Development of the complex human heart is tightly regulated at multiple levels, maintaining multipotency and proliferative state in the embryonic cardiovascular progenitors and thereafter suppressing progenitor characteristics to allow for terminal differentiation and maturation. Small regulatory microRNAs (miRNAs) are at the level of post-transcriptional gene suppressors, which enhance the degradation or decay of their target protein-coding mRNAs. These miRNAs are known to play roles in a large number of biological events, cardiovascular development being no exception. A number of critical cardiac-specific miRNAs have been identified, of which structural developmental defects have been linked to dysregulation of miRNAs in the proliferating cardiac stem cells. These miRNAs present in the stem cell niche are lost when the cardiac progenitors terminally differentiate, resulting in the postnatal mitotic arrest of the heart. Therapeutic applications of these miRNAs extend to the realm of heart failure, whereby the death of heart cells in the ageing heart cannot be replaced due to the arrest of cell division. By utilizing miRNA therapy to control cell cycling, the regenerative potential of matured myocardium can be restored. This review will address the various cardiac progenitor-related miRNAs that control the development and proliferative potential of the heart. At the cellular level, response to environmental stimuli requires tight regulation of gene expression. This control is spread amongst a number of key players that play regulatory roles at different stages of gene expression. MicroRNAs (miRNAs) are a class of non-coding RNAs (~ 23 nucleotides) that regulate gene expression post-transcription of their associated messenger RNAs (mRNA), termed RNA interference (RNAi). Significant progress in our understanding of miRNA regulation and mechanism has been made within the past decade. To date, miRNAs have been shown to play roles in a large number of biological events, such as embryonic development wherein miRNAs regulate transcription factor expression, setting up morphogen concentration gradients as well as contributing to the dynamic processes of stem cell maintenance, early cell migration and proliferation , 2. ScieIn the field of cardiovascular research, various miRNAs have been shown to contribute to cardiac fate specification and maintenance of cardiac progenitor during development. As would be expected, dysregulated miRNA expression in embryonic cardiovascular progenitors during development would lead to numerous structural congenital heart defects as a result of poorer cell proliferation, migration and specification into appropriate cell types . These sThe topic of miRNA biogenesis and mechanism of action has been reviewed extensively as the understanding of the field progressed over the years –14. WithRISC carries out post-transcriptional silencing via the guide miRNA base pairing to miRNA response element (MRE) sequence on the mRNA , 18. MREDicer1 knockout in mice resulted in embryonic lethality and heterozygous mutants stained for Oct4 showed much reduced protein expression, indicating a loss of the stem cell population in the embryo. Later research unveiled key miRNAs highly expressed in embryonic stem cells involved in pluripotency maintenance, such as miRNA-34a and the miR-290 family  [Looking at the bigger picture, miRNA control over gene expression has been shown to be crucial for pluripotency maintenance and embryonic development. A detailed review was previously provided by Carnell et al. . BrieflymiR-295) –30. AmonmiR-295) .sdf1a transcripts in all cells such that only cells actively transcribing new sdf1a will express it [Further down the pipeline, miRNAs also play a key role in regulating the downstream differentiation and large-scale cell migrations into the different embryonic tissue and organs. Early stages of embryogenesis involve the generation of distinct cell types from a single totipotent cell, via the setting up of morphogen gradients to convey positional information within the embryo . The prepress it , 35. TheMesp-1/2, Hand1/2, Wnt/β-catenin signalling, Bmp2 and FGFs in inducing cardiac progenitor migration and setting up the heart fields [Nkx2.5 and Isl1 expressing cardiac progenitor cells proliferate and contribute to the initial formation of the heart tube and eventual chamber structures, conduction channels and nodes [Dicer knockouts in mice cardiac tissue, resulting in perturbed cardiac development and various cardiac pathological phenotypes such as hypertrophy, fibrosis, biventricular enlargement and ultimately heart failure [Dicer in zebrafish led to embryonic lethality due to heart developmental defects amongst several gastrulation associated issues [Coming to the cell population of interest, embryonic heart development similarly requires tight regulation. In the mammal, the heart is the first functional organ to develop with multiple stages in the cardiac developmental timeline. Firstly, mesodermal precursors are specified into cardiovascular progenitors which migrate during gastrulation to form the two heart fields. Subsequently, the cardiac crescent and tubular heart are formed. Lastly, respective cells are recruited to the poles of the heart, and differentiation into the cardiac chambers occur (Abu-Issa 2007). Development of the heart has been extensively studied, uncovering the roles of t fields –39. Nkx2nd nodes –43. It i failure . Homozygd issues . These fFurther research identified a number of cardiac-specific miRNAs, most notably miR-1 and miR-133, which are highly abundant and conserved in orchestrating cardiac development . In the Isl1 and Tbx1 progenitor gene expression, which contain the MREs in their 3′ UTRs [Other prominent cardiac miRNAs have been identified within the cardiac differentiation lineage, highlighted in Table  3′ UTRs . In mice 3′ UTRs . In zebr 3′ UTRs . TherefoDrosophila or zebrafish do not develop mammalian four-chambered hearts and may not be an accurate depiction of human developmental defects. In reality, there is little characterizing done on miRNAs present in the developing human heart apart from an expression profiling study which has identified upregulated miRNAs likely to be involved in the heart chamber, septum and outflow tract development [A significant amount of our understanding of miRNAs implicated in heart development has been derived from animal models, as portrayed in Fig. elopment . There ielopment .Fig. 3PUsing in vitro differentiation of human embryonic stem cells, researchers are able to derive cardiac progenitors, cardiomyocytes, endothelial cells and smooth muscle cells to carry out miRNA profiling. Notably, miR-1 and miR-499 were identified in derived cardiomyocytes, and their role in cardiomyocyte fate commitment was looked into , 60. miRDysregulation in miRNAs related to cardiac progenitors and development will inevitably lead to structural defects of the heart. In the past, diagnosis of cardiovascular diseases used to be mainly through echocardiography or clinical syndromes. However, molecular biology advancements have uncovered diagnostic biomarkers which could be potentially used to detect cardiac diseases, providing insights into the possible pathological mechanisms underlying these diseases . IdentifCongenital heart diseases (CHDs) encompass a broad range of phenotypes resulting from cardiac structural defects at birth and are the most common type of birth defect present amongst newborns, accounting for about 25% of infant morbidity within the first month . As the In an effort to study TOF, O’Brien et al. compared the non-coding RNA (which includes miRNAs) expression level between non-syndromic TOF infants and healthy infants. From the analysis, approximately 61 miRNAs involved in cardiac development pathway were reported to exhibit an altered expression in the right ventricular myocardium of TOF infants as compared to healthy infants . TOF is GJA1 and SOX9. The upregulation of both genes is correlated with VSD incidence, implicating them in VSD pathogenesis [VSD itself has been well studied in relation to miRNAs. To date, several animal studies revealed the involvement of miR-1, miR-133a and other miRNAs in the manifestation of VSD. As mentioned previously, 50% of miR-1-2 knockout mice suffered from embryonic lethality, largely due to VSD and chamber dilation as a result of dysregulated Hand2 levels , 52. Conogenesis .Similarly, miRNA dysregulation was also found to associate with syndromic congenital heart diseases such as Down syndrome. Down syndrome is characterized by trisomy 21, and about 40–60% of babies born with it also suffer from CHD, making it one of the most common genetic syndrome of CHD. Five miRNAs, present on chromosome 21—mir99a, let-7c, miR-125b-2, miR-155 and miR-802—were identified to be highly expressed in cardiac tissue of patients suffering from Down syndrome . TherefoEach of the congenital heart diseases described is structural defects of the heart that can develop via impairment of various cardiomyocytes proliferation or migratory pathways. As a result, the different pathologies express distinct miRNA transcriptomes alluding to the affected pathways. The distinct transcriptome landscape was evident from a genetic profiling conducted on 67 patients by Ikeda et al., based on three heart pathologies—ischemic cardiomyopathy, dilated cardiomyopathy and aortic stenosis. Forty-three out of 87 miRNAs were expressed differentially in at least 1 of the disease group . AdditioExtending our knowledge of dysregulated miRNAs can plausibly help to fix the structural defects of the heart. However, another pertinent issue with cardiac tissue is its lack of regenerative capacity. Cardiac cell death occurs over the human lifespan and can be accelerated due to cardiovascular complications and myocardial infarction. Acute myocardial infarction is characterized by large-scale cardiac cell death, resulting in a scarred heart with impaired cardiac contractility and remodelling of the left ventricle. Artificially altering miRNA levels can confer a cardioprotective effect as shown by Gu et al. wherein miR-21 overexpression in mice reduced myocardial infarction levels by 36.9% and lowered levels of apoptosis . In addiThe accumulation of cardiac tissue damage and myocardial scarring over time add increasing mechanical burden to the heart. As heart failure is the leading cause of death, there is a demand for biomarkers to aid in heart failure risk assessments. In this regard, certain miRNA expression levels have recently been identified to be differentially expressed in heart failure cases. The miRNA transcriptome of a failing heart was found to be highly similar to foetal cardiac tissue, with more than 80% of both induced and repressed miRNAs regulated similarly . In addiAs summarized in the previous sections, miRNAs have been shown to play roles in cell cycling and proliferation as well as the onset of cardiac pathologies and therefore can potentially be a therapeutic option. When considering methods to control and modify gene expression, post-transcriptional silencing via miRNA