of breeding teams with climate scientists and crop modelers is perceived as necessary to address the challenges of climate change cultivars suitable for the target environment Trait phenotyping and crop growth models are evolving to the point where breeders can access mechanistic information on the physiological determinants of plant adaptation for precise selection of Outstanding QuestionsWill genetic gain match the challenges imposed by the global  change for the coming decades? Genetic gain is stagnated in many regions and the promises of a new ‘Green Revolution’ as the natural result of biotechnological advances have not yet been realized.Even though high-throughput phenotyping is perceived as a bottleneck in breeding, it has yet to deliver. Is the dilemma of controlled versus field phenotyping in terms of yield or adaptation to abiotic stresses already solved considering that research and breeding application are not the same?Will low-cost, high-throughput phenotyping tools be adopted regularly by breeders in the next decades? If so, are RGB cameras, mobile apps, and drones the natural candidates?To what extent will the size of breeding programs need to be increased, considering improvements in accuracy of selection, ensuring adequate genetic variation, and acceleration of breeding cycles?How will decision support systems and simulation models contribute to the breeding pipeline to predict future target environments in breeding, a component in molecular breeding, or design ideotypes?As the public sector breeding moves toward informal networks such as the proposed ‘European Consortium for Open Field Experimentation’


Chronic kidney disease (CKD) patients are among the groups at the highest risk for cardiovascular disease and significantly shortened remaining lifespan. CKD enhances oxidative stress in the organism with ensuing cardiovascular damage. Oxidative stress in uremia is the consequence of higher reactive oxygen species (ROS) production, whereas attenuated clearance of pro-oxidant substances and impaired antioxidant defenses play a complementary role. The pathophysiological mechanism underlying the increased ROS production in CKD is at least partly mediated by upregulation of the intrarenal angiotensin system. Enhanced oxidative stress in the setting of the uremic milieu promotes enzymatic modification of circulating lipids and lipoproteins, protein carbamylation, endothelial dysfunction via disruption of nitric oxide (NO) pathways, and activation of inflammation, thus accelerating atherosclerosis. Left ventricular hypertrophy (LVH) and heart failure are hallmarks of CKD. NADPH oxidase activation, xanthine oxidase, mitochondrial dysfunction, and NO-ROS are the main oxidative pathways leading to LVH and the cardiorenal syndrome. Finally, a subset of antioxidant enzymes, the paraoxonases (PON), deserves special attention due to abundant clinical evidence accumulated regarding reduced serum PON1 activity in CKD as a contributor to the increased burden of cardiovascular disease. Future, meticulously designed studies are needed to assess the effects of antioxidant therapy on patients with CKD. Strikingly, in patients with mild to moderate CKD, the incidence of cardiovascular mortality is much higher than the incidence of end-stage renal disease (ESRD) [Chronic kidney disease (CKD) patients are among the groups at the highest risk for cardiovascular disease. There is abundant evidence in the literature regarding the inverse association between kidney function and cardiovascular risk –4. Premae (ESRD) , 7.The spectrum of cardiovascular disease in the CKD population extends from arterial vascular disease including atherosclerosis and arteriosclerosis to left ventricular remodeling with concentric hypertrophy, left ventricular dilation, and subsequent heart failure, with nonatherosclerotic cardiovascular disease becoming dominant at more advanced stages of CKD . TraditiIncreased oxidative stress in CKD, similarly to other pro-oxidant states, is characterized by excessive generation of oxidant compounds due to activation and upregulation of various enzymatic pathways including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, lipoxygenase, xanthine oxidase, uncoupled nitric oxide synthase (NOS), and the mitochondrial respiratory chain , 13. InsThe aim of this review is to describe the available links between experimental and clinical evidence regarding the intricate mechanisms via which CKD enhances oxidative stress in the organism with ensuing cardiovascular damage and as a consequence amplification of cardiovascular morbidity and mortality. Similar backgrounds are required so as to find common pathophysiological denominators which shall serve as future targets of therapy.Progressive renal dysfunction is characterized by amplification of oxidative stress. This has been indicated by several clinical studies which showed a gradual augmentation of levels of different oxidative stress markers including plasma F2-isoprostanes, advanced oxidation protein products, and malonyldialdehyde (MAD) as well as oxidized LDL in CKD patients in parallel with advancing stages of renal dysfunction , 16, 17.The pathophysiological mechanism underlying the increased ROS production which is invariably present in all forms of CKD is at least partly mediated by the pathological upregulation of the intrarenal angiotensin system, as evidenced by marked upregulation of angiotensin II receptors (AT1 and AT2) and simultaneous increases in angiotensin II-producing cells in the diseased kidney. It has been shown that activation of the AT1 receptor by angiotensin II increases superoxide production in the kidney and vasculature via NOX isoforms , 19. SpeEnhanced oxidative stress in the setting of the uremic milieu modifies circulating lipids and lipoproteins causing essential alterations of their biological properties. Thus, covalent modifications of lipids and lipoproteins generate several lipid end products such oxidized phospholipids (oxPLs), fatty acid peroxidation products (FAPPs), oxysterols, and F2-isoprostanes . Lipid pPatients with kidney disease and most markedly ESRD patients have been shown to display not only higher rates of pro-oxidant activity but impairment of antioxidant defenses as well . NuclearFurthermore CKD is usually associated with the presence of other pro-oxidant states such as diabetes mellitus, dyslipidemia, hypertension, and aging. Hyperglycemia and advanced glycation end products (AGEs) promote ROS generation by the mesangial cells. AGEs are a heterogeneous group of compounds which are derived from the nonenzymatic glycation of proteins, lipids, and nuclear acids through a complex sequence of reactions. They are produced not only in the presence of hyperglycemia but also in the setting of increased oxidative stress, as it occurs in CKD . Nox4 anκB, which regulate expression of gene proinflammatory cytokines and chemokines [Inflammation is the most common outcome of oxidative stress, and both processes are deeply interrelated in kidney diseases in a vicious cycle as both amplify each other , 36. Oxiemokines . Likewisemokines . Accordiemokines .The process of atherogenesis involves the interplay of oxidized plasma lipids and inflammatory cells of the immune system within a dysfunctional vascular wall. The elucidation of the intricate mechanisms through which the pro-oxidant systems promote atherogenesis has been the subject of extensive and ongoing research and is beyond the scope of our review.The major ROS-producing systems expressed in the vascular wall, with some of them being expressed by the immune cells as well, include the Nox1- and Nox2-reduced isoforms of NADPH, xanthine oxidase, uncoupled endothelial NOS (eNOS), and enzymes of the mitochondrial respiratory chain . The supSeveral protective antioxidant mechanisms such as glutathione, peroxidase, and catalase are available within the vascular wall in order to detoxify hydrogen peroxide –42. eNOSClassical risk factors for atherosclerosis are considered to induce the pro-oxidant pathways as well as eNOS uncoupling in the vascular wall thus creating a vicious circle where oxidative stress begets more oxidative stress. Accordingly, increased oxidative stress is directly linked to endothelial dysfunction by reducing ΝΟ availability with subsequent alterations in the vascular permeability and entrance of LDL cholesterol within the vascular intima where it is oxidized as well as to migration of inflammatory cells in the subendothelial space after expression of adhesion molecules by the dysfunctional endothelium , 46.There is abundant experimental evidence available, which has shed light on the implications of oxidative stress, chronic inflammation, and endothelial dysfunction as well as their crosstalk in the pathogenesis of atherosclerosis , 47. NevThe specific processes via which CKD-associated oxidative stress promotes accelerated atherogenesis have been extensively studied . The enzΟxidated lipids are important players in the initiation and progression of atherosclerotic changes. The oxidation of LDL by HOCl generated in MPO-catalyzed reaction is thought to be a proatherogenic event which precedes the formation of foam cells, a hallmark of atherosclerotic plaque development. Oxidized epitopes of LDL can initiate an immune response and lead to the formation of antibodies directed against oxLDL. Both OxLDL and antibodies against oxLDL have been correlated with carotid atherosclerosis and cardiovascular events in HD patients , 59. HigA recent clinical trial showed that renal transplantation does not correct impairment of uremic HDL particles. Accordingly, HDL cholesterol acceptor capacity and antioxidative activity were profoundly suppressed in kidney transplant recipients independent of graft function and were comparable with levels in patients with ESRD, suggesting that pathologic modification of HDL may contribute to the residual cardiovascular risk in the transplant population . The carCarbamylation, which is a posttranslational modification of proteins or amino acids, results either from