Dry eye disease (DED) is one of the most frequent presentations to optometrists with over 16 million US adults (6.8% of adult population) diagnosed as having this disorder. The majority of associated marketed products offer relief from symptomatology but do not address aetiology. DED harbours many distinguishing features of a chronic inflammatory disorder. The recent explosion in human microbiome research has sparked interest in the ocular microbiome and its role in the preservation and extension of ocular surface health and in the contribution of the gut microbiome to chronic systemic inflammation and associated “Western life-style” diseases. With a significant lack of success for many patients using currently available DED treatments, in this era of the microbiome, we are interested in exploring potential novel therapies that aim to reconstitute healthy bacterial communities both locally and distally (in the gut) as a treatment for DED. Although this direction of investigation is in its infancy, burgeoning interest makes such a review timely. This paper considers a number of studies into the use functional foods and associated products to ameliorate dry eye. Dry eye disease, a multifactorial disorder characterized by a loss of homeostasis of the tear film and hyperosmolarity, visual disturbance and ocular surface inflammation  encompasAltered immunity is a significant factor in DED. As articulated by Stern and colleagues , increasIn addition to the localized autoimmunity, it has long been recognized that systemically delivered antibiotics can ameliorate ocular surface inflammation . A numbeThe purpose of this review is to explore the use of antibiotics in treating dry eye; as antimicrobial agents and/or anti-inflammatory mediators and to showcase the emerging field of DED treatment that seeks to redress the autoimmune dysfunction of DED through pro-biotics, pre-biotics and functional foods with a view to perhaps eventually moving beyond antimicrobial therapy with all its associated disadvantages.Beyond antimicrobial agents, there are many over the counter treatments on the market for DED that are directed against symptoms, offering immediate but transient relief. A Cochrane review from 2016 studied 43 randomized trials (comprising 3497 dry eye subjects). The article was critical of the lack of homogeneity in variables such as diagnostic criteria, interventions, outcome measures and so forth, to the extent that the authors highlighted these constraints as potential confounders in their meta-analysis. Given these limitations  the authors concluded that overall there is little certainly that over-the-counter (OTC) drops offer any greater relief than placebo.Dry eye ophthalmic drugs aim to provide relief in both signs and symptoms. A recent, elegant review by Holland and colleagues  providesMucin secretogogues (that stimulate mucin or mucin-like substance production) appear to reduce corneal staining  but attenuate few other signs of dry eye while (subjective) comfort appears to improve ,20,21. MBiocidal action of antibiotics is seen as helpful in blepharitis- and MGD- induced dry eye through reduction of lid margin bacterial burden. Topical application of biocidal concentrations of fusidic acid and fluroquinolones have been advocated, with systemic cloxacillin and rifampin for recalcitrant disease, cautioned however by disadvantages of increasing antibiotic resistance .Physical therapies aimed at stimulating meibum production  mediates the inflammatory cascade but products aimed at antagonizing IL-1 have had mixed results in clinical trials as reported by Holland and colleagues . One proThe use of systemic antibiotics for DED is well established. Anti-inflammatory effects of bacteriostatic tetracyclines such as oxytetracycline, doxycycline and minocycline are advocated for meibomian gland dysfunction induced DED though treatment breaks after three months are recommended . Anti-inMacrolides such as azithromycin and tacrolimus exert similar anti-inflammatory activities as evidenced in trials to treat blepharitis and MGD ,36,37. TAdditionally, low dose antimicrobials inhibit production of bacterial lipases and cognate free fatty acids that exacerbate inflammation, leading to improved meibomian gland function and stable tear film . The relEven more so than biocidal use of antibiotics, such treatment is open to all the attendant disadvantages of chronic antibiotic use with low dosage more likely to lead to increased antibiotic resistance.As useful as antibiotics, corticosteroids and other agents employed to modulate inflammation have been for DED, we need to move beyond merely keeping inflammation in check but rather, determining and ameliorating the source of inflammation .In relation to DED, antibiotics used to induce gut dysbiosis have resulted dry eye in experimental animal models . FurtherBut perhaps a greater issue to be addressed with all these products is the design concept of stimulating or initiating local responses for amelioration rather than addressing the aetiology of DED. As articulated by Goldstein and colleagues : “DED reAs has been established from the above, a large component of DED is inflammatory. Perhaps it is time we move beyond attempts to address only localized ocular inflammatory cascades and expand our search for a dry eye “cure” by consulting the chronic inflammatory disease literature. This review posits a case for a more holistic approach to combating signs and symptoms of DED.+ T [Modern lifestyle choices appear to be an important factor in many inflammatory diseases, particularly those with an autoimmune or metabolic component including obesity, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, eczema, allergy, and psoriasis ,49. Thes+ T . DED oftHence, if we view DED as a localized autoimmune disease originating from an imbalance in the protective immunoregulatory and proinflammatory pathways of the ocular surface  we can dPoor Western diets, typified by low fibre and high fat, have been shown to affect gut microbial composition and immune system functions and consequently trigger disease ,54,55. GLactobacillus reuteri which, in turn, exacerbated systemic autoimmunity [Perturbation of the gut microbiome illustrated by germ-free or antibiotic-treated mice has demonstrated the regulation of lymphocytic development and inflammatory responses in the gut ,60. The immunity . The autimmunity . Such stIn an effort to address aetiology of DED, our group is interested in exploring alternative treatments for dry eye that re-establish a healthy ocular microbiome both through topical and systemic means; constraining ocular and gut dysbiosis and cognate local or systemic inflammation through the use of functional foods and related bioactive substances.This article reviews a number of potential avenues of treatment for dry eye along the lines of functional foods and related products to work to effect healthier gut and ocular bacterial communities. Research in this arena in relation to dry eye is in its infancy but no doubt will be exploding in the next few years.For this narrative review, a literature search was performed between January and May 2019 using electronic databases PubMed and Google Scholar. Included studies were clinical trials and meta-analyses and reviews of such, on the oral supplementation of probiotics, prebiotics and supplements in the form of functional foods for the treatment or prevention of DED and related disorders. Exclusion criteria compromised articles for which there was no full text available, articles written in a language other than English and grey literature. For inclusion into the review, as this is yet an emerging field of research, we identified 27 articles specific to this search from the first round and additional references were identified from related studies cited within these articles.Ocular surface pain and discomfort are prominent symptoms of chronic dry eye and as such, often provide the impetus for presentation to a specialist . ConjuncBacillus spp. and Klebsiella oxytoca, as well as an overall increased bacterial count (CFU/swab) in participants with dry eye [Staphylococcus epidermidis was present in 100% of samples. Later works have suggested that S. epidermidis, as an integral member of epithelial microflora, rather than merely providing a benign presence, might actually exert a probiotic function by preventing colonization of more pathogenic bacteria [In 2007 Graham and colleagues  compared dry eye . Staphylbacteria .On the ocular surface, goblet cells are responsible for mucin production and hence reduction in these cells will reduce mucin production and disturb the healthy tear film. Graham et al.  showed iThere is precedence in the literature for the use of probiotic lysates, vitamins and omega-3 fatty acids to treat comorbid ocular, enteral and affective symptoms that comprise a disorder called “irritable eye syndrome” . The autPrebiotics were defined about twenty years ago as “a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and thus improves host health” . ThroughAntibiotics, minerals and vitamins can alter the gut bacterial composition but these are not considered as prebiotics. Prebiotics harbour distinct features. First, they are selectively utilised which means not utilised by the pathogenic microorganisms; second, evidence supports their beneficial effects on host; and third, the host enzymes should not degrade the compound .Clostridia and Escherichia coli [Prebiotics alter the composition of the gut microflora and studies have shown the modification of faecal microbiota by adding non-digestible energy sources in the diet . Clinicahia coli ,78,79. Enterobacteriaceae.Traditionally, non-digestive carbohydrates have been considered prebiotics and much research has focussed on three major groups; inulin, fructooligosaccharides (FOS) and galactooligosaccharides (GOS). According to the recent definition of prebiotics, particular non-carbohydrate compounds such as plant polyphenols may fit the prebiotic definition although more investigation needs to be done regarding this matter . FurtherIn the last decade, evidence of prebiotics effect on the