Optical neuropathies are neuro-ophthalmologic disorders, the main symptoms of which are the decrease of visual acuity and the alteration of the color vision. Optical coherence tomography has been one of the most important innovations in ophthalmology, which offered the possibility to analyze specific structures of the retina. Optical coherence tomography performs in vivo, real-time, noncontact scanning and provides cross-sectional and volumetric images with a resolution approaching that of histology. Optical coherence tomography offers the opportunity to study neurological diseases in an objective and non-invasive manner. The measurements of retinal nerve fiber layer can be an objective measurement of nerve swelling or nerve atrophy. By analyzing the ganglion cell complex, optical coherence tomography can help detect early axonal damage and may predict the visual outcome. It can be useful for diagnosis and follow-up of optic nerve and chiasmal compressive diseases. Furthermore, optical coherence tomography is useful in patients with multiple sclerosis in distinguishing macular disease from optic neuritis and in monitoring the treatment. Multiple studies and clinical observations support the importance of optical coherence tomography in the diagnosis, treatment, and follow-up of optic neuropathies.Abbreviations: OCT = optical coherence tomography, VA = visual acuity, RNFL = retinal nerve fiber layer, GCL = ganglion cells layer, MS = multiple sclerosis, ON = optic neuropathy, NAION = non-arteritic ischemic anterior optic neuropathy, LHON = Leber hereditary optic neuropathy, RE = right eye, LE = left eye Optic neuropathy refers to damage to the optic nerve due to multiple causes. Damage and death of these neurons leads to characteristic features of optic neuropathy. The main symptoms are a decrease of visual acuity (VA) and the alteration of the color vision, with colors appearing subtly washed out in the affected eye. On clinical examination, the optic nerve head appears edematous in early stages. A pale disc is characteristic of long-standing optic neuropathy. Optic atrophy is the end result of any disease that damages nerve cells anywhere between the retinal ganglion cells and the lateral geniculate body. 1]. The operation of the OCT is based on an optical measurement technique called “low coherence interference”. When light emitted by the source of the device is directed to the eye, it is reflected by intraocular structures with different optical properties. The OCT uses coherent laser light to sweep the retina and analyze the light reflected by the retinal layers. This way, an in vivo “biopsy” of the retina provides information of high quality resolution about all its layers . The OCT allows qualitative  and quantitative analyzes  [4].Optical Coherence Tomography (OCT) is a non-invasive method that makes the introduction to high-tech medicine, a technique that revolutionizes the early detection of ocular and central nervous system disorders . OCT is a method capable of producing two-dimensional cross-sections of the retina with very good spatial resolution. OCT has a wide use in ophthalmology, especially in the diagnosis and monitoring of a wide variety of retinal diseases affecting the macula, as well as calculates the thickness of the retina [Retinal layers:1. Retinal pigment epithelium layer2. Layer of the external segment of photoreceptor cells3. External limiting membrane4. The external nuclear layer5. External plexiform layer6. The internal nuclear layer7. Internal plexiform layer8. Ganglion cells layer (GCL)9. Nerve fibers layer10. Internal limiting membraneOCT in different optic neuropathies1. Optic neuritis in multiple sclerosis5]. While visual recovery from ON is considered as a first demyelinating event [6], studies showed that patients would have deficits that are not well captured by high-contrast visual acuity alone [7]. Optic neuropathy (ON) may be the first clinical demyelinating event in up to 20% of the patients with multiple sclerosis (MS) and the probability of developing clinically-definite MS was 50% by 15 years after the onset of acute ON [8]. The OCT measurements showed both axonal loss and retinal ganglion cells loss.The OCT exam allows a correlation between the structural aspect  and visual dysfunction. The OCT exam shows a thinning of the RNFL and the ganglion cells layer GCL, and can be used as a marker in the follow up of multiple sclerosis patients. Trip et al. reported a 33% reduction in peripapillary RNFL thickness in eyes with a history of ON and incomplete recovery. There was a 27% reduction in the affected eyes compared to the unaffected fellow eyes [9] .The decrease in the peripapillary RNFL thickness by approximately 10-40μ is maximal at 3-6 months after the acute episode, and a stabilization is observed at 7-12 months [10]. In 2006, Fisher et al. compared the RNFL thickness among MS eyes with a history of ON, MS eyes without a history of ON and disease-free eyes. The RNFL thickness was reduced significantly among MS eyes as a group overall (92 μ) vs. controls (105 μ) and particularly reduced in MS ON eyes (85 μ) [11].OCT was able to identify mild and even clinically undetectable optic disc edema in eyes with acute ON [12], while INF-β can cause exudates and retinal hemorrhage [13]. An OCT exam is recommended before treatment, and one at every 3-4 months .OCT plays a role in monitoring the treatment with fingolimod and INF-β. Fingolimod can cause macular edema (0.3-1.2% of the patients) and uveitis [14].Patients with DeVic disease can present microcysts in the internal nuclear layer on OCT - signs of inflammation, autoantibodies against aquaporin 4, microglial activation, neurodegenerative changes . In time, the edema reduces, and we can observe a thinning of the RNFL corresponding to optic nerve atrophy, on OCT. At 2 months post acute episode, the RNFL thickness in the affected eye is similar to that of the congenital eye and at 3-4 months, there is a 40% decrease in the thickness of the RNFL compared to the healthy eye. At 12 months, the RNFL thickness stabilizes  .At presentation, the OCT shows the RNFL edema. Histopathological studies have shown a doubling of RNFL in NAION [18]. Hedges observed that 8 out of 76 eyes with NAION had subfoveal fluid, at 4 weeks after the acute phase [18]. In NAION, it appears to be a more significant damage to the maculopapillary fibers, which corresponds to a central defect. Papchenko found a strong correlation between macular thickness and visual field sensitivity. Macular thickness may be a surrogate marker for determining the extent of optic nerve damage [19]. Ganglion cells thickness correlates with visual field parameters, both in acute and chronic phase. Ganglion cells thickness measured by OCT can detect axonal damage when the RNFL is edematous [20] .Certain patients with secondary papillary edema NAION develop subretinal fluid. OCT shows peripapillary subretinal hyporeflectivity adjacent to RNFL thickening and subfoveolar hyporeflectivity . RNFL thickening is correlated with axonal edema, with mitochondrial redistribution at dysfunctional ganglion cells, affecting the prelaminar unmyelinated portion of the optic nerve axons [22].In healthy carriers, the OCT test showed RNFL thickening in temporal quadrants and in asymptomatic men in the inferior sector [23] . At the atrophic stage, a RNFL thinning appears in all segments . Patients who recover from VA have a higher RNFL thickness than those who do not recover [23]. In the acute phase and in the first 6 months of onset, the RNFL analysis shows thickening in the upper and lower segments, compared to the control group [24]. 6% of the patients had microcystic degeneration and were associated with focal retinoschisis induced by vitreomacular tractions in combination with RNFL atrophy [25]. The OCT analysis of macular thickness showed a thinning of the GCL at the early stage in the sectors of the inner ring  . Meningioma occurs with the progressive decrease of VA. The OCT exam is important in predicting the visual prognosis: when the VA decrease occurs in a patient with minimal RNFL loss, the prognosis is favorable. Loo evaluated 14 eyes of 12 patients with pretreatment and post-treatment OCT, along with clinical examination. After treatment, the group with normal RNFL experienced improvement in VA, color vision, visual field, compared with the group with altered RNFL [27]. Fard et al. included 38 eyes from 23 children with optic nerve glioma and they correlated the OCT exam with clinical and radiological changes. The RNFL and macular OCT measurements were significantly different in patients with tumor progression [28]. Glioma - Avery et al. studied 62 patients with NF-1 and optic nerve glioma. OCT showed a reduction in RNFL thickness, which significantly associates with VA decrease and sensitivity to contrast [29]. After decompression, an improvement in VA and visual field in patients with normal retinal integrity was marked, as assessed by OCT [29] .Graves Orbitopathy is an autoimmune inflammatory disorder that affects extraocular muscles and orbital fat, which are responsible for proptosis, diplopia, congestive signs, and dysthyroid optic neuropathy. Because prognosis is significantly improved when early diagnosis is made, OCT can be used to detect high-risk patients. The OCT measurements of the RNFL can predict axonal losses. Forte et al. observed RNFL losses in 30% of the patients with dysthyroid optic neuropathy and intraocular hypertension [30] . Danesh-Meyer evaluated 40 patients with chiasmatic lesions, with OCT and visual field, pre and post decompression treatment. Patients with thin RNFL did not demonstrate significant improvement in VA and visual field as compared to those with normal RNFL [31].Chiasmal lesions may be caused by pituitary adenoma, craniopharyngioma, meningioma, and aneurysm. Chiasmatic lesions can cause changes ranging from minimal to severe, with optic nerve atrophy and ganglion cells loss. The OCT measurements of retinal thickness can detect neuronal loss from onset. Patients with bitemporal hemianopsia show RNFL losses in all optic nerve quadrants and a macular thinning nasal from the fovea [32].Using OCT to quantify axonal losses by measuring RNFL and macular thickness can be considered a predictor for vision recovery. Jacob et al. showed that the increase in RNFL thickness correlated with visual field improvement [5. Nutritional and Toxic Optic Neuropathies33]. The OCT measurements show a thickening of the RNFL, followed by a more pronounced thinning in the temporal sector [34].Nutritional Optic Neuropathy is common in patients with chronic diseases and malnutrition, being associated with deficiency of B1, B12, folic acid, Cu. Deficiency in these elements affects mitochondrial oxidative phosphorylation [35].Toxic Optic Neuropathies occur due to accidental ingestion of substances such as alcohol , anti-tuberculosis drugs, antimalarial drugs, antiarrhythmic , and antibiotics. They present with painless, progressive, bilateral visual acuity decrease, dyschromatopsia, and central scotoma. The site can be variable, including the retina, intraocular nerve fiber axons, and chiasm. The symptomatology is due to the damage of the papillomacular bundle, which is more susceptible due to the large unmyelinated segment and thin gauge .In early stages, toxicity accumulates in the mitochondria of the ganglion cells, causing axonal thickening, which is detectable in OCT. In the subacute stage, the OCT shows RNFL thinning of the papillomacular bundle in the inferior-temporal sector. In the late stage, RNFL thinning is observed in all layers [37]. In the late-stage, the OCT changes are the thinning of the RNFL in the inferior-temporal sector and significant thinning of the ganglion cells layer [38]. Toxic optic neuropathy to ethambutol may become clinically manifested between 2 and 12 months after the onset of treatment. It is reversible if it is recognized on time. Permanent toxic effects may occur at standard doses of 15-25 mg/ kg/ day. Early animal experiments have demonstrated ethambutol toxicity affecting the retinal ganglion cells, the optic nerve, chiasm, and optic tracts [39] .Methanol optic neuropathy is due to a cytotoxic edema at the retinal level and at the optic nerve, which appears at 48h post ingestion. OCT is used for the exploration of the macula and peripapillary retina, showing early peripapillary RNFL edema, subsequent optic nerve atrophy, and intraretinal fluid accumulation [40].Toxic optic neuropathy to amiodarone can be confused with NAION. It has an insidious onset with a less severe VA decrease and optic nerve edema. As a mechanism, we note the accumulation of cytoplasmic inclusions in lysosomes. OCT changes show RNFL thickening in early stages, followed by axonal loss and optical atrophy [41].Tobacco toxic optic neuropathy progressively affects the papillomacular bundle, which correlates to central and cecocentral scotomas. In early stages, the optic nerve appears normal, with hemorrhages and dilated vessels [OCT is a major step forward in ophthalmology, allowing the obtaining of in vivo images of the retina and optic nerve with high resolution. OCT offers the opportunity to study neurological diseases in an objective and non-invasive manner. Multiple studies and clinical observations support the importance of the OCT in the diagnosis, treatment, and follow-up of optic neuropathies, which has become an important tool in the management of this group of diseases.