nonenzymatic reactions of amino acid residues with urea decomposition products such as isocyanic acid, or through a MPO-catalyzed pathway. Recently, it has been shown that carbamylated LDL induces endothelial dysfunction and increased ROS production, leading to eNOS uncoupling . AdditioIn subjects with angiographic evidence of coronary artery disease, peripheral artery disease, myocardial infarction, stroke, or previous revascularization, protein carbamylation as assessed by plasma protein-bound homocitrulline level was found significantly higher than healthy controls. It should be noted that in clinical studies, protein-bound homocitrulline levels predicted cardiovascular disease even after extensive adjustments for traditional cardiovascular risk factors, renal function, and both MPO levels and high sensitivity C-reactive protein concentrations . The plaκB (NF-κB) in the vascular endothelial cells [Indoxyl sulfate, one of the uremic toxins associated with accelerated progression of CKD, has been suggested to induce vascular disease via amplification of oxidative stress. Experimental studies have shown that indoxyl sulfate stimulates proliferation of rat vascular smooth muscle cells . Indoxylal cells . Subsequal cells –83. Howeal cells .Clinical research has aimed at providing unifying models of oxidative stress markers in CKD. In a recent study, antioxidant defenses were measured together with markers of oxidative stress and inflammation markers in patients with CKD experiencing cardiovascular distress and were compared to normal age and sex-matched individuals. Levels of SOD, catalase, and glutathione as well as NO levels were decreased in patients with CKD whereas MDA levels and IL-1 levels were increased, further supporting the fact that impairment of antioxidative capacity of the organism accompanied by a proinflammatory state is associated with cardiovascular risk in CKD .β, interleukin-6, and tumor necrosis factor (TNF) were increased in the calcified lesions of the aortic media as were the expression of NADPH oxidase subunits whereas the expression of antioxidant enzymes was reduced in CKD high-calcium-phosphate diet rats. Additionally, oxidized peroxiredoxin, a sensor of ROS generation, was significantly increased and ROS-sensitive signaling pathways were activated in the aorta from CKD high-calcium-phosphate diet rats [Coronary artery calcifications, a hallmark of the atherosclerotic burden, are a ubiquitous finding in patients with advanced stages of CKD and more so in ESRD . Vasculaiet rats . Few cliiet rats –93. Addiiet rats .Increased common carotid artery intima-media thickness is considered an early phase of atherosclerosis. It has been associated with cardiovascular risk and risk of coronary artery disease events and has been largely used as a marker of atherosclerosis for clinical studies. Cross-sectional studies which have investigated the possible association between biomarkers of oxidative stress and intima-media thickness of the carotid artery in CKD patients have shown controversial results –102.Similarly, the clinical studies which have evaluated possible association between oxidative stress and the prevalence of cardiovascular disease in hemodialysis patients yielded conflicting results as well –109. HowNevertheless, the efficacy of antioxidant therapy in terms of prevention of cardiovascular disease in people with CKD remains controversial. A large meta-analysis included approximately 2000 dialysis and nondialysis CKD patients and kidney transplant recipients treated with various antioxidants, including different doses of vitamin E, coenzyme Q, acetylcysteine, bardoxolone methyl, human recombinant SOD, and multiple antioxidant therapy . CompareThe prevalence of left ventricular hypertrophy (LVH) significantly increases with deterioration of CKD stages . Thus, pThe relatively recent term cardiorenal syndrome has been applied as a unifying concept to the bidirectional interaction between heart disease and kidney disease as either acute or chronic dysfunction of the one organ can, respectively, cause acute or chronic dysfunction in the other , 126. SpκB (NF-κB) and they play an important role in G protein-coupled hypertrophic stimulation by angiotensin II [κB to stimulate hypertrophy [NADPH oxidase activation, xanthine oxidase, mitochondrial dysfunction, and NO-ROS are the main pathways through which increased oxidative stress is generated in CKD leading to LVH and cardiorenal syndrome –130. Whiensin II , 132. Inertrophy . ROS alsertrophy , 131.N-terminal kinase (JNK) and p38-mitogen-activated MAPK promoting both cardiomyocyte autophagy and mitochondrial autophagy, eventually leading to apoptosis [β thus contributing to cardiac and renal fibrosis [2− production and has a growth-promoting effect on the wall of renal vasculature mediated by ROS production [Accordingly, a growing body of evidence suggests that in the setting of heart failure (HF), ROS production within the myocardium and the vasculature is substantially increased . ROS deppoptosis –136. Chrpoptosis . Angiotefibrosis –140. In oduction , 142.α, which in experimental models have been shown to further stimulate angiotensin II-mediated ROS production in cultured rat aortic smooth muscle cells [The chronic inflammatory state, present in both CKD and HF, is associated with increased levels of proinflammatory cytokines such as IL-1b, IL-6, and TNF-le cells , 144.Vascular endothelial dysfunction due to uncoupling of the NOS, activation of vascular and phagocytic membrane oxidases, or mitochondrial oxidative stress may lead to increased vascular stiffness, further compromising cardiac performance in the failing myocardium . The norβ-oxidation of fatty acids to glycolysis; thus, free fatty acids are not transported into the mitochondria and accumulate into the cytosol, thus activating the oxidative cascade [Heart failure is associated with a shift in energy metabolism form  cascade . Patholo cascade –149.There is a lack of clinical studies regarding direct correlation of oxidative stress markers and LVH in patients with CKD. However, a recent clinical trial, conducted in children with CKD, showed a positive correlation between serum levels of oxidized LDL and protein carbonyl group and left ventricular mass .β-blockers, and statins have been shown to reduce the burden of oxidative stress in the organism whereas most clinical trials which utilized administration of specific antioxidants vitamin C and vitamin E failed to show a benefit for the treatment of cardiovascular diseases, including HF [Likewise, despite the abundant experimental evidence regarding the interplay of different oxidative stress pathways in HF and CKD, there is a relative paucity of clinical research regarding this issue. A recent study conducted in diabetic patients with end-stage CKD undergoing hemodialysis showed that serum carbamylated albumin was strongly associated with the 4-year risk of death from congestive HF, thus suggesting a direct link between carbamylation and uremic cardiomyopathy in patients with diabetes mellitus and kidney disease . Most cluding HF , 152. Nouding HF –155. Finuding HF –159.The paraoxonases (PON), another family of enzymes with shared distinct properties, has increasingly been the subject of extensive investigation due to its potential discrete albeit substantial role in the antioxidant mechanism of the organism. Thus, we believe that considering the substantial clinical evidence accumulated, the PON merit special attention.The PON family includes 3 enzymes, PON1, PON2, and PON3, which belong to the armamentarium of the antioxidant defenses of the organism, with PON1 being the most extensively studied . They arPON2 and PON3 modes of action, despite similarities to that of PON1, have not been completely clarified. The antiatherogenic effect of PON2 could be attributed to the protection of mitochondria against oxidative stress, while PON3 has been shown to be a protective factor against obesity .βII, followed by inhibition of eNOS-activating pathways and decreased NO production with ensuing impaired endothelial repair [The PON family appears to play an important protective role in the process of the atheroma plaque formation, but pathogenic mechanisms are hard to establish , 163. Gel repair .There is general agreement in the literature that serum PON1 paraoxonase activity is decreased in patients with coronary artery disease or acute coronary syndromes, as compared to healthy individuals , 167. LoIt is well established that reduced serum PON1 activity in CKD contributes to the increased burden of cardiovascular disease in this patient group, being more prominent in end-stage CKD. PON1 concentration and activity are lower in patients with chronic renal failure and in patients under renal replacement therapy as compared to healthy subjects whereas the effect of renal transplantation on PON1 activity remains unclear –176. HowThere are only few recent publications both experimental and clinical, regarding PON and its association with LVH and HF of either ischemic or nonischemic etiology. A novel antihypertrophic role for the PON gene cluster has been suggested by experimental data . AdditioIn the setting of the hostile uremic milieu, there exists only fragile steady-state equilibrium, with only minor precipitating factors tipping the balance towards decompensation and major cardiovascular events. Thus, it cannot be overemphasized that further elucidation of the oxidative pathways involved in cardiorenal damage will provide substantial aid so as to improve prevention and treatment strategies in patients with CKD who are at highest risk for cardiovascular complications. The continuously increasing information regarding oxidative stress and its mechanisms of disease, the oxidative pathways activated in CKD, and consequent cardiovascular damage requires translation into clinical evidence-based medicine. Further, appropriately powered and meticulously designed clinical studies with long follow-up are needed to reliably assess the effects of antioxidant therapy in patients with CKD.