host-microbial community and the benefits for health has gained traction ,82,83. BPlant oligosaccharides, flavonols and polyphenols confer numerous benefits including anti-oxidative and anti-inflammatory actions . Often tThe impact of prebiotics on human health and different diseases has been explored in a number of studies. The efficiency of galactooligosaccharide (GOS) has been reported in irritable bowel syndrome (IBS) . Also, IFimicutes/Baceroidetes ratio, can reduce endotoxmeia associated with low grade inflammation—a distinguishing feature of chronic conditions including insulin resistance, obesity, IBD, anxiety, mood and depression, RA and cancer [Faecalibacterium and Bacteroidetes. Additionally, omega-3 PUFAs can influence butyrate-producing genera such as Bifidobacterium, Lachno-spira, Roseburia and Lactobacillus, many of which are reported to be in dysbiosis in IBD subjects [Omega-3 fatty acids (FA) are essential polyunsaturated fatty acids obtained through diet. Short chain  fatty acids are found in walnuts, soy, flaxseed and linseed, while long chain [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) subtypes are derived from oily fish or produced in the body from desaturation/elongation of short-chain FAs . There id cancer . Extant subjects . Omega-3subjects . Animal subjects .Gut dysbiosis has been demonstrated in many inflammatory and autoimmune ocular diseases such as uveitis, which is an inflammatory condition of ocular tissues. A recent clinical study compared the gut microbiome of people with uveitis and control group, showing a reduction of gut microbiome diversity in people with uveitis. This reduction was shown to be more significant in anti-inflammatory bacterial strains .Sjögren syndrome (SS) is an autoimmune disease with a variety of ocular complications. This disease affects lacrimal glands, the primary source of production of watery layer of the tear film and triggers DED and other ocular surface disorders. The microbiome shifts in SS have been previously reported, with suggestions for both prebiotic and probiotic interventions .Apart from the extensive literature on omega-3 PUFAs in treatment of DED , there are few reports addressing the use of prebiotics for DED. However, in a recent review of diet and rosacea (which included ocular symptoms)  a prebioIn relation to dry eye, there has been robust investigation into the oral consumption of omega-3 fatty acids in the treatment of DED with mixed results initiated by the observation from the 32,000 participant Women Health study that drew an association between DED and low consumption of omega 3 PUFA . By way Turmeric, one of the most important herbal medicines in many Asian countries, is derived from Curcuma longa rhizome. Curcumin is a well characterized derivative of turmeric but fewer studies have explored the use of the more complex turmeric extract . TurmeriStaphylococcus aureus colonization with a concomitant increase in Staphylococcus epidermidis colonization of the ocular surface.Based on the idea of the eye as an ecosystem in which inflammation and autoimmunity are linked to the microbiota, Chisari and colleagues  recruiteThe patent literature cites use of Aloe Vera and a prebiotic, fructan derived from chicory root, for amelioration of neurological symptoms, chronic pain, inflammatory bowel disease and dry eye, amongst other disorders . InvestiUnique to milk are a structurally diverse group of oligosaccharides that rather than providing energy to the newborn, serve instead as prebiotics . Back inAs a member of the flavonol class of flavonoids, quercetin is well known for its anti-oxidant and anti-inflammatory actions. Quercetin is found in high abundance in apples, grapes, onions, artichoke, fennel, celery, beans, chickpeas, plum, turnips, peppers, strawberries, tomatoes and broccoli . HoweverAkkermansia spp., bifidobacteria and lactobacilli [Similar to quercetin and curcumin, resveratrol has been shown in mouse models to act as a prebiotic, leading to increased levels of obacilli . Topicalobacilli . Lactobacillus and Bifidobacterium with a few strains of other lactic acid bacteria, Bacillus spp., Saccharomyces spp. and E. coli [Roseburia, Eubacterium or Faecalibacterium spp. that might mitigate intestinal inflammation and enhance gut barrier defences [The term probiotic was introduced by Lilly and Stillwell in 1965  as an ag E. coli ,126,127.defences ,128. MarProbiotics and cognate products of metabolism effect equilibration of anti- and pro- inflammatory responses by various means . CommuniLactobacillus paracsei spp., paracasei, Bifidobacterium animalis ssp., Lactis and heat-treated Lactobacillus planatarum has been shown to improve immune function [Epithelial cells protect the host from assault by pathogenic organisms and toxic agents by forming a mucosal barrier. Probiotics help maintain intestinal barrier integrity by competing with pathogens for nutrients and competing for attachment sites on epithelial cells, inhibiting attachment of pathogenic bacteria , and modfunction . In colifunction . Variousfunction ,145 therL. rhamnosus also suppresses Th2, TH17 and ameliorates clinical signs relation to atopic dermatitis, allergic asthma and rhinitis [Various probiotic strains reduce Th17 inflammatory cells production and expression of downstream cytokines TNF-α and INFγ  L. rhamnrhinitis .In relation to ocular uses of probiotics, a comprehensive systematic review and meta-analysis of allergic rhinitis conducted in 2016 considered ocular symptoms in twenty-two randomised, double-blind, placebo-controlled studies and reported an overall positive effect of probiotics in reduction of ocular symptom scores in allergic rhinitis .Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae were more evident in healthy individuals compared with people suffering from bacterial keratitis. They also found more pathogenic fungus in BK groups. Their result strongly suggests the imbalance of gut bacterial community in people with BK [Recently, Jhaveri Microbiology Centre of Brien Holden Eye Research Centre in India has examined the association between gut bacterial and fungal dysbiosis and bacterial keratitis. They analysed the bacterial and fungal composition of faecal samples of 21 healthy participants versus 19 patients with bacterial keratitis (BK). Anti-inflammatory bacteria including  with BK  and indiLactobacillus casei, Lactobacillus acidophilus, Lactobacillus reuteri, Bifidobacterium bifidum and Streptococcus thermophiles) on management of autoimmune uveitis (EAU) and autoimmune dry eye in mice was explored. Monitoring inflammatory score of retinal tissues for uveitis and corneal staining and tear secretion for dry eye, the authors report that probiotics can be effective in decreasing clinical signs of autoimmune dry eye [+ Th1/Th17) along with cognate inflammatory mediators [Immune responses to commensal bacteria and opportunistic pathogens have been suggested as triggering Sjögren syndrome . In a re dry eye . Such da dry eye  and to p dry eye . It is sediators . As suchLactobacillus acidophilus, Streptococcus thermofhilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacteriumlactis, Zn, Vitamins B1, B2, B6 and niacin.In 2016, a group of researchers examined the effect of a combination of probiotics plus vitamins in people with DED. Their results suggest that synbiotics, which combine probiotics and prebiotics in a form of synergism, can decrease some signs and symptoms of DED while also modulating gut function . This grEnterococcus faecium WB2000 on dry eye reported significant improvement in clinical symptoms at 4 and 8 weeks [Another dry eye study examining the effect of a combination of fish oil, lactoferrin, zinc, vitamin C, lutein, vitamin E, γ-aminobutanoic acid and  8 weeks .Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus reuteri, Bifidobacterium bifidum and Streptococcus thermophiles) on autoimmune dry eye. IRT-5 was efficacious in reducing dry eye through attenuation of autoreactive T cells, suggesting as the authors report, relevance to treatment of Sjögren syndrome with IRT-5. The working theory behind this research suggesting the severity of Sjögren syndrome is correlated with microbial dysbiosis [+IFNγhi cells, leading to clinical improvement in SS.A 2017 report from Korea  exploredysbiosis , is thatEnterococcus faecium and Saccharomyces boulardii. Their results showed reduction in disease severity through improvements in Schirmer tear test and tear film break up time in addition to alteration of the ocular microbiome.In 2017, Chisari and colleagues  reportedOther studies have reported mixed results with use of probiotics with a recent systematic review reporting studies showing an increased risk of allergic conjunctivitis with administration of probiotics perinatally and other studies offering evidence for efficacy of probiotics .Many people who suffer from glaucoma and use associated medications complain of dry eye and have a higher prevalence of ocular surface disease than the general population ,157. TheHoney features as a remedy in ancient civilisations, ancient Ayurvedic texts and teachings of Aristotle and Hippocrates as well as ancient Arab physicians . Honey pAnthocyanins, known collectively as flavonoids (part of the greatly diverse plant polyphenols) are the largest group of water-soluble pigments in the plant kingdom with reported protective effects including antioxidant, anti-allergic, anti-inflammatory, anti-viral, anti-proliferative and anti-microbial . A 2017 Camellia sinensis) is one of the most widely consumed drinks worldwide. Green tea contains numerous polyphenols  including low molecular weight phenols (such catechins), which account for approximately 25% of the dry weight of green tea [Tea , including grape derived proanthocyanidins, monomeric flavonoid, aglycones and related glycosides , suggestOral evening primrose oil has been shown to reduce contact lens discomfort and associated dry eye . The mecS. aureus [Staphylococcal spp. that have been implicated in rosacea, blepharitis etc.) without disturbing the microbiota [Through the increased intake in the modern Western diet over the last 