Chronic kidney disease (CKD) is a global public health problem associated with high rates of morbidity and mortality due to end-stage renal disease and cardiovascular disease. Safe and effective medications to reverse or stabilize renal function in patients with CKD are lacking, and hence it is important to identify modifiable risk factors associated with worsening kidney function. Environmental pollutants, including metals, air pollutant, phthalate and melamine can potentially increase the risk of CKD or accelerate its progression. In this review, we discuss the epidemiological evidence for the association between environmental pollution and kidney disease, including heavy metals, air pollution and other environmental nephrotoxicants in the general population. Chronic kidney disease (CKD) is a global public health issue The pathophysiology and mechanisms of worsening renal function are complex and multifactorial. In addition to the well-known risk factors for renal injury, such as aging, diabetes mellitus and hypertension, some environmental chemicals have also been shown to be important risk factors for renal injury http://www.ncbi.nlm.nih.gov/pubmed) database to find published studies from January 1988 to June 2020 that investigated the relationships between environmental exposure to toxins and pollutants and CKD and/or markers of kidney injury. We performed the search using the following terms: 'environmental pollution and chronic kidney disease', 'environmental pollution and proteinuria', 'environmental pollution and albuminuria', 'environmental exposure and chronic kidney disease', 'environmental exposure and proteinuria', 'environmental exposure and albuminuria', 'air pollution and chronic kidney disease' , 'air pollution and proteinuria', 'air pollution and albuminuria, 'metals and chronic kidney disease', 'metals and proteinuria', 'metals and albuminuria'. The search was limited to research articles involving humans and those published in English. Unpublished data and abstracts were not included in this review.In this review, we summarize the current data regarding environmental exposure to toxins and pollutants and kidney disease. Environmental nephrotoxicants can be classified as follows: (1) metals, (2) air pollution, and (3) other non-metal exposure. We searched the PubMed  is a highly toxic metalloid that occurs ubiquitously in the environment 2 was increased by around 2-fold in people with a urine As level > 75 μg/g creatinine compared to those with a urine As level ≤ 35 μg/g creatinine 2/year). Another cross-sectional study conducted in China also found that a plasma As concentration > 0.93 μg/L was associated with eGFR < 60 mL/min/1.73 m2Exposure to As through the environment, occupation and diet has been reported to cause renal injury and the development of renal disease Cadmium (Cd) is known to be nephrotoxic environmental pollutant Clinically, Cd nephrotoxicity presents with symptoms including low molecular weight proteinuria, glucosuria, aminoaciduria, low molecular weight proteinuria, hypercalciuria and renal stones Lead (Pb) is found throughout the environment, primarily due to human activity. Pb compounds are commonly used in gasoline, batteries, pipes, ammunition, paints, ceramic glazes, water contaminated by Pb pipes, food contaminated during processing, Pb-adulterated alcohol, and other industrial applications Pb is the most common environmental nephrotoxicant, and exposure can cause oxidative stress in tubular and glomerular cells and lead to the generation of free radicals, potentially contributing to cellular apoptosis and subsequent changes in renal structure and function Two cross-sectional studies analyzed the US NHANES from 1999 to 2002 and 1999 to 2006 2An increasing number of longitudinal studies have supported that Pb exposure contributes to an increased risk of kidney disease. Yu et al. explored the association between low-level environmental Pb exposure and renal function among 121 patients with non-diabetic CKD in Taiwan Several studies have explored the relationship between environmental Pb exposure and nephrolithiasis. Hara et al. recruited 1302 Flemish participants and reported that environmental Pb exposure was a risk factor for nephrolithiasis Exposure to mercury (Hg) compounds occurs via occupational, dietary and environmental sources, including contaminated water, fresh water fish from a contaminated source, predatory ocean fish, gold mining, smelting, burning fuel, incineration and whitening creams  pars recta of proximal tubules has been reported to be most sensitive to Hg, and it is usually the first nephron segment to be affected by exposure to Hg compounds 2) with increasing tertiles of blood Hg level , compared with the lowest tertile (blood Hg < 0.66 mg/L). In addition, Nuyts et al. conducted a case-control study of occupational exposure to Hg in 272 patients with CKD and 272 controls matched for age, sex and area of residence, and found that Hg exposure was independently associated with an increased risk of CKD  Hg readily accumulates in the kidneys and can contribute to both tubular and glomerular damage Environmental exposure to uranium (U) is mainly through the ingestion of contaminated groundwater, soil, and food Oral exposure to U from drinking contaminated water and occupational exposure have been associated with glucosuria, aminoaciduria, microalbuminuria, β2 microglobulinuria, phosphaturia, and hypercalciuria Particulate matter (PM) is a mixture of suspended liquid and solid particles in the air. It is a common air pollutant which varies widely in terms of size and chemical composition 2 per year 3 increase in PM2.5 concentration was associated with a 6% increased risk of developing CKD  Increasing epidemiologic evidence suggests that PM is a risk factor for CKD 2 and SO2 was a significant risk factor for mortality due to ESRD regardless of the length of exposure Few studies have examined the association between PM and the progression of CKD in patients with kidney damage In summary, experimental and clinical findings indicate the biologic plausibility and support the hypothesis that environmental exposure to elevated levels of PM2.5 is associated with an increased risk of kidney disease. More evidence from a variety of regions and populations is needed to clarify the effects of PM on renal health. These findings support the global strategy to reduce air pollution and prevent the development of CKD.In 2011, a major health scandal involving phthalate-tainted foodstuffs occurred in Taiwan Recent studies have investigated the relationship between phthalate exposure and renal function including early renal injury markers such as urine albumin/creatinine ratio and urinary β2-microglobulin (β2M) in different populations. Trasande and colleagues investigated the relationship between exposure to phthalates and renal function in 667 children aged 6-19 years who participated in the 2009-2010 NHANES by measuring metabolites in their urine 2 from 2006 to 2010 in Taiwan, and