Osteoarthritis (OA) is a common age-related disease with complex pathophysiology. It is characterized by wide-ranging tissue damage and ultimate biomechanical failure of the whole joint. However, signs of tissue adaptation and attempted repair responses are evident in OA-affected osteochondral tissues. Highlighted in this review article is the role of bone-resident mesenchymal stem cells (MSCs) in these bone remodeling responses, and a proposal that targeting MSC activities in OA subchondral bone could represent a novel approach for intrinsic joint regeneration in OA. The development of these therapies will require better understanding of MSC proliferation, migration and differentiation patterns in relation to OA tissue damage and further clarification of the molecular signaling events in these MSCs during disease progression. Osteoarthritis (OA) is a joint disorder, in which the cartilage, the underlying bone and other joint tissues are affected. Recent evidence demonstrating attempted repair responses in these OA tissues challenges the traditional view of OA as a degenerative disorder. Signs of tissue regeneration are particularly evident in the bone located directly underneath the damaged cartilage, where increased stem cell activity has been observed. Targeting these stem cells could represent a novel approach for intrinsic joint regeneration in OA. To progress with developing these novel therapies, a better understanding of stem cell function in normal and OA joint tissues is needed. Osteoarthritis (OA) is the most common form of chronic joint disease in man and represents the leading cause of pain and disability with increased prevalence due to aging populations . It has OA has long been considered a disease of the cartilage but now it is acknowledged as a disease of the whole joint, including closely linked alterations in cartilage, synovium and subchondral bone . The synIt is now recognized that the OA process can start in various joint structures including ligaments, menisci or in the bone where the latter has been termed as osteogenic OA . WhetherDuring disease progression, the aforementioned structural changes in OA bone are a predictable consequence of alterations in the cell-mediated bone remodeling process. Bone remodeling is an active and dynamic process that depends upon the tightly regulated balance between bone formation and bone resorption . The keyMSCs were first described as a rare BM-derived adherent population of colony-forming cells with high proliferative capacity . BM MSCsin vivo counterparts, notably with regard to bone-related signaling pathways [There are internationally recognized criteria for defining culture-expanded MSCs . Howeverpathways , adhesiopathways  and homipathways . So far pathways . In termpathways ,32. In Opathways , particuMSCs cultured from mouse BM or compact bone share the same surface markers, being positive for CD29, CD44, CD105 and Sca-1 expression . PDGFR-αMSCs are common residents of all joint tissues, including synovial tissue and fluid  as well et al. which reported that MSCs in OA BM remote from the site of damage had reduced chondrogenic and adipogenic potentials [et al. study, Campbell et al. have recently shown that the number of resident MSCs in affected human OA bone (in the BML areas) was also increased compared with non-BML areas but their proliferation and mineralization capacities were reduced [One of the earliest studies on this issue was carried out by Murphy tentials . A more tentials . The higtentials   [More recent evidence suggests that migration of MSCs along chemokine gradients created along thinned cartilage fissures and other breaks in the osteochondral junction, may acquire undesirable features such as the loss of chondrogenic potential , thus ex SDF-1α) , suggest SDF-1α)  and it i SDF-1α) . Future Aberrant migration and escape of endogenous subchondral bone MSCs into the synovial fluid can be also achieved with the use of barrier membranes in procedures such as matrix-assisted chondrocyte implantation , which min situ concentrations of PRP constituents in subchondral bone areas, allowing more direct action on subchondral bone MSCs. Active PRP components that may exert beneficial effects on MSCs could include platelet-derived growth factor, known to enhance MSC proliferation in vivo [Intra-articular injections of platelet-rich plasma (PRP) have been used in sports medicine for a great number of years and have shown mixed results, primarily due to a lack of consistency in PRP preparation, characterization and administration ,86. An i in vivo ,89, as w in vivo , which cAs MSC abnormalities appear to be most confined to the BML areas of OA bone, these BMLs may be targeted for direct administration of therapeutic agents into OA bone. While it may be seen as rather invasive in the OA rheumatology community, MSC and scaffold delivery to the lesional areas of bone is a common procedure for the treatment of avascular necrosis of the femoral head in the orthopedic field . FurtherIt is known that BML volumes change over the course of repeated MRI investigations . This coCurrent therapies for OA treatment focus either on the addition of exogenous MSCs to the affected joint or the use of scaffolds, with or without MSCs, in order to plug cartilage defects and restore joint surface continuity . While rThese therapies are likely to act on a broader range of cells, not only MSCs, therefore future efforts should present a holistic approach to subchondral bone OA treatment whereby both mesenchymal- and hematopoietic-lineage stem cells and their descendants are all considered for simultaneous modulation, taking into account their native niches and three-dimensional environments.In contrast to osteoporosis, which is a systemic bone disease, OA is characterized by local and temporal alterations in bone remodeling activity, in which MSCs appear to have a prominent role. Future joint regeneration strategies should not only aim at restoration of the damaged cartilage, but also at target subchondral bone MSC abnormalities, by means of pharmacological and biological agents to correct their altered migration, proliferation and differentiation capacities. These interventions may be most successful in conjunction with novel surgical techniques such as subchondroplasty.There are spatial and temporal alterations in the bone remodeling processes of osteoarthritis (OA) subchondral bone that can be therapeutically targeted, in order to restore osteochondral tissue homeostasis.Recent evidence demonstrates significant changes in mesenchymal stem cell migration, proliferation and differentiation potentials in OA subchondral bone, particularly in its damaged areas.Correcting mesenchymal stem cell functionality in disease-affected areas of OA bone can represent a novel strategy for joint regeneration in OA.