treatment is highly attractive. Apart from post-transcriptional silencing, gene expression can be altered by two main methods. Firstly, transcription of target genes can be reduced via epigenetic modifications, reduction of transcription factor levels or entire gene knockouts –84. SecoMicroRNAs, on the other hand, are more versatile and offer a transient handle on gene expression levels. Once miRNAs linked to a potential drug target have been identified, synthesizing complementary oligonucleotide sequences is trivial and acts as a good start point to develop drugs for treatment. Current miRNA-based drugs utilize stable nucleic acid analogues, termed as locked nucleic acids, to generate an antisense oligonucleotide sequence to target mRNA sequences, acting as synthetic miRNA sequences to exert gene silencing . New miROne main issue with the adult heart is its innate lack of proliferative and regenerative capacity, resulting in damage to myocardial tissue accumulating as a result of ageing, increasing heart failure risks over time. In the context of this review, it is relevant to look at the potential of miRNA therapy in restoring the proliferative capacity of matured myocardium.Researchers have utilized animal models to show that miRNAs are able to induce regeneration in other tissue types—miR-124 and miR-302 induced regeneration in the brain and lung tissue, respectively , 94. SimIn a recent attempt to bring regenerative research closer to human therapy, the Giacca group utilized a more clinically relevant porcine model to show the effects of human miRNA has-miR-199a treatment . Their fTo make up for the adult human myocardium lacking regenerative capacity, miRNA therapies can potentially enhance regeneration by providing a proliferative niche via the upregulation of cardiac progenitor miRNAs (miR-302-367) or downregulation of cell cycling/proliferation inhibiting cardiac miRNAs (miR128/miR-15). These miRNAs therapeutics could work by inducing dedifferentiation locally wherein the terminally differentiated cardiomyocytes change their gene expression pattern, losing their contractile, electrophysiological and other cardiomyocyte properties, and express genes that enable proliferation and migration characteristic of a more multipotent progenitor. In this case, the adult myocardium undergoing mitotic arrest can be reverted towards the proliferative cardiovascular progenitors within the same lineage to enable tissue repopulation and repair. MGN-1374 is one such drug currently being developed, being an anti-miR targeting the miR-15 family seed region, such as to enable post-myocardial infarction remodelling and induction of cardiomyocyte proliferation .Wt1) expression in the epicardial layer bordering the myocardial infarction, inducing a proliferative and neovascularization response [Dicer knockout mice result in embryonic lethality due to cardiac defects and impaired coronary vessel development in mice, confirming the relevance of miRNAs in epicardial development [On the other hand, miRNA therapeutics could also utilize the epicardium to encourage myocardium repair. Successful models of regeneration induced cardiomyocyte proliferation and repair in injured myocardium via activation of the epicardial cells , 100. Firesponse , 101. Wtresponse , 103. Hoelopment .WT1 and EMT-related genes as it presents an attractive stem cell-based therapy to enable cardiac repair post-myocardial infarction. In the context of miRNA control, Let-7 microRNA expression has been found in the heart and in vitro embryonic stem cell-derived matured cardiomyocytes. Let-7 inhibition post-myocardial infarction has shown to attenuate myocardial remodelling, improve cardiac function, increase the expression of EMT-related genes in the epicardial cells and increase the expression of pluripotency genes Sox2 and Oct4 in the epicardial-derived fibroblasts [For cardiac repair post-myocardial infarction, Wt1+ epicardial cells consist of multipotent cardiac stem cells that can give rise to de novo cardiomyocytes  and vascroblasts , 108. AsThe biological importance of miRNAs has been conclusively determined over the past two decades. With roles in stem cell maintenance to the terminal differentiation of cells, these miRNAs behave as molecular rheostats that are highly targetable by synthetic oligonucleotides. In the field of cardiovascular research, significant effort and funding are being poured into regenerative therapy to look for a solution to cardiac regeneration. The reversion of adult cardiomyocytes into a progenitor-like state or activation of the progenitor properties of epicardial WT1+ cells are both potential methods to enable cardiac proliferation. To that end, our increasing understanding of the control miRNA has over pluripotency and proliferation can be utilized. While significant amounts of miRNA modelling have already been carried out in animal models, further validation needs to be carried out in in vitro human cardiomyocyte or 3D cardiac organoid cultures in the near future.


Liver pathophysiology can, directly and indirectly, impose morphological or biochemical abnormalities of the platelets. Conversely, platelets are also able to regulate the promitogenic and profibrotic signals on liver pathobiology. Platelet contribution to the liver pathophysiology is typically facilitated by the platelet-derived growth factors that are sequestered in different subsets of alpha and dense granules, and the release of these growth factors is synchronized according to the stage and type of liver disease or injury. Thus, platelets harbor clinically relevant information with potential diagnostic and prognostic implications in liver diseases. Hepatocellular carcinoma (HCC) largely influences the platelet kinetics, and a growing body of evidence has recognized its association with HCC occurrence or prognosis. This narrative review summarizes the progress made on implicating platelet as a diagnostic and prognostic tool for HCC; the review also dissects the contradictory results from earlier studies and reflects how combining platelet-based information may enable more reliable test for diagnostic and prognostic evaluation of HCC. Hepatocellular carcinoma (HCC) is the leading cause of cancer deaths worldwide with the highest incidence in Asia and Africa, where the endemic prevalence of hepatitis B (HEP-B) and hepatitis C (HEP-C) strongly predisposes to the development of chronic liver disease (CLD) and consequent occurrence of HCC. In 2015 alone, HCC accounted for estimated 470,000 deaths globally . The lifTransplant or curative resection of HCC offers the best outcome in a selected group of patients; however, the long-term recurrence-free survival (RFS) rates are limited to 40 percent or less . AlthougThere is already a pile of evidence that highlights the role of platelets in liver homeostasis and inflammation , 10. PlaA literature search was performed for the current study on the benchmark of searches for a narrative review article. The electronic search comprised mainly two databases, PubMed and Google Scholar, and used two search terms: “platelet and hepatocellular carcinoma”. Besides, additional references were obtained by manual search. The inclusion criteria were all related articles displayed in PubMed and Google Scholar. Articles without full text and not in English were excluded.Platelets, nonnucleated cell elements, born out of the cytoplasmic fragment of bone marrow megakaryocytes, inherit some very important properties of typical cells. These small fragments, sometimes called pollutants or even cell debris, are equipped with the sense to discriminate several types of danger signals and adjust their response . This reA closely interlaced relationship between hemostasis and hepatology brings platelets always inside the ring during liver injury or inflammation. Platelets accumulate in the injured liver and interact with liver cells in a disease-specific and stage-specific manner . PlateleA majority of HCC is diagnosed in patients with underlying cirrhosis or CLD. Distortion of the normal liver architecture in patients with cirrhosis increases resistance to blood flow, resulting in portal hypertension, splenomegaly, and resultant thrombocytopenia. Thus, the platelet count has its own classical merits in predicting cirrhosis , 18 and Association between platelet count and prognosis is established in different solid tumors. A reduced risk for cancer metastasis with aspirin administration at an antiplatelet dose reported recently corroborates the long-established relationship between thrombocytosis and cancer progression , 23. MorVarious studies have explored the link between platelet count and HCC; in a recent meta-analysis, Pang et al. clinched a conclusion suggesting an overall lower pretreatment platelet count as a risk factor for the overall and tumor-free risk of death . Yılmaz Platelet count has also pulled some attention for its predictive relevance in HCC recurrence after curative resection. Several studies suggest thrombocytosis as a predictive factor for HCC recurrence after liver resection; however, there is also contrasting evidence that reports thrombocytopenia as a risk factor for postresection HCC recurrence. In one recent study, the postresection recurrence rate was stratified by the postresection platelet count, demonstrating an inverse relationship between the recurrence and postresection platelet concentration . In patiPlatelet-lymphocyte ratio (PLR), an inflammation-based score, has lately gained a substantial attention as a potential prognostic marker in different solid tumors. A wealth of studies has investigated the prognostic significance of PLR in HCC; the conclusions drawn from the studies are distinctly contradictive. A positive association was observed between high PLR and poor prognosis in patients with transarterial chemoembolization (TACE); high PLR was also able to predict OS , 31. In Besides PLR, the prognostic value of platelet, neutrophil, and lymphocyte ratio (PNLR) has also been examined. In a retrospective study conducted in patients with Barcelona clinic liver cancer (BCLC) stage A HCC, increased postoperative PNLR was associated with higher incidence of postresection recurrence and poor OS .A large portion of serotonin, secreted by enterochromaffin cells of the intestine, is taken up by platelets; thus, circulating platelets act as a warehouse for serotonin. Platelets sequester about 95% of total plasma serotonin in their dense granules . An overGrowth promoting effect of serotonin was found in different liver cancers including HCC. Serotonin promotes survival and growth of HCC cells by activation of serotonin receptor 2B. Two serotonin receptors 1B and 2B were found overexpressed in the liver of patients with HCC . SerotonMonitoring IP serotonin served as a sensitive marker and provided relevant clinical information for diagnosis of some tumors . ElshayePadickakudy et al. have recently discovered a bivalent property of platelet-sequestered serotonin in liver regeneration and HCC recurrence; high perioperative platelet serotonin concentration was associated with an increased incidence of early tumor recurrence, and excessively low platelet serotonin levels correlated with a higher rate of morbidity in patients who underwent liver resection for liver tumors . Beyond A major pool of vascular endothelial growth factor (VEGF), a key driving force for physiological and pathological angiogenesis, is largely sequestered in the blood platelets ; thus, pDiagnostic and prognostic role of VEGF in HCC has been extensively explored. Higher serum VEGF per platelet count was observed in patients with HCC . Serum Vβ, and Raf kinase. A meta-analysis pooled from 9 different studies concluded that high VEGF concentrations after sorafenib administration would suggest poor survival and poor clinical efficacy [Serum VEGF, derived from platelets, is also considered a potential biomarker of sorafenib efficacy in HCC not amenable to locoregional therapy, including surgery, RFA, or TACE. Sorafenib is a small tyrosine kinase inhibitor that targets VEGF receptor (VEGFR), PDGF receptor-efficacy . Strikinefficacy . Since Vβ1) and platelets derived growth factor-BB (PDGF-BB). Serum concentration of PDGF-BB was able to reflect degree of liver damage and degree of liver fibrosis in patients with chronic hepatitis B (CHB) [Evidence from rodents has been translated in human studies substantiating the profibrotic effect of transforming growth factor beta-1 (TGF- B (CHB) ; PDGF le B (CHB) . The lat B (CHB) .