found that urinary melamine level was significantly associated with deterioration in kidney function in patients with early-stage CKD Melamine is a synthetic organic base used in many commercial products including dry erases boards, cleaning supplies, and other plastic goods. Despite the 2008 melamine baby formula scandal in China which resulted in kidney-related disease in children Melamine has been shown to crystallize in distal renal tubules, and this may explain the reported cases of nephrolithiasis and acute kidney injury. It has also been hypothesized that melamine-induced oxidative stress contributes to renal tubular injury Bisphenol A (BPA) is a synthetic chemical comprised of two phenol rings connected by a methyl bridge, to which two methyl groups are attached. Environmental BPA exposure can occur via absorption through the skin, ingestion and respiration, and detectable levels of BPA have been reported in the urine of >93% of adults Previous studies have reported inconsistent associations between BPA and kidney function. Urinary BPA levels have been positively associated with ACR among US children Environmental pollutants including heavy metals, PM, and other chemicals such as phthalates, melamine and BPA are important factors in the etiology of CKD, especially in developing countries in which environmental pollution is prevalent. The pathogenic mechanisms by which most environmental nephrotoxicants induce CKD have been elucidated. Most studies on the pathogenic mechanisms of environmental pollutants have focused on systemic inflammation and oxidative stress, and the detailed mechanisms of the pathogenesis of specific kidney diseases are still not fully understood. Studying interactions between environment pollutants and genetic factors may help to elucidate disease susceptibility.Most of the epidemiological evidence regarding the association between environmental pollution and kidney diseases discussed in this study comes from cross-sectional studies. To establish causal relationships and dose-response associations between exposure to environment pollutants and kidney disease for a wide range of exposure levels, more detailed longitudinal studies and also experimental designs with specific and quantified measurements of environmental exposure are required. It is crucial to implement environmental protection strategies and establish safe exposure levels of environmental pollutants, such as air quality standards. In summary, our findings support the need for regulatory strategies for the control of pollution and reduction or prevention of exposure to environmental health risks. Clinicians should be aware of the adverse renal effects induced by environmental exposure to pollutants. Detailed exposure assessments based on the sources of exposure for potential nephrotoxicants should be performed on a patient-by-patients basis.


FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22  FANCA to FANCW) that participate in this pathway have been identified. Germline pathogenic variants (PV) in any one of these genes are the origin of this disease . Th. Th41]. The heterogeneous clinical phenotype observed in the patients with FA contrasts with their highly constant cellular and cytogenetic phenotype. The homogeneous cellular phenotype observed in FA indicates that failure in any stage of the FA/BRCA pathway results in the incapacity for repairing ICLs and DSBs in an error-free manner. Misrepaired DSBs in particular, which arise after initial ICL processing, are the main source of chromosomal aberrations (CA) present in FA cells, and this sensitivity has been critical for the diagnosis of FA, which is largely based on the detection of CA observed in cell cultures treated with Diepoxybutane (DEB) and mitomycin C (MMC) .In FA cells, chromosome breakage is the result of initiated but unfinished ICL repair. Most of the breaks in FA cells are of the chromatid type b,c, indiSince the FA/BRCA pathway is not functional in FA cells, the presence of chromosome breaks, when FA cells are treated with ICL inducing agents, suggests that endonucleases, alternative to the canonical FA/BRCA pathway, unhook the ICL and generate a DSB. This DSB however is not channeled to HR by the downstream modules of the FA/BRCA pathway and might remain unrepaired; when a cell reaches metaphase, the sites of these unrepaired DSBs can be visualized as chromatid breaks. Of note, the piece of broken chromatid usually remains adjacent to its chromosome due to the mitotic chromatin structure and the cohesin proteins that hold together the sister chromatids and prevent their separation until anaphase  Figure c.In FA cells, the presence of translocations, dicentrics and radial figures makes evident the relevance of FA/BRCA pathway in the protection against SCAs, since all of these aberrations originate by ligation of two or multiple broken DNA ends with little or absent homology. If the end joining pathways cNHEJ and MMEJ operate during S/G2 phases of the cell cycle, when the replicated chromosome is composed by two sister chromatids, the joining of one of these chromatids with a non-sister chromatid from a different chromosome (homologous or non-homologous) will lead to SCA formation .Radial figures are formed when at least two DSBs from non-sister chromatids are joined together. In these two DSBs, four DNA ends are available; therefore, if the four DNA ends are joined by an error prone DNA repair machinery, a closed tetraradial can be generated. However, an open tetraradial figure will be originated if only two DNA ends are rejoined d. A trirTranslocations, dicentric chromosomes and chromosome deletions may be directly originated during the abnormal processing of ICLs or arise as a consequence of the extremely abnormal segregation that radial figures undergo during mitosis. Depending on the type of radial chromosome, the transition through anaphase will result in the segregation to the daughter cells of translocated chromosomes, dicentric chromosomes, acentric fragments and deleted chromosomes . ImportaAll of these SCA can be accompanied by numerical alterations, such as aneuploidies (gains or losses of whole chromosomes) and polyploidization. In metaphase spreads of patients with FA, it is relatively common to find tetraploid cells and mitotic figures with endorreduplicated chromosomes, with four instead of two chromatids. FA cells are also known to have alterations in the duration of the cell cycle phases (explained below) or in the transition from one phase into another. These might provoke new DNA replication cycles in the absence of mitosis and cytokinesis, leading to endorreduplicated chromosomes in the next mitosis a 47]47]Presence of SCAs is a hallmark of the FA cellular phenotype, therefore the analysis of the number and type of SCA is used in the diagnosis of FA. An approximate 10-fold increase in the DEB-induced frequency of SCA and the presence of radial figures are indicative of FA . In someDNA replication in the human chromosomes starts at