Advanced pancreatic cancer (PC) is an aggressive malignancy with few effective therapeutic options. While the evolution of precision medicine in recent decades has changed the treatment landscape in many cancers, at present no targeted therapies are used in the routine management of PC. Only a minority of patients with PC present with surgically resectable disease, and in the remainder obtaining high quality biopsy material for both diagnosis and molecular testing can prove challenging. Endoscopic ultrasound-guided fine needle aspiration (EUS FNA) is a widely used diagnostic procedure in PC, and allows tumour sampling in patients with both early and late stage disease. This review will provide an update on the role of EUS FNA as a diagnostic tool, as well as a source of genetic material which can be used both for molecular analysis and for the creation of valuable preclinical disease models. We will also consider relevant clinical applications of EUS FNA in the management of PC, and the path towards bringing precision medicine closer to the clinic in this challenging disease. Pancreatic cancer (PC) is a highly lethal malignancy and ranks as the fourth most common cause of cancer-related death worldwide ,2. The pBRAF mutation in melanoma which predicts responsiveness to BRAF/MEK inhibitor therapy [KRAS wild-type tumours in colorectal cancer which are sensitive to anti-epidermal growth factor receptor (EGFR) therapies [EGFR mutations and ALK gene rearrangements in non-small-cell lung cancer, which are sensitive to EGFR and ALK inhibitors, respectively [The last three decades have seen the evolution of precision medicine, which is now applied to many different cancers. Precision medicine involves reserving specific treatments for patients who have tumours with amenable genetic profiles. Examples include: HER2 amplification in breast cancer which is responsive to trastuzumab ,9; BRAF  therapy ,11,12; Kherapies ,14 and Eectively ,16. Giveectively .In recent years, the genomic landscape of PC has become increasingly well characterized, leading to a better understanding of the pathogenesis of PC as well as the identification of a number of potential therapeutic targets ,22,23,24CDKN2A, TP53 and SMAD4 [An activating somatic mutation of the KRAS gene has long been identified as a critical event occurring in the vast majority of human PCs . An earlnd SMAD4 . However, recent studies have demonstrated that beyond these common mutations there is a high degree of heterogeneity among PC tumours, especially those of the pancreatic ductal adenocarcinoma type which represents ~90% of all PC, highlighting a significant challenge when considering the application of precision medicine in this disease ,23,24,25BCAR1, KLF14, PDX1, CHEK2, TERT) [KRAS, RBM10), but also identified patients who may respond to targeted therapies  [On a larger scale, a recent multi-stage, genome-wide association study on over 7000 PC patients with over 14,000 control individuals identified numerous susceptibility loci for PC lying in close proximity to a variety of genes, some of which have previously been implicated in oncogenesis  . Whole-e PIK3CA) .The high genetic diversity of PC tumours provides a potential explanation for the relatively slow progress in the development of novel and effective chemotherapies for PC, especially since new treatments have previously been tested on unselected PC patient populations ,7,25,26.Of note, these studies used surgical resection specimens to provide tissue for the isolation of genetic material. This reliance on surgical resection specimens effectively excludes the possibility of real time genetic analysis for many patients with advanced disease. Consequently, one of the major obstacles to the introduction of precision medicine in PC has been the difficulty in recruiting patients with high quality tumour-derived genetic material (genomic DNA and/or RNA) in sufficient quantities for subsequent molecular profiling, as recently reported by the Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) Trial. The IMPaCT trial was designed to identify subsets of patients with advanced metastatic disease who could be targeted, based on mutations within their tumour genome, with currently available therapies . A majorThe above observations highlight a potential role for EUS FNA to isolate genetic material to direct precision medicine. Up to 50% of PC patients present with locally advanced disease and undergo EUS FNA to establish a tissue diagnosis. Although EUS FNA has been used to provide tissue for the genetic analysis for PC and other cancers, the clinical utility of this technique has been hampered by concerns about low tissue quantities leading to a suboptimal yield of genetic material, as well as sample contamination with non-malignant cells ,48,49,50Indeed, EUS FNA is possible at all stages of disease and can be repeated more easily than other biopsy techniques, further highlighting its potential utility. For example, it could be used to guide selection of neo-adjuvant therapy in operable patients, while in those patients receiving palliative chemotherapy it allows for repeat sampling of the tumour, which might allow assessment of whether a particular therapy is having the desired effect on the specific molecular pathway targeted by the treatment. Here we review the use of EUS FNA as a source of genetic material from PC tumours and examine the potential to use this technique in guiding precision medicine in PC.®later to preserve RNA [A number of groups have examined the use of EUS FNA-derived DNA for the detection of KRAS mutations in order to improve diagnostic sensitivity of EUS FNA ,55,56,57erve RNA .Many studies have relied upon formalin-fixed and paraffin-embedded (FFPE) samples for the isolation of gDNA. The amount of FFPE tissue available from EUS FNA is often very limited and the fixation process can result in nucleic acid fragmentation, which can reduce the yield and quality of gDNA and substantially degrades RNA, thereby reducing the potential for this method to detect mutations in tumoural DNA and eliminating the ability to measure gene expression. This suggests that the sensitivity of EUS FNA and KRAS mutations could be further improved if more reliable DNA extraction techniques were used, such as snap freezing of biopsies. The added utility of KRAS mutation assay in the diagnosis of cystic lesions within the pancreas has also been examined. Overall, cytological analysis has a much lower sensitivity in cystic lesions of the pancreas compared to solid lesions, only ranging from 35–63% ,63. In aTo date, only a limited number of studies have reported the use of EUS FNA-derived RNA in PC, including one study from our institution ,66,67. AIn an earlier study by Bournet et al. , EUS FNABerry et al., attempted to use transcriptome profiling from metastatic and localised PC to determine whether there was a difference in the genetic profiles of these two clinical phenotypes . InteresNevertheless, these studies suggest that highly sensitive sequencing studies are indeed possible with EUS FNA-derived material, and therefore that this technique has the potential to guide precision medicine for PC patients. A recent study attempted to assess the potential for this strategy using the Human Comprehensive Cancer GeneRead™ DNAseq Targeted Panel V2 , a panel which detects mutations across the exome (coding regions) of 160 genes frequently mutated in malignancy . IncludeThe overwhelming majority of work to characterize the genetic phenotypes of PC has been performed with surgical resection specimens. This makes the interpretation of novel phenotypes identified using EUS FNA problematic. Although it has been demonstrated that allelic frequency is comparable between EUS FNA-derived and surgically-derived DNA, it is unclear what impact the sampling method has on non-tumoural cell content and gene expression. For instance, EUS FNA samples typically contain blood, inflammatory cells and even intestinal wall epithelial cells; whereas, surgical specimens contain large amounts of stromal tissue but no contamination with intestinal wall cells. Consequently, gene expression studies using EUS FNA-derived material need to address these differences. In studies using surgical resection specimens, samples are usually acquired in the presence of the surgeon and pathologist and a frozen section is often performed to quantify the tumour cellularity within the sample. Alternatively, micro-dissection can be performed to maximise tumour cellularity ,42,71. TWe have already alluded to the similarities between the two sample types in terms of mutational concordance, but what of the differences? Diagnostic gene signatures have been used for both resection specimens and EUS FNA samples to distinguish PC from non-malignant tissue . Bhasin et al.  performeIn a pre-clinical setting, precision therapy can be tested using patient-derived xenograft (PDX) models or organoid culture for in vivo and in vitro drug testing ,74,75. PRecently, the growth of patient-derived tumour tissue in vitro using specifically defined media and conditions (organoid culture) provides a complementary tool to PDXs to test the anti-cancer efficacy of new compounds ,75,85. TThere are a number of potential ways to integrate molecular analyses