Acute Kidney Injury (AKI) is an independent risk factor for mortality in hospitalized patients. AKI syndrome leads to fluid overload, electrolyte and acid-base disturbances, immunoparalysis, and propagates multiple organ dysfunction through organ “crosstalk”. Preclinical models suggest AKI causes acute lung injury (ALI), and conversely, mechanical ventilation and ALI cause AKI. In the clinical setting, respiratory complications are a key driver of increased mortality in patients with AKI, highlighting the bidirectional relationship. This article highlights the challenging and complex interactions between the lung and kidney in critically ill patients with AKI and acute respiratory distress syndrome (ARDS) and global implications of AKI. We discuss disease-specific molecular mediators and inflammatory pathways involved in organ crosstalk in the AKI-ARDS construct, and highlight the reciprocal hemodynamic effects of elevated pulmonary vascular resistance and central venous pressure (CVP) leading to renal hypoperfusion and pulmonary edema associated with fluid overload and increased right ventricular afterload. Finally, we discuss the notion of different ARDS “phenotypes” and the response to fluid overload, suggesting differential organ crosstalk in specific pathological states. While the directionality of effect remains challenging to distinguish at the bedside due to lag in diagnosis with conventional renal function markers and lack of tangible damage markers, this review provides a paradigm for understanding kidney-lung interactions in the critically ill patient. Acute respiratory distress syndrome (ARDS) is a life-threatening condition and a leading cause of mortality in critically ill patients causing nearly 200,000 deaths in the United States each year . The devThe implications of AKI are broad and include fluid overload, electrolyte abnormalities, immunoparalysis, and multiple organ dysfunction through organ crosstalk. Respiratory complications are a key marker of increased mortality in patients with AKI, which points to a biologically plausible mechanistic link between AKI and Acute Lung Injury (ALI). Animal models show AKI causes ALI . ConversThere are many challenging and complex interactions between the lung and kidney in critically ill patients with AKI and ARDS . These iElucidation of the pathophysiology of AKI has revealed a complex, multisystem disorder with clinically important implications for patients with AKI. Important among these is the relationship between AKI, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Data from studies in multiple clinical settings and animal models support a dynamic interplay between the kidneys and lungs with each set of organs having the potential to influence and disrupt the structure and function of the other . In the Several populations of immune cells, including T-cells, neutrophils and macrophages, have been shown to mediate lung injury in the setting of AKI. Evidence for the involvement of T-cells in distant organ effects in the setting of AKI comes initially from investigation of the actions of T cells following organ specific injury. For example, in a mouse model of ischemia-reperfusion AKI, T-cell trafficking to the injured kidney has been shown to occur within 1 hour of the time of insult and to result in the local release of proinflammatory mediators . FurtherSimilar to T-cells, both neutrophils and macrophages have been shown to be involved in the mediation of organ specific injury in both the kidney and the lung. In several models of AKI, including ischemia and nephrotoxin associated AKI, resident renal macrophages are involved in the initial response to injury and are targeted to the sites of injury through the release of damage associated molecular pattern (DAMP) molecules and hypoxia inducible factors (HIFs) from injured renal tubular epithelial cells , 18. ThiSimilar macrophage and neutrophil driven processes occur in ARDS, and highlight the importance of the balance between initial rapid pro-inflammatory phase, and the subsequent regulation of inflammation and shift to a more reparative milieu. One important neutrophil related intersection of AKI and ALI could be at the formation of neutrophil extracellular traps (NETs). NET formation is a unique form of bacterial killing and is important to the normal response to microbial infection, but it also has been shown to occur at sites of sterile inflammation including the development of AKI and ALI/ARDS , 24. IntIn addition to cellular mediators of tissue injury, several soluble factors have been reported to trigger ARDS in the setting of AKI. Similar to the cell driven inflammatory responses outlined above, these inflammatory molecules have been shown to mediate damage not only locally but also have been implicated in distant organ effects including causing ALI in the setting of AKI .Important among the molecular mediators of lung injury associated with AKI is interleukin-6 (IL-6). Clinically, serum IL-6 levels have been shown to increase in patients following AKI, and also have been associated with prolonged mechanical ventilation in the setting of AKI . RegardiSimilar to IL-6, interleukin-8 (IL-8) has been studied with regard to its involvement in tissue specific injury in AKI and ALI as well as in AKI mediated lung injury. There is evidence that IL-8 is increased in the serum of patients with AKI following cardiac surgery, and that this elevation in IL-8 is associated with prolonged mechanical ventilation . Also siIn contrast to IL-6 and IL-8, other circulating cytokines appear to play a more anti-inflammatory role in modulating lung injury following AKI. In a mouse model of AKI using bilateral nephrectomy, treatment with IL-10 was shown to decrease pulmonary edema, neutrophil infiltration and BAL fluid protein, indicating a protective effect of IL-10 on the lungs of these experimental animals with AKI . ConflicTNFa, IL-6, and CXLCL-1  has been identified as a mediator of vascular permeability in patients with sepsis and ARDS, and it could play a pivotal role in kidney-lung crosstalk –53. Ang-ith ARDS , 56, 57.ith ARDS , 58, 59.Available data support the concept of distant organ effects on the lungs in AKI caused by a complex interplay between multiple cellular and molecular modulators of the immune system, which can initiate and perpetuate damage to the lungs. This is all under the assumption that the initial site of injury is the kidneys, and it is important to recognize the bidirectionality of kidney-lung crosstalk. Despite the high incidence of AKI in patients with ARDS, the impact of ALI on this ARDS-AKI construct and the effects of mechanical ventilation on the kidneys are not well understood . The leaALI and ARDS often lead to hypoxemia and hypercapnia, both of which affect renal blood flow and can lead to direct renal ischemia and subsequent injury. While the hallmark of ARDS is hypoxemic respiratory failure, hypercapnia can result from either the primary disease process, lung protective strategies limiting ventilation, or frequently, a combination of the two. Specifically, the current recommendations for treatment of ARDS include targeting lung protective strategies with lower goals for oxygen levels  and permissive hypercapnia . Both hyAn additional suggested mechanism of lung-kidney crosstalk involves extracellular nucleotides released by injured pulmonary epithelial cells. Nucleotides are molecules that modulate numerous functions including vascular tone, apoptosis, membranous ion conductance, and trans alveolar fluid regulation in the lungs. Nucleotides are released by pulmonary epithelial cells in response to physical stimuli including shear stress during mechanical ventilation. In addition, nucleotides induce the synthesis and release of cytokines, specifically IL-6, which is a proinflammatory mediator described in the pathogenesis of AKI and the generation of renal tubular injury , 73. Douin vitro with elevated blood urea nitrogen and creatinine levels as well as in vivo in rabbit models with injurious ventilator strategies; hypothesized to be secondary to soluble Fas ligand (sFasL), an important mediator of renal cell injury via apoptosis , and various cytokines, and chemokines . In factMost recently, the changing epidemiology of AKI among the pandemic SARS-CoV-2 related ARDS cases worldwide has further highlighted the potential mechanistic link between a therapeutic approach and remote organ injury propagation. Many centers have reported a high incidence of renal replacement therapy requiring AKI in the setting of COVID-19 early on in the pandemic with dismal outcomes . The useAlthough our understanding remains incomplete, awareness of continuous interaction and organ crosstalk highlights the importance of vigilance related to lung protective ventilation strategies in the management of ARDS in order to limit lung mediated remote organ injury, such as AKI. In addition to inflammatory consequences, mechanical ventilation has a number of hemodynamic effects and influences neurohormonal factors that contribute to AKI. Elevated pulmonary vascular resistance and right ventricular strain in the setting of ARDS might impact renal perfusion . ElevateKnowledge of the pathophysiologic links between AKI and ARDS could be leveraged to identify patients with AKI who are at risk of developing ARDS and can be used to develop targeted therapies that prevent or treat this complication. Unfortunately, the complex interplay between both organ systems discussed thus far makes this endeavor less attainable in clinical practice. Lung protective mechanical ventilation and fluid management to restore effective arterial blood volume are the cornerstones of ARDS management. Currently, avoiding positive fluid balance remains the most important predictor of pulmonary complications in patients with AKI and is the primary target for intervention. Restrictive fluid management in ARDS has indeed become the standard of care , 87. DesCalfee et al. have described two different subphenotypes ARDS in patient cohorts enrolled in two randomized controlled trials based on clinical and biomarker variables derived from latent class analysis , 67. ImpIt is easy to speculate that pediatric AKI could very well represent a heterogenous syndrome with varied responses to fluid exposure significantly impacting lung-kidney interactions. Stratified analysis of a prospective study showed that mortality was associated with greater cumulative fluid balance on Day 3 of ARDS with concomitant AKI . In addiCritical illness is largely a state of organ crosstalk and interaction; therefore, successful management requires a thorough understanding of its management. Changing epidemiology of pediatric AKI has clearly placed this syndrome in the setting of multiple organ dysfunction especially as it relates to the most severe forms of AKI with the poorest outcomes. Similarly, refinement of the pediatric ARDS definition has improved our understanding of the disease pathophysiology. As such, pediatric AKI and ARDS are the two most common organ failures intensivists deal with on a daily basis. Recent evidence, especially around mechanistic pathways in each syndrome, has enhanced our appreciation of the actual scale of organ crosstalk that extends beyond simply fluid accumulation and its management in AKI and ARDS. Multiple disease-specific molecular mediators and inflammatory pathways are involved in organ crosstalk in the AKI-ARDS construct, and the reciprocal hemodynamic effects of elevated pulmonary vascular resistance and central venous pressure (CVP) augment both renal and pulmonary congestion and impair renal oxygenation. Future research  should bAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Extensive searches and literature reviews have shown that a large amount of data exists on the pathological process underlying the effects of cigarette smoke and its components on PAD through various mechanisms. Cigarette smoke extracts (CSE) induce endothelial cell dysfunction, smooth muscle cell remodeling and macrophage phenotypic transformation through multiple molecular mechanisms. These pathological changes are the molecular basis for the occurrence and development of peripheral vascular diseases. With few discussions on the topic, we will summarize recent insights into the effect of smoking on regulating PAD through multiple pathways and its possible pathogenic mechanism.Smoking is one of the most important preventable factors causing peripheral artery disease (PAD). The purpose of this review is to comprehensively analyze and summarize the pathogenesis and clinical characteristics of smoking in PAD based on existing clinical,  With changes in global population epidemiology, the incidence of PAD may increase in the future . In a brNumerous studies have confirmed the link between active smoking and PAD. A systematic review showed that half of PAD cases are due to smoking . CompareThe harm caused by smoking cannot be underestimated, but researchers have not clearly determined whether quitting smoking effectively reduces the incidence of cardiovascular disease. Although quitting smoking reduces the risk of cardiovascular morbidity and mortality in patients with a history of smoking, the continuing effects of smoking are still relatively high. Therefore, smoking cessation is never too late to reduce the cardiovascular risk . At the In terms of the effect of smoking on PAD after surgery, studies have shown that smoking intensity exerts an unusually large effect on the prognosis of patients with symptomatic lower extremity revascularization. Compared with patients who continue to smoke, the mortality rate of patients who quit smoking is lower and these patients have a higher survival rate without amputation , 12.Smoking has been widely regarded as the leading risk factor for clinical cardiovascular disease that directly affects atherosclerosis. Active smoking and passive smoking directly correlate with the occurrence of atherosclerosis , 14. StrWith the transformation of tobacco marketing methods, in addition to traditional smoking and its methods, some new types of tobacco substitutes  have gradually become new consumption methods and are more commonly used by young people , 19. HunAtherosclerosis is the primary pathological basis of PAD. Its main feature is the formation of atherosclerotic plaques, which are characterized by the accumulation of lipids and immune cells in the blood vessel wall covered by a collagen fiber cap. The abnormal metabolism and distribution of lipids play an important role in the occurrence and development of atherosclerosis , 26. StuIn addition to abnormal lipid metabolism, various cells are also involved in the occurrence of atherosclerosis . At pres2+  plays a crucial role in regulating endothelial dysfunction , 37 Fig. The mon2+ , 43. HOS2+ . CSE-der2+ .Oxidative stress is the interaction between the body's response to oxidation caused by various pathological factors and antioxidants. Normally, it is in a balanced state. However, when the effect of oxidation is greater than the antioxidant effect, the infiltration of inflammatory cells occurs, the secretion of various proteases and cytokines increases, and a large amount of oxidative intermediate products are generated, which in turn cause a series of physiological and pathological changes , 47. Stuchondria . CSE redthe body . Meanwhibody  are significantly higher in smoking patients than in non-smokers . ADMA, aIn vitro studies have shown that CSE reduces protein synthesis by inducing protein misfolding in the endoplasmic reticulum. At the same time, a large number of unfolded proteins interact with each other and lead to the phosphorylation of PERK and eIF2a, which will prolong endoplasmic reticulum stress and activate autophagy-related programs, resulting in endothelial cell dysfunction  is a member of the transcriptional regulator bHLH (basic helix-loop-helix)-PAS (Per-ARNT-Sim) family, which mainly regulates various developmental and physiological functions , 87. NorMonocytes and macrophages that enter the endothelium release large amounts of MPO that forms hypochlorous acid (HOCl) and hypothiocyanic acid (HOSCN). Macrophages exposed to HOSCN exhibit increased release of inflammatory cytokines and chemokines, including MCP-1, TNF-α, and IL6, and the subsequent of amplification of the inflammatory cascade. However, HOSCN-induced cytokine/chemokine expression and cell death are reduced by inhibiting the NF-kB signaling pathway . Further2+. When the concentration of Ca2+ in the cytoplasm is further increased, it will cause a conformational change in calpain-1 that triggers its proteolytic activity. Therefore, calpain promotes remodeling of the actin cytoskeleton in VSMCs to form podosomes by activating PKC, accompanied by the degradation of extracellular matrix (ECM)  . On the ix (ECM) , 108. Seferation .Nicotine also participates in the functional and phenotypic changes of smooth muscle cells through nAChR-induced regulation of multiple related downstream signaling pathways. Knockdown of nAChRα1 significantly reduces the CSE-induced phosphorylation of STAT3, Akt, and mTOR, while overexpression of nAChRα1 exerts the opposite effect. STAT3 directly interacts with the nicotine receptor nAChRα1 to regulate the nuclear translocation of STAT3 and its binding to the Akt promoter region, thereby promoting the proliferation and migration of VSMCs and inflammation mediated by macrophages, which in turn causes atherosclerosis. STAT3 inhibition significantly reduces nicotine-induced atherosclerosis . Nicotin2+ signaling channels -induced vasodilation. This mechanism may be caused by the hyperpolarization of VSMCs induced by stimulating endothelial cells to reduce the production of NO and PGI2 . Based oin vivo experiments, many studies have also confirmed the relationship between smoking and atherosclerosis in animals , or Buerger's disease, is a group of unexplained non-atherosclerotic inflammatory diseases mainly caused by small and medium-sized arteriovenous vascular lesions. It is characterized by the involvement of vascular thrombosis , infiltration of inflammatory cells (in the tissue surrounding the thrombus or the adventitia), fibrosis of the intima and media, and changes in the internal elastic layer. Among people with a low socioeconomic status, smoking history is an important risk factor for this condition, and a strong correlation has been observed between heavy tobacco use and thromboembolic vasculitis , 150. AtCurrently, although a large number of studies have confirmed that smoking is closely related to the occurrence and development of TAO, its specific mechanism is not yet clear. Under physiological or pathological conditions, the role of NO in maintaining vascular wall tension is mainly regulated by eNOS . HoweverIn addition, studies have shown that long-term cigarette exposure will lead to damage to the antioxidant defense system, which in turn increases the expression of MMP-9 and high mobility group box 1 (HMGB-1); high expression of MMP-9 and HMGB-1 also plays a role in TAO-related vascular lesions , 137. Inin vitro and clinical research. Although animal models of TAO (induced by sodium laurate) have been generated, the establishment and confirmation of these models are only confirmed by pathological examinations  is an abnormal enlargement of the infrarenal aorta. The pathophysiology of AAA causes the aortic wall to become increasingly thinner, which may rupture and cause life-threatening bleeding. Various factors may cause AAA. According to the classification of risk factors for AAA, smoking is the most important risk factor leading to aneurysm-related hospitalization and death, followed by the male sex and hypertension –173. A lIn addition, smoking may exert a direct effect on perioperative and long-term complications after AAA. In terms of the effect of smoking on post-operative AAA, quitting smoking more than 8 weeks before opening and repairing AAA significantly reduces the incidence of post-operative pulmonary complications. However, elective open AAA repair after smoking cessation for <8 weeks has no correlation with the perioperative prognosis , 179. AlAt present, the pathogenesis of AAA is mainly attributed to inflammatory cell infiltration, the destruction of elastin and collagen in the media and adventitia, biological changes in smooth muscle cells and angiogenesis , 181. StHow does smoking affect animal models of AAA? At present, the three most commonly used animal models of AAA are calcium chloride-induced AAA, subrenal abdominal aortic perfusion with elastase, and subcutaneous injection of angiotensin II (AngII) , 187. Smin vitro studies, cigarettes and their substitutes can regulate the occurrence and development of PAD through a variety of pathways. In vivo studies, numerous studies confirm the association between smoking and cardiovascular disease. However, due to the advantages and limitations of different animal models, as well as the differences in the components of cigarette smoke and intervention techniques, the mechanism of smoking on cardiovascular diseases cannot completely mimic the pathogenesis of humans. Therefore, in the study of animal models of smoking and peripheral arterial disease, we should select appropriate animal models for related research based on the purpose of the research and combining the pathophysiological mechanisms of human diseases. At the same time, with the development of gene editing technology, we also look forward to the development of genetically modified animal models related to PAD, which can better simulate the occurrence and development of human PAD.With aging of the population, the incidence of PAD is increasing annually. Many factors promote the development of this disease, and smoking is an independent risk factor for PAD. The effect of tobacco exposure on PAD is continuous. Quitting smoking can partially reverse or delay disease progression, but it is not completely eliminated. As for the pathogenesis of smoking and PAD, WW and TZ designed the study and searched literature. WW, KG, and GY contributed to drafting of the manuscript. All authors have read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy and integrity of any part of the work are appropriately investigated and resolved.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.


The gut microbiota metabolizes the nutrients to produce various metabolites that play crucial roles in host metabolism. However, the links between the microbiota established by different nutrients and the microbiota-influenced changes in the plasma lipids remain unclear. Diets rich in cornstarch, fructose, branched chain amino acids, soybean oil (SO), or lard established a unique microbiota and had influence on glucose metabolism, which was partially reproduced by transferring the microbiota. Comparison of plasma lipidomic analysis between germ-free and colonized mice revealed significant impacts of the microbiota on various lipid classes, and of note, the microbiota established by the SO diet, which was associated with the greatest degree of glucose intolerance, caused the maximum alteration of the plasma lipid profile. Thus, the gut microbiota composed of dietary nutrients was associated with dynamic changes in the lipids potentially having differential effects on glucose metabolism.  •Diets with different nutrient compositions differentially affect glucose metabolism•Gut microbiota established by soybean oil-rich (SO) diet impairs glucose metabolism•Gut microbiota established by diets has dynamic effects on the plasma lipid profile•SO diet has the greatest impact on the plasma lipid profile through gut microbiota Microbiology ; Microbiome ; Lipidomics In recent years, it has become clear that the gut microbiota are one of the major regulatory factors of the host metabolism . BacteriBoth genetic and environmental factors determine the host-specific composition of the gut microbiota ;however,To investigate the effects of dietary interventions on the metabolic profiles in mice, we designed five different diets, namely, diets rich in cornstarch, fructose, BCAA, soybean oil, and lard  . In the TLR4 in the SO group  revealed that the recipient mice colonized with bacteria from the SO diet group showed significantly worse glucose tolerance than those that were colonized with bacteria from the Lard group A. To invSO group E. The chSO group F. Thus tFirmicutes and Bacteroidetes. The Fru group showed increased counts of Proteobacteria and decreased numbers of Actinobacteria, whereas the BCAA group showed lower counts of Deferribacteres. The BCAA, SO, and Lard groups showed higher populations of Firmicutes (Firmicutes/Bacteroidetes (F/B) ratio was elevated in both the high-fat diet groups, associated with a decrease in the numbers of Bacteroidetes , and the presence of Peptococcaceae, Lactococcus, Peptostreptococcaceae, and Clostridiales showed positive correlations with these parameters  colonized with the bacteria of chow-fed mice (Col mice) A. After adiation C and S5Eadiation C. To undadiation . Overalladiation , it is padiation C. This madiation D. Plasmaadiation , our finadiation C and 5E.adiation , suggestrameters F. These CD36 that facilitates FA uptake and decreased the expressions of some sterol transporters, such as ABCA1, ABCG5, and ABCG8 , proteins (BCAA diet), or lipids (SO and Lard diets). Among the major findings of the study was that both the high-fat diets (the SO and Lard diets) caused obesity and glucose intolerance with insulin resistance. However, the glucose intolerance in the SO group was worse than that in the Lard group, which was reproduced by FMT to GF mice, suggesting that the gut microbiota established by the SO diet had a more severe adverse impact on the host glucose metabolism than that established by the Lard diet. On the other hand, no significant difference was observed in the degree of chronic inflammation, the plasma leptin and ghrelin levels, or food intake between the SO and Lard groups, suggesting that there are mechanisms other than chronic inflammation and appetite regulation that might also have contributed to the worse glucose metabolism in the SO group. Plasma lipidomic analyses in GF mice and their littermates colonized with bacteria (Col mice) at the start of a 2-week dietary intervention revealed that the plasma lipid profiles were significantly affected by the presence of the gut microbiota established in response to different nutrient compositions. The SO diet increased the plasma levels of multiple types of lipids, including campesterol, sitosterol, neutral lipids, and PCs in the presence of the gut microbiota, whereas the effects of the Lard diet, also a high-fat diet, were not identical. The Col mice fed the Fru diet (Col-Fru group) showed higher plasma levels of phospholipids, such as LPE, LPI, PG, and alpha-hydroxy FA when compared with the GF mice fed the same diet (GF-Fru group), whereas those fed the BCAA diet (Col-BCAA) showed increased plasma levels of carnitine and N-acyl phosphatidylethanolamine with ether linkages when compared with the GF mice fed the same diet (GF-BCAA group). Although the percentages and major sources of lipids were similar in the CS, Fru, and BCAA diets, the three diets produced different alterations of the plasma lipid levels, presumably under the influence of the different microbiota established by the three diets, suggesting that the intestinal microbial composition and activities have unique impacts on the plasma lipid profile. The differences in the bacterial biomass and activities established by different dietary compositions may also be affected by the amount of energy intake. However, in our study, the CS and Fru or SO and Lard groups showed differences in the lipid profile derived from their microbiota despite similar levels of energy intake. Thus, not only the amount of energy intake but also differences in the nutrient compositions affect the microbial composition of the intestinal microbiota, with differential effects on the host lipid profile.Prevotella copri and Bacteroides vulgatus. Administration of P. copri or BCAA worsened the degree of insulin resistance in a mouse model of diet-induced obesity , was demonstrated to exert anti-inflammatory and anti-obesity effects in mice .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shiho Fujisaka (This study did not generate new unique reagents.The data that support the findings of this study are available from the lead contact upon reasonable request.All methods can be found in the accompanying


The fundamental framework of steroidogenesis is similar across steroidogenic cells, especially in initial mitochondrial steps. For instance, the START domain containing protein-mediated cholesterol transport to the mitochondria, and its conversion to pregnenolone by the enzyme P450scc, is conserved across steroidogenic cells. The enzyme P450scc localizes to the inner mitochondrial membrane, which makes the mitochondria essential for steroidogenesis. Despite this commonality, mitochondrial structure, number, and dynamics vary substantially between different steroidogenic cell types, indicating implications beyond pregnenolone biosynthesis. This review aims to focus on the growing roles of mitochondria, autophagy and lipophagy in cholesterol uptake, trafficking and homeostasis in steroidogenic cells and consequently in steroidogenesis. We will focus on these aspects in the context of the physiological need for different steroid hormones and cell-intrinsic inherent features in different steroidogenic cell types beyond mitochondria as a mere site for the beginning of steroidogenesis. The overall goal is to provide an authentic and comprehensive review on the expanding role of steroidogenic cell-intrinsic processes in cholesterol homeostasis and steroidogenesis, and to bring attention to the scientific community working in this field on these promising advancements. Moreover, we will discuss a novel mitochondrial player, prohibitin, and its potential role in steroidogenic mitochondria and cells, and consequently, in steroidogenesis. Steroid hormones are an important class of regulatory molecules, which are synthesized mainly in the adrenal glands, the ovary, and the testis, in response to steroidogenic stimuli, and regulate growth and drive a variety of physiological processes, such as reproduction and metabolism . The impThe first step in the biosynthesis of steroid hormones is the enzymatic cleavage of a six-carbon unit side chain of cholesterol molecule by the 20–22 desmolase/lyase activity of the cytochrome P450 side chain cleavage (P450scc) enzyme system located in the inner mitochondrial membrane (IMM) ,4. SteroFor example, mitochondrial features and the distribution in adrenocortical cells and Leydig cells are relatively more prominent in comparison to ovarian and placental steroidogenic cells . While sThe steroid hormones are not stored in secretory vesicles like peptide hormones, but released into the blood upon their biosynthesis . This inThus, an expanding role of cell-intrinsic processes  in steroidogenesis have created a need for a timely review article for the benefit of the scientific community engaged in this field, and to facilitate research to answer fundamental and emerging unanswered questions. In this review, we will focus on the cell-intrinsic attributes of different steroidogenic cells involved in cholesterol handling and mitochondrial activities. We have two interconnected goals: first, to discuss the importance of various processes involved to maintain a readily available pool of cholesterol for the varying need of steroidogenic demands, and second, to review the growing role of mitochondria, autophagy, and lipophagy, and other related activities in a steroidogenic cell type-specific manner to meet the diverse physiological demand of each steroid hormone . While sA cell is classified as “steroidogenic” if it expresses the enzyme P450scc and therefore can catalyze the first reaction of steroidogenesis . Many cells can transform steroids produced in other cells , but only cells expressing P450scc are steroidogenic . As P450The precursor cholesterol for steroidogenesis is known to come from at least three sources, such as the mobilization of cholesterol from the lipid droplets (LDs), the uptake of circulating cholesterol esters, and the de novo synthesis of cholesterol, which have been described extensively in many review articles ,11. HoweIn addition to steroidogenic cells, the scavenger receptor class B type I (SR-BI) that are involved in cellular cholesterol uptake are found in many non-steroidogenic cell types, such as macrophages and endothelial cells . In testStAR and Cyp11A1a knockout mouse models are consistent with this notion. For instance, the mitochondria of the luteal cells of Cyp11A1 transgenic mouse model are elongated from their normal spherical shape [Cyp11A1 and StAR knockout on the mitochondria of zona fasciculata cells differ from each other, which is more severe in the former than the later [Structural changes in the mitochondria appears to be an integral feature of the differentiation of steroidogenic cells, which involve an acquisition of steroidogenic capability due to the expression of steroidogenic enzymes during development ,15. For he later ,19. As CTropic hormone-induced steroidogenesis in the adrenals and the gonads have been studied extensively. However, the context-dependent potential contributions of cell-intrinsic events remain largely unexplored. New evidence demonstrating the importance of autophagy and lipophagy in steroidogenesis in combination with our previous knowledge of cholesterol homeostasis and mitochondrial dynamics in steroidogenic cells have created an opportunity to explore these aspects and attain new insights. For example, preclinical models mimicking congenital adrenal hyperplasia (CAH) phenotype provides an excellent example to investigate cell-intrinsic processes and the physiological needs of cell type-specific steroidogenesis on the varying impacts of the loss-of-function mutation in StAR.The characteristic features of lipoid CAH are hypertrophied adrenals with enlarged lipid droplets because of high ACTH level and renin activity, and substantially reduced serum levels of steroid hormones . Bose etThe initiation of the biosynthesis of steroid hormones occurs in the mitochondria, which are known to undergo dynamic changes called mitochondrial fission and fusion . TherefoThe differences between placental trophoblasts and syncytiotrophoblasts that sustain progesterone production during human pregnancy is accompanied by mitochondrial fragmentation and cristae remodeling . SubsequWhile the final steroid hormone product differs in different steroidogenic cell types, the first step in the steroidogenic pathway is precisely similar, which is catalyzed by the enzyme P450scc located in the IMM. In addition to the common first step of the steroidogenic pathway, the final steps in the biosynthesis of glucocorticoids and mineralocorticoids are also catalyzed by two closely related mitochondrial enzymes: CYP11B1 and CYP11B2 , located in the IMM . Both enSteroidogenic mitochondria, particularly in adrenal and gonadal steroidogenic cells, are affected differently by the actions of the pituitary tropic hormones during the acute and the chronic response . The acuStructural changes in steroidogenic mitochondria have been reported during the development and differentiation of steroidogenic cells. For instance, the differentiation of cytotrophoblasts into syncytial trophoblasts is marked by a substantial increase in the expression levels of CYP11A1 and vesicular cristae . As discIn addition to the P45scc that is present in all steroidogenic cells, adrenocortical mitochondria contain two additional P450 enzymes: 11β–hydroxylase in zona fasciculata and aldosterone synthase in zona glomerulosa, which also localize to the IMM. The former catalyzes the conversion of 11-deoxycortisol to cortisol, while the latter catalyzes the conversion of deoxycorticosterone to aldosterone ,30,31. ADespite several commonalities with adrenal and gonadal steroidogenesis , placental steroidogenesis displays certain unique arrangements and characteristics, which are shared among placentae from different species. These include differences in regulatory mechanisms that control the expression of steroidogenic enzyme genes from other steroidogenic tissues, an interplay between maternal and fetal compartments to support placental steroidogenesis, and the regulatory influences on maternal hypothalamic-pituitary-gonadal axis and fetal adrenal corticosteroids . ImportaLike the adrenal cortex and the gonads, progesterone production by the human placenta requires pregnenolone synthesis from cholesterol by cytochrome P450scc ,34,35,36A unique aspect of placental steroidogenesis is its close relationship with maternal and fetal steroidogenesis. For example, E2 and P4 produced from maternal ovaries play important roles in placenta formation and function during early stages of pregnancy. Subsequently during later stages, the placenta itself acquires the ability to produce the P4 required to maintain pregnancy . In thisPregnancy in women is marked by substantial changes in their endocrine system . For insMany placental species, including human, does not express 17α-hydroxylase ,52,53. TCholesterol, which is an essential component of all animal cell membranes, plays a critical role in defining membrane’s biochemical and biophysical characteristics . NotablyCholesterol has many versatile characteristics. On the one hand, cholesterol plays a critical role in determining the biochemical and biophysical properties of cellular membranes, whereas on the other hand, cleavage of its six-carbon unit side chain from cholesterol molecule (at the beginning of enzymatic step in the steroidogenic pathway) in combination with its oxygenation at certain residues changes its chemical properties and functions . This chCholesterol may be produced de novo from acetate in steroidogenic cells through a series of enzymatic pathways mainly located in the endoplasmic reticulum (ER) . HoweverCholesterol functions as the precursor substrate for the biosynthesis of all steroid hormones, which begins in the IMM by the cytochrome P450scc enzyme system leading to the formation of pregnenolone, the first steroid in the steroidogenic pathway. To ensure that this essential step proceeds normally, the steroidogenic cells have evolved a robust mechanism to maintain cholesterol pool in the cytoplasm and then traffic to the steroidogenic mitochondria when needed . This isCholesterol is a versatile lipid, which is synthesized by animal cells and is an integral component of their subcellular membranes . MoreoveAutophagy is an evolutionarily conserved, cellular degradative pathway that involves the systematic degradation of select cytoplasmic components and organelles . During Lipophagy is a subtype of autophagy where the mobilization of lipids from lipid droplets are intimately linked with autophagy to deliver contents of lipid droplets to lysosomes . LipophaFurthermore, the knockdown of Beclin-1  was found to decrease LH-stimulated StAR expression and testosterone production in mouse Leydig cells, leading to the conclusion that autophagy plays a role in the maintenance of steroidogenesis in Leydig cells. The decline in testosterone was found to be caused by a defect in cholesterol uptake in autophagy-deficient Leydig cells. Further investigations revealed that disruption of autophagic flux leads to the downregulation of the SR-BI receptors leading to insufficient cholesterol supply. Notably, in both studies, the disruption of autophagy by pharmacological or genetic approaches led to a decrease in LH-stimulated StAR expression, suggesting a link between LH-induced signaling events and autophagy in the regulation of testosterone production, which may involve cholesterol trafficking to the mitochondria, as the StAR protein plays a central role therein.In addition to the Leydig cells and adrenocortical cells, a positive effect of autophagy has been reported in porcine granulosa cell steroidogenesis in response to FSH . MechaniIn summary, these findings have shown the importance of autophagy and lipophagy in lipid regulation and steroid production. Future experiments should explore the relative importance of autophagy and select autophagy  in the basal, acute, and chronic regulation of steroid production in different steroidogenic cell types. The elucidation of these functions will be important to understand how cell-extrinsic and cell-intrinsic factors and processes coordinate to maintain various states of steroidogenesis across steroidogenic cells, which vary substantially.Of note, most of the work on autophagy and lipophagy has been reported in relation to Leydig cell steroidogenesis ,96,97, wAutophagy is an evolutionarily conserved process in cell physiology, from organisms such as yeasts to mammals, which raises the question of whether autophagy’s steroidogenic role, which has been reported in many mammalian species, is also involved in other species. Recently, Texada et al.  showed tMitochondria are emerging as cellular-signaling platforms deeply integrated into diverse cellular processes. Prohibitin  is a hallmark protein of the IMM, which is involved in mitochondrial biogenesis and modulates mitochondrial dynamics . PHB andBoth PHBs belong to a group of proteins family, which are thought to function as lipid and protein scaffolds in the IMM that affect the lateral distribution of the membrane lipid and protein components ,105. PHBCholesterol serves as the metabolic precursor of all steroid hormones, and as such, steroidogenic cells and tissues can be seen as highly specialized lipid-processing cells and tissues. Since PHB has been implicated in lipid metabolism and homeostasis across species, including in mitochondrial phospholipids, and in autophagy/mitochondrial proteases, we speculate that PHB might be involved in steroid biosynthesis via lipid/cholesterol homeostasis across steroidogenic cells/tissue types. During autophagy, LC3-I is conjugated into phosphatidylethanolamine (PE) to form the LC3-PE conjugate, which is then tightly bound to the autophagosomal membranes . NotablyPHB not only interacts with LC3 but is also associated with the biology of mitochondrial phospholipids, including PE . It is tIn addition to mitochondrial biology and autophagy, the PHB family member proteins Erlin-1 and Erlin-2 are shown to be highly enriched in the detergent-soluble, buoyant fraction of sucrose gradients in a cholesterol-dependent manner . HoweverMoreover, the PHB family member protein MEC-2 and Podocin have both been found to bind cholesterol to regulate the activity of associated ion channels . This biFurthermore, PHB has been reported to play a role in granulosa cells . HoweverMost of the arguments on the putative role of PHB in steroidogenesis in this section are hypothetical and are based on previous research findings on PHB in mitochondrial biology and lipid metabolism. Almost none of PHB’s mitochondrial and lipid metabolism attributes has been reported in relation to steroidogenesis. However, new unpublished findings from our laboratory are suggestive of PHB playing an important role in autophagy/lipophagy, cholesterol homeostasis and in mitochondrial dynamics in steroidogenic cells. It is our hope that others will see many research opportunities here, and that they will carry out studies that test these ideas.The emergence of the role of mitochondria from the site of initiation of steroidogenesis to the regulator of cholesterol mobilization, trafficking, and homeostasis to support the body’s physiological levels of steroid hormone production have provided new insights and created exciting future research directions. One such example is the putative role that PHB plays in integrating various aspects of steroidogenic mitochondria, because of many fitting attributes it possesses related to mitochondrial biology and lipid metabolism . However


The advancement of precision medicine critically depends on the robustness and specificity of the carriers used for the targeted delivery of effector molecules in the human body. Numerous nanocarriers have been explored in vivo, to ensure the precise delivery of molecular cargos via tissue-specific targeting, including the endocrine part of the pancreas, thyroid, and adrenal glands. However, even after reaching the target organ, the cargo-carrying vehicle needs to enter the cell and then escape lysosomal destruction. Most artificial nanocarriers suffer from intrinsic limitations that prevent them from completing the specific delivery of the cargo. In this respect, extracellular vesicles (EVs) seem to be the natural tool for payload delivery due to their versatility and low toxicity. However, EV-mediated delivery is not selective and is usually short-ranged. By inserting the viral membrane fusion proteins into exosomes, it is possible to increase the efficiency of membrane recognition and also ease the process of membrane fusion. This review describes the molecular details of the viral-assisted interaction between the target cell and EVs. We also discuss the question of the usability of viral fusion proteins in developing extracellular vesicle-based nanocarriers with a higher efficacy of payload delivery. Finally, this review specifically highlights the role of Gag and RNA binding proteins in RNA sorting into EVs. Recent developments in precision medicine are largely due to the successful implementation of specific delivery of biologically active cargos to the target tissues. This should significantly decrease the possible side effects of therapeutic agents, while maintaining an appropriate efficacy. The successful implementation of this approach into clinical use would be beneficial for the treatment of a broad spectrum of various diseases, ranging from cancer, to bone fractures, to monogenic diseases. Targeted delivery may be implemented by using various carriers such as viral vectors, liposomes, nanoparticles, extracellular vesicles (EVs) and other artificial vectors. Natural carriers such as viruses or EVs provide an effective alternative to artificial nano-carriers, as they have innate tropism to particular tissues. The best example of such carriers is recombinant non-replicating viral vectors. They have been extensively studied as promising tools for transgene delivery. Depending on the serotype, they possess a natural tropism to different organs and tissues . ParticuNotwithstanding the recent advances in the development of new biomaterials and nanofabrication, there are still many problems to be solved before the next generation of “smart” nanocarriers may be applied in everyday clinical practice. Their surface should be modified appropriately by various targeting molecules that would confer the affinity of nano-vehicles for specific tissues. One of the major obstacles is that higher vertebrates have various barriers that prevent the invasion of foreign materials, including nanosized delivery systems. Immune cells efficiently recognize and phagocyte the foreign biomaterial. At the same time, blood enzymes alter the structure and biological properties of these exogenous particles . AlthougMany studies in this field have been focused on the development of lipid-based systems such as liposomes and lipid nanoparticles. One of the main problems with such systems is their failure to naturally fuse with cell membranes. Usually, they enter a cell via an endocytosis-based pathway. Natural carriers such as exosomes, on the contrary, have a complex surface composition with a specific set of membrane proteins that assist efficient targeting and entrance into the cells. When enriched with small non-coding RNAs (including miRNAs), mRNAs, fragments of DNA, or proteins ,12,13, eThe most common way for nanoparticle vectors to enter the cell is via the endosomal pathway. In this case, loaded therapeutic molecules interact with the endosomal, and then the lysosomal, environment. The latter is characterized by high acidity and high protease activity, which may alter the structure or properties of the delivered therapeutic molecules . The endCell to cell communication is carried out through direct interaction, secretion of various soluble factors or different cell vesicles that can have an impact on other cells. Such vesicles regulate fundamental biological properties in multiple ways, merging their membrane contents into the recipient cell plasma membrane and delivering effectors including transcription factors, oncogenes, small and large non-coding regulatory RNAs, mRNAs and infectious particles into recipient cells. Among the various articles considering exosomes as delivery vehicles, the emphasis is usually on the content of vesicles and their distribution throughout the body, but not on the mechanism of their entry into cells ,24,25. AThe lipid bilayer of liposomes, lipid nanoparticles, and biological membranes consists of charged polar head surrounded by a hydration layer, and neutral tails oriented towards each other. Regardless of the complexity of the system, the fusion of two membranes is accompanied by the interaction of various surface forces: hydration repulsion, hydrophobic attraction, and the Van der Waals force . When puThe energy barrier to the formation of the water-free lipid conformation is directly proportional to the intermembrane distance. Shortening the distance between the membranes leads to an attenuation of the repulsive forces between them. Membranes must stay in close juxtaposition and become destabilized to allow the transition from the bilayer to the non-bilayer conformation. During the membrane interaction, the lipid bilayer undergoes perturbation and destabilization, which causes high local curvature and membrane fusion . When thFrom an evolutionary point of view, membrane fusion has been developed as a well-controlled process, while random fusions could lead to serious problems for the multicellular organism ,31,32. SThe energy barrier for hemifusion is estimated to be ≈50 kcal/mol. Therefore, membranes are unable to fuse under normal conditions ,38. In bThe onset of the fusion process initiates conformational changes in the virus fusion protein. A trimeric intermediate brings together the target cell and viral membranes and then folds back to a hairpin conformation. The trimer penetrates the target membrane via a hydrophobic loop domain, which leads to the deformation of the two membranes  and to tSeveral mechanisms for the activation of viral fusion proteins have been described. They include receptor binding , receptor binding to a separate attachment protein (paramyxoviruses), the binding of a receptor with a coreceptor (HIV), low pH , and receptor binding under low pH (avian sarcoma/leukosis virus). All these signals trigger the thermodynamically favored process of the refolding of the fusion protein into a stable final post-fusion form . In the Importantly, similar to the viral fusion proteins described above, eukaryotic cells have their own special proteins that are able to merge membranes. Several genes that are involved in the process of membrane fusion during embryo differentiation and development were originally acquired from viruses. Endogenous retroviral genes are involved in syncytium formation and cell fusion. Inside the syncytium, all cells are connected through channels that have high permeability for macromolecules, allowing them to spread between neighboring cells. In terminal differentiation, the lens fibers are deprived of organelles and form syncytia, while adjacent immature fibers ensure the diffusion of nutrients . During The common way to transmit viruses among the host’s cells is to assemble particles intracellularly and then release them into the extracellular environment. However, during the infection, virus-infected cells can fuse and form syncytia. Furthermore, viruses can exploit existing intercellular communication pathways mediated by EVs to infect new populations of cells . They caExosomes, being part of the cell–cell cargo delivery system, potentially co-evolved together with viruses ,62. ThisViruses exploit the Endosomal Sorting Complex Required for Transport (ESCRT) pathway to intercept cell membrane traffic. Viral structural proteins, such as retroviral Gag proteins, arenaviral Z proteins, and filoviral VP40 proteins, act as ESCRT adaptors. They interact with ESCRT components via specific sequence motifs similar to cell proteins . SeveralViral attachment and subsequent cell entry are mediated by a number of membrane molecules located on the surface of the host cell . SeveralViral pseudogenes are involved in the building of syncytia in muscles, osteoclasts, the lens, placenta, embryonic ovaries, testes, sexual reproduction. However, there is only a limited number of organs with syncytia, which restricts the use of human fusion proteins for the vesicle-mediated targeted delivery. Therefore, the idea of targeting EVs and exosomes with synthetic viral fusion proteins seems to be worthy of experimental testing. If this approach proves efficient, it would help to expand the application and efficacy of these systems by allowing them to escape degradation in the course of the endosomal pathway .Altogether, numerous variants of viral capsid proteins provide a new platform for using viral tropism as a tool for selective tissue-specific recognition and merging with the target cells. As a new approach in cancer immunotherapy, it was proposed to use tumor xenogenization (a process of addition of the pathogenic antigen that increases the visibility of cancer cells for the host immune system) through fusogenic exosomes with special fusogenic viral antigens . Ideally, this approach would enhance the recognition and uptake by dendritic cells, which would then recruit T-lymphocytes and ultimately result in the suppression of tumor growth ,159. TheAnother way of increasing the specificity of exosome targeting can be achieved via the insertion of specific integrins into membranes of exosomes. Integrins are heterodimeric cell membrane proteins consisting of α and β subunits. The major biological role of integrins is to mediate cell–cell contacts at specific focal adhesion points. Importantly, integrins transmit transmembrane signals bi-directionally thereby modulating the intracellular events in response to specific ligands. During the formation of exosomes, integrins are captured on their surface. Consistent with their functions, exosomal integrins have been shown to be capable of guiding exosomes to specific tissues. In the pioneering work, Lyden’s group have shown that exosomes derived from breast and pancreatic cancer cells ectopically expressing integrin α6β4 were preferentially targeted to the lung tissue since the latter expresses laminin, the ligand for the α6β4 integrin. On the contrary, those exosomes that expressed integrin αVβ5, which specifically recognizes fibronectin, were preferentially distributed to the liver cells, which are known to abundantly express this ECM protein . Thus, oExosomes, especially the ones derived from cancer tissues, are deemed to affect the physiology of target cells . It is kLoading of RNA into EVs can occur in several ways: randomly, due to the abundance of certain RNA species in the cytosol or selectively, or by RNA-binding proteins (RBPs) that recognize specific motifs/secondary structures in the RNAs to be sorted. These RBPs recognize specific : the matrix (MA) domain, which is primarily associated with membrane-binding capability; the capsid (CA) domain, which mediates numerous Gag–Gag interactions; the nucleocapsid (NC) domain, which is involved in specifically packaging the genome RNA (gRNA); and p6, which contributes to the release of the assembled particle from the host cell by interacting with the cellular endosomal sorting complexes required for transport (ESCRT) machinery . The NC Recombinant Gag proteins can assemble into particles in vitro, but this assembly requires the addition of nucleic acids; nearly any single-stranded nucleic acid longer than ~20–30 nucleotides can support assembly under these conditions . Using gThere are ~100 retroviral Gag-like genes in the human genome that can self-assemble into RNA-carrying capsids . NotablyAn understanding of the mechanisms of membrane fusion and RNA sorting promises new approaches to control the loading of EVs with specific RNAs. More specifically, the recognition of the RNA packaging signal by the RNA-binding domain of synthetic viral-like proteins may assist the loading of mRNA and miRNA of interest into the EVs . To thisGiven the fact that the HIV genome (9.8 kb) can be packed within the volume of the exosome size, one should potentially be able to pack 500 copies of miRNAs into a single exosome . FurtherWe hypothesize that the expanding knowledge of different mechanisms that viruses utilize to interact with host cells, together with the ability to manipulate and engineer hybrid EVs, could be the basis for the development of future therapies. In fact, effective targeting delivery systems have already been developed by viruses in the course of evolution, which can specifically navigate through the human body. Therefore, the success of future studies relies on the ability to repurpose the already available delivery systems for the benefit of human health.


Based on a weak  recommendation, the panelconcludes that “In elderly (75 years or older) patients living in Latin America withsevere symptomatic aortic stenosis (AS) candidates for transfemoral approach, the panelsuggests the use of TAVI over SAVR”. This guideline has been put together and funded bythe Sociedad Latinoamericana de Cardiologia Intervencionista (SOLACI) with theendorsement of the Sociedad Interamericana de Cardiologia (SIAC). As stated by theauthors, the rationale for this document emerges as a response to their disagreementwith the decision made by the cost-effectiveness technology assessment agency inArgentina, which defined the use of transcatheter aortic valve replacement forinoperable patients. The aim was “to develop a high-quality and transparent guideline tohelp physicians and other stakeholders concerning the use of TAVI versus SAVR in LatinAmerica”. Despite the authors’ thorough job reviewing the existing internationalevidence according to the Grading of Recommendations, Assessment, Development andEvaluations (GRADE) criteria and the relevant systematic review, these recommendations,which stem from Latin-American authors, are not specifically for Latin America.The Heart journal, from the British Medical Journal and the British CardiovascularSociety, has recently published a review titled “Clinical practice guideline fortranscatheter (TAVI) versus surgical aortic valve replacement (SAVR) in patients withsevere aortic stenosis in Latin America”The Latin American Association of Cardiac and Endovascular Surgery (LACES) nucleates over400 members of the Latin-American continent. It started in 2019 and is currently themost inclusive cardiac surgical association in Latin America, recognized by the EuropeanAssociation of CardioThoracic Surgery, Society of Thoracic Surgeons, AmericanAssociation of Thoracic Surgery, and Association of Thoracic and Cardiovascular Surgeonsof Asia. Based on the following issues, this association does not support theSOLACI/SIAC recommendations..Extrapolating data from a different socioeconomic reality with different hospital volumeexperiences to our continent is dangerous for our patients and the health economicsystem. When the aim is to perform regional guidelines, as in the title of this review,it is critical to consider the regional context; if not, this review may well apply toAfrica, Europe, America, Asia, or Oceania. The authors explicitly disregardedLatin-American evidence under the assumption that the conclusions were “erratic”. Notincluding regional evidence is a significant flaw and suggests serious selection bias inthe evidence process.A guideline to help physicians and stakeholders decide the best course of action in LatinAmerica must come from Latin-American effectiveness data. It is impossible to recommendsomething for our region when we do not know what is going on. The effectiveness of aprocedure depends not only on the procedure, per se, but also on the socioeconomicreality, which directly affects our patients and is the basis of the cost-effectivenessanalysis. Several reports have highlighted the importance of hospital and technicalvolume to assure good outcomes-.To begin with, the document fails to examine critical and recent evidence and makerecommendations in line with the goals of treating heart valve disease. The treatment ofAS should be accomplished aiming to restore long-term life expectancy and improvequality of life, avoiding detrimental late events reversing the early success of theprocedure. For now, only SAVR is established to restore the prognosis of patients withsymptomatic severe AS, with long-term postoperative survival becoming comparable to anage- and sex-matched general population without AS in patients over 65 yearsold. The report of thefive-year outcomes of the Placement of Aortic Transcatheter Valve (PARTNER) 2 cohort Atrial found a higher risk of death or disabling stroke between two and five years afterTAVI than after SAVR, with a hazard 27% higher,. Themeta-analysis by Barili et al. with Kaplan-Meier-estimated individual patient dataevaluating the effects of TAVI and SAVR on the long-term all-cause mortality raterevealed a lower incidence of death in the first year after TAVI. In contrast, there wasa reversal of the endpoint after 40 months favoring SAVR over TAVI. The mortality ratesin trials of TAVI vs. SAVR are affected by treatments with atime-varying effect and TAVI is related to better survival in the first months afterimplantation whereas, after 40 months, it is a risk factor for all-causemortality.The SOLACI/SIAC document implicitly assumes an equipoise between long-term outcomes ofTAVI and SAVR, which has been challenged by recent evidence. In this way, the results ofrandomized controlled trials, metanalyses, and national databases show theopposite.In the PARTNER 3 trial, the event-rate lines for death and disabling strokes, whichsignificantly favored TAVI in the one-year analysis, from the two-year follow-up, thecurves are converging over time, and reversal of fortunes may become tangible in thelonger-term.The German Aortic Valve Registry (GARY), assessing the long-term outcomes of aortic valvereplacement with TAVI and SAVR, revealed that after propensity score matching, TAVI withearly generation prosthesis was associated with significantly higher five-year all-causemortality than SAVR, reaffirming earlier data from Italian and French nationwideregistries. Additionally, the curves for survival probability keep diverging over time,hinting at a longer-term worse prognosis for patients who underwent TAVI procedure. Thefinding from the GARY registry is worrisome as it raises the further question of whatcould be done to low- and intermediate-risk patients who had TAVI implanted and now mayface shorter life expectancy.The recommendation for TAVI in patients over 75 years old is unrealistic, given that thelife expectancy for men and women aged 75 is a further 11.18 and 12.97 years,respectively, therefore the long-term benefit of the procedure would be adverselyaffected cutting short late survivalDuring all these years, different centers from Latin America have generated essentialdata to produce high-quality effectiveness results and therefore adapt recommendationsto our reality. We consider this is critical to produce trustworthy recommendations andinform stakeholders on the best action to take. Our association has always been open toworking in a multidisciplinary team objectively to produce and analyze these data. Webelieve this is the initial step before making any regional guideline.Severe conflicts of interest (COI) generate undisputed bias. Despite the fact thatsurgeons have proficiently been treating aortic valve stenosis for more than 60 years,no cardiovascular surgical association was invited to take part in this document. Theauthors mention that no surgical association exists in Latin America, which shows anapparent disrespect to LACES and the concept of Heart Team..In fact, surgical cardiovascular societies have been recurrently excluded from makingTAVI guidelines, raising the suspicion of primary stealth financial interest behindthis. The methodologist and principal investigator, Dr. Lamelas, hasheavy COI with Edwards, Medtronic, and Boston Scientific. The voting panel included fourmembers who are proctors for these companies as well. The methodology team, led by Dr.Lamelas, decided that “TAVI proctoring may not be a strong financial conflict ofinterest”. We strongly disagree with this decision and believe that Latin America hassurgeons and interventional cardiologists with robust academic experience and withoutCOI who are not proctors for the industry with the capacity to evaluate the evidence andprovide an objective vote.Citing Gordon Guyatt from GRADE, “We believe that the key to developing conflict-freerecommendations is that panel members without conflicts and, in particular, themethodologist chapter editor, bear responsibility for the final presentation of evidencesummaries and rating of the quality of evidence. The chapter editor is also responsiblefor ensuring that, during the discussion of evidence, panel members with conflicts donot take an aggressive advocacy role”The authors mention that they did not involve SOLACI authorities in the process of theguidelines. This assertion is unclear since the principal investigator (PL) is thecoordinator of research in SOLACI, and one of the external reviewers, Oscar Mendiz (forwhom the COI are not disclosed), is part of the counseling committee of SOLACI.The authors have mentioned but not analyzed the role that costs have on TAVIimplementation in Latin America. They propose to waive taxes on TAVI as a solution forthis limitation. Considering that the GRADE approach requires a multidisciplinary teamwith specific skills, it would have been critical to have experts from healthcare andhealth technology assessment from Latin America among the guideline team..National per capita expenditures on health vary widely in Latin-American countries, witha mean of USD PPP 1,025, ranging from less than USD PPP 500 in some countries of CentralAmerica and the Andean region , to aroundUSD PPP 1,138 in Mexico, USD PPP 1,280 in Brazil, USD PPP 1,907 in Argentina, and USDPPP 2,484 in Cuba. These figures are in sharp contrast with the average Organisation forEconomic Co-operation and Development (OECD) countries and United States of America 2019health spending per capita of USD PPP 4,223 and USD PPP 11,071, respectively.In times of extreme pressure on health resources, regardless of the countries’ economicposition, adopting a new technology that is five to 10 times more expensive than theexisting standard, with inferior results, seems illogical and requires seriousreflectionIn summary, despite the extensive review of the current evidence of TAVI and SAVR, webelieve the authors failed to examine the existing evidence from Latin America,comprehensively. Heavy COI in members of the voting panel and from the chair of theguideline (PL) make the process and recommendations unrealistic and biased. We believeguidelines should meet three essential requisites:Be based on the reality of the region where it intends to be appliedBe led, as GRADE suggests, by an independent group of investigatorsFollowing GRADE recommendations, including experts from all criticaldisciplinesFurthermore, accurate and trustful information to patients and their family should bemade available - the risks involved, the benefits afforded, and the expected long-termprognosis.The extensive financial COI may unduly influence professional judgments, interfere withthe appraisal and recommendation of the Heart Valve Team, and discredit the integrity ofscience, the quality of care, and public confidence in medicine. There is a debate thatsurgical and medical societies should compulsorily embrace and coordinate their effortsregarding the response to flawed trials and unrealistic guidelines.LACES is open to working jointly for this and future guidelines under GRADE methodologyand led an independent GRADE expert group. The stand taken by the Heart journalpublishing the pretense SOLACI/SIAC guidelines with all the raised biases istroublesome. This document has been submitted as a Letter to the Editor to the Heartjournal in response to its publication and, since no response was received afterrepeated e-mail contacts to the editorial office, a decision was made to submit it tothe Brazilian Journal of Cardiovascular Surgery.


Cutis rhomboidalis nuchae was assessed in a 65-year-old patient. Optical microscopy showed basophilic agglomerations in the reticular dermis with decreased elastic fibers. Transmission electron microscopy showed elongated, curved and fragmented structures, and in their interior the presence of electron-dense lumps was reduced and irregular, similar to modified elastic fibers, whereas the collagen fibers had a normal aspect. Scanning electron microscopy showed deposits between the bundles of collagen, resembling pebbles or stones. These findings demonstrate that, at one stage of the disease, the collagen remains normal and the alterations are seen in the elastic tissue. Cutis rhomboidalis nuchae (CRN) is a type of solar elastosis identified especially in patients with a history of chronic sun exposure while working. The skin of the posterolateral cervical region acquires the typical appearance of “leather”, with a yellowish and ridged surface.A small fragment of CRN skin was obtained from a 65-year-old patient with a history of chronic exposure to sunlight . Part ofOptical microscopy with hematoxylin and eosin staining showed flattening of the epidermis, with basophilic clumps in the reticular dermis A; on higTransmission electron microscopy of the reticular dermis showed findings that were similar to those observed on high power in optical microscopy, with the presence of elongated, curved, and fragmented structures A. ObservScanning electron microscopy of the dermis surface showed deposits between the collagen bundles A and B aCRN is a frequent finding in physical examinations and a marker of chronic photo exposure, being correlated with the prevalence of malignant and premalignant lesions.4Our optical microscopy findings showed a basophilic deposition in the upper dermis, called solar elastosis, and basophilic degeneration of collagen.Transmission electron microscopy, where tissue sections are examined, allowed us to observe that the collagen fibers were normal in appearance, unlike the elastic fibers, which showed an irregular contour and were fragmented, demonstrating that at least during one phase of the disease, the collagen remains normal, in agreement with previous publications.The three-dimensional findings with scanning electron microscopy also showed fragmented tissue in the dermis together with collagen bundles. These findings of deposits resembling small stones or pebbles have already been described in other dermal conditions, demonstrating the superiority of TEM in the analysis of elastic fibers, with a better demonstration of the internal changes in fibers in tissue sections.Considering these results, the histological expression basophilic degeneration of collagen should be replaced by basophilic dermal degeneration, while the expression, solar elastosis, should be utilized as a clinical term and not as a histological one.None declared.Ângela Faistauer Torre: Approval of the final version of the manuscript; drafting and editing of the manuscript; critical review of the literature; critical review of the manuscript.Hiram de Almeida Jr: Approval of the final version of the manuscript; drafting and editing of the manuscript; critical review of the literature; critical review of the manuscript.Valeria Magalhães Jorge: Approval of the final version of the manuscript; drafting and editing of the manuscript; critical review of the literature; critical review of the manuscript.Antônia Larangeira de Almeida: Approval of the final version of the manuscript; drafting and editing of the manuscript; critical review of the literature; critical review of the manuscript.None declared.