thousands of replication origins that are “licensed” by the minichromosome maintenance proteins (MCM2-7) before entering S-phase. Not all the licensed replication origins will fire, most of them will remain as dormant origins . The firFA proteins play a central role in protecting DNA replication both under normal and replication stress conditions. In this scenario, the functions of the FA proteins are independent from their role in the repair of ICLs and change depending on the severity of the replication stress . FANCD2 In conditions of high replication stress, components of the FA/BRCA pathway are activated, particularly RAD51/FANCR binds to the ssDNA exposed in the stalled replication fork and form a nucleofilament that is protected by BRCA1/FANCS and BRCA2/FANCD1; in addition, the nascent DNA is protected by FANCC, FANCJ, FANCM and the FA core complex ,21. MonoRecently, it has been found that FANCM regulates the correct repair of stalled fork, selecting the HR pathway, which protects fork-remodeling and elicits error-free repair, instead of alternative error-prone repair pathways. Specifically, FANCM, aided by BRCA1/FANCS, BRCA2/FANCD1 and RAD51/FANCR, is able to select the conservative repair mechanism that generates “short-tract” gene conversion and suppresses error-prone “long-tract” gene conversion. On the other hand, FANCM-BLM and BRCA1/FANCS (independently of BRCA2/FANCD1 and RAD51/FANCR) suppress the formation of tandem duplications arising by error-prone replicative responses, thus highlighting the function of FANCM as a key mediator of repair pathway choice at stalled replication forks, to preserve the genomic stability .Collectively, all this information evidences an important role for the FA proteins in protecting DNA replication, beyond ICL repair. Nucleotides are essential components for DNA replication and may affect FA cells ; depletiCollision of the replication and transcription machineries has also been associated with replication stress. During transcription, a DNA:RNA hybrid, known as R-loops, can be formed between the template DNA and nascent RNA. R-loops can interfere with replication fork progression and induce replication fork stalling and collapse. R-loops usually occur at sites encoding large genes of more than 800 Kb or can also be induced by the aldehydes generated during normal cellular metabolism . FA/BRCAChromosomal regions with highly repetitive sequences, such as centromeres, telomeres and CG-rich DNA, are difficult to replicate and susceptible to present replicative stress due to the formation of complex DNA secondary structures such as stem-loops, G-quadruplexes (G4) structures and DNA catenates. All of these structures have the potential to interfere with the progression of the replication fork and consequently generate under-replicated DNA regions, formation of UFBs, chromosome breakage and impaired cytokinesis. A functional FA/BRCA pathway is fundamental to prevent replication stress and maintain genomic stability ,60.Common Fragile Sites (CFS) are late-replicating repetitive sequences that may contain tumor suppressor genes or proto-oncogenes. These defining characteristics make them genetically unstable chromosomal regions that are highly susceptible to replication stress and make them hotspots for structural chromosomal aberrations, notably due to: (a) DNA repetitive sequences prone to form secondary structures; (b) transcription–replication collisions (mainly in the presence of active oncogenes); and (c) scarcity of replication initiation  61]. Mo. Mo61]. In the absence of exogenous stress, the FA pathway and specifically FANCD2 and FANCI are involved in the protection of CFS ,61. ImpoAs mentioned in the previous sections, the DNA repair deficiency that characterizes FA leads to accumulation of unrepaired DNA damage with cellular and physiological consequences . When faThe normal and timely progression through the cell cycle is controlled by several cell cycle checkpoints functioning during the G1, S and G2 phases ,65. ThesThe prominent cell cycle arrest to which FA cells are subjected has been ascribed to p53, a cell cycle master regulator . p53 is γH2AX to signal the end of the DNA damage response [Additional mechanisms that allow the escape of FA cells from the strong cell cycle checkpoints are starting to be elucidated. These include the checkpoint recovery, a whole system of phosphatases, led by PPM1D/WIP1, that dephosphorylates ATM, CHK1, p53 and the histone response . When thresponse . HoweverThree main clinical features of FA have long been recognized: (1) developmental alterations; (2) bone marrow failure; and (3) an increased risk to develop cancer. The clinical presentation among patients is highly heterogeneous: not all patients develop all features and there is important variability in the severity of each documented feature.The complete FA pathway is only present in mammals, but can be found in a reduced version in other organisms  The effeA recent literature review of published cases, which analyzes the reported physical features of the largest number of confirmed patients with FA, found that almost 80% of them had at least one physical feature, the more frequent ones were: short stature, upper limb radial ray abnormalities, skin pigmentation changes, renal malformations and central nervous system findings [Morphogenesis is a highly regulated process, there are critical moments during development where different organs can be particularly sensitive to insults .Aldehydes are also a byproduct of alcohol metabolism, alcohol has been shown to rearrange chromosomes and kill cells  and its FANC gene genotype and similar genetic background among siblings) [It could be speculated that the hardship found by patients with FA during development to take care of the ICL resulting from endogenous acetaldehydes could contribute to the physical phenotype of patients with FA. It has been hypothesized that the overlapping features in FA and FASD are the consequence of aldehyde susceptibility of somatic stem and progenitor cell populations . ALDH2 giblings) .Fancd1 and Fancn) die early during embryonic development due to increased apoptosis [FANCD1 and FANCN genes, but has been linked to genotypes of downstream genes [Anthropometric features are key components of the classical FA physical phenotype. Short stature is reported in half of the patients and low birth weight is also a commonly reported feature of patients with FA . Evidencpoptosis . Nevertham genes . The assam genes , may refam genes ,84.Upper limb radial ray abnormalities are also a pivotal feature that brings the FA diagnosis into the minds of clinicians. It has been estimated that up to 1% of patients with congenital thumb malformations have FA . The uppThe relevance of stochastic factors in developmental phenotypes is also illustrated by renal malformations. The patterns of abnormalities found in these paired organs point to disruption of migration patterns of embryonal organs to their final position occurring at an early developmental stage, suggesting that the FA pathway may have a role in this . A literA more flagrant misestimation of occurrence is found for skin pigmentation changes, which have been described in almost 40% of published cases of patients with FA . HoweverBone marrow failure (BMF) is the more characteristic feature of patients with FA. It has been estimated that the cumulative incidence of severe BMF reaches 70% by age 50 years, with a median age at presentation of seven years ,94.