While normal-pressure hydrocephalus (NPH) is most commonly diagnosed in older adulthood, a significant body of literature has accumulated over half a century documenting the clinical phenomenon of an NPH-like syndrome in pediatric patients. As in adult NPH, it is likely that pediatric NPH occurs due to a heterogeneous array of developmental, structural, and neurodegenerative pathologies, ultimately resulting in aberrant cerebrospinal fluid (CSF) flow and distribution within and around the brain. In this review, we aimed to systematically survey the existing clinical evidence supporting the existence of a pediatric form of NPH, dating back to the original recognition of NPH as a clinically significant subtype of communicating hydrocephalus. Leveraging emergent trends from the old and more recent published literature, we then present a modern characterization of pediatric NPH as a disorder firmly within the same disease spectrum as adult NPH, likely with overlapping etiology and pathophysiological mechanisms. Exemplary cases consistent with the diagnosis of pediatric NPH selected from the senior author’s neurosurgical practice are then presented alongside the systematic review to aid in discussion of the typical clinical and radiographic manifestations of pediatric NPH. Common co-morbidities and modern surgical treatment options are also described. The existence of a syndrome in children and young adults with presentation and clinical findings similar to normal-pressure hydrocephalus (NPH) has been acknowledged in the pediatric population ,13,14,15Despite decades of recognition in the published literature, classification and diagnosis of NPH in the pediatric population remains controversial. The term “compensated ventriculomegaly” has been a misnomer applied in associated conditions which are linked to the development of chronic hydrocephalus which may or may not necessitate shunt treatment. Such conditions include prematurity, post-traumatic hydrocephalus, spina bifida, Chiari malformation, suboccipital pathologies , benign extra-axial fluid collections in infancy, craniosynostosis, Down Syndrome or other syndromic conditions, storage diseases, meningitis, vascular malformation  or other vascular complications ,13,15,22If one considers that the clinical hallmarks of NPH include the clinical triad  with symptomatic progression in the presence of chronic ventriculomegaly, or progressive cognitive and motor decline with or without urinary incontinence but in the absence of progressive ventriculomegaly, the literature collectively supports an NPH-equivalent diagnosis in the pediatric population. Most such situations where a similar clinical pattern is seen among children, often including failure to meet developmental milestones in lieu of marked deterioration of function per se, are associated with the above chronic conditions. Among both adults and children, presentation of cognitive dysfunction as well as treatment responsivity is widely variable, perhaps due to generally high prevalence of comorbidity with other brain pathology. Indeed, the possible importance of comorbid neurodegenerative pathology has recently emerged in the adult NPH literature ,24,25,26In this article, we aimed to systematically survey the literature evidence of pediatric NPH as a unique diagnostic entity which has emerged over the past five decades. Trends regarding etiology and diagnosis are qualitatively analyzed and discussed. Literature findings are coupled with a series of representative case presentations from the senior author’s clinical practice to support the concept of pediatric NPH applied in a modern cohort. Special consideration is paid to etiological circumstances or comorbidities of NPH in the pediatric population, and implications for clinical diagnosis and surgical management are highlighted.We conducted a systematic search of the PubMed database  to identify pertinent literature on the topic of pediatric NPH. Search terms included variants of “pediatric,” “child,” “infant,” or “newborn” in combination with “normal pressure hydrocephalus” or “idiopathic hydrocephalus,” contained within the title and/or abstract of the article. The citation lists of relevant articles identified by these criteria were additionally reviewed for studies which may have been excluded based on search criteria alone. This search methodology yielded a total of 75 PubMed-indexed articles for further examination. Abstracts and available full texts of identified articles were then individually reviewed for possible inclusion in this systematic review according to predefined criteria . ArticleOur single-institutional case series spans eleven years of the senior author’s neurosurgical practice in pediatric hydrocephalus, spanning from 2009 to 2020. Electronic medical records were reviewed for cases consistent with the diagnosis of pediatric NPH, as described in the summarized literature. Nine cases were identified as illustrative examples of pediatric NPH, and key details including suspected or confirmed etiology, clinical presentation, radiographic findings, and treatment outcomes were tabulated. Six cases were selected for narrative case presentation vignettes (‘Case Presentations’).Review of the literature revealed 15 clinical studies describing unique cases of pediatric NPH, comprising a total of 146 patients ranging in age from infancy to teenage years. Included articles, along with key methodological details and results, are tabulated in Etiologically, pediatric NPH has been identified by the existing literature as emerging from a diverse range of degenerative, congenital, traumatic, and genetic conditions . Common The literature suggests that the presentation of children with NPH is similar to that of adults with NPH, frequently demonstrating at least two of the three of the classic triad of symptoms: progressive cognitive impairment, motor/gait disturbance, and urinary sphincter dysfunction. In children, progressive cognitive decline associated with NPH is frequently seen as failure to meet developmental milestones, sometimes such that overall progression lags behind verbal development if detailed neuropsychological testing is performed ,10,14,15Nine illustrative cases from the senior author’s practice demonstrating the etiologic diversity and typical clinical presentation of NPH are tabulated in A 6-year-old female patient with a known history of Chiari I malformation (CM1) with inferior displacement of the cerebellar tonsils presented with recent, progressive deterioration of school performance, increased distraction and irritability, and frequent falling, as well as recent worsening of occipital headaches attributed to CM1. The patient had also experienced recent occasional episodes of incontinence. MRI demonstrated ventriculomegaly . On examA 17-month-old male patient with cerebral palsy presented with a life-long history of developmental delay secondary to chronic lung disease and tracheobronchomalacia after premature birth at 24 weeks gestation. During a six-month stay in the neonatal ICU, the patient struggled with respiratory failure requiring months of respiratory support and was believed to have suffered anoxic brain injury early in life. The patient also had a history of episodic breath holding with cyanosis. The patient did not have any associated intraventricular hemorrhage based on serial CT evaluation. However, by 18 months, there was concern for incongruent motor and cognitive developmental delays, so VPS was offered in the setting of ventriculomegaly, and webbing of the cerebral aqueduct visualized on MRI. The patient had an uncomplicated procedure but experienced an inpatient course complicated by continued breath holding spells with episodes of bradycardia and was eventually discharged home after 1 week. The patient experienced continued psychomotor developmental delay and unfortunately was diagnosed with shunt infection and underwent VPS removal at 18 months following placement. The patient’s parents opted not to pursue further shunt placement given that the patient did not have any improvement. The patient required extensive support for ongoing cerebral palsy with associated spasticity and dystonia over the subsequent decade, but hydrocephalus remained stable with mild ventriculomegaly persistent through most recent MRI follow-up .An otherwise healthy 18-month-old female patient with history of premature birth (36 weeks) and being the smaller fraternal twin presented with delayed motor development relative to the twin first noticed as frequent tripping, falling, and unsteady gait. Mild spastic diplegia was noted on exam. Ultrasound and MRI studies both demonstrated ventricular enlargement. MRI additionally confirmed diagnosis of communicating hydrocephalus with possible partial aqueductal webbing . After dTwo male siblings with history of Hunter’s syndrome (including a third sibling who was diagnosed and passed away) presented at 10 and 12 years of age, respectively with progressive ventriculomegaly, cerebral atrophy consistent with neurodegeneration, and functional and motor deterioration. VPS was recommended in both cases. Both surgeries were completed uneventfully; in one, a Rickham reservoir was placed, and in the other, a Codman cylindrical valve. Both patients had post-operative difficulty with airway secretions and management but improved gradually and were each discharged within 3 days of surgery. The 12-year-old patient (case #7) was re-admitted two months later for headache, during which time he was diagnosed with an elbow abscess positive for staph aureus. Both patients experienced continued progressive decline as expected with Hunter’s syndrome over subsequent years but did not experience acute worsening of hydrocephalus or require shunt revision .An 18-month-old male patient with history of intrauterine left-sided hemispheric stroke with residual cerebral ischemia and porencephalic cyst presented with interval history of slowing of motor development but continued verbal development. MRI demonstrated communicating hydrocephalus with massive left lateral ventricular enlargement with marked porencephaly, possibly dysplastic left-sided basal ganglia, and left-to-right midline shift . VPS wasTaken together, the results of the systematic literature review and institutional series comprise 155 total patients under the age of 18 years who underwent diagnosis and treatment of NPH-like syndromes. The findings of this systematic review help to establish the clinical phenomenology of a pediatric NPH-like syndrome. The 155 patients found through this review likely represent an under-estimation of the true number of pediatric cases published to date which are consistent with a diagnosis of NPH, as NPH diagnosis in this age group remains poorly defined and likely not often explicitly mentioned. Literature on this subject has also interestingly waned over the last two decades. These trends further underscore the need for additional study in support of evidence-based recommendations for this population. Based on published articles, the diagnosis and treatment of both secondary and idiopathic pediatric NPH is, in practice, comparable to that for adults, though without the same degree of standardization. That individual case series exhibit between-article variability in both etiology distribution and specific diagnostic criteria for NPH also highlights lack of standardization as far as when to suspect NPH in children. Establishment of pediatric NPH guidelines is warranted, similar to the guidelines that have been previously proposed for the adult NPH population ,21.While pediatric NPH is less likely to be mentioned explicitly in the more recent literature, some of the articles published in the last decade have provided novel mechanistic insights into potential genetic and circulatory pathophysiology present in children, as well as in adults, which may contribute to the development of NPH across ages. Two pediatric articles have implicated genes pertinent to ependymal ciliary structure and function; coupled with a third recent study by Yang et al. 2021) which observed a separate genetic defect also related to ependymal cilia function in 8/53 (15%) of adult idiopathic NPH patients who underwent full exosome DNA sequencing, this relatively new line of research provides insight into a potential mechanism of NPH related to cerebrospinal fluid production and circulation dynamics  which ob. SeparatBiomechanically, the impact of altered brain structure and neurodegeneration due to underlying comorbidities may also significantly predispose patients to developing NPH early in life, perhaps conceptually related to the high comorbidity of Alzheimer’s Disease-like pathology which has been appreciated in adult (but not pediatric) NPH ,38,39,40As in adults, variation in CSF pressure dynamic profile across pediatric hydrocephalus etiologies has been documented previously ,8. ContiContinuous recordings have been previously applied to model and categorize communicating hydrocephalus in young adult patients, including those with congenital structural abnormalities underlying altered CSF dynamics such as myelomeningocele and leading to so called “arrested hydrocephalus” ,45. GiveClinical and surgical decision making should be driven primarily by symptomatology, with clear evidence of deterioration or failure to meet milestones coupled with ventriculomegaly taken as a sign that surgical intervention may be indicated. In this series, all patients underwent VPS due to presentation including deterioration in some symptom domain consistent with NPH. Appropriately diagnosed and treated infants and young toddlers may be more likely to present chiefly with reduced global activity and progressive macrocephaly  while slightly older toddlers and children older than 4 may present with motor development outpacing verbal development, developmental slowing, or worsening of baseline neurologic symptomatology . Older children are more likely to have more classic NPH-like symptomatology, such as in our 6-year-old female patient with Chiari I malformation who had deterioration of school grades (suggestive of cognitive slowing), balance, and urinary continence over a period of months (case #1). This patient was also diagnosed with ADHD and, leading up to VPS, reportedly became increasingly irritable. These symptoms were present at baseline and originally attributed to her Chiari I malformation but seemed to be exacerbated by progressing NPH. Somewhat similarly, case #7, a 12-year-old male with Hunter syndrome and baseline seizure activity, experienced an increase in seizure frequency prior to VPS. Such patients may benefit from close attention to progressive symptomatic worsening, and even mild early ventriculomegaly should be tracked closely with serial neuroimaging so as to discern more subtle but clinically significant presentations of NPH alongside existing neurologic comorbidities. Both of these patients experienced improvement after surgery, which consisted of ventriculoperitoneal shunt placement. It should also be noted that, as seen in adult hydrocephalus due to the neurodegenerative nature of NPH and frequently associated pathologies, patient symptoms can sometimes worsen despite VPS. This was unfortunately seen in two of our presented patients with baseline genetic comorbidities (cases #3 and #5), also with associated morbidity and mortality. While all nine pediatric NPH cases in this institutional series, and most cited from the literature, underwent VPS, endoscopic third ventriculostomy (ETV) is a viable evidence-based alternative in obstructive pediatric hydrocephalus, though evidence specifically in pediatric NPH is quite limited.Normal-pressure hydrocephalus (NPH) remains a pertinent clinical diagnosis which is likely heterogeneous in pathophysiology and can occur in patients across the life cycle, including both older adults and children. The diagnosis of pediatric NPH is backed by less substantial clinical evidence than adult NPH. However, diagnosis and treatment across over 150 known patients, including those presented in this case series, has substantiated presenting clinical features similar to those in adult NPH including delay of cognitive, motor, and urinary developmental milestones during childhood. Ongoing study of genetic risk factors and neurodegenerative pathologies associated with NPH-like syndromes in children and adults may produce near-term mechanistic insight into this relatively common but likely underdiagnosed disorder. Ventriculoperitoneal shunting is a viable treatment option for patients with symptomatic pediatric NPH and the impact of endoscopic third ventriculostomy should be studied.


Cordycepin consistently repressed cell migration (26 papers) and cellular inflammation (53 papers). Evaluation of 76 papers on signal transduction indicated consistently reduced PI3K/mTOR/AKT and ERK signalling and activation of AMPK. In contrast, the effects of cordycepin on the p38 and Jun kinases were variable, as were the effects on cell cycle arrest (53 papers), suggesting these are cell-specific responses. The examination of 150 animal studies indicated that purified cordycepin has many potential therapeutic effects, including the reduction of tumour growth (37 papers), repression of pain and inflammation (9 papers), protecting brain function (11 papers), improvement of respiratory and cardiac conditions (8 and 19 papers) and amelioration of metabolic disorders (8 papers). Nearly all these data are consistent with cordycepin mediating its therapeutic effects through activating AMPK, inhibiting PI3K/mTOR/AKT and repressing the inflammatory response. We conclude that cordycepin has excellent potential as a lead for drug development, especially for age-related diseases. In addition, we discuss the remaining issues around the mechanism of action, toxicity and biodistribution of cordycepin.We conducted a systematic review of the literature on the effects of cordycepin on cell survival and proliferation, inflammation, signal transduction and animal models. A total of 1204 publications on cordycepin were found by the cut-off date of 1 February 2021. After application of the exclusion criteria, 791 papers remained. These were read and data on the chosen subjects were extracted. We found 192 papers on the effects of cordycepin on cell survival and proliferation and calculated a median inhibitory concentration (IC Cordyceps militaris [Cordycepin is the adenosine analogue 3’-deoxyadenosine, which is isolated from the caterpillar fungus ilitaris . The catilitaris . This inCordyceps militaris and characterised in 1950 [Cordycepin was first isolated from  in 1950 ,4,5,6,7.Early in the study of cordycepin, it was observed that high doses can kill cells in culture and affect cell proliferation ,9. The iA total of 103 papers contained data on effect of cordycepin on cell numbers in vertebrate tissue culture, 65 of which attributed this cytotoxicity at least in part to the induction of apoptosis (see the Methods section). In eight cases, autophagy was reported in addition to apoptosis ,15,16,1750) is commonly used to compare the effects of cytotoxic drugs on cell lines. This number incorporates both effects on cell death and on proliferation during the incubation time. The IC50 concentrations for cordycepin treatment of various cell lines ranged from 15 µM to 2 mM, with the incubation time usually being 24 or 48 h. By collecting IC50 data for 126 experiments from 74 papers (see the Methods section), we found that the average IC50 is 194 µM, with a standard deviation of 250 µM, reflecting the very large variability. However, as can be seen in 50 data cluster around the median at 135 µM, with a small number of almost resistant cell lines skewing the data. A caveat of this survey is that if a cell type does not reach the IC50 within the dose range tried, it will not appear in our analysis, so these data are likely to have the most validity for cell types and under conditions that do exhibit cordycepin sensitivity.The 50% inhibitory concentration . The doses ranged from 40 nM to 500 µM and had a median of 50 µM. Moreover, 19 of these studies indicated that cordycepin can actively promote cell survival or prevent senescence under conditions such as ER stress, oxidative stress, radiation, etoposide exposure and other disease-related cellular stresses ,42,43. ICordycepin is known to affect the cell cycle in oocytes and early embryos, presumably through its well-characterised effects on the cytoplasmic polyadenylation of mRNAs encoding cell cycle regulators . Indeed,50 (median 135 µM). However, as dose-response data were not widely available, it is not possible to be sufficiently quantitative for a firm conclusion. As can be seen in The role of cytoplasmic polyadenylation in normal mitotic cell division is, however, less clear ,62. ThisCell migration plays a large role in normal tissue development and the function of the immune system, but it is also associated with the metastasis of cancer cells and the progression of inflammatory diseases. We surveyed the papers with data on cordycepin in vertebrates for effects of cordycepin for data on cell migration and found 27 such papers; 26 of these papers reported a repression of cell migration in a variety of cell types including cancer derived cell lines, macrophages, smooth muscle cells and endothelial cells at doses between 0.4 and 400 µM and with a median of 100 µM (see the Methods section). Only one paper reported no effect of cordycepin on cell migration in myeloid leukemic cells, but the relevant data were not included in this paper . In eighAt the cellular level, the inflammatory response is a well-characterised set of gene expression programmes that is activated by signals indicating tissue damage or infection . The indWe found 38 papers which described effects of cordycepin on inflammatory gene expression (see the Methods section). Of these papers, 36 reported a reduction of inflammatory products by cordycepin. A single paper reported inhibition of iNOS but induction of TNFα  and onlyTGFβ is a cytokine family which is normally involved in stem cell maintenance, wound healing and resolution of the inflammatory response . ChronicNFĸB is a transcription factor with key roles in activating genes during inflammation and wound healing . ActivatIn addition, when we were examining animal studies (further discussed below) we found 28 papers that indicated that inflammatory processes were inhibited by cordycepin in a variety of animal models of disease ,134,135.A key goal of this systematic review was to obtain a clearer picture of the effect of cordycepin on signal transduction. We collected the papers studying the effects of cordycepin on signal transduction. After screening of the content, 79 papers were retrieved for detailed assessment of the effect of purified cordycepin on specific signal transduction pathways in tissue culture . Our assThe phosphatidylinositol 3-kinase (PI3K) and its downstream effectors mammalian target of rapamycin (mTOR) and protein kinase B (AKT) pathways are closely intertwined pathways that regulate biological processes such as metabolic balance, growth, differentiation, cell migration and angiogenesis. They play a key role in a variety of human diseases, such as cancer, type 2 diabetes mellitus and neurodegenerative diseases, and have been implicated in aging,139,140.2448 [2448 [2448 [The effect of cordycepin on mTOR activity is commonly assessed by its effect on the phosphorylation of S6 kinase (S6K), which is a major mTOR downstream substrate. This phosphorylation results in an increase in S6K activity. Conversely, it has been shown that S6 kinase phosphorylates mTOR at Ser2448 , which l2448 ,145,146.48 [2448 ,44,142. 48 [2448  . Most studies (nine articles) demonstrated an inhibitory effect of cordycepin on Akt phosphorylation at Ser473 [308; one concluded that cordycepin has no effect on Akt activation at this site [473.AKT phosphorylation is the most widely studied modification in cordycepin-treated cells. AKT is a family of serine/threonine protein kinases that plays a key role in cellular proliferation, apoptosis and migration. AKT kinases consist of three conserved domains: an N-terminal PH domain, a central kinase CAT domain and a C-terminal extension (EXT) containing a regulatory hydrophobic motif (HM). AKT kinases are phosphorylated by their activating kinases at a threonine residue in the activation T loop  and a Serine residue in the C-terminal HM  ,148. AKT activity,148. In  studied  papers. t Ser473 ,152,153.t Ser473 . These dhis site  and the his site . In addihis site . Overall172 residue [5’-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a highly conserved serine/threonine kinase. The AMPK kinase is a heterotrimeric protein complex containing a catalytic alpha subunit, an AMP-binding gamma subunit, and a scaffolding beta subunit. It is considered to be the master regulator of cellular energy homeostasis and is activated by upstream kinases, such as LKB1, CaMKKβ and Tak1 ,157. AMP residue . In the  residue ,160,161.172, as depicted in 172 [108 of the beta subunit plays a key role in cell cycle regulation and promotion of pro-survival pathways in response to energy stress [108 residue is auto-phosphorylated after prior phosphorylation at Thr172 residue, this residue is also the phosphorylation target of kinases other than AMPK such as ULK1 [172 and Ser108, indicating activation in all cases [The best-studied mechanism of AMPK activation is phosphorylation of the alpha subunit at Thrd in 172 . It has y stress . Althoug as ULK1 . The litll cases ,166,167.Various extracellular stimuli activate the mitogen-activated protein kinase (MAPK) cascade pathways . Three d180/Tyr182 [P38 proteins are a main subgroup of mitogen-activated protein kinases (MAPKs) that have been implicated in diverse biological processes, such as proliferation, differentiation, apoptosis and migration. Recent studies demonstrated that dysregulation of p38 has a key function in a wide variety of pathological conditions, such as solid tumours, arthritis and inflammation of the liver, kidney, brain and lung ,171,172.0/Tyr182 ,176,177.0/Tyr182   belong to the superfamily of the mitogen-activated protein kinase (MAPK) family involved in regulating eukaryotic cell reaction to a diverse range of cellular stress insults. They also coordinate essential physiological processes, including neuronal plasticity, immunological reactions, and embryonic development through their influence on gene expression, cytoskeletal protein dynamics, and cellular senescence ,180. Bas3/Tyr185 ,103,177,3/Tyr185 ,176  [202/Tyr204 [The extracellular signal-regulated kinases (ERK1 and ERK2) are evolutionarily conserved, highly regulated serine-threonine kinases that control cellular processes such as proliferation and differentiation. ERK plays a key role in development, and its upregulation is associated with the development and progression of diseases such as cancer. The ERK cascade is activated by signals such as growth factors, cytokines, viruses and G-protein-coupled receptor ligands . The litan ERK2) . Seven o2/Tyr204 ,177,178.2/Tyr204 , and ano2/Tyr204   and also inhibiting mTOR indicated by reduced the phosphorylation at Ser2448 (two articles), confirming the effects observed in tissue culture [In reviewing the effect of cordycepin on signalling cascades in animal models (detailed below), eight articles were identified with data on signal transduction. Five of these met the eligibility criteria (see the Methods section). The outcomes were activation of AMPK indicated by increasing phosphorylation at Thr culture ,183,184.One of the first animal studies on the effects of cordycepin examined the effect of cordycepin on the growth of tumour cells in mouse ascites , but theConcerning the cardiovascular system, 15 articles were found for a variety of animal models. Cordycepin was reported to have beneficial effects for cardiac hypertrophy , ischemiFourteen articles reported that cordycepin alleviates inflammation and pain in different animal models. Furthermore, cordycepin is described to have anti-inflammatory  and analgesic effects in osteoarthritic animal models and in other conditions associated with pain and inflammation. Different mechanisms were proposed to potentially explain the effect of cordycepin on pain behaviour, such as inhibition of inflammatory signalling as well as direct effects on the primary afferent nociceptors ,218,219.T. evansi-infected animal models and confer antiparasitic activity. In addition, the in vivo and in vitro activity of synthetic cordycepin derivatives has been studied in order to explain the structure–activity relationship [Several studies strongly suggest that cordycepin can be used for treatment of trypanosomiasis, but that it needs to be stabilised to do so. Treatment with cordycepin, when combined with an inhibitor of adenosine deaminase, can prolong the survival of tionship ,230,231.We found 10 papers indicating that cordycepin can have protective effects on the brain in various models of brain dysfunction. For instance, it reduces the effects of chronic unpredictable mild stress in animal models and improves depression-like behaviour ,232. SomEffects of cordycepin on animal models of respiratory disease were reported in eight papers. Multiple studies have shown an anti-asthmatic effect, which appears to be mediated by the inhibition of inflammation ,124,128.The effect of cordycepin was also studied in animal models of metabolic diseases, including diabetes and obesity. Cordycepin has been shown to reduce body weight and change fat metabolism in animal models of obesity ,239,240.Eight studies indicated the effects of cordycepin on reproductive disorders ,250,251.Our systematic review of the literature shows that cordycepin has significant effects in many animal models of disease and is an anti-inflammatory compound in both tissue culture experiments and animal models. The repression of inflammation is likely to be one of the key events in the therapeutic effects of cordycepin. While the mechanism by which cordycepin inhibits inflammation is not fully elucidated, it appears likely that the effects on signal transduction at least contribute. mTOR/AKT and AMPK are known to be involved in the regulation of inflammation and link metabolic changes to the inflammatory response in macrophages ,254,255.The cordycepin literature is overall of modest quality, as can be observed from common study flaws, such as the lack of information on which phosphorylation sites were examined, a frequent lack of primer sequence data and the few cases of image duplication (see the Methods section). This is likely due to the combination of meagre funding but high public interest for research into natural products, leading to large numbers of relatively low-budget studies and publications which are sometimes insufficiently critically reviewed. Nevertheless, this systematic review shows that the volume of studies indicating promising biological effects is now so large that the number of replicates is making up for any noise in the data. Our meta-analysis clearly shows that cordycepin has anti-proliferative and anti-inflammatory effects and that it activates AMPK, represses phosphorylation of AKT by mTOR and often reduces phosphorylation of ERK by MEK. While it can be argued that many of the animal models that have been used do not accurately replicate human disease, there can be no doubt that cordycepin has clear beneficial effects in many animals with a variety of disease-related symptoms.A weakness of all systematic reviews is that conclusions can only be drawn on subjects that are widely researched and the choice of these subjects is dependent on the interests of the research community. It is, therefore, possible that the key biological effects or highest therapeutic potential are not summarised in this review. Nevertheless, the findings discussed above strongly indicate that cordycepin is an excellent lead compound for drug discovery, especially in cancer and age-related diseases. Indeed, we found 18 publications describing drug development projects tackling cordycepin modification and formulation ,266,267.A major outstanding issue that is hampering the development of cordycepin as a lead compound is the lack of a clearly identified cordycepin-binding target molecule and a mechanism of action that connects this binding with the therapeutic effects. Proposed binding targets include poly (A) polymerases, adenosine receptors, CDK2, PARP1, AKT, AMPK, FGFR2 and RuvB-like ATPase 2 (RUVBL2) ,277,278.Several studies showed that cordycepin is efficiently converted to cordycepin triphosphate and trapped in cells, leading to accumulation. The inhibition of import and phosphorylation of cordycepin has been shown to reduce its effects, suggesting that intracellular and phosphorylated cordycepin is indeed at least one of the active metabolites of cordycepin ,284,285.Cordyceps militaris and only 98% pure. As cordycepin is generally used in micromolar quantities, it is possible that there are contaminants active in the nanomolar range that contribute to the biological effects of cordycepin. The fact that similar effects have been observed over time and with different suppliers suggests that, if there are important bioactive contaminants, these should be very consistently present. Thankfully, in a few cases, purer and synthetic cordycepin preparations have been shown to have similar effects to those observed with the standard preparations [Another complicating factor in cordycepin research is the lack of commercially available highly purified and/or synthetic preparations. The most widely used preparation from Sigma (now Merck) is isolated from arations ,239,291.The toxicity of cordycepin in animals has been reported to be low in the absence of adenosine deaminase inhibitors ,231,292,Promising effects in animal models do not always translate into good medicines for human patients, and we will have to await the reports of clinical trials to know if cordycepin and its derivatives are also bioactive in people . A trial of cordycepin in combination with the adenosine deaminase inhibitor pentostatin for acute lymphocytic leukaemia issued a preliminary account in 2000 , but unfIn conclusion, cordycepin has clear biological effects in a large number of animal models of disease. It has inhibitory effects on the PI3K/AKT/mTOR signalling pathway and activates AMPK. Most therapeutic effects of cordycepin are consistent with them being mediated by these effects on signal transduction. Moreover, the wide range of therapeutic effects reported in animal models is similar to other AMPK activators and mTOR inhibitors. Remaining challenges are the obscure mechanism of action of cordycepin, the lack of commercial availability of high purity cordycepin and the incomplete understanding of cordycepin biodistribution. Nevertheless, cordycepin appears to be an excellent drug lead for many common diseases and deserves to be further investigated.Only papers recorded in PubMed were considered. A PubMed search on “cordycepin” found 1167 publications entered in PubMed by 1 February 2021. We found 37 additional papers on the subject through references in other papers and by examining the abstracts for a PubMed search for “3’-deoxyadenosine NOT cordycepin” for relevant papers, yielding 1204 papers for initial examination.Cordyceps extracts and chemical synthesis of cordycepin. We also excluded papers only containing data on chemical derivatives of cordycepin, with the exception of studies of the intracellular metabolite cordycepin triphosphate and animal studies. These filters removed 204 papers.We removed 39 articles not written in English from consideration. For the remaining papers, abstracts were then read to exclude publications solely containing data on cordycepin preparation, fungal production of cordycepin, crude A total of 33 previous reviews not containing original data were also removed from consideration. Two corrections on included papers were removed from the database. The remaining publications were collated in an excel spreadsheet. Full articles were obtained from online sources, the University of Nottingham library and through the British Library. We failed to obtain full text copies for eight papers. Data on the biological effects of cordycepin were extracted from each paper by a member of the team using a spreadsheet.A total of 910 papers potentially containing data on the biological effects of purified cordycepin remained at this stage.After initial reading, we classified these papers according to the system studied . Viruses were classified with their host. If more than one system was studied, the earlier listed system (as above) was entered. Notes were entered on the characteristics and species studied and themes were extracted for further analysis.We decided to exclude the papers on plants, as cordycepin is primarily used as a transcription inhibitor to elucidate contributions of this process to biological changes (123 papers). Some differences to other transcription inhibitors have been noted in seed germination, but the nature of this difference is unclear .A total of 791 other papers remained containing data on effects of cordycepin , gene expression (inflammatory and other) and other effects. For papers describing animal experiments, notes were added on the following subjects: animal model, dose, administration route, inflammation, other immune system effects, bone and cartilage, tissue remodelling and fibrosis, tumour growth, neuronal effects, metabolic effects and other effects.The original 1204 papers were used to explore the publication year of cordycepin papers. Publication year was used as stated by PubMed.First affiliation of the last author was determined from the PubMed entry, or if absent, by consulting the paper if it was available to us. The continent of the affiliation was entered. If the last author had affiliations in more than one continent, the first stated was used. We determined the origin for 932 papers after excluding papers with unclear affiliation.We found 196 papers containing data on the effects of cordycepin on cell viability, death or proliferation and these were examined further for effects on cell number, cell survival and cell cycle.50) for cells in conventional (2D) culture was determined or could be estimated from the data for the 24 h or 48 h timepoint for one or more cell lines (48 h was selected if both were available). These studies were used for the graph in A total of 103 papers contained data on the effects of cordycepin on cell numbers in vertebrate tissue culture. Data for modified cordycepin, cordycepin with other compounds such as pentostatin, cordycepin in protective or slow-release formulations or for cells in 3D culture were excluded. Of these 103, 74 papers included cell viability experiments which used colorimetric assays with tetrazolium salts -2,5-diphenyltetrazolium bromide) or employed a live/dead cell counting method, e.g., trypan blue or propidium iodide, for which the 50% inhibitory concentration . A total of 29 articles were finally included in the systematic review to study the effect of cordycepin on the following kinases: mTOR ,44,142, We initially retrieved 160 papers studying the effect of cordycepin in diverse animal models. Ten articles were excluded because they did not use purified cordycepin or employed cordycepin derivatives only. Papers studying combination treatment with adenosine deaminase inhibitors were not excluded. Notes were entered from these papers including animal species, type of human diseases models, dose in mg/kg and route of administration. Data were also gathered regarding effects on inflammation, neuronal function and metabolism. While gathering data from papers studying the effect of cordycepin on signal transduction in animal models, we excluded any papers that did not report the precise phosphorylation site , and we became aware of the problem of so-called paper mill publications. To try to avoid including any so-far undetected paper mill publications, we examined all articles we had selected for inclusion in the review on duplicated images. No problems on a scale as large as the first paper were encountered, but we found three more papers with clear duplication of one panel. While these may be due to honest mistakes, it shows insufficient care and these papers were removed from consideration. We notified the publishers of our concerns. In two other cases, two images were very similar, but not identical, and we decided to include these but also notify the publishers of these papers.


The hepatotoxicity of irinotecan is drawing wide concern nowadays due to the widespread use of this chemotherapeutic against various solid tumors, particularly metastatic colorectal cancer. Irinotecan-induced hepatotoxicity mainly manifests as transaminase increase and steatosis with or without transaminase increase, and is accompanied by vacuolization, and lobular inflammation. Irinotecan-induced steatohepatitis (IIS) increases the risk of morbidity and mortality in patients with colorectal cancer liver metastasis (CRCLM). The major risks and predisposing factors for IIS include high body mass index (BMI) or obesity, diabetes, and high-fat diet. Mitochondrial dysfunction and autophagy impairment may be involved in the pathogenesis of IIS. However, there is currently no effective preventive or therapeutic treatment for this condition. Thus, the precise mechanisms underlying the pathogenesis of IIS should be deciphered for the development of therapeutic drugs. This review summarizes the current knowledge and research progress on IIS. Irinotecan, also termed as CPT-11 or 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecine Figure 1Irinotecan, which is a semi-synthetic and water-soluble camptothecin-derivative cytotoxic drug . It inhiUnfortunately, irinotecan can non-specifically damage any rapidly proliferating cell, including both tumor cells and non-tumor cells, such as bone-marrow cells and intestinal basal cells, as well as the commensal bacteria in the body . ConsequLong-term or high-dose administration of irinotecan may impair the liver parenchyma, thus leading to hepatotoxicity with or without transaminase increase , 13–16. IIS is associated with the disruption of lipid homeostasis and with inflammation in hepatic cells. It may progressively increase the risk of fibrosis, cirrhosis and liver failure , 25, bec2 under irinotecan-based chemotherapy have a 2.03-fold risk of IIS compared with those with BMI < 25 kg/m2 (2 (Multiple studies have found that confounding factors could impact the development of chemotherapy-associated steatohepatitis (CASH) (including IIS) –28. High25 kg/m2 . Anotherkg/m2 , which are the main form of lipid storage in the cell . Thus, iIn general, there is still a lack of effective preventive and therapeutic strategies against IIS due to its complex mechanism of pathogenesis. However, several preclinical studies for IIS have suggested potential interventional drugs or measures, as described below.Silybum marianum  and polyunsaturated fatty acids (PUFA) in diet may be effective in suppressing non-alcoholic fatty liver disease (NAFLD) or IIS. Isoflavones can be beneficial against NAFLD by reducing the lipogenesis, lipolysis, and fat deposition in adipocytes , 53. PUFOverall, IIS is a crucial adverse effect of irinotecan and can increase the risk of morbidity and mortality in patients with CRCLM. The major risks and predisposing factors for IIS include high BMI or obesity, diabetes, and high-fat diet. Although mitochondrial dysfunction and autophagy impairment may be involved in the pathogenesis of IIS, the exact causal mechanisms of IIS have not been fully elucidated. Till now, liver biopsy is the gold standard in diagnosing hepatotoxicity, including IIS, but it is a highly invasive, complex, and painful operation. Thus, biomarkers of IIS, are urgently needed to precisely evaluate irinotecan-induced hepatotoxicity. Besides, we should explore more risk factors for the development of IIS which can help oncologists to identify the patients at risk. Furthermore, effective preventive and therapeutic approaches are still lacking. Potential interventional drugs or measures have been reported in multiple preclinical studies and the medications susceptible to be active in steatosis such as fibroblast growth factor 21 agonists, obeticholic acid or glucagon-like peptide-1 agonists deserve considerations. Thus, further investigations involving humans, especially clinical trials, are required to develop feasible preventive and therapeutic approaches against IIS.CZ designed this work. JH and JZ wrote this manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the scientific research project of Wuhan NO.6 hospital (No. LX19013 to JH).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.


Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 10 Virus-like particles (VLPs) are recognized as a promising strategy in recombinant vaccine development due to their ability to mimic native viruses with the lack of a viral genome. Their highly organized and repetitive antigen-presenting structure has been shown to stimulate both cellular and humoral immune responses ,2. MoreoAlthough the generation of these particles has been described in the literature for more than three decades, there are still challenges for their clinical-grade production ,6. Gag VAnother factor that contributes to this challenging purification is the need for appropriate analytical methods to corroborate the structural integrity of the VLPs, as well as its separation from other nanoparticles . AlongsiFinally, the preservation of purified Gag VLPs is another general concern in the field. While cold chain storage is still the classical approach, the instability associated with this step prior to administration can affect the efficiency of the candidate . EngineeIn this work, the stability, purification, and preservation of Gag VLPs have been studied. The combination of new membrane-based filtration and chromatographic methods together with lyophilization is proposed here. Specifically, a purification process consisting of depth filtration to obtain the clarified supernatant, ion-exchange chromatography (IEX) chromatography to concentrate the VLPs, and size-exclusion chromatography (SEC) to polish and prepare the purified material for lyophilization has been developed. VLP, EV, double-stranded DNA (dsDNA), and total protein concentration were quantified during the different unit operations. The obtained data on Gag VLP purification and preservation will contribute to define strategies that will facilitate their manufacturing and distribution worldwide .2, 80% relative humidity at 110 rpm in Multitron shakers  with HyCell™ TransFx-H medium from HyClone™ . HyCell™ culture medium was supplemented with 0.1% Kolliphor188  and 4 mM GlutaMax . Cell density and viability were routinely assessed with Vi-Cell XR .The mammalian cell line used in this work is a serum-free suspension-adapted HEK 293 cell line . Cells were cultured in disposable PETG flask  at 37 °C, 5% COHEK 293 cells were transfected using 25 kDa linear polyethylenimine (PEI)  as previously described . Gag-eGFQuantification of Gag-eGFP in sample supernatants was assessed using an in-house developed and validated spectrofluorometry assay . Green fFlow virometry experiments were performed with a CytoFLEX (Beckman Coulter Inc.) with violet side scatter (V-SSC) 405 nm filter configuration. In all cases, samples were diluted with PBS 1 X until the abort rate value was below 2%. A total of 300,000 events per sample were analyzed at a flow rate of 10 µL/min. V-SSC vs. B525-FITC density plots were used to gate the different particle populations. Gating was manually adjusted for each channel. The results were analyzed with the CytExpert software (Beckman Coulter). Nanoparticle concentrations were calculated with Equation (2):® dsDNA kit (Life Technologies). Since Gag-eGFP VLPs emit at the same range as the Quant-iTTM PicoGreen® reagent, the native fluorescence was measured prior to the reagent addition and later subtracted to the fluorescence after the reaction. Assays were performed in a 96-well plate format. The 96-well plates were read and analyzed with Synergy HTX multi-mode reader and Gen5 software, respectively .Total protein concentration was determined using RC/DC assay  or Micro BCA assay (Thermo Fisher) following the manufacturer’s instructions. The calibration curve was obtained using bovine serum albumin (BSA) standards (Thermo Fisher) diluted in distilled water with a concentration range of 50–250 µg/mL. The concentration of HEK293 HCP was determined by the HEK293 host cell protein ELISA kit  according to the manufacturer’s instructions. DsDNA quantification was performed with the Quant-iTTM PicoGreenDLS experiments were performed using a Zetasizer Nano ZS instrument  with a He/Ne 633 nm laser at 173°. The hydrodynamic diameter and polydispersity index (PDI) were calculated with cumulative fit correlation at 25 °C and 0.8872 cP as previously described [® NS300 device was used  as previously described [® NS300 is equipped with a blue laser module (488 nm) and a neutral density filter that was used to quantify GFP fluorescent nanoparticles and total particles by light diffraction, respectively. Gag-eGFP VLP concentrations were calculated as the total fluorescent particles, and the concentration of EVs was calculated as the difference between light scattering particles and fluorescent particles.Gag-eGFP VLPs and total particle content were analyzed by NTA. A NanoSightescribed . NanoSigCryo-TEM analyses were performed at Servei de Microscòpia at Universitat Autònoma de Barcelona  using a Leica EM GP cryo workstation and observed in a Jeol JEM-2011 TEM electron microscope operating at 200 kV  as previously described .v/v of 1.4 M DTT. Each sample was incubated at 95 °C for 20 min. Precision Plus Protein™ Standards All Blue (Bio-Rad) was used as a protein marker. Samples were diluted 1:4 with distilled water before its addition to the gel. The gel was run at 200 V (400 mA). Coomassie staining solution was used to stain the total protein in the SDS-PAGE. For the western blot analysis, proteins were blotted using Trans-Blot® turbo system (Bio-Rad) with 0.2 μm nitrocellulose membranes and blocked with 5% (w:v) skimmed milk overnight at 4 °C. Detection of Gag-eGFP protein was performed by incubation with primary mouse monoclonal antibody against HIV-1 p24 , diluted 1:1000 in PBS-T for 2 h. Anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Abcam), diluted 1:1000 in PBS-T was used as a secondary antibody. Finally, the membrane was incubated with enhanced chemiluminescence (ECL) reagent (Bio-Rad) and visualized using a ChemiDoc imager (Bio-Rad).Precast TGX 4–15% Midi Gel  were used in a Tris-Glycine buffer system. The protocol was adapted from previous studies ,27. BriePreliminary thermostability studies of Gag-eGFP VLPs at different temperatures were performed prior to DSP development. Gag-eGFP VLPs were produced in HEK 293 cells by PEI-mediated transient gene expression as previously described . Thermos2 effective surface area . Filters with 5, 3, 1.2, 0.65, and 0.45 µm nominal pore size were used. A total volume of 40 mL of cell culture supernatant containing Gag-eGFP VLPs were sequentially filtered at 266 LMH (liter per meter hour), corresponding to a flow rate of 2 mL/min at RT in sterile conditions with the different filters. MasterFlex® pump with MasterFlex® 96410-13 silicon tubes  and pressure gauge  connected to the filter inlet were used in the filtration experiments. A total of 2 mL of sample was taken between serial filtration for analysis. The same experiment was run for negative control for comparison.Serial filtration experiments were performed with SartoScale PP3 polypropylene filter membrane of 25 mm and 4.5 cm®+ D0HC µpod filter with 23 cm2 effective surface area , adapted from [−2) of filters in the screening experiments was evaluated until feed pressure reached a maximum of 14.5 psi (1 bar). Filtration was performed at an average LMH of 135.5 ± 34.3 in sterile conditions. Filters were flushed and conditioned with 50 mL of 50 mM HEPES, pH 7.4, 300 mM NaCl buffer. MasterFlex® pump with MasterFlex® 96410-13 silicon tubes (Cole-Parmer) and pressure gauge (WIKA Alexander Wiegand SE & Co. KG) connected to the filter inlet were used for the filtration experiments.Preparative primary clarification of Gag-eGFP VLPs from HEK 293 cell cultures was carried out at 72 hpt. A total of 1 L of cell culture was incubated for 1.5 h at RT where flocculation of cells was obtained, followed by dead-end filtration with MilliStak® Mustang® QXT membrane adsorber . Before loading, the column was equilibrated with 50 mM HEPES, 100 mM NaCl, pH 7.2 (5% buffer B). After the loading phase, the column was washed with equilibration buffer (5% buffer B) for 5 membrane volumes. In the linear-gradient purification, a salt linear gradient from 100 to 1000 mM NaCl (5% to 50% buffer B) in 20 membrane volumes was used adapted from [Clarified cell culture supernatant containing Gag-eGFP VLPs was loaded into a 0.86 mL XT Acrodiscted from . For the2, 2% sucrose at pH 7.5. Then, 4–6.4 mL of Gag-eGFP VLPs were loaded and eluted with the equilibration buffer in the void volume. UV absorbance at 280, 260, and 488 nm and pH were measured online in AKTA Explorer . The different fractions obtained were pooled and analyzed according to the chromatogram.Polishing step after IEX at bench scale was performed with pD10 desalting column according to manufacturer’s instructions . Gag-eGFP VLPs were eluted in PBS (Hyclone). Preparative chromatography was performed with a 43 mL sepharose 4 Fast Flow (4FF) packed column at a constant flow rate of 1 or 2 mL/min. Briefly, SEC column was equilibrated with 20 mM NaH2PO4, 50 mM NaCl, 2 mM MgCl2, 2% sucrose at pH 7.5). Aliquots were frozen at −80 °C and subsequently lyophilized at −60 °C at 100 µbar for 24 h in a VirTis BenchTop 6K . Lyophilized material was stored at RT for two months until analysis. Lyophilized samples were resuspended in distilled water prior to analysis. Cryo-TEM, DLS, flow virometry, and NTA were used to evaluate particle integrity in purified samples.Purified Gag-eGFP VLPs were aliquoted in 2 mL glass vials  for their lyophilization. The purified Gag-eGFP VLPs were previously buffer exchanged into lyophilization buffer , standard cold chain (4 °C), and freezing temperatures (−20 and −80 °C) were tested. Two different techniques were combined to analyze VLP stability over time: spectrofluorometry and fluorescent nanoparticle tracking analysis (NTA) . Spectro10 VLPs/mL was quantified in crude harvested supernatants by both techniques  was obtained in Gag-eGFP quantification, while a loss of 47% ± 16% was quantified by NTA after 10 days of storage. The PSD analysis of −20 °C condition showed a loss of nanoparticles in the range of 100 to 200 nm and the appearance of larger aggregates with time  calculated as follows:g is the acceleration of gravity (9.807 m/s2), d is the particle diameter, and n is the medium viscosity. Assuming a similar 3 [3,and a n of 0.001 kg/(m s) at 20 °C, a settling velocity in the order of few cm per hour is obtained from Equation (3) [®D0HC was performed. Feed pressure and fluorescence in the clarified volume were monitored during the experiment . The clarified material was further characterized by cryo-TEM where the presence of VLPs (black arrows), but also the naturally co-produced EVs of different sizes were identified  . Thus, tperiment A. A totaperiment C,D. A coectively C, while As a proof of concept, settled cells were resuspended with the addition of 50 mL of PBS (Hyclone) and loaded to the filter. The addition of concentrated cells to the depth filter immediately increased the feed pressure to 16 psi (1.1 bar), over the defined limit of 1 bar. Thus, the settling of cells prior to depth filtration allowed to increase the filter capacity in an unlimited way since feed pressure only increased when cells were loaded. Similar improvements in terms of filter capacity were reported by Westoby and co-workers when CHO cells flocculation at acidic pH was performed before microfiltration .The concentration and purification of Gag-eGFP VLPs were assessed by means of IEX chromatography. Initial linear-gradient experiments were performed with clarified supernatant to evaluate the suitability of the Mustang Q membrane to purify these nanoparticles. A total of 20 mL of conditioned medium, as negative control and Gag-eGFP VLP-containing supernatant, were loaded into a 0.86 mL Mustang Q in two separate experiments C. The prFrom the total protein loaded, 37% ± 5% and 14% ± 0% were collected in the flowthrough (FT) and wash (W) fractions, respectively, while the rest was distributed in the other two peaks (E1 and E3). In the case of dsDNA, 52% ± 10% was recovered in the third peak (E3); meanwhile, it was below LOD in the other pooled fractions E. The sa11 VLPs was loaded, according to flow virometry results. A second run was then performed, with a total volume of 413 mL of supernatant loaded  and optimizing the elution by applying a step gradient to concentrate the Gag-eGFP VLPs in the elution phase. A total volume of 150 mL representing 7.5 times more supernatant than in the previous run was loaded to study the DBC. Thereafter, an initial three-step gradient of 15%, 45%, and 60% of buffer B was tested, based on the results observed in the linear gradient B when a t loaded E–G. In t11 VLPs/mL and a total recovery of 38% of the VLPs . In all