Environmental or occupational exposure to pesticides is considered one of the main risk factors for the development of various diseases. Behind the development of pesticide-associated pathologies, there are both genetic and epigenetic alterations, where these latter are mainly represented by the alteration in the expression levels of microRNAs and by the change in the methylation status of the DNA. At present, no studies have comprehensively evaluated the genetic and epigenetic alterations induced by pesticides; therefore, the aim of the present study was to identify modifications in gene miRNA expression and DNA methylation useful for the prediction of pesticide exposure. For this purpose, an integrated analysis of gene expression, microRNA expression, and DNA methylation datasets obtained from the GEO DataSets database was performed to identify putative genes, microRNAs, and DNA methylation hotspots associated with pesticide exposure and responsible for the development of different diseases. In addition, DIANA-miRPath, STRING, and GO Panther prediction tools were used to establish the functional role of the putative biomarkers identified. The results obtained demonstrated that pesticides can modulate the expression levels of different genes and induce different epigenetic alterations in the expression levels of miRNAs and in the modulation of DNA methylation status. Pesticides are a heterogeneous group of chemical compounds widely used in agriculture and livestock to kill pests and weeds . Due to Observational studies on workers exposed to pesticides have shown how these chemicals represent an important risk to public health, with about 3 million cases of pesticide poisoning every year ,4.A direct link has been well established between the environmental or occupational exposure to pesticides and the development of several chronic-degenerative diseases, including tumors, neurological disorders, and autoimmune diseases ,6,7. TheAlthough pesticide-associated genetic alterations are well known and characterized ,10, the It should be noted that epigenetics is the study of heritable changes in gene expression that occur without a change in the DNA sequence . EpigeneDNA methylation in a promoter region causes gene silencing, resulting in the down-regulation of the gene itself; on the contrary, intragenic hypermethylation is associated with gene up-regulation ,18.Regarding histone modifications, all histones are subject to post-transcriptional modifications, including acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, and ADP-ribosylation .The epigenetic alterations of histones have important roles in transcriptional regulation as histones can regulate the conformation of the DNA structure into actively transcribed euchromatin and transcriptionally inactive highly condensed heterochromatin. The histones forming the euchromatin are characterized by high levels of acetylation, while heterochromatin is generally characterized by low levels of acetylation .Regarding miRNAs, these are small noncoding RNA fragments of 20-22 bp able to bind specific messenger RNAs (mRNAs) by inducing their silencing. In particular, when the complementarity between the miRNA sequence and mRNA target is complete, the RNA-induced silencing complex (RISC) induces the degradation and permanent silencing of the targeted mRNA; otherwise, the RISC complex induces a transient inhibition of gene translation in the case of an imperfect match between miRNA and mRNA. .Some studies have already associated pesticide exposure with the aforementioned epigenetic alterations ,22,23; hOn these bases, the aim of the present study was to identify a panel of genetic and epigenetic alterations induced by the exposure to pesticides that could be predictive for the development of diseases associated with these toxicants. For this purpose, the bioinformatics data reported in the literature and related to both genetic and epigenetic alterations mediated by pesticides were analyzed in order to establish the molecular alterations associated with the exposure to pesticides and predisposing the development of oncological and neurodegenerative diseases.www.ncbi.nlm.nih.gov/geo/, accessed on 15 August 2021), was used to select the DNA methylation, gene expression, and miRNA expression datasets of individuals with and without pesticide exposure.The Gene Expression Omnibus (GEO) DataSets database, publicly available on NCBI  AND ‘Homo sapiens’ [porgn: txid9606]).’ In this way, only datasets containing the expression or methylation data from individuals or human cells exposed to pesticides were collected.The selected datasets included datasets published up to February 2020 using the following inclusion and exclusion criteria to select only relevant datasets:Inclusion criteria: (i) datasets containing expression data on human samples, human cell lines, or animal models investigating potential biomarkers for humans; (ii) case-control studies, with cells or individuals exposed or not to pesticides.Exclusion criteria: (i) toxicity studies of pesticides in animals or vegetables, studies conducted on human cell lines or in vivo with less than 10 samples; (ii) datasets with genes, methylation probesets, or miRNAs not correctly annotated.After selecting and downloading the methylation, gene expression, and miRNA expression data matrices from GEO DataSets, differential analyses were performed between exposed and nonexposed samples to identify genetic and epigenetic factors dysregulated by pesticides. For this purpose, the GEO2R bioinformatic tool available on the GEO DataSets database was used. In particular, GEO2R allows the clustering of the samples of each dataset into a ‘nonexposed control group’ and ’exposed samples group,’ performing the differential analysis between expression levels of each gene, miRNA, and methylation hotspot in every dataset. Thus, it was possible to identify a panel of up-regulated or down-regulated genes, miRNAs, as well as hypo- and hyper-methylated hotspots altered as a result of exposure to different pesticides.In order to better understand the modulating effects of pesticides on the expression levels of miRNAs, as well as to predict the functional role of the candidate genetic and epigenetic biomarkers previously identified, several bioinformatic analyses were performed to identify the gene molecular pathways actively modulated by the selected miRNAs. For this purpose, first, bioinformatic analysis was performed by using the miRTargetLink tool, as previously described . This sohttps://string-db.org/, accessed on 13 July 2021) and GO Panther v14.0 , were used to analyze the interaction network and the biological and cellular roles of the genes identified through GEO2R, the genes targeted by the selected miRNAs, and the genes with alterations in the DNA methylation levels. In particular, STRING analysis was used to establish the molecular link between differentially expressed genes. The same analysis was performed on hypo- and hyper-methylated genes after pesticide exposure and genes targeted by the differentially expressed miRNAs [Subsequently, two bioinformatics software, STRING v11.0 , as well as within the two normal cell lines (HepRG and hESC), together with samples obtained from individuals professionally exposed to pesticides (GSE30335).It should be noted that the GSE35642 datasets containing gene expression data obtained from neuroblastoma cell lines exposed or not to pesticide reported a significant down-regulation for all of the 66 genes identified. A possible explanation of these data may rely on the type of cells but may also be due to the type of pesticide tested (rotenone) and the microarray platform used by the authors.These first analyses have allowed the identification of a panel of genes significantly modulated by different pesticides. In addition, these results revealed that pesticides are able to alter the expression levels of the same genes in different histological contexts; however, the effects of pesticides are different according to the type of cells . A further selection of dysregulated genes was also performed, selecting only the dysregulated genes with concordant expression levels in the three datasets of normal cells and the two datasets of tumor cells; however, this further selection criterion implicated a significant loss of data; therefore, the subsequent analyses were carried out on the 66 genes shown in In the same manner, the differential analyses performed for the two datasets containing DNA methylation data have allowed the identification of a panel of 62 genes with differential methylation patterns as a consequence of pesticides exposure. Of these 62 genes, 31 showed concordant methylation alterations as they were hypermethylated or hypomethylated in both datasets .The differential analyses revealed that the highest methylated genes were FGFR1 and A4GALT, while the lowest methylated ones were TMPRSS2, SLC4A11, and NEK3 .p < 0.05) after atrazine exposure, of which five miRNAs were up-regulated and 15 miRNAs down-regulated. In particular, the miRNAs hsa-miR-3149 and hsa-miR-4505 were the most up-regulated, while the miRNAs hsa-miR-10b-3p, hsa-miR-4515, hsa-miR-4313, and hsa-miR-126-3p showed the highest rate of down-regulation  targeting 22 different genes  B.It should be noted that some of these genes were targeted by three different miRNAs, particularly VEGFA and BCL2, suggesting how pesticide exposure might strongly modulate the expression of these genes through epigenetic mechanisms mediated by miRNAs. Interestingly, among the 22 genes targeted by the seven down-regulated miRNAs, there were genes notoriously involved in the development and progression of different diseases such as PTEN, KRAS, VEGFA, BCL2, FOS, DNMT1, MCL1, DNMT3A, and FOXO3. In addition, these data also suggested that pesticide exposure can profoundly alter human epigenetics through the alteration of miRNAs and, in turn, through the modulation of genes involved in DNA methylation such as DNMT1 and DNMT3A.After identifying the two miRNAs–genes interaction networks, a second prediction analysis was performed to identify the molecular pathways directly modulated by the 20 selected miRNAs. For this purpose, the DIANA-miRPath v.3 software was used to predict the pathways modulated by the five up-regulated and the 15 down-regulated miRNAs following pesticides exposure.For the five up-regulated miRNAs, the DIANA-miRPath analysis revealed eight different modulated pathways and a total of 75 modulated genes, including some genes identified through the previous miRTargetLink Human analysis  .Similarly, the DIANA-miRPath analysis allowed the identification of genes and related pathways modulated by the 15 miRNAs down-regulated after exposure to pesticides. In this latter case, only nine of the 15 down-regulated miRNAs showed interaction with specific pathways. Overall, these miRNAs were able to target a total of 3096 genes and 62 different molecular pathways. Of these 62 pathways, ten were related to infectious diseases and, therefore, were not directly correlated with pesticide exposure. Thus, DIANA-miRPath analysis demonstrated that almost all of the selected miRNAs were strongly involved in the modulation of several cellular and molecular pathways underlying the development of several pathologies.Due to the high number of genes and pathways modulated by the down-regulated miRNAs, it was also possible to obtain a dendrogram summarizing the strongest interaction between the 15 down-regulated miRNAs and the pathways identified .To further unveil the effects of both genetic and epigenetic alterations induced by pesticide exposure, the protein-protein interaction (PPI) and functional role of the identified putative biomarkers were assessed by using STRING and GO Panther bioinformatics tools, respectively.First, the PPI network of the genes targeted by the five up-regulated miRNAs identified through miRTargetLink Human was identified. Of the 12 genes previously identified, only four were interconnected. Despite the low number of gene interactions, this further analysis highlighted again how the PRKCA gene might be involved in pesticide-associated molecular alterations, as suggested by the miRTargetLink Human and DIANA-miRPath analyses A. In parA more complex PPI network was obtained by analyzing the 22 genes modulated by seven of the 15 down-regulated miRNAs, as predicted by miRTargetLink Human analysis. In this case, STRING analysis revealed that 20 out of 22 genes were involved in an integrated PPI network B. In parThis first STRING analysis, performed on the genes altered by the up-regulated and down-regulated miRNAs associated with pesticide exposure, highlighted how pesticides could induce a strong epigenetic modulation in key genes involved in different pathological processes. Therefore, the evaluation of both miRNAs and targeted gene expression levels could be used for diagnostic purposes to predict the degree of exposure to these toxic substances and consequently predict the risk of the onset of pathologies related to prolonged exposure to pesticides.To further uncover the functional role of the genes altered by the selected miRNAs, GO Panther enrichment was performed. The GO Panther analysis conducted on genes altered by up-regulated and down-regulated miRNAs showed that they are all firmly involved in different molecular functions, biological processes, and molecular pathways. For all the genes identified, the type of coding protein was highlighted to better understand its action at the cellular and molecular levels.As for the molecular functions, the GO Panther analysis has highlighted how almost 50% of the identified genes (15 genes) were involved in the molecular binding of other proteins, while a large part of them, ten genes, were involved in fundamental cellular catalytic activities A.The same analysis performed for the ‘Biological Process’ category revealed that the majority of genes were involved in cellular processes and in the regulation of various biological processes, including cell communication, cell death, and cell homeostasis B.Regarding the type of proteins codified by the previously identified genes, these were mainly enzymes involved in the cell metabolisms and detoxification, as well as protein-modifying enzymes such as SIRT1 and DNA methyltransferases C.The same STRING and GO Panther analyses were also performed for the methylation and genetic markers previously identified by differential analysis.Regarding the PPI network between the 31 methylated and demethylated genes, the STRING analysis showed how these genes form a moderate network where NRXN3 and PAX6 genes, both hypermethylated after pesticide exposure, represented two key nodes . These tIn order to establish the functional role of this second panel of genes, the Gene Ontology analysis allowed us to establish the biological processes, molecular functions, and protein classes of the 31 genes with an altered DNA methylation status.Similar to what was observed for the genes targeted by the selected miRNAs, pesticide-associated demethylated genes were mainly involved in the molecular functions of protein binding , and in some catalytic functions mainly represented by redox reactions A.Analyzing the functional category of biological processes, GO Panther analysis highlighted a strong grouping of demethylated genes involved in different cellular processes and, in particular, cellular metabolic processes, the organization of cellular components, and the regulation of various biological functions B. In addp-value < 0.01 , receptor molecules and proteins involved in the degradation of the extracellular matrix , and proteins with several ontologies.With regard to the molecular functions, the 66 differentially expressed genes were mainly involved in catalytic activities (hydrolases and transferases) and in the binding of proteins, organic heterocyclic compounds, and organic cyclic compounds A.The subsequent analysis of the ‘Biological Process’ category highlighted a strong prevalence of genes involved in the metabolic and biosynthesis cellular process, suggesting how the alteration of these genes induced by pesticides could be related to the incomplete detoxification of xenobiotics with the consequent harmful accumulation of toxic metabolites for the cells B.The analysis of ‘Protein Class’ has demonstrated that most of the 66 dysregulated genes were protein-modifying enzymes, translational proteins, and transcriptional regulators C.These further bioinformatic analyses allowed us to establish the functional roles of the genetic and epigenetic biomarkers predictive for pesticide exposure and potentially used as early diagnostic biomarkers for both tumor and neurodegenerative diseases.The integrated bioinformatics analyses of gene expression, miRNA expression, and DNA methylation datasets performed here have allowed the identification of genetic and epigenetic alterations associated with pesticide exposure that could be predictive for the development of both tumor and neurodegenerative diseases. Several studies have demonstrated that environmental and occupational exposure to pesticides represents a significant risk factor for the development of neurodegenerative, neoplastic, immunological, and hormonal disorders ,37,38,39Besides the epigenetic alterations, gene modulation after pesticide exposure was also observed, highlighting how the effects of these toxic substances differ depending on whether pesticide exposure occurs in tumor cell lines, normal cell lines, or biological samples obtained from individuals professionally exposed to pesticides.The results obtained demonstrated that 20 miRNAs were significantly altered by pesticide exposure, of which five were up-regulated and 15 were down-regulated. Among these miRNAs, hsa-miR-210-3p is of particular interest as it has already been associated with the development of Parkinson’s disease, associated with exposure to pesticides and other toxic substances. In particular, Zhang and colleagues (2017) demonstrated that the alteration of the expression levels of hsa-miR-210-3p contributes to the dopaminergic damage observed in meperidine (MPTP)-induced Parkinson’s disease . Other sOther studies demonstrated an indirect involvement of the identified miRNAs in different tumors and neurological diseases, suggesting how the alteration of specific miRNAs induced by pesticides may be associated with an increased risk of disease onset. In this context, hsa-miR-210 and hsa-miR-29 have been associated with the development of breast cancer and uveal melanoma ,44,45. IRelevant results were also obtained by analyzing the miRNA–mRNA interaction. It was observed how the selected miRNAs were able to modulate numerous genes involved in the development of tumors, in the detoxification of harmful substances, and in the neurodevelopment and neurotransmission. Among the miRNA-targeted genes, PRKCA appears one of the most modulated. This gene is sensitive to the action of various pesticides, insecticides, and herbicides that, when altering its expression, can cause neurotoxic damages .Other fundamental genes targeted by the selected miRNAs and strongly involved in the epigenetic alterations predisposing the development of tumors are DNMT1 and DNMT3, responsible for the alteration of the DNA methylation status, and VEGFA, BCL2, KRAS, SIRT1, and FOXO3, involved in different physio-pathological processes. In particular, it was demonstrated that VEGFA expression is increased by hexachlorobenzene favoring angiogenesis in breast cancer models . SimilarAll these data about the impact of pesticide exposure on these genes are strongly supported by the results of the STRING and DIANA-miRPath prediction analyses performed here.It should be noted that a single dataset on miRNA dysregulation after pesticide exposure was available. In addition, the data generated in the dataset GSE78805 were related to zebrafish larvae treated with atrazine as a single toxicant. However, miRNAs are highly conserved between species, and different studies have demonstrated the validity of zebrafish as an optimal model to study the alterations of human miRNA expression after exposure to toxic agents ,56,57; tWith regard to the data obtained from the analysis of the DNA methylation datasets, these allowed the identification of genes with an altered methylation status suggestive of pesticide exposure. Among the most hyper- and hypo-methylated genes, there were RYR1 and PAX6, already associated with pesticide exposure. In particular, RYR1 is a protein constituting the muscle calcium release channel, and the pesticide-induced methylation affecting its intron is associated with a lower gene expression and consequently with alterations of neurotransmission and muscle contraction . RegardiThe evaluation of the modulation of gene expression after pesticide exposure has generated heterogeneous results, whose interpretability could be complex. Overall, the analysis of the gene expression datasets revealed that pesticides have different modulating effects on the same genes depending on the cell type examined. In particular, the expression levels of genes were concordant among the group of cancer cells and the group of normal cells, independently, while a general inverse pattern of expression was observed comparing the expression levels of the same genes in the groups of cancer and normal cells. For both normal and cancer cells, the PPI and Gene Ontology analyses have shown once again the existence of a close network of interaction between the various genes that are significantly dysregulated following pesticide exposure. These further analyses have highlighted how pesticide exposure might influence different cellular mechanisms, including DNA mismatch repair, extracellular matrix degradation, and the alterations of signal transduction pathways, all mechanisms involved in the development and progression of different diseases, including tumor and neurodegenerative disorders ,61,62,63In this context, the analysis of circulating biomarkers, including circulating DNA alterations, miRNAs, and lncRNA, ,65,66,67These further analyses will make it possible to accurately identify the iatrogenic power of pesticides and to identify novel candidate biomarkers for the prediction of occupational or environmental pesticide exposure and for pesticide-related oncological and neurodegenerative diseases.To the best of our knowledge, this is the first study assessing both the genetic and epigenetic alterations induced by pesticides. Through the computational analysis of gene expression, miRNA expression, and DNA methylation datasets obtained from the GEO DataSets database, key genetic and epigenetic alterations associated with pesticide exposure were identified. In addition, the functional role of the genetic and epigenetic factors identified here was further elucidated by using different prediction analysis tools. Overall, the present study provides a comprehensive overview of the molecular effects induced by a group of pesticides. It should be noted that the results obtained here are only exploratory and show in broad terms the pathways and genetic and epigenetic factors significantly modulated by pesticides; therefore, our findings represent the starting point for further studies that will be performed on a large number of in vitro and in vivo models, as well as exposed individuals, to investigate the molecular alteration induced by a specific pesticide in order to identify reliable factors associated with the early diagnosis of pesticide-related diseases.