γH2AX [Multiple reports suggest a direct role for p53 hyperactivation as an important mechanism of BMF in FA patients ,95,96,97γH2AX . It is wγH2AX .FA cells also overexpress other cell cycle regulators such as the ATM and CHK1 kinases which are important checkpoint overseers. Both these kinases have the capacity to phosphorylate p53. Moreover, ATM is also capable of phosphorylating CHK1  in response to DNA damage ,103. TheUntil recently, bone marrow failure in FA has been thought as the consequence of excessive growth suppressive pathways that in addition to p53/p21 include the hyperactivation of the potent growth inhibitory TGFβ pathway . HoweverPatients with FA have a significant risk to develop cancer. The cumulative incidence of leukemia has been estimated to be under 5% by age 30, while the myelodysplatic syndrome (MDS) cumulative incidence was found to be 50% by age 50. Regarding solid tumors, the cumulative incidence is about 20% by age 65, with a hazard rate that increases exponentially after the age of 30. This translates into a reduced median overall survival of patients with FA of 39 years .Non-transplanted patients with FA have an outstanding risk to develop MDS, which has been shown to be over 5500-fold that of the general population. The hazard rate of MDS reaches 1% by age 10 , and theLeukemia has been found to occur in 3% of patients with FA . In overUp to 40% of children and young adults with FA exhibit signs of clonal evolution in the bone marrow , while uProgression to MDS and AML in FA is associated with the presence of clonal and non-clonal chromosomal alteration, and both are valuable biomarkers for detecting progression to cancer. This agrees with the theory of CIN as a driver of the evolution to cancer. In fact, as shown in RUNX1 and RAS [Cytogenetic and next generation sequencing analysis of MDS and AML bone marrow samples from patients with FA have identified gross chromosomal abnormalities. The most frequent findings include partial duplication of chromosome 1q , partial duplication of chromosome 3q+ (41.3%), duplications in 21q+ (20.7%), monosomy of chromosome 7 or deletion of chromosome 7q− (17.2%) and 11q+ (13.8%), whereas mutations are more commonly in the genes  and RAS ,118.Although some of the chromosome abnormalities mentioned above are shared between patients with FA and MDS/AML from the general population, mutations in MDS/AML oncogenes and tumor-suppressor genes classically found in MDS/AML samples are rarely found in FA. On the other hand, chromosomal lesions that seem to be specific to FA include 1q+ and 3q+; of note, 1q+ has been observed in the BM of patients with FA in all MDS/AML stages and even in normocellular bone marrow or hypoplastic bone marrow without signs of transformation, suggesting that 1q+ clones might confer a survival advantage to the HSPCs from FA patients without being a part of the malignant transformation process ,118.Solid tumors were found in 12% of patients with FA. Major cancer sites have consistently been reported as head and neck squamous cell carcinoma (HNSCC), vulva, esophagus and brain. Their cumulative incidence is about 20% by age 65, with a hazard rate that increases exponentially after the age of 30. This translates into a reduced median overall survival of patients with FA of 39 years. Overall, a 19 observed/expected ratio for all solid tumors in non-transplanted patients with FA was found, with a median age of presentation of 34 years. An increased risk of solid tumors in transplanted patients was confirmed . MoleculSkin cancer is also extremely frequent in patients with FA. In the NCI cohort, the overall number of non-melanoma skin cancers in both non-transplanted and transplanted patients is larger than the number of solid tumors, and over 70% (8/11) of patients who had skin cancers had more than one . The pubFANCD1 (BRCA2) or FANCN (PALB2), in whom cancer usually occurs in the first decade of life [Besides the more frequent solid tumors such as HNSCC, there are around 40 reported cases of FA patients in whom primary solid tumors have been found during childhood. The known genotypes of these patients are either  of life ,124. The of life .FANC genes and the hereditary breast and ovarian cancer (HBOC) genes BRCA1 and BRCA2 [FANCD1 (BRCA2), FANCJ (BRIP1), FANCN (PALB2), FANCO (RAD51C) and FANCS (BRCA1)   are at iThe ability to perform DNA repair is a central mechanism for the protection of genome stability. It is of no surprise then that inappropriate genome maintenance conducts to cancer. Genes from the FA/BRCA pathway are of course susceptible to acquiring mutations in the course of malignant transformation for a number of cancers. The sporadic forms of cancers typically found in patients with FA  have been reported to have an assortment of somatic mutations in genes from the FA/BRCA pathway. This somatic susceptibility for mutations in genes of the FA/BRCA pathway is not limited to such cancers, but can also be found in infrequent cancers in FA such as melanoma, evidencing the importance of this pathway for genomic stability in a myriad of cell types .The lack of a functional FA/BRCA pathway can have important and adverse consequences in the gonadal function, and not surprisingly fertility issues have been reported in both female and male patients with FA. On the one hand, ICL mis-repair would lead to gross chromosomal rearrangements that will affect the mitotic divisions that germ cells undergo before entering meiosis , leading to death of cells with chromosome breaks and genomic imbalances that provoke oligospermia or azoospermia in male FA patients. On the other hand, if cells with balanced chromosome rearrangements (translocations or inversions) move into the prophase I of meiosis, the pairing that the homologous chromosomes undergo during zygotene might be impaired, thus preventing the synaptic events needed for recombination of the homologous chromosomes or generating complex meiotic figures during pachytene, which subsequently could stop the meiotic division and trigger apoptosis . The thiFANC genes have been found in patients with non-syndromic POI [Due to gonadal dysfunction, pregnancies are rare events in FA; to our knowledge, there are fewer than 50 pregnancies reported in the literature. Although both transplanted and non-transplanted patients have become spontaneously pregnant , their pomic POI , and malomic POI , substanMice models have proven essential to further understand the mechanisms that result in subfertility in FA. The FA/BRCA pathway is needed