β1 [β1 is incorporated into the alpha granules of the platelets. Some functions of TGF- β1 in the liver include stimulation of extracellular matrix and production of fibrous tissue. TGF-β1 concentration in serum reflects the total platelet TGF-β1 [β1 exerts a negative effect on hepatocyte proliferation via induction of apoptotic mechanisms [β1 is documented in patients with CHC that is presumed to have some roles in the pathogenesis and chronicity of these diseases [β1 concentration and a higher risk of incidence HCC [β1 in identifying high-risk patients who are likely to develop HCC, especially among HCV-positive patients. Serum TGF-β1 was not only elevated in patients with HCC but also correlated with the tumor progression [β1 concentrations improved the detection of AFP-negative HCC in high-risk patients [β1 was evaluated by analyzing downstream molecules involved in the progression of HCC in a large cohort of HCC patients in different disease stages; the study concluded that, rather than indicating advanced HCC, TGF-β1 corresponds to intrinsic biological properties of the tumor, affecting progression and the clinical outcome of the disease [β1 concentration has also been implicated to predict outcome in HCC patients treated with sorafenib; high pretreatment serum TGF-β1 levels were linked with poor PFS and OS. There are still ample possibilities for investigating and validating the diagnostic or prognostic implications of these profibrotic growth factors in HCC.Megakaryocytes are the major sites of production and storage of TGF-β1 ; TGF-β1 t TGF-β1 . Contrarchanisms . An elevdiseases . In a neence HCC . Their rgression . Intrigupatients . In anot disease . Serum TThe megakaryocytes synthesized epidermal growth factor (EGF) is largely present in the alpha granules of human platelets . EGF is Fibroblast growth factors (FGF), multifunctional proteins, possess angiogenic property stronger than VEGF or PDGF . StromalPossibilities are increasingly apparent for building platelet-based diagnostic or prognostic system in HCC. With all the above-mentioned platelet-based information, the multifaceted platelets may serve as a minimally invasive form of liquid biopsy enabling a valid test for detecting and monitoring the prognosis of HCC .Differential expression of platelet alpha or dense granule proteins, such as IP serotonin and IP VEGF, in HCC patients has influenced the horizon of platelet-based diagnostics. Concurrently, divergent results on the prognostic potential of platelet growth factors in HCC may relate to multiple factors. As discussed in the manuscript, the platelet growth factors were typically assayed in serum. Serum analysis may not include entire analytes found in the granules of platelets; some IP growth factors are presumed to be released into the serum during agonist (thrombin or ADP) stimulation during serum clot formation, but a substantial amount still remains associated with the platelets and is likely lost with the hematocrit , 79. ThuThe paradoxical platelet kinetics has made it more complex to mechanistically comprehend its property as a diagnostic marker in HCC. Platelet biomarkers were not assessed at a uniform time point in the aforementioned studies. Platelets are believed to affect the liver pathophysiology in disease-specific, stage-specific, and site-specific manner ; we prevThe introduction of tumor-educated platelets (TEP) in platelet-based cancer diagnostic is another intriguing progress. Based on platelet RNA analyses, Best et al. distinguished cancer patients from healthy subjects with greater than 95% accuracy . They haKanikarla-Marie et al. describe “circulome” as an entity that integrates all the circulating factors in the bloodstream . BesidesUnlike several other malignant diseases, diagnosis of HCC does not necessarily require a tissue biopsy or histological verification. Platelet can offer itself as a liquid biopsy with a potential revolution in HCC diagnostics and prognostics. Substantial progress has been made in HCC biomarkers discovery, but only a modest number of markers have shown their relevance in clinical settings. Moreover, the existing classical biomarkers are less promising in predicting prognosis and HCC recurrence. Platelets patrolling the injured liver, interacting with hepatic sinusoidal endothelium, and influencing effector cell recruitment and activation no more remain the mystery . We are


Anti-VEGF drugs are as the first-line therapies for diabetic macular edema (DME). In this study, we investigated the association between hyperreflective foci in the outer retinal layers and functional efficacy in DME patients who received intravitreal ranibizumab (IVR) injections. We retrospectively reviewed 77 eyes of 71 patients with DME treated with pro re nata IVR injections for at least 12 months. We evaluated how baseline hyperreflective foci in the outer retinal layers on spectral domain optical coherence tomography images were associated with an improvement in logarithm of the minimum angle of resolution visual acuity (logMAR VA) at 12 months. Forty-three eyes with hyperreflective foci in the outer retinal layers had greater VA improvement than 34 eyes without such foci at 12 months. Multivariate analyses demonstrated that both logMAR VA and hyperreflective foci in the outer retinal layers at baseline were associated with VA improvement. Structural analyses revealed that the central subfield thickness was decreased and that the ellipsoid zone of photoreceptors was improved more significantly in eyes with hyperreflective foci in the outer layers than eyes without such lesions. Baseline hyperreflective foci in the outer retinal layers predict the functional efficacy of IVR injections for DME. Both basic and clinical researches have shown that vascular endothelial growth factor (VEGF) is a main regulator in the pathogenesis of DME, and anti-VEGF treatment has great effects on anatomical and functional outcomes in DME5. Two major drugs neutralizing VEGF, ranibizumab and aflibercept, have been used as the first-line therapies for DME in many countries since the approval by the administrations. However, the magnitude of efficacy varies among each patient, indicating a need to find the prognostic factors for anti-VEGF treatment7.Diabetic macular edema (DME) leads to visual impairment in diabetic patients, mediated via the breakdown of blood-retinal barrier (BRB) and concomitant neuroglial dysfunction13. Post hoc analyses of a clinical trial found that the presence of subretinal fluid, one of the qualitative OCT findings, predicts better visual outcomes after intravireal ranibizumab (IVR) injections for DME7. The qualitative or quantitative parameters in foveal cystoid spaces are also correlated with the short-term anatomical responses to medical treatments15. Vitreoretinal abnormalities are a predictor of lower efficacy of IVR, whereas epiretinal membrane peeling is associated with greater visual acuity (VA) improvement in eyes that underwent vitrectomy16. The ellipsoid zone of photoreceptors (EZ) line (or the junction of photoreceptor inner and outer segments[IS/OS]), a marker of foveal photoreceptor status, is restored at 12 months under anti-VEGF treatment, which may partly explain the functional efficacy17.Spectral domain optical coherence tomography (SD-OCT) has been introduced into the clinical setting and can be used to evaluate the anatomical changes in neuroglial tissues of the retinas18. An initial report hypothesized that these foci may correspond to the precursor of hard exudates or lipid-laden macrophages, and other publications reported that they may also be the debris of photoreceptors or pathological changes in the retinal pigment epithelium (RPE) in the outer layers in chorioretinal diseases20. Additionally, the amount and distribution of hyperreflective foci or hard exudates change after treatment for DME24. Hyperreflective foci within the subretinal fluid predict poor visual function after intervention in eyes with serous retinal detachment (SRD), and the foci in the outer retinal layers than the external limiting membrane (ELM) are associated with photoreceptor damage and visual impairment in eyes without SRD25. Such foci are associated with poor visual outcomes in eyes that receive vitrectomy for DME26.Hyperreflective foci, which appear as highly reflective dots on SD-OCT images, are a sign of vascular hyperpermeability27.In the current study, we investigated the changes in hyperreflective foci in the outer retinal layers and their association with functional efficacy at 12 months in eyes with center-involved DME treated with pro re nata (PRN) IVR injectionsP = 0.010 and P = 0.015, respectively; Figs. After the exclusion of 10 eyes at baseline and 63 eyes that were lost to follow-up before the 12-month visit, we retrospectively reviewed 77 eyes of 71 patients whose characteristics are shown in Table P = 0.284) or at 12 months  between eyes with and without hyperreflective foci in the outer retinal layers , although there were no differences at 1month or later time points  line was longer in eyes with such foci at baseline , whereas the length did not differ between eyes with and without these lesions at 12 months  lines during IVR treatment. Eyes with hyperreflective foci in the outer retinal layers had a greater disruption in EZ (IS/OS) lines at baseline and their greater improvement at 12 months than eyes without such foci. A recent publication demonstrated the association between VA improvement and a decrease in disrupted EZ (IS/OS) lines after anti-VEGF treatment for DME17. This allows us to hypothesize that greater VA improvement can be partly explained by foveal photoreceptor restoration in eyes with hyperreflective foci