50 years of partially hydrogenated vegetable oils and trans fatty acids, such as linoleic acid, there has been a concomitant loss of dietary lauric acids (such as glycerol monolaurate or monolaurin) . This sh. aureus . In vitrcrobiota . It is ncrobiota . GML is used widely used in the food industry as a surfactant and preservative and this raises the possibility of ingested GML as a potential agent for reducing systemic inflammation, in addition to topical ocular application, given its good safety profile on skin and mucosal surfaces  and negaPrevious studies conducted in our laboratory explored the efficacy of GML on inhibition of production of bacterial lipase from ocular isolates ,183. FurA combination nutraceutical comprising fish proteins, rice carbohydrates, aloe Vera, papayam curcuma, green tea, omega3/6 fatty acids and other nturients has been used to treat dogs with keratoconjunctivitis sicca (KCS) . DeliverDevelopment of novel therapies that provide a cure for dry eye is the holy grail of DED research. In rising to meet this challenge, perhaps we should revisit the very questions we are asking about DED to stimulate alternative hypotheses of disease aetiology that might give rise to rigorous, consistent, repeatable clinical research methodologies, effecting easier comparison of data. In this way we might even begin to close the treatment gap between signs and symptoms.There is an emerging understanding of the efficacy of probiotics, prebiotics, synbiotics and other functional foods on gut and local ocular microbiota in modulation of ocular inflammation and ocular surface health and homeostasis. Just as antibiotics can have far reaching effects beyond purely antimicrobial efficacy, prebiotics, probiotics and functional foods can manifest their benefits in a number of ways. Re-envisioning the mechanism of action of these various agents which have featured strongly in the DED literature with often wildly differing outcomes, as agents that modify the microbiome, in addition to more direct effects, might focus clinical study design to tease out the various mechanisms of such agents and disambiguate extant data.If DED is indeed on the spectrum of chronic inflammatory disorders, lifestyle changes along with medications aimed to reduce inflammation could be an effective strategy. Adherence to lifestyle changes for chronic disease languishes around 10% while medication adherence is around 50% . As such


Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts  Förster resonance energy transfer (FRET) is an electromagnetic phenomenon, by which the energy of a light-excited fluorophore molecule (FRET donor) is transferred in a non-radiative way (by dipole–dipole coupling) to another molecule (FRET acceptor) located in very close proximity ,2,3. FREEFRET) depends on the orientation of donor and acceptor dipole moments (κ2), the quantum yield of the donor (Φ0), the extinction coefficient of the acceptor (Aε), the overlap integral between the normalized donor emission and acceptor excitation spectra (J), and, very importantly, the sixth root of the distance between donor and acceptor molecules (R) [n the refractive index of the medium, and λ the wavelength. R0 is the Förster radius (in nm), a distance specific to every donor–acceptor pair, at which an excited donor releases half of its energy by FRET (EFRET = 0.5).As described in the following three fundamental equations, FRET efficiency (ules (R) ,2,3,4: studies on spatiotemporal organization of cellular multiprotein assemblies using FRET as a molecular ruler, and (ii) analyses of biochemical and biophysical reactions in cells using FRET biosensors. We will also narrow our focus to FRET studies performed with two important unicellular model organisms: budding yeast To strengthen the application of FRET microscopy in yeast, we first provide an overview of FRET experiments performed to map yeast multiprotein assemblies in vivo. Second, we list FRET biosensors applied to study biochemical or biophysical events in yeast. Third, we describe suitable FRET techniques and fluorescent proteins for FRET microscopy in yeast, discussing their advantages and potential shortcomings. Finally, we provide several practical tips for successful FRET imaging of yeasts cells. We assume that also readers not directly working with yeast will find this review helpful, as many topics covered  are broadly applicable to FRET experiments with other uni- and multicellular organisms.The characterization of the nanoscale architecture of cellular supramolecular assemblies is critical for the understanding of their function and mechanism of action. Because of its unique spatial sensitivity, FRET has been repeatedly used to elucidate the organization of many crucial multiprotein complexes in yeast. In a general scheme of these experiments A, yeast The molecular architecture of several large multiprotein assemblies, which consist of dozens of subunits , has been systematically analyzed by FRET in yeast: the organization of the nuclear pore complex and its associated transport factors ,12, kineThese successful efforts illustrate the power of FRET-based mapping of protein assemblies in yeast. The main advantage is the simplicity by which yeast strains expressing FP-tagged pairs of complex subunits can be generated. The protein fusions can be made either N- or C-terminally, and very importantly, are usually expressed from endogenous loci, assuring near-native protein levels. Though this could be eventually reached in other cell types too , the following tests for functionality of constructed protein fusions are very straightforward in yeast haploid cells.FRET-based protein proximity mapping in yeast can also encounter some difficulties. Aside from constraints not directly related to FRET , the densely packed yeast cytoplasm and highly mobile intracellular membrane compartments could be disadvantageous for FRET mapping of certain protein complexes. Fluorescently tagged subunits of low abundance, or inversely, highly ubiquitous protein complexes, might give an insufficient signal-to-noise ratio of fluorescence signal, making their FRET analyses difficult. In addition, protein complexes of mobile membrane organelles might be too dynamic for reliable FRET measurements. Nevertheless, further developments of fluorescent microscopy tools might soon provide better spatiotemporal resolution for FRET analyses of these low-abundant or dynamic yeast protein complexes.Genetically-encoded FRET biosensors became popular tools for monitoring various molecular events in vivo soon after the first application of fluorescent proteins . Though Though development of FRET biosensors is often a complex endeavor , they ofCandida albicans homolog Amt1), and Yap1, respectively [ectively ,54,55. Tectively ,57.As mentioned in the previous section, FRET can be efficiently used to study medically related prion aggregation in yeast. In a pilot study, an elegant FRET biosensor (AmFRET) was designed to analyze nucleation kinetics of several yeast prion-like proteins . It consAside from tracing individual molecules, FRET biosensors can also monitor crucial cellular processes. Transcriptional activity of different RNA polymerase II promoters was followed by the expression of RNA aptamers (IMAGEtags), which mediate FRET of its specific Cy3/5-labeled ligands . ActivitFinally, the biophysics of several cellular processes has been studied by sophisticated FRET-based tension/force biosensors in yeast  of the fluorescence emitted by the donor/acceptor when the polarized light is used for its excitation. Techniques following fluorescence anisotropy are highly sensitive for FRET and offer fast readout (useful for high-throughput screens), but they are not quantitative. Importantly, they can be applied on a pool of molecules tagged with a single FP, where individual FP molecules work both as donor and acceptor because of an overlap between FP’s excitation and emission spectra. This special technique, called “homoFRET”, is often used to analyze clustering or homo-oligomerization of tagged molecules, as further detailed in .Finally, in the presence of a FRET acceptor, donor molecules stay shorter in their excited state , which is connected to the shorter lifetime of their fluorescence. This can be followed by sophisticated techniques of fluorescence lifetime imaging microscopy , which compare changes in donor fluorescence lifetime in the presence or absence of an acceptor D. FRET-FThe advantages and potential drawbacks of discussed FRET methods, as well as instrumental and analytical requirements, are summarized in The choice of FPs used as the FRET donor and acceptor is very important for a successful FRET experiment. As already mentioned, FRET depends on the quantum yield of the donor and the extinction coefficient of the acceptor, two key characteristics constituting the in vitro brightness value of FPs. In addition, a substantial overlap between the donor emission and acceptor excitation spectra is critical for FRET. Several other FP characteristics are, however, important for its in vivo performance, especially for FRET measurements ,86,87. Fwww.fpbase.org), a curated database of available FPs, which also provides an intuitive, user-adoptable FRET calculator [Advantageously, yeast biologists have already tested and implemented many FPs for yeast in vivo imaging studies. These efforts culminated in the recent comprehensive study that compared important characteristics of many advanced versions of FPs in yeast . Based olculator .Because of their good spectral overlap and advantageous match of their brightness, cyan fluorescent proteins (CFPs) and yellow fluorescent proteins (YFPs) have often been used for FRET analyses, not only in yeast see . This apTo avoid the interference with yeast cytosolic autofluorescence and to better visualize less abundant donor proteins, combinations of green and red fluorescent proteins (GFPs and RFPs) can be used as FRET donor–acceptor pairs. Though EGFP still has good fluorescent characteristics to serve as a bright FRET donor, mNG offers substantially higher brightness and better spectral overlap with RFP-based