into clinical decision-making, including for the identification of new molecular targets, to guide the selection of therapy, and in monitoring disease. The identification of known molecular targets allows for treatment with currently available therapeutics; examples include treating HER2-amplified tumours with trastuzumab or KRAS wild-type tumours with EGFR inhibitors . Other pA phase III clinical trial of gemcitabine with or without erlotinib, an EGFR inhibitor, as the first line therapy in unselected patients with locally advanced or metastatic PC demonstrated a statistically significant improvement in overall survival in the combination arm, albeit only in the manner of approximately two weeks . Given tThe use of immunotherapy agents such as antibodies against programmed-death-1 receptor (PD-1) has dramatically improved treatment responses in some malignancies such as advanced melanoma , but to Despite recent advances, translating promising pre-clinical results into meaningful clinical benefits remains elusive, as reflected in the disappointing results of early phase clinical trials utilising novel agents such as hedgehog inhibitors in unselected PC patients ,94, and At present, chemotherapy remains the standard of care for the management of advanced PC , althougWhile treating a specific molecular abnormality with a targeted therapy is appealing, there may be a complex array of interacting genes contributing to treatment susceptibility and resistance mechanisms ,24,98. TTogether, these applications can have an immediate impact on patient outcomes by directing clinicians to use targeted therapies in a personalised manner. In addition, the future implications in terms of drug discovery offer exciting new directions in molecular oncology research.However, despite recent advances, in clinical practice the translation of promising data from the bench to the bedside remains challenging. The IMPaCT trial, mentioned above, clearly demonstrated some of these hurdles and highlighted the need for flexible, pragmatic study designs for personalised medicine trials in PC . DespiteThere is a clear unmet clinical need for the advancement of therapeutic options in advanced PC. EUS FNA has only recently emerged as a candidate for isolating genetic material from PC and therefore as a means for biomarker identification, as well as in the establishment of valuable pre-clinical disease models such as PDX and organoid cultures. A major advantage of EUS FNA compared to surgical resection specimens is the ability to include patients with unresectable disease, who make up the overwhelming majority in PC. In addition, the ability to obtain tissue early in the clinical course of the disease facilitates the procurement of chemo-naïve tissue. Given the success in obtaining both gDNA and RNA and translating these into meaningful genomic and transcriptomic data, EUS FNA is poised to identify and characterise known and novel therapeutic biomarkers. As such, new trials of personalised therapy in PC should endeavor to use EUS FNA to direct treatment as well as, or instead of, surgical specimens. By doing so, we may overcome some of the practical barriers which currently limit the clinical application of precision medicine for PC.


The phenological development of plants can be broadly divided into 4 stages, embryo/juvenile, adult , reproductive (the generative stage), and senescent. This research topic focusses on the transition from vegetative growth to reproductive development, commonly referred to as floral transition. Plants have coordinated the seasonal timing of flowering and reproduction with the prevailing environmental conditions. In agriculture, flowering is a prerequisite for crop production whenever seeds or fruits are harvested. In contrast, avoidance of flowering is necessary for harvesting vegetative parts of a plant such as tubers or roots. Late flowering also severely hampers breeding success due to long generation times. Thus, flowering time regulation is of utmost importance for genetic improvement of crops.Arabidopsis thaliana . Several of these pathways are activated by different environmental factors such as low temperature, day length, light intensity, or stress. Conservation of major flowering-time regulators and regulatory pathways between different species as well as increased availability of genome sequences and improvements in computational biology offer a unique opportunity to study flowering time genes across species. In general terms, the central elements that perceive day-length signals to control floral transition are conserved across the plant kingdom. CONSTANS (CO)-like genes and PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN (PEBP) encoding genes play major roles in these pathways and were first identified by genetic analysis in Arabidopsis. CO-like sequences seem to exist in all plants. Recent evidence indicates that CO of Arabidopsis arose from a family-specific duplication and similar events might have occurred independently in many plant families , and GCN-like (GNL) previously identified as growth regulators mediating control by several phytohormones have emerged as repressors of flowering, acting via SOC1 . During the juvenile to adult phase transition, a gradual decrease in miR156 and a reciprocal increase in miR172 ultimately leads to the activation of FLOWERING LOCUS T (FT) .New components have been added to the network of flowering time regulators mostly working upstream of key regulator elements, e.g., GATA transcription factors, small RNAs, in particular microRNAs (miRNAs) or sugar molecules. The GATA transcription factors INDETERMINATE flowering gene was cloned from maize by transposon tagging . For instance, whole transcriptome analysis revealed the circadian clock homolog of EARLY FLOWERING 3 (ELF3) and mapping-by-sequencing applied on exome-capture data from phenotypic bulks identified PHYTOCHROME C as important components of photoperiodic flowering in barley  and HvSDW1 (GA20ox2) were linked to both, developmental and yield-related traits. In a salinity tolerance study with HEB-25, the wild barley allele at the HvCEN locus  promoted flowering and maturity, resulting in a higher harvest index and a higher yield under salt stress in the field  describe flowering time genes from oilseed rape where the vernalization pathway with its central element FLOWERING LOCUS C (FLC) is essentially the same as in Arabidopsis. In contrast, an FLC homolog from beet was proven not to be a major regulator of vernalization response in biennial beets Vogt et al. New sequence variation has been induced in rapeseed by EMS mutagenesis which gave rise to plants with altered flowering time in spite of the polyploid nature of this species. Mutations within a single gene can have a big impact on flowering time even if there are several paralogs of an Arabidopsis flowering time gene present in the rapeseed genome.Flowering time regulation is strongly conserved among the Brassicas to which Arabidopsis also belongs. Two articles . The authors discuss molecular pathways that control seasonal flowering responses in the Pooideae sub-family and how variations in flowering time gene activities contributed to the adaptation to different environments. Refined flowering time regulatory pathways have been identified from rice  and TERMINAL FLOWER 1 (TFL) of Arabidopsis attracted the highest attention. As highlighted for tomato  and for rice  ratio is an important process for patterning plant architecture. Lifschitz et al. explain how the ratios between FT-like and TFL1-like genes control the patterning of the shoot systems across many different plants. In tomato, an increase of the florigen protein SINGLE FLOWER TRUSS , relative to the anti-florigen protein SELF PRUNING  induces growth arrest and termination of meristems across the tomato shoot, while high relative levels of SP promote the formation of an indeterminate vegetative inflorescence. Consequently, SFT/SP ratios determine the number of flowers and eventually, tomato fruits per inflorescence. Naturally occurring mutations have been selected in both gene families to adapt crops to different environments and to increase productivity. TFL1 mutants have been frequently used in breeding . Likewise, mutations of FT-like genes were selected in sunflower, sugar beet, rice, potato, and wheat. Moreover, single point mutations within FT- and TFL1 orthologs can drastically alter their function from floral inducers to floral repressors, as demonstrated for sugarcane Coelho et al. and beet .