in the response to replication stress; it becomes essential in situations of rapid proliferation such as expansion and maintenance of primordial germ cells (PGC) . Mice stMany of the features discussed in the previous section can also be found in individuals from the general population, but they develop at later stages of life when the individuals belong to the elder population . PatientAging in the tissues from FA patients is associated to ineffective cell divisions, especially in the HSPCs compartment, leading to impaired supply of new functional cells and tissue attrition A; howeveParadoxically, although cancer and aging are considered antagonists, age is the most significant risk factor for cancer development with the majority of cancers being diagnosed after the age of 65. However, in patients with FA, cancers typically appear at a remarkable young age ,109,111.The hallmarks of aging are grouped into three main categories: (1) primary hallmarks, considered to be the origin of cellular damage; (2) antagonistic hallmarks, considered to be compensatory or antagonistic responses to the damage, they initially mitigate the damage but might eventually become deleterious themselves; and (3) integrative hallmarks, responsible for the functional tissue decline associated with aging . At the Primary hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations and loss of proteostasis (the mechanisms that maintain correctly folded proteins and correct protein turnover) . For allAntagonistic hallmarks of aging include deregulated nutrient sensing, mitochondrial dysfunction and cellular senescence . MitochoFinally, integrative hallmarks of aging, considered the culprits of the phenotype, are the exhaustion of tissue specific stem cells and altered intercellular communication . ExhaustAcute inflammation is a transient response to infection or tissue damage that is beneficial and facilitates tissue repair; however, sterile inflammation is a chronic sustained process, probably promoted by incomplete resolution of the initial stimuli and might ultimately result in tissue remodeling and dysfunction. Sterile inflammation is thought to result from exposure to various endogenous and environmental insults throughout the entire lifespan of a person ,141,142.Several agents can activate sterile inflammation including debris from macromolecules, microbial components or extracellular and cytoplasmic DNA fragments, collectively known as damage-associated molecular patterns (DAMPs). DAMPs can activate innate immune cells  and non-immune cells  through the transmembrane pattern-recognition receptors of the Toll-like receptor (TLR) family. TLRs in turn activate the NF-kB transcription factor that upregulates various pro-inflammatory cytokines, including TNFα, IL-1β, IL-12 and Interferons .Although DAMPs-mediated sterile inflammation has not been coined in FA, a pro-inflammatory phenotype has been very well described and includes increased production of pro-inflammatory cytokines, including TNFα and IFNγ, and increased C-Reactive protein (CRP) ,145,146.In FA, however, adaptation of HSPCs to the harsh bone marrow microenvironment can lead to survival and selection of clones resistant to the pro-apoptotic and pro-senescent mechanisms. For example, the inflammatory episodes mentioned above can serve as “selective sweeps” that get rid of non-fitted HPSCs and permit the evolution of clones with the capacity to tolerate the stressors ,149. TheThis selection can fine-tune, similar to aging, a clonal drift in the composition of HSPCs populations in the FA bone marrow; this drift is typically characterized by a decline in the frequency of lymphoid committed HSPCs and an increase in the frequency of myeloid committed HSPCs ,151. DifAlthough the above-mentioned adaptations would extend the survival of HSPCs under hostile microenvironmental conditions, the clonal progeny might acquire subsequent abnormalities in mechanisms controlling growth and differentiation, thus diverting from the original clone and giving rise to MDS and AML.FANC genes render this pathway non-functional, the FA phenotype arises. Clinically, it is characterized by a highly variable presentation including any or a combination of developmental alterations, bone marrow failure and an extremely high risk to develop cancer. In sharp contrast, the cellular phenotype is markedly constant; it is characterized by a prolonged G2 cell cycle phase, proclivity to apoptosis and most notably by CIN. This cellular phenotype is true irrespective of the role performed by the affected FANC protein in the intricated FA/BRCA pathway; it serves as a reminder that FANC proteins function comprehensively to protect the integrity of the genome.The FA/BRCA pathway coordinates the repair of ICLs through the error-free DNA repair mechanism known as HR. When constitutional PV in A non-functional FA/BRCA pathway translates at the cellular level into the continuous generation of DSBs, which promote the formation of SCA that are themselves susceptible to further generate other chromosomal alterations, bolstering a vicious cycle of CIN. The resulting load of DNA damage accumulated in FA cells leads to hyperactivation of cell cycle checkpoints that impede cellular division. Unsatisfied checkpoints can concurrently drive increased apoptotic rates or the activation of the senescence program. However, a proportion of FA cells manage to either ignore the cell cycle checkpoints or to override apoptosis giving rise initially to cells with non-clonal chromosomal aberrations that can eventually promote the formation of genomic rearrangements that propel the evolution of malignant clones.MYC oncogene. MYC overexpression is thought to preserve the population of HSPCs that allows the survival of patients with FA (In brief, the two alternatives for cells that cannot repair their damaged DNA are cell death and survival despite genomic damage; these two cellular fates are at the center of the phenotypic presentation of patients with FA. On the one hand, loss of pluripotent stem cells, particularly when subjected to replicative stress during expansion, affects myriad tissues and partly accounts for features such as short stature, microcephaly, infertility and the more extensively studied bone marrow failure, which is currently more thoroughly understood. In the bone marrow, HSPCs with genomic damage can activate the p53 dependent apoptotic program, stop proliferating in a TGFβ dependent manner or divide, mediated by the overexpression of the  with FA B. Nevert with FA A,B, coulThe lack of animal models that fully recapitulate the human FA phenotype have particularly added difficulty to the study of mechanisms that give rise to the malformative phenotype of FA; today, its pathophysiologic basis remains mainly unknown. Stochastic events, including aldehyde exposure at particular developmental stages, appear to be important; however, mechanistic investigations into the physical phenotype of patients with FA is an area awaiting to be explored.