in the outer retinal layers.Preoperative hyperreflective foci in the outer retinal layers were related to VA improvement at 12 months under IVR injections for DME. Structural analyses using OCT images revealed that such foci also predicted a greater decrease in CSF thickness at 12 months. CSF thickness is a main surrogate marker during DME management, and these functional and structural efficacies of anti-VEGF treatment are consistent with each other20. The former publication discussed that hyperreflective foci may correspond to deposited lipoproteins or lipid-laden macrophages, which represent extravasation and neuroinflammation. Hyperreflective foci in the inner and outer layers were reduced at later time points. This decrease may be explained by the blockade of vascular hyperpermeability to some extent2. However, photoreceptor damage and retinal thickening were restored in both early and late time points. Anti-VEGF treatment thus might inhibit VEGFR1-dependent activation of microglia/macrophages and concomitantly recover structural damage regardless of the presence of these inflammatory cells31. Another possibility might be that eyes with hyperreflective foci in the outer retinal layers had reversible and half-broken photoreceptors, which were restored under reduced hyperpermeability by anti-VEGF treatment.Hyperreflective foci in the outer retinal layers might correspond to deposited lipoproteins, lipid-laden macrophages, debris of photoreceptors, and RPE hyperplasia32. Especially, Kang and associates declared that hyperreflective foci predict poor visual outcomes after PRN intravitreal bevacizumab (IVB) injections23. In the current study, logMAR VA did not differ at 12 months between eyes with and without hyperreflective foci in the outer retinal layers, whereas eyes with such foci had greater VA improvement. This discrepancy might be partly explained by the different durations of follow-up, the different drugs, and the different methods used to assess hyperreflective foci. Additionally, hyperreflective foci in the inner retinal layers were not associated with VA improvement in this study, in contrast to those in the outer layers. Signal transduction from foveal photoreceptors is mediated via bipolar cells and ganglion cells in the inner layers of the extrafoveal areas rather than of the fovea. Consequently, future studies should elucidate how hyperreflective foci in the extramacular inner layers can predict the efficacy of IVR injections for DME. Another explanation might be the different macrophage polarization between inner and outer retinal layers33. Hyperreflective foci in the inner retinal layers might correspond to anti-inflammatory macrophages and rescue neuroretinas in a VEGF-independent manner. Hyperreflective foci in the outer retinal layers, as proinflammatory macrophages, might exacerbate neuroinflammation, which is reversed by anti-VEGF drugs.Hyperreflective foci in both the inner and outer retinal layers were decreased at the later time points under PRN IVR injections in this study. This result is consistent with the decreased number of hyperreflective foci or spots or the decreased cases with hard exudates after anti-VEGF treatment found in previous studies36. In particular, the post hoc analysis of the RISE/RIDE studies demonstrated that the presence of subretinal fluid predicts better visual gain after two years of monthly IVR injections7. This result agrees with the greater VA improvement in eyes with subretinal fluid found in the univariate analysis of this study. Seventeen eyes (40%) with hyperreflective foci in the outer retinal layers had subretinal fluid, whereas only 2 eyes (6%) without such foci were accompanied by SRD. In addition, there was an association between the disrupted EZ (IS/OS) line and hyperreflective foci in the outer retinal layers, and several publications reported photoreceptor damage as a predictor of poor visual outcomes37. Significantly, multivariate analysis designated subretinal fluid and the disrupted EZ (IS/OS) line as confounders of hyperreflective foci in the outer retinal layers. Thus, hyperreflective foci in the outer retinal layers represent vascular hyperpermeability and can be an upstream regulator of these structural changes in neuroretinas.Several publications advocated foveal morphologies as predictors of functional outcomes after anti-VEGF treatment for DME39. The number of IVR injections during 12 months did not differ between eyes with and without hyperreflective foci in the outer retinal layers. It suggests that this OCT finding is feasible to optimize the indication of anti-VEGF treatment, independent of socioeconomic concerns. The other predictor is the duration of DME or symptoms, as shown in several publications39. Since most patients were referred to our institutes, we could neither know the exact onset of DME nor investigate the relevance of this parameter. Future study should elucidate the relationship between the duration of symptoms and hyperreflective foci in the outer retinal layers.Previous publications have suggested that the treatment frequency and duration of DME are also associated with functional efficacy under anti-VEGF treatment. The frequent injections of expensive anti-VEGF drugs are a socioeconomic burden. The comparisons of visual outcomes between several clinical trials have suggested that VA improvement is positively correlated to the treatment frequency41. Several OCT parameters were subjectively evaluated, and further objective and automated detection of retinal lesions should be developed to improve the reproducibility. Ultimately, future comparative studies to other interventions would help guide us to personalized medicine.This retrospective study with a small number of cases had several potential limitations. All participants were from a single center, Asian and treated according to PRN dosing for only 12 months. Additional multi-center studies should confirm the reproducibility in other races managed using other regimens after longer follow-upIn conclusion, we demonstrated that, among several OCT parameters, preoperative hyperreflective foci in the outer retinal layers are a novel predictor of VA improvement at 12 months under PRN IVR injections for DME.In this retrospective study, we reviewed 77 consecutive eyes of 71 patients who had a baseline visit at Kyoto University Hospital from March 2014 to February 2017 as the baseline visit and received PRN IVR injections for DME. The inclusion criterion was center-involved DME that was treated with IVR administration according to the 3 + PRN regimen for at least 12 months. The exclusion criteria were eyes with media opacity leading to visual reduction, other chorioretinal diseases, treatment for DME within 6 months, previous intraocular surgery other than cataract surgery, or cataract surgery within 3 months. We additionally excluded the cases which dropped out during 12-months follow-up due to a patient’s desire to terminate treatment or switch to other treatment, patients’ inconvenience, drug tachyphylaxis, or additional treatments, i.e., panretinal photocoagulation, cataract surgery, vitreoretinal surgery (for vitreous hemorrhage), or focal/grid photocoagulation.All research and measurements adhered to the tenets of the Declaration of Helsinki after the approval of the study protocol by the Kyoto University Graduate School and Faculty of Medicine, Ethics Committee. All participants provided written informed consent before study enrollment.38. Ranibizumab (0.5 mg) was injected 3.5 mm posterior to the limbus after disinfection. After three monthly loading doses, PRN IVR injections were considered at monthly visits according to the retreatment criteria of the RESTORE study, which is the standard-of-care therapy for DME.In this study, IVR administration was performed according to the 3 + PRN regimen described in the Ranibizumab Monotherapy or Combined with Laser versus Laser Monotherapy for Diabetic Macular Edema (RESTORE) study42. Three-dimensional images were obtained using the raster scan mode, and two-dimensional maps were constructed, which enabled us to measure the CSF thickness of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid , as previously described42.The best-corrected decimal VA was measured and converted to logMAR VA at every monthly visit. After the comprehensive ophthalmic examinations, we obtained retinal sectional images using SD-OCT . Vertical and horizontal retinal sectional images dissecting the fovea were acquired using the 30-degree cross-hair mode, and 20 to 100 images were averaged to create better images as previously described26. When the ELM was disrupted at the fovea, we drew an imaginary straight line connecting the ELM line in the parafovea and determined the presence or absence of hyperreflective foci in the outer layers  line within the central 1 mm on both vertical and horizontal sectional images, and the average was calculated. We further assessed three qualitative findings, i.e., cystoid abnormalities, and subretinal fluid, as previously described6. Two retinal specialists evaluated these OCT parameters. Disagreements were discussed until a consensus was reached regarding qualitative OCT findings, and the average of the quantitative parameters was applied for further analyses.We qualitatively and quantitatively evaluated several parameters within the central 1 mm in both vertical and horizontal sections. Hyperreflective foci between the ELM and the surface of the RPE were defined as those in the outer retinal layers, as previously describeders Fig. . The fov24. After mydriasis, we obtained 40-degree fundus photographs centering on the fovea using a color fundus camera . Two retinal specialists assessed the severity of hard exudates based on the standard photographs on a diagnostic monitor 44. The central circle  was defined as the CSF on fundus photographs24. ‘Definite’ or more severe grades of hard exudates in the CSF were categorized into ‘hard exudates in the CSF’.Hard exudates in the CSF were evaluated on color fundus photographs according to the modified methods described previouslyP < 0.10) were applied for multiple linear regression analysis. We used SPSS version 24.0 for statistical analyses .The results are expressed as the mean ± standard deviation. After test of normal distribution, the Student’s t-test or Mann-Whitney U-test was used for the parametric or non-parametric datasets. Fisher’s exact test or chi-square test was applied to evaluate the sampling distribution. The Kappa coefficient or intraclass correlation coefficient (ICC) was employed to assess the concordance in the qualitative or quantitative parameters, respectively. Linear regression analyses were applied to evaluate whether baseline characteristics , pseudophakia, panretinal photocoagulation, hard exudates in the CSF, CSF thickness, cystoid abnormalities, subretinal fluid, disrupted EZ (IS/OS) line, hyperreflective foci in the inner retinal layers, and those in the outer retinal layers) were associated with the improvement of logMAR VA at 12 months. Significant factors in univariate analyses (Supplementary information.