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reading frame altering genomic variants can impact gene expression levels and the structure of protein products, thus potentially inducing disease phenotypes. Current annotation approaches report the impact of such variants in the context of altered DNA sequence only; attributes of the resulting transcript, reading frame and translated protein product are not reported. To remedy this shortcoming, we present a new genetic annotation approach termed Codon Consequence Scanner (COCOS). Implemented as an Ensembl variant effect predictor (VEP) plugin, COCOS captures amino acid sequence alterations stemming from variants that produce an altered reading frame, such as stop-lost variants and small insertions and deletions (InDels). To highlight its significance, COCOS was applied to data from the 1000 Genomes Project. Transcripts affected by stop-lost variants introduce a median of 15 amino acids, while InDels have a more extensive impact with a median of 66 amino acids being incorporated. Captured sequence alterations are written out in FASTA format and can be further analyzed for impact on the underlying protein structure.https://github.com/butkiem/COCOSCOCOS is available to all users on github:  To alleviate these limitations, we introduce COCOS, CodonConsequenceScanner, a VEP plugin to provide additional information about the altered reading frame and translated sequence resulting from stop-lost variants and small InDels.Annotation of the functional consequences of DNA sequence variants is increasing in importance for human genomic studies. Specifically, variants that are thought to be highly disruptive to protein sequence  and are presumed to be a primary driver of disease pathogenesis have garnered special attention. The designation ‘Loss-of-Function’ varies between studies, but generally involves the insertion of a stop-codon, disruption of a splice site and insertions/deletions (InDels) that disrupt the translational reading frame , but recThe primary functionality of the COCOS plugin is to capture AA sequence alterations stemming from variants that produce an altered reading frame, such as stop-lost variants and InDels. Though their potential to dramatically change the ultimate protein sequence is high, some of these variants may be benign, introducing only a few altered AAs in the final coding sequence. The coding sequence is terminated when a subsequent stop-codon terminates the translation process. The COCOS algorithm is outlined in viable translation sequence alterations. We accessed data from the 1000 Genomes project (phase 3) . A subsein silico annotations of deleterious variants, and thus help interpret implications in a biological context.The COCOS plugin for VEP captures variant induced codon alterations and presents this altered sequence as translated AAs, allowing for improved interpretation or scoring of predicted variant consequences in a protein product disruption context. As seen in


Drug combination is a powerful and promising approach for complex disease therapy such as cancer and cardiovascular disease. However, the number of synergistic drug combinations approved by the Food and Drug Administration is very small. To bridge the gap between urgent need and low yield, researchers have constructed various models to identify synergistic drug combinations. Among these models, biomolecular network-based model is outstanding because of its ability to reflect and illustrate the relationships among drugs, disease-related genes, therapeutic targets, and disease-specific signaling pathways as a system. In this review, we analyzed and classified models for synergistic drug combination prediction in recent decade according to their respective algorithms. Besides, we collected useful resources including databases and analysis tools for synergistic drug combination prediction. It should provide a quick resource for computational biologists who work with network medicine or synergistic drug combination designing. Complex diseases such as cancer are often caused by a collective of abnormalities of correlated genes or biology processes. The traditional paradigm of “one gene, one drug, one disease” has been challenged by the increasing rate of drug failure and the huge costs of time and money in drug development and research , 2. What in silico methods and in vitro methods have been applied to screen synergistic drug combinations. The most straightforward methods for screening synergistic drug combination are in vitro methods.To tackle the problem of drug resistance and reduce the great expenses of time and money in drug discovery, researchers have made great efforts to discover synergistic drug combinations. Synergistic drug combination means that the overall therapeutic effect of the combination is larger than the sum of effects independently caused by individual component . Synergi in vitro methods, namely, the highest single agent (HSA) model [ in vitro, especially in validation of novel drug combinations from various kinds of computational models. The in vitro models mentioned above are all based on drug-treated dose-response curve. Dose-response curve based model can achieve good performance for low-throughput data and be used to validate novel drug combinations. Nevertheless, without involving any molecular level data such as drug-treatment transcriptional expression profile, these models may not help to discover the underlying mechanism of drug synergy [ in vitro methods which screen all possible combinations by experimental trials are time and money consuming, and usually only a small number of synergistic drug combinations can be identified.There are three popular reference modelsA) model , the LoeA) model , and theA) model . The difA) model . More imA) model , 12. The synergy . Besides in vitro methods in identifying synergistic drug combinations, in silico methods based on “omics” data have become more and more popular [To address this limitations of popular –17. With popular . Network popular –23. WithInspired by the rapid development of biomolecular network-based synergistic drug combination discovery, here we present a brief review on the prediction models of synergistic drug combination. All the models are divided into three classes according to the type of drug pairs used to train the prediction model, namely, unsupervised learning prediction models depending on hypothesis of drug synergy and unlabeled drug pairs, semi-supervised learning prediction models involving few labeled drug pairs, and many unlabeled drug pairs in model training and supervised learning prediction models using labeled drug pairs to train models. Labeled drug pairs denote that effective drug combinations have been approved by FDA or validated by experiments, while unlabeled drug pairs denote drug pairs without synergistic effect evidence. The general work flow of identification of novel synergistic drug combinations based on biomolecular network is shown in  in silico. Recently, with the rapid development of next generation sequencing (NGS) technologies and rapidly accumulation of “omics” data , there ahttp://dx.doi.org/10.1155/2016/8518945). With these data, the performance of prediction models based on compound/agent-treated transcriptional expression profile can be evaluated without conducting experiments.The Connectivity Map (CMap) collects a genome-wide transcriptional expression data from cultured human cells treated with various bioactive small molecules, providing a bridge to connect drugs and genes . Drug CoThe therapy strategy of “one drug, one gene” is not always successful to treat disease because cells can often find alternative ways to compensate the function after a gene or protein is perturbed by drug treatment . Thus toUnsupervised learning models were mainly based on various features of drugs, targets, drug-treated cellular response data, and functional networks following various hypotheses. Drug-treated cellular response data can provide an insight of drug mechanism of action . TranscrDrugComboRanker was built based on the hypothesis that effective drug combinations can inhibit major modules of disease signaling networks simultaneously and drugs often have multiple active target genes or proteins . Based oJin et al. built a model called enhanced Petri-net (EPN) to predict the synergistic effect of pairwise drug combinations from genome-wide transcriptional expression data by applying Petri-net to identify drug targeted signaling network . They asWu et al. utilized information of protein interactions, protein-DNA interactions, and signaling pathways to construct a molecular interaction network . Their aHomo sapiens” pathways, and edges are pathway-pathway interactions. Then for each drug pair they defined a score which indicated the connectivity of pathways perturbed by the individual drug of drug combinations on the WWI network and drug targets on the PPI network. Finally, all the query drug pairs were ranked in descend order of the score and the top-ranked ones would be more likely to be synergetic.Similarly, a model named pathway and pathway interaction (WWI) was based on the assumption that drugs targeting one same pathway or related pathways will be more likely to be synergistic drug combinations . The resAnother group of unsupervised learning prediction models were built according to the DEARM Challenge data. In 2012, the Dialogue on Reverse Engineering Assessment and Methods (DREAM) consortium designed an open competition for researchers all over the world to rank the effect of all 91 pairwise combinations on OCI-LY3 human diffuse large B-cell lymphoma (DLBCL) cell line from the most synergistic to the most antagonistic . This prAmong all of those 31 groups participating in the project, Drug-Induced Genomic Residual Effect (DIGRE) model achieved the best performance on prediction of synergistic drug combinations . DIGRE wFrom above, we can see that drug-treated transcriptional expression data is commonly used in unsupervised learning prediction models because