Cancer remains one of the leading causes of death worldwide. The advancements in 3D tumour models provide in vitro test-beds to study cancer growth, metastasis and response to therapy. We conducted this systematic review on existing experimental studies in order to identify and summarize key biomimetic tumour microenvironmental features which affect aspects of cancer biology. The review noted the significance of in vitro hypoxia and 3D tumour models on epithelial to mesenchymal transition, drug resistance, invasion and migration of cancer cells. We highlight the importance of various experimental parameters used in these studies and their subsequent effects on cancer cell behaviour.The development of biomimetic, human tissue models is recognized as being an important step for transitioning in vitro research findings to the native in vivo response. Oftentimes, 2D models lack the necessary complexity to truly recapitulate cellular responses. The introduction of physiological features into 3D models informs us of how each component feature alters specific cellular response. We conducted a systematic review of research papers where the focus was the introduction of key biomimetic features into in vitro models of cancer, including 3D culture and hypoxia. We analysed outcomes from these and compiled our findings into distinct groupings to ascertain which biomimetic parameters correlated with specific responses. We found a number of biomimetic features which primed cancer cells to respond in a manner which matched in vivo response. Cancer remains one of the leading causes of death despite notable advances in modern medicine . In vitrSeveral previous literature reviews of experimental studies involving 2D and 3D in vitro tumour models, the microenvironmental features of different tumour models and drug responses have drawn conclusions that 3D in vitro tumour models mimic physiological characteristics and display biomimetic response to chemotherapy ,10,11,12In this review we have systematically analysed the effect of the addition of biomimetic features, including physiological hypoxia and 3D culture, on cancer cell behaviour. We have focused in particular on cancer cell growth, invasion, EMT and response to therapeutic interventions within biomimetic 3D cultures.The systematic review was conducted using a number of search engines including “Medline“, “Embase“ and “Web of Science“ with the same initial search terms: “cancer“ and “hypoxia“, then narrowed down further using terms including “2D“ and “3D“ for all databases used. The databases above were all accessed on 28 May 2020. All publications in English language until 28 May 2020 were included for screening. Spelling variations and similar search terms were included for thorough inclusion of existing publications .Inclusion and exclusion criteria were decided and a total of 475 citations were identified through the initial search, excluding duplications, non-full text and non-eligible publications according to the exclusion criteria, 141 publications were deemed eligible for systematic qualitative analysis .Lists of materials, methods, outcomes and conclusions from each publication were recorded via an Excel spreadsheet. To minimize outcome reporting bias, data collection was conducted by two independent investigators, with a third investigator comparing the two data sets for discrepancies and any identified were further analysed by a fourth investigator before final outcome from the specific publication was recorded for the qualitative analysis.2 or partial pressure of oxygen in the overlaying media. The pO2 within 3D cultures can drop to 0% causing cell migration away from the core of 3D cultures where this is measured directly within 3D tissue models [Pathological hypoxia plays an established role in tumour growth, invasion and migration, involving mechanisms such as EMT, angiogenesis and glucose metabolism. Tumour masses of over 500 micrometer diameters display gradients of oxygen concentration, affecting aspects of tumour growth as described above [e models .2 probes to measure the media and or 3D tumour models for dissolved oxygen; stains such as pimonidazole [In this review a total of 51 publications ,62,63,64nidazole  as a tumnidazole . The hyp2 of arterial blood is a measure of the effectiveness of transfer of oxygen from the atmosphere (air) by the lungs. Once dissolved, this oxygen is measured using mmHg units, and 7.8 mmHg is roughly equivalent to 1% atmospheric oxygen. The majority of studies in this review have not directly measured hypoxia in 3D cultures, but instead utilised hypoxia chambers to control or “set“ the free atmospheric oxygen. Many researchers will rely upon the equilibrium achieved between free atmospheric oxygen of the chamber and dissolved oxygen in the culture media, and thus this was difficult to verify for each study. There are issues around how quickly this process occurs, especially given aspects such as: (i) the constant temperature of the culture; (ii) the opening and closure of hypoxia chambers, which can cause fluctuations in oxygen tension; (iii) an understanding of the oxygen diffusion coefficient of the 3D scaffold materials used in 3D culture and; (iv) the multiple monitoring of the media pO2 and the 3D culture pO2, which should preferably be done in the core and at the surface [When directly measuring oxygen in tissues, this is done so by measuring the partial pressure of oxygen (mmHg) and therefore reported levels of tissue oxygenation use this measure. The pO surface .2, is distinct from atmospheric oxygen concentration, which is 21% in air [Tissue normoxia, measured as pO% in air . Existin% in air . Taking % in air , one wou3D tumour models are considered to better recapitulate the in vivo tumour microenvironment compared to standard 2D cultures. In this review we have identified a total of 37 publications ,96,97,98These studies used a variety of measurements to determine the extent of biomimicry within in vitro cultures, using a range of criteria including: Genetic expression of angiogenic and matrix remodelling genes, protein synthesis of angiogenic, invasive and matrix remodelling molecules,; morphological basis involving regulators such as expression of hypoxia induced factors, proangiogenic factors, EMT markers, and factors associated with tumour cell stemness; increased IL-8 expression; measured increases in growth of cancer and invasion.3D culture and the presence of hypoxia, measured by the various methods outlined in EMT is a process identified as one of the key features in tumour invasion and metastasis. We identified a total of 19 publications ,106,107 We identified a total of 42 publications ,122,123 Overall, 85% of experimental outcomes showed 3D tumour cultures displayed increased resistance to anticancer therapies and 15% showed increased sensitivity in 3D when compared with 2D cultures. Of those which showed increased sensitivity in 3D, the therapies tested were predominantly proapoptotic agents, novel targeted therapy, hypoxia activated drugs, radiotherapy or natural compounds .Furthermore, we investigated the specific response from each cancer type to multiple therapeutic interventions. We categorized the cell lines into 11 organ systems. All cancer types showed a trend of overall increased resistance in 3D compared to 2D in vitro tumour cultures. Most cancer types showed similar responses ranging from 72% to 88% of studies displaying enhanced resistance to chemotherapy in 3D. Studies involving urological and head & neck cancers reported 100% chemo resistant outcome in 3D, whereas prostate cancers and sarcomas showed a significantly lower proportion for chemo resistant outcomes in 3D compared to other cancer types at 57% and 56% prospectively .We identified 42 publications from our cohort that specifically studied chemotherapy resistance in 3D cultures, and similar numbers of studies used in scaffold-based cultures and non-scaffold-based cultures (53% vs. 47%). On further investigation, the studies where scaffold-based 3D cultures were used, a much greater percentage of studies used native materials (72%) compared to non-native materials (28%). Similar to the cohort of studies concerning biomimicry of tumour culture, collagen and alginate appear to be the most frequently used materials for native and non-native scaffolds. This noteworthy finding could be due to native ECM proteins being deemed more biomimetic as a 3D scaffold, with materials such as collagen used extensively. Collagen type I is found in the dense fibrotic tissue and the tumour microenvironment, and by using this material, studies report the use of native materials to be more consistent between in vitro and in vivo findings .When describing cancer progression and metastasis, invasion and migration often go hand in hand and are described as parts of a process facilitating cancer spread. We investigated the evidence within the scope of this review for the definition and measurable markers used for invasion and migration, and whether these were distinct to one or the other process. 