Pancreatic fistula occurs as a result of pancreatic duct disruption during acute pancreatitis. An external or pancreatico-cutaneous fistula is defined as a leakage of pancreatic secretion through an abdominal wound or previously inserted drain. If the extravasated pancreatic juice is walled-off by the fibrous or granulation tissue, a pseudocyst is formed. Fistulization of the pancreatic pseudocyst into the different surrounding hollow viscera is reported. However, we present a patient with spontaneous cutaneous fistulization of the pancreatic pseudocyst into the lumbar region successfully treated conservatively. Such an extremely rare presentation is only reported twice and thus highly unexpected during the follow-up of patients after recovery from acute pancreatitis.The patient presented with 5-days intermittent fever and a tender, fluctuant, and erythematous swelling of the left lumbar paravertebral region with black necrotic skin spot on the top of it.Abdominal computed tomography scan revealed retroperitoneal pseudocyst originating from the pancreatic body and tail and extending to the left flank.Incision of the swelling evacuated dark amylase rich fluid. Colostomy disc and bag were applied to collect further spontaneous outflow of pseudocyst content.Symptoms instantly resolved and the patient was managed conservatively with ambulatory follow-up of the daily volume of fistula discharge. Over the next 37 days daily fistula output gradually reduced to nil with the spontaneous closure of the external skin fistula opening.Frequent follow-ups of patients after severe acute pancreatitis are necessary for early detection and timely successful treatment of pancreatic pseudocysts with such unusual and rare presentation. Herein, we present a very rare case of spontaneous cutaneous fistulization of the pancreatic pseudocyst into the lumbar region successfully treated conservatively. Such an extremely unusual pseudocyst presentation must be kept in mind during the follow-up of patients after pancreatitis as it may have important implications for further treatment decision-making. All the procedures, data collection and presentation were approved by the hospital Ethics Committee (Decision No: 452/11) and with the patient's informed consent.Pancreatic pseudocysts are collections of pancreatic juice leaked out of a disrupted pancreatic duct, localized with non-epithelialized fibrous wall, and usually appear as a complication of acute or chronic pancreatitis. Internal fistulization of pancreatic pseudocysts into the adjacent hollow viscus or cavity such as colon or pleural space is known and well-described.2A 65-year-old woman presented with 5-day intermittent fever (up to 38 °C) and a 70 × 50 mm tender, fluctuant, and erythematous swelling of the left lumbar paravertebral region with black necrotic skin spot on the top of it Fig. . Previou3 They are located intra-abdominally and when they erode adjacent hollow organs or spaces internal pancreatic fistulas occur. An external pancreatic fistula, or pancreatico-cutaneous fistula is, by definition, leakage of the pancreatic juice through the abdominal wound or inserted drain persisting for >7 days and most commonly occurs as a complication of pancreatic surgery or percutaneous drainage of pseudocysts. However, spontaneous external cutaneous fistulization of the pancreatic pseudocyst is extremely rare. To our knowledge only 2 such cases were reported: one with external opening at umbilicus, treated conservatively and second with free communication with the left flank managed at surgery.,4Pancreatic pseudocysts are associated with 10% of patients with acute and 20% to 38% of patients with chronic pancreatitis.,6 Urgent management of pancreatic pseudocysts is mandatory if complications occur, such as infection, bleeding, rupture or internal fistulization, and mass effect  and treatment modalities include operative, endoscopic, or percutaneous drainage. Nevertheless, initial treatment is conservative as spontaneous resolution is expected in the majority of cases including those with low-output (<200 mL per day) external fistulas such as our patient. Severe complications such as hydroelectrolyte disturbances, organ failure, or sepsis may occur in patients with high-output external drainage of pancreatic fluid and require more intensive therapeutic measures: nil per os, parenteral nutrition, IV fluids, electrolyte correction, naso-gastric tube placement, analgesics, and antibiotics. Due to good general status and fast resolution of symptoms our patient did not require these measures with exception of daily skin care and protection for mild skin excoriation. Also, gradual reduction and spontaneous closure of the external pancreatic fistula after 37 days was due to the unobstructed downstream flow of the pancreatic juice through the pancreatic duct. Therefore, early endoscopic retrograde pancreatography and stent insertion or surgery including Roux-en-Y pancreatico-jejunostomy or resection were not mandatory to ensure fistula closure. Frequent follow-ups of patients after severe acute pancreatitis are necessary for early detection and subsequent successful treatment of pancreatic pseudocysts with such unusual and rare presentation.The preferred imaging method for such patients is computed tomography with fistulogram.Conceptualization: Milan Radojkovic.Data curation: Milan Radojkovic, Predrag Kovacevic, Danijela Radojkovic.Formal analysis: Milan Radojkovic, Predrag Kovacevic, Danijela Radojkovic.Supervision: Predrag Kovacevic, Danijela Radojkovic.Validation: Predrag Kovacevic, Danijela Radojkovic.Writing – original draft: Milan Radojkovic.Writing – review & editing: Milan Radojkovic.


For many decades, researchers have studied how plants use bet-hedging strategies to insure against unpredictable, unfavourable conditions. We improve upon earlier analyses by explicitly accounting for how variable precipitation affects annual plant species’ bet-hedging strategies. We consider how the survival rates of dormant seeds (in a ‘seed bank’) interact with precipitation responses to influence optimal germination strategies. Specifically, we incorporate how response to resource availability (i.e. the amount of offspring (seeds) generated per plant in response to variation in desert rainfall) influences the evolution of germination fractions. Using data from 10 Sonoran Desert annual plants, we develop models that explicitly include these responses to model fitness as a function of precipitation. For each of the species, we identify the predicted evolutionarily stable strategies (ESSs) for the fraction of seeds germinating each year and then compare our estimated ESS values to the observed germination fractions. We also explore the relative importance of seed survival and precipitation responses in shaping germination strategies by regressing ESS values and observed germination fractions against these traits. We find that germination fractions are lower for species with higher seed survival, with lower reproductive success in dry years, and with better yield responses in wet years. These results illuminate the evolution of bet-hedging strategies in an iconic system, and provide a framework for predicting how current and future environmental conditions may reshape those strategies. In such environments, natural selection will favour traits or strategies that buffer against uncertainty, usually by spreading risk over time or space. This is often achieved through bet-hedging, in which strategies or traits reduce variance in reproductive success at some cost to mean reproductive success –3. EmpirBet-hedging strategies interact with other traits to determine fitness, so the degree of bet-hedging should depend on other traits that influence whether an organism can survive until reproduction ,12. For In desert annual plants, low-resource tolerance (i.e. stress tolerance) and resource-use capacity  are likely to influence the fitness consequences of annual germination rates. One approach to assess the fitness consequences of such functional responses is through relating resource availability to two quantities: the probability of producing any offspring (reproductive success) and the number of offspring (yield upon reproductive success). The set of intercepts and slopes from these per-species relations explain performance upon low-resource availability and responsiveness to increased resource availability, respectively, and can be incorporated into models for understanding the adaptive value of germination timing. Of course, abiotic conditions, such as temperature or precipitation, are not the only sources of risk in uncertain environments. The fitness consequences of risk-spreading strategies also depend on biotic conditions, making traits that affect response to competition or other density-dependent processes important as well ,15. Undeper capita yield, conditioned on reproductive success, as functions of precipitation and intraspecific density. We incorporate these relationships into a density-dependent seed bank model to predict evolutionarily stable germination strategies. To distinguish the relative importance of density, seed survival and functional responses in driving the evolution of germination strategies, we then build a statistical regression model and use it to explore how well it predicts observed and model-predicted germination strategies.Here, we build upon a model of Gremer & Venable  to estim2.a) introduced by Gremer & Venable . O. Oa) [11a) –29, whicOur analyses also indicate differences in what is driving predicted ESS values versus observed germination rates. While the general intercept, reproductive yield and seed survival rates within the seed bank affect predicted ESS values (see equation (3.1)), reproductive success is a better predictor of observed germination fractions (see equation (3.2)). The general intercepts of the two regressions account for the largest difference in fit between the statistical model and the ESS models’ predicted germination fractions. This major difference partially stems from the variance in our predicted yield values generally having lower variance than that of the estimated low-density yield values of Gremer & Venable . Larger e models ,30,31.As a model for displaying the relative importance of survival rates, reproduction rates and conditional seed production rates, the statistical regression for observed germination fractions suggests that we are not fully capturing variance in yield and reproductive values. Thus, discerning these biological uncertainties and modelling them will be an important step toward understanding the reasons why desert annuals hedge their bets as much as they do. Moreover, our ESS model can be a significant springboard to predicting germination rates for desert annuals: not only can one fine-tune the ESS estimates we have predicted by incorporating variable factors such as temperature and predation, one may also use this same ESS machinery to predict germination rates of desert annuals outside of the 10 studied here. Finally, as 30 years of data did not capture the whole picture in mean and variation in precipitation, it will be equally important to investigate how differences in either of these affect ESSs.