acceptors. Compared with many RFPs tested in yeast, mCherry and mScarlet-I  represenThe power of yeast genetics and available tagging techniques offer multiple ways to fuse a suitable FP to a protein of interest. These techniques allow endogenous tagging of proteins, either N- or C-terminally , in a fast and efficient way, often directly providing short linkers needed for the random orientation of the FP in the protein fusion. The full coverage of these techniques is beyond the scope of this review, so we list here only selected original works focused on FP tagging ,97,98,99We conclude with several practical tips regarding cell and microscope preparation for FRET imaging in yeast. As autofluorescence of yeast cytosol and vacuoles can seriously interfere with the fluorescence signals of especially CFPs and RFPs, respectively, we suggest minimizing it by using a low-fluorescence minimal medium  and working with freshly grown early logarithmic cells. Also, movement of cells during imaging should be prevented by their stable attachment to the microscopy slide . For extensive FRET experiments, it is also worth optimizing the optical configuration of the microscope (used excitation or emission filters and dichroic mirrors) to achieve the highest performance of your chosen FRET pair . Finally, considering that in vivo FRET signal often constitutes only a small percentage of the total registered fluorescence, a careful control of its specificity is always desirable. This is imperative especially when the FRET donor and acceptor are two molecules of different abundance or mobility, as this can influence apparent FRET values (discussed in more detail in ). All neThe outstanding ability to sensitively and non-invasively analyze molecular proximities and reactions directly in living cells has made FRET microscopy an invaluable tool for modern biomedical research. The importance and application of FRET in yeast will probably be ever-increasing, especially if it is coupled to current single molecule localization microscopy approaches , to whic


Impulse control behaviors (ICB) are recognized as non-motor complications of dopaminergic medications in patients with Parkinson's disease (PD). Compelling evidence suggests that ICB are not merely due to the PD-related pathology itself. Several risk factors have been identified, either demographic, clinical, genetic or neuropsychological. Neuroimaging studies have yielded controversial results regarding ICB correlates in PD and still it is not clear whether they can be triggered by the PD biology or the dopaminergic treatment stimulation. We provided an overview of the imaging studies that offered the most relevant insights into the debate about the role of drugs and disease in ICB pathophysiology. Understanding neural correlates and potential predisposing factors of these severe neuropsychiatric symptoms will be crucial to guide clinical practice and to foster preventive strategies. Impulse control behaviors (ICB) are neuropsychiatric symptoms characterized by impulsive acts, which are performed compulsively and are potentially detrimental to the person itself or others, severely affecting subjects' quality of life . ICB areInterestingly, ICB also occur in patients with restless legs syndrome , 8 or prModern neuroimaging techniques have been widely tested to support PD diagnosis  as well as to provide further insights into motor and non-motor symptoms pathophysiology, complications and treatment-related effects  .PET and SPECT studies have been extensively applied to analyze neural correlates underpinning ICB in PD –14. By mStructural MRI changes have been also observed in PD patients with ICB. Gray matter atrophy as well as corticometric changes across several brain areas involved in behavioral modulation  are the most frequent findings related to the ICB presence and severity in PD , 16. HowFunctional MRI studies –21 were The DMN encompasses mainly precuneus and posterior cingulate, bilateral inferior-lateral-parietal and ventromedial frontal cortices. It is involved in cognitive processing and mind-wandering and becomes deactivated during specific goal-directed behaviors. The CEN is involved in executive control and decision-making and operates through medio-frontal areas, including anterior cingulate and para-cingulate cortices. The SN is a limbic-paralimbic network that plays an important role in orienting attention toward salient stimuli and facilitating goal-directed behaviors, reward processing and interoceptive awareness . It encoOverall, most imaging studies applied cross-sectional designs, yielding controversial results. Thus, it is not possible to rule out whether these findings reflect the effect of chronic dopaminergic treatment or represent a neural pattern predisposing to ICB .Here, we aim to review the most relevant imaging studies that provide a contribution to the debate about the role of drugs and disease in the pathogenesis of ICB in PD.n = 984), excluding duplicates and articles judged irrelevant by title and abstract screening. The same raters performed the quality check of selected studies and the most relevant ones for the topic were finally included in the review  evaluated the results  density has been reliably reported in PD-ICB patients compared to PD patients without ICB , 28. ThiIn a functional MRI (fMRI) study, patients with PD and hypersexuality exposed to sexual cues had higher activity within the SN compared to patients with PD without ICB . MoreoveIn summary, different neuroimaging techniques have been used to analyze the integrity of striatal and extra-striatal dopaminergic pathways in PD patients with and without ICB. The presence of a specific “hyperdopaminergic” state in the brain of patients experiencing ICB have been consistently highlighted. An important limitation is that these studies have mainly enrolled PD patients with a long history of PD as well as ICB, which may both influence the reward and impulse-control pathways themselves. Indeed, after ICB emergence, progressive neuroplasticity processes involving mainly dopaminergic circuitries may occur, eventually leading to consolidation of pathological habits . Thus, eDopaminergic medications can influence rewarding processing, by enhancing learning from positive feedback and impairing learning from negative feedback , 40. MorReward processing changes after dopaminergic drugs administration has been studied in healthy subjects as well as in patients with restless legs syndrome in order to describe pharmacological effects not biased from neurodegenerative pathology. Pessiglione et al.  performeAlong with levodopa, the effect of DAA treatment on the reward processing was tested in both healthy and non-healthy subjects as well. A double-blind study compared results from a probabilistic reward task performed after either a single low dose of pramipexole (0.5 mg) or placebo , revealiThese results parallel with several evidence coming from PD patients with ICB. A task-related and pharmacological fMRI study performed before and after DAA treatment, showed that PD-ICB patients under DAA present enhanced sensitivity to risk compared to PD patients without ICB in the same experimental condition . DAA intHowever, it should be noted that PD results from the degeneration of dopaminergic projections involved in the reward processing itself. By contrast, dysfunctions within the reward system are difficult to study in PD as most patients are treated with dopaminergic drugs. In this context, a fMRI task-related paradigm was used in a small group of drug-naïve PD patients performing a simple two-choice gambling task . In thisIn summary, even in the absence of manifest ICB symptoms as well as PD pathology, chronic dopaminergic medication was shown to severely impair the reward processing. Although limited, neuroimaging evidence of altered reward-processing in PD patients even in the absence of DAA treatment have been provided.To date, only a few studies have been designed to find potential neuroimaging biomarkers able to predict future development of ICB in PD. This is crucial, as previous studies did not allow to disentangle the complex interplay between drugs and disease in ICB pathophysiology. Vriend et al.  performeMore recently, resting-state fMRI was used to analyze the intrinsic functional connectivity within and between the major neurocognitive networks in a cohort of drug-naïve PD patients that developed ICB (ICB+) after treatment initiation compared with PD patients who did not (ICB–) . In physIn summary, longitudinal neuroimaging studies on premorbid ICB population are limited. However, they support the hypothesis that a pre-existing vulnerability to ICB development may be present in a specific subset of PD patients, likely related to PD-pathology, involving both dopaminergic signaling and reward processing, which are in turn affected by dopaminergic medications.The relationship between PD pathology, DAA treatment and ICB development is complex. Neuroimaging studies have provided crucial insights to support the presence of increased dopaminergic firing in response to reward stimuli in the cortico-striato-cortical pathway in PD patients more prone to develop ICB. Dopaminergic treatment exposure may overdrive this pathway and also induce further dopamine receptors changes, leading to the development of such behavioral disturbances. Future multimodal imaging studies able to look at several aspects of the dopaminergic cortical and subcortical signaling, as well as prospective longitudinal designs, will allow to disentangle how drugs and disease may interplay to trigger these relevant neuropsychiatric symptoms. Understanding neural correlates and potential predisposing factors of these severe behavioral symptoms will be crucial to guide clinical practice and to foster preventive strategies.RD study concept and design, acquisition of data, drafting the article. AT drafting the article and revising it critically for intellectual content. AR critically revision of manuscript for intellectual content. GT critically revision of manuscript for intellectual content.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.


Orbivirus , Dianthovirus  and Alphainfluenzavirus . Despite their distinct genomic features and diverse host ranges  each of these viruses uses trans-acting RNA–RNA interactions (tRRIs) to facilitate co-packaging of their segmented genome. The tRRIs occur between different viral genome segments and direct the selective packaging of a complete genome complement. Here we explore the current state of understanding of tRRI-mediated co-packaging in the abovementioned viruses and examine other known and potential functions for this class of RNA–RNA interaction.RNA viruses represent a large and important group of pathogens that infect a broad range of hosts. Segmented RNA viruses are a subclass of this group that encode their genomes in two or more molecules and package all of their RNA segments in a single virus particle. These divided genomes come in different forms, including double-stranded RNA, coding-sense single-stranded RNA, and noncoding single-stranded RNA. Genera that possess these genome types include, respectively,  RNA viruses are important pathogens that infect a wide variety of hosts. The RNA genomes of these viruses can exist in different forms . Those ttrans-acting RNA–RNA interactions (tRRIs), could also facilitate co-packaging. Indeed, the latter strategy appears to be involved in this process for a number of segmented RNA viruses . B. B37]. BCollectively, these results provide a solid foundation for identifying additional BTV interactions and exploring the assembly pathways involved. Future studies will need to integrate all of the existing validated tRRIs into a unified model. Currently, most of the larger segments have not been analyzed extensively, thus their proposed roles in later stages of tRRI network assembly warrant further investigation. Also, it remains to be determined whether accurate assembly requires a tRRI network that includes all ten segments and, if so, how is a complete ensemble of ten “sensed” by the virus.Rotavirus is in the same Sedoreovirinae subfamily as orbiviruses, but these viruses contain eleven dsRNA genome segments. Similar to BTV, in vitro studies on rotaviruses (RVs) support the involvement of mRNA tRRI networks in segment packaging [The genus ackaging ,40, howeackaging . In vitrackaging . It has ackaging . VP6 of ackaging . InsteadTombusviridae is a large plant virus family whose members contain (+) RNA genomes [Dianthovirus. Red clover necrotic mosaic virus (RCNMV) is the best characterized species in this genus [ genomes . All memis genus . Its genis genus . RNA2 coAlthough RNA1 is capable of genome replication on its own, the transcription of the sg mRNA during infections requires the presence of both RNA1 and RNA2, signifying a communication requirement between the viral genome segments . Furthertrans-activator (TA). Base pairing of this TA loop with its complementary partner sequence in RNA1, the trans-activator binding site (TABS), forms a higher-order bimolecular RNA structure that halts progression of the RdRp , which can affect both RNA-protein and RNA–RNA interactions . FinallyA variety of approaches including computational, biochemical, genetic, and cellular techniques have been used to identify and study the functions of viral tRRIs. The initial identification of putative interacting genome segments and corresponding complementary sequences involved computer-assisted RNA structural analyses , RNA–RNAThe tRRIs that form in different viruses are predicted to be disrupted at different stages after assortment. In BTV, inter-mRNA links are presumably lost when the messages are converted to dsRNA genomes during virion assembly, making them relatively transient. In contrast, tRRIs in IAV are maintained in virions and may even persist following fusion-mediated delivery into the cytosol of newly infected cells, possibly mediating codelivery of vRNPs to the nucleus . For RCNThe presence of tRRIs in coding regions is also relevant to experiments that assess virus protein function by modifying its corresponding coding sequence, as substitutions or deletions can concomitantly disrupt network connections. The same knowledge may also be pertinent to certain applications, such as optimizing the design of expression vectors by integrating appropriate partner tRRIs with introduced foreign genes. As one would expect, tRRIs also influence the efficiently of reassortment, as demonstrated experimentally for IAV . LikewisThe study of tRRIs in segmented RNA viruses is a new and growing field in virology. Understanding the roles of this class of interaction will provide important basic knowledge on how selective packaging occurs in segmented RNA viruses. Information on tRRIs will also be useful in assessing the likelihood of new viral strains emerging via reassortment, and could guide the development of antiviral treatments based on inhibition of these interactions. Moreover, as observed for RCNMV, the possibility exists that the functions for tRRIs extend beyond their established role in genome assortment. As the field expands, it will be interesting to see if novel functional tRRIs are active in other types of segmented RNA viruses.


Plants accumulate and tolerate Se to varying degrees, up to 15,000 mg Se/kg dry weight for Se hyperaccumulators. Plant Se accumulation may exert positive or negative effects on other species in the community. The movement of plant Se into ecological partners may benefit them at low concentrations, but cause toxicity at high concentrations. Thus, Se accumulation can protect plants against Se-sensitive herbivores and pathogens  and reduce surrounding vegetation cover via high-Se litter deposition . While hyperaccumulators negatively impact Se-sensitive ecological partners, they offer a niche for Se-tolerant partners, including beneficial microbial and pollinator symbionts as well as detrimental herbivores, pathogens, and competing plant species. These ecological effects of plant Se accumulation may facilitate the evolution of Se resistance in symbionts. Conversely, Se hyperaccumulation may evolve driven by increasing Se resistance in herbivores, pathogens, or plant neighbors; Se resistance also evolves in mutualist symbionts, minimizing the plant’s ecological cost. Interesting topics to address in future research are whether the ecological impacts of plant Se accumulation may affect species composition across trophic levels (favoring Se resistant taxa), and to what extent Se hyperaccumulators form a portal for Se into the local food chain and are important for Se cycling in the local ecosystem. Mammals and many other animals, as well as many prokaryotes and some algae, require Se for their essential metabolism, as a structural component of selenoproteins , which hSelenium is toxic to organisms at elevated tissue levels, firstly because inorganic forms of Se can cause oxidative stress, and secondly because the organic selenocompounds selenocysteine (SeCys) and selenomethionine (SeMet) can get non-specifically incorporated into proteins, replacing their sulfur (S) analogues, which can compromise protein function . For aniIn plants, Se typically stimulates growth and stress resistance at 1–10 mg Se/kg DW , and theAll plants readily take up and accumulate Se in all their tissues, quite similar to their utilization of the analogous element S . SelenatWhen plants grow in a Se-containing environment, they bioconcentrate the locally available Se in their tissues and, depending on the species, also biotransform the inorganic Se to organic Se to a varying degree . This acThere is broad evidence  from fieStanleya pinnata (Brassicaceae) pretreated with or without Se were planted in a naturally seleniferous habitat in Colorado, USA, and herbivory and survival were scored. The plants pretreated with Se (containing 50 mg Se/kg DW) survived better and suffered less herbivory, both in the course of several months when grasshoppers were the predominant herbivore [Most studies on the effects of Se on plant–herbivore interactions were carried out in a controlled laboratory setting, in the form of choice and non-choice feeding experiments. In addition, a few field manipulative studies were carried out in which Se hyperaccumulator erbivore , as wellerbivore .The finding that Se at low concentrations (< 10 mg/kg DW) is already protective against herbivory and that the protective effect is correlated with Se concentration provides insight into the evolution of Se (hyper)accumulators from nonaccumulators, and the likely selection pressures that continuously favor increasing Se levels . At someS. pinnata and Astragalus bisulcatus (Fabaceae) collected in their native seleniferous habitat both were found to harbor different beetle, wasp, or chalcid herbivores [(Plutella xylostella) variety found on S. pinnata in its natural seleniferous habitat in Colorado (USA) with a diamondback moth variety from a low-Se area, the CO diamondback moth appears to have lost its aversion to feed or oviposit on high-Se plants, as well as its capacity to break down methyl-SeCys to more toxic forms of Se [The finding that Se-containing plants deter generalist herbivores and are toxic when consumed also offers insight into the potential ecological effects of high-Se plants in the field, either in natural settings or when cultivated on seleniferous soils. In agricultural settings, the protective effects of Se likely will reduce the need for pesticide application. In natural settings, the high-Se plants may be expected to affect local herbivore abundance and composition and may change herbivore load on local nonaccumulator plant species. On the one hand, the volatile Se produced by high-Se plants may deter herbivores across a certain area of influence that may also benefit neighbors, but on the other hand, the herbivores deterred from high-Se plants may pose extra herbivory pressure on low-Se neighbors. A field survey comparing invertebrate load and composition between two Se hyperaccumulator and two non-accumulator species growing in the same seleniferous area found overall lower herbivore load on the hyperaccumulators as well as lower and altered species diversity . This firbivores . While trbivores ,38. The rbivores ,37. BaseIn summary, these collective plant–herbivore interaction studies indicate that Se accumulation in plants negatively affects generalist herbivores (via deterrence and toxicity), thereby protecting the plants, but also favoring the evolution of Se resistance in herbivores, which as a result can exclusively utilize high-Se plants as a food source . PotentiS. pinnata) and non-hyperaccumulator  plants with leaf and floral Se concentrations up to 4000 mg/kg DW were visited to equal extent as their non-Se treated counterparts by potential pollinators in the field in Colorado, which included both native bumble bees and non-native honey bees [Plants accumulate Se in all their organs, including flowers. Hyperaccumulators even contain the highest Se concentrations in their flowers and fruits, including the pollen, nectar, and seeds . Consideney bees . Similarney bees . Bumble ney bees . TherefoAn important question that still awaits further study is whether and how the collected plant Se affects the health of the pollinators. From feeding studies with Se-spiked sugar water, it is clear that inorganic and organic Se are toxic to honey bees at the levels found in high-Se plants . There mIn summary, plant–pollinator interaction studies so far indicate that Se accumulation in plants does not deter pollinators and does not affect pollinator visitation . WhetherThe plant microbiome consists of prokaryotic and fungal symbionts that live inside the plant (endosphere), or on or near the plant’s shoot or root surface , where they benefit from the plant’s release of organic carbon compounds . Many ofB. juncea from two fungal pathogens, one a leaf fungus (Alternaria brassicicola) and the other a stem/root fungus (Fusarium sp.). Indeed, these fungi showed sensitivity to Se levels like those found in the plants when grown on medium spiked with Se. Thus, Se may protect plants from generalist Se-sensitive fungal pathogens. However, there are leaf fungal pathogens in seleniferous areas that can utilize Se hyperaccumulators as a host: an Albugo species is regularly observed on leaves of wild S. pinnata containing 2000 mg Se/kg DW in Colorado, USA .Hanson and coworkers  showed tA. bisulcatus and S. pinnata contained up to 30% elemental Se (Se0) in their natural habitat, but only accumulated organic Se when grown from surface-sterilized seed in a greenhouse [A. bisulcatus root nodules harboring a N2 fixing bacterial endosymbiont was Se0, while the adjacent roots accumulated only organic Se [Astragalus species showed no significant differences in nodulation between the groups. Thus, there is no evidence for a cost of Se hyperaccumulation in Astragalus through negative effects on nitrogen fixing endosymbionts. It is possible that hyperaccumulator-associated Rhizobia have evolved enhanced Se resistance; their isolation and characterization will be interesting to pursue in future studies.A study by Wangeline and coworkers  found theenhouse . Similarganic Se . The intganic Se ,49, who Sura-de Jong et al.  and CochProkaryotic isolates from the endosphere and rhizosphere of hyperaccumulators, or from various other environmental sources have also been studied for their effects on plant growth and Se accumulation. Enhanced plant growth and Se accumulation was found after bacterial inoculation in many different studies using host and non-host species, hyperaccumulators, and non-hyperaccumulators, and wild and crop species ,55,56,57In summary, there is no evidence that high plant Se levels have an evolutionary cost by negatively affecting the plant’s beneficial symbiotic interactions with fungi or prokaryotes. However, there is evidence that Se can protect plants from certain pathogenic fungi. Prokaryotes generally appear to be highly Se resistant, and thus likely will not suffer toxicity at the Se levels found in plants. Nevertheless, Se accumulation by plants appears to affect the composition of the plant microbiome. While this likely does not reflect differences in Se tolerance, it is possible that some microbes can benefit more from the plant-associated Se than other ones. Thus, Se may affect competitive interactions and microbial fitness. Microbial species richness may also be positively affected by plant Se accumulation, because it creates more variation (heterogeneity) and thus more niches to occupy. There is ample evidence that fungi and bacteria can positively affect plant growth and Se accumulation and volatilization, and these effects are not restricted to their natural host. This may have applications in Se biofortification  and phytoremediation  ,58. ThisThere is evidence that high-Se plants can enhance the tissue Se levels in neighboring plants in seleniferous natural areas: different individuals of the same species showed up to 20-fold higher tissue Se concentrations when they were growing in close proximity to hyperaccumulators ,60. The The high-Se soil surrounding hyperaccumulators and concomitant increase in tissue Se concentration in neighboring plants may affect these neighbors differently, depending on their Se tolerance. To Se-sensitive plant species  forms. These practices may benefit Se phytoremediation, but also may create Se biofortified plant products that may help alleviate Se deficiency in humans or livestock elsewhere.The aim of this section is to summarize and interpret the various ecological implications that Se (hyper)accumulation may have on the plant itself as well as the surrounding local ecosystem. There are some general trends emerging from the various studies on the ecological effects of plant Se accumulation, which were already noted in our earlier review in 2012 , and havThe presence at different trophic levels of Se-tolerant species that can accumulate Se to substantial concentrations likely contributes to the movement of Se in the local food chain, and ultimately to Se cycling in the local ecosystem. The Se hyperaccumulator plants form the entry point into this movement, and may be an important factor. At this point, not very much is known about the relative contribution of terrestrial vegetation in global Se cycling . This sh


Heart failure is a progressive deterioration of cardiac pump function over time and is often a manifestation of ischemic injury caused by myocardial infarction (MI). Post-MI, structural remodeling of the infarcted myocardium ensues. Dysregulation of extracellular matrix (ECM) homeostasis is a hallmark of structural cardiac remodeling and is largely driven by cardiac fibroblast activation. While initially adaptive, structural cardiac remodeling leads to irreversible heart failure due to the progressive loss of cardiac function. Loss of pump function is associated with myocardial fibrosis, wall thinning, and left ventricular (LV) dilatation. Surgical revascularization of the damaged myocardium via coronary artery bypass graft (CABG) surgery and/or percutaneous coronary intervention (PCI) can enhance myocardial perfusion and is beneficial. However, these interventions alone are unable to prevent progressive fibrotic remodeling and loss of heart function that leads to clinical end-stage heart failure. Acellular biologic ECM scaffolds can be surgically implanted onto injured myocardial regions during open-heart surgery as an adjunct therapy to surgical revascularization. This presents a novel therapeutic approach to alter maladaptive remodeling and promote functional recovery. Acellular ECM bioscaffolds have been shown to provide passive structural support to the damaged myocardium and also to act as a dynamic bioactive reservoir capable of promoting endogenous mechanisms of tissue repair, such as vasculogenesis. The composition and structure of xenogenic acellular ECM bioscaffolds are determined by the physiological requirements of the tissue from which they are derived. The capacity of different tissue-derived acellular bioscaffolds to attenuate cardiac remodeling and restore ECM homeostasis after injury may depend on such properties. Accordingly, the search and discovery of an optimal ECM bioscaffold for use in cardiac repair is warranted and may be facilitated by comparing bioscaffolds. This review will provide a summary of the acellular ECM bioscaffolds currently available for use in cardiac surgery with a focus on how they attenuate cardiac remodeling by providing the necessary environmental cues to promote endogenous mechanisms of tissue repair. According to the American Heart Association (AHA), the prevalence of congestive heart failure (CHF) is expected to increase by 46% from 2012 to 2030 with a staggering 960,000 new CHF cases reported annually . CHF is There are a plethora of pharmacological interventions, including antianginal medications such as beta blockers, which may be employed to improve the symptoms and prognosis of heart failure patients. However, pharmacotherapy alone is unable to elicit functional recovery of the infarcted myocardium . RevascuIn the past, the field of cardiovascular tissue engineering has focused on leveraging a stem cell-based or gene therapy-based approach as a solution to restoring cardiac function post-MI. However, the road to clinical translation of these approaches has largely been fraught with challenges, specifically uncertainty regarding clinical efficacy and feasibility . Acellulin-vitro  inflammatory stage, (2) proliferative stage, and (3) maturation stage . Initialin-vivo, and plays a central role in infarct expansion  and PhotoFixTM is cross-linked using dye-mediated photo-oxidation  the tissue source from which they are derived, and (2) the fixation method, or lack thereof, implemented in processing the ECM bioscaffold . Acellulxidation . Furtherxidation .® - SIS-ECM  for cardiac repair  and in-vivo using a rat MI model   .® highlights the therapeutic potential of CorMatrix®, but also underscores the need for further elucidation of its specific surgical indications , found no detectable calcification of the bioscaffold in patients at reoperation, follow-up imaging, or medium-term histological analysis after explanation  where acellular ECM bioscaffold therapy, CorMatrix®, is surgically implanted at the time of CABG surgery.A recent systematic review of the initial preclinical and clinical findings of CorMatrixications . For inslanation . As calc epitope . It also epitope . Extracaitope  ECM bioscaffolds have a wide variety of uses in cardiac surgery, including but not limited to pericardial closure, valve repair and replacement, and septal reconstruction. Glutaraldehyde cross-linked BP-ECM bioscaffolds, such as Peri-GuardTM , found that at follow-up 3.2 ± 1.6 years post-implantation there were no deaths related to PhotoFixTM bioscaffold failure  clinical trial (NCT03669042). While this study is powered to assess outcomes in patients undergoing vascular repair or reconstruction surgery, it may provide additional insight into the use of PhotoFixTM as an adjunct therapy to the surgical revascularization of infarcted myocardium post MI.A variety of alternative strategies have emerged to neutralize or remove glutaraldehyde from cross-linked BP-ECM bioscaffolds, or to replace the use of glutaraldehyde entirely. PhotoFixp models . In addiantation . Recentl failure . Moreovefication . Current® , is a BP-ECM bioscaffold that is cross-linked using a proprietary fixation technique known as ADAPT®    . Notablysitions) . Given i®  exhibits greater stiffness or less elasticity compared to CorMatrix®, PhotoFixTM, and CardioCel®  , of an acellular ECM bioscaffold for use as an adjunct to CABG remain unknown. For example, Peri-Guardctively) . Further®, our group has shown its bioinductive properties are responsible for stimulating endogenous mechanisms of vasculogenesis and functional recovery post-MI (® has been shown to improve contractility and exhibit positive electrical conductivity in a porcine model of MI sixty-days after surgical implantation (Additionally, the influence of acellular ECM bioscaffold therapy on the electrophysiology of the heart must also be considered. While the specific details are beyond the scope of this review, cardiac fibrosis is known to potentiate cardiac arrhythmia . By redu post-MI , 2017. Fantation . Clearlyin-situ. Alternatively, as the placenta is a highly vascularized structure, it may be that an acellular ECM bioscaffold derived from placental tissue holds the necessary bioinductive cues to promote neovascularization and integration of the bioscaffold into the native cardiac ECM microenvironment itself. Overall, as the composition and structure of ECM bioscaffolds are a function of the physiological requirements of the tissue from which they are derived, future work should consider the therapeutic potential of these alternative tissue-sources in developing acellular ECM bioscaffolds for use in cardiac surgery.Beyond those discussed above, there are many other acellular ECM bioscaffolds used or in development for use not only in cardiac surgery, but across a variety of other surgical applications . In addiUltimately, our research group is interested in investigating the use of acellular ECM bioscaffolds to influence and direct the local cardiac ECM microenvironment at the site of MI. The goal is to leverage this innovative technology to drive endogenous mechanisms of repair, such as angiogenesis or vasculogenesis, and to attenuate the activation of cardiac myofibroblasts. In doing so, the maladaptive structural cardiac remodeling that ensues following ischemic injury to the heart may be better managed. There is a gap in knowledge regarding the application of currently available acellular ECM bioscaffolds as an adjunct at the time of CABG surgery. Similarly, little is known about the ability of these bioscaffolds to influence the dysregulated ECM microenvironment of the damaged myocardium, possibly by way of retained bioactive properties and/or mechanical support. Therefore, there is an opportunity to characterize the potential therapeutic benefits of acellular ECM bioscaffold-based infarct repair.in-vitro testing. These studies should be complemented with well-designed pre-clinical investigations, which can be used to assess specific indications of ECM bioscaffolds. Finally, there is a need for larger scale prospective clinical trials to evaluate the possible therapeutic benefits of ECM bioscaffolds.We describe the varied clinical outcomes of currently available acellular ECM bioscaffolds in various cardiac surgery applications. We believe that initial investigations of the utility of acellular ECM bioscaffolds for infarct repair should begin with rigorous A unique opportunity exists to leverage acellular ECM bioscaffolds for infarct repair. By implementing ECM bioscaffold therapy to modulate the local ECM microenvironment at the site of an MI, we could prevent maladaptive structural cardiac remodeling that often leads to irreversible heart failure. Future investigations should focus on the ability of these bioscaffolds to drive endogenous mechanisms of repair and to attenuate cardiac fibrosis. An ideal acellular ECM bioscaffold for infarct repair will be able to elicit functional recovery of the damaged myocardium. Acellular ECM bioscaffolds could have a significant clinical benefit and improve the prognosis of patients suffering from cardiac fibrosis and heart failure.All authors designed, drafted, and revised the manuscript.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.