of its informative properties, such as reflecting the mechanism of drug action underlying a biological pathway. However, there is no standard rule to process transcriptional expression data. Thus, selection of significant differential expression genes is largely depended on individual researchers and significant gene lists can be quite diverse according to different algorithms. For models based on transcriptional expression data, different networks will be constructed by different researchers depending on diverse processing methods of the same microarray data, which can lead to difficulty in the final interpretation .Traditional classifier uses only labeled data to train the prediction model. However, it is both time and money consuming to collect labeled data by experts. Semi-supervised learning solves this problem by using large amount of unlabeled data together with a limited number of labeled data . To the Sun et al. constructed a model called Ranking-system of Anticancer Synergy (RACS) based on semi-supervised learning which was used to rank drug pairs according to their similarity to the labeled samples in a specified multifeature space . FirstlyChen et al. developed an algorithm termed Network-based Laplacian regularized Least Square Synergistic drug combination prediction (NLLSS) based on their observation that principal drugs which obtain synergistic effect with similar adjuvant drugs are often similar , where pAs can be seen from above, RACS and NLLSS share common features. Firstly, they both have a small number of labeled data, such as 26 labeled data compared with 502 unlabeled data for RACS training set and 75 labeled data compared with 4079 unlabeled data for NLLSS training set. Secondly, they were developed based on known and complex machine learning algorithms for manifold regularization which is a technique for using the shape of a dataset to constrain the functions that should be learned on that dataset .Supervised learning is a machine learning algorithm of inferring a function from training data. The training data consists of a set of training samples which consist of an input object and a relevant label. Then with the inferred function, new sample can be labeled . Thus wiZhao et al. developed a model based on features of US Food and Drug Administration (FDA) approved drug combinations including drug features such as drug target proteins and corresponding downstream pathways, medical indication areas, therapeutic effects as represented in the Anatomical Therapeutic Chemical (ATC) Classification System, and side effects . They peXu et al. proposed a model called Drug Combination Predictor (DCPred) . With thp value based on random distribution. After performance evaluation of PEA using 10-fold cross-validation scheme accompanied with the receiver operating characteristic (ROC) curve analysis, integrative analysis of side-beneficial effects for drug combinations, external literature validation, and experimental validation, tens of effective drug combinations were confirmed.Similarly, Li et al. proposed another model called probability ensemble approach (PEA) based on drug-based similarity features including drug chemical structure, ATC code, target side effect, and target-based similarity features include target sequence, target-target interaction in PPI, and Gene Ontology (GO) semantic . The sixC152 = 105) among 15 drugs. Then drug interaction data measuring synergistic and antagonistic effect together with chemogenomics data were put into INDIGO as training data. INDIGO only requires chemogenomics data of individual drugs to output novel drug combinations.Chandrasekaran et al. developed a computational model entitled INferring Drug Interactions using chemo-Genomics and Orthology (INDIGO) which used chemogenomics data to predict antibiotic drug combinations . The corThe supervised learning models tend to take advantage of drug property information like drug target, ATC code, and chemical structure, while drug synergy is strongly context-dependent, disease type  and drug in silico modeling for synergistic drug combination represents both an opportunity and a challenge for medicine research. Combined with knowledge of mathematics, computer science, and biology, analysis of complex molecule interactions based on biomolecular networks can greatly accelerate the discovery of synergistic drug combinations [ in vitro methods should be used to get dose-response curves. Finally, either Chou-Talalay method or reference models such as Loewe additivity model and Bliss independent model can be used to determine the effect of drug combination .With the explosive growth of high-throughput data,inations . To builBased on the review above, we can see that the number of biomolecular network-based unsupervised learning models is much bigger than that of semi-supervised learning or supervised learning. The possible reason is that only small number of drug combinations has been approved by regulatory agency, which limits the use of machine learning methods such as semi-supervised learning method and supervised learning method. Several significantly different features between synergistic drug combinations and random drug combinations have been identified. The average shortest distance in PPI network of targets between synergistic drug combinations is significantly smaller than that in random drug combinations , 73, 79.There are several limitations in effectively applying these network-based models. Firstly, synergistic drug combination modeling utilizing high-throughput data based on biomolecular network is in its infancy and most of these models have not been fully validated in practice. Secondly, despite the complexity of these prediction models, the massive noise of high-throughput data at different levels from different contributors can play an import role in model construction and in turn lower the performance of the prediction model. For instance, prediction models can perform very well in one test set, while poor result may be achieved when the model is applied to another test set. For example, Sun et al. applied the transcriptional expression profiles and target information of drug treatment on the human lung adenocarcinoma cell line A549 to RACS . ResultsFuture models for synergistic drug combination prediction should pay more attention to incorporating comprehensive information including disease signaling pathways and drug targeting pathways as well as drug metabolism processes such as drug absorption, transportation, metabolism, and clearance.Pairwise drug combinations in Drug Combination Database (DCDB), components of which are both in Connectivity Map.


Autophagy, the main lysosomal degradative machinery, plays a major role inmaintaining cellular homeostasis and thus a healthy state in an organism. Thisprocess recycles unnecessary or damaged material, therefore, not only providingnutrients to maintain vital cellular functions in times of starvation but alsoeliminating potentially harmful cellular material 2As sensors of the current environmental status, nutrient signaling pathways representcentral aging regulators. For instance, individual interventions in theinsulin/insulin-like growth factor 1 (IGF-1), Ras, protein kinase A (PKA), target ofrapamycin (Tor), or protein kinase B SCH9/Akt) pathways have been shown to extendlifespan in various organisms, including mammals 78/Akt path10111518S cerevisiae, glucose is the preferred carbon sourcefor fueling energy metabolism and its fermentation produces acetate and ethanol thatis used for subsequent respiration after the diauxic shift. Both intermediates arereleased into the medium and have been attributed a role in limiting yeastchronological life span 23232527In the yeast The levels of polyamines, a class of ubiquitously occurring small basic polycations,decline with progressing age in various organisms, including humans In line with the vast importance of nutrient signaling during aging, amino acidmetabolism has an important impact on eukaryotic aging and its related diseases. Thelevels of specific amino acids like tryptophane, methionine, arginine, or leucinehas often been suggested to (positively or negatively) influence the autophagypathway and impact aging in different eukaryotes 394041in vitro and in vivo.This might bring up metabolomics as a future trend for aging analyses A variety of potential metabolic controllers of autophagy and health span havealready been proposed. However, precise strategies to target the correlatingpathways  remain to be elucidated in more detail. Forexample, it would be of great interest to determine if special diets that includethe limitation of (defined) amino acids or the uptake of certain polyamines, likespermidine, influence the metabolism towards improved cellular conditions duringaging. It also remains elusive how certain diets may affect the microbiome and inturn impact the levels of certain metabolites that have been shown to regulatecellular fitness, such as citrate, pyruvate, butyrate, or acetate. The investigationof metabolites as powerful rheostats in aging and autophagy is supported by theimprovement of technologies that have opened up new possibilities to detect andtrace even small molecules


Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two human cancers, Kaposi’s Sarcoma (KS) and primary effusion lymphoma (PEL), and a lymphoproliferation, Multicentric Castleman’s Disease (MCD). Progression to tumor development in KS is dependent upon the reactivation of the virus from its latent state. We, and others, have shown that the Replication and transcriptional activator (Rta) protein is the only viral gene product that is necessary and sufficient for viral reactivation. To induce the reactivation and transcription of viral genes, Rta forms a complex with the cellular DNA binding component of the canonical Notch signaling pathway, recombination signal binding protein for Jk (RBP-Jk). Formation of this Rta:RBP-Jk complex is necessary for viral reactivation to occur. Expression of activated Notch has been shown to be dysregulated in KSHV infected cells and to be necessary for cell growth and disease progression. Studies into the involvement of activated Notch in viral reactivation have yielded varied results. In this paper, we review the current literature regarding Notch dysregulation by KSHV and its role in viral infection and cellular pathogenesis. Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 is a γ-herpesvirus discovered in 1994 from Kaposi’s sarcoma tissue by the identification of unique viral sequences  and fromRecent studies have implicated the manipulation of the Notch signaling pathway in KSHV and primary effusion lymphoma (PEL) pathogenesis ,7, addinKSHV belongs to the rhadinovirus genus of the herpesviridae family of viruses. Similarly to all other members of this genus, the KSHV virion is enveloped with an icosahedral capsid protecting the genome . The genThe KSHV envelope consists of a lipid bilayer studded with viral glycoproteins. These viral glycoproteins play a role in viral recognition of host cells and entry. The glycoprotein gB interacts with the cellular receptor integrin α3β1 to mediate viral entry , while KThe envelope is connected to the capsid via cellular and viral proteins organized in an intricate network of protein–protein interactions ,22. ThisTegument proteins regulate viral immune evasion by inhibiting: type I interferon (IFN)-mediated immune responses ,26, actiAs previously mentioned, this tegument layer is connected to the viral capsid. Similarly to α and β herpesviruses, KSHV’s capsid is arranged in a T = 16 icosahedral lattice with an identical inner radius to that of HSV-1 . Within KSHV identifies and infects cells through the use of viral glycoproteins that are found on the envelope surface . The binViral entry is facilitated by the cellular E3 ligase, c-Cbl (Casitas B-lineage Lymphoma), which translocates extracellular KSHV along with the host’s cell receptors to lipid rafts to promote the association of integrins with ephrin receptors. This results in the formation of a complex between integrins, c-Cbl, and myosin, which induces the creation of endocytic blebs , allowinOnce viral DNA has entered the host cell nucleus via the nuclear pore, the genome expresses viral lytic genes involved in host cell immune evasion . For insUpon entrance into the latent phase, the linear genome is circularized into an episome and tethered to the host cell genome . The virOnce the virus switches to its lytic state, and mature virions are produced, the virus leaves infected cells via exocytosis . During In this model, viral DNA is packaged into preformed capsids in the nucleus ,55. ThisIn the cytoplasm, the nucleocapsid acquires both its final tegument and final envelope ,55. ThisIt is believed that the principal site of KSHV latency is in B lymphocytes due to the isolation of the virus from the bone marrow of infected individuals ,61 and tThere is still some debate as to how KSHV is transmitted from one individual to another. The disagreement arises in part from variations in the prevalence of KSHV and KS incidence based on age, sex, and geographical region. It is generally agreed upon that saliva ,70 is liAs previously mentioned, KSHV is the causative agent of two human cancers, Kaposi’s sarcoma  and MultThere are an estimated 1863 to 2050 expected cases of Castleman’s disease in the U.S. per year, with 294 occurring among individuals infected with HIV . CastlemA condition similar to MCD, KSHV inflammatory cytokine syndrome (KICS), was recently identified. Patients with KICS exhibit similar symptoms as those with MCD, but no pathologic nodal changes are observed, nor is lymphadenopathy prominent . BecauseKSHV infection is not uniformly associated with MCD development, however KSHV is the causative agent of all cases of PEL. Malignant PEL cells are always infected with KSHV , and comKSHV also causes KS, the disease in which the virus was discovered and identified as a separate entity from HIV . The lesClassic KS predominantly occurs in older, HIV-negative men of Mediterranean and Eastern European decent . BecauseUnlike classic KS, endemic or African KS is very aggressive, is regularly seen in HIV-seronegative children and young adults, and is a leading cause of death in Sub-Saharan Africa . IndividTransplant-associated or iatrogenic KS is found in patients seropositive for KSHV taking immunosuppressant therapy. This form of KS causes a significant number of deaths in countries with high KSHV seroprevalences . IndividThe most aggressive and common variant of KS is AIDS-associated or epidemic KS . This vaKSHV contributes to KS progression using two molecular phases: latent and lytic. While the majority of infected cells within a KS tumor are latently infected , the expDuring latent, or non-productive, KSHV infection, the 160–170 kb viral DNA genome  replicatde novo infection, LANA mutants with defective RBP-Jk interactions fail to support episome persistence. These observations highlight the importance of Notch dysregulation by LANA in controlling latency and lytic reactivation. Another viral protein, vIRF4, also binds and sequesters RBP-Jk to hinder Notch signaling -l-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) [To determine the pathologic consequences of increased Notch receptor expression and activation on KSHV infection and disease progression, labs have evaluated the effects of treating cells with gamma secretase inhibitors (GSI) such as r (DAPT) ,12. The r (DAPT) . The samr (DAPT)  and infer (DAPT) . In lympBecause of Notch’s overexpression in KSHV-infected cells and tumors, coupled with the apoptosis and necrosis observed in GSI treated samples, many labs have attempted to determine the mechanism by which Notch might contribute to KSHV progression. Multiple studies have shown a link between Notch signaling and the endothelial to mesenchymal transition (EMT), a step considered to be a hallmark of KSHV infection ,197,200.Lymphatic endothelial cells (LEC) infected with KSHV express endothelial precursor markers such as CD133 and late mural cell markers . In thesAdditional studies showed that DAPT treatment of KSHV-infected LECs decreased sprouting, a key EMT process . MoreoveBuilding upon these studies, Notch was also shown to stimulate the EMT in KSHV-infected dermal microvascular endothelial cells (DMVEC) . Again, These three studies conclusively show that KSHV stimulates the EMT in a Notch-associated manner. Each of these studies implicated different mechanisms by which Notch regulated this transition, highlighting a primary role for Notch in disease progression in KSHV-infected cells ,197,200.Other studies suggest that Notch isoform 1 drives oncogenesis in KSHV infected cells, as its inhibition leads to necrosis . KSHV-inBased on the knowledge that there are approximately 81 consensus and additional non-consensus RBP-Jk binding sites within the KSHV genome  , and Notch inhibitor, DAPT, and scored reactivation based on the expression of the late gene K8.1. Using immunofluorescence, we observed a decrease in K8.1 expression upon treatment with DAPT , but no decrease in Rta expression .All of these provocative, yet contradictory results hint at the complexity of interactions of Notch with KSHV and demand further studies to fully understand the effects of Notch on viral infection.Many viruses manipulate host signaling pathways to regulate viral gene expression. KSHV employs this biologic strategy by manipulating the Notch pathway. Due to the presence of RBP-Jk sites within lytic gene promoters  and NotcStudies reporting the overexpression of activated Notch in KS lesional tissue and infected cells  corroborThe contribution of the Notch signaling pathway in KSHV disease progression remains a vital topic in virology. A consensus on whether Notch is sufficient to “flip the switch” and reactivate KSHV from latency has not yet been reached. Conflicting answers to that question support potential cell-specific effects for Notch activity in reactivation. Because the literature also reports promoter-specific effects of activated Notch for the virus and host cell, the means of quantitating reactivation could also influence the conclusions of different studies. Superficially, Rta appears to mimic activated Notch, but detailed studies highlight significant mechanistic differences between the proteins. Continued investigation of the mechanism by which Notch and Rta function in B cells during KSHV reactivation is required to grasp the complexity and pathologic consequences of KSHV–Notch interactions and regulation.