In this review, we describe the insights and progress that have been gained from the investigation of direct cardiac reprogramming. We focus on the use of key transcription factors and cardiogenic genes as well as on the use of other biological molecules such as small molecules, cytokines, noncoding RNAs, and epigenetic modifiers to improve the efficiency of cardiac reprogramming. Finally, we discuss the development of safer reprogramming approaches for future clinical application.The human adult heart lacks a robust endogenous repair mechanism to fully restore cardiac function after insult; thus, the ability to regenerate and repair the injured myocardium remains a top priority in treating heart failure. The ability to efficiently generate a large number of functioning cardiomyocytes capable of functional integration within the injured heart has been difficult. However, the ability to directly convert fibroblasts into cardiomyocyte-like cells both  Heart failure (HF), the leading cause of death and hospitalizations worldwide, results from a myriad of cardiovascular diseases that lead to the death or dysfunction of cardiomyocytes. With a prevalence of 38 million people worldwide, it places a significant financial burden on health care systems, with an estimated $30 billion of annual spending in just the United States alone , 2. DespThe human adult heart lacks endogenous repair mechanisms to fully restore cardiac function after an insult; thus, the ability to regenerate and repair the injured myocardium remains a top priority in treating HF. However, the ability to efficiently generate a large number of functioning cardiomyocytes capable of functional integration within the injured heart has remained an obstacle. Current cell therapies are focused on three main approaches: (1) induction of endogenous cardiomyocytes to undergo proliferation and repopulate the damaged myocardium, (2) transplantation of cardiovascular progenitor cells (CPCs) or cardiomyocytes generated through the differentiation of pluripotent stem cells, and (3) direct reprogramming of somatic cells to cardiomyocytes or expandable CPCs without transitioning through a pluripotent intermediate. This review is focused on the last approach. Direct reprogramming was first reported in 1987 when a single cDNA encoding MyoD was transfected into fibroblasts converting them into muscle myoblasts . A few yIn this review, we describe the insights and progress that have been gained from the investigation of direct cardiac reprogramming, with a focus on the use of key transcription factors and other cardiogenic genes. Furthermore, we discuss the use of other biologics and small molecules to improve the efficiency of cardiac reprogramming and the development of safe reprogramming approaches for clinical application.Gata4, Mef2c, and Tbx5 (GMT) in mouse cardiac and tail-tip fibroblasts [Direct reprogramming of fibroblasts into cardiomyocyte-like cells was first reported in 2010 using viral overexpression of three important cardiac developmental transcription factors (TFs), roblasts . Ieda etroblasts . One sucroblasts . MEF2c, roblasts . After tMyoD was fused to Mef2c, a 15-fold increase in reprogramming efficiency was observed [One of the first TFs added to GMT was the bHLH TF HAND2 (referred to as GMHT). In cardiac development, HAND2 plays an important role in the formation of the ventricular chambers through interaction with GATA4 and NKX2–5 . GMHT trobserved . Other Tobserved .Tbx5, Mef2c, and Myocd, a developmental regulator of cardiomyocytes and smooth muscle cells, were able to induce a more complete cardiac phenotype than GMT in mouse fibroblasts [Myocd and Srf, a TF important in mesoderm formation, or GMT plus Myocd, Srf, Mesp1, another mesoderm-inducing TF, and Smarcd3, a chromatin structure-altering protein, enhanced reprogramming and the expression of cardiac sarcomeric proteins over GMT alone [Additionally, an alternative screening approach that surveyed triplet combinations of 10 important cardiac TFs revealed that roblasts . LikewisMT alone .ETS2 and MESP1 proteins to induce reprogramming of human dermal fibroblasts to cardiac progenitors [MESP1 and MYOCD in human cardiac and dermal fibroblasts was sufficient to induce expression of multiple cardiac-specific proteins, increase a broad range of cardiac genes, and exhibit spontaneous calcium transients [ESSRG , MESP1, MYOCD, and ZFPM2 (a modulator of GATA proteins) in human fetal cardiac fibroblasts and neonatal skin fibroblasts enhanced cardiac reprogramming, sarcomere formation, calcium transients, and action potentials [Despite the successes of TF overexpression to reprogram murine cells, similar approaches to reprogram human somatic cells have been more difficult to achieve. Only a few studies have reported successful reprogramming of human cells to iCMs using TFs alone. The first of these studies reported a combination of the E26 transformation-specific (ETS) TF family member genitors . Anotheransients . The thitentials . ResultsDespite the successes of direct reprogramming using forced expression of cardiac TFs, the efficiency remains low. The initial report on direct conversion of fibroblasts to cardiomyocyte-like cells noted an efficiency of 4.8%. In an effort to improve reprogramming efficiency, many methods have been developed using additional molecules. These additives can be classified into three major categories: inhibitors/cytokines, noncoding RNAs, and epigenetic modifiers. A summary of these reprogramming experiments is presented in β pathway. TGF-β has diverse and pleotropic effects through its activation and signaling. The downstream effect of the TGF-β signaling pathway involves phosphorylation of receptor-regulated SMADs that ultimately activate TFs that participate in the regulation of target gene expressions, many of which are critical in fibroblast activation and proliferation. Since inhibition of TGF-β has been shown to increase mouse embryonic stem cell differentiation to cardiomyocytes [β inhibition could improve reprogramming. The TGF-β inhibitors SB431542 and A83-01 have been added to various reprogramming combinations and have shown an increase in reprogramming efficiency. SB431542 is a selective and potent inhibitor of the TGF-β pathway through suppression of the activin A receptors ALK5, ALK4, and ALK7. A83-01 is also a selective inhibitor of ALK5, ALK4, and ALK7 but is more potent than SB431542 in its inhibition and effectively blocks phosphorylation of Smad2. When SB431542 was combined with GMHT, a 5-fold increase in reprogramming efficiency was observed in both MEFs and mouse adult cardiac fibroblasts [β pathway, other profibrotic and intracellular signaling pathways such as the Rho-associated kinase, JAK/STAT, Notch and Akt pathways have been targeted to improve reprogramming [A potential approach to improving reprogramming is to inhibit the endogenous signaling pathways and gene programs that maintain the distinct properties of fibroblasts. One of the major signaling pathways active in fibroblasts is the transforming growth factor- (TGF-) myocytes , 25, it roblasts . Likewisroblasts . In addigramming –31.in vitro, and thus, the activation of the important developmental pathway during reprogramming warrants further research.The utility of other molecules to enhance cardiac reprogramming has been inspired by using cytokines and/or modulators that are critical in mammalian cardiac development, many of which are commonly used in the differentiation of cardiomyocytes from pluripotent stem cells. Fibroblast growth factor-2 (FGF2), FGF10, and vascular endothelial growth factor (VEGF) in combination with GMT or GMHT in MEFs and mouse tail-tip fibroblasts showed an increase in the number of iCMs that spontaneously contract . These ain vivo [MicroRNAs (miRNAs) are small noncoding RNAs that induce degradation or inhibit translation of target mRNAs. miRNAs are an attractive additive to reprogramming since they play important roles in the posttranscriptional regulation of cardiac gene expression and have critical function in almost every stage of heart development. miRNA-1, miRNA-133, miRNA-208, and miRNA-499 have been shown to be cardiac- and muscle-specific and play important roles in cardiac development and function. miRNA-1, contributing to ~40% of total miRNAs in the heart, has been shown to promote cardiomyocyte proliferation and suppress apoptosis during development; however, its function in cardiac reprogramming remains unknown . It alsoin vivo . The potin vivo .Mesp1, and Myocd or GHT, Myocd, and miRNA-1, the reprogramming efficiency was increased in both human and mouse fibroblasts by repressing Snai1 and silencing fibroblast gene signatures [α-actinin+ iCMs [HAND2 and MYOCD in GMT direct reprogramming experiments using human and porcine fibroblasts [in vivo clinical application.Similarly, when miRNA-133 was used in conjunction with GMT, gnatures , 37. Zhain+ iCMs . miRNA-5roblasts , 39. WhiBvht) and Fendrr play a critical role in cardiac lineage commitment by regulating the transition from mesoderm to CPCs through activation of key cardiac development genes and TFs including some of those studied above [Hotair, Chaer, and other lncRNAs have also been shown to regulate the epigenetic landscape in cardiac development by regulating proteins involved in histone modification at targeted promoters [Long noncoding RNAs (lncRNAs) are a heterogeneous group of transcripts longer than 200 nucleotides that exert major regulatory roles in gene expression during development and disease through many different mechanisms. Recent advances in sequencing and analysis technologies have allowed many lncRNAs to be identified, but due to their complex and multiple mechanisms of action as well as to the low interspecies conservation, it has been difficult to decipher biological functions of many lncRNAs . A list ed above –44. Hotaromoters , 46. lncBmi1 activity was knocked down, the active histone mark, H3K4me3, was increased while the repressive H2AK119ub mark was reduced, leading to increased cardiac gene expression at important loci [in vivo reprogramming experiments discussed later in this review, other epigenetic modifiers that inhibit histone methyl transferases and histone demethylase have been used. The importance of epigenetic landscape and changes that happen during reprogramming have recently begun to be unraveled using a single-cell transcriptomic approach by Liu et al. [Reprogramming of one somatic cell type to another requires the activation and repression of multiple sets of genes, leading to vast genomic changes. The epigenetic landscape plays an important role in determining the reprogramming efficiency as accessibility of TFs to their DNA targets is critical. During reprogramming, epigenetic marks such as histone methylation, acetylation, and ubiquitination must be added and removed from fibroblast- and cardiac-specific genes. These modifications will suppress expression of fibroblast genes while activating cardiac genes by remodeling chromatin structure to allow or restrict the access of TFs to their target genes. It has been shown that during cardiac direct reprogramming, the trimethylated histone H3 of lysine 27 (H3K27me3), which marks inactive chromatin, increases at fibroblast promoters and decreases at cardiac promoters while the activated chromatin mark H3K4me3 shows the opposite pattern at important loci , 47. Morant loci . In noniu et al. . These rin vitro that can then be expanded in culture before transplantation into the injured heart. Upon transplantation, the CPCs will differentiate into three major cells of the heart; cardiomyocytes, endothelial cells, and smooth muscle cells. Two groups were able to successfully generate expandable CPCs using unique reprogramming cocktails containing a variety of biomolecules described above. Lalit et al. generated CPCs from fibroblasts using Mesp1, Tbx5, Gata4, Nkx2–5, and Baf60c, a chromatin remodeling protein. In vitro expansion and maintenance of a CPC state were achieved using a Wnt activator, BIO, and a JAK/STAT activator, LIF [in vitro and in vivo when transplanted. Direct reprogramming of fibroblasts to CPCs represents a scalable method for the generation of multiple cardiac cell types for clinical applications; however, this approach has not yet been applied to human cells.Another recent approach of clinical promise is the generation of expandable CPCs by direct reprogramming. The goal of this approach is to safely create CPCs tor, LIF , 51. On tor, LIF . Both grin vivo. Other studies have used in vivo transplantation of reprogrammed cells to test their regenerative potential. However, cell transplantation is complicated by many factors such as cell retention, viability, structural and functional integration, and immune rejection. Therefore, in situ repair of the heart is best studied by targeting endogenous cardiac fibroblasts through viral transfection of the infarct zone. This approach was attempted in 2012 by Song et al. and Qian et al., in which local delivery of GM(H)T viruses induced reprogramming of nonmyocytes into iCMs by 4 weeks postsurgery [β4 and GMT viruses further improved ejection fraction and reduced scar formation. Interestingly, it has been reported that the in vivo cardiac niche may improve the efficiency of reprogramming; however, the mechanisms underlying this observation remain elusive [in vivo reprogramming efficiency even further by optimizing polycistronic expression vectors to control the stoichiometry of TF expression or by the addition of small molecules delivered with TFs [The ultimate goal of direct reprogramming is to be able to repair the damaged myocardium after injury. Direct reprogramming offers two potential approaches for heart regeneration: (1) transplantation of reprogrammed fibroblasts into the infarcted heart and (2) reprogramming resident cardiac fibroblasts directly to cardiomyocytes. The first attempt at cardiac regeneration using direct reprogramming was carried out using cardiac fibroblasts that were transduced with GMT for 1 day and then transplanted into mouse hearts . Analysitsurgery , 54. Add elusive , 53, 54.with TFs , 55–57.in vivo reprogramming has been reported to be more efficient than in vitro reprogramming, despite the fact that upon injury, cardiac fibroblast expresses TGF-β, which has been shown to be inhibitory to reprogramming in vitro. Furthermore, our lab has shown that cardiac fibroblasts are a heterogeneous population from different embryonic origins [Cardiac fibroblasts are ideal targets for direct reprogramming as they are the most prominent cell type within the heart and play key roles in regulating normal myocardial function as well as adverse remodeling following injury. Various mouse and/or human fibroblast sources have been tested, including mouse embryonic fibroblasts, tail-tip fibroblasts, and dermal fibroblasts, with varying results, suggesting the importance of the starting cell type for direct reprogramming. It is also interesting to note that  origins . It is pin vivo applications. Adeno-associated virus (AAV) vectors are attractive tools for TF delivery, but the limited capacity of about 4.5 kb complicates the expression of multiple TFs in a single vector and still involves the use of an integrative viral system. Sendai virus reprogramming is an appealing alternative to AAV since it does not integrate into the host genome and has been successfully used to reprogram many different cell types to pluripotency; however, its use in direct reprogramming has not yet been explored. A recent study showed that acute expression of GMT in nonintegrating adenoviral vectors was as efficient as lentiviral vectors at reprogramming in a rat infarct model, which has increased the clinical applicability of in vivo reprogramming. As described in two recent reports, the temporal control and stoichiometric control of TFs are also important in determining reprogramming efficiency [in vivo viral reprogramming tools are unable to control dosage and temporal expression of TFs but warrant further investigation to improve reprogramming efficiency.The reprogramming results shown thus far suggest that direct reprogramming of fibroblasts can be a feasible therapeutic approach to repairing the injured myocardium. However, relatively safe methods for the delivery of various reprogramming factors need to be explored for ficiency , 59. Unfin vitro. A combination of ascorbic acid, RepSox (a TGF-β inhibitor), forskolin, valproic acid, and CHIR99021 (a WNT pathway activator through the inhibition of glycogen synthase kinase 3) was shown to reprogram MEFs and mouse tail-tip fibroblasts to iCMs in vitro [in vitro before transplantation in injured murine hearts [β inhibitor), and JNJ10198409 (a PDGF receptor tyrosine kinase inhibitor). However, the use of small molecules for in vivo reprogramming poses some unanswered questions. Small molecules can enter the blood stream and spread to other organs with unknown consequences. Additionally, the ability of timely uptake into specific target cell type remains a challenge. Development of novel biomaterials for local delivery, controlled release, and retention of small molecules is still needed.Compared to TFs and miRNAs, small molecules have many advantages such as more effective cell delivery and being nonimmunogenic and less expensive and are generally safer. Moreover, it is more convenient to control the process of reprogramming through varying small molecule concentrations and combinations in vitro . Cao et e hearts . Among tin vitro transcription system to generate mRNAs that contain a synthetic 5′ guanine cap and poly-A tail, which improves half-life and stability, as well as modified nucleotide bases that reduce the innate immune response of the host cell. This technology is endowed with a number of attractive properties that would make it a potentially powerful platform for direct cardiac reprogramming. ModRNAs can mediate robust and dose-titratable expression of key TFs over a specified time and in a particular sequence. Previous studies outlined in this review have highlighted the fact that direct cardiac reprogramming is a complex process that may require sequential treatments to better overcome the reprogramming barrier. ModRNAs may be ideal for direct reprogramming as they have a relatively short half-life; therefore, distinct factors can be expressed for a short period of time and then removed from the reprogramming cocktail or added again to continue expression. ModRNAs may open the door to following a more developmentally relevant sequence of TFs to improve transcription. It is also foreseeable that modRNAs could be combined with other small molecules, cytokines, and noncoding RNAs discussed in this review.Another promising nonviral method of direct cardiac reprogramming is the use of modified mRNAs (modRNAs) . ModRNAsin vitro for transplantation. One avenue that has the potential to generate the number of cells needed for transplantation is reprogramming to CPCs, which can be expanded in vitro before transplantation. The retention, integration, and maturation of iCMs or CPCs after transplantation remain a concern. Multiplex immunostaining and patch clamp analysis have also revealed the presence of all three cardiomyocyte types  in iCMs, therefore increasing the risk of arrhythmias [in vitro and in vivo direct reprogramming. A safe and effective approach to delivering and targeting reprogramming factors in vivo will be needed to circumvent in vitro reprogramming completely.There has been significant progress in recent years with direct cardiac reprogramming through important discoveries in understanding the mechanism of reprogramming and the biology of cardiac development. However, several challenges must be addressed prior to clinical translation of this technology. The reprogramming efficiency must be increased in order to generate enough cells hythmias . There iαMHC) versus GCaMP activity, and the method of measurement, flow cytometry versus immunofluorescence (IF), make comparisons among studies difficult. A myriad of criteria and stringency that have been used to evaluate reprogramming efficiency have been summarized by Addis and Epstein and are presented in Transcription factors, inhibitors, cytokines, noncoding RNAs, and epigenetic modulators have been shown to be important for direct cardiac reprogramming. However, studies have uncovered variable reproducibility between different labs, leading to wide differences in reprogramming efficiency, maturity, and characteristics of the iCMs. These inconsistencies can be attributed to many factors other than the reprogramming factors themselves. First, the components of culture media used during reprogramming widely vary from group to group along with the duration of reprogramming before analysis. Additionally, the induction time, the type of fibroblasts, and the amount and sequence of factors used along with the time exposed to reprogramming factors are different between protocols. Moreover, the criteria used to measure the outcome and success are inconsistent and not standardized in the field. Reprogramming success is measured by some as the presence of cardiac-related structural proteins on immunostaining, while others employ a much more detailed approach including appearance of spontaneous beating along with gene and protein expression data. Even differences in the cardiac markers used to analyze the reprogramming efficiency, cardiac troponin T (cTnT) versus alpha myosin heavy chain . Differences in fibroblast populations may be attributed to differences in epigenetic landscape, which can be influenced by many factors such as the cell environment or developmental origin. Further research on the heterogeneity of fibroblast epigenetic landscapes is warranted and will be of great benefit to direct reprogramming.Optimization of the minimal yet sufficient combination of factors to improve reprogramming requires further research. Studies presented here have revealed that simply expressing a few core transcription factors is not sufficient for efficient cardiac reprogramming . There mIn this review, we discussed the reprogramming of fibroblasts into cardiomyocyte-like cells and expandable CPCs using transcription factors, small molecules, noncoding RNAs, and other biologics for the treatment of heart failure . Despite


Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. All of them participate in one or more of eight cell cycle events. Seventeen of them have not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers. One notable exception was the apparent effect of TP53 on CRL4 activity. Depletion of NEDD8, DTL or DDB1 produced higher levels of EDR in the presence of TP53, suggesting that TP53 promotes EDR when CRL4 activity is suppressed.Simply knowing which genes are essential for preventing EDR reveals the pathways that are modified in cancer cells to promote genomic instability and its associated aneuploidy and consequently allow cancer cells to evolve so as to evade anti-cancer treatments.A high throughput screen (HTS) that was originally developed to identify small molecules that induced accumulation of excess DNA in cancer cells  was adap5 cells per well to insure they were undergoing exponential proliferation until collection. Cells were transfected with a single siRNA (40nM) at one and two days post-seeding using RNAiMAX per manufacturer's instructions. During each medium replacement, floating cells were collected and added back to the wells in order to include detached apoptotic cells in the analysis. Cells were harvested by trypsinization three days after the first transfection and combined with detached cells.Cells were seeded in 6-well dishes at 0.7×105 cells were stained with propidium iodide and subjected to FACS analysis [Cells were washed in phosphate buffered saline, and then 2×10analysis  using a MLN4924, BI2536, Etoposide, VX680, Nocodazole, Paclitaxel, and Z-VAD(OMe)-FMK (ZVAD) were purchased from Cayman Chemical Company, USA.Equal numbers of cells were collected, lysed in Laemmli sample buffer, and subjected to electrophoresis and protein immuno-blotting. Filters were probed with specific antibodies and the signals visualized with enhanced chemiluminescence (ThermoScientific).


Bicuspid aortic valve (BAV) is the most common type of congenital cardiac malformation. Patients with a BAV have a predisposition for the development of thoracic aortic aneurysm (TAA). This pathological aortic dilation may result in aortic rupture, which is fatal in most cases. The abnormal aortic morphology of TAAs results from a complex series of events that alter the cellular structure and extracellular matrix (ECM) composition of the aortic wall. Because the major degeneration is located in the media of the aorta, most studies aim to unravel impaired smooth muscle cell (SMC) function in BAV TAA. However, recent studies suggest that endothelial cells play a key role in both the initiation and progression of TAAs by influencing the medial layer. Aortic endothelial cells are activated in BAV mediated TAAs and have a substantial influence on ECM composition and SMC phenotype, by secreting several key growth factors and matrix modulating enzymes. In recent years there have been significant advances in the genetic and molecular understanding of endothelial cells in BAV associated TAAs. In this review, the involvement of the endothelial cells in BAV TAA pathogenesis is discussed. Endothelial cell functioning in vessel homeostasis, flow response and signaling will be highlighted to give an overview of the importance and the under investigated potential of endothelial cells in BAV-associated TAA. Bicuspid aortic valve (BAV) is the most common congenital cardiovascular malformation with a prevalence of 0.5–1.5% in the general population and a male predominance of about 3:1 Roberts, . In thisWhile smooth muscle cells (SMCs) in the healthy media have a contractile phenotype, they are not terminally differentiated. This ensures the ability to regenerate the vessel wall after injury. This flexible change between cellular phenotypes is called “phenotypic switching,” with the contractile and synthetic SMCs on opposite sides of the spectrum. After phenotypic switching the synthetic SMCs can migrate towards a wounded area by secreting proteinases to break down the ECM. Synthetic SMCs also proliferate and produce ECM to repair the wall. When the vessel wall is repaired, synthetic SMCs will re-differentiate toward a contractile phenotype. TAA is characterized by phenotypic switching of contractile to synthetic SMCs and fragmentation of elastic lamellae Figure . The BAVACTA2, MYH11. Furthermore, in BAV patients multiple mutations have also been found in genes related to signaling proteins such as NOTCH1 and genes related to the TGFβ signaling pathway  or that an intrinsic cellular defect contributes to the formation of BAV as well as to the dilation of the aorta in these patients  and the main structural component of the aorta (the media) the function of endothelial cells is to communicate the signal between these two layers. Upon flow and stimuli such as inflammatory cytokines, signaling pathways like TGFβ, angiotensin, and nitric oxide (NO) allow endothelial cells to directly target the contraction status of SMCs or indirectly target the SMC contractile phenotype to influence vessel wall functioning Figure . PrimaryThe flow pattern of blood from the heart into the aorta is altered by a BAV , which is important for maintaining blood pressure. By constriction/relaxation of blood vessels and adjusting water retention of the kidneys, the blood pressure is regulated. The juxtaglomerular cells in the kidney and baroreceptors in vessel wall can sense arterial blood pressure. Upon a drop in pressure, renin is released by the juxtaglomerular cells and renin then converts angiotensinogen into angiotensin I (ANGI), which in turn is converted by angiotensin converting enzyme (ACE) into angiotensin II (ANGII). Amongst others, ANGII can cause contraction of the SMCs to increase blood pressure. This contraction is caused by the binding of ANGII to the angiotensin II type 1 receptor (AT1) on the SMCs, resulting in activation of the CaApoE−/− mice demonstrated to cause progressive TAAs and AAAs  demonstrated promising results for preventing and even reversing aortic dilation  inhibited aneurysm formation  is produced when NO synthase (NOS) converts arginine into citrulline, releasing NO in the process. NOS was originally discovered in neurons (nNOS/NOS1), after which inducible NOS (iNOS/NOS2) and endothelial NOS (eNOS/NOS3) were found. eNOS phosphorylation increases NO production and is induced by factors such as shear stress, acetylcholine and histamine. NO has a very short half-life of a few seconds, making it a local and timely signal transducer. Endothelial secreted NO diffuses into the SMC where it relaxes the cell by increasing the calcium uptake into the sarcoplasmic reticulum: NO stimulates the sarco/endoplasmic reticulum ATPase (SERCA), and thereby decreases cytoplasmic CaApoE−/− mice and HPH-1 mice 1 and ALK5. Upon ligand binding, ALK5 can phosphorylate SMAD2 or SMAD3 and ALK1 can phosphorylate SMAD1, SMAD5 or SMAD8. The phosphorylated SMADs translocate into the nucleus with SMAD4 to induce the canonical signaling pathway. TGFβ can also signal via non-canonical pathways by activating PI3K/AKT, MAPK, or NF-kB. Via the canonical and non-canonical pathways, TGFβ influences cell cycle arrest, apoptosis, inflammation, proliferation, and more.In endothelial cells, TGFβ signaling can either inhibit or stimulate the cell growth and function depending on the context  is highly expressed by endothelial cells and plays a role in the ALK1 and ALK5 signaling balance  mesenchyme of the developing AV cushions. Intriguingly, a single-nucleotide polymorphism in ENG was found in BAV patients, indicating that in BAV patients endothelial TGFβ signaling might be altered, potentially promoting a phenotypic switch in the underlying SMCs . PAH and HHT are 2 major genetic diseases in which the role of the endothelial cells is well recognized. Two recent advances in these research fields worth mentioning for future perspectives in BAV TAA research, will be discussed in the next paragraphs.−/− mice developed pulmonary hypertension, a phenotype that was rescued by restoring miR-143 levels  causes narrowing and occlusion of the lumen, leading to an increased pressure in the lungs and increased load of the right ventricle  or ALK1  and endothelial colony forming cells (ECFCs) will provide a new study model, facilitating patient specific analysis of the endothelial contribution to the disease (Asahara et al., Although few studies on the role of endothelium in BAV disease and its associated TAAs have been performed in the last decade, some seminal papers have been published. In this review we have created an overview of the recent studies implicating endothelial cells as a pivotal player of vascular homeostasis, and their underappreciated role in TAA pathogenesis in patients with a BAV. Figure VvdP, KK, and M-JG conceptualized the review. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasticity is the hallmark of stem cells. At the same time, stem cells, like any other cell type, are influenced by their microenvironment and respond to it accordingly. A specific microenvironment is defined by a variety of factors, including biological and chemical factors, cell-cell interactions, but also metabolic and mechanical cues. Such dynamic and specialized microenvironment where the stem cells reside is considered a stem cell niche. Tissue injury as well as malignant tissue alterations lead to changes in the niche influencing the plasticity and biology of residing stem cells. Similarly, the niche changes upon tissue damage, which eventually induces differentiation of stem cells and ultimately regeneration of the tissue.Within this research topic, different aspects of microenvironment-derived stem cell plasticity are discussed including techniques to detect niche-residing stem cells, cell communication within the niche and the influence of oxygen tension and inflammation on stem cell plasticity. Furthermore, disease-associated microenvironments are addressed. Finally, two contributions focus on the use of micro environmental cues for therapeutic application of stem cells.Morikawa and Takubo concisely summarize methods used for visualization, tracking and localization of hematopoietic stem cells within their microenvironment, and review recent advances in our understanding of stem cell/niche biology through examples of imaging-based studies of various physiological and pathological hematopoietic activities.Interactions of hematopoietic stem/progenitor cells with their microenvironment is central for regulation of hematopoiesis, both in steady state and in disease. Development of imaging techniques brings about powerful new methodologies to enlighten these very complex and dynamic processes. Dostert et al. The two-way interaction has been considered, describing MSCs as donors and recipients of EVs. A number of factors discovered in EVs have been distinguished as protein effectors and micro RNAs involved in cell-cell communication, specifically focusing on EV-mediated tissue regeneration, immunomodulation and cross talk with cancer cells.An overview of the communication between mesenchymal stem cells (MSCs) and other cells in their microenvironment via extracellular vesicles (EVs) is provided by Miclau et al. have summarized the current literature on how oxygen directs MSC fate in the context of fracture healing. There is clear evidence from preclinical and clinical studies, that a disrupted vasculature and/or ischemic conditions due to co-morbidities have a negative impact on fracture healing. However, on a cellular level, it is yet not clear how the tissue oxygen level is associated with stem cell differentiation; unexpected results have also been reported in the context of tissue revascularization indicating the need to better understand the regulation of oxygenation in fracture healing.Oxygen tension is an important aspect of the tissue microenvironment. Lerrer et al. demonstrate in their study that these cytokines cooperatively induce a pro-inflammatory fate in MSCs. This implies that the complex composition of the niche-specific secretome is of critical importance for the immunomodulatory fate of stem cells. Michael et al. provide in their mini review very interesting aspects on the specific outcomes of inflammatory incidents affected by direct regulation of stem cells. During tissue damage caused by either pathogen infection or physical/chemical damage (sterile induced inflammation), the inflammatory response acts as a regulator of tissue stemness either by directly affecting tissue stem cells or by shifting differentiated cells toward a stem-like cell character. Menendez and Alarcón present a threshold model for aging and cancer, based on regulation of cellular reprogramming by senescence-inflammatory signaling, and offer new therapeutic strategies for these ailments. According to this model, a transient cellular reprogramming induced by activation of innate immunity or senescence-associated inflammatory components brings cells to a state of epigenetic plasticity, enabling adaptive phenotypic adjustments in order to promote damage repair and tissue regeneration. However, if this de-differentiation is not accompanied by re-acquisition of the original or alternative differentiated cell fate, the resulting tissue plasticity impairs the replacement of damaged cells and leads to chronically unresolved tissue damage.It has been shown that MSCs have immunomodulatory properties and the ability to adapt to the tissue environment. Tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) are two cytokines present in different niches, which may have opposing roles (pro- and anti-inflammatory). Johnson et al. who analyzed the expression of the monocarboxylate transporter 1 (MCT1) in a significant number of breast cancer tissue samples with variable characteristics in terms of hormone-dependence. They showed that MCT1 level of expression varies between cancer subtypes and that it can be correlated to breast cancer recurrence, thus suggesting MCT1 as a prognostic biomarker as well as a target worth exploring for cancer therapy.Malignant tissue alterations such as cancer are associated with drastic changes in the tissue environment and its metabolome. In this context, the metabolic plasticity of cancer cells has been reported by Ghosh et al., who investigated how TGF-β pretreatment affects MSC-mediated wound healing. In the mouse model, TGF-β-pretreated MSCs distribute more evenly in the wound and improve wound closure. Based on in vitro findings, the authors propose a time-dependent model of TGF-β-stimulated bone marrow MSCs that helps the wound healing process. They suggest that increased MSC adhesion and migration properties are regulated via integrin/focal adhesion kinase activation which is essential for both SMAD3-dependent and -independent signal transduction after TGF-β treatment.Stem cell plasticity can also be used as a tool to improve the regenerative capacity of cells. Such approach is presented by Ottoboni et al. review the literature on neural stem cell (NSC) plasticity and methods for manipulation of their plasticity for treatment of central nervous system (CNS) diseases. They particularly emphasize the double-side effect that plasticity can have, even threatening the NSC therapeutic capacity, because manipulation of NSCs or of their trophic microenvironment might induce unwanted side effects, such as senescence. The authors conclude that it is necessary to increase the knowledge on (i) the influence of cell metabolism on NSC behavior during CNS diseases; (ii) the ex-vivo maintenance of NSCs; (iii) the most suitable window time for transplant to best leverage graft survival and disease specific environmental conditions. While this review was focused on neural stem cells, the mentioned aspects similarly present a challenge in other research fields.Finally, All authors JK, MH, IG, and SM contributed equally in writing the Editorial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This similarity included the concentration-dependency and biphasic pattern of glucose-induced insulin secretion, the dynamics of the responses to non-glucose stimuli and metabolic amplification of these responses. The insulin content was not different, but fractional insulin secretion rates were lower in infant than adult islets irrespective of the stimulus. However, the stimulation index was similar because basal secretion rates were also lower in infant islets. In conclusion, human β-cells are functionally mature by the age of one year, before expansion of their mass is complete. Their responsiveness (stimulation index) to all stimuli is not smaller than that of adult β-cells. Yet, under basal and stimulated conditions, they secrete smaller proportions of their insulin stores in keeping with smaller in vivo insulin needs during infancy.Insulin secretion has only exceptionally been investigated in pancreatic islets from healthy young children. It remains unclear whether those islets behave like adult islets despite substantial differences in cellular composition and higher β-cell replication rates. Islets were isolated from 5 infants/toddlers (11–36 month-old) and perifused to characterize their dynamics of insulin secretion when subjected to various stimuli and inhibitors. Their insulin responses were compared to those previously reported for similarly treated adult islets. Qualitatively, infant islets responded like adult islets to stimulation by glucose, tolbutamide, forskolin (to increase cAMP), arginine and the combination of leucine and glutamine, and to inhibition by diazoxide and CaCl Inadequate insulin secretion disrupts glucose homeostasis at all ages, including infancy. Much attention has been paid to the mechanisms causing excessive insulin secretion in congenital hyperinsulinism –3 and inNormal neonates display higher plasma insulin/glucose ratios than infants and children , 13, andThree-to-four decades ago, insulin secretion by the human fetal pancreas has been extensively studied in vitro. The consensus that emerged was that between 14 and 22 weeks of gestation, human fetal β-cells poorly responded to stimulation by glucose alone, but that a response could be unmasked by cAMP-raising agents, and that amino acids and sulfonylureas were more effective than glucose –28. In cDuring a previous in vitro study of insulin secretion by islets from normal adult donors , we receIn brief, after culture in RPMI medium containing 5mmol/l glucose for about 38 h, similar portions of each islet preparation were transferred into perifusion chambers and perifused with a bicarbonate-buffered salt-balanced solution supplemented with glucose and test agents as required . BecauseResults in infant islets were compared with those previously obtained in adult islets that were isolated, purified, counted, cultured and eventually perifused in exactly the same way and over the same time period . CharactThe insulin content of islets from infants was estimated by summing insulin secreted during experiments and insulin extracted from islets at the end of experiments. Values for the five individual cases are given in Stepwise increases in the glucose concentration progressively increased insulin secretion in infant islets . Alone, Total insulin secretion over the 240 min of stimulation by glucose alone or with forskolin is shown for each of the five cases in ATP channels with diazoxide abolished insulin secretion, whereas subsequent closure of the channels with tolbutamide completely reversed the inhibition, and addition of forskolin strongly amplified the secretory response. Complete reversibility of the stimulation was observed on returning to G1 alone ATP channThe dynamics of insulin secretion in infant islets  were thu2 from the medium 2 from the medium .2+ (tolbutamide) and augmentation of the response to Ca2+ (glucose) [A 10-min pulse of arginine (10 mmol/l) slightly increased insulin secretion in infant islets perifused in G3 . Tolbutaglucose) . They arglucose) . HoweverATP channels in β-cells are closed by a high concentration of tolbutamide [Metabolic amplification of insulin secretion can be evidenced by increasing the concentration of glucose when all Kbutamide . As showbutamide , but totIn vitro studies of insulin secretion by β-cells from normal young children are extremely rare owing to the exceptional availability of the necessary material. This rarity at least partly originates from the idea that children islets are suboptimal for transplantation and hence infrequently isolated from very young organ donors .2 omission. This similarity included the biphasic pattern of glucose-induced insulin secretion and the dynamics of the responses to other stimuli. Amplification of insulin secretion by glucose when all KATP channels are closed by a sulfonylurea was operative as in adult islets, as was amplification of arginine-induced insulin secretion by tolbutamide and by glucose [Our study shows that the characteristics of insulin secretion by islets from normal infants are similar to those previously defined in similarly treated islets from normal adults . Qualita glucose .Morphometric studies of the intact pancreas have shown that the average diameter of islets is smaller in children than adults , 11, butMany β-cells are scattered single or form small clusters in the pancreas of neonates, but most of them reside in well-structured islets after 6 months of age , 11, 40.ATP channels [One recent study compared in vitro β-cell function in islets from adults and from 3 infants  . Using hchannels . In the channels  were quaIn conclusion, islets isolated from 11–36 month-old children secrete insulin virtually like adult islets when tested in vitro. The only significant difference is quantitative, not qualitative. Whereas the dynamics of their responses to an array of stimuli and inhibitors are similar, infant islets consistently secrete lower proportions of their insulin stores than adult islets, a difference that is attenuated by cAMP. Yet, because unstimulated secretion rates are also lower, the amplitude of their responses above baseline (stimulation index) is not reduced. Human β-cells therefore have reached functional maturity by the age of 1 year, before expansion of their mass is complete. Closer timing of postnatal β-cell maturation and elucidation of its mechanisms will require studies of islets from infants between birth and the age of 1 year, particularly around weaning . IdeallyS1 FigIndividual responses of the five preparations of infant islets whose mean response is shown in (PDF)Click here for additional data file.S2 FigIndividual responses of the five preparations of infant islets whose mean response is shown in (PDF)Click here for additional data file.S3 FigIndividual responses of the five preparations of infant islets whose mean response is shown in (PDF)Click here for additional data file.


Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location  of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These “weak” segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination. Genetic recombination was demonstrated to occur in practically all viruses investigated in this respect , althougThese strains were generated by Albert Sabin by selection from three serotypes of wild-type (wt) polioviruses . Upon thAiming to understand these reasons, in this study we have analyzed some aspects of a unique natural mega-experiment when a mixture of three well-studied and related viruses  was given to billions of people and a plethora of progeny isolates were available for investigation. Specifically, we have sequenced, fully or partially, 98 genomes of intertypic poliovirus recombinants and analyzed these data together with the previously published results to infer common patterns of organization of the recombinant genomes. As a result, some important conclusions have emerged relevant not only for the understanding of the nature of Sabin viruses and their penchant for recombination but also of some general issues of biology of RNA viruses.Virus strains derived from Sabin polioviruses were mainly collected in Russia and countries of the former Soviet Union from patients with acute flaccid paralysis (AFP), patients with other diseases as well as from healthy individuals and environment. Isolation, identification and intratypic characterization were performed according to the standard WHO polio surveillance protocol .Nucleotide sequences were determined by full-genome deep sequencing as follows. Cell culture supernatants were treated with micrococcal nuclease to digest free nucleic acids, and RNA was isolated by RNeasy kit . It was then fragmented by using focused ultrasonicator  to produce 200–400 nt-long molecules. cDNA was prepared by reverse transcription using Superscript III . Illumina adapters with index oligonucleotides (barcodes) were added and the resulting DNA library was amplified by PCR. Sequencing was done using Illumina Miseq in multiplex format with 10–12 barcoded samples analyzed in one run. For most samples treated this way, poliovirus-specific sequence reads represented the majority of all reads (50–95%), and the depth of coverage was between 10,000 and 200,000 reads per genomic position. All new nucleotide sequences were deposited in GenBank (accession numbers MG212427-MG212495).Bioinformatic analysis was performed using two in-house software packages: ‘swarm’ program running on MacPro  computers in UNIX (Darwin) environment, and cloud-based HIVE  system . The anaThe location of crossovers in some recombinants investigated years ago and reported for the first time here were determined as follows. Viral RNA was extracted from the cultural medium of virus-infected cells with the Trizol reagent  and reverse transcribed with avian myeloblastosis virus reverse transcriptase (Promega) using random hexamer primers . Approximate positions of the crossover were mapped by using the restriction fragment length polymorphism (RFLP) assay ,21. To tThe set of intertypic recombinants between the Sabin vaccine polioviruses analyzed here, 189 samples, included 98 viruses collected mostly in Russia and countries of the former Soviet Union since the 1980s, whose genomic crossover sites were determined in this study either by full genome deep sequencing  sequences were regularly lost, in some cases together with more or less extended parts of the P3 region and sometimes also distal portions of the P2 region  or poorly compatible with other genome parts (epistasis). Some ground for choosing the more likely explanation can provide a comparison of data of Thus, the presence of homotypic 5′- and 3′-adjacent sequences in the type 1 recombinants A could bThe notion of the existence of presumably low-fit serotype-specific segments of poliovirus genomes is also supported by the structures of recombinants between Sabin strains and non-polio enteroviruses. Thus, for example, such recombinants of type 1, which triggered polio outbreaks in the Dominican Republic and Philippines in 2000–2001, demonstrated a replacement of a significant portion of their P2 region ,26; the A plausible assumption is that the observed regularity of losing/gaining distinct genomic segments is linked to their fitness-modulating capacities. Are they somehow related to the type-specific attenuating mutations acquired upon derivation of the vaccine strains? To approach this point, we used an arbitrary classification of presumably low-fit regions based on the frequency of their removal upon intertypic recombination. Those that were lost in 90% or more recombinants of serotypes 2 and 3 and in at least 75% of serotype 1 were considered as “weak” (heavier shading in ts) phenotype-modulating) mutations are located upstream of the recombination-affected parts of the genomes situated, as demonstrated above, in our set of viruses downstream of the VP1/2A border. Moreover only a single known Sabin 1-specific attenuating mutation  are scattered throughout the entire genome , and thecis-element (oriR), which in poliovirus RNA is composed of two hairpins linked to each other by a tertiary (“kissing”) interaction [oriR. Whether one or both of these features could be linked to fitness loss remains unknown. It may also be noted that the distal part of the 3D protein invariably lost in the serotype 2 recombinants differs from its counterparts in types 1 and 3 at only one position, amino acids 362 and 425, respectively. The loss of this segment could, or could not, be simply due to a kind of “passenger effect” because of its proximity to the 3′UTR.Thus, it is reasonable to look at the intrinsic  peculiarities of the “weak” genomic segments. The regular absence of the type 2-specific 3′UTR in the investigated recombinants could be due to certain peculiarities of the structure of this replicative eraction . Althoug15 in 3A. On the other hand, as noted above, it cannot be ruled out that a nearly universal loss of the relevant short fragment resulted merely from the partial overlap of two low-fitness regions located in P2 and P3, respectively.The presence of Arg at position 15 of 3A protein of Sabin-3 could also be related to the removal of this region from nearly all type 3 recombinants. Indeed, none of the wild polioviruses deposited in the GenBank (except the Sabin-3 parent Leon/37 and its descendants) contain ArgThus, the above results are compatible with the hypothesis that the fitness-related features of the genome segments that are acquired/lost upon intertypic recombination between Sabin polioviruses might be determined by not only vaccine-specific mutations but could also be inherited from their wild type progenitors.To better evaluate the choice of the observed crossover sites, it is appropriate to briefly remind what is known about the mechanisms of RNA recombination . The widAnother, nonreplicative, mechanism of RNA recombination implies end-to-end joining of independently synthesized RNA fragments without participation of viral RNA-dependent RNA polymerase (RdRP) and does not require any similarity between the partners involved ,41. AlthAlthough the choice of the replaced segments appears to be related to fitness, there should be mechanistic circumstances to allow recombination events to occur at certain distinct loci. The available large collection of recombinants originating from known parents may be useful for a search for such mechanistic preferences/limitations, if they exist at all. In our set of recombinants, the crossovers were distributed throughout the entire region encoding nonstructural proteins, though clearly non-randomly with relatively “hot” and “cold” areas . RemarkaThe above pattern of crossover distribution was generated manually using 50 nt-long windows by comparing aligned type-specific sequences. To assess the real level of homology around crossover points in the recombining partners, we looked at the sequences of relevant genomes. It turned out that in the overwhelming majority of cases (81%), this homology indeed spanned for at least 4 nt and in 68% cases it was 7 nt-long or longer . This maThe data reported here revealed previously unknown serotype-specific peculiarities of distinct genomic segments of poliovirus vaccine strains, which are likely related to their effects on viral fitness. Although polio eradication campaign launched by the WHO in 1988  met with a marked success, with only a handful of poliomyelitis cases caused by wild poliovirus reported globally in 2017 , the neeUnfortunately, phenotypic differences between Sabin viruses and their intertypic recombinants have not been systematically investigated. In a small-scale experiment, Savolainen-Kopra et al.  have demTo decrease the ability of vaccine strains to recombine it was suggested to introduce certain mutations into the viral RNA-dependent RNA polymerase  and to uA likely pattern emerging from the results presented here is that naturally circulating (“wild”) RNA viruses may contain lineage-specific more or less extended genomic segments with a relatively low fitness. Indeed, our observations suggest that Sabin viruses (at least of serotype 2 or 3) might have inherited from their wt parents certain “weak” RNA sequences not related to the attenuating (and other) mutations acquired during their selection by A. Sabin. Such low-fitness segments could well arise (and probably indeed are not infrequently arising) upon error-prone RNA replication caused by low fidelity of the RdRP  and The analysis of the pattern of inheritance of distinct genomic segments upon intertypic recombination between the Sabin strains of poliovirus allowed us to draw several conclusions and hypotheses. It suggests that Sabin strains contain, in addition to known attenuating mutations, distinct serotype-specific genomic segments with low-fitness. The genetic determinants responsible for their low fitness could be derived not only during the laboratory attenuation of these strains but also could be inherited directly from their wild-type parents. The presence of these segments underlies the propensity of these viruses to recombine with each other and with other enteroviruses as well as makes a significant contribution to the choice of crossovers sites. Thus, our results clearly demonstrate a role for RNA recombination as an efficient tool of adaptive viral evolution. Also, the knowledge of localization of low-fitness segments in Sabin viruses opens additional possibilities for the design of more genetically stable and/or more attenuated vaccine strains.Our results also provide new insights into the problem of fitness, genetic stability and evolvability of wild polioviruses and, by generalization, of other RNA viruses. We propose that the genomes of such viruses may also harbor hidden low-fitness segments, which cannot be readily rehabilitated and can (nearly) exclusively be removed by recombination.Finally, the absence of sufficiently long homologous sequences around crossover sites in the relatively large pairs of recombining partners may be considered as an indirect evidence for possible involvement, in certain cases, of recombination mechanisms other than the classical copy-choice replicative mode. However, more direct data to support this notion are needed.


During development, the organization of the auditory system into distinct functional subcircuits depends on the spatially and temporally ordered sequence of neuronal specification, differentiation, migration and connectivity. Regional patterning along the antero-posterior axis and neuronal subtype specification along the dorso-ventral axis intersect to determine proper neuronal fate and assembly of rhombomere-specific auditory subcircuits. By taking advantage of the increasing number of transgenic mouse lines, recent studies have expanded the knowledge of developmental mechanisms involved in the formation and refinement of the auditory system. Here, we summarize several findings dealing with the molecular and cellular mechanisms that underlie the assembly of central auditory subcircuits during mouse development, focusing primarily on the rhombomeric and dorso-ventral origin of auditory nuclei and their associated molecular genetic pathways. Cre- and Flp-recombinase specific mouse lines coupled to reporter lines has become a key tool in labeling genetically defined cell populations and identifying the embryonic origin of auditory hindbrain nuclei. Conditional knock-in and knock-out transgenic mice have allowed testing the function of individual genes during the construction of the auditory system and the characterization of their pathological role during hearing loss or deafness. Dissecting the anatomical components and functional role of auditory circuits in both normal and pathological conditions contributes to decipher the complexity of sound encoding processes. Moreover, identifying novel genes and molecular pathways specific to distinct rhombomere-specific subcircuits of the developing auditory system is crucial in finding novel players involved in different patterning aspects of hindbrain circuit formation and in the identification of genetic mutations causative of human hearing loss disorders. Below, we will first describe our knowledge about early hindbrain patterning and general anatomy of the auditory system. Then, we will summarize the progress made in the characterization of the rhombomeric and DV origin of auditory hindbrain nuclei and in the identification of genes that along the AP and DV axes are involved in rhombomere-specific formation of distinct functional auditory subcircuits.Hearing depends on the transmission and transduction of sound stimuli in the inner ear and on processing sensory information in the central nervous system. Proper development and function of both peripheral and central components are necessary for normal sound perception. In this review, we will recapitulate the anatomical, molecular and functional mechanisms involved in the assembly of the central auditory system in the developing mouse. It is known that the unique molecular identity of each rhombomere along the antero-posterior (AP) and the shared genetic pathways of dorso-ventral (DV) domains confer a positional pattern on neuronal progenitors, determining their neuronal subtype identities, migratory pathways, and axonal projections to their proper targets. Several studies have started to correlate early rhombomeric territories and specific DV domains to adult auditory neuronal identity, migration and connectivity patterns , named crypto-rhombomeres  and rhombomere-like hidden segments called pseudo- or crypto-rhombomeres along the AP axis, and which will give rise to the prepontine region including the cerebellum , the pons , the retropontine region , and the medulla oblongata (r7–r11) in the adult brain Puelles, . RhombomHox genes are the earliest genes that delimit rhombomeric boundaries along the AP hindbrain axis. Indeed, the overt inter-rhombomeric boundaries of r1/r2 to r6/r7 correlate with the anterior boundary of Paralog Groups (PG) 1-3 Hox genes following the principle of 3′-5′ spatial collinearity . Inductive signals and specific transcriptional pathways confer a positional identity on neural progenitors, in accordance with their position along the AP and DV axes, and determine the future neuronal fate of distinct neuronal subtypes, specific migratory pathways and axon projections to their corresponding targets  are an important efferent component of the auditory system. In particular, lateral OC (LOC) motor neurons innervate afferent sensory neurons in synaptic contact with the IHCs, modulate cochlear nerve excitability and protect the cochlea from acoustic injury Simmons, , whereasIn this section, we will discuss the evidence regarding the origin of sensory and motor auditory nuclei from distinct rhombomeres along the AP axis Figure  and diffThe cochlear nuclear (CN) complex is subdivided into ventral (VCN) and dorsal (DCN) cochlear nuclei. While the DCN exhibits a laminar structure that contains the molecular (ML), the fusiform cell (FCL) and the deep (DL) layers, the VCN is further subdivided into anterior (AVCN) and posterior (PVCN) regions  , DCN and shell  and contributes to distinct auditory nuclei. Atoh1 is expressed in cycling cells of the ventricular zone (VZ) and in presumed progeny neurons in the immediately lateral mantle zone (MZ)  and DCN, as well as to cochlear granule cells  caudal to r1, contributes to all divisions of the cochlear nuclear complex: the magnocellular core regions of the AVCN, PVCN and DCN, as well as granule cells of the microneuronal shell . Both cochlear granule cells and their precursors express the homeobox gene bHLH transcription factor Atoh7  is expressed in migrating cells of the LRL Atoh1-lineage and is also regulated by Atoh1  encodes for a basic-helix-loop-helix (bHLH) protein expressed in neural progenitors of the A4 and B1 dorsal domains in the mouse hindbrain  neurons derive from the Ptf1a-expressing neuroepithelial domain  derive from the same DV neuroepithelial domains but differentiate into distinct subtypes of inhibitory  or excitatory  neurons. This depends most probably on the integration of the same DV molecular profile with different AP rhombomere-specific genetic pathways. To support this issue, the intersectional genetic fate mapping using subtype- and rhombomere-specific transgenic lines will represent the most appropriate tool to characterize molecular complex mechanisms that generate the vast diversity of CN neurons, and correlate their origin to their neuronal fate and function.The n Figure . Ptf1a-dn Figure . SimilarBhlhb5  is a neural-specific transcriptional repressor expressed in the DCN during embryonic and postnatal development by the unipolar brush (excitatory) and cartwheel (inhibitory) cells  has revealed that Bhlhb5 expression is upregulated in r4 and restricted to the r4 ventral motor neuron domain and to longitudinal stripes in alar r4  complex, one of the major stations along the hearing path, is subdivided into ventral (VLL), intermediate (ILL), and dorsal (DLL) nuclei. The VLL receives inputs mainly from the contralateral ear and project to the ipsilateral inferior colliculus (IC) , whereas the DLL receives inputs from both ears mainly through connections with the superior olive and projects to both IC    for glutamatergic neurons, and of the Vesicular Inhibitory Amino Acid Transporter VIAAT  for both GABAergic and glycinergic neurons  is a group of brainstem nuclei that act in multiple aspects of hearing and is an important component of the ascending and descending pathways of the auditory system. The nuclei that form the SOC are: the lateral (LSO) and medial (MSO) superior olive, the medial (MNTB), ventral (VNTB), and lateral (LNTB) nuclei of the trapezoid body, the superior paraolivary (SPO) nucleus and the periolivary nuclei Figure . While rProper spatial information on the horizontal (azimuth) and vertical (elevation) directions and distance of the sound source from the listener is required for sound localization. Sound localization in vertical plane and in horizontal front/back position is a monaural pathway, which uses modifications in the sound spectra produced by the interactions of sound with the asymmetric external ear    and the Bosley-Salih-Alorainy Syndrome (BSAS) characterized by sensorineural deafness due to the absence or aplasia of the cochlea  or conditionally (Hoxb1lateCKO) inactivated in r4, Hoxb2 is reduced and Hoxa2 is increased at expression levels similar to those in r3  and to the ectopic formation and functional acquisition of neuronal populations that normally derive from r3  , whereas nuclei generated after Hoxb1 inactivation, such as the CN, have similar defects in all mutants.Differently from Hoxb1 is an important determinant gene in the r4-derived CN development in part by inhibiting a r3-like genetic pathway. One of the key Hox genes involved in patterning the AVCN in r3 is Hoxa2, which is instead expressed at low levels in r4 differentiating cells, playing thus only a minor role in patterning r4-derived CN  and to ectopic production of cochlear Atoh1-derived excitatory populations (such as cochlear granule cells and Atoh7+ neurons)  results in a less severe reduction of the VLL  , while a second population follow a trigeminal-like pattern of migration , FBM and IEE  and in Hoxb2 mutant mice until E10.5 and E11.5, respectively. Consequently, MNv generated before Hoxb1 downregulation retain r4 characteristics, while late-born neurons adopt a serotonergic neuronal fate, demonstrating that Hoxb1 expression is required over time allowing progenitors to maintain r4-positional identity and to acquire their appropriate neuronal fate  and Gata3 (for IEE) in ventral r4 and repressing thus a r3-like serotonergic fate.Besides regulating Hoxb1 mutant embryos, OHCs degenerate mainly in the apical cochlear portion, causing the altered hearing threshold described in adult mice  reflexes, as well as LOC neurons, which cannot protect the organ of Corti from noise-induced hearing damages. Accordingly, Hoxb1 conditional mice, whose efferent neurons  are partially preserved have a less severe increase of auditory threshold with age compared to Hoxb1null mice in which all efferent motor neurons are completely absent  syndrome  are lost with a duplication of the malleus and incus primordia and enlargement of the muscles of the malleus, the tensor tympani . In the hindbrain, Hoxa2, strongly expressed in r3 and at lower levels in r2, plays a key role in the segmental identity of these rhombomeres and is required in patterning the r2/r3-derived AVCN , connectivity patterns and molecular profile of several auditory neuronal populations. The intersectional strategy using both All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rln+/+) mice at estrus, it evoked the production of Cxcl1 mRNA and its secreted protein product CXCL1 in four of eight animals. Mating experiments were then conducted using mice with a null mutation in the relaxin gene (Rln−/− mice). qRT-PCR analysis of mRNA expression in wild-type females showed diminished uterine expression of several cytokine and chemokine genes in the absence of male relaxin. Similar differences were also noted comparing Rln−/− and Rln+/+ females mated to wild-type males. Quantification of uterine luminal fluid cytokine content confirmed that male relaxin provokes the production of CXCL10 and CSF3 in Rln+/+ females. Differences were also seen comparing Rln−/− and Rln+/+ females mated with Rln−/− males for CXCL1, CSF3, and CCL5, implying that endogenous relaxin in females might prime the uterus to respond appropriately to seminal fluid at coitus. Finally, pan-leukocyte CD45 mRNA was increased in wild-type matings compared to other combinations, implying that male and female relaxin may trigger leukocyte expansion in the uterus. We conclude that male and/or female relaxin may be important in activating the uterine cytokine/chemokine network required to initiate maternal immune adaptation to pregnancy.The hormone relaxin is important in female reproduction for embryo implantation, cardiovascular function, and during labor and lactation. Relaxin is also synthesized in males by organs of the male tract. We hypothesized that relaxin might be one component of seminal plasma responsible for eliciting the female cytokine response induced in the uterus at mating. When recombinant relaxin was injected into the uterus of wild-type ( In the female mammal the peptide hormone relaxin is secreted into the circulation principally from the ovary and, during pregnancy, probably also from the endometrium and placenta  to devise controlled matings between wild-type and homozygous null mutant males and females in all combinations, followed again by analysis of uterine tissue and uterine fluid lavage. There is no a priori evidence to suggest any implantation defect in these mice and litter sizes appear normal  in the seminal plasma of most mammals  at the University of Adelaide from founders generously provided by Dr. Chrishan Samuel , as previously described in Zhao et al.  adult (8–12 weeks) virgin female mice in estrus were identified by daily vaginal smearing  were mated overnight with either Rln−/− or Rln+/+ males of proven fertility. After checking for copulatory plugs the following morning, the females were killed between 10:00 and 12:00 h by cervical dislocation. Uterine lavage and uterine tissue were collected as above.In the second experiment, adult virgin females  were euthanized as above and testes, epididymides, seminal vesicles, vas deferens, prostate, preputial, and coagulating glands were dissected for extraction of RNA (see below). Also testes and epididymides from adult sibling wild type (+/+) and relaxin deleted (−/−) male mice were similarly processed. Animal experimentation was approved by the Animal Ethics Committee of the University of Adelaide  or by the Animal Ethics Committee of Mecklenburg-Vorpommern, Germany (no. LALLF M-V/TSD/7221.3-1.1-033/06).Rat anti-mouse mAb hybridoma supernatants recognizing CD45  and Ly-6G  were used to determine the density and distribution of leukocytes within the mouse endometrium  according to the manufacturer's instructions. Because it proved difficult to process prostate tissue without incurring significant RNA degradation, RNA from CD1 and CD57BL/6 mouse prostate was also obtained from Amsbio . cDNA was prepared from 3 μg total RNA per sample using oligo(dT) priming and Superscript II (Life Technologies) reverse transcriptase, again according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was carried out using a Corbett Rotor-Gene 3000  and the ExTaq Sybr-Green pre-mix , with oligonucleotide primers and PCR conditions as listed in Supplementary Table Rps27a; Lee et al., Rln−/− female mice mated with Rln−/− males (n = 4) or Rln+/+ males (n = 4). Samples were selected on the basis of RNA integrity and quality, assessed by UV spectrophotometry (A260/A280 ratio > 2) and gel electrophoresis (28S/18S integrity) as well as by high consistency in the CT-values for housekeeping transcripts. Results were normalized against internal Gapdh, Actb, and Rn18s ribosomal RNA housekeeping targets.RNA was prepared from uterine tissue and from various male organs as previously  was assessed using a commercial ELISA kit  exactly as per the manufacturer's instructions. For experiment 2, all samples were analyzed for cytokine and chemokine content using the Luminex multiplex system  with a panel of 15 targets . The manufacturer's protocol was followed precisely.post-hoc test or by Student's t-test to check individual comparisons. Because of marked skewness in the data, the Luminex cytokine data were log transformed prior to statistical analysis. p < 0.05 was considered significant.All data sets were analyzed by ANOVA followed by Neumann-Keuls Rln transcripts possibly depending on mouse strain. Our results are very similar to those presented by Ganesan et al. , or frozen in OCT medium for immunohistochemical evaluation (right horns). qRT-PCR analysis was carried out to quantify mRNA for Cxcl1, which encodes a typical pro-inflammatory cytokine, CXCL1 (mouse KC), which is known to be secreted from the mouse uterine epithelium , expressing ovarian relaxin, as well as Rln−/− female mice were mated with either Rln+/+ or Rln−/− males. These mating combinations provided the opportunity to evaluate the relative contributions of both seminal relaxin and endogenous ovarian relaxin in regulating the female post-mating cytokine response. Unlike the first experiment, analysis has made use of a more unbiased approach using LDAP cytokine mRNA arrays as well as cytokine/chemokine Luminex immunoarrays.In the second set of experiments, we investigated the effect of genetic relaxin deficiency in either male semen and seminal fluid or the female reproductive tract. Wild-type female mice . This initial analysis was used to identify potential candidates for in-depth qRT-PCR measurement.The preliminary cytokine mRNA LDAP analysis was restricted to comparing gene transcripts which were either up- or down-regulated when uterine tissue was derived from es Table . Only 14Ccl2, Cd80, IL6, and Ptgs2, in that these four gene transcripts also showed a similar trend toward reduction caused by mating the Rln−/− females with Rln+/+ males, compared to Rln−/− males, though this was just not statistically significant. Measurement of cytokines in the uterine lavage from these matings showed a significant reduction on mating to Rln+/+ males only for CCL5, though similar trends were apparent also for CSF1 and IL1b  reduction upon mating with Rln−/− males, reflecting that seen at the mRNA level, as also a strong trend to reduction for CXCL1 (p = 0.08).Uterine flushings from the same mating combinations , Ptgs2, and Smad3 , and Smad3  and the qRT-PCR results , at least in the cross-mating experiment, implying that there may indeed be a net increase in uterine leukocytes following mating with CSF3 and CXCL1 are identified as regulators of neutrophil recruitment and activation, while CXCL10 targets monocyte macrophages, dendritic cells, and lymphocytes  between peptide and mRNA expression strongly implies that the variance in this model is not of a technical nature, but is due to the biological variability in mouse responsiveness. Histological examination indicated no obvious morphological correlates possibly indicative of infection. Moreover, in this experiment, no differences were noted in terms of leukocyte recruitment to the uterus as assessed by immunohistochemistry and qRT-PCR  was chosen as indicator, since it has been previously shown to be a reliable marker of specific uterine epithelial secretion in the mouse  males resulted in a significantly diminished uterine expression in the absence of endogenous relaxin of transcripts for Ccl2, Cd80, Csf3, Cxcl1, Cxcl10, Il6, Ptgs2, Ptprc (CD45), and Smad3. This suggests that pre-exposure of the female to endogenous relaxin in some way potentiates the female response to seminal fluid introduced during mating. This was not reflected in any change in secreted cytokines at the peptide level . However, since the experimental approach evaluated only proteins secreted at the apical surface of the epithelium or by cells within the lumen, but not basally secreted proteins or stromal cell products, the possibility that more detailed analysis would reveal protein differences cannot be discounted.In addition to the effects of male relaxin discussed above, significant effects of endogenous female relaxin were also evident in experiments comparing Rln−/− males were evaluated. qRT-PCR analysis showed no significant effect of the presence or not of female relaxin in this context. At the level of uterine lavage, however, differences were seen for CSF3, CXCL1, and CCL5, with the presence of relaxin in the female leading to a diminution in these peptides. One interpretation of this data, taken together with data from matings with Rln+/+ males, is that the potentiating effect of relaxin on female tract responsiveness particularly affects the response to the relaxin component of the seminal fluid signaling activity. Whilst, it could be expected that chronic relaxin absence in null mutant females might lead to a change in the expression of the relaxin receptor, RXFP1, or otherwise alter the relaxin signaling cascade, no differences in Rxfp1 mRNA expression were observed , Smad3, Bcl2). No gene products are significantly down-regulated in this mating combination. Importantly, for Csf3 and Cxcl10 gene products the up-regulation is significant at both mRNA and secreted peptide levels, providing confidence in the interpretation that these factors are indeed regulated by seminal relaxin.This is quite different where the female mice express endogenous relaxin. On the Rln−/− males (Samuel et al., Rln+/+ males of Cxcl10 and the tolerogenic dendritic cell marker Cd80.There is considerable evidence to show that one of the main components of mouse seminal plasma, TGFß, has a significant influence on the immune competence of the inseminated uterus (Rodríguez-Martínez et al., per-se, and in particular the TGFß component of this, are important for the recruitment of leukocytes to the uterus and their involvement in the development of immune tolerance to potential paternal alloantigens expressed by the fetus (Robertson et al., Rln−/− mice, uterine mRNA for the pan-leukocyte antigen CD45, as also the dendritic cell marker CD80, were indeed significantly altered in direct association with the changes in specific cytokine transcripts. Future studies to evaluate the precise phenotypes and functional competence of leukocytes in activities including antigen presentation and fate determination of activated T cells would be warranted. It is relevant that previous studies in relaxin-deficient mice have not included analysis of allogeneic matings, where fetal-maternal MHC disparity might reveal a more profound role in maternal immune regulation than is suggested by existing reports.It has been shown that seminal plasma Rln−/− compared to a female Rln+/+ background. The relaxin contributed by the female may act in some way to “prime” or sensitize the immune response within the uterus. This female effect may also be simply additive or synergistic in regard to effects of relaxin in the female tract, whereby the uterine lumen could be considered as an independent compartment separated from the endometrium by the tight junctions of the luminal epithelium (Robertson et al., One of the more interesting findings was the observation that the effect of seminal relaxin was markedly different on a female in vitro to promote the formation of Th1 cells, which produce the anti-inflammatory cytokine IFNγ, rather than Th2 cells (Piccinni et al., Finally, there is the question of whether, besides activating cytokines of epithelial origin, relaxin is also able to stimulate immune cells directly. It has been long established that relaxin is able to stimulate PKA-dependent pathways within human monocytes (Bartsch et al., Rln−/− mice are essentially fertile shows that there is a degree of redundancy in such informational processing. These findings are of necessity preliminary and will require more intensive study in the future, making use of other time-points and possibly including animals with inactivated relaxin receptors.In conclusion, these studies in mice show for the first time that there is an important role for relaxin of male origin to act in concert with other proinflammatory agents in seminal fluid, such as TGFß, in order to initiate the cytokine response underpinning maternal immune adaptation for embryo implantation and development. Furthermore, as a corollary, female relaxin may act to sensitize the uterus to such effects, since both male and female relaxin appear to be required for maximal cytokine expression and effect. However, the fact that RI was responsible for the overall conception and management of the project, and contributed significantly to writing the manuscript. DG, KH, DR, DS, and RA all contributed to the experimental work and interpretation of results. RA and SR shared responsibility with RI in the writing and overall interpretation of results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.