In the adult organism, reservoirs of MSCs can be found in almost all tissues where MSCs contribute to the maintenance of organ integrity. The use of these different MSCs for cell-based therapies has been extensively studied over the past years, which highlights the use of MSCs as a promising option for the treatment of various diseases including autoimmune and cardiovascular disorders. However, the proportion of MSCs contained in primary isolates of adult tissue biopsies is rather low and, thus, vigorous ex vivo expansion is needed especially for therapies that may require extensive and repetitive cell substitution. Therefore, more easily and accessible sources of MSCs are needed. This review summarizes the current knowledge of the different strategies to generate human MSCs  Among the adult stem cells, MSCs are supposed to be the most promising stem cell type for cell-based therapies –4. Compahttp://www.clinicaltrials.gov) for disease treatments including graft versus host disease, chronic obstructive pulmonary disease, Crohn's disease, or even multiple sclerosis [Once therapeutically applied, MSC can either act directly by homing to particular anatomical sites after transplantation and differentiating into specific cell types to locally restore the damaged tissue. Even more important, MSCs can support tissue regeneration by a paracrine (“hit and run”) mechanism of action, such as secretion of multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation –14. In aclerosis –20. Geneclerosis –23.in vitro expansion of ex vivo isolated adult MSCs: a decline of their plasticity and in vivo potency over time was reported, as well as accumulated DNA abnormalities and replicative senescence [in vivo effectiveness of MSCs [The classical known reservoir of MSCs is the bone marrow, but nowadays, MSCs are effectively isolated from almost every organ such as adipose tissue, cartilage, muscle, liver, blood, and blood vessels , 24–29. nescence –35. In a of MSCs –39. Therin vitro were established. MSCs for autologous cell replacement therapy can be derived from immune-compatible somatic cells, which possesses huge clinical potential. However, the large-scale production of human MSCs for regenerative cell therapies depends on well-defined, highly reproducible culture and differentiation conditions. This review will focus on the different methods to generate therapeutically active MSCs in vitro.To circumvent many of these issues, alternative methods to generate therapeutically sufficient numbers of MSCs MSCs can be derived from different donor cells via 2 primary strategies: (1) direct conversion or induced transdifferentiation of patient-specific somatic cells or (2) differentiation from reprogrammed (pluripotent) somatic cells (iPSCs) . No mattThe first reports on MSCs generated from pluripotent stem cells were performed with pluripotent embryonic stem cells before iPSCs came into focus. Xu et al. isolated human embryonic fibroblast-like cells (HEF1 cells) from pluripotent human embryonic H1 stem cells after induction of differentiation by small aggregate formation (embryonic bodies) and subsequent cultivation in HEF1 medium (knockout- (KO-) DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and nonessential amino acids) . The remin vitro generation of MSC differentiated from pluripotent stem cells which followed the classical MSC characteristics was made.Only one year later, a more directed approach for the in vitro generation of MSCs was reported. Barberi et al. used the same embryonic H1 stem cell line (together with H9 embryonic stem cells) and cocultured these cells with the mesoderm embryonic cell line OP9  to induce mesodermal differentiation in the presence of 20% heat-inactivated FBS  for approximately 40 days prior flow-activated cell sorting (FACS) for the MSC marker CD73 . This siA number of reports followed to derive MSCs from human embryonic stem cells. A more specific approach was provided by Lian et al. who established a protocol for the derivation of clinically compliant MSCs, which were derived from Hues9 and H1 human embryonic stem cells without the use of animal products . MesoderThis issue was addressed in the following study. Karlsson et al. established an optimized protocol resulting in the simple and reproducible derivation of mesenchymal progenitors from xeno-free, undifferentiated human embryonic stem cell lines , 48. TheConclusively, MSCs can either be derived from human embryonic stem cells spontaneously, upon less stringent culture conditions, and in particular upon culturing in medium which is deprived of pluripotent signals, or by a specific stimulus  which directs MSC differentiation. Most of these protocols were consistent and cost-effective, but inefficient, as the MSC population yielded by the unspecific differentiation methods yielded only approximately 5% MSCs. Pure cultures could then be established upon prolonged culturing, by fluorescence-activated cell sorting or by manual selection of cell populations.Human iPSCs constitute a well-characterized, generally unlimited cell source for the mass generation of lineage- and patient-specific MSCs without any ethical concerns because of their theoretical unlimited growth and differentiation potential. Human iPSC-derived MSCs were already shown to display similar features with mature MSCs at the genetic and functional levels , 50–52. bone morphogenetic protein 4, activin A/nodal, bFGF, and glycogen synthase kinase 3 inhibitors or WNT ligands) in chemically defined monolayer systems. Successive treatment with MSC-specific growth factors and/or sorting for MSC-specific cell surface markers using flow cytometry or immunomagnetic separation allows then the isolation and expansion of the selected MSCs under chemically defined cell culture conditions.The classical method for differentiating iPSCs towards MSCs is the use of media that contains a high serum concentration or MSC-typical growth factors such as bFGF after mesoderm induction , 53–55. KCNH1-encoded human ethera`-go-go 1 (hEAG1) potassium channel was identified being responsible for higher cell proliferative rate in iPSC-derived MSCs using the Lian protocol [A clinically compliant protocol for the MSC differentiation of human iPSCs was established by Lian et al. . Accordiprotocol , 58.β pathway inhibitor SB431542 [In vitro, iPSC-MSC-like cells displayed the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes [A new method to rapidly derive MSC-like cells from human iPSCs in one step using thin, fibrillar, type I collagen as matrix that mimics the structure of physiological collagen was reported for the effective differentiation of MSC from the human dermal fibroblast-derived HDFa-YK26 iPSCs . ResilieSB431542 . iPSCs  for the strong positive effect on the proliferation of MSCs [Most of the protocols used fetal bovine serum as supplement which provides multiple growth factors with nonspecific signals to the cultures. In contrast, providing a chemically defined medium with known morphogens that fosters the MSC differentiation is supposed to increase the yield and homogeneity of the derived MSCs. In addition to the more specific and defined medium supplements, the use of xeno-free supplements  would allow the generation of highly identical and clinically compliant MSC cultures from human iPSCs. Luzzani et al. used H9 human embryonic stem cells and iPSCs reprogrammed from human foreskin fibroblasts in combination with platelet lysate as a media supplement to produce pluripotent-derived MSCs (PD-MSC) within 3 to 4 weeks in a robust and consistent way . The aut of MSCs , 68. A p of MSCs .in vitro generation of vascular wall-typical MSCs from iPSCs, based on a vascular wall MSC-specific gene code, was reported by our group [in vitro and in vivo [In summary, robust protocols have been established to obtain patient-specific, therapeutically active MSCs from iPSCs in large amounts which will potentially open avenues towards novel, MSC-based therapies. Another straight forward strategy would be the ectopic overexpression of MSC-related genes or transcription factors in human iPSCs to generate MSCs. The ur group , 70. Her in vivo . HoweverThe main limitation for a possible therapeutic use of pluripotent stem cells and/or their derived MSCs is the medical risk to generate teratomas. Although already robust selection markers and refined experimental protocols have been established to guide human iPSCs reproducibly to MSCs, an additional negative selection against remaining pluripotent cells could be an additional option, to limit the risk of teratoma formation and foster clinical safety. As an alternative, somatic donor cells that have reached a particular differentiation stage could be used.in vitro and in vivo, and were not tumorigenic [β inhibitor)) with or without the addition of three growth factors ) efficiently generated functional iMSCs from human primary dermal fibroblasts  within 6 days (average rate of 38%)). The generated MSCs shared similar molecular signatures with bone marrow MSCs and fulfilled all of the MSC criteria defined by ISCT, including plastic adherence, marker expressions, and multipotency differentiation. In vivo, a markedly attenuation of endotoxin-induced acute lung injury, which was paralleled by a decrease of the amounts of proinflammatory cytokines, was reported [Meng et al. used CD34-positive cord blood and adult peripheral blood cells in combination with a single factor, namely, OCT4 to demonstrate a direct programming of patient-specific somatic cells into MSCs . An episorigenic , 72. Conorigenic . A cocktreported . Thus, areported .Conclusively, chemical-induced conversion or direct programming of somatic cells into MSCs is possible and augurs strong clinical potential for respective MSCs but the protocols up to now are limited.Global gene and miRNA profiling of human iPSC- and embryonic stem cell-derived MSCs demonstrated a high degree of similarity between the derived MSCs, in particular as compared to bone marrow MSCs , 46, 49.According to the general guidelines, MSCs from distinct tissue origins have a large number of similarities concerning their characteristics, but the isolated MSCs remain a heterogeneous cell population until a clonal expansion. Up to now, it is not clear, whether tissue-resident MSCs are the progenies of one ancestor cell lineage or the results of parallel cell developmental events , 84. TheThe tissue-specific homing and the activities of isolated and cultured MSCs prior to transfusion are supposed to be based on an underlying transcriptional code caused by epigenetic memory allowing them to home back to the tissue they originally were derived from . In lineThe age of MSCs may also have a major impact on their therapeutic outcome, as the differentiation potential of MSCs decreases with age . Aged MSFinally, the use of pluripotent stem cell-derived MSCs could be limited due to allogenic rejection or teratoma formation. In particular, the clinical use of the successful generated MSCs from human iPSCs and embryonic stem cells has been hampered by the tumorigenic potential elicited by undifferentiated iPSCs potentially remaining in the differentiated cell population, the lengthy and inefficient differentiation process, and genomic instability due to suboptimal culture conditions. Observed differences and efficiencies in MSC generation might be based on a somatic memory of the different cell types used for iPSC generation –100. In in vitro is the direct conversion of somatic cells, for example, easily accessible fibroblasts or blood cells. However, respective studies and standardized protocols used to prepare large-scale MSC as well as useful tests to compare their potency are limited up to now. The significant molecular and functional differences in the properties of the generated MSCs according to their different origins influence the respective therapeutic potential. Therefore, the identification of the (tissue-specific) nature of in vitro generated MSCs from pluripotent stem cells needs further investigations as well as the establishment of protocols which would allow the generation of tissue-specific MSCs. Finally, the somatic memory of iPSCs should be carefully considered before clinical translation. Up to now, there are no reports on the direct comparison of MSCs generated by different approaches, which must be investigated in future studies together with the clinical safety of different MSC sources. Concerning the patient-derived autologous MSCs generated in vitro, the respective genetic background should be a benefit for (disease) modeling studies, but the same genetic or acquired abnormalities that predisposed a patient to a particular disease will be persisted in the respective MSCs which might result in dysfunctional MSCs with reduced therapeutically activities.Beside the abundant origins but low frequencies of tissue-specific MSCs, the potentials to generate patient-individualized MSCs with comparable properties that bypass the immunogenicity and ethical issues in therapeutically relevant numbers are central advantages of using somatic cells and iPSCs as MSC source. Concerning the proliferative and regenerative potency of generated MSC, iPSC-derived MSC may be superior to somatic cell-derived MSC because MSCs differentiated from iPSCs might be closer to fetal MSCs, since pluripotent stem cells represent the early time point in development. In contrast, the tumorigenic potential of undifferentiated iPSCs in a population of iPSC-derived MSCs is a significant safety concern for iPSC-related clinical applications. This risk might be further reduced by the use of MSCs derived from integration-free iPSCs. An alternative quite promising method to gain MSCs


Correction to:Translational Psychiatry10.1038/s41398-019-0391-z published online January 2019After publication, the author’s noted that the original Article contained clerical errors in the Results section.The following passage from the original text:“For example, we observed significant differences in body weight , most OFT indices ), crossing counts , and rearing counts  starting in the 3rd week of exposure to CUMS. We did not observe significant changes in sucrose preference rate  or grooming time of OFT  until the last week of CUMS modeling, although an isolated notable change was evident in the 1st week.”Has been updated to:“For example, we observed significant differences in body weight , two OFT indices ), and rearing counts  starting in the 3rd week of exposure to CUMS. We did not observe significant changes in sucrose preference rate , grooming time  or crossing counts  of OFT until the last week of CUMS modeling, although an isolated notable change was evident in grooming time  at the 1st week.”


Self-expanding removable stents are used for the treatment of esophageal strictures. Partially covered metal stents become embedded in the esophageal wall due to mucosal tissue reaction providing good anchorage. This can also lead to extreme difficulty in the removal of such stents. Several different individual techniques have been used in literature for removal of these esophageal stents. Ours is the first case using a combination of cryoablation and stent-in-stent technique for removal of an extremely difficult case of embedded esophageal stent. Self-expanding removable esophageal stents have been used in a variety of conditions including refractory or recurrent benign esophageal strictures, malignant obstructions, tracheoesophageal fistula, perforations, or leaks . Self-exFully covered SEMS (FC-SEMS) are appropriate for benign disease because the complete stent coating prevents tissue ingrowth and allows for reliable endoscopic removal of the stent. Partially covered SEMS (PC-SEMS) with its proximal and distal exposed bare metal flares and uncovered SEMS typically develop reactive mucosal tissue hypertrophy causing tissue ingrowth into the stent interstices, which can help lower migration rates, but render such stents often unremovable –3; thus A 72-year-old man with history of localized esophageal carcinoma with a history of neoadjuvant chemoradiation and esophagectomy presented to a local hospital with dysphagia four months after the surgery. Endoscopy revealed a benign-appearing esophageal stricture at the site of the anastomosis, and biopsies confirmed benign tissue. He was treated with a series of esophageal dilations and temporary placement of a FC-SEMS by his surgeon. This stent was removed after 3 months of placement; however, the patient developed recurrent symptoms after several weeks. Given no evidence of cancer recurrence, a 100 mm × 23 mm Wallflex PC-SEMS  was placed by his surgeon as an attempt for a permanent solution to the patient's dysphagia.The patient developed recurrent dysphagia after 3 months. A CT chest with oral contrast demonstrated the PC-SEMS in appropriate position at the anastomosis but demonstrated evidence of circumferential soft tissue extending approximately 2 cm in length and 7 mm in depth nearly occluding the proximal side of the stent. An upper endoscopy confirmed the above finding, and biopsies which concluded this represent benign and hypertrophic tissue.This patient was then referred to our institution for further management and consideration of stent removal. At the time of referral, the PC-SEMS had been in place for almost 5 months. Repeat endoscopy demonstrated a benign-appearing stricture in midesophagus, beyond which the standard 9.8 mm endoscope could not pass. An ultraSlim 5.5 mm gastroscope was then advanced through the stricture, and the stent was identified beneath the tissue ingrowth and extending across the anastomosis. The proximal edges of the stent were not visible and fully covered by tissue ingrowth . CryoablVarious techniques have been described in the literature for PC-SEMS retrieval such as using rat-tooth forceps, argon plasma coagulation (APC) , 5, piecCryoablation has been used for the treatment of benign, dysplastic, and neoplastic lesions of the esophagus –9. The mHirdes et al.  reportedLow and Kozarek  have proChandrasekhar et al.  describeIn cases like ours with tissue embedding at both the proximal and distal ends, stripping of the stent using forceps or invagination techniques was difficult with a significant risk of complications. Reducing the tissue burden with a combination of cryoablation and stent-in-stent technique provided greater chances of successful stent retraction and should be considered a valid clinical option for removal of embedded esophageal stents.


Acute aortic syndromes comprise a group of potentially fatal conditions that result from weakening of the aortic vessel wall. Pre-emptive surgical intervention is currently reserved for patients with severe aortic dilatation, although abundant evidence describes the occurrence of dissection and rupture in aortas with diameters below surgical thresholds. Modern imaging techniques (such as hybrid PET-CT and 4D flow MRI) afford the non-invasive assessment of anatomic, hemodynamic, and molecular features of the aorta, and may provide for a more accurate selection of patients who will benefit from preventative surgical intervention. In the current review, we summarize evidence and considerations regarding predictive aortic imaging and highlight evolving imaging modalities that have shown promise to improve risk assessment for the occurrence of dissection and rupture.• Guidelines for the preventative management of aortic disease depend on maximal vessel diameters, while these have shown to be poor predictors for the occurrence of catastrophic acute aortic events.• Evolving imaging modalities (such as 4D flow MRI and hybrid PET-CT) afford a more comprehensive insight into anatomic, hemodynamic, and molecular features of the aorta and have shown promise to detect vessel wall instability at an early stage. CardThe aorta is the largest artery in the body and runs from the aortic valve until the abdominal bifurcation. From proximal to distal, it consists of the aortic root, ascending aorta, aortic arch, descending thoracic aorta, and abdominal aorta Fig. . Cross-sAn aneurysm is defined as a localized arterial dilatation of ≥ 2 standard deviations (SDs) above the expected vessel diameter . The undProgressive aortic dilatation is a well-acknowledged risk factor for the occurrence of both acute dissection and rupture. While these natural complications are rare in ascending aortic aneurysms of moderate size , a sharp step-up in their occurrence—to 6.9% yearly—is observed when the diameter exceeds 60 mm  have been associated with abnormal development of the aortic valve [The role of hemodynamics in the pathogenesis of aortopathy has been subject of a long-standing debate. Especially in BAV patients, who exhibit enlarged aortic diameters even in the absence of valvular dysfunction, there has been controversy regarding the origin of aneurysm formation . Two theic valve , 47. Givic valve , 49. Iniic valve . Howeveric valve , 51.Over the past few years, the hemodynamic theory has become increasingly popular, along with the evolvement of functional imaging modalities that afford a more comprehensive insight into aortic hemodynamics. In flow MRI, phase-contrast (PC) techniques are used to generate image contrast between moving protons (such as in blood) and stationary protons (most soft tissues). The underlying concept is based on the feature of protons to accumulate an MRI phase shift that is proportional to the speed at which they move along a magnetic gradient field. Phase-contrast MRI is traditionally performed using a manually positioned two-dimensional (2D) acquisition that encodes velocity in one principal direction. This approach generates time-resolved velocity maps, which can be used to quantify flow rates and velocities of blood moving through the imaging slice. As such, it enables evaluation of a broad spectrum of cardiovascular diseases, including assessment of shunt fractions and valvular regurgitant volumes. Over recent years, methodological advances have facilitated the acquisition of time-resolved, three-dimensional, three-directionally encoded velocity data. This technique, commonly known as 4D flow MRI, affords a uniquely detailed flow visualization within the heart and large vessels and allows post hoc flow quantification at any location within an acquired volume . FurtherFlow displacement is another 4D flow MRI parameter that has shown potential to predict progression of aortic disease. It provides a quantitative measure of flow eccentricity by calculating the distance between the location of peak systolic flow and vessel center in a 2D imaging plane . Like WS+ and CD68+ cells were found throughout the medial layer of TAAs [Immunohistochemical studies have reported extensive inflammatory activity within the aneurysmatic vessel wall . Whereas of TAAs . Of note of TAAs . Since l of TAAs , 62. A s of TAAs , 64. Fur of TAAs –70. As y of TAAs . Of the In conclusion, no clear relation between FDG-PET and clinical outcomes in thoracic and abdominal aneurysm patients has yet been demonstrated, although it should be noted that the inconsistency between studies partly relates to their small and heterogeneous study populations. Prospective studies with larger patient cohorts are warranted to assess the actual clinical value of FDG-PET. Also, future work could address the clinical value of novel radiotracers that have shown potential to target other processes within the inflammatory pathway of aneurysmal disease, such as expression of matrix metalloproteases (MMPSense 680), active vascular calcification (18F-NaF), and chemokine receptor 4 expression (68-Ga-pentixafor) –74. The Excessive aortic dilatation (diameter ≥ 5.5 cm) poses a significant risk for the occurrence of acute aortic events, and patients with aneurysms beyond this size should be referred for prophylactic surgical intervention. However, the majority of acute aortic events occur in aortas with diameters below surgical thresholds. Therefore, these patients would not have qualified for preventative surgery, even when screened appropriately prior to event onset. Several modern imaging techniques (such as hybrid PET-CT and 4D flow MRI) have shown promise to detect vessel wall instability at an early stage. Studies that aim to demonstrate a causal link with the occurrence of acute aortic syndromes are currently ongoing and could lead to the integration of these techniques into clinical practice guidelines.