three cases, a mean hydrodynamic diameter between 100 and 200 nm was found, such as in the loaded supernatant (S), whereas biochemical analyses confirmed Gag-eGFP presence in all samples (~87 kDa) by means of SDS-PAGE and Western blot anti-p24 .IEX fractions A, E1–E4 11 VLPs and represented 75% of the Gag-eGFP polyprotein-loaded material , as shown by the presence of a large peak of 1126 mL·mAU in SEC2 pooled fraction. This peak corresponded to 1.08 × 10material D. As forTo validate these results, a complete purification process was performed, combining the clarification, IEX, and SEC steps. The mass balance of this complete validation run is summarized in Finally, first attempts in the lyophilization of Gag-eGFP VLPs were performed in views to optimize its storage and transportation. Gag-eGFP VLPs were lyophilized in 1 mL aliquots stored at RT for two months. Alternatively, purified Gag-eGFP VLPs were frozen at −80 °C, in the same buffer, as the control.Concentrated and purified Gag-eGFP VLPs are clearly observed in cryo-TEM micrographs , compare2 were achieved in membrane-based processes. Previous works analyzed different filters for primary and secondary clarification for influenza VLPs produced with baculovirus expression vector system (BEVS), where a maximum load of 150 L/m2 was reported for primary clarification with MilliStak®+ D0HC [A four-step process has been developed for Gag-eGFP VLPs. Load capacities and flux rates are summarized in k®+ D0HC . Here, t2. From the different optimizations performed, a maximum load capacity of 842.9 L/m2 was achieved  per square centimeter of adenoviruses, more than 8.7 × 1010 transduction units (TU) of lentiviral vectors, and 1.27 × 108 infective units (ifu) of γ-retorviruses per mL of bed volume have been reported with membrane adsorbers [12 of total particles per mL in QA monolith in the purification of Gag VLPs [IEX chromatography was performed with a Mustang Q composed of 16 layers of Mustang Q membrane with a bed volume of 0.86 mL and a surface area of 4.9 cmachieved D, corresdsorbers ,35,36. SGag VLPs . On the SEC chromatography has been used as a desalting column after IEX chromatography to perform a buffer exchange. A maximum 6.4 mL load is allowed in a 43 mL SEC column for desalting purposes. Thus, several rounds are required to purify higher volumes, as for the E2 fraction here. Despite the simplicity of the method, a dilution factor of 1.7 and larger operation times are needed. Alternative membrane-based ultrafiltration and diafiltration have been described in the literature to overcome such limitations. Specifically, tangential flow filtration (TFF) with 300 and 1000 kDa cutoff membranes has been used for the concentration of influenza VLPs with higher recoveries ,38. More9 VLPs have been purified from HEK 293 clarified supernatants. More than 30 µg/mL of Gag-eGFP polyprotein are obtained after purification, which represents the 98% of the total protein (Lyophilized Gag-eGFP VLP vials containing 2.2 × 10 protein . Of note protein , whereas protein . The preDespite the obvious advantages of lyophilized material in comparison to cold chain storage, the process of freeze-drying is still a challenge in viral-based vaccines, especially for enveloped viruses. Lipid and protein damage, changes in the pH or osmolarity, or aggregation are some of the reported drawbacks . Here, GOverall, there is no established process in the purification of viral-based products . In a re, respectively [9 VLPs, the present strategy meets the required quality attributes and offers great promise for the further use of this platform in the development of novel vaccine candidates.A 23% recovery of highly purified VLPs from the crude supernatant was achieved . While cectively ,38. Howeectively . Regulatectively ,40. ConsGag VLPs are a very promising platform for antigen and drug delivery strategies. A complete purification train from crude supernatants to highly purified Gag VLP preparations has been developed in this work. Thermostability and particle aggregation studies of harvested Gag-eGFP VLPs showed these particles to be stable at 4 and −80 °C for up to three months. A nimble DSP consisting of clarification, IEX chromatography, SEC, and lyophilization was developed. The separation of Gag-eGFP VLPs from EVs was observed in the final purified samples, where Gag-eGFP polyprotein represented more than 98% of total protein. In summary, lyophilized vials were obtained and stored at RT for up to two months. These preliminary results in enveloped VLP lyophilization open the window for developing new formulations increasing nanoparticle stability for its use and transportation worldwide.


Using cryotomography, we show that unlike immature HIV virions, in the Gag lattice of VLPs the CA-CTD and SP1 regions are significantly less ordered. Our observations suggest that within immature HIV virions, other viral factors in addition to Gag, contribute to ordering in the CA-CTD and SP1 regions.Immature HIV virions harbor a lattice of Gag molecules with significant ordering in CA-NTD, CA-CTD and SP1 regions. This ordering plays a major role during HIV maturation. To test the condition in which the Gag lattice forms in vivo, we assembled virus like particles (VLPs) by expressing only HIV Gag in mammalian cells. Here we show that these VLPs incorporate a similar number of Gag molecules compared to immature HIV virions. However, within these VLPs, Gag molecules diffuse with a pseudo-diffusion rate of 10 nm Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). AIDS has claimed upward of 30 million lives over the past 30 years and while the disease can be kept in check using potent antivirals, there remains no cure for AIDS. HIV-1 developing resistance to antivirals is an increasingly significant problem which highlights the need for continued development of new antivirals that exploit novel targets to inhibit HIV-1 replication ,3. The aImmature HIV virions released from infected cells, package ~2000 copies of Gag . The GagTomogram averaging cryotomography has resolved major interactions between Gag molecules within the lattice, which shows each molecule having interactions with at least 5 other Gag molecules. Specifically each Gag molecule is stabilized within the lattice through interactions in its CA-NTD domain which forms the basis of the Gag hexagonal structure ,17. FurtHIV Gag expressed within mammalian cells is sufficient for the release of Gag virus- like particles (VLPs) . These pWe previously established a method based on iPALM  to furthThe 293 cells were grown in DMEM media supplemented with 10% FBS at 37 °C. The designated plasmids are transfected using Lipofectamin 2000 . The supernatant is collected 36 h post transfection. The cells in the supernatant were separated through centrifugation  at 5000 rpm for 15 min. The supernatant was filtered using 0.4 um filters. The VLPs were segregated from the supernatant by centrifugation at 10,000 rpm for 2 h in Beckman L8-70M Ultracentrifuge and SW41 rotor  at 4 °C through 1 mL of 10% sucrose. The VLP pellet was resuspended in PBS and stored at 4 °C.The VLPs are diluted in PBS from the stock solution in the ratio 1:1. The VLPs were treated with heterodimerizer HAXS8  at a concentration of y is the percentage of complex formed over time (x), α reports the pseudo diffusion coefficient, k and n are scaling factors that solely depends on the percentage of Gag molecules that has been tagged with SNAP or Halo. The value of α was fixed to 1 in the simulation data to obtain the values k and n. Then these values were fed in the experimental data to obtain the value of the pseudo diffusion coefficient α.A 2-dimensional hexagonal lattice with a size of 8 nm and ~5000 lattice points were generated. The lattice sites were randomly populated with 2000 Gag molecules. The initialization was biased to one part of the lattice such that the other part of the lattice is completely empty, to replicate the void space present in a HIV lattice. In one of the models 10% of the total molecules were tagged with Halo and the other 10% were tagged with SNAP. In the second model 20% of the total number of molecules were tagged with Halo and the other 20% with SNAP. In the third model we had 40% of the total number of molecules tagged with SNAP and the other 40% with Halo. The molecules hopped on the lattice following a Monte Carlo algorithm and with spherical boundary conditions imposed. The move is rejected if three molecules already occupy the nearest site. Out of the three available neighboring sites the molecule chose its moving site based on an unbiased random number (rand in MATLAB). No binding interactions between the Gag molecules has been taken into consideration except when a Gag-SNAP and Gag-Halo met on a lattice site. It was then treated as a covalently bonded complex, and they hopped as a single entity but always considered to be 2 molecules. The move of a Gag-SNAP-Halo-Gag was allowed to the nearest neighbor provided they did not infract the rule that a lattice site cannot be occupied by more than 3 molecules. The number of Gag-SNAP-Halo-Gag complexes was observed over time. The hopping in each of the simulations was performed for 1000 frames. The timeframe in the simulations were scaled to real experimental time using Hill’s equation:The simulation code was written in MATLAB and performed on the compute nodes with two Intel Xeon Gold 6130 CPUs, 32 CPU cores and 96 GB of RAM per node . The code has been attached in the A 25 mm #1.5 glass coverslip  and a 18 mm #1.5 glass coverslip  were used to prepare the sandwich for imaging. The glass coverslips were washed with 70% ethanol and then in 1 M NaOH for 20 min. They were then thoroughly washed in miliQ water and blow-dried with ultra-high-pressure nitrogen. The coverslips were plasma cleaned for 10 min. The 25 mm coverslip was coated with poly-D-lysine from Sigma-Aldrich at a concentration of 50 mg/mL and incubated in a humid atmosphere for 45 min. After the incubation period, the coverslip was thoroughly washed with mQ water and blow-dried with ultra high-pressure nitrogen. The VLPs were then added to the 25 mm coverslip and incubated in a humid atmosphere for 30 min. The coverslip was then washed gently with PBS so as to remove the VLPs that were not immobilized on the coverslip. This 25 mm coverslip was then sandwiched with the 18 mm coverslip and sealed with Clear Weld quick setting epoxy (by J-B Weld) before imaging. For fixation of the VLPs, freshly prepared disuccinimidyl suberate (DSS)  was added to the VLPs to a final concentration of 5 mM and incubated at room temperature for 30 min. Then, a quench solution (1 M Tris (pH 7.5)) was added to a final concentration of 25 mM to quench the reaction and incubated at room temperature for 1 h before the fixed VLPs were immobilized on the 25 mm coverslip with the same protocol outlined above.The VLPs were visualized with iPALM on a prototype setup by Thermo Fisher Scientific. The sandwiched sample was secured onto a micro-positioning stage and illuminated by a 315 mW 561 nm laser. Two 60× Apo TIRF objectives  focused on the sample from top and bottom. The custom three-way beam splitter was adjusted to get the interference and maintain a 120° phase difference between the 3 cameras . The system was calibrated focusing the gold fiducials that are embedded in the 25 mm coverslip so as to get a resolution below 10 nm in both optical and axial directions. The sample was illuminated using a 561 nm laser in a pseudo TIRF condition so that the entire VLP was illuminated uniformly. The Dendra2 molecules, used as a tag for the HIV Gag, were activated by a 405 nm laser. To ensure uniform activation and avoid multiple activations at the same time point the exposure of the 405 nm laser was increased progressively.The HIV Gag was tagged with Dendra2 in between MA and CA domain. The VLPs were harvested from 293 T cells as per the protocol described in materials and methods. During the imaging of the HIV GagDendra2 lattice using iPALM  and Gag VLP particles were applied to a thin (2 nm) carbon surface supported by thicker holey carbon (2 µm holes 1 µm apart) and a copper mesh grid . The specimen was blotted to make thin and plunged into liquid ethane. A Vitrobot (ThermoFisher) was used to prepare these cryogenic specimens. Next, cryo-specimens were imaged in a Titan Krios transmission electron microscope (ThermoFisher) equipped with a Gatan BioQuantum K3 energy filter and direct electron detector. Tilt series from −60° to +60° were recorded at 3° steps over holes in the carbon film via SerialEM software . The micThree-dimensional reconstructions were computed from the −7 µm defocus tilt series by means of the IMOD software package . Images Central sections of cryotomography reconstructions were analyzed using circular Hough transform to fit the exterior circumference of each particle’s image. By nature of Hough transform, such fits favored regions of higher ordering of the Gag lattice . Radial iPALM microscopy combines the stochastic activation and localization of single fluorescent proteins with an interferometric detection path which allows localization of single molecules with 8 nm resolution in all three dimensions . Given tOne simple way to improve our resolution of localizing Gag molecules within the VLP, was to move the Dendra2 molecules further towards the membrane, allowing localization of molecules on a sphere with a larger radial distance. In this study, therefore, we designed a Gag construct by inserting Dendra2 in between the MA and CA domains of Gag, Gag(MA-Dendra2-CA), utilizing similar design as the iGFP HIV backbones  as detaiPurified Gag(MA-Dendra2-CA) VLPs were immobilized on glass coverslips and imaged in iPALM as previously described in  and in m the VLP .To verify the dynamics observed with iPALM, we have imaged Gag(MA-Dendra2-CA) in the presence of a membrane permeable fixative (DSS) as described in methods. As evident from this section, iPALM analysis is more complex than simple imaging approaches and heavily dependent on statistics for proper analysis. To have enough localizations to decipher a patch of Gag molecules and maintain temporal distance between localizations required for individual single molecule localizations iPALM experiments take ~90 min and can generate approximately two frames capturing the statistical localization of Dendra2 molecules within the cavity. While this approach is sufficient for a statistical estimate of presence of dynamics within the Gag(MA-Dendra2-CA) lattice, it cannot quantify the rate of Gag(MA-Dendra2-CA) dynamics within the VLP.If Gag molecules are undergoing significant dynamics within the Gag VLPs, as is suggested by our data presented in We have previously developed a biochemical kinetic assay to measure dynamics within the Gag(VLPs) through measurements of hetero-dimerization of a small fraction of Gag-SNAP and Gag-Halo molecules added to otherwise untagged Gag within Gag VLPs . SNAP anAs intuitively expected, increasing the percentage of Gag-SNAP and Gag-Halo within the VLP results in faster kinetics for hetero-dimer formation. The biochemical analysis has the advantage of providing multiple measurements in the 0–200-min span. Since the western blots are performed on a pool of VLPs simultaneously; the results are reproducible and show kinetics of the hetero-dimer formation from 0–200 min. While, from observations of the raw data presented in In principle, Gag dynamics within the VLPs can be analyzed using molecular dynamics simulations which can take into account all known interactions of Gag with both membrane and other Gag molecules. However, such a detailed model is computationally expensive and therefore cannot be used easily to model the experimental biochemical data presented in m parameter for each condition. Using these fits, we report pseudo diffusion coefficients of ~10 nm2/s for WT Gag, ~4 nm2/s for E2Q and ~2 nm2/s for E2A mutations. Specifically, the E2A mutant is expected to increase the probability of helix formation within the SP1 domain, which should in principle increase the avidity of Gag within the Gag lattice inside VLPs.Having established a Monte Carlo model to analyze the biochemical kinetic hetero-dimerization curves, we performed the hetero-dimerization experiments at three different concentrations of Gag-SNAP and Gag-Halo incorporation  in VLPs created by expressing WT Gag in If Gag molecules are indeed moving within the interior of the VLP, given the strong MA domain membrane interactions that recruit Gag to bud from the plasma membrane of the infected cells, one would expect that all Gag molecules within the VLP should be anchored within the membrane through their MA interactions. This is supported by the measured radial distribution of Dendra2 molecules as presented in The effects of melittin were first quantified on the dynamics of Gag(MA-Dendra-CA) within Gag(MA-Dendra-CA) VLPs. As data presented in previous sections demonstrates, there is clear evidence of Gag dynamics within Gag VLPs. The rate of rotocols . To testrotocols  to creatWe have further calculated the radial density function for HIV(D25N) virions and compared them to Gag VLPs as shown in Here we show significant evidence that Gag molecules assembled into VLPs incorporating similar numbers of Gag molecules to HIV virions, fail to create a stable Gag lattice. Given that significant Gag-Gag interactions have been previously identified, we did not expect the observed results. Here we will first discuss all experimental conditions followed by possible ramifications of our observed results.Since Gag-Gag interactions are essential for formation of the immature lattice, we will first discuss all experimental conditions relevant to formation of Gag VLPs. The Gag VLPs used in this study have been highly purified in equilibrium sucrose gradients and float at the same density as immature HIV virions. The counting of Dendra2 molecules within each VLP is methodical and precise . It takeWe have used Monte-Carlo simulations to obtain pseudo-diffusion of Gag molecules within isolated VLPs. A full model of Gag dynamics requires parameters describing all of the Gag interactions within the immature lattice (“on” and “off” rates) and the varied mechanisms of diffusion of Gag. Such a model would have a large number of free parameters and would not be properly constrained with the biochemical data presented in this manuscript. We have therefore introduced the pseudo-diffusion of Gag which is a combination of all “on” and “off” binding rates as well as purely diffusive behavior. The pseudo-diffusion only contains a general description of dynamics within the VLPs. To dissect all the parameters involved in Gag dynamics, additional novel methods for detecting interactions within the VLPs have to be developed.Incorporation of IP6 has been shown to play a role in the formation of immature HIV lattice . Both thWhile none of the HIV accessory proteins, which include Nef, Vpr, Vif, and GagPol, are expected to have any structural role in the assembly of the immature lattice, they are technically absent during the assembly of Gag VLPs; therefore, one cannot rule out however unlikely at least an indirect role for these proteins in the structural stability of the immature lattice. Incorporation of the ENV protein within the envelope of the immature HIV virions, was shown to affect the overall stiff ness of the immature virions as measured by atomic force microscopy  The incrThe main difference between the HIV virions and Gag VLPs, however, is the incorporation of the gRNA in the immature Gag lattice of the HIV virions. While the full picture of the gRNA interaction with Gag is very complex, it has been shown using crosslinking-immunoprecipitation (CLIP) sequencing that gRNA interacts with Gag along all its length within the immature HIV virions  and Gag Previous comparisons between HIV Gag and other retroviral Gag VLPs have been carried out using both negative staining as well as cryotomography ,47. The A lattice of Gag molecules lining the inner leaflet of the membrane has been identified in all retroviral genera including alpharetroviruses, betaretroviruses, deltaretroviruses, epsilonretroviruses, gammaretroviruses and lentiviruses (which include HIV) ,46,48. R


This article summarizes synchrotron wide-angle x-ray diffraction (WAXD) patterns, polarized attenuated Fourier transform infrared spectroscopy (ATR-FTIR) data and differential scanning calorimetry (DSC) data of differently stress-annealed poly(3-hydroxybutyrate) (P3HB) fibers. Additionally, in-situ polarized ATR-FTIR data has been measured under tensile drawing of pre-annealed P3HB fibers under low annealing stress. Modifications to the ATR-FTIR setup and sample holders for performing measurements on P3HB fibers are explained in the experimental section. For more information see ‘Reversible mesophase in stress-annealed poly(3-hydroxybutyrate) fibers: A synchrotron x-ray and polarized ATR-FTIR study'  Fiber (II)s-polarized ATR-FTIR spectra for low-stress annealed (1.6 MPa at 115°C) fiber (II) measured during the first tensile drawing cycle.1.3DSC was performed only on a selected number of fibers, since not enough material remained for several annealing conditions after the WAXD and ATR-FTIR studies. Note that all the raw data files are given as (.txt) files in the Mendeley repository. The measured DSC heating curves of P3HB fiber (I) always showed two melting peaks, a small one at about 55°C, which corresponds to the melting of PCL (plasticizer in fiber (I)) The calculated crystallinities from DSC are plotted vs. the annealing stress in 2The materials and the description of experimental methods to measure the structure of stress-annealed P3HB fibers have already been partially described in the article by Perret et al. 2.1"Two P3HB fibers were melt-spun from modified P3HB (density 1.2 g/cm3), provided by Biomer , on a customized pilot melt-spinning plant originally built by Fourné Polymertechnik . Fiber no. 974  was melt-spun from a pelletized P3HB compound (Mw=0.5 MDa), whereas raw P3HB powder (Mw=1.6 MDa), mixed with 20 wt.% plasticizer (tri-nbutyl citrate (TBC)), was used to melt-spin fiber no. 1108. The P3HB pellets, used for fiber 974, contained a nucleating agent (boron nitride (BN)), 20 wt% TBC and various other processing aids, including low molecular weight poly-ε-caprolactone (PCL). The fiber diameters are about 90 μm for both fibers. Fiber 974 was melt-spun with a draw ratio (DR) of 7.0, and fiber 1108 with DR = 6.0. More detailed information about materials, properties and melt-spinning parameters can be found in our previous publications 2.2“The stress-annealing of aged (eight years at 23°C) P3HB fibers was carried out by attaching different weights  to the fibers, corresponding to applied stresses of 1.6, 8.0, 16.0, 25.6, 32.0 and 40.0 MPa. The annealing was performed in a furnace with hot air circulation at different temperatures  for 60 minutes. Above 100 and 115°C, respectively, fibers (I) and (II) were only annealed at low stress (1.6 MPa). Applying higher stresses at elevated temperatures resulted in fiber breakage within a few minutes. The length of each monofilament was measured at room temperature, with an applied weight of 1g (pre-stress of 1.6 MPa), before and after annealing, in order to determine the change in filament diameter and thus the linear mass density.” For further details we refer the reader to the article by Perret et al. 2.3“In-situ WAXD measurements were performed at the cSAXS beamline at the Swiss Light Source synchrotron of the Paul Scherrer Institute in Switzerland. Differently annealed monofilaments have been mounted on a sample holder  and WAXD patterns were measured at the center of the fibers with 5 second exposures using a Pilatus 2M detector 2.4“Polarized ATR-FTIR spectra have been recorded from differently stress-annealed P3HB fibers with a Bruker Tensor 27 FTIR spectrometer , using a single reflection attenuated total reflectance (GladiATR™) accessory from Pike Technologies . The FTIR spectrometer uses a mid-infrared (MIR) Globar source and a narrow-band mercury cadmium telluride (MCT) detector. The ATR accessory is equipped with a monolithic diamond ATR crystal but does not have the option to add polarizers.” The setup modifications and fiber holders are described in the following sections. For further details we refer the reader to the article by Perret et al. 2.4.1TM from Pike Technologies  was modified in order to be able to make polarized measurements. The added polarizer slot is shown in The GladiATR2.4.22.4.32.5The thermal properties and average crystallinity values of the P3HB fibers were determined using DSC. All measurements were carried out in nitrogen atmosphere (40 mL/min) using the instrument DSC 214 Polyma . The fibers were chopped into small pieces (∼2 mm long), and about 5-10 mg of cut fibers were put into small crucibles. The crucibles were then heated from 25°C to 200°C and cooled from 200°C to 25°C at a ramping rate of 10°C/min. The data analysis was carried out using the NETZSCH Proteus thermal Analysis software .The melting peaks of P3HB have been integrated in order to calculate the P3HB crystallinities from the melting enthalpies, The authors followed generally expected standards of ethical behavior in scientific publishing throughout article construction.E. Perret: Software, Formal analysis, Investigation, Data curation, Writing original draft,Visualization. K. Sharma: Data curation. S. Tritsch: Data curation. R. Hufenus: Supervision,Project administration, Writing review & editing.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Pirfenidone is an orally bioavailable drug with antifibrotic and anti-inflammatory properties already approved for the treatment of idiopathic pulmonary fibrosis. Pirfenidone has been recently tested in animal models of myocardial fibrosis with promising results. Here we will review the rationale underlying the potential therapeutic effect of Pirfenidone in HFpEF.Heart failure with preserved ejection fraction (HFpEF) is a major public health problem with growing prevalence and poor outcomes, mainly due to the lack of an effective treatment. HFpEF pathophysiology is heterogeneous and complex. Recently a “new paradigm” has been proposed, suggesting that cardiovascular and non-cardiovascular coexisting comorbidities lead to a systemic inflammatory state, perturbing the physiology of the endothelium and the perivascular environment and engaging molecular pathways that ultimately converge to myocardial fibrosis. If inflammation and fibrosis are the “ Myocardial fibrosis precedes the clinical diagnosis of HFpEF and is strongly associated with disease severity and adverse outcomes (Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome characterized by typical symptoms and signs of heart failure (HF) with normal or near-normal left ventricular ejection fraction LV EF ≥ 50%), echocardiographic features of diastolic dysfunction and/or structural heart disease and elevation of natriuretic peptides . The pre0%, echocoutcomes .Based on these evidences, a mechanistic overlap between HFpEF and other fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) seems likely . PirfeniHFpEF is a systemic syndrome, driven by accumulated risk factors and comorbidities, which, in vulnerable subjects, trigger pathways leading to increased ventricular stiffness, diastolic dysfunction and abnormal ventricular-arterial coupling , 9. The The classic risk factors for developing HFpEF include age, female gender, hypertension, diabetes, overweight/obesity, renal dysfunction, metabolic syndrome (present in the 85% of patients) and physical inactivity , 13. AccIn acute HFpEF, changes in levels of inflammatory markers such as pentraxin-3, TNF- receptor1a, myeloperoxidase, and lymphotoxin β receptor are correlated to outcome . The “neThe role of CMD at the basis of HFpEF pathogenesis and evolution has gained growing consent over years . CMD is both diastolic dysfunction and impaired CFR demonstrated a > 5-fold increased risk of HFpEF hospitalization, providing evidence that CMD, alongside myocardial stiffness, may play an important role in the pathophysiology of HFpEF.In the multi-national PRevalence Of MIcrovascular dySfunction in Heart Failure with Preserved Ejection Fraction (PROMIS-HFpEF) study , CMD hasMyocardial fibrosis is an endogenous response to different cardiac insults that may become maladaptive over time and contribute to the onset and progression of HF. ECM expansion, secondary to excess collagen accumulation, is considered a key pathophysiological mechanism of HFpEF, a common pathway that exists regardless of aetiology. There is strong evidence demonstrating both the primary aetiological role of myocardial fibrosis in HFpEF, but also the adverse impact that ECM expansion has on myocardial mechanical, electrical and microvascular function, confirming that the myocardial fibrotic burden is strongly and independently associated with adverse outcome –33. HoweInterestingly, myocardial and pulmonary fibrosis share some characteristics and molecular mediators, with TGF-β and Ang II being the major regulating factors. The activation of the AT1 receptor in fibroblasts by Ang II leads to the secretion of TGF-β, which stimulates fibroblast proliferation and ECM synthesis  in an autocrine manner and induces cardiomyocytes hypertrophy in a paracrine manner . TGF-β tTo date, unlike HFrEF, HFpEF is still orphan of treatments proven to significantly reduce major CV events. Beta-blockers, angiotensin-converting enzyme inhibitors, Ang-II receptor blockers, mineralocorticoid receptor antagonists and angiotensin receptor-neprilysin inhibitors have all failed to reach the pre-specified primary endpoints in trials testing their effects on CV outcomes  49–71),–71,49–71Over the last years, the new insights on HFpEF pathophysiology have increased the interest in testing new drugs specifically targeting the molecular mediators involved in HFpEF. Given the role of the oxidative stress and NO metabolism perturbation, it seemed reasonable to propose organic NO donors as potentially useful therapeutic tools. Unfortunately, results from the early studies are at best inconclusive and in some patients, paradoxically, a tendency to reduce the total physical activity was observed , 63. HowDrugs specifically targeting inflammation have been tested in HFpEF: in animal models chemokine antagonists  and immuno-modulatory cytokines  showed promising results . Anakinrin vivo and direct effects on dapagliflozin on the activity of this exchanger in endothelial cells demonstrated in vitro (The sodium–glucose cotransporter 2 (SGLT2) inhibitors  were found to reduce hospitalizations as well as mortality in HF , 74, butin vitro . These dDue to the key pathophysiological role of myocardial fibrosis in the development and progression of HFpEF, there is a growing interest about the potential beneficial effects of anti-fibrotic drugs, commonly used in other fibrotic disease such as IPF, also in HFpEF such as Pirfenidone.Pirfenidone is an orally bioavailable small synthetic molecule, with proven anti-inflammatory and anti-fibrotic properties and it is authorized by the European Commission for the treatment of adults with IPF .Pirfenidone is rapidly absorbed in the gastrointestinal tract and its half-life is about 3 h , 79. It The precise mechanism of action of this drug remains still unclear . PirfeniThe available data on cardio-protective effects of Pirfenidone are still in a preclinical phase, but there are several evidences pointing to that direction .cardiac fibroblasts migratory ability, inhibits their proliferation and the process of myofibroblast differentiation (by inhibition of α-SMA expression) as well as the myocardial fibroblast synthesis and secretion of TGF-β1. Pirfenidone also regularizes ratios of myocardial MMPs and tissue inhibitors of metalloproteinases, enhancing myocardial renin-angiotensin system imbalance and cardiac fibroblast synthesis and secretion of IL-10, an anti-fibrotic cytokine  trial . This isIn humans, only two retrospective studies have investigated the effect of Pirfenidone on LV structure and function in patients with IPF treated with this drug.In the first one, Alansari et al.  hypothesIf cardiac fibrosis and microvascular inflammation are the common pathophysiological substrates in this conundrum, Pirfenidone could have a powerful role in the treatment of all the different HFpEF subtypes, targeting the “core mechanisms” they all share. However, differences in timing of fibrogenesis and in fibrotic burden in each HFpEF-related comorbidity are still poorly understood, being influenced by many co-factors. Nowadays circulating biomarkers are considered a powerful tool to depict the patients pro-inflammatory and pro-fibrotic profile. Moreover, CMR offers the unique possibility to non-invasively estimate the amount of myocardial fibrosis. We can speculate that a “risk stratification” strategy, using inflammation and fibrogenesis biomarkers and CMR, might identify patients who will benefit most from Pirfenidone: those showing an intense inflammatory and fibrogenetic activation before the evolution toward an advanced, irreversible, stage of diffuse myocardial fibrosis. Trials specifically testing Pirfenidone effects on patients with this profile might help to optimize treatment in HFpEF.fil rouge in the HFpEF conundrum, thus they can be considered reasonable targets treatment. Pirfenidone is a well-established drug for the treatment of IPF and in animal studies it showed its anti-inflammatory properties and its ability to reverse cardiac fibrosis. Taken together these data suggest that Pirfenidone could have a role in the treatment of HFpEF by targeting inflammation and myocardial fibrosis, however, at present, clinical trials are lacking. Large, specifically-designed studies with hard end-point in this setting are needed.Up to now, HFpEF treatment has been borrowed from the HFrEF experience, with poor results. The complex and not completely understood HFpEF pathophysiology is probably the key to develop a tailored effective treatment. The new HFpEF paradigm states that the coronary microvascular endothelial inflammation is the main driving factor, activating complex molecular pathways that eventually converge to myocardial fibrosis. Coronary microvascular inflammation and myocardial fibrosis can be considered the FG and RL wrote sections of the manuscript. FC contributed to manuscript revision. All authors read and approved the submitted version.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.