During an animal’s life span, terminally differentiated cells must be resilient to fluctuating environmental and physiological stresses to assure proper function of a tissue and prevent disease. A key component of this resiliency is the maintenance of protein homeostasis. Our study reveals that the AMPylating enzyme filamentation induced by cyclic-AMP (Fic) is required in differentiated tissue for the regulation of the unfolded protein response (UPR) during both physiological and pharmacological stresses. We also propose that cells with high regenerative capacity may not require this level of regulation, as new cells will bypass the need for long-term survival. We predict that Fic is important in mitigating deleterious effects of UPR activation in a variety of tissues with UPR-associated diseases and thus holds promise as a new therapeutic target. fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, −/−Fic mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both −/−fic flies and −/−Fic mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of  Protein homeostasis is regulated by proper synthesis, folding, modification, and degradation of proteins and is vital to cellular health. In the endoplasmic reticulum (ER), when the load of unfolded proteins is excessive, the unfolded protein response (UPR) is activated, triggering signaling pathways that result in changes to protein synthesis, modification, and degradation until the load of unfolded proteins is resolved. If the burden of unfolded proteins is prolonged and/or remains high, proapoptotic pathways can be activated. The activation of the UPR is, in part, regulated by the Hsp70 protein chaperone binding immunoglobulin protein (BiP), a protein that binds and helps fold proteins as they pass through the ER checkpoint and into the secretory pathway. Depending on the level of unfolded protein, complex signaling networks are activated and respond in accordance to the severity. Mild to moderate levels of UPR signaling are prominent with cell recovery and cell survival, whereas strong and prolonged UPR signaling leads to apoptosis , 2. Thesadaptive phase and the maladaptive phase  called AMPylation . AMPylatDrosophila as an animal model, we found that Fic is responsible for reversible AMPylation of BiP , the gross morphology of the fly eyes appeared normal, albeit they exhibited mild vision defects. When acute physiological ER stress was induced in fly eyes by exposure to continuous light, photoreceptors in wild-type flies, but not in −/−fic mutants, could adapt , elevated IRE1 activity, and reduced neurotransmission , in which two LoxP sites were integrated upstream of and within exon 3 . Expression of Cre leads to Cre-Lox recombination that results in a nonfunctional Fic gene (Fic−) that is deleted for both the tetratricopeptide repeat (TPR) and Fic domains that are required for the targeting and AMPylation of BiP, respectively. The remaining truncated gene encodes only a small N-terminal peptide with the transmembrane sequence . fl/-Fic mice were intercrossed to obtain sibling cohorts of fl/flFic and −/−Fic mice that were subsequently used in this study. −/−Fic mice were indistinguishable compared to fl/flFic and fl/-Fic littermates in viability, appearance, and weight .To determine the role of Fic-mediated AMPylation in the mammalian system, we chose to generate a conditional knockout line of n exon 3 A. In addsequence A. Ficfl nic mice  to generFic transcript corresponding to exon 3 of Fic, validating the Fic knockout . As an alternative, we used immunofluorescence in various tissues known to express Fic and were able to detect a signal for Fic in coronal brain sections, strikingly in vimentin-positive cells lining in the third ventricle of the hypothalamus . In corresponding sections from −/−Fic mice, we were unable to detect a signal for Fic protein, supporting the genetic and messenger RNA expression data that Fic is not expressed in −/−Fic mice.Using qPCR analysis of liver complementary DNA, we confirmed loss of knockout B. Levelsfl/flFic and −/−Fic littermates were analyzed for the presence of AMPylated BiP. Both Western blot analysis  indicate that BiP is no longer AMPylated in −/−Fic mice.We next analyzed tissue for the presence or absence of Fic-mediated BiP AMPylation. Whole-cell lysates of livers from analysis D and mas−/−Fic mice would have dysfunction in tissues that rely heavily on UPR to maintain proteostasis. Although many tissues are known to require this regulation, we decided to focus our initial study on Fic in the pancreas, a tissue that is well documented to rely on the UPR for proper exocrine and endocrine function  with unrestricted access to water before sacrifice. Compared to fl/flFic and fl/−Fic controls, fic null mice have normal weights  and appear generally healthy. However, fasted serum levels of amylase were found to be significantly elevated in our −/−Fic cohorts . Of note, serum lipase levels and fasting glucose levels were not affected in our fl/flFic and −/−Fic mice. Since high amylase has been linked to hepatic disfunction . Histopathological analysis of both the pancreas and liver revealed no detectable defect in either tissue .We predicted that function , 25. Usifunction , we look−/−Fic mice, we predicted that −/−Fic mice would show changes in UPR activation in the exocrine pancreatic tissue. To determine if this was the case, we utilized a mild physiological stress of fasting-feeding to activate the UPR in the exocrine pancreas. Fasting-feeding is a well-established method used to activate mild UPR in the pancreas through the stimulation of pancreatic digestive enzyme synthesis  confirmed that BiP AMPylation is present during fasted conditions and lost during fasted-fed conditions. Of note, AMPylation of BiP returns in the fasted-fed-recovery group, albeit reduced . Western blot analysis performed on Phos-tag gels indicated that activation of IRE1 was observed to be stronger in fasted-fed −/−Fic mice compared to fl/flFic controls .Compared to −/− mice B and C.  reduced D. WesterSI Appendix, Fig. S10). In fl/flFic mice, levels of Fic were found to increase during fasted-fed conditions and then diminish during fasted-fed-recovery . Notably, in −/−Fic mice, many UPR-induced transcripts were elevated compared to fl/flFic controls. Levels of BiP, Atf4, Xbp1s, and Chop were all found to be significantly increased in −/−Fic mice during fasted-fed conditions . As previously reported, UPR-induced transcripts are elevated upon caerulein-induced injury and resolve over the course of injury recovery . However, at 4 h after the first injection, Ask1 and Egr1 levels were comparable in wild-type and −/−Fic mice, and Fgf21 transcript levels were significantly lower than in wild type. Similarly, at 4 h after the first injection, levels of Atf3, Atf4, and Hmox1 transcripts were elevated in −/−Fic mice compared to fl/flFic . By 8 h after the first injection, transcript levels of Atf3 and Atf4 were comparable between fl/flFic and −/−Fic mice, whereas Hmox1 transcript levels were significantly lower in −/−Fic mice. BiP transcript levels also dropped significantly in −/−Fic mice at 8 h . Expression patterns of Chop were also significantly altered, with reduced transcript levels for −/−Fic both at 4 and 8 h after the first caerulein injection . Levels of Xbp1s appeared unaffected in −/−Fic mice . Of note, the changes in Fic transcript levels in caerulein-treated mice mirrored the changes seen in Xbp1s transcript levels . With the exception of Chop and Xbp1s, UPR transcripts appeared to peak and diminish in the pancreas of caerulein-treated −/−Fic mice early compared to fl/flFic.One hour after the first injection of caerulein, levels of the UPR-induced transcripts Drosophila as a model, we found that −/−fic flies were maladaptive to light stress. In this system, 3 d of constant light caused damage to ommatidial structures found in the −/−fic compound eye and enhanced UPR activation  for 3 d, followed by 3 d of recovery (LD) on the same cohort of flies and scored for presence or lack of intact deep pseudopupils (DPPs), a visual indicator for disruption of ommatidial structure A. As pre−/−Fic mice, we hypothesized that −/−Fic mice might also exhibit more severe and prolonged histopathological changes in the pancreas upon caerulein-induced pancreatitis. The severity of pancreatitis in each saline- and caerulein-treated mouse was scored by severity of pancreatic edema, inflammatory infiltrate, and necrosis, as previously described , revealing no detectable difference in pancreatitis severity tissue after one round of acute caerluein injury.Based on the changes in UPR response kinetics and increased serum amylase and lipase levels in caerulein-treated escribed . Based ofl/flFic and −/−Fic mice recovered from caerulein-induced pancreatitis. A cohort of 6- to 8-week-old fl/flFic and −/−Fic male mice was induced for pancreatitis with seven hourly IP injections of saline or caerulein and left to recover for 7 d. Mice were then split into two groups: a fasted and fasted-fed group as previously described .The UPR is crucial for the maintenance of protein homeostasis during physiological stress of cells with high secretory capacity. When cellular stress levels reach maladaptive levels, recovery from stress becomes challenging due to prolonged attenuated protein synthesis of nonstress-responsive genes and activation of apoptotic machinery. Thus, regulation of the UPR must be tightly coordinated to meet the needs of the tissue. To understand the role of reversible AMPylation of BiP in protein homeostasis in a mammalian system, we generated a conditional knockout of Fic in mice. We speculated that tissues reliant on secretion might be most affected by the deletion of Fic and therefore focused our initial efforts on the pancreas. Using both a fast-feeding and a caerulein-induced pancreatic injury model, we observed changes to UPR signaling and physiology of the pancreas suggestive of exocrine pancreas disfunction in Drosophila eyes and mouse pancreas, respectively, indicates that loss of Fic reduces recovery from ER stress-associated tissue damage in both animal models . Repetitive stress in −/−Fic tissues would result in amplified UPR, leading to progression into the maladaptive phase of the UPR and tissue damage over the lifetime of the tissue. Our data point to evidence of this in the exocrine pancreas, where elevated UPR signaling and serum amylase indicate functional disruption. As elevated serum amylase is one of the first key clinical indicators of pancreas dysfunction, we suspect additional and continued physiological stresses to −/−Fic tissue will lead to increased prevalence of disease.Cells without Fic regulation of BiP lack this pool of chaperone on standby, resulting in prolonged and elevated UPR signaling, as a delayed response requires more chaperone via transcription and translation to accommodate the increased physiological stress. This is supported by our qPCR analysis of UPR-responsive genes in −/−fic eyes are challenged over time and lose the potential to regenerate rhabdomere integrity. Analogously, we observe more scarring in the injured −/−Fic pancreas.The pancreas primarily comprises terminally differentiated professional secretory cells with limited regenerative capability. Therefore, the pancreas must employ mechanisms to ensure resilience to repetitive stress in order to last and properly function for the lifetime of the animal. Herein, we provide evidence that Fic provides one such mechanism through the moderation of the UPR during physiological stress. Similarly, a wild-type fly eye has the capacity to recover from the physiological stress of continuous light. In the absence of the Fic rheostat, the Many studies to date have used tissue culture cell lines as a model to study the UPR in which a chemical stress is applied to cells resulting in a very strong, and frequently irreversible, induction of the UPR . Under t−/−Fic mouse. We propose the presence of Fic rheostat allows for tempering of the UPR response by maintaining a window for reversible UPR response that is critical for maintenance of protein homeostasis. The importance of this window has been highlighted with the treatment of UPR stress with the pharmacological agent ISRIB (integrated stress response inhibitor) where it is only observed to be efficacious during the adaptive phase of UPR (−/−Fic mice will be useful for understanding the importance of the Fic-mediated rheostat and treatment of disease.Whereas this study focuses on this one tissue only, we speculate that other tissues with professional secretory, terminally differentiated cells that must adapt to fluctuating stress will be similarly affected in the e of UPR . Future For the health of an animal, it is critical to maintain resilience in terminally differentiated cells during repeated physiological stress to prevent disease. We predict that Fic regulation of the UPR will play a role in mitigating the deleterious effects of UPR activation in a variety of tissues with UPR-associated diseases, including retinal degeneration, atherosclerosis, metabolic syndrome, and various neurodegenerative disorders. Our future studies will focus on the identification of tissues in which Fic plays a role in the regulation of the UPR and the physiological consequences of the absence of Fic-mediated regulation of the UPR.SI Appendix, SI Materials and Methods. These include mouse protocols, generation of conditional Fic knockout, histology, quantitative real-time PCR, immunofluorescence of brain sections, mass spectrometry analysis, Western blot analysis, fly stocks and rearing conditions, and deep pseudopupil analysis.Detailed descriptions of the experimental methods are provided in Supplementary File


However, thyroid cancer clinical practice recommendations already encouraged physicians to reduce “low-value” care: in particular, to avoid screening of general population, to reduce the number of unnecessary biopsies, and to adopt a conservative approach to indeterminate thyroid nodules and low-risk thyroid cancers [The outbreak of COVID-19 pandemic has acted as a significant stress test for healthcare systems worldwide, due to the need for hospitalization of an increasing number of infected patients. The shift of massive resources to the acute needs of the pandemic led to an upheaval of the usual diagnostic and therapeutic pathways of chronic diseases, including thyroid cancer disease. The In 2020, the European Society for Medical Oncology published an Interdisciplinary Expert Consensus on the management of cancer patients. The experts recommended that cancer care intensity be adapted to the pandemic scenario, to local R0 index and to health facilities and resources [This was soon after followed by specific guidance documents on single cancer types, palliative, and supportive care. As for thyroid cancer treatment prioritization, newly diagnosed patients with clinical T4, bulky lymph node involvement, or aggressive histotypes  were considered high priority for primary surgery; clinical T2 and T3 with no extensive lymph node involvement as medium priority; and clinical T1 or indeterminate cytology nodules were classified as low priority [When treatment for low-risk, early-stage cancer is likely to be postponed or avoided altogether, interest in cancer screening programs is expected to diminish. Using the Google Trends tool, Cohen et al. found that there was a significantly reduced public interest in six malignancies used as a benchmark (including thyroid cancer) at the beginning of the COVID-19 pandemic. However, online search volumes returned to pre-pandemic levels in the second half of 2020 . An updaWhile this may be worrying for some cancer types, it is not for thyroid cancer. According to the World Health Organization, cancer screenings are justified if the target cancers are frequent; a high proportion of patients is diagnosed in advanced stages; early detection methods are cost-effective and easily accessible for individuals at risk; diagnosis, treatment, follow-up, and quality assurance procedures can be implemented; and the benefits of early detection outweigh the risks (such as complications and negative effects). According to the US Preventive Services Task Force (USPSTF), this is not the case for asymptomatic adults with differentiated thyroid cancer. USPSTF recommends against screening since the potential risks outweigh any potential benefits . FurtherIn summary, the decreased interest in thyroid cancer screening is likely to reduce overdiagnosis and overtreatment of indolent thyroid cancer.The COVID-19 pandemic impact on cytopathology practice was investigated both at national and international levels. An international, multicenter study found a persistent reduction in the cytological specimen volume both during the lockdown , definedThe thyroid case is, however, peculiar. In fact, scientific societies have released a number of recommendations to better stratify the malignancy risk of thyroid nodules, aiming at an overall reduction of unnecessary fine-needle aspiration biopsies . InteresIn this context, surgical activity for thyroid disease was also reduced . During A North American survey showed that many endocrine surgeons (37.7%) were reassigned to other duties during the pandemic; most surgeons reported decreased clinical volume (74.6%) . A multip = 0.040), extrathyroidal extension , and lymph node metastases  [In a Chinese cohort, patients who experienced a COVID-19-related delay in surgical treatment had more aggressive cancer behavior. Patients treated after lockdown were more likely to have multiple lesions  .p = 0.036), according to the ATA risk stratification. In any case, the response to treatment 1 year after the initial treatment was not affected by the delay, which actually led to a better risk-stratification and risk-benefit balance. All patients underwent a risk-stratification triage, which prompted to active surveillance programs in those cases harboring low-risk papillary microcarcinomas, as already suggested by recent evidence and clinical practice guidelines [In our experience , delayedidelines .Even if a delay in treatment of newly discovered thyroid cancers that pose no clinical risks is completely acceptable by physicians who are aware of the recent evidence and recommendations, the patients’ experiences may be negative, as the perceived risk may be overestimated.During the COVID-19 pandemic, thyroid cancer survivors reported increased anxiety compared to a pre-COVID cohort . In our A similar UK survey identified a negative impact of COVID-19 outbreak and lockdowns on psychological wellbeing of thyroid patients (including patients with benign diseases such as primary hypothyroidism and hyperthyroidism). These patients reported low overall satisfaction rates, due to cancellations of appointments and planned treatments .To deliver comprehensive care, clinicians should listen and understand patient concerns and improve their communication about risk-benefit balances of different treatment plans.The COVID-19 pandemic might therefore have started a virtuous process and be an opportunity to reduce the unnecessary diagnostic and therapeutic burden for thyroid cancer patients. However, there is still a way to go toward full implementation and acceptance of recommendations within the wider endocrine and surgical communities to reduce the unnecessary interventions. Furthermore, this requires better physician–patient communication and truly shared decision-making process.


Tumor necrosis factor (TNF) is widely recognized as a pivotal player in both systemic and local inflammatory processes. Due to the critical role this molecule has in driving both chronic and acute inflammation, it was among the earliest therapeutic targets utilized for patients with autoimmune (AI) diseases. While inflammation in the prostate is commonly observed, the organ has not previously been considered a target of systemic inflammation associated with some AI diseases. In patients with benign prostatic hyperplasia (BPH), chronic inflammation is common, and immune cells represent a significant proportion of cells in the organ. Accumulation of inflammatory cells may be a response to an initial insult and/or a factor in driving BPH pathogenesis. Certainly, inflammation can limit the efficacy of existing medical therapies in these patients. We previously showed that a pattern of gene expression in BPH tissues from patients who had progressed to indication-specific surgery was consistent with the changes seen in AI diseases. Recently, we demonstrated that patients with AI disease have an approximately 50% increase in BPH prevalence compared to patients without AI disease. Treatment of AI disease patients, specifically with TNF-antagonists, reduces BPH incidence back to, or in some diseases, below, the baseline population BPH diagnosis rate. Treatment of AI disease patients with the broad spectrum anti-inflammatory methotrexate did not elicit this reduction in diagnoses. Systemic treatment with TNF antagonists reduces epithelial proliferation and macrophage accumulation in the prostate tissues from two mouse models of prostatic hyperplasia as well as human patients. These studies suggest that TNF is a potential therapeutic target in BPH patients. Figure 1).BPH becomes more prevalent in men with increasing age and is associated with prostatic enlargement and/or lower urinary tract symptoms (LUTS). Treatment strategies include either 5α-reductase inhibitors (5ARIs), which limit androgen receptor activity and cause the prostate to shrink due to increased apoptosis, or α-adrenergic receptor antagonists (α-blockers), which reduce smooth muscle tone and improve urine flow. Both treatment strategies can reduce LUTS in patients, thereby treating the clinical manifestation of BPH. Unfortunately, roughly one-third of BPH patients have no improvement in LUTS with therapy and some patients have recurring symptoms after a short-term improvement with treatment. There is a limited understanding of the mechanisms associated with BPH treatment failure, but chronic inflammation in the prostate has been associated with reduced therapeutic success in the Medical Therapy Of Prostatic Symptoms (MTOPS) and the REduction by DUtasteride of prostate Cancer Events (REDUCE) cohorts. In light of our recent studies, targeting inflammatory pathways with agents such as TNF-antagonists may provide an alternative or complementary therapeutic strategy for BPH/LUTS patients  use in BPH/LUTS patients, suggesting a need for a deeper understanding of both the cellular and molecular connection(s) between prostatic inflammation and BPH.The complete mechanism of TNF-mediated promotion of prostatic hyperplasia is unknown. Several driving factors for BPH development have been proposed as men age, including reduced testosterone:estrogen (T:E2) ratio, tissue remodeling, and loss of epithelial barrier integrity. Additional driving factors may lead to BPH and chronic inflammation concurrently, such as a response to damage-associated molecular patterns (DAMPs) or the development of an autoimmune response in the prostate . Anti-inAfter TNF-antagonist treatment in NOD mice, bulk RNA-seq of prostate tissues identified a reduction in inflammatory and anti-apoptotic pathways in treated versus control samples. TNF-antagonist treatment results in diminished inflammatory signaling. This was evident by a reduction in antigen presentation and differentiation of various T cell subsets, determined by KEGG pathway enrichment analysis. Similarly, TNF-antagonist treatment reduces signaling downstream of TNF receptor 2 (TNFR2), with a reduction in anti-apoptotic signals such as Jun1, IKK, and Akt. In a chronic inflammatory environment, activation of these factors may contribute to the enhanced proliferation observed in BPH. Thus, inhibition of these proteins through blockade of upstream factors such as TNF can restrict cell proliferation and overall prostatic growth and/or de-repress apoptosis and allow shrinkage of the gland. Multiple cell types can express TNF receptors, but scRNA-seq of all human prostatic cell types indicates the highest expression of TNFR2 in macrophages. Further investigations will determine if protein expression of TNFR2 matches the expression observed in the scRNA-seq data.in vitro, whereas TNF-antagonists reduce epithelial proliferation in vivo. It is likely that TNF derived from inflammatory cells  functions either indirectly or in combination with other secreted factors to drive epithelial cell proliferation . Further work is necessary to elucidate mechanisms of inflammation-mediated regulation of prostatic hyperplasia.Our recent studies indicate that TNF can directly stimulate stromal, but not epithelial, proliferation An important aspect of patient management is recognizing the potential for co-morbidities in patients, in particular those generally seen by different medical specialists. In this regard, AI disease patients who have an elevated risk of developing BPH should perhaps be screened for urinary symptoms. Patients with psoriasis, rheumatoid arthritis, and type I diabetes are significantly more likely to be diagnosed with BPH after treatment with methotrexate versus no medication. On the other hand, psoriasis patients treated with TNF-antagonists have a significant reduction in BPH diagnosis rate compared to patients without AI disease. Together, these data indicate that specific AI disease therapies could either increase or decrease the likelihood of BPH diagnosis, even within the same disease. Of course, diagnosis of each AI disease may have a preferred therapeutic option that could differ from other AI diseases. The limited number of patients with some AI diseases in our recent studies provides an opportunity for a greater understanding of the connection with these diseases and BPH in larger patient cohorts. An additional evaluation of whether patients without AI disease but with other systemic inflammatory processes like obesity or type 2 diabetes could benefit from anti-inflammatory treatments requires further study. Our recent evaluation was conducted in a patient population that was primarily Caucasian, with only 4% African American representation. Considering possible health disparities across patient race/ethnicity, further work is needed to address potential differences.It is not clear whether prostatic inflammation directly influences LUTS in BPH patients. Although many patients with very large prostates have moderate-severe LUTS, prostate size and symptoms do not directly correlate. Our recent studies did not include an evaluation of LUTS in animal models treated with or without TNF-antagonists (etanercept), so further studies are needed to evaluate if voiding function is improved after anti-inflammatory therapies. LUTS questionnaires developed by the American Urological Association (AUA) or the Lower Urinary Tract Dysfunction Research Network (LURN) provide beneficial clinical information, but are not able to provide insight into cellular changes within lower urinary tract tissues. Knowing if a BPH patient has chronic inflammation in the prostate could guide therapy. Identifying BPH patients who may benefit from anti-inflammatory therapies needs to be improved, perhaps through the use of imaging modalities or secreted biomarkers.Figure 2). There is plenty of reason for optimism due to the advances in technology that allow for a cellular description of the disease within each patient, so in conjunction with expanding BPH biorepositories we have many opportunities to gain a greater understanding of this disease. Investigating whether TNF-antagonists have clinical benefit to a broad range of BPH patients is likely the first of many novel therapeutics to treat inflammation-associated BPH.The hypothesis that BPH could be an autoimmune disease has been proposed previously. Our current studies do not specifically address this question, but do show a correlation between BPH and AI disease diagnoses. It is clear that inflammatory signaling plays a role in the hyperplastic response in the prostate, but more studies are required to determine whether inflammatory cell populations are autoreactive to prostate-specific proteins. BPH is both histologically and clinically variable among patients, so even if BPH is determined to have an AI disease phenotype, this may only be relevant to a subpopulation of patients. Indeed, in the medium to long-term, “BPH” is likely to become a term that describes a disease spectrum with variability in the hyperplastic cell type, symptoms, inflammatory milieu, presence of fibrosis, and prostatic enlargement (


Fibrosis is a pathological manifestation of wound healing that replaces dead/damaged tissue with collagen-rich scar tissue to maintain homeostasis, and complications from fibrosis contribute to nearly half of all deaths in the industrialized world. Ageing is closely associated with a progressive decline in organ function, and the prevalence of tissue fibrosis dramatically increases with age. Despite the heavy clinical and economic burden of organ fibrosis as the population ages, to date, there is a paucity of therapeutic strategies that are specifically designed to slow fibrosis. Aryl hydrocarbon receptor (AhR) is an environment-sensing transcription factor that exacerbates aging phenotypes in different tissues that has been brought back into the spotlight again with economic development since AhR could interact with persistent organic pollutants derived from incomplete waste combustion. In addition, gut microbiota dysbiosis plays a pivotal role in the pathogenesis of numerous diseases, and microbiota-associated tryptophan metabolites are dedicated contributors to fibrogenesis by acting as AhR ligands. Therefore, a better understanding of the effects of tryptophan metabolites on fibrosis modulation through AhR may facilitate the exploitation of new therapeutic avenues for patients with organ fibrosis. In this review, we primarily focus on how tryptophan-derived metabolites are involved in renal fibrosis, idiopathic pulmonary fibrosis, hepatic fibrosis and cardiac fibrosis. Moreover, a series of ongoing clinical trials are highlighted. Fibrosis is a pathological extension of the wound healing process with high mortality that is majorly characterized by the formation and deposition of excess fibrous connective tissue resulting in progressive architectural remodeling in nearly all tissues and organs, eventual organ deformation and functional failure , 2. It iAryl hydrocarbon receptor (AhR) is a highly conserved environment-sensing transcription factor that integrates dietary, microbial, environmental, and metabolic cues to regulate complex transcriptional programs in a ligand-specific and cell-type specific manner -9. It isAhR ligands are considerably selective in their activity, and the pharmacokinetic processes of ligands may be very different . Well-knAhR is implicated in various aging-related diseases and tissue fibrosis, as it modulates numerous genes that are involved in many molecular pathways and cellular processes , 26. ForTryptophan is an essential amino acid for humans that is acquired exclusively from dietary intake. Tryptophan and its metabolites have great effects on diverse physiological processes in which they serve as signaling molecules and building blocks of proteins to coordinate body responses to dietary and environmental cues -41. GeneChronic kidney disease (CKD) affects 8-16% of the population worldwide and is recognized as the 16th leading cause of death . Aging lApart from the abovementioned factors, tryptophan metabolism is also highly associated with the development of complications in CKD, and a vicious cycle exists since CKD complications could exacerbate renal fibrosis. Cardiovascular disease is a global health threat, the risk of which is evidently increased in CKD . MountinIPF is a fatal age-associated disease characterized by progressive and irreversible lung scarring, affecting approximately 130,000 people in the US and 3 million people worldwide, the prevalence and incidence of which dramatically increase with age . In addiInflammatory aging also contributes to the development of pulmonary disease in elderly individuals . It is wHepatic disease is the fifth leading cause of premature life lost in the Western world, with irreversible damage caused by liver fibrosis, and ultimately cirrhosis, a primary contributor to mortality . Aging i4 treatment accompanied by the activation of AhR signaling, whereas IDO2 inhibition attenuates hepatic fibrosis, hinting that kynurenine produced by IDO2 exacerbates liver injury by activating AhR [Kynurenic acid exerted a hepatoprotective effect on thioacetamide-induced liver failure partly by mediating AhR, and the use of a diet containing high kynurenic acid can be beneficial to the recovery of liver injury, as kynurenic acid is a constituent of foods and is found in considerable amounts in selected products . Indole-ting AhR . Astonisting AhR , 109 (FiCardiac fibrosis is a hallmark of myocardial remodeling and is deeply implicated in the development of heart failure . Aging iadenomatosis polyposis coli [TGF-β/Smad signaling is widely recognized as the core pathway of fibrosis, and AhR may attenuate lung fibrosis by inducing Smad4 degradation in the proteasome under non-ligand conditions . Wnt/β-csis coli . AhR cousis coli . Serotonsis coli , 120 butsis coli . In addisis coli . Sirtl asis coli , 124, ansis coli  and Sirtsis coli . Moreovesis coli . FurtherWorldwide estimates indicate that approximately 700 million individuals suffer from CKD, which contributes greatly to diminished quality of life and shorter life expectancy . DespiteIn addition, higher plasma marine n-3 fatty acids were inversely associated with renal fibrosis development and independently associated with better survival in 1990 Norwegian renal transplant recipients, suggesting that marine n-3 fatty acid supplementation may assist renal function recovery . HoweverThe development of pirfenidone and nintedanib has been an exciting landmark in the treatment of IPF, and both of them are recommended for clinical use in the current clinical practice guidelines . UnfortuMoreover, mesenchymal stromal cells , pamrevlNon-alcoholic fatty liver disease is the most common liver disease with complex pathophysiology that is characterized by hepatic steatosis and can potentially progress to advanced fibrosis or end-stage liver disease, affecting approximately 24% of the population worldwide . ElafibrHeart failure is a major public health problem with substantial morbidity and mortality, the burden of which has increased to 23 million people worldwide . EmpagliFibrosis is a common sequela of organ injury that results in organ dysfunction and potential death. Although it accounts for a considerable burden of diseases globally, there are no effective therapeutic strategies that successfully mitigate fibrosis. Aging-related tissue fibrosis, including renal fibrosis, IPF, hepatic fibrosis and cardiac fibrosis, has become more acute with the aging of populations. Moreover, COVID-19 is spreading rapidly across the world with substantial morbidity and mortality, which has given rise to a global pandemic of unprecedented proportions in the modern era since it is highly contagious and has an enormous impact on human health -219. As Accumulating evidence indicates that the gut microbiota is an important contributor to the metabolic health of humans, the dysregulation of which has a pathogenic effect on multiple diseases, including tissue fibrosis -228. AhR


Glaucoma is a progressive, irreversible loss of retinal ganglion cells (RGCs) and axons that results in characteristic optic atrophy and corresponding progressive visual field defect. The exact mechanisms underlying glaucomatous neuron loss are not clear. The main risk factor for glaucoma onset and development is high intraocular pressure (IOP), however traditional IOP-lowering therapies are often not sufficient to prevent degeneration of RGCs and the vision loss may progress, indicating the need for complementary neuroprotective therapy. This review summarizes the progress for neuro protection in glaucoma in recent 5 years, including modulation of neuroinflammation, gene and cell therapy, dietary supplementation, and sustained-release system. Glaucoma is a progressive, irreversible loss of retinal ganglion cells (RGCs) and axons that results in a characteristic optic atrophy and a corresponding progressive visual field defect. The most common types of glaucoma are primary open-angle glaucoma and primary angle closure glaucoma (PACG) . Acute PAlthough some scholars believe that it is a neurodegenerative disease , it is nThe main risk factor for glaucoma onset and development is high intraocular pressure (IOP), and the current treatments available target the lowering of IOP . HoweverImmune system dysregulation is increasingly being attributed to the development of a multitude of neurodegenerative diseases . In receAstrocytes perform critical non-cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Astrocyte-derived lipoxins A4 and B4 promote neuroprotection from acute and chronic injury neuroprotective signal . StatinsGene therapy, which uses a viral vectors to deliver genetic material into cells, is a promising approach to directly target pathogenetic molecules . The retCell therapy provides a new therapeutic strategy for glaucoma. Stem cell therapy mainly involves the transplantation of cells to replace the dead and lost RGC. However, it is associated with a number of major challenges besides ethical issue. Regeneration of RGCs requires full synaptic integration of host inner retinal stem cells and the development of long-distance axons, which project to the brain and accurately form effective synaptic connections with corresponding targets to complete signal transmission. Up to now, the replacement of RGCs has not made a breakthrough . SeveralIn animal models of glaucoma, various diet-related treatments were found as non-IOP-related neuroprotective mechanisms. High VitK1 intake  , CoenzySeveral preclinical studies demonstrate that neurotrophins (NTs) prevent RGCs loss . NTs canSunitinib is a protein kinase inhibitor with activity against the neuroprotective targets dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK). It was found to enhance survival of RGCs for neuroprotection. Recently, a hypotonic, thermosensitive gel-forming eye drop  and a suCannabinoids (CBs) was found to target several factors that related with the progression of glaucoma, it promotes neuroprotection, abrogates changes in ECM protein, and normalizes the IOP levels in the eye .In summary, various neuroprotective therapy  can help


The COVID-19 pandemic has highlighted shortfalls in the delivery of vaccine programmes to older migrant groups. Guidelines exist, however, little is known around care pathways and engagement of these older cohorts in routine vaccinations in primary care, including catch-up programmes. We explored the views of primary care professionals around barriers and facilitators to catch-up vaccination in adult migrants  with incomplete or uncertain vaccination status.We did a qualitative interview study with purposive sampling and thematic analysis in UK primary care  with 64 primary care professionals (PCPs): 48 clinical staff including GPs, Practice Nurses and healthcare assistants (HCAs); 16 administrative staff including practice managers and receptionists .Participants highlighted direct and indirect barriers to catch-up vaccines in adult migrants who may have missed vaccines as children, missed boosters, and not be aligned with the UK's vaccine schedule, from both a personal and service-delivery level, with themes including: lack of training and knowledge of guidance around catch-up vaccination among staff; unclear or incomplete vaccine records; and lack of incentivization  and dedicated time and care pathways. Adult migrants were reported as being excluded from many vaccination initiatives, most of which focus exclusively on children. PCPs noted that migrants expressed to them a range of views around vaccines, from positivity to uncertainty, to refusal.Vaccine uptake in adult migrants could be improved through implementing new financial incentives, strengthening care pathways and training, and working directly with local community groups to improve understanding around the benefits of vaccination at all ages.There are direct and indirect barriers to catch-up vaccines in adult migrants who may have missed vaccines as children, missed boosters, and not be aligned with the UK’s vaccine schedule.Primary care teams have a key role to play in implementing WHO’s new Immunization Agenda and to better consider catch-up vaccination to under-immunised groups across the life-course.


The nutritional content of food plants can, to a large extent, determine the physical attributes of herbivorous insects, from growth rates to the need for defenses against predators. In forests, tree species richness may influence these plant-mediated effects through increasing variation in the nutritional quality that herbivorous insects encounter. Seasonal progression can also shape the plant–herbivore relationship, with lowered leaf quality in later seasons. It is expected that specialist herbivores fare better than generalists in poorer nutritional-quality host plants, whereas generalists can benefit from dietary mixing in more variable neighborhoods. However, a clear understanding of how these factors interact to influence the diversity and functionality across multiple traits of herbivorous insect communities is lacking. In this study, we found support for the expectation of higher generalism of caterpillars in high-nutrition content trees, which also promoted higher abundance but lowered caterpillar species richness and smaller and less defended individuals. Increasing tree richness led to higher caterpillar species sharing between tree species, decreased trait variation, and increased caterpillar species richness per tree species. Our findings shed light on how leaf traits and changes in tree richness interact to influence the trait composition of key herbivores through fine-scale habitat partitioning in host plant neighborhoods.Nutritional content of host plants is expected to drive caterpillar species assemblages and their trait composition. These relationships are altered by tree richness-induced neighborhood variation and a seasonal decline in leaf quality. We tested how key functional traits related to the growth and defenses of the average caterpillar hosted by a tree species are shaped by nutritional host quality. We measured morphological traits and estimated plant community-level diet breadth based on occurrences from 1020 caterpillars representing 146 species in a subtropical tree diversity experiment from spring to autumn in one year. We focused on interspecific caterpillar trait variation by analyzing presence-only patterns of caterpillar species for each tree species. Our results show that tree richness positively affected caterpillar species-sharing among tree species, which resulted in lowered trait variation and led to higher caterpillar richness for each tree species. However, community-level diet breadth depended more on the nutritional content of host trees. Higher nutritional quality also supported species-poorer but more abundant communities of smaller and less well-defended caterpillars. This study demonstrates that the leaf nutritional quality of trees shapes caterpillar trait composition across diverse species assemblages at fine spatial scales in a way that can be predicted by ecological theory. Herbivorous insect abundance and species richness tend to increase with increasing plant richness ,2. BesidThe Resource Concentration Hypothesis  predictsCaterpillars represent a substantial component of total insect diversity in forest ecosystems  and are The positive effect of high nutritional quality on herbivorous insect preference has been demonstrated to be strong . NitrogeLeaf traits and plant species richness can also influence caterpillar traits that are indirectly linked to resource use. Plants or older leaves with lower nutritional quality can reduce the growth rate of caterpillars and, thus, prolong development time and increase their vulnerability to predation, which, in turn, may demand greater investment in defense mechanisms ,16,48. HIn this study, we aimed to test how the leaf nutritional content of trees affects the trait composition of caterpillar communities and how this relationship is altered by the surrounding tree richness and seasonal progression in a subtropical tree diversity experiment. We tested what kind of caterpillar fauna, on average, a tree species hosts at a given point in time, along with increasing tree neighborhood richness, by sampling over spring, summer, and autumn during a single year. Presence-only sampling units for trait analysis were formed by averaging the caterpillar traits across species for each tree species per tree richness level per season. Changes in trait variation were tested as functional diversity of caterpillar fauna within each tree species and between the caterpillar trait averages among tree species. We expected that: (i) tree richness increases sharing of caterpillar species between tree species (measured as tree richness level–specific beta diversity), which also shows as increased caterpillar generalism  and as an increase in caterpillar richness per each tree species; (ii) increased caterpillar species-sharing among tree species at higher tree richness results in reduced caterpillar trait variation. Similarly, it reduces within-tree species functional diversity ; (iii) higher nutritional content (N and Mg), which is promoted in early seasons, leads to lower body weights by favoring earlier instars and smaller species, and lower defensive traits due to faster development. Carbon content (C) is expected to have the opposite effect of N and Mg on body weight due to tougher and palatability-reducing structural carbohydrates and tannins, but, alternatively, can lead to increased growth rates because of the higher amount of shorter, more usable carbohydrates. Similarly, the effect of C on head size may be positive or negative, either by promoting reduced head size and selective feeding or by increasing head musculature. By linking tree richness and leaf nutritional traits, and the functional composition of a highly diverse herbivore larval community in a controlled tree richness setup, our study provides insights into how bottom-up effects shape caterpillar communities through fine-scale habitat selection.2). Within each plot, 400 trees were planted in a 20 × 20 (rows by columns) design. The selection of species followed a random broken stick design for extinction scenarios with mixtures of 24, 16, 8, 4, and 2 species and monocultures. The 24-species mixtures are an additional treatment on top of the gradual extinction scenario design starting from the 16-species mixture [Castanea henryi Rehd. and Wils., Choerospondias axillaris Roxb., Koelreuteria bipinnata Franch., Liquidambar formosana Hance, Nyssa sinensis Oliver, Quercus fabri Hance, Quercus serrata Murray, Rhus chinensis Mill., Sapindus mukorossi Gaertn., and Triadica sebifera L.; and of evergreen species: Castanopsis eyrie Champ. ex Benth., Castanopsis sclerophylla Lindl. and Paxt., Cyclobalanopsis glauca Thunb., Cyclobalanopsis myrsinifolia Blume, Lithocarpus glaber Thunb., and Schima superba Gardner and Champ. No living individuals of Rhus chinensis Mill. were found in 2-species mixtures, so these plots were reassigned as Schima superba Gardner and Champ. monocultures. Some individuals of Castanopsis eyrei Champ. were recognized to belong to Castanopsis fargesii Franch. and were excluded from the data. The number of sampled trees per species per richness level varied from 1 to 16 with an average of 5.9 (SD = 2.5)  as part of the BEF-China biodiversity experiment. BEF-China is currently the largest tree diversity experiment in the world, where tree richness and tree species composition of individual plots were manipulated following a strict design . The st mixture . The ric2.5) see . The caterpillar samples were collected three times in 2019 , from 449 tree individuals in spring, 465 in summer, and 463 in autumn, with the campaign lasting 12, 16, and 16 days, respectively. Caterpillars were collected by beating the tree crown seven times with a padded stick over a suspended white sheet (1.0 m × 1.0 m) that was lifted as close as possible to the branches under collection by a telescopic pole reaching a maximum of 8 m height. Due to this collection method, the caterpillars represent only external feeders. All dislodged caterpillars were collected and stored separately in tubes with 99.5% ethanol.Given the throughput limitations in the morphological identification of caterpillars, DNA barcodes were sequenced from all caterpillar individuals and clustered into Molecular Operational Taxonomic Units  for further analyses. Threshold-based hierarchical clustering with BLASTclust   samples were retained for subsequent analyses.One sample was excluded from the data, as leaf traits were unavailable for We measured the diet breadth of each caterpillar species as occurrences across all tree species within the plant community . Body weight, head capsule width, hair coverage, and aposematism were measured from each caterpillar individual. Body weight (with an accuracy of ±0.1 mg) was measured from the ethanol-stored caterpillars first air-dried on Petri dishes. Head capsule width was measured under a stereoscopic microscope with a measuring scale with 10× to 40× magnification. An increase in head capsule width is highly correlated with an increase in body weight. In addition, head size may be hypoallometric (decrease relatively as the body grows) because body weight increases continuously during the instar development, but the sclerotized head capsule size increases when molting and is assumed to follow a geometric growth ratio along instars . TheHair coverage and aposematism were evaluated by using similar methods to those in other studies of caterpillar defenses, e.g., ,27, but Specific leaf area (SLA), leaf dry matter content (LDMC), nitrogen (N), carbon (C), magnesium (Mg), calcium (Ca), potassium (K), and phosphorus (P) were measured on all trees used in the study  only tree species identity as a random variable and (2) with the following non-hypothesis covariates: tree replicates for caterpillar species richness, tree replicates and caterpillar species richness for abundance, caterpillar species richness and body weight for head capsule width, and only caterpillar species richness for the other trait models and FDis (see below). All models were evaluated by comparing the AIC-values   against Further, sensitivity comparisons of the effect of the most common and widely spread caterpillar species between tree species were conducted. This was conducted for all caterpillar traits and FDis by removing all species that appeared on more than half of the tree species. This accounted for 11 species with a total of 621 individuals .Functional dispersion (FDis), which works without abundance-weighing as a measure of functional diversity , was useIn addition to functional trait diversity within tree species, the caterpillar trait variation between tree species was estimated by calculating the standard deviation (SD) of the caterpillar trait values between sampling units. The change in standard deviation was analyzed with linear models with the function ‘lm’ in the R environment. Explanatory variables used were tree richness, sampling season, and caterpillar species richness. Body weight averages for the respective tree richness levels were also included in the relative head capsule width model to test if systematic changes in body weight lead to increasing variation in the head capsule width. Caterpillar beta diversity between tree species for each tree richness level was estimated for comparison to caterpillar trait variation between tree species and species richness per tree species. The change in community assembly was tested using Sørensen dissimilarity and, separately, its two components, species turnover using Simpson’s dissimilarity and nestedness, with the R package ‘betapart’ . These rBecause caterpillar traits can vary both between and within species, a robust comparison of intra- and inter-specific trait variation was conducted in order to test their relative effect on the underlying trait patterns. Overall, caterpillar intra-specific trait variation was estimated for each caterpillar trait as the average of standard deviations (SD) of each species that had more than one individual. The inter-specific variation was estimated as the standard deviation between caterpillar trait averages of each caterpillar species across all sampling units.In total, we analyzed 1020 successfully sequenced and trait-evaluated caterpillar samples. Sequencing failed for 15 samples of 1092 caught. The caterpillars were delineated to 176 species by BLASTClust, of which 146 were retained for further analysis after the exclusion of missing trait values and incorrect tree species. Caterpillar species richness and abundance with respective tree replicates for each sampling unit are shown in Caterpillar species richness was positively affected by the number of tree replicates and tree richness and negatively by leaf Mg content see a, with iGeneralism of caterpillar species varied between 1 and 16 host tree species, but with the vast majority of species being found only from one (92 species) or two (20 species) tree species . AverageBody weight was higher in spring and especially in low Mg content tree species that, in turn, hosted slightly smaller caterpillars in autumn d see Tab. Head capsule width increased strongly with increasing body weight and decreased with increasing C, N, and Mg content. The negative effect of Mg was especially strong in spring, with a weak positive trend in autumn e. Head cThe relative head capsule width was negatively affected by body weight and caterpillar species richness. Relative head capsule width decreased with increasing Mg and its interaction with the season f, with aHair coverage and aposematism were strongly positively correlated with each other see b. IncreaFDis of the caterpillar traits was strongly positively affected by caterpillar species richness see h.Our sensitivity comparison, with the most common species removed, led to similar overall results but with several of the weaker explanatory factors being dropped . AdditioIncreasing caterpillar species richness had a positive effect on variation of abundance, and a negative effect on other traits, with significant trends on body weight, head capsule width, hair coverage, aposematism, and FDis  in a tree diversity experiment with controlled tree richness and freely assembled insect fauna, our study provides insight into how functional traits determine interactions between consumer insects and primary producers through bottom-up effects. Our study answers what kind of caterpillar fauna, on average, is observed in tree species with varying nutritional quality at a given point in time along a tree richness gradient, and how tree richness affects caterpillar trait variation between the tree species. Caterpillar traits, especially body size, may change along the caterpillars’ development, and it should be noted that the scope of the study is limited to comparisons between tree species at specific time points without extending to what ultimate body sizes or other trait values would be reached by specific caterpillar species on variations of diets. However, seasonal and body size-dependent trait patterns show that caterpillar functional traits are dynamic and deserve consideration beyond simple species-to-species connections. The tree richness gradient and leaf traits in this study reflect habitat quality at fine spatial scales, enabling a better understanding of how local conditions shape the community assembly of the caterpillar fauna via trait matching.The direct effects of tree richness on caterpillar functional traits remained weak in our study. This is in concordance with earlier results from the same field site, which demonstrated the higher importance of tree functional and phylogenetic composition on herbivore assemblage . NeverthFor our fine spatial-scale approach, the decrease in beta diversity at higher tree richness can be seen as support for the Resource Concentration Hypothesis , statingWhether higher plant richness increases predation pressure (Enemies Hypothesis ) has beeOverall, how herbivorous insects are affected by tree richness has focused more on the important role of plant defenses  than on Unexpectedly, our results showed overall higher effects of leaf Mg on caterpillar abundance and trait composition than leaf N content. Some studies have demonstrated the importance of micronutrients such as Mg ,46 on heNutritional effects did not remain stable over the seasons. Even though a reduction in leaf quality towards later seasons is expected to be common among plant species ,38,41, uIn our study, hair coverage was connected to lower nutritional content, similar to aposematism, promoting the interpretation that lower nutrient content leads to slower growth rates and a higher need for defenses ,48. InteAdditionally, beta diversity and variation of generalism and aposematism between tree species increased towards autumn. This, along with generalism being more common in spring and aposematism in autumn, shows the species pool was more strongly separated along these traits when the differences between tree species in leaf nutritional content are expected to be highest. Similarly, within-tree species FDis was also negatively affected by nutritional content (Mg) in spring and summer, demonstrating the unifying effect of nutritional content on the caterpillar species pool. In addition, Mg also increased abundance despite reducing species richness. Predation can shape the caterpillar community in low nutrient content, favoring more specialized species if they are better defended , but comThe observed trends with respect to increasing C content were bi-directional, with, on the one hand, higher aposematism  and lower generalism in autumn, which can be expected by higher structural carbohydrate and tannin concentrations on caterpillar growth . HoweverAs we analyzed the caterpillar fauna time point specifically, the results are influenced, besides inter-specific variation, by intra-specific variability due to caterpillar trait ontogenetic change. The inter-specific variation was shown to be clearly higher than the intra-specific variation for all traits except relative head capsule width. However, this comparison does not capture all of the ontogenetic effects, as many of the caterpillars were caught only at a certain life stage. Indication of ontogenetic changes was seen with aposematism, as it was more common in larger caterpillars, supporting the expectation of their higher visibility due to body size itself and the accompanied higher mobility ,52. AddiSome method-related correlations unavoidably arose in our experimental design of manipulated tree richness. However, these effects were, in most cases, weaker than those of tree richness, leaf traits, and caterpillar body weight. The models that include leaf traits also had clearly better AIC values compared to random variable- and covariate-only models. In addition, sensitivity comparison revealed that the used presence-only analysis structure balances the strong abundance differences between caterpillar species, though the trends became somewhat weaker with the loss of the most common species. It is not surprising that the number of tree replicates had a strong effect on caterpillar species richness. This demonstrates, in part, the role of asymmetrical self-thinning caused by tree species competition with high planting densities . Self-tStudies investigating bottom-up effects on multiple caterpillar traits at the community level have been sparse (but see ) despite


Post-transplant lymphoproliferative disease (PTLD) is the most common malignancy complicating solid organ transplantation (SOT) in adults and children. PTLD encompasses a spectrum of histopathologic features and organ involvement, ranging from benign lymphoproliferation and infectious-mononucleosis like presentation to invasive neoplastic processes such as classical Hodgkin lymphoma. The predominant risk factors for PTLD are Epstein-Barr virus (EBV) serostatus at the time of transplant and the intensity of immunosuppression following transplantation; with EBV-negative recipients of EBV-positive donor organs at the highest risk. In children, PTLD commonly presents in the first two years after transplant, with 80% of cases in the first year, and over 90% of cases associated with EBV-positive B-cell proliferation. Though pediatric kidney transplant recipients are at lower risk (1–3%) for PTLD compared to their other SOT counterparts, there is still a significant risk of morbidity, allograft failure, and an estimated 5-year mortality rate of up to 50%. In spite of this, there is no consensus for monitoring of at-risk patients or optimal management strategies for pediatric patients with PTLD. Here we review pathogenesis and risk factors for the development of PTLD, with current practices for prevention, diagnosis, and management of PTLD in pediatric kidney transplant recipients. We also highlight emerging concepts, current research gaps and potential future developments to improve clinical outcomes and longevity in these patients. Post-transplant lymphoproliferative disease (PTLD) is the most common malignancy complicating solid organ transplantation (SOT) in adults and children . IncidenWhile reduction of immunosuppression is a cornerstone of therapy, patients often require additional treatments, including rituximab, chemotherapy, and adoptive immunotherapy, with radiation and surgical therapy reserved for select cases. Current literature in pediatric PTLD is limited to case series and small studies and many practice patterns for treatment are adopted from the adult literature, as outlined in this review. Importantly, PTLD is associated with significant morbidity and mortality, and at present, there is no consensus for monitoring of at-risk patients or optimal management strategies for pediatric patients with PTLD. Early recognition of risk factors with close attention to prevention efforts and prompt intervention is paramount for improving survival.Here we review pathogenesis and risk factors for the development of PTLD, with current practices for prevention, diagnosis, and management of PTLD in pediatric kidney transplant recipients. We also highlight emerging concepts, current research gaps and potential future developments to improve clinical outcomes and longevity in these patients.Though there are several reported risk factors for the development of PTLD, the two major risk factors in pediatric kidney transplant recipients are: EBV-serostatus at the time of transplant and the intensity of T-cell immunosuppression following transplantation .Over 90% of pediatric PTLD is due to EBV positive B-cell proliferation in the setting of immunosuppression and decreased T-cell immune surveillance. Typically, acute EBV infection results in a polyclonal expansion of B-cells containing the virus. In healthy, immunocompetent hosts, EBV infected B-cells are rapidly eliminated by EBV-specific cytotoxic T-cells. However, in the setting of high-dose T-cell immunosuppression (such as thymoglobulin), cytotoxic T-cell function is diminished, resulting in uncontrolled proliferation of EBV-infected B-cells and the development of PTLD , 12.de novo infection or when it is acquired from passenger lymphocytes in the graft  involved. Patients will typically have non-specific constitutional symptoms such as fever, fatigue and weight loss , 45. FrePatients may also present with laboratory abnormalities similar to non-transplant patients with lymphoproliferative disorders, such as: anemia, thrombocytopenia, leukopenia, elevated serum lactate dehydrogenase, hypercalcemia or hyperuricemia . There mAlthough an elevated EBV viral load should raise suspicion for EBV-positive PTLD, this is not sufficient for diagnosis. In patients with a high clinical suspicion for PTLD , diagnosis and classification require tissue biopsy, with excisional biopsy of a suspicious lesion when possible . And, whThe most recent World Health Organization guidelines divide PTLD into four main categories based on morphologic, immunophenotypic, genetic and clinical features . IdentifThe first category of PTLD is non-destructive and encompasses three patterns: plasmacytic hyperplasia, infectious mononucleosis-like and florid follicular hyperplasia. These are considered early lesions with benign proliferations.The remaining three categories are considered neoplastic processes and the presence of a lymphoid tumor in combination with two of the three following features confirm a diagnosis of PTLD: disruption of underlying tissue architecture by a lymphoid proliferation, presence of mono- or oligoclonal lymphoid cell populations, and EBV infection of many cells.Monomorphic PTLD meets the criteria for a non-Hodgkin B cell or T/NK cell lymphoma with monoclonal malignant cells. The majority are B cell lymphomas (most commonly diffuse large B cell lymphoma) but can include Burkitt lymphoma, plasma cell neoplasm, peripheral T-cell lymphoma not otherwise specified or EBV + T/NK cell lymphoma.Polymorphic PTLD has a pleomorphic lymphoid infiltrate that does not fill criteria for one of the aforementioned B cell or T/NK cell lymphomas above.Classical Hodgkin lymphoma-like PTLD fulfills the criteria required for the diagnosis of classic HL and is the least common form of PTLD.The overarching goals for PTLD management are eradication of the PTLD and preservation of allograft function. These priorities often involve conflicting treatment approaches, and typically one goal will take precedence over the other based on the specific needs of the patient. For example, a mainstay of PTLD eradication involves reduction of immunosuppression, but this increases the risk of graft rejection and failure.The main approaches to PTLD management largely depend on the PTLD subtype but nuances may vary from institution to institution. Here we review the most common PTLD treatment strategies, their respective indications, and the risks and benefits to each approach.Reduction of immunosuppression (RIS) is the cornerstone of management for all types of PTLD, with the goal of restoring EBV-specific cellular immunity without increasing the risk of acute rejection. This remains the most common practice at diagnosis of PTLD in pediatric SOT recipients , 24. GenRetrospective studies regarding the efficacy of RIS in adults and children are challenging to interpret due to study design and small sample sizes with conflicting results. Some studies suggest resolution of early PTLD lesions with RIS alone, but other studies suggest that many patients treated initially with RIS go on to require additional therapy, and up to 50% may have evidence of organ rejection during their reduced immunosuppression phase . ProspecRituximab is an anti-CD20 monoclonal antibody and a reasonable therapeutic option for patients with CD20+ PTLD with residual disease despite RIS or for those who are not candidates for RIS.Rituximab can be used alone or in conjunction with chemotherapy depending on the clinical circumstance. There are no current consensus guidelines for whom would benefit from both therapies and whether to administer them concurrently or sequentially. Patients with minimal symptoms and/or those who are not candidates for initial chemotherapy can begin with rituximab alone at a typical dosing regimen of 375 mg/m2 weekly for 3–4 doses. Adult SOT studies demonstrate response rates of 44%–79% with rituximab monotherapy and complete remission rates in 20%–55%, obviating the need for chemotherapy in many patients , and onePatients with CD20 + PTLD may receive chemotherapy in conjunction with rituximab in a regimen known as R-CHOP . The ideal time course of these therapies remains unknown; however, many authors suggest the addition of chemotherapy in patients who don't achieve an adequate response or complete remission after initial treatment with rituximab –58. ThesPatients with CD20- PTLD or those who are not candidates for rituximab may receive CHOP chemotherapy alone; and patients with classic Hodgkin lymphoma (HL) like PTLD will receive chemotherapy in accordance with HL protocols. There are no randomized trials comparing chemotherapy regimens in PTLD and some patients may require other regimens at the discretion of their physician or based on their personal risk of side effects. Indication for and selection of additional agents is beyond the scope of this review.Local therapy by surgery or radiation is limited to rare situations, such as treatment of local disease, symptomatic control, or palliative care. For disseminated or central nervous system disease, radiation therapy may be indicated in conjunction with chemotherapy , 61.Adoptive cell therapy with EBV-cytotoxic T-lymphocytes (EBV-CTLs) involves transferring naturally occurring EBV-specific CTLs that can kill EBV-transformed B cells into recipients with EBV + PTLD . Up to tOne systematic review of 36 adult patients demonstrated a 66% response rate, with rare adverse effects limited to mild, nonspecific symptoms  . Use of Previous limitations surrounding the time required to generate autologous EBV-CTLs (2–3 months) in critically ill patients at risk for rapid progression of disease have now been addressed with availability of third party, HLA compatible EBV-CTLs with favorable safety profile and outcomes , 65. WhiPublished data in all pediatric transplant recipients suggest a multitude of parameters associated with poor prognosis, including: advanced disease, multifocal and extra-nodal disease, CNS involvement, allograft involvement and high EBV load at the time of diagnosis. Morphologically, CD20-negative, EBV-negative, monomorphic subtype and late-onset PTLD are all associated with less favorable outcomes ; as wellThere are currently two major gaps in research of PTLD. First, not all EBV is the same. Many patients are chronic EBV carriers and never develop PTLD. A better understanding of the viral and host factors and immune responses that determine whether EBV-positive PLTD develops is critically needed . In addiOverall, PTLD is a rare but serious complication after kidney transplantation, and many questions remain unanswered given the wide spectrum of disease presentation with variable phenotypes, organ involvement and the complex interplay among factors that determine treatment and prognosis. Until vaccines are proven safe and effective and become readily available, attention must continue to be focused on prevention and management.


Fungal communities (mycobiome) have an important role in sustaining the resilience of complex microbial communities and maintenance of homeostasis. The mycobiome remains relatively unexplored compared to the bacteriome despite increasing evidence highlighting their contribution to host-microbiome interactions in health and disease. Despite being a small proportion of the total species, fungi constitute a large proportion of the biomass within the human microbiome and thus serve as a potential target for metabolic reprogramming in pathogenesis and disease mechanism. Metabolites produced by fungi shape host niches, induce immune tolerance and changes in their levels prelude changes associated with metabolic diseases and cancer. Given the complexity of microbial interactions, studying the metabolic interplay of the mycobiome with both host and microbiome is a demanding but crucial task. However, genome-scale modelling and synthetic biology can provide an integrative platform that allows elucidation of the multifaceted interactions between mycobiome, microbiome and host. The inferences gained from understanding mycobiome interplay with other organisms can delineate the key role of the mycobiome in pathophysiology and reveal its role in human disease. In contrast, the mycobiome  study remains in its formative years.4–6 To date, there are substantially fewer studies focused on fungi within the microbiome due to difficulties in nucleic acid extraction, rudimentary fungal genome annotations and limited bioinformatic tools to analyse these data or established standards. Early studies using culture-dependent studies suggested that only 70% of the population harboured a fungal population.7 A total of 267 fungal taxa have been identified in the human gut mycobiome by several studies. However, the validity of some of these fungal species (e.g. Penicillium) is unclear as they cannot be grown under human gut conditions in the laboratory.8 The long-term residence of fungi in the gut microbiome is thus still the subject of debate as the longevity of fungal species within a mycobiome is undetermined.8 Further, fungal genes within the microbial metagenome have been estimated to only make up 0.1% of the total microbial genes.9 However, the importance of fungi to the host cannot be determined by measures of the overall relative abundance of their genetic material within the microbiome. Production of metabolites and small molecules by fungi potentially has a more significant impact in the microbiota and host compared to bacterial production, owing to the greater fungal biomass and associated higher occupation of surface area, however, this remains largely unproven.9–11 A summary table of mycobiome studies associated with dysbiosis highlights the increasing relevance of fungi  tract is a contributing factor to IBD onset.31–36 Further, a shift in Ascomycota and Basidiomycota proportions potentially drives inflammatory processes in IBD.37 The distal effect of mycobiome dysbiosis is chronic immune-mediated inflammation and appears to be related to central nervous system (CNS) infection associated with neurological diseases.38–40 Fungal products have been found in Alzheimer patients,41 whilst another study confirmed the presence of 14 fungal species, including Malassezia, Candida, Alternaria and Botrytis,42 in different brain regions of Alzheimer’s patients.43 Similarly, fungi have been associated as triggers of autoimmunity and multiple sclerosis,44 whilst the presence of C. albicans and S. cerevisiae antibodies in schizophrenia patients has been linked to the gastrointestinal disorder, supporting the hypothesis of a gut-brain axis.45Fungal dysbiosis potentially affects a significant portion of the population, being implicated in irritable bowel syndrome (IBS) and Crohn’s disease (CD) and a direct contributing factor in IBD30 see . For exa40 For example, the mycobiome is associated with cancer development and progression,46 where Malassezia spp. are increased in pancreatic ductal adenocarcinoma (PDA) in an in vivo (murine) study.46 In contrast, Candida tropicalis, another commensal fungus, is associated with slower or lack of tumour development.46 This study, among others, indicates that mycobiome composition is paramount to host health and disease status. However, specific fungal species do not exclusively drive these interactions. In order to survive, fungi adapt to their local environment; this includes adjusting to acidic environments through morphology switching and metabolic plasticity. Biofilms are a classic example of fungi changing their growth pattern in response to host responses and environmental cues. Fungi-microbe interactions play a key role in adaptation. Fungi cooperate with bacteria to increase fungal biofilm potential, resulting in significant increases in the severity of inflammatory diseases – most notably in IBD.37 Alternatively, Pichia spp. interaction with Candida results in the reduction of biofilm due to competition in the environment.47 Thus, mycobiome composition plays a significant role in the maintenance of the homeostatic relationship within the microbiota. Determining whether fungal-bacterial-host relationships drive compositional changes and whether controlling each element in these networks could potentially prevent disease is a fundamental goal for future microbiome research.Mycobiome dysbiosis is a predisposing factor in other diseases as well.48 or modulate immunological responses.49 These host immune responses to fungal infection have been reviewed extensively elsewhere.50–54 To creates symbiotic relationship with microbiota for host immune-tolerance, there is a positive exchange of compounds and nutrients. With evidence of fungi producing vitamins and fatty acids,55–57 there is an opportunity and potential for these exchanges with the mycobiome that remains to be investigated.The interplay between the mycobiome and host immune responses involves physical interactions at the cell surface, metabolic reprogramming, regulatory pathways, and secretion of small molecules. Exposure to host responses and environmental stressors provides a platform for metabolic plasticity to manipulate host interactions and promote invasion58–60 For instance, short-chain fatty acids (SCFA), tryptophan and branched-chain amino acids have all been identified as playing a role in immune and homeostatic responses.61 Short-chain fatty acids (SCFAs) available within the host are necessary for regulating some fungal growth as well as modulating immune responses. Fungi are capable of producing vitamins, notably including vitamin D and B6, that can impact the immune system.62–66 The different fungal moieties, such as cell wall polysaccharides and melanin,67 drive metabolic reprogramming of innate immune cells to confer protective inflammation and activate antimicrobial defences.68–70 Equally, contact with host cells drives shifts in fungal metabolism.52 For instance, host recognition of ß-glucan from fungal cell walls reprogrammes macrophage glucose metabolism towards aerobic glycolysis, promoting more rapid ATP creation.67 Importantly, however, C. albicans undergoes a similar metabolic reprogramming, meaning that local glucose is rapidly depleted, leading to the premature death of macrophages.52 As C. albicans has a significant degree of metabolic plasticity, it can then revert to using alternative carbon sources to survive. Melanin produced by Aspergillus fumigatus is recognised by macrophages via MelLec (a C-type melanin-sensing receptor) via calcium sequestering in phagosomes, leading to activation of antifungal defences within the host.71A. fumigatus responds further by reducing melanin for fungal persistence, leading to conidia germination and increased glycolysis allows metabolic repurposing of macrophage response for effective fungal defence.70 Hosts defensive metabolic responses and the parallel evasive mechanistic pathways in fungi demonstrate a need to understand metabolic networks in host mycobiome in health and disease.52Microbial metabolism, including fungal metabolism, has been associated with driving inflammation, various diseases and even cancer progression.in vivo data set sources is listed in 72–74 The interaction of fungi and bacteria occurs on several different levels. Physical contact can occur between members of microbial communities leading to the exchange of secreted products and communication between the two cell surfaces. Cell signalling between microbes also provides regulation and trade of metabolites and genetic materials through different mechanisms, such as horizontal gene transfer, signal transduction and interaction with the host.76 This exchange allows for an interplay between microbes that can maintain the ecosystem’s resilience or drive a dysbiosis state. This interplay within the host means there is a knock-on effect for the host, potentially resulting in disease pathology. For example, C. tropicalis is significantly more abundant in Crohn’s disease (CD) patients in comparison to non-CD relatives.37 This increased abundance correlates with increased abundance of some bacterial species and related improved biofilm formation and disease severity. These interactions can potentially be bi-directional. For example, Escherichia coli, Serratia marcescens, and Candida tropicalis together exhibit enhanced biofilm formation, giving a thicker, more robust film.37 Furthermore, the production of bile acids (e.g. cholic acid) from the microbiota and environment have been shown to promote the growth and morphogenetic plasticity of C. albicans,77 whilst hexadecanedioic and caproic acids have demonstrated antifungal activity.29In a series of animal model studies, the interaction between host-mycobiome and fungus-bacterium has been linked to the health status of the host  affect both confirmation and community structure of the mycobiome, potentially leading to dysbiosis.Alteration of the bacteriome composition drives diverse changes in metabolite levels in the gut and effects on the mycobiome. For example, antibiotic-treated mice have increased levels of 29 The natural ability of fungi to produce this cornucopia leads to ripple effects in their environment.97 The metabolic activity of fungal communities has a pivotal role in microbiome modulation, contributing to disease pathogenesis and pathology. Fungi can catabolise a panoply of different substrates, including carbohydrates, amino acids, lipids, proteins, and vitamins utilising both primary and secondary metabolism. Primary metabolism is involved in the growth and development of the cell, whilst secondary metabolism increases fungal viability in the environment. Fungi produce a variety of secondary metabolites, including acids, toxins, polyols and sugars that mediate cross-talk and are considered a by-product of other reactions.98Fungi produce an expansive variety of metabolites with varied impacts on their survival, as well as host and microbiota interactions. For example, the fungal metabolite N-acetyl-L-glutamic acid has hypotensive effects.99–102 Polyketide (PKS) class consists of derivatives of fatty acids and the output of acetyl CoA pathways that allow for various structures and functions.103 Non-ribosomal peptides (NRPS) are derived from amino acids and cannot perform RNA transcription.104 Terpenes class are generated from terpene synthases, a product of the mevalonic acid pathway and produced as steroid compounds.105 Terpenoids are a modified class of terpenes. Dimethyallyl tryptophan (DMATs) class is comparable to the ergot alkaloid class and are usually pharmacologically active compounds responsible for producing indoles.106–108 As the detection of growing numbers of metabolites progresses, methods in finding genes responsible for secondary metabolites are also being developed. These genes are called biosynthetic gene clusters (BGCs) and are based on the chromosomal arrangement of the synthesis of secondary metabolites. These BGCs ranges from smaller clusters relating to synthases to a larger cluster of genes that include transcription factors, as detailed in Keller et al. (2019). These genes are globally regulated by transcriptional activity, not all necessarily from within the cluster, thus indicating that transcription and epigenetic activation of BGCs is dependent on external factors. These factors include environmental stimuli, metal presence, nutritional availability, temperature, pH and light, triggered by regulatory pathways.76Secondary metabolites are key by-products of primary reactions and play a crucial role in shaping the interaction within a niche. In the elucidation of enzymatic processes, fungal secondary metabolites have been classed into several groups depending on the biosynthesis of these products. The most notable involves polyketide synthases (PKS), non-ribosomal peptides synthases (NRPS), fatty acid derivatives, terpenoids, and steroids.Candida, Penicillium and Aspergillus species and their possible metabolic pathways have been illustrated in The mycobiome includes metabolic processes with an extensive pool of secondary metabolite production that can be key in driving or preventing a pathogenic switch. For instance, metabolites and small molecules can activate the host immune system via recognition of foreign material or the release of toxins, driving inflammatory responses. Potential effects of known metabolites produced by Aspergillus species is naptho-γ-pyrones. This natural metabolite has various biological effects, including anti-bacterial and anti-oxidant activity.120–124 The wide variety of fungal metabolite functions makes them ideal targets for therapeutic use. This can be done by targeting metabolic pathways to derive benefits. For example, by driving immunometabolic shifts that affect glucose competition and redirecting pathways for cancer treatment.125 As well as direct impacts on the host whether as colonisers or during infection, these metabolites are ideal potential drugs for treating the microbiome directly. For example Penicillium produces a mycotoxin called citrinin that has antibiotic properties against bacteria and can have a toxic effect on kidney and liver.126–128 Secondary metabolites can thus conceivably both provide diagnostic markers for the detection of disease and manipulate metabolic pathways, altering host health status. System biology contributes useful tools in investigating metabolic pathways in fungi in the context of the human mycobiome.Secondary metabolites can have a knock-on effect on host metabolism, and this has been used extensively for therapeutic effects . One met4 The development of molecular techniques such as 18S ribosomal RNA and inter transcribed spacer (ITS) sequencing has profoundly impacted the detection of fungi and the characterisation of the mycobiome. However, these techniques are limited in understanding the mycobiome community’s overall structure, function, and dynamic existence on a large-scale platform.129 Many mycologists face the obstacle of limited bioinformatic tools and databases available compared to bacteriologists. With improving next generation sequencing (NGS) technologies and lower costs, multi-omics data generation is increasing rapidly. Multi-omics data includes metagenomics, transcriptomics, proteomics, metabolomics and fluxomics that impart in-depth information that has enabled us to learn about microbiota interactions  has proven to enable the genotype-phenotype relationships through a series of connected gene-protein-reaction links, providing a powerful predictive toolkit.135 GSMM have been applied in different microbial and human studies as a discovery platform to propose novel markers, treatments and bioproduction enhancement.138–141 GSMM predictions and discovery can be further evaluated and validated using synthetic biology, such as CRISPR-cas systems  for genome editing to test and manipulate molecular and metabolic pathways.142To date, mycobiome studies have primarily been grounded in culture-based techniques that are hostage to the diverse requirements of each community member, meaning many fungi remain undetected and undiscovered.ractions .130–132 S. cerevisiae model is the benchmarking fungal GSMM with a comprehensive network structure including the 3D structure and protein kinetics.143 This GSMM is a reference model used to generate the scaffold of other fungal species models.30 Other well-constructed fungal reference models that have been generated include Scheffersomyces stipitis,144C. glabrata146 and Pichia pastoris.147 Fungal GSMM are mainly used to inform the metabolic engineering experiments, increase the efficiency of the process and markers, such as modelling yeast suggested methods for increasing vanillin production by 5-fold.148 Similarly, the model’s biological interpretation of Yarrowia lipolytica metabolic pathways identified that this fungal species is an ideal producer of di-carboxylic acid using a lipid pathway.149The 139 This has led to a recent increase in studies predicting biological behaviour, the use of biotics for metabolic diseases, personalised medicine, and drug targets for pathogens.149–154 Modelling has implicated the production of SCFAs from microbial fermentation in the host in CD aetiology; thus, adjusting diet can control the presence of SCFAs in patients and modulate disease.155 Exploring IBD compared to healthy cohorts showed an increase in vitamin metabolism in IBD patients, whilst a closer inspection of species in the microbiome using GSMMs unveiled vitamin and biotin producing gut organisms in those suffering from IBD,156 whilst using modelling of the microbiome in neurological diseases including Parkinson’s Disease157–160 and Alzheimer’s disease162 has identified microbiome perturbations, causing flux changes in pathways and production of SCFAs that ultimately potentially cause disease. Studies on community modelling of the microbiome predict metabolic interactions between species with the overall metabolic contribution of each species determined.163–165 Research in this area remains in its infancy as the development of algorithms and tools used to explore community modelling continues, but the potential is beyond doubt.The application of GSMM technologies to the human microbiome has identified cross-feeding of metabolites between host and microbiota in health and disease.C. albicans have been used to investigate its regulatory system in the clinical context.166As there are several available fungal GSMM, they could be implemented in studying the mycobiome to elucidate the complex interactions between mycobiome, bacteriome and host, thereby explaining the metabolic role of fungi in disease pathophysiology. Exploring the mycobiome with GSMM will significantly improve our understanding of fungal interactions with the host in health and disease. Synthetic biology can then be used to validate and assess genetic circuits and metabolic frameworks for complex interactions. Recently, CRISPR-based systems for repression and activation in 167The difficulties lie in exploiting fungal models and synthetic biology due to annotation accuracy with high plasticity of metabolic pathways, diverse morphology, and evolutionary adaptive traits. There is a lagging development of fungal GSMM and applying CRISPR system in clinical and healthcare settings due to a lack of quantitative tools to study fungal genomes a lack of annotation and validation in the model. The global overview provided by this approach offers the potential to drive strategies to manipulate the mycobiome to make predictions, test hypotheses, propose clinical areas for manipulation, identify new diagnostics target areas with better understanding, and discover novel therapeutic targets.in silico models as a predictive tool to validate current datasets and knowledge. In doing so, we will generate a platform to answer how mycobiome metabolism affects the host in health and disease.Understanding the behaviour of fungi, both individually and as a community, is essential in elucidating the role of the mycobiome in health and disease. Untethering the distinct metabolic interactions and effects of secondary metabolism provide an opportunity for disease intervention, determining the point at which dysbiosis arises, identifying biomarkers for better diagnostics and novel therapeutic targets. Metabolism is a holistic and integrative subject that combines genetics, molecular pathways, signalling and environmental factors. Multi-disciplinary tools and methods such as system biology are needed to develop in silico while sustaining full metabolic flux functionality of the system. The functionality of a model refers to the natural ability of the model to undertake reactions, a realistic rate of energy consumption, rate of energy production and apply physio-chemical laws and environmental input to create a system that is true-to-life.168 The conversion of the reactions into a stoichiometric matrix allows mathematical inferences to integrate data into a predictive biological framework called constraint-based modelling.169 This developmental process of integration requires automated and manual curation for efficient quality standards. The GSMM community has developed a MEMOTE suite package to ensure the models’ standardisation and functional feasibility.170 With synthetic biology approach of genome engineering techniques pave the way for validating fungal species’ biological mechanisms underlying these systematic alterations in response to different environmental changes such as response to anti-fungal drug and the host organism’s reaction during fungal infection .142Genome-scale metabolic models (GSMM) are a systematic and curated method to establish genotype-phenotype relationships. GSMM aim to bridge together the complex network of genes, reactions and thousands of metabolites 171 The current approach to reconstructing GSMM combines top-down and bottom-up approaches using automated tools such as the RAVEN toolbox .172 GSMM can be used in constraint-based modelling to predict the flux distribution and secretion of metabolites within the organism under certain constraints such as the intake of substrates and governing specific objective functions such as growth, thereby providing information that would be difficult to generate in vitro experiments .169 As of 2019, there are reconstructed GSMM for 434 bacteria, 40 archaea and 117 eukaryotes .30 The majority of these GSMM are used in the simulation of a single organism. However, their use can go further to study the metabolic cross-talk of microbial communities.The creation of a GSMM includes two methods: firstly, a top-down approach, which integrates experimental data into a mathematical model. This method makes it possible to create a network of reactions and metabolic outputs through pathways. The study of metabolism can provide more accuracy and specificity with further predictive analysis and assessment. Secondly, the bottom-up approach includes defined knowledge and information sourced from readily available studies and the input of current literature .173 What makes Type-II CRISPR-Cas9 a well-established CRISPR approach in the genome-editing field is the ability of manipulation at genome level using just chimeric single guide RNA, making double-strand breaks to insert-deletion gene (indel) using cas9 nuclease, followed by homologous/non-homologues recombination and multiple genes indel .175CRISPR  is a robust and customised method of gene editing which is adapted from the immune mechanism in bacteria and archaea against viral (bacteriophage) invasion .Click here for additional data file.


Spinal cord injury (SCI) is a very serious clinical traumatic illness with a very high disability rate. It not only causes serious functional disorders below the injured segment, but also causes unimaginable economic burden to social development. Exosomes are nano-sized cellular communication carriers that exist stably in almost all organisms and cell types. Because of their capacity to transport proteins, lipids, and nucleic acids, they affect various physiological and pathological functions of recipient cells and parental cells. Autophagy is a process that relies on the lysosomal pathway to degrade cytoplasmic proteins and organelles and involves a variety of pathophysiological processes. Exosomes and autophagy play critical roles in cellular homeostasis following spinal cord injury. Presently, the coordination mechanism of exosomes and autophagy has attracted much attention in the early efficacy of spinal cord injury. In this review, we discussed the interaction of autophagy and exosomes from the perspective of molecular mechanisms, which might provide novel insights for the early therapeutic application of spinal cord injury. More than 1 million people worldwide are disabled due to SCI  are present in almost all human cells and transmit beneficial or harmful information from cell to cell  mechanism Hurley . ESCRT iRecently, exosomes derived from different cells have been extensively studied in SCI. Exosomes are involved in intercellular communication by carrying informational substances from the external environment or cells. Exosomes can be extracted by different methods such as differential ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polyglycerol, and immunocapture  and other related cells. First, MSCs-exosomes have anti-inflammatory effects, inhibit neuronal apoptosis, promote axonal regeneration, improve angiogenesis, and enhance macrophage/microglia polarization in the model of SCI , macrophages, astrocytes, and Schwann cells also play a significant role in SCI , were first discovered in yeast  lacks an amino acid signal sequence in the cytoplasm and is activated by the inflammasome  from MVBs, thereby avoiding the acidification of MVBs lumen  produced through an unconventional exosome release pathway. ANXA2 is captured by autophagosomes with the participation of autophagy-related proteins LC3β and ATG5. Following, RAB11 stimulated the fusion of ANXA2-loaded autophagosomes with MVBs to form ANXA2-loaded amphisome, which further fused with the plasma membrane with the participation of RAB8A and RAB27A to release ANXA2-loaded exosomes  to regulate autophagy in various cells, or to use exosomes with specific components to repair autophagy-induced damage after SCI. Here, we briefly summarize the applications between autophagy and exosome biogenesis in recent years and propose new possible ideas for the early treatment of SCI.