Eosinophilic lung diseases represent a heterogeneous group of disorders with prominent infiltrate of eosinophils in lung interstitium and alveolar spaces. Peripheral blood eosinophilia is often present. Infections, drugs, allergens, toxic agents have to be evaluated as possible causes of eosinophilic lung infiltrates. The category of myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, FGFR1 and PCM1-JAK2 represents an uncommon cause of eosinophilic lung infiltrate.We report the case of a 70-year old man complaining of dry cough and dyspnea. Ground glass-opacities were seen on imaging studies and peripheral blood eosinophilia was present. A thorough step-wise patient’s evaluation led to identify the clonal nature of eosinophilia and the diagnosis of myeloid/lymphoid neoplasm with eosinophilia and rearrangement of PDGFRA was made.Correlation with clinical history, laboratory tests and imaging studies is essential to achieve the correct diagnosis when facing with eosinophilic lung infiltrates. A prolonged eosinophilia can cause life-threatening organ damage. Identification of PDGFRA rearrangement, as in the present case, is particularly critical given the sensitivity and excellent response to imatinib, which has completely changed the natural history of this neoplasm. Eosinophilia comprises a heterogeneous group of disorders that, except for the eosinophilia feature itself, have few things in common . As eosiEosinophilia is more often secondary to a broad variety of both non-neoplastic and neoplastic disorders –6. ClonaThe present challenging case of eosinophilia clarifies the progressive workup, which led to the diagnosis of myeloid/lymphoid neoplasm with eosinophilia and rearrangement of PDGFRA, a rare disease with less than 1 case per 1000000 persons per year . In the A 70-year old man presented with dry cough and dyspnea on exertion over the preceding 8 months. He was afebrile, with no history of allergies, asthma, drug intake or travelling. Physical examination revealed a moderately enlarged spleen; wheezes were present at pulmonary auscultation. Blood tests showed an increasing leukocytosis 17000/mmc) with up to 2000 eosinophils/mm3. Stool, urine and blood were negative for parasitic infections. Pulmonary function tests, with forced expiratory volume in 1 s (FEV1) of 60%, showed moderate small airway obstruction. High resolution computed tomography (HRCT) scan of the thorax revealed patchy ground-glass opacities bilaterally, predominantly in the lower pulmonary lobes (Fig. 7000/mmc Eosinophilia is defined as a peripheral blood eosinophil count greater than 1500/mm3 . It can The interest of our case resides mainly on the progressive thorough step-wise patient’s study leading to diagnosis and adequate treatment. The clinical presentation together with radiological pulmonary findings led to think of an interstitial lung disease. BAL evaluation identified a high percentage of eosinophils. In BAL specimens eosinophils are frequently degranulated or show cytoplasmic vacuolization and may be overlooked and misinterpreted either as neutrophils or macrophages . In the After secondary causes of eosinophilia were excluded, our patient’s work-up proceeded to evaluate a primary bone marrow disorder. Bone marrow aspirate and trephine biopsy examination in conjunction with FISH analysis identified the presence of PDGFRA rearrangement.The category of myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, FGFR1 and PCM1-JAK2 includes a group of disorders characterized by an aberrant tyrosine kinase activity resulting either from a fusion gene or a mutation . This grIn conclusion, recognizing the category of myeloid/lymphoid neoplasms with eosinophilia is crucial for treatment –6. The a


Cutaneous adverse reactions to medications are extremely common and display characteristic clinical morphology. A fixed drug eruption is a cutaneous adverse drug reaction due to type IV or delayed cell-mediated hypersensitivity. Ivermectin, a broad-spectrum anti-parasitic compound, has been an essential component of public health campaigns targeting the control of two devastating neglected tropical diseases: onchocerciasis (river blindness) and lymphatic filariasis.We report the case of a 75-year-old Cameroonian man of the Bamileke ancestry who developed multiple fixed drug eruptions a few hours following ivermectin intake that worsened with repeated drug consumption. Discontinuation of the drug, counselling, systemic steroids, and orally administered antihistamines were the treatment modalities employed. Marked regression of the lesions ensued with residual hyperpigmentation and dyschromia.Keen observation on the part of physicians is mandatory during the administration of ivermectin for quick recognition and prevention of this adverse drug reaction. A fixed drug eruption (FDE) is a cutaneous adverse drug reaction (CADR) due to type IV or delayed cell-mediated hypersensitivity . It can Many cases of FDE are reported in clinical practice today with several variants being described based on their clinical features and distribution of the lesions . It has Ivermectin via mass distribution has become essential for health campaigns in the elimination of two disfiguring and devastating tropical diseases: onchocerciasis (river blindness) and lymphatic filariasis . HundredFDE secondary to ivermectin is reported extremely rarely, emphasizing that there are few reports of this condition. Here we present a rare case of multiple FDE in a 75-year-old man following repeated ivermectin intake.A 75-year-old man from the South-West Region of Cameroon (an endemic zone for onchocerciasis) and of Bamileke ancestry presented to our clinic with skin lesions that had been evolving for over a year. The eruptions were first noticed a few hours after he took 12 mg of ivermectin (Mectizan) during mass drug administration (MDA) campaigns carried out every 3 months  during the ensuing campaign resulted in worsening of the old lesions with development of multiple new lesions over his face, back, and extremities. His family and medical history were not remarkable for any previous drug or cross-reactivity reactions.On physical examination, he looked well with vital signs within normal limits. There were multiple well-defined circular erythematous hyperpigmented plaque lesions of sizes ranging from 1 × 3 cm to 7 × 10 cm on his face, neck, groin area, and both extremities  serology, urine analysis, and biochemistry (liver and kidney function tests) were normal. Erythrocyte sedimentation rate was at 65 mm/hour after the first hour, while punch biopsy of the skin, and antinuclear antibodies (ANA)/antineutrophil cytoplasmic autoantibody (ANCA) were requested but were unavailable.A working diagnosis of FDE was made based on clinical signs and patient history despite the lack of histopathological findings.Discontinuation of ivermectin (plus counselling on avoidance of other possible culprits), a short course of systemic corticosteroids (prednisone 60 mg daily for a week), and orally administered antihistamines (hydroxyzine 75 mg daily) were employed as treatment modalities. Close patient follow-up revealed marked regression of lesions within a fortnight with residual hyperpigmentation Figs.  and 3.FiIvermectin, a semi-synthetic derivative of avermectin, was discovered in 1975 and is a broad-spectrum anti-parasitic compound with the ability to kill parasites both inside and outside the body; hence the term endectocide , 9. It iSide effects from ivermectin are common, and include mostly abdominal pain as well as asthenia, hypotension, and dizziness. Prolonged liver dysfunction in a patient with scabies has been reported . CADRs tSevere flares of FDE may closely resemble other forms of erythroderma including atopic dermatitis, erythema multiforme (EM), SJS, and hypersensitivity vasculitis, both clinically and histologically. Biopsy of skin specimens may be required to aid the diagnosis in selected situations.Our patient reported lesions a few hours following ingestion of ivermectin and he had multiple sharply marginated, annular, itchy, erythematous plaques which would make FDE more likely than EM and SJS where lesions are atypical, irregular, purpuric macules with occasional blistering. This was further confirmed by the fact that discontinuation of the offending agent led to resolution of symptoms. The absence of palpable purpura, nodular lesions, ulceration, and racemosa coupled with no other system involvement made vasculitis less likely with the negative laboratory results.Our diagnosis was based mainly on our patient’s history and we concluded that it was probable FDE based on the Naranjo Adverse Drug Reaction Probability Scale : our patAn orally administered drug re-challenge test was not done on this patient as he denied consent, however, the fact that he reported lesions each time after the consumption of ivermectin made it the likely culprit here. Re-challenging the patient to the suspected offending drug is the only known test to possibly distinguish the causative agent, however, patch testing of suspected drug can also be helpful in checking cross-sensitivities to medications , 17.Given the severity of this condition (approximately two-thirds of TBSA), a short course of systemic corticosteroids and orally administered antihistamines were administered alongside discontinuation of the drug with education on other possible culprits (mainstay of treatment). Follow-up was marked by regression of lesions with apparent dyschromia .We have presented, to the best of our knowledge, a very rare case of FDE secondary to ivermectin from Cameroon. Although a non-life-threatening complication of CADRs and bearing in mind that other life-threatening complications like SJS have been recently reported , the gen


V600mutBRAF metastatic melanoma, the combination of BRAF and MEK inhibitors  has undergone multiple resistance mechanisms, limiting its clinical benefit and resulting in the need for response predicting biomarkers. Based on phase III clinical trial data, several studies have previously explored baseline genomic features associated with response to BRAFi + MEKi. Using a targeted approach that combines the examination of mRNA expression and DNA alterations in a subset of genes, we performed an analysis of baseline genomic alterations involved in MAPK inhibitors’ resistance in a real-life cohort of V600mutBRAF metastatic melanoma patients. Twenty-seven patients were included in this retrospective study, and tumor samples were analyzed when the BRAFi + MEKi therapy was initiated. The clinical characteristics of our cohort were consistent with previously published studies. The BRAFi + MEKi treatment was initiated in seven patients as a following-line treatment, and had a specific transcriptomic profile exhibiting 14 genes with lower mRNA expression. However, DNA alterations in CCND1, RB1, and MET were only observed in patients who received BRAFi + MEKi as the first-line treatment. Furthermore, KIT mRNA expression was significantly higher in patients showing clinical benefit from the combined therapy, emphasizing the tumor-suppressor role of KIT already described within the context of BRAF-mutant melanoma.In  BRAFV600-mutated (V600mutBRAF) lesions are currently described in approximately 50% of patients with metastatic melanoma, and are known to constitutively activate the MAPK (mitogen-activated protein kinase) pathway  for quantitative variables and in terms of number (%) for qualitative variables. PFS was defined as the time between BRAFi + MEKi initiation and disease progression or death of any cause under treatment, whichever occurred first. Patients were censored at the time of BRAFi + MEKi interruption. PFS was estimated using the Kaplan–Meier method, and comparisons were performed with the log rank test. Copy number variations were dichotomized as amplifications (>5) and as deletions (<0.5). The number of DNA alterations between groups was compared using a opackage . ΔΔCt, dV600mutBRAF metastatic melanoma patients receiving this combined therapy as a first-line treatment. KIT was found to be overexpressed in patients with an improved clinical course, suggesting its potential predictive value. Further studies on larger cohorts are needed to support our findings.Despite the small number of patients included in our study, our targeted genomic approach in a real-life cohort of patients initiating the BRAFi + MEKi treatment revealed a differential baseline transcriptomic profile with higher mRNA expression in