Dopamine transporter (DAT) single-photon emission tomography (SPECT) with (123)Ioflupane is a widely used diagnostic tool for patients with suspected parkinsonian syndromes, as it assists with differentiating between Parkinson’s disease (PD) or atypical parkinsonisms and conditions without a presynaptic dopaminergic deficit such as essential tremor, vascular and drug-induced parkinsonisms. Recent evidence supports its utility as in vivo proof of degenerative parkinsonisms, and DAT imaging has been proposed as a potential surrogate marker for dopaminergic nigrostriatal neurons. However, the interpretation of DAT-SPECT imaging may be challenged by several factors including the loss of DAT receptor density with age and the effect of certain drugs on dopamine uptake. Furthermore, a clear, direct relationship between nigral loss and DAT decrease has been controversial so far. Striatal DAT uptake could reflect nigral neuronal loss once the loss exceeds 50%. Indeed, reduction of DAT binding seems to be already present in the prodromal stage of PD, suggesting both an early synaptic dysfunction and the activation of compensatory changes to delay the onset of symptoms. Despite a weak correlation with PD severity and progression, quantitative measurements of DAT binding at baseline could be used to predict the emergence of late-disease motor fluctuations and dyskinesias. This review addresses the possibilities and limitations of DAT-SPECT in PD and, focusing specifically on regulatory changes of DAT in surviving DA neurons, we investigate its role in diagnosis and its prognostic value for motor complications as disease progresses. Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized primarily by the selective degeneration of dopaminergic neurons in the pars compacta of the substantia nigra (SN). Intraneuronal inclusions composed of aggregates of a-synuclein (α-syn), called Lewy bodies (LBs), are the other neuropathological hallmark . Loss ofSome attempts have been performed to use DAT-SCAN for a differential diagnosis between PD and atypical parkinsonisms, such as an evaluation of the asymmetric index and the caudate/putamen ratio, with no significant success in clinical practice ,40,41. IDAT activity is under the control of different presynaptic proteins, including DA autoreceptors, that can promote both trafficking and the availability of the transporter at the plasma membrane  Figure . In animSelective serotonin reuptake inhibitors (SSRIs) are the mainstay treatment for major depressive disorder, and their effect on DAT is not fully understood yet . CitalopThe density of DAT on presynaptic terminals is considered a surrogate marker for dopaminergic nigral cell counts and vitality, but a clear, direct relationship between nigral loss and DAT decrease has, so far, been controversial. Reduced DAT striatal availability parallels loss of striatal DA and loss of nigrostriatal fibers in monkeys chronically treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) . RecentlDirect visualization of the SN is one of the unmet needs in neuroimaging of parkinsonisms, with controversial results obtained from the various attempted approaches . By usinThese observations could also have implications in the potential detection of the premotor phase of PD in order to evaluate at-risk subjects. In this respect, carriers of dominant PD genes, such as leucine-rich repeat kinase 2 (LRRK2) mutations, or subjects with REM sleep behavior disorder (RBD) are ideal candidates to evaluate the detection of preclinical/premotor PD by means of nuclear medicine or MRI. The potential role of DAT imaging has been demonstrated ,91,92,93Emerging interest has pointed towards the mutual relationship between SN MRI anatomical changes and the assessment of dopaminergic nigrostriatal functions, as assessed by DAT imaging. A good agreement between SN-MRI abnormalities and [123I]FP-CIT-SPECT has been reported . HoweverAt the molecular level, loss of dopaminergic axonal terminals seems to precede SN cell body loss . SpecifiNeuronal loss in the SN, with or without LBs, occurs with normal aging in a spectrum that spans from age-associated brain changes to structural alterations of neurodegenerative diseases like PD . In geneSimilarly, Lewy pathology has been found at autopsy in the brains of elderly individuals without clinical PD in association with intermediate SN neuron loss between PD cases and controls, supporting the concept that subjects with incidental LB pathology represent preclinical PD . Indeed,Redundancy in basal ganglia functions and mechanisms of functional compensation could serve to delay the clinical onset of PD motor signs during early stages. From this point of view, a late appearance of parkinsonian symptoms would be due to failure of compensatory mechanisms to maintain dopaminergic control over striatal cell function . It is pMore recently, adaptive changes in other neural structures than the nigrostriatal pathway, such as the subthalamic nucleus and the globus pallidus pars internalis, have been invoked to play a central role in the compensatory mechanisms that sustain dopaminergic functions in the early phases of PD . It is pIndeed, presynaptic and postsynaptic mechanisms have also been observed to persist in more advanced stages of the disease and have been predicted to promote the development of motor complications as the disease progresses . SpecifiSeveral longitudinal observations indicate that the magnitude of DAT binding in early PD, especially in the posterior putamen, could be predictive for the development of dyskinesias  and motoIf the loss of DA storage due to presynaptic dopamine neuronal degeneration is preferentially associated with the development of motor fluctuations, dopamine neurons in the anterior putamen could have a greater role in storing DA . HoweverSimilarly, the relationship between DAT binding and PD progression remains unclear. Bearing in mind that reduced DAT expression might indicate neuronal and axonal dysfunction without alterations in dopaminergic neuronal counts, several authors investigated whether the baseline dopaminergic striatal uptake could predict the development of clinically important long-term motor and nonmotor outcomes. Ravina and colleagues reported that lower [123I]b-CIT SPECT at baseline is independently associated with motor-related disability as well as with nonmotor features such as cognitive impairment, psychosis, and depression . Accordi11C-PE2, a highly specific DAT radioligand suitable for PET imaging, as a potential alternative surrogate biomarker for studies of PD progression for its ability to track changes in motor performance over time in 33 PD subjects [The emergence of new DAT tracers promises to result in the development of powerful markers for PD. Furthermore, it is noteworthy to mention that SPECT imaging is limited by poor spatial and temporal resolution and has a worse quantitative capacity than PET. Recently, Li et al. identified subjects . Overallsubjects  and bodysubjects  have alsDAT Imaging is a reliable tool for investigating the presynaptic dopaminergic nigrostriatal pathway, and it is useful in clinical practice as proof of degenerative parkinsonisms. However, there are several findings that DAT expression is not merely related to the vitality of the nigral neurons, since synapsis involvement could antedate nigral cellular degeneration and DAT density could be modulated as a compensatory mechanism in preclinical/early PD. All of these points, along with several potential external interferences, make DAT imaging a less reliable marker of disease progression.


It is encouraging to witness the recent price reduction and expanded access to next generation sequencing platforms, the increasing number of investments and publications on new targets and respective targeted drugs, as well as the worldwide excitement with anti-cancer personalised therapies. This editorial aims to highlight the limitations regarding the small proportion of solid cancers potentially eligible for the use of molecular-based targeted drugs until now. It also covers the expected clinical benefits in refractory patients treated by matched therapies, and detailed cost-effectiveness analysis of the use of DNA sequencing analysis oncology practice in an academic and large-scale community. The major goal of personalised cancer therapy is to identify druggable tumour genomic alterations that can improve patients’ quality of life and survival. The feasibility of large-scale clinical use of precision medicine, the impact of actionable matched treatments and the most adequate value-based genomics strategies are not yet well established .The implementation of targeted therapies was a revolutionary landmark in oncology. Significant gains in clinically relevant outcomes have been achieved in several types of cancer such as gastrointestinal stromal tumours (GIST), EGFR-mutated and ALK-fused non-small cell lung cancer, all-RAS wild-type colorectal cancer, BRAF V600 melanoma, ovarian BRCA-deficient, HER-2 breast and gastroesophageal cancers, agnostic high microsatellite instability solid tumours .However, targeted therapy development requires several years of intense multidisciplinary effort, from understanding the cancer biology to the goal of testing a new drug during a phase III study. As a result, it is important to recognise that at this point the magnitude of predictive biomarkers is restricted to certain subtypes of solid tumours. Even in countries where international regulatory agencies have more liberal incorporation policies, the coverage of next-generation sequencing (NGS) molecular testing and personalised treatments is limited. For example, it is estimated that for all US patients diagnosed with advanced or metastatic cancer in 2018, only 8.33% would be eligible for genome-driven drugs and only 4.9% would benefit from using personalised therapies [Two meta-analyses with 346 phase I and 560 phase II studies suggested statistically significant gains in progression-free survival (PFS) and overall survival (OS), respectively, when biomarker based treatment strategies were used [Interpretation and validation of precision oncology studies are especially challenging because of their inherent pre- and post-analytical complexity: epidemiological and genetic variability between populations, individual tumour heterogeneities, sample process requirements, NGS sequencers techniques, genomic panels covered, and bioinformatics analysis differ widely between the trials and commercially available platforms. In this context, we believe that a multidisciplinary molecular tumour board integrative case discussion should be standard practice in order to minimise pitfalls, maximise holistic data interpretations and optimise matched-therapy guided orientation . A betteLast but not least, we must consider the cost effectiveness of the required infrastructure necessary to enable routine practice of precision cancer medicine from the academic, community and individual perspectives. Among community treated advanced NSCLC, broad-based genomic sequencing–tested patients did not benefit from improved survival in comparison to routine EGFR- and ALK-tested patients . In an iIn conclusion, it is encouraging to witness the recent price reduction and expanded access to NGS platforms, increasing number of publications on new targets and respective targeted drugs, investments by the pharmaceutical industry and excitement within the oncology community over personalised therapies. Our greatest challenge, however, continues to be adequate patient selection, development and access to clinical trials, tumour board decision process, incorporation of cost effectiveness analysis and being honest about patients’ and family members’ expectations and frustrations regarding the heterogeneity of our precision. Precision oncology is in the earliest stages of development and in expanding our knowledge in this field we look forward to a time when our expectations surpass current limitations.We declare that we have no conflicts of interest.