Severe bronchiolitis  during infancy is a heterogeneous condition associated with an increased risk for childhood asthma development , 2. BronTo further address the issues outlined above, our Research Topic congregates evidence from observational and interventional studies exploring the severe bronchiolitis to childhood asthma causal pathway with the aim to identify severe bronchiolitis endotypes that can guide predictive  and prognostic  enrichment strategies see Figure 1Pischedda et al. compare the methylome of children who have been hospitalized with respiratory syncytial virus (RSV)-induced bronchiolitis and are followed-up for three years. Their study assessed differentially methylated positions (DMPs) in genes in peripheral blood samples and associated with risk for recurrent wheeze development. The lead methylation position (cg24509398) identified in this study falls at the gene body of Eyes absent protein 3 (EYA3), a tyrosine phosphatase connected with pulmonary vascular remodeling, a key underlying pathogenetic mechanism in asthma. Interestingly, atopic asthma after rhinovirus-induced severe wheeze has been associated with different type of DNA methylation changes,   and Misshapen-like kinase 1 (MINK1) in T-helper (Th)17-dominant asthma . The MBD2 and MINK1 genes were silenced or overexpressed by small interfering RNA and plasmids and the study showed that MBD2 and MINK1 regulate Th17 cell differentiation and IL-17 release. It is of note that other in vivo models of chronic airway inflammation have showcased that Th17 responses contribute to airway remodelling too, independent of the Th2 responses . The first study shows that infants with decreased expression of a thymus and activation-regulated chemokine (TARC) or increased expression of interleukin 13 (IL-13) in anti-CD3/anti-CD28 stimulated PBMC during a rhinovirus-induced acute respiratory infection are associated with relapses within a 2-month period of follow-up. The second study shows that genetic variants in the interleukin 33 gene in infants with severe bronchiolitis, regardless respiratory virus type etiology, is associated with increased risk for asthma development. Although these studies identify possible biomarkers for asthma development, we still need to understand whether pro-Th2 or Th2 mediators are more strongly associated with subsequent asthma development in comparison to respiratory virus type.To further approach the question around the role of respiratory viruses, two observational studies submitted at this research collection attempt to identify genetic variants and cytokine expression patterns in severe bronchiolitis and relate to subsequent asthma development . However, meta-analysis was not possible due to small number of studies. However, evidence points toward non-RSV viruses being associated with higher risk for asthma and increased effectiveness with corticosteroids and warrants further trials. Possibly, other than respiratory virus exposures  can act as more sensitive biomarkers too. The relevant evidence is presented in the following reviews .Toward this direction, a systematic review submitted at this research collection explores the potential for use of respiratory virus testing in guiding acute treatment with corticosteroids and prediction of subsequent long-term respiratory sequela . In response to non-protective environmental exposures , disruption of lung and gut microbiota diversity is associated with the activation of leukocytes and secretion of mediators that further promote Th2 differentiation. Therefore, as discussed at the description of this Research Topic, the interaction between exposures in severe bronchiolitis is complex, and analytical integration of data deriving from these epidemiological studies may be required.In addition to respiratory virus types as possible exposures associated with increased risk for asthma development, this research collection is bringing to the fore a comprehensive review around the role of microbial dysbiosis in asthma development . In this study, 749 infants with severe bronchiolitis underwent both genotyping and nasopharyngeal metabolome profiling. Through an integrated analysis, sphingomyelins, genes on chromosome 19p13 , and 1,2-dioleoyl-GPG were associated with increased risk for asthma development. Although these genes are known to be associated with asthma development, their biological functional potential is further enhanced through confirmation of metabolites expression too.This integrative approach is highlighted through the last study of this research collection presenting the first integration of genomic and metabolomic data in severe bronchiolitis (In brief, this Research Topic highlights that severe bronchiolitis is a heterogeneous condition. Different respiratory viruses, in interaction with the host genome, methylome, proteome and microbiome, can possibly drive risk for asthma development. However, the exact mechanisms are still unknown and therefore there is lack of validated predictors for severity in the acute phase as well as risk predictors for asthma. There is no doubt that, with the use of novel sampling methods and novel omics analytical approaches followed by mechanistic studies, we can advance our knowledge on bronchiolitis endotyping and identify novel therapeutic targets and biomarkers for asthma development.HMa: composed and distributed the first draft of the manuscript, designed the figure. HMo: revised the drafted manuscript and the figure. EN: revised the drafted manuscript and the figure. TJ: revised the drafted manuscript and the figure and provided detailed feedback around the included studies he co-authored. All authors contributed to the article and approved the submitted version.


Kidney transplantation is the most common solid organ transplant and the preferred treatment for pediatric patients with end-stage renal disease, but it is still not a definitive solution due to immune graft rejection. Regulatory T cells (Treg) and their control over effector T cells is a crucial and intrinsic tolerance mechanism in limiting excessive immune responses. In the case of transplants, Treg are important for the survival of the transplanted organ, and their dysregulation could increase the risk of rejection in transplanted children. Chronic immunosuppression to prevent rejection, for which Treg are especially sensitive, have a detrimental effect on Treg counts, decreasing the Treg/T-effector balance. Cell therapy with Treg cells is a promising approach to restore this imbalance, promoting tolerance and thus increasing graft survival. However, the strategies used to date that employ peripheral blood as a Treg source have shown limited efficacy. Moreover, it is not possible to use this approach in pediatric patients due to the limited volume of blood that can be extracted from children. Here, we outline our innovative strategy that employs the thymus removed during pediatric cardiac surgeries as a source of therapeutic Treg that could make this therapy accessible to transplanted children. The advantageous properties and the massive amount of Treg cells obtained from pediatric thymic tissue (thyTreg) opens a new possibility for Treg therapies to prevent rejection in pediatric kidney transplants. We are recruiting patients in a clinical trial to prevent rejection in heart-transplanted children through the infusion of autologous thyTreg cells (NCT04924491). If its efficacy is confirmed, thyTreg therapy may establish a new paradigm in preventing organ rejection in pediatric transplants, and their allogeneic use would extend its application to other solid organ transplantation. Kidney transplantation is the treatment of choice for pediatric patients with end-stage renal disease. In children, congenital anomalies of the kidney and urinary tract and glomerular diseases are the most frequent indications for kidney transplantation . AlthougThe existing scientific evidence supports that only the induction of immunological tolerance, re-educating the recipient's immune responses, will allow the indefinite survival of the graft. In this sense, one of the most promising alternatives to increase the life expectancy of transplant patients is to induce tolerance through cellular immunotherapy without using pharmacological immunosuppression, thus eliminating the toxic effects of these therapies and maintaining a competent immune system . Along tThere are many cell types involved in the preservation of tolerance, both of innate and adaptive immunity, such as tolerogenic dendritic cells (DC) , tolerogTregs are characterized by the high and stable expression of the α chain of the IL-2 receptor (CD25) and the transcription factor Foxp3 (forkhead box protein 3), which is critical for the development and maintenance of the function and the Treg phenotype . Tregs cIn animal models, Treg cells have demonstrated their ability to delay/prevent graft rejection and promote transplant tolerance and indefinite graft survival –19. SpecImmunosuppressive treatment of transplant patients aims to limit the recipient's immune response against the graft. The treatment begins in the perioperative period, continues after transplantation with high levels of immunosuppressants, and later, the doses can be reduced to a maintenance therapy administered throughout the patient's life. There is a wide variety of immunosuppressive drugs available that allow the establishment of different treatment combinations that vary according to the transplanted organ, the patient, the risk of rejection, and also between countries and institutions . In mostin vitro experiments indicate that MMF does not alter the phenotype of Tregs or can even promote the predominance of Tregs on Th17 cells , completely blocking the interactions between CD25 and IL-2. Its use aims to block IL-2 uptake by activated Teff, harmful in the context of transplantation, thus preventing their proliferation . However17 cells ; and it 17 cells . However17 cells . Finally17 cells . In the 17 cells .Considering the evidence supporting the role of Tregs in preventing rejection in transplanted children, monitoring of Treg and Teff cell values and the imbalance between both cell subsets could constitute markers of early risk of rejection that can help to avoid graft damage before it occurs. New strategies aimed at preventing the decrease in Treg or restoring their frequencies could impact the prevention of graft rejection. Interventions such as: (i) the choice of immunosuppressive drugs with less impact on Tregs; (ii) the supplementation with vitamin D, which has been shown to increase Treg survival  and reduOne of the most promising alternatives to provide indefinite graft survival and thus increase the life expectancy of transplanted patients without morbidity is to induce immune tolerance by cellular immunotherapy. Establishing tolerance in transplantation aims to deliberately abate the detrimental allograft-specific immune response while avoiding the toxicity of long-term immunosuppression and maintaining a competent immune system .In the search for this “Holy Grail” of transplantation tolerance, all efforts are currently directed toward developing new immunomodulatory approaches with advanced cellular therapies mainly based on immune regulatory or suppressive cells to reduce or eliminate immunosuppressants. Among them, different cell therapy approaches are being explored, such as mesenchymal stem cells , 45, 46 ex vivo followed by the re-infusion of expanded or induced Treg. However, others consider the possibility to expand them in vivo to limit the ex vivo cellular manipulation. Some preclinical models showed that activating a tumor necrosis factor receptor superfamily member 25 (TNFRSF25) with or without IL-2 could reduce the graft-vs.-host disease or prevent allergic lung inflammation in mice recipients  reject human skin grafts, while those that also receive Tregs show stable survival of human skin transplantation together with a reduction of human CD8+ T cells in the infiltrates of the skin graft . In the After animal models, the safety and potential efficacy of the Treg therapy to re-establish the immune tolerance is being evaluated in clinical trials in adult transplanted patients. Currently, ongoing clinical trials are using a therapy with Treg cells obtained from peripheral blood to prevent rejection of the transplanted organ in adult patients, most of them in the context of kidney and liver transplantation ex vivo expanded autologous Tregs using a medium rich in deuterated glucose to label and track cells in vivo. Patients received a single infusion of 320 × 106 and maintained their immunosuppressive regimen with TAC, MMF and Pred. The infused Tregs peaked in circulation the first week after infusion, with detectable signals during the first month falling at 3 months after infusion. None of the patients presented infusion reactions, and no infections or malignancies were observed in the 1-year follow-up period.In 2017, the results of the TASK trial (NCT02711826) conducted at the University of California (UCSF)  were pub9ex vivo expanded autologous Treg cells at 60 days post-transplantation  were published in 2018 by Mathew et al. from the Northwestern University, Chicago, including a total of 9 kidney transplant patients that were divided into three groups of 3 patients who received 0.5, 1, and 5 × 10antation . In this6 cells/kg of patient's weight and were administered in a single infusion 10 days post-transplantation. Patients were routinely monitored for the primary endpoint biopsy-confirmed acute rejection (BCAR) within 60 weeks post-transplantation. The results were published in mid-2020 and showed good safety data. However, the efficacy results are not entirely conclusive, as BCAR rates were comparable between the standard immunosuppressive and cell therapy groups (12 vs. 16%). Subsequently, results from two more clinical trials within the ONE Study consortium have been published. The ONEnTreg13 phase I-IIa trial (NCT02371434) conducted at the Charité-University Hospital in Berlin compared 11 living donor kidney transplant recipients who received the autologous Treg therapy with 9 patients from the reference group . The other published clinical trial part of The ONE Study consortium was conducted in the United Kingdom as a phase I trial, including 12 living donor kidney transplant recipients divided into four groups receiving 1, 3, 6, or 10 × 106 Treg/kg at 5 days post-transplantation, and 19 patients in the reference group  in living donor kidney transplant patients . Two autce group . Tregs wce group . No safein vivo Treg's mode of action as their location. However, the clinical efficacy results are not entirely encouraging, presenting a low/short therapeutic effect that would be not enough to provide the indefinite survival of the graft.A possible risk of Treg therapy could be associated with the fact that high numbers of Treg cells can suppress antitumor responses favoring the tumor progression . Indeed,ex vivo activation/expansion of Tregs to obtain an appropriate number of cells; and (d) infusion of Tregs obtained in the patient himself (autologous use)  drawing peripheral blood from the patient; (b) purification of Tregs from peripheral blood; (c) ous use) , 68, 69.6 Tregs  can lose their Treg phenotype due to presenting more methylated  limited . Further limited , 75.ex vivo may lose Foxp3 expression and its Treg phenotype, Treg cells with naïve phenotype can maintain Foxp3 expression and suppressive capacity after repeated stimulation and expansion (Some studies consider the population of naïve Treg cells (CD45RA+) as the optimal subpopulation for therapy since these cells have more remarkable survival and maintain their suppressive capacity and phenotype stability , 77. Whixpansion . In pedi6 Tregs. However, since the yield in cellular isolation is <20% of the theoretical number (16.2%)  , only 1 6 Tregs can be obtained . This clinical trial represents the first Treg cell therapy performed in transplanted children and, importantly, the first worldwide employing thyTreg from thymic tissue. In addition, the large number of thyTreg cells obtained with this strategy opens the possibility for the administration of successive doses at different time intervals or when signs of rejection appear to induce graft tolerance. If its efficacy is confirmed, the thyTreg therapy may establish a new paradigm in preventing organ rejection in solid organ transplantation. At present, we are also exploring the allogeneic use of thyTreg, which would allow us to extend the use of this therapy to kidney-transplanted children or children transplanted with other organs.Treg cells play a crucial role in the balance between immune tolerance and graft rejection. Immunosuppressive drugs addressed to limit the immune responses against the graft could also impair the intrinsic tolerance mechanism promoted by Tregs. Restoring the balance between Treg and Teff cells by increasing Treg reserves through cell therapy can be an effective strategy to induce immune tolerance and prevent graft rejection. The ongoing clinical trials employing Treg therapy to prevent solid organ rejection in adults use autologous Treg cells obtained from peripheral blood. However, this strategy cannot be employed in transplanted children due to the limited number of Treg cells available in peripheral blood. The therapeutic strategy developed in our group that employs thyTreg purified from pediatric thymic tissue represents an innovative approach that could make Treg therapy available in transplanted children. From here, a wide range of possibilities opens up to exploit the potential of this therapeutic arsenal that constitutes the thyTreg in the prevention of organ rejection and the treatment of other diseases associated with phenomena of hyperactivation of the immune system.EB-d-Q, MP, MM-B, and RC-R have organized and discussed the review and contributed to the manuscript's elaboration. All authors have participated sufficiently in this work to take public responsibility for the content. All authors contributed to the article and approved the submitted version.This work was supported by grants from Fundación Familia Alonso (FFA- FIBHGM 2019), Instituto de Salud Carlos III (ISCIII) cofinanced by FEDER funds . EB-d-Q was supported by a grant from Comunidad de Madrid (EXOHEP-CM. B2017/BMD3727). MM-B was supported by the Sara Borrell Program from ISCIII (CD18/00105) and Marie Skłodowska-Curie Actions (H2020-MSCA-IF-2020- 101028834).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Diabetes is a common metabolic disease whose hyperglycemic state can induce diverse complications and even threaten human health and life security. Currently, the treatment of diabetes is restricted to drugs that regulate blood glucose and have certain accompanying side effects. Autophagy, a research hotspot, has been proven to be involved in the occurrence and progression of the chronic complications of diabetes. Autophagy, as an essential organismal defense mechanism, refers to the wrapping of cytoplasmic proteins, broken organelles or pathogens by vesicles, which are then degraded by lysosomes to maintain the stability of the intracellular environment. Here, we review the relevant aspects of autophagy and the molecular mechanisms of autophagy in diabetic chronic complications, and further analyze the impact of improving autophagy on diabetic chronic complications, which will contribute to a new direction for further prevention and treatment of diabetic chronic complications. Autophagy is an essential catabolic process in which broken organelles and proteins in cells are wrapped by a double layer of vesicles to form autophagosomes, which then combine with lysosomes to constitute autolysosomes and are finally degraded by a variety of enzymes in the autolysosomes . In addiAutophagy, a cellular degradation pathway, has been proposed for decades. Another cellular degradation pathway, the ubiquitin-proteasome system, is interconnected and even has the same molecules as autophagy . The mulIn mammalian cells, autophagy occurs within the lysosome and can be divided into three types according to the route of transport of substances to the lysosome: microautophagy, macroautophagy and CMA . MicroauAutophagy-associated genes (ATGs) are central to the molecular machinery of autophagy and are involved in the encoding of key proteins that play an active role in signal transduction pathways. Furthermore, ATG, whose aberrant expression can induce disease, play important functions in different autophagic processes such as cytoplasmic encapsulation, autophagic vesicle formation and lysosomal fusion . ATG5 anβ cells on the one hand, but increases insulin sensitivity by reducing ER stress in insulin-responsive cells on the other (Phosphorylated Akt can inhibit autophagy by activating mTOR complex 1 (mTORC1) and inhibiting FOXO1, playing an important role between insulin signaling and autophagy regulation. In animal experiments using mice with adipocyte-specific innate knockout of Atg7 and mice with adipocyte-specific knockout of Atg1611 or Atg3, respectively, it was concluded that innate inhibition of autophagy affects adipocyte differentiation, promotes adipocyte browning and increases insulin sensitivity, but selective inhibition of autophagy in mature adipocytes leads to insulin resistance . Studieshe other .Diabetes is a common metabolic disease with hyperglycemia. According to statistics, the global incidence of diabetes has been increasing since 1980 and has now doubled . HyperglIn addition to the major complications of diabetes, Alzheimer’s disease (AD) is closely associated with T2D, which is referred to by some as type 3 diabetes. Insulin resistance and insulin signalling dysfunction in T2D are important risk factors for dementia and AD. Peripheral and central insulin resistance leads to reduced insulin signalling, increased Aβ toxicity, oxidative stress and even the development of neurodegeneration. Even in non-diabetic people, insulin resistance increases the risk of dementia and AD .DR is a common diabetic complication associated with microangiopathy, which can result in further health impairment in diabetic patients with other vascular diseases . The latAbnormal autophagy due to high glucose promotes the progression of DR. High glucose facilitates ROS production by impacting mitochondrial function, and the resulting high ROS levels and damaged mitochondria can induce oxidative stress. Oxidative stress is one of the molecular mechanisms of autophagy, which can activate autophagy in DR. Appropriate stress can be used to improve cellular status by regulating autophagy, whereas excessive stress has the opposite effect in facilitating disease progression . High glDN, a chronic complication of diabetes mellitus, is impacted by glycemic, lipid, lifestyle and genetic factors, for which proteinuria, neutrophil gelatinase-associated adiponectin, cystatin C and plasma growth differentiation factor 15 are several representative markers . The hypAbnormal autophagy caused by high glucose promotes DN progression. The homeostasis of podocytes is related to autophagy in which the removal of Atg5 in podocytes would impact the cellular state of podocytes contributing to the inability of cells to resist stimuli induced by high glucose, a change which also exists in endothelial cells. The high glucose state in diabetes increases the levels of the autophagy marker LC3B II in podocytes with the consequent activation of autophagy which is uncertain whether it protects the kidney . HoweverLesions of blood vessels in diabetic patients can induce heart-related problems such as heart failure through oxidative stress and inflammatory stimulation, which greatly increase mortality in diabetic patients. Vascular disease is even the most common cause of death in patients with T2D . The symvia suppressing the expression level of miR-204-3p  and different stresses. Autophagy is dramatically increased in response to cellular stresses, such as oxidative stress and ER stress. It provides assistance to cells in handling stress and restoring stability to the internal environment by removing faulty proteins as well as harmful substances .β cells. Overactivation of mTORC1 initially promotes increased β cell mass, increased insulin levels, and improved glucose tolerance, but over time this promoting effect becomes inhibitory and β cell function declines more rapidly , a signal that maintains autophagy, which functions in the cell depending on the concentration. Low concentrations of ROS can act as REDOX signals, while high concentrations of ROS can injure cells . High le2+ signaling pathways   . ER strese (UPR) . UPR helse (UPR) . It has se (UPR) . The ER se (UPR) .With the epidemic of diabetes, a large number of novel hypoglycemic agents have been developed, some of which can regulate autophagy through varying signaling pathways and are useful in the treatment of chronic complications of diabetes .Dispo85E has been shown to be a potential drug for the treatment of diabetic vascular complications, and the mechanism by which it acts is associated with autophagy. In a diabetic mouse model, Dispo85E facilitates the expression of hepatocyte growth factor in non-platelet cells, thereby promoting the degradation of AGEs by hepatocyte growth factor-induced autophagy, which has a therapeutic effect on diabetic complications . Pioglitβ cell loss in type 2 diabetes by restoring lysosomal function and autophagic flux and facilitates the maintenance of glucose homeostasis. Exendin-4 also alleviates tacrolimus-induced hyperglycaemia, oxidative stress and other states of autophagic overload by activating autophagosomal clearance , an inhibitor of glycolysis, its expression level is decreased in high glucose state. High expression of TIGAR mitigates autophagy damage induced by high glucose and positively regulates autophagy. TIGAR protects neuronal cells through autophagy in diabetic neuropathy and attenuates cognitive impairment . Increasvia the TAK1-IKKα/β-Hsc70 signalling pathway and effectively reduces the accumulated brain Aβ plaques, providing a therapeutic effect in AD (In a study of newly diagnosed T2D patients in Korea, there were 1675 AD cases with 8375 control cases. The study suggests that metformin use is associated with an increased incidence of AD in patients with type 2 diabetes and that the duration of diabetes increases the risk of AD . Interesct in AD . Based oct in AD . In addict in AD .in vivo with a vital role in the chronic complications of diabetes. However, its specific mechanisms have not been fully explored, with relevant theories requiring further research. Although some studies have shown that complete remission is possible in T2D patients treated with intensive lifestyle changes, this study has many limitations and further improvements are needed in diabetes research (Autophagy is an essential metabolic pathway research . For diaOverall, therapeutic approaches to diabetes targeting autophagy need to be better designed. In this review, we describe the molecular mechanisms associated with autophagy and diabetic complications and summarise the latest autophagy-related drugs to improve diabetes and its complications. These results highlight the role of autophagy in the amelioration of diabetic complications and contribute to the research and application of glucose-lowering drugs related to the molecular mechanisms of autophagy. Currently, common approaches to modulating autophagy in diabetes are the use of autophagy inhibitors or knocking out autophagy-related genes, which have shown good modulation in animal experiments but still lack clinical trials. Therefore, further research is still needed on drugs that modulate autophagy, and modulating autophagy may be a potential research direction for the treatment of chronic complications of diabetes.


The amnion is an extraembryonic tissue that evolutionarily allowed embryos of all amniotes to develop in a transient and local aquatic environment. Despite the importance of this tissue, very little is known about its formation and its molecular characteristics. In this review, we have compared the basic organization of the extraembryonic membranes in amniotes and describe the two types of amniogenesis, folding and cavitation. We then zoom in on the atypical development of the amnion in mice that occurs via the formation of a single posterior amniochorionic fold. Moreover, we consolidate lineage tracing data to better understand the spatial and temporal origin of the progenitors of amniotic ectoderm, and visualize the behaviour of their descendants in the extraembryonic–embryonic junctional region. This analysis provides new insight on amnion development and expansion. Finally, using an online-available dataset of single-cell transcriptomics during the gastrulation period in mice, we provide bioinformatic analysis of the molecular signature of amniotic ectoderm and amniotic mesoderm. The amnion is a tissue with unique biomechanical properties that deserves to be better understood.This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’. In the first amniotes that evolved, around 340 million years ago during the Carboniferous Period , embryosa). Notably, in the mouse, the ‘chorion’ is the name given to the structure that gives rise to the placental plate and does not surround the embryo (a); the membrane that interfaces with the mother in mice is the parietal yolk sac, consisting of trophectoderm and parietal endoderm . T. Ta)   and provide a heatmap of some of the most relevant ones b. The pas in pig . From thllantois .Wnt6 and Lrp2 [c), suggested that the population of amniotic ectoderm may be contained within cluster 2 (surface ectoderm). This subpopulation of cluster 2 was also positive for Krt7. At the developmental stage analysed (E6.5–E8.5), the cells in the surface ectoderm and amniotic ectoderm may not be sufficiently different to partition into separate clusters. Notably, a strong similarity between surface ectoderm and amniotic ectoderm has also been recently reported in a unique human embryo of 16–19 days post-fertilization analysed by single-cell transcriptomics [Isl1, a marker recently associated with amniotic ectoderm in primates and in differentiated human amnion ectoderm-like cells [Isl1 in the Wnt6 + Krt7 + Lrp2 + population.Exploring the expression of the few known markers of amniotic ectoderm, such as and Lrp2   indicated that the cluster 8 may correspond to the amniotic mesoderm. Other markers highly expressed in this cluster were Pitx1 and Pitx2 (c). Interestingly, smooth muscle cells markers such as Tagln and Acta2 were not only highly expressed in amniotic mesoderm (c), but also in the population of Wnt6 + Krt7 + Lrp2 + cells, suggesting that the amniotic ectoderm may undergo some degree of epithelial-to-mesenchymal transition. Furthermore, we observed high expression of Bmp4 in extraembryonic mesoderm (c), in agreement with our previous observations of Bmp signalling activity in that tissue [Epcam and Cldn7 and mesenchymal markers Fn1 and Foxf1 [Fn1 and Foxf1 lead to pronounced defects in extraembryonic mesoderm [Foxf1 leads to a small and inflexible (closed) amnion among other defects, the absence of Fn1 resulted in several defects in mesodermal tissues and although there was a undersized closed amnion, the amniotic cavity seemed to lack pressure [Plotting the expression of amniotic mesoderm markers such as Postn  and Bmp2tn [Bmp2  , they were expressed in ISL1 + amniotic ectoderm cells in human (b), but to a lesser extent in monkey probably owing to the reduced amount of cells or absence of that cell population of interest in the dataset (d). As in human, amniotic ectoderm in monkey has been shown to express high levels of ISL1 [POSTN and PITX2) were expressed in a specific cluster of extraembryonic mesoderm in both human (cluster 11) (b) and monkey (cluster EXMC) (d), suggesting a degree of similarity regarding the molecular signature of these two cell types between mice, monkey and human.Although the amnion in primates and the amnion in mice forms via different mechanisms , we have plotted available single-cell datasets from gastrulating human  and cynoin human b, but to dataset d. As in  of ISL1 . Moreovester 11) b and moner EXMC) d, sugges. 7in vitro models using human pluripotent stem cells have sparked debate as it remains unclear whether human pluripotent stem cells are capable of differentiating into trophoblast, suggesting totipotency, or whether that is actually bona fide amniotic ectoderm that has been differentiated [in vivo human trophectoderm and amniotic ectoderm at the time of implantation. However, our knowledge on gene expression in early human amnion is currently restricted to one human embryo of 16–19 days post-fertilization recently used for single-cell transcriptomics [The amnion remains an elusive tissue. Its small size and its well-hidden development within the mouse conceptus hampers studying its early development. Review of clonal analysis data provides, however, a comprehensive view on the origin and growth of amniotic ectoderm cells. Recently, several entiated ,46. Thisiptomics . The molIn contrast with mouse, the primate amnion has recently been gaining more attention. Interestingly, several studies on early primate embryogenesis and stem cell-based embryonic models have shown that the amnion in primates may act as signalling centre for normal embryonic development ,24,47–49


Diabetes is a chronic disorder that affects more than 500 million individuals worldwide. It is a life-long disease with complications that attack nearly all other systems within the body. Although there is a slight increase in the prevalence of diabetes in males, ocular surface complications are equally present in males and females.This review provides a discussion on preclinical studies related to the dysregulation of a biological pathway that appears to be causally related to diabetic ocular surface complications including dry eye, delayed corneal epithelial healing, and decreased corneal sensitivity. Most basic science and clinical studies focus on male sex in animal models in order to avoid confounders related to hormonal cycling. However, with approximately 10.2% of all women in the US aged 18-44 being diagnosed with diabetes and nearly 4% additional women having undiagnosed disease, it is prudent to examine the onset of these dysregulations also in females and to note any sex-related differences in the timing of onset or severity of ocular surface complications.Data from several well-controlled investigations have documented that female rats with type 1 diabetes develop ocular surface complications before male rats. In part, this finding may be due to the increase in the inhibitory peptide Opioid Growth Factor (OGF) that occurs within 2 weeks of the induction of hyperglycemia in female animals in comparison to the changes in OGF levels in male rats which occur at 4 weeks. It was noted that estrogen levels drop within weeks of induction of hyperglycemia and could serve as another marker for the onset of disease activity and/or its complications. Finally, insulin does not appear to protect against early changes in OGF levels or estrogen secretion in diabetic female rats, setting the stage for a distinction in the disease profile of diabetes between males and females. These data encourage further studies on both sexes in order to establish a complete understanding of the underlying pathologies associated with complications associated with diabetes. Diabetes is a global epidemic with type 1 and type 2 diabetes affecting more than 95 million individuals in the western hemisphere . The CDCdb/db mice that modeled type 2 diabetes (T2D) confirmed that ocular surface complications were present in female animals and able to be modulated by the OGF-OGFr axis [Data accumulated from several decades of research on streptozotocin-induced (STZ) type 1 diabetic (T1D) rat models suggest that the Opioid Growth Factor (OGF) - OGF receptor (OGFr) axis is present in the eye and is involved in growth regulation and homeostasis of corneal surface epithelium -12. The GFr axis .ex vivo studies on rat and human corneal explants [Our research involving explants ,19, whicexplants  or topicexplants ,12,14, eexplants ,20 suggeexplants  followinOur research has shifted to focus on the timing of the onset of corneal complications in T1D rats, with follow up studies exploring whether prophylactic administration of NTX beginning at the appearance of hyperglycemia can prevent or delay the onset of dry eye, deceased corneal sensitivity, and delayed corneal epithelial wound healing ,15. GiveIn a side-by-side study of male and female Sprague Dawley rats rendered diabetic with intraperitoneal injections of STZ, a number of interesting comparisons were noted . In addiWith regard to tear volume, female T1D rats displayed a deficit in tear volume within 3 weeks of being hyperglycemic, which was 2 weeks prior to effects recorded in male rats. Insulin implants at the time of initial hyperglycemia were protective against low tear production in female rats, but not in male rats, as the T1D-INS male animals had significantly reduced tear volumes beginning 6 weeks after hyperglycemia was measured. These data suggest that the dysregulation of the OGF-OGFr pathway, along with the elevated OGF blood levels may be an independent predictor of dry eye. Maintenance of normal glycemic blood levels and control of sex differences in the susceptibility to diabetic complications and/or response to insulin therapy may influence the magnitude of diabetic complications, but not prevent or reverse them.Corneal tissue levels of OGF and OGFr were measured by confocal microscopy and optical density readings in male T1D rats at 8 weeks of hyperglycemia. Both OGF and OGFr corneal tissue levels were statistically  elevated above normal levels. Male rats treated with systemic NTX or topically with just one drop of NTX also had elevated OGF and OGFr tissue levels relative to their baseline or normal values. Only when NTX was administered topically twice daily for 8 weeks did OGF levels in corneal tissue match those of normal male rats. Understandably, OGF serum values were not altered by topical treatment with NTX, but were reduced following systemic NTX. OGFr levels in the serum of male diabetic rats did not respond to systemic or topical (2 drops) NTX treatment. Female diabetic rats also had substantially higher OGF and OGFr optical density measurements in ocular tissue than non-diabetic female rats. Insulin did not impact these tissue levels, but twice daily NTX drops did significantly reduce both OGF and OGFr ocular tissue levels to those observed in control female animals.Sex hormone levels were measured in an effort to determine whether the sex differences in onset and magnitude of corneal surface complications were related to blood levels of testosterone and estrogen ,21. InteOGF blood levels were not altered in either male or female rats on week 2 following STZ, but by week 3, female T1D and T1D-INS rats had elevated levels. OGF levels in males were elevated by week 5 and remained high throughout the 8-week study. Of note, OGF levels in normal and diabetic female rats were higher than their male counterparts. Beginning on week 4, female diabetic rats had blood OGF values ranging from 125 – 160 pg/ml in comparison to normal females at 50-75 pg/ml, and normal male rats with levels consistently below 25 pg/ml. The highest T1D male OGF level was approximately 60 pg/ml. Increases in enkephalin levels in diabetes have been documented in humans  and miceMultiple studies in male and female rat models of T1D confirm our hypothesis that elevated OGF and OGFr levels in serum and tissue following induction of hyperglycemia play a role in the onset and magnitude of diabetic complications involving the ocular surface, resulting in dry eye, depressed corneal surface sensitivity, and prolonged corneal epithelial wound healing. Investigations using T1D models, as well as genetic T2D female mice, confirm that treatment of this dysregulated OGF-OGFr axis using the receptor antagonist NTX effectively diminishes the complications. The timing of NTX administration is important. Early administration of NTX – as soon as hyperglycemia is detected – delays the onset and reduces the magnitude of complications, and in some cases, prevents the appearance of diabetic ocular surface complications altogetherMoreover, regulation of hyperglycemia with insulin seems to be independent of the effects of the OGF-OGFr pathway as both T1D and T1D-INS rats had elevated serum levels of OGF, and both groups responded to blockade of the regulatory pathway with NTX. As with most investigations, more questions are left unanswered. Could NTX be used prophylactically in humans with pre-diabetes? If so, would a NTX patch that slowly released the drug be feasible? Should pre-diabetic patients be screened for OGF serum levels? If OGF levels begin to increase, possibly intervention with NTX would be warranted. Limitations to the overall synopsis of data include the reliance on only female, and no male, type 2 diabetic mouse models, and no inclusion of high-fat diet diabetes animal models. Future investigations are needed to address the mechanism(s) underlying the dysregulation of the OGF-OGFr axis in diabetes, and to identify potential genetic markers in order to facilitate moving this research into the clinic so that pre-diabetes and diabetes could benefit from individualized treatment modalities.


Heart murmurs are a common finding in cats and, in many cases, the presence of an audible murmur on cardiac auscultation does not necessarily imply the presence of an underlying heart disease. Several studies have been published in the past to address the prevalence and clinical significance of heart murmurs in cats, but very few have looked into the exact origin of the blood flow turbulence responsible for this finding. We retrospectively reviewed clinical records and echocardiographic examinations of 856 cats with heart murmur and found that the majority of murmurs are caused by systolic anterior motion of the mitral valve (SAM) (39.2%) and dynamic right ventricular outflow tract obstruction (DRVOTO) (32%). These causes of murmur do not appear associated with a structural cardiac abnormality in 56.1% (SAM) and 85.0% (DRVOTO) of murmurs. This study also demonstrated that some heart murmur characteristics  can occasionally discriminate between normal cats and cats with underlying heart disease, with the exception of loud and palpable murmurs, which are inevitably associated with significant cardiac abnormalities. However, since the majority of heart murmurs in cats appear to be systolic and mild–moderate in loudness, echocardiography should always be considered following identification of a heart murmur on physical examination in cats.Background: Cardiac auscultation is one of the most important clinical tools to identify patients with a potential heart disease. Although several publications have reported the prevalence of murmurs in cats, little information is available in relation to the exact origin of the blood flow turbulences responsible for these murmurs. The aim of this study was to determine the prevalence and clinical significance of murmurs detected during physical examination in cats. Methods: Retrospective evaluation of clinical records and echocardiographic examinations performed in cats for investigation of heart murmurs; Results: Records of 856 cats with full clinical information were available for review. The cause of murmur was identified in 93.1% of cases . Systolic anterior motion of the mitral valve (SAM) was the primary cause of murmur in this population (39.2%), followed by dynamic right ventricular outflow tract obstruction (DRVOTO) (32%) and flow murmurs (6.9%). Most cats with a murmur (56.7%) did not present any structural cardiac abnormality. Conclusions: This study indicates that some heart murmur characteristics  can potentially predict the presence of an underlying cardiac disease. In people, heart murmurs are the most common reason for referral to a cardiologist and, especially in children, approximately 50–70% of these murmurs are clinically insignificant . SimilarTherefore, the main aim of this retrospective study was to determine the prevalence of heart murmurs detected during routine clinical examination in cats, with a particular emphasis on obtaining the exact echocardiographic identification of the origin of the flow turbulence deemed responsible for those murmurs. This study also sought to potentially identify patient and murmur characteristics that might predict a diagnosis of significant congenital or acquired cardiac disease.We hypothesized that the majority of cats with a heart murmur detected on cardiac auscultation do not have an identifiable cardiac abnormality on echocardiographic examination as previously observed in other studies ,3 and thClinical records and echocardiographic examinations performed on cats between June 2009 and June 2022 for diagnostic investigation of a heart murmur at 2 institutions, namely Specialist Veterinary Cardiology Consultancy  and The Ralph Veterinary Referral Centre , were reviewed retrospectively. The relevant information included examination date, cat’s signalment, heart murmur characteristics, echocardiographic identification of blood flow turbulence (single or multiple) associated with the detected heart murmur, echocardiographic measurements, presence of structural abnormalities, electrocardiographic (ECG) findings and whether or not the cat underwent sedation prior to the echocardiographic examination. Cats were excluded from the study if they had incomplete clinical information, including the full description of the detected heart murmur or if the full native echocardiographic examination was not available for review.Essential description of heart murmurs included timing , loudness/intensity  , point oAll echocardiographic examinations were performed by a board-certified cardiologist (LF) or a RCVS certificate holder in veterinary cardiology (HF) using an Esaote MyLab 30 Gold Cardiovascular or MyLab Omega  (SVCC Ltd.), or Esaote MyLab X8   echocardiographic machine and a 7.0–10.0 MHz or a 2.0–9.0 MHz phased array transducer depending on the size of the cat. All echocardiographic examinations were reviewed using dedicated offline ultrasound imaging software  to verify the correct identification of significant blood flow turbulence and confirm the final diagnosis.Complete transthoracic echocardiographic examination  was performed using routine echocardiographic views . All catSimultaneous ECG monitoring was obtained by applying non-traumatic ECG clips just above or below the elbows and just above or below the patella, using ultrasound gel as a conductive medium . WheneveLeft ventricular dimensions were measured from the right parasternal short axis view at the level of the papillary muscles using B-mode images from previously acquired high frame-rate cine-loops. For the purposes of this study, left ventricular hypertrophy was defined as a measurement of either interventricular septum in diastole (from leading to trailing edge) or left ventricular free wall in diastole (from leading to leading edge) greater than 6.0 mm, avoiding prominent false tendons, focal myocardial thickening at their insertion or punctual hyperechogenicity ,12. LeftDiagnosis of SAM was characterized by the concurrent presence of motion of the septal mitral valve leaflet towards the interventricular septum during systole, observed on a right parasternal short-axis M-mode view of the left ventricle at the level of the mitral valve, turbulent flow in the left ventricular outflow tract with simultaneous mitral regurgitation in mid-systole systole  and a dagger-shaped (scimitar-like) flow profile on spectral Doppler interrogation of the left ventricular outflow tract ,14,15,16The presence of DRVOTO was confirmed as systolic color Doppler aliasing in the right ventricle (RV) just cranial to the tricuspid valve and extending into the RV outflow tract, associated with spectral dispersion and a scimitar-like appearance of the RV outflow profile on spectral Doppler study, as previously described ,19. InduDiagnosis of flow (often named “innocent”) heart murmurs was based on the absence of any detectable structural or functional abnormalities on echocardiographic examination, despite the presence of a soft heart murmur on auscultation ,21.Mitral valve and tricuspid valve regurgitation were diagnosed following identification of a systolic turbulence directed towards the left and right atrium, respectively. The valvular regurgitation was confirmed in the left apical view using spectral Doppler interrogation of the abnormal flow. Conversely, mitral valve stenosis was diagnosed by demonstrating a diastolic turbulence directed towards the left ventricle and a typical prolonged deceleration time of the E wave on spectral Doppler inflow studies .Mid-left ventricular outflow obstruction, also called mid-cavitary outflow obstruction, was defined as a systolic turbulent flow originating at the middle of the left ventricle as a result of segmental mid-septal hypertrophy and misplacement of the papillary muscles and chordae tendineae in cats with left ventricular hypertrophy ,24.Pulmonic stenosis (PS) and aortic stenosis (AS) were deemed to be valvular unless otherwise stated and were confirmed by identification of partially fused cusps and increased systolic blood flow across the pulmonic and aortic valve respectively. Sub-aortic stenosis and supravalvular aortic stenosis were diagnosed after identification of a systolic blood flow turbulence in correspondence of a membrane just below or above the aortic valve respectively, causing a fixed obstruction. Double-chambered right ventricle (DCRV) was diagnosed when the blood flow turbulence and associated increased flow velocity was originating from a mid-cavitary obstruction dividing the right ventricle into a high-pressure proximal portion and a low-pressure distal portion. Aortic insufficiency (AI) was diagnosed after identification of a significant diastolic flow from the aorta towards the left ventricular cavity, mostly associated with aortic valve endocarditis or aortic valve dysplasia ,26.Heart murmurs associated with VSD and ASD were diagnosed following visualization of an incomplete septum and identification of a systolic flow turbulence across the defect .Blood flow turbulence associated with tetralogy of Fallot was characterized by a combined pulmonic stenosis and right-to-left shunting VSD .Diagnosis of patent ductus arteriosus (PDA) was based on the identification of the ductus arteriosus and an associated continuous blood flow turbulence across the defect directed toward the main pulmonary artery, since all PDAs in this cohort were left-to-right shunting .The blood flow turbulence associated with atrioventricular canal defect, also called cushion defect, was mainly characterized by a systolic shunt of the blood passing from the left ventricle to the right ventricle and out the pulmonary artery .Heart murmur in coronary artery to pulmonary artery fistula was associated with a continuous left-to-right blood flow turbulence across the abnormal vascular communication .Collected data were transferred to an electronic spreadsheet  and verified by 3 investigators  for accuracy. Quantitative variables were analyzed for normal distribution using the Shapiro–Wilk test and subsequently reported as mean ± standard deviation if normally distributed or median and range if non-normally distributed. Qualitative variables were summarized using absolute and relative frequencies.To test the hypothesis that in the majority of cats with a heart murmur detected on cardiac auscultation the murmur was not associated with a structural cardiac disease, we simply calculated the percentage of cats with a heart murmur without echocardiographic evidence of structural heart abnormalities.p value of <0.05 was considered to be statistically significant. All statistical analysis was performed with a commercially available statistical program .Univariable logistic models were used to test the hypothesis that patient signalment  and heart murmur characteristics  can predict the presence of a structural cardiac dis-ease. For this purpose, cat’s age, gender, breed as well as heart murmur characteristics were assessed as categorical variables, and murmur intensity was reclassified as “soft” (grade 1/6 or 2/6), “moderate” (grade 3/6), “loud” (grade 4/6) or “palpable” (grade 5/6 and 6/6), following the scheme proposed by Rishniw in 2018. This system is described as a simpler and more intuitive grading scheme that should provide the same clinical information as that obtained with the more complex Levine scheme but with potentially less confusion -phenotype) were 6 months old and, in both cases, the echocardiographic changes were associated with suspected acute myocarditis, which was followed by a complete reverse cardiac remodeling (confirmed on repeat echocardiographic examination) after a few weeks and complete disappearance of their heart murmur.Systolic anterior motion of the mitral valve (SAM) was the most common cause of heart murmur identified in this feline population (39.2%), followed by dynamic right ventricular outflow tract obstruction (DRVOTO) (32%) and flow murmurs (6.9%). All different causes of blood flow turbulence responsible for heart murmurs in this feline population are reported in n = 485; 56.7%). These were mainly cases where the blood flow turbulence was associated with SAM (50.3%), DRVOTO (19.9%) and flow murmurs (14.6%). However, when these types of murmurs were analyzed individually as a sole cause of murmur, 56.1% of cats with SAM and 85.0% of cats with DRVOTO did not display any structural cardiac abnormalities on echocardiographic examination. None of the cats with flow murmurs had structural abnormalities, by definition. Most cats with DRVOTO were older individuals, with a median age of 9.3 years .The majority of cats that underwent full echocardiographic examination to identify the cause of a previously detected heart murmur did not present any structural cardiac abnormality  or acquired cardiac conditions (29.3%), with a relative frequency depending on the underlying condition, as reported in Physical characteristics of the cats  and heart murmur characteristics  were analyzed using univariable and multi-variable logistic regression analyses to assess the probability of identifying a structural heart disease on echocardiography. All palpable, continuous, diastolic and to-and-fro murmurs were associated with a significant congenital cardiac disease (100%). Conversely, a negative association was found between soft/moderate systolic heart murmurs, intermittent heart murmurs, murmurs with a PMI over the right parasternal area and the presence of a cardiac abnormalities on echocardiography .A group of cats with an audible heart murmur on cardiac auscultation did not reveal any significant blood flow turbulence on echocardiographic examination until a provocative maneuver was performed and such murmurs were classified as inducible heart murmurs . These were characterized by inducible DRVOTO (8.3%) and inducible SAM (2.6%). In more detail, a provocative maneuver was necessary to reveal DRVOTO in 20.3% of cats and SAM in 5.1% of cats, respectively.To the best of the authors’ knowledge, this is the largest and most comprehensive study looking into the etiology and clinical characteristics of heart murmurs in cats and, unlike most of the published research on feline heart murmurs, is the first attempt to systematically identify the precise origin of the blood flow turbulence responsible for these murmurs.The median age and body weight of cats referred to our clinics for cardiac investigation were very similar to other studies performed in cats referred to a cardiologist for further cardiac investigations ,6.The majority of murmurs detected on auscultation were systolic, soft or moderate and with a PMI over both parasternal areas  with no apparent association with the underlying heart murmur etiology or the severity of the underlying heart condition. This suggests that heart murmur characteristics in the majority of cats may have a limited diagnostic relevance, although loudest murmurs are more likely associated with a pathological condition, mainly severe congenital cardiac defects, as previously reported . The sigEchocardiographic identification of the blood flow turbulence responsible for the detected heart murmur was obtained in 93.1% of cases. In the remaining 6.9% of cases, echocardiographic examination failed to identify any significant turbulence or structural abnormality, which led to an echocardiographic diagnosis of flow (innocent) murmurs, adopting the same diagnostic criteria described in humans ,21.Approximately one quarter of cats had multiple blood flow turbulences identified on echocardiography, which were deemed responsible for the murmur detected on auscultation. This is, in our opinion, another important finding that reinforces the above observation that detection of heart murmurs in cats, in the absence of an echocardiographic examination, cannot provide sufficient information towards an etiological diagnosis, with the exception of intermittent heart murmurs inducible by chest compression and unmasked by varying the pressure of the stethoscope on the chest wall  or heartUnlike other studies where heart murmurs in cats appeared to be mostly associated with a cardiac disease ,3,5,7, tThe most common origin of heart murmurs in these cats was dynamic left ventricular outflow obstruction associated with SAM, which represents approximately 40% of all murmurs detected in this feline population. Eighty percent of cats with HCM-phenotype and a heart murmur on auscultation had SAM. Systolic anterior motion of the mitral valve was also present in other forms of cardiomyopathy, such as in 27.3% of restrictive cardiomyopathy (RCM)-phenotype, 33.3% of non-specific cardiomyopathy and 22.2% of end-stage HCM, based on the classification proposed by Luis Fuentes et al. ,14.The second most common cause of heart murmur in this feline population was represented by a dynamic right ventricular outflow tract obstruction (DRVOTO), which was identified in approximately one third of cases. DRVOTO did not seem to be associated with any cardiac disease in 85.0% of cases, and since the echocardiographic abnormalities observed in the remaining 15.0% of cases did not appear to affect the right outflow tract, we believe that the presence of DRVOTO in such cases represents a pure incidental finding.Flow (Innocent) heart murmur represented the third most common cause of murmurs in this study (6.9%). However, it may be possible that the number of innocent murmurs was overestimated since a provocative maneuver was not performed in all echocardiographic examination to reveal the presence of inducible SAM or iatrogenic DRVOTO . It is aBlood flow turbulences associated with various congenital defects were considered responsible for an audible heart murmur in the remaining 21.9% of cases. However, it was impossible from our data to establish whether ASDs were responsible for an audible heart murmur since they were always associated with other significant blood flow turbulences.The importance of this finding is particularly relevant for the clinical management of these cases, where an early diagnosis may change the clinical outcome by prompting corrective or palliative interventions  or regular monitoring of the sleeping respiratory rate to allow early identification of clinical signs associated with congestive heart failure ,35.Owing to the retrospective nature of this study, some data were missing in a number of patients, including blood pressure measurement, hematocrit and serum concentrations of thyroxine hormone, which may have had a potential impact on the genesis of the heart murmurs. Given the lack of data on systemic status of cats, it is not possible for the authors to conclusively exclude that, at least in some patients, the actual main or concurrent trigger of the murmur could have been misdiagnosed.Another limitation was the inability to determine if outflow obstructions (namely SAM and DRVOTO) could have been potentially induced in some cases of “innocent” murmurs since the provocative maneuvers explained above were not always performed, especially when these techniques were still unknown or poorly described. Furthermore, limited characterization of the type of murmur for each echocardiographic abnormality may also represent a possible bias.Another potential limitation is represented by the unknown effect of the various sedative protocols on the structural and functional echocardiographic findings, although a relatively small percentage of cats were sedated in this study. Similarly, information about the use of cardioactive drugs was missing in many cases, and therefore it was not possible to analyze their effect on the clinical and echocardiographic findings. Finally, different clinicians were involved in the diagnosis and management of cats included in this study and this could have affected the accuracy of the results, although all clinical records and echocardiographic examinations were retrospectively reviewed by a board certified cardiologist to double-check the accuracy of measurements and diagnoses.More than half of cats referred for a cardiac evaluation of an audible heart murmur on thoracic auscultation do not have any echocardiographic evidence of heart disease, suggesting that most heart murmurs in cats are benign or may be potentially associated with a subclinical form of heart disease not detectable echocardiographically. This study also demonstrates that some heart murmur characteristics  can occasionally discriminate between normal cats and cats with underlying cardiac disease, with the exception of loud (mostly systolic) and palpable murmurs inevitably associated with a significant heart disease. However, since the majority of heart murmurs in cats appear to be systolic and mild-moderate in loudness, echocardiography, ideally performed by an experienced cardiologist, should always be considered following identification of a heart murmur on routine physical examination in cats, especially when they do not present any clinical signs referable to a heart disease.


Firmicutes along with decreased abundance of Bacteroidetes in newDM. At the genus/species levels, decreased abundance of Faecalibacterium prausnitzii, Roseburia, Dialister, Flavonifractor, Alistipes, Haemophilus and Akkermansia muciniphila and increased abundance of Lactobacillus, Streptococcus, Escherichia, Veillonella and Collinsella were observed in the disease groups in at least two studies. Lactobacillus was also found to positively correlate with fasting plasma glucose (FPG), HbA1c and/or homeostatic assessment of insulin resistance (HOMA-IR) in four studies. This renders a need for further investigations on the species/strain-specific role of endogenously present Lactobacillus in glucose regulation mechanism and T2DM disease progression. Differences in dietary intake caused significant variation in specific bacterial abundances. More studies are needed to establish more consistent associations, between clinical biomarkers or dietary intake and specific gut bacterial composition in prediabetes and early T2DM.Evidence of gut microbiota involvement in regulating glucose metabolism and type 2 diabetes mellitus (T2DM) progression is accumulating. The understanding of microbial dysbiosis and specific alterations of gut microbiota composition that occur during the early stages of glucose intolerance, unperturbed by anti-diabetic medications, is especially essential. Hence, this systematic review was conducted to summarise the existing evidence related to microbiota composition and diversity in individuals with prediabetes (preDM) and individuals newly diagnosed with T2DM (newDM) in comparison to individuals with normal glucose tolerance (nonDM). A systematic search of the PubMed, MEDLINE and CINAHL databases were conducted from inception to February 2021 supplemented with manual searches of the list of references. The primary keywords of “type 2 diabetes”, “prediabetes”, “newly-diagnosed” and “gut microbiota” were used. Observational studies that conducted analysis of the gut microbiota of respondents with preDM and newDM were included. The quality of the studies was assessed using the modified Newcastle-Ottawa scale by independent reviewers. A total of 18 studies  were included. Low gut microbial diversity was generally observed in preDM and newDM when compared to nonDM. Differences in gut microbiota composition between the disease groups and nonDM were inconsistent across the included studies. Four out of the 18 studies found increased abundance of phylum  It is expected that by 2030, 578 million people worldwide will have diabetes, with type 2 diabetes mellitus (T2DM) accounting for about 90% of this staggering figure . With anIndividuals with T2DM have an increased risk of developing complications such as kidney failure, retinopathy, neuropathy, cardiovascular disease and limb amputations . Recent A systematic review of observational studies was performed according to a protocol published in the International Prospective Register of Systematic Reviews (PROSPERO)   and repoA systematic search of published literature from inception to February 2021 was conducted using electronic databases including PubMed Central by the National Center for Biotechnology Information (PMC-NCBI), MEDLINE Complete and CINAHL (EBSCO Host). Briefly, main keywords included in the search were “type 2 diabetes”, “prediabetes”, “newly-diagnosed” and “gut microbiota”. The complete search strategy is provided in Studies that met the following criteria were included: (1) observational studies including case-control, cohort and cross-sectional study design; (2) studies on adult participants with newDM or preDM (3) studies that included a nonDM control group and (4) studies in which faecal samples were collected for gut microbial analysis. We excluded studies in which the participants were receiving treatment or dietary intervention prior to the investigation, studies in which participants provided microbiota samples from other body sites, animal studies and studies in which participants had other types of diabetes besides T2DM. Review articles, unpublished data, and articles in other languages were also excluded.All search results were exported to a reference manager software (Endnote X9.3.1). Two authors (GL and SGP) selected the articles based on their titles and abstracts. Any disagreements between the reviewers were resolved through discussion with the third reviewer (NA). The full texts of eligible studies were assessed, and studies deemed irrelevant were excluded. A standard form was used to extract the data of included studies  and characteristics of methodology used by selected studies (microbiota quantification methods and diversity indices used) and the outcome measured. The primary outcomes were gut microbial abundance and differences between study groups at the phylum, class, order, family and genus taxonomic ranks. Species were grouped according to their respective genus. The secondary outcomes included clinical biomarkers, dietary intake and other parameters measured along with their correlation with the gut microbial composition.The study quality was assessed using the modified Newcastle Ottawa scale . The scaOf 3,994 articles identified, 18 studies were included in this systematic review See , four crThe 18 studies consisted of 5,489 participants. In 15 of the 18 studies, 43% of the participants were male  and 57% were female . Three studies  did not Atopobium cluster, Bacteroides fragilis, Bifidobacterium, Clostridium coccoides, Clostridium leptum, Clostridium perfringens, Enterobacteriaceae, Enterococcus, Lactobacillus and Prevotella  , metagenThe α-diversity or the average microbial diversity within a single sample was reported by 13 of the 18 studies and the key measures used were Shannon, Simpson, observed species richness, abundance-based coverage estimator (ACE), Chao1 and phylogenetic diversity indexes Table 3.Ruminococcaceae was the most positively correlated with α-diversity while Fusobacterium, Flavonifractor, and Parasutterella were the most negatively correlated with α-diversity , principal component analysis (PCA) of either weighted or unweighted UniFrac or Bray Curtis dissimilarities distance matrices Table 3.Six studies found a significant difference in β-diversity among preDM , newDM  and in bAll eighteen studies provided information on microbial abundance by genus/species ranks. Eleven studies  reportedBacteroidetes and Firmicutes were the predominant phyla in all groups studied, i.e., preDM and/or newDM and nonDM  among newDM . SCFAs, namely acetate, propionate and butyrate are metabolic products of fibre fermentation by bacteria in the gut. They are shown to exert many beneficial effects on human metabolism and immune system  and Collinsella (an Actinobacteria). Streptococcus, Escherichia and Collinsella are all known gut inhabitants whose abundance are associated with several inflammatory diseases including T2DM   , howeversubjects . Althouge models , it is lLactobacillus abundance. Similarly, no consistent findings between studies were noted in correlation between dietary intake and specific gut bacterial composition. However, the studies were able to conclude that a less healthy food pattern including increased carbohydrate, fat or energy intake or a reduced fibre intake correlated with prevalence of preDM or newDM and differences in dietary intake caused significant variation in specific bacterial abundance.Overall, the increased levels of glycaemic and pro-inflammatory markers along with low diversity of gut bacteria among the preDM and newDM generally observed in this review suggest the possibility of gut bacteria- associated inflammation-induced environment preceding the development of T2DM. Although the studies included in this review found significant association between clinical biomarkers and the abundance of specific bacterial groups, no consistent findings were observed between studies, with the exception of the correlation between glycaemic markers and One of the limitations of the present review is that it only summarises studies investigating gut microbiota based on composition found in faecal samples. There is a need for more studies to look into microbiota of mucosal biopsies in addition to faeces, in order to differentiate mucosa-associated bacteria from the total composition present in faeces. This is because mucosa-associated bacteria may have a more pertinent role in the pathogenesis of disease. It is also important to emphasise that findings from observational studies selected herein do not conclusively establish whether changes seen in microbiota composition were a cause or effect of glucose intolerance. Hence, bacteria found to be decreased or increased in the disease groups could not be conclusively termed as being ‘protective’ or ‘pro-diabetic’, respectively.Studies involving taxonomic analyses of microbiota have several biases. One of it is that compositional analyses of microbiota requires that the microbial proportions to be summed to a 100% total, thus reporting the relative abundance and not the absolute composition of the microorganisms present in the faecal samples of participants. Thus, if one bacterial group appears to be reduced, the others will naturally appear to be increased and vice versa. Differences in sampling fractions may also tend to introduce false positives and false negatives .Another challenge in analysing gut microbiome data across studies is the biases caused by the heterogenous methodology adapted. This limits the comparability of studies and leads to ambiguous results across similar studies. A meta-analysis was also not workable for the same reason. Although all studies targeted the 16S rRNA gene, factors that differed across the studies i.e. the choice of sequencing region, pipelines and databases used for bioinformatics analysis influenced the results produced. Moreover, the usage of different diversity indexes and statistical analysis affects the comparability of microbial diversity between the studies. The choice of the diversity index affects the interpretation of the microbiome data and leads to a lack of generalizable results across the studies. Besides methodological limitations, the use of alpha diversity as a biomarker in health and disease may be confounded by colonic transit time  and stooMore importantly, the taxonomic diversity exhibited by these analyses does not take into account, the functional redundancy among members of the microbiota. Taxonomically distinct bacterial species are able to perform similar metabolic functions and as such, taxonomic variation does not reflect functional variation . TherefoLactobacillus, Streptococcus, Escherichia, Veillonella and Collinsella) and/or decreased  in preDM and newDM. These alterations were however not consistent across all studies included, hence emphasising the uncertainty that lies in this field of study. The increased presence of Lactobacillus in preDM and newDM along with its positive correlation with glycaemic markers were also inconsistent observations. This renders a need for more investigation on the species/strain-specific role of this genus in T2DM disease progression and glucose regulation mechanism. Healthier food intake inversely correlated with prevalence of preDM and newDM, while differences in dietary intake caused significant variation in specific bacterial abundances. More studies should investigate the correlation of clinical biomarkers and dietary intake with gut bacterial composition in prediabetes and early T2DM to establish more consistent associations.The 18 studies included herein were found to have heterogeneity in methodology and inconsistencies in the findings on gut microbial changes observed among preDM and newDM when compared to nonDM. By focusing on changes that were similarly reported in two or more studies, it was evident that certain bacteria were found to be increased . Geetha Letchumanan is supported by the same grant.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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In this Outlook, Marino discusses a study in this issue by Youn et al. that describes defined and diverse roles of primary cilia in molecularly distinct medulloblastoma subgroups, highlighting once again the importance of designing subgroup-specific therapeutic approaches for this tumor. Genes & Development by Youn et al. (pp. 737–751), which describes defined and diverse roles of primary cilia in molecularly distinct medulloblastoma subgroups, highlighting once again the importance of designing subgroup-specific therapeutic approaches for this tumor.Here I discuss the study in this issue of  Medulloblastoma, the most common malignant primary brain tumor in children, is a paradigm of deregulated developmental mechanisms leading to tumor formation . It is cIn the brain, primary cilia—microtubule-based cellular structures anchored to a basal body that serves as a template for the assembly of the ciliary microtubule —are critThe presence of primary cilia is known to be associated with the molecular subgroups of human medulloblastoma; they are found in SHH and WNT subgroups but not in G3 and G4 subgroups, a finding that is well mirrored by mouse models of these subgroups . In partGenes & Development has now significantly expanded our understanding of the multifaceted role of cilia in medulloblastoma pathogenesis. The investigators show that in mice, primary cilia are required to enhance the proliferation and survival of ectopically accumulated dorsal brainstem cells; i.e., the cells of origin of WNT medulloblastoma. Moreover, tumors that formed in mice lacking cilia showed decreased proliferation and increased cell death compared with WNT medulloblastomas arising in control mice. Fully transformed WNT medulloblastoma cells also require primary cilia for continuous proliferation and optimal survival. Mechanistically, cilia control β-catenin synthesis rate, as assessed by cycloheximide/proteasome inhibitor treatments in cell lines, via promoting nuclear accumulation of ELAVL1. Consequently, cilium loss increased stress granules containing β-catenin mRNA, the translation of which became disrupted. Hence, primary cilia promote WNT medulloblastoma by facilitating the synthesis of β-catenin, the major oncogenic driver in 90% of WNT medulloblastomas.The elegant study of Interestingly, the investigators show a very different scenario in G3 medulloblastoma. Here, cilia are present in the progenitor cells, giving rise to G3 medulloblastomas; however, they are progressively lost during neoplastic transformation, and this significantly shortened the lives of mice with tumors. In particular, proliferation was increased via altered cell cycle dynamics, as assessed by double-thymidine analog labeling; the G2 phase was affected via abnormal CDK1 activation in the absence of cilia, which shortened the S phase and triggered premature G2/M transition. DNA damage and genome instability contributed to tumorigenesis in this scenario.This study has expanded the repertoire of ciliary functions and further highlighted their context dependency. More work will be required to assess whether and how these mechanisms can be exploited therapeutically. However, this study emphasizes once again the importance of a detailed molecular understanding of the characteristics of medulloblastoma subgroups if novel patient-tailored more effective and less toxic therapies are to be developed for these tumors.


Leber’s Hereditary Optic Neuropathy (LHON), is one of the most frequent mitochondrial diseases characterized by Retinal Ganglion Cells degeneration. Pathogenic gene mutations in LHON induces mitochondrial impairment, which in turn leads to insufficient mitochondrial ATP production. The pathologic hallmark of the disease is primary degeneration of retinal ganglion cells, which results in optic nerve atrophy. The paper reviews some of the recent advances in the understanding of LHON: new genetics discoveries and novel therapeutic approaches.  The first point mutation was discovered by Wallance et al., one century later, in 1988, in the mitochondrial DNA (mtDNA) (m.11778G>A in MT-ND4) [2] and further other two-point mutations were described (m.3460G>A in MT-ND1 and m.14484T>C in MT-ND6) . These three mtDNA mutations are responsible for approximately 90% of LHON cases (mtLHON) . Another 5% of LHON cases are produced by rare mtDNA mutations, the so-called „exotic mutations”, including variants associated with systemic pathology  . All these mtDNA mutations disrupt subunits of mitochondrial complex I, impairing the electron transfer and the ATP synthesis [1].Leber’s Hereditary Optic Neuropathy (LHON), the most frequent mitochondrial disease, was described as a clinical entity by Theodor Leber in 1871 [1]. The mutation was identified in a nuclear encoded gene, DNAJC30 [1]. The DNAJC30 proved to be a chaperone protein needed for the efficient complex I function [1]. In arLHON, all characteristics of mtLHON were present, including: incomplete penetrance , male predominance and significant idebenone responsivity [1].Stenon et al. recently described an autosomal recessive model of inheritance for LHON (arLHON), resolving the problem of undiagnosed patients with LHON manifestations and no proof of pathogenic mtDNA mutations . Except for some few pairs and triplets of identical haplotypes that were observed, the large majority of the LHON mutations were due to independent mutational events [8]. The analysis showed that the primary mutations 11778, 3460, and 14484 were recent and were due to multiple mutational events [7]. However, there are other putative secondary/ intermediate LHON mutations  that are ancient polymorphisms and are associated in specific combinations, especially in haplotype group J that is European-specific [7]. The 11778 and 14484 primary mutations also showed a strong preferential association with haplogroup J, this meaning that one ancient combination of haplogroup J-specific mutations increases both the penetrance of the two primary mutations 11778 and 14484 and the risk of disease expression [7].High-resolution restriction-endonuclease analysis was performed in LHON patients to define the phylogenetic relationships between the mtDNA haplotypes and the LHON mutations [9]. The skeletal muscle biopsy and the urinary sediments are the gold standard, but the skeletal muscle biopsy is invasive and might not be justified in some cases.A key issue in LHON molecular diagnosis is the tissue used for sequence analysis. Some mtDNA mutations are counterselected in a fast turnover tissue like the cells from the blood sample, and disappear in an age-related fashion. Therefore, different tissues should better be collected for the LHON molecular diagnosis. Except for the blood, the total DNA may be extracted from tissues including skeletal muscle, buccal epithelia, and urinary sediments [10]. in 2016, Catarino et al. reported a LHON family with multiple patients harboring two primary LHON mutations, m.11778G>A homoplasmic and m.14484T>C heteroplasmic [10]. Some atypical findings were registered in this family [10]. The male-to-female ratio was unusually low as all three affected family members were females [10]. The index patient had a very late onset of symptoms (at 75 years) and low visual acuity, but her two daughters had both onset in childhood (6 and 9 years), with moderate to mild visual impairment [10]. A higher degree of heteroplasmy of the m.14484T>C mutation was found to correlate with an earlier age at onset in this family [10]. A rare possibility is to demonstrate two primary mutations in the same patient [11]. OPA1 is a nuclear gene that regulates the mitochondrial fusion and dynamics and is involved in the oxidative phosphorylation . Nowadays, other genes involved in DOA for patients negative for OPA1 mutations have been described [11]. AFG3L2 and SPG7 genes showed to have a crucial role in the maintenance of the optic nerve physiology and must be screened when suspecting DOA patients [14].When considering the children with LHON or patients with early onset of the disease, the specific acute/ subacute phase may be absent or overlooked, as for a child it is difficult to assess the moment when visual acuity decreases. When examining pediatric patients, the ophthalmologist may find optic nerve atrophy from the first presentation of the patient. Therefore, differential diagnosis between pediatric LHON and Dominant Optic Atrophy (DOA) may be challenging. The diagnosis of DOA can be ruled out by genetic analyses, OPA1 mutations covering 70% of DOA cases [12]. Therapeutic strategies are now limited to LHON, but hopefully they will also be offered soon in other mitochondrial optic neuropathies [12]. The Retinal Ganglion Cells (RGCs) degeneration is the common pathogenic event of LHON, DOA and others Hereditary Optic Neuropathies . This anatomical particularity is associated with an increased energy requirement, when the saltatory conduction of action potentials is absent. Therefore, the macular RGCs axons demonstrated to have mitochondrial clustering and a special sensitivity to energy depletion and reactive oxidative species (ROS) accumulation as it happens in mitochondrial dysfunctions . The optic nerve has normal myelinated axonal structure posterior to the lamina cribrosa .In order to avoid blocking the light to reach to the retinal pigment epithelium, in the macular region and particularly in the papillomacular bundle, the RGCs have unmyelinated narrower caliber axons [12]. These include: antioxidants, anti-apoptotic drugs, activators of mitobiogenesis and gene therapy [12].Therapeutic options for Hereditary Optic Neuropathies and innovative drugs acting on different molecular pathways are nowadays under study [12]. Idebenone acts as a strong antioxidant that restores the ATP synthesis at mitochondrial level in LHON patients by direct electrons transfer to complex III of the electron transport chain, thereby bypassing the unfunctional complex I . The efficacy and safety of idebenone was assessed by Klopstock et al. in 2011 in the RHODOS study . The experts’ consensus of idebenone recommends that the treatment should start in the first year from visual loss onset (in the acute stage), as early as possible, the administration of the idebenone dose of 900 mg/ day being given into three daily doses of 300 mg with meals . Treatment should be given for at least 12 months to assess a therapeutic response, then discontinuation of the drug should be considered after 12 months once a plateau recovery is reached, or no improvements are observed . Two others multicenter studies (LEROS and PAROS) were launched after the idebenone commercial approval, being designed to assess the long-term efficacy and safety of the drug [12]. The results are now being processed and will be published soon. Pump et al. recently published new promising results with visual improvement after treating patients in both early chronic phase  and late chronic phase (>5 years) with idebenone [30].Idebenone (Raxone®) was the first molecule approved for LHON in Europe in 2015 and due to the low prevalence of the LHON it is an orphan drug [12]. In 2012, Sadun et at. demonstrated the arrest of the disease progression and improvement of visual acuity in four of five patients with LHON treated with Epi-743, three times daily, with an oral dose of 100-400 mg per dose, for 90 days [31]. Since then, the Epi-743 was not used any more in LHON patients [31]. In 2018, Zesiewicz et al. demonstrated the Epi-743 efficacy in the improvement of both the neurological function and the disease progression in 63 patients with Friedreich’s ataxia, but, in this study, the Epi-743 failed to demonstrate any significant visual benefit in the two-thirds of the treated patients who associated optic atrophy . Epi-743 (vatiquinone) is an antioxidant drug acting on oxidoreductase enzymes . Elamipretide is a Szeto-Schiller antioxidant peptide that targets cardiolipin selectively in the inner mitochondrial membrane, this way preventing conversion of cytochrome c into a peroxidase, preserving its function as simple electron carrier, and promoting OXPHOS functioning [12]. Sadun et al. conducted a Phase II Clinical Study (ReSIGHT) to evaluate the safety, tolerability, and efficacy of the Topical Ophthalmic Solution of Elamipretide for 12 patients with LHON [12]. The results are not yet published, but a press release announced promising results [12].Elamipretide (MTP-131) is a molecule that increases the ATP synthesis and reduces the ROS production, regardless of the type of mitochondrial abnormality [12]. So far, only the Cyclosporine A, an inhibitor of the opening of the mitochondrial permeability transition pore, has been inefficiently tested in five LHON patients . Other molecules inhibiting different apoptotic events may represent future therapeutic agents for hereditary optic neuropathies in future [12].Cell degradation and self-destruction of the mitochondria are activated by caspases that are specific enzymes in the apoptosis cascade .Photobiomodulation by near-infrared light-emitting diode arrays (NIR-LED) is a promising therapy in LHON as it was demonstrated that it attenuates the optic nerve degeneration after acute mitochondrial injuries with different toxins in a rat model. Human research is necessary but the only study set by now failed to recruit enough patients [12]. To date, there are several phase I, II and III studies for LHON gene therapy [12]. Promising results were obtained on LHON patients with m.11778G>A mitochondrial DNA mutation [12]. The gene therapy consisted in a wild-type of ND4 subunit that was packaged into an adeno-associated-virus and injected into the vitreous [12]. RESCUE and REVERSE are two randomized, double-masked, sham-controlled, multi-center, phase III clinical studies in which an intravitreal administration of rAAV2/ 2-ND4 (GS010) was delivered in one eye and an intravitreal administration of sham injection was delivered in the fellow eye . 37 patients with visual acuity loss duration of 180 days or less were included in the RESCUE study and 39 patients with the onset of the disease between 6 and 12 months were included in the REVERSE study . In both studies, a statistically significant visual acuity improvement was surprisingly demonstrated both in the GS010-treated eyes and in the sham-treated eyes . A non-clinical trial on primates suggested a possible retrograde, trans-chiasmatic transit of the vector from the GS010-treated eye to the sham-treated eye as explanation for the unexpected bilateral visual improvement found in the RESCUE and REVERSE studies [12]. Recently, in 2019, the enrollment of 98 patients in a new Phase III clinical trial (REFLECT) was completed . The REFLECT study will evaluate the efficacy and safety of bilateral intravitreal injections of GS010 in subjects with 11778 LHON mutation .Gene therapy is the goal in hereditary optic neuropathies treatment [Advances in the diagnosis and treatment of LHON are currently in a positive dynamic direction. New pathogenic mechanisms, novel therapeutic molecules and gene therapy are the near future approach in LHON and other mitochondrial optic disorders.Conflict of Interest statementThe authors state no conflict of interest. AcknowledgementsNone.Sources of FundingNone. DisclosuresNone.


Metabolic rewiring is now considered a hallmark of cancer. Pancreatic ductal adenocarcinoma (PDAC) extensively rewires its metabolism especially for the utilization of glucose and glutamine that are mainly used to provide the biosynthetic intermediates for nucleotides, protein, and lipid synthesis and for redox homeostasis. In this regard, mitochondrial solute carriers play a key role in metabolic compartmentalization and hence metabolic rewiring. At least five mitochondrial transporters are involved in the metabolic reprogramming of PDAC. Targeting these transporters may provide an attractive strategy to combat this aggressive cancer. This review dissects the current knowledge about the role of these transporters in PDAC in order to explore the therapeutic potential of their targeting.Pancreatic cancer is among the deadliest cancers worldwide and commonly presents as pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming is a hallmark of PDAC. Glucose and glutamine metabolism are extensively rewired in order to fulfil both energetic and synthetic demands of this aggressive tumour and maintain favorable redox homeostasis. The mitochondrial pyruvate carrier (MPC), the glutamine carrier (SLC1A5_Var), the glutamate carrier (GC), the aspartate/glutamate carrier (AGC), and the uncoupling protein 2 (UCP2) have all been shown to influence PDAC cell growth and progression. The expression of MPC is downregulated in PDAC and its overexpression reduces cell growth rate, whereas the other four transporters are usually overexpressed and the loss of one or more of them renders PDAC cells unable to grow and proliferate by altering the levels of crucial metabolites such as aspartate. The aim of this review is to comprehensively evaluate the current experimental evidence about the function of these carriers in PDAC metabolic rewiring. Dissecting the precise role of these transporters in the context of the tumour microenvironment is necessary for targeted drug development. Pancreatic cancer is the fourteenth most common cancer and the seventh leading cause of cancer-associated death worldwide  and it iPancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer accounting for more than 90% of diagnosed cases . PDAC ar+ regeneration [Although the first observation of the metabolic changes in cancer highlighting that tumour cells consume more glucose to produce lactate and energy even in the presence of oxygen (known as the “Warburg effect”) was made a century ago, metabolic reprogramming in cancer has gained its importance only in the past two decades. Currently, metabolic reprogramming is considered a major hallmark of cancer  and it ineration . Lipid mneration ,24). Canneration . The uptneration . The metneration ,28  found in the brain, and a short isoform  usually overexpressed in cancers [GLS2 encodes two transcripts named GAB and LGA, arising from transcription driven by surrogate promoters. GLS2 is transcriptionally regulated by TP53 and it is hypermethylated in several cancers such as glioblastoma and hepatocellular carcinoma, suggesting that it is a tumour suppressor gene [2O2-mediated oxidative stress by suppressing the PI3K/AKT pathway [Glutamine, the most abundant amino acid in human plasma, is used by cancer cells for versatile metabolic roles including the synthesis of proteins, nucleotides, lipids, and non-essential amino acids, or it acts as an exchanger for the transport of other amino acids ,37. Glut cancers . Similarsor gene ,41. Inde pathway .In PDAC, the GLS isoform GAC plays a key role in upregulating glutaminolysis and thus glutamine metabolic rewiring . GLS seeDespite reports showing that GAC is localized in the inner mitochondria membrane, the exact localization of its catalytic domain has not yet been elucidated ,47,48. CThe search for the mitochondrial glutamine transporter began in the 1970s . HoweverThis transporter, similarly to the mitochondrial pyruvate carrier (MPC) ,53, lackSLC25A22 and SLC25A18 genes, respectively [+, and ameliorates redox homeostasis in favour of cell growth, migration, and invasion [The glutamate present in the cytosol obtained either from diet or protein degradation and/or from the interconversion of other amino acids is transported into mitochondria by glutamate carriers (GC). In humans as in other organisms, two different isoforms are known, GC1 and GC2, encoded by the ectively ,64. Bothectively ,65  and SLC25A13  [2+-dependent manner mitochondrial aspartate for cytosolic glutamate  plus one proton [2+-dependent stimulation of both aralar1 and citrin is enabled by the presence of a unique long N-terminal extension harbouring a number of EF-hand motifs and facing the intermembrane space [The aspartate–glutamate carriers AGC1 and AGC2 can also transport glutamate into the matrix. They belong to the mitochondrial carrier family SLC25 and are transcribed from two different nuclear genes, namely  citrin) ,72. Bothe proton   that maps to chromosome 6 and it has a transcript made of 8 exons and 7 introns giving rise to a 109 amino acid-long protein. MPC2 is encoded by the MPC2 gene  mapped to chromosome 1 and it has five exons and four introns producing a 127 amino acid-long protein [The mitochondrial pyruvate carrier (MPC) is highly conserved throughout evolution from yeast to humans . In huma protein ,53.Unlike other known canonical mitochondrial carriers such as SLC25, MPC belongs to a new class of mitochondrial carriers, SLC54 . AlthougCancer cells dramatically reprogram glucose metabolism through increased glucose flux via the glycolytic pathway and lactate efflux to the extracellular microenvironment, as well as decreased pyruvate influx to mitochondrial oxidative phosphorylation even in the presence of oxygen and functional mitochondria ,143. ThePDAC is characterized by extensive metabolic rewiring including enhanced aerobic glycolysis and glutaminolysis or glutamine addiction. In this context, glucose is metabolized through aerobic glycolysis for rapid energy production and to provide the building blocks for biosynthetic pathways including the pentose phosphate pathway, hexosamine pathway, and serine biosynthesis . On the To favour metabolic rewiring, PDAC cells change the transport of metabolites through the inner mitochondrial membrane towards the matrix by decreasing the flux of pyruvate, increasing that of glutamine and glutamate, while increasing the efflux of aspartate. Hence, the mitochondrial solute transporters MPC, SLC1A5_var, SLC25A12, SLC25A22, and UCP2 play a central role in these metabolic reprogramming scenarios . Data obGenipa americana fruit) has shown promising effects against PDAC cell lines by inducing GAPDH nuclear translocation and autophagy [From a patient treatment perspective, PDAC metabolic rewiring offers new attractive therapeutic targets. To date, most efforts have been put into the design and development of potent inhibitors of glucose and glutamine metabolism, such as those targeting glutaminase (CB-839) and hexokinase (Benitrobenrazide), for example. On the other hand, studies aimed at the treatment of PDAC targeting the mitochondrial carriers are very limited, likely because of the very recent discovery of their role in the pathogenesis of the disease and the challenges and limitations intrinsic to in vitro inhibition assays. However, preliminary evidence shows that targeting mitochondrial carries with small molecules, particularly those of natural origin, could provide powerful tools to fight PDAC . For insutophagy , increasutophagy , and redutophagy . Therefoutophagy . Finallyutophagy ,167. Fur