16 publications ,112,124 Migration imaging assays describe the process as the movement of tumour cells from one position to another, for example across porous membranes, movement within the tumour spheroids or out into the surrounding matrix in an in vitro setting. The morphology of cancer invasion is described in a more aggressive manner, with terms such as invasive, spiky projections and invadopodias into the surrounding ECM. Fibronectin coding genes were found to be up-regulated in studies describing cancer cell migration and various EMT markers such as vimentin, E-cadherin, MMP2 and MMP9, which were all associated with and used to measure cellular invasion. Only invasion is described in an in vivo setting, which has been duly noted in multiple publications. Florczyk et al. described the two cellular processes as, “invasion in vivo and migration in vitro by effective degradation of ECM” . Therefo2, and is distinct from ambient atmospheric oxygen, which is 21%. Tissue normoxia, or physiological hypoxia, ranges between 3–7%, thus when conducting comparisons of hypoxic vs normoxic cultures in cancer studies, a more appropriate choice for hypoxia level should be no higher than 2% oxygen and normoxia control samples should be conducted within the “normal” physiological oxygen levels to truly represent the in vivo tumour and stromal microenvironments [It is important to consider the average range of oxygen concentration in physiologically and pathologically hypoxic tissues when conducting in vitro tumour cultures. Normal tissue oxygen saturation is based on pOronments .To highlight the significance of ranges of hypoxia, over a fifth 22%) of studies included in this review explored tumour hypoxia using 3% oxygen or higher for the hypoxic samples, and furthermore 5% of studies % of stud,47,51 usApart from the level of hypoxia involved in these in vitro studies, the length of exposure also plays a vital role for the biomimicry of these tumour models. There is evidence to suggest that acute hypoxia is more likely to contribute to the malignant progression of tumour cells when compared with chronic hypoxia, meaning that an exposure period of 72 h or less used by the majority (71%) of tumour models included in this review is a sufficient period for hypoxic exposure for malignant progression of most cancer cells. HIF1’s activation has been shown to upregulate EMT within 18hrs of exposure to 1% oxygen and upregulation of angiogenesis related markers can be seen within 2 to 4 h . One stuIn this review we can deduce that 3D tumour models have shown to be a better representation of the in vivo tumour microenvironment regardless if they are scaffold based or rely upon cell-generated ECM deposition to develop 3D matrix, when compared to 2D cultures. Multicellular spheroids and ECM/scaffold-based tumour cultures have similarities in their shared three-dimensional nature in terms of presence of hypoxic regions and their effects on tumour growth and metastasis. Both scaffold and non-scaffold-based tumour cultures demonstrate that tumour cells play a significant role in the secretion of various ECM proteins such as collagen and fibronectin. There is significant data to suggest that non scaffold based in vitro cancer cell cultures, with an absence of extracellular ECM have a less significant effect on the biomimetic properties of the tumour models in terms of hypoxia driven gene expressions, cell ECM interaction driven growth and invasion, and malignant progression. The role of ECM in facilitating tumour invasion and migration is still not fully understood, however evidence has shown that ECM remodelling and infiltration of ECM is strongly primed by various soluble factors by the infiltrating tumour cells . This woHowever, when we analysed the studies exclusively exploring the role of 3D tumour models on EMT, the majority of studies involved scaffold based cultures in comparison with studies looking at biomimicry as a generic theme. ECM components such as collagens, fibronectins, and hyaluronic acid have been shown to play a crucial regulatory role in the EMT signalling pathways. Studies have shown growth factors such as TGF-B, which is present in or sequestered by ECM components, via the TBG-B-ECM axis increases expression of pathways for EMT execution in a dependent manner. Overall, ECM not only provides a structurally supportive role in cancer growth and invasion, but also ECM derived signals provide critical anchor points involved in EMT during the tumorigenesis process and the fine-tuned interaction between the ECM and tumour cells have a vital role in pathogenesis of cancer . TherefoCSCs have been isolated and characterised from numerous types of solid cancers such as breast, colorectal, and hepatocellular carcinomas. Their role in tumorigenesis and metastasis has been described in previous literature and their self-renewal, replicative and differentiative abilities via common signalling pathways have been hypothesised to be the basis for metastatic tumour growth . This reThe preference of native material over non-native material for scaffold based in vitro tumour models in drug resistance studies is the logical approach since the purpose of these experiments are for prediction of in vivo therapy response in an in vitro laboratory setting. Therefore, in order to recapitulate the natural in vivo tumour microenvironment, the choice of natively occurring scaffold material is an obvious preference.Response to all therapy classes conveyed a trend of increased resistance to therapy in 3D in vitro tumour cultures compared to 2D cultures. Furthermore, five out of six of the chemotherapy classes expressed congruent response of increased resistance to therapy in all experimental setups in 3D compared to 2D. Pro-apoptotic agents stood out showing almost even-handed response. We uncovered that 9/10 experimental setups expressing sensitivity to proapoptotic agents used the drug triapizamine. Tirapazamine, a hypoxia- activated agent, showed increased sensitivity in 3D tumour in all the studies which involved the drug. Due to the presence of intrinsic hypoxic cores in 3D tumour constructs, tirapazamine targets hypoxic regions of the tumour, therefore is highly sensitive in such tumour cultures compared to 2D tumour cultures which lack hypoxic regions . The tarThe proportional increase in chemosensitivity of prostate cancer and sarcomas can be accounted for by the multiple specific enzymes targeting novel chemotherapy agents tested in each of the publications compared to other cancer types studied in other publications. When sarcoma cell lines responses to novel targeted therapy DZNep compared to a classical chemotherapy agent cisplatin, all cell lines displayed chemosensitivity to DZNep and resistance to cisplatin . The limThe increased drug resistance in 3D tumour cultures compared to 2D culture systems is not only owing to its oxygen diffusion gradient. The presence of hypoxia is a well-known factor responsible for drug resistance via several difference mechanisms such as hypoxia induced G0 cell cycle arrest . The incNature Research stating invasion as “the ability of cells to become motile and to navigate through the ECM within a tissue or to infiltrate neighboring tissues“ [Cell migration in the non-malignant setting usually refers to the orchestrated movement of cells in a particular direction to a specific location . Cell intissues“ . Neverthtissues“ . TherefoIt is crucial to recognise possible reporting biases which may have resulted during the course of this comprehensive review. Publication, time lag and language biases were noted since this review deemed only full text publications up until 28 May 2020 in English language eligible for analysis. Outcome reporting bias was also taken into account and two independent investigators were involved in the data collection process separately, without influence from external parties. Nevertheless, taking into account of possible biases, this report should still show a relative comprehensive analysis of the topic due to the inclusive search and analysis methods.To conclude, this comprehensive systematic review has analysed in depth the published outcomes in existing literature of the role of 3D cell culture techniques and the corresponding physiological hypoxia on cancer cell behaviour, in particular focusing on growth, invasion, EMT and response to therapeutic interventions. Hypoxia level and duration is a crucial consideration when partaking in in vitro tumour hypoxia studies and must reflect the tissue oxygen concentrations. Whether to use scaffold-based tumour models should be carefully considered when exploring the effects of 3D tumour models on EMT and furthermore the choice of scaffolds in drug resistance studies highlights the importance for in vitro tumour models to recapitulate the in vivo tumour microenvironment. Migration and invasion are distinct but related processes both play crucial roles in cancer progression and metastasis. The development of and optimisation of in vitro 3D tumour models using a variety of microenvironments is essential in the study of cancer growth and progression, and advances in cancer treatments.


That certain cell types in the central nervous system are more likely to undergo neurodegeneration in Parkinson’s disease is a widely appreciated but poorly understood phenomenon. Many vulnerable subpopulations, including dopamine neurons in the substantia nigra pars compacta, have a shared phenotype of large, widely distributed axonal networks, dense synaptic connections, and high basal levels of neural activity. These features come at substantial bioenergetic cost, suggesting that these neurons experience a high degree of mitochondrial stress. In such a context, mechanisms of mitochondrial quality control play an especially important role in maintaining neuronal survival. In this review, we focus on understanding the unique challenges faced by the mitochondria in neurons vulnerable to neurodegeneration in Parkinson’s and summarize evidence that mitochondrial dysfunction contributes to disease pathogenesis and to cell death in these subpopulations. We then review mechanisms of mitochondrial quality control mediated by activation of PINK1 and Parkin, two genes that carry mutations associated with autosomal recessive Parkinson’s disease. We conclude by pinpointing critical gaps in our knowledge of PINK1 and Parkin function, and propose that understanding the connection between the mechanisms of sporadic Parkinson’s and defects in mitochondrial quality control will lead us to greater insights into the question of selective vulnerability. Parkinson’s disease (PD) is a late-onset neurodegenerative disease characterized by a core triad of symptoms; resting tremor, bradykinesia, and elevated resting tone . While 1The realization that PD pathology is not solely confined to the SNpc and can spread across the CNS has profoundly altered our understanding of PD pathogenesis and disease progression. However, despite evidence of widespread pathology, not all cell populations are equally resilient. Certain populations of neurons remain more vulnerable to developing LB pathology and to neurodegeneration in PD, suggesting that cell-intrinsic factors can gate selective vulnerability. The most vulnerable neuronal subpopulations include SNpc DA neurons; cholinergic neurons in the pedunculopontine nucleus, nucleus basalis of Meynert, and dorsal motor nucleus of vagus; noradrenergic neurons in the locus coeruleus; and serotoninergic neurons in the raphe nucleus , 13. Whi2+; regulate cellular redox balances; control apoptosis; and serve as key anchoring scaffolds for intracellular signaling networks PINK1/PRKoduction , 203 – bBeyond a neuron-centric view of PD pathogenesis, recent and ongoing studies of non-neuronal MQC defects and neuroinflammation will bolster our understanding of non-cell autonomous mechanisms of neurodegeneration. Finally, further elucidating the mechanistic interaction of α-synuclein aggregation and PINK1/Parkin MQC inactivation will be critical for establishing the role of MQC in sPD and synthesizing a more unified understanding of PD pathogenesis.


They trigger cilia to move faster in response to elevated cytoplasmic Ca2+ originated from intracellular sources or replenished from extracellular space. Well-known cilia-stimulatory effect of Ca2+ ions can be abolished or even reversed by modulating the phosphodiesterase (PDE)-mediated breakdown of cyclic adenosine monophosphate (cAMP) since the overall change in ciliary beating has been dependent on the balance between Ca2+ ions and cAMP. Moreover, in chronic respiratory diseases, high ATP levels may contribute to cAMP hydrolysis and thus to a decrease in the ciliary beat frequency (CBF). The role of PDE inhibitors in airway cilia-driven transport may help in prevention of progressive loss of pulmonary function often observed despite current therapy. Furthermore, administration of selective PDE inhibitors by inhalation lowers the risk of their systemic effects. Based on this review we may conclude that selective  or dual PDE inhibitors (PDE3/4) increase the intracellular level of cyclic nucleotides in airway epithelial cells and thus may be an important target in the development of new inhaled mucokinetic agents. Further research is required to provide evidence of their effectiveness and feasibility regarding their cilia-modulating properties.Mucociliary clearance is an essential airway defense mechanism dependent predominantly on the proper ciliary function and mucus rheology. The crucial role of cilia is evident in `a variety of respiratory diseases, as the ciliary dysfunction is associated with a progressive decline in lung function over time. The activity of cilia is under supervision of multiple physiological regulators, including second messengers. Their role is to enable a movement in coordinated metachronal waves at certain beat frequency. Ciliary function can be modulated by various stimuli, including agents from the group of beta This exmportant  and imprCilia are microtubule-based organelles that have diverse motility and sensory functions. Although nearly all of cilia possess sensing role and transmit extracellular signals into intracellular biochemical responses, only some of them are able to move.Motile cilia perform rotary motion or create wave-like planar beating based on the presence or absence of a central pair of microtubules surrounded by nine microtubule doublets of axoneme. Rotary motion additionally requires the absence of radial spokes . AdenosiImmotile cilia  act as sensory organelles of the cell, and their crucial role can be observed in various ciliopathies including retinal degeneration, anosmia, deafness, hepatobiliary, pancreatic cyst, and many others .Cilia can be classified into four basic types .Motile cilia of airway epithelial cells contain a central pair of single microtubules surrounded by nine peripheral microtubule pairs (9 + 2) and protein complexes necessary for ciliary beating, such as axonemal dynein arms and radial spokes . The dynin vitro models to investigate mucociliary clearance.Normal ciliary ultrastructure can be visualized using a transmission electron microscopy (TEM). High-speed digital video analysis has improved measurement and quantification of CBF, while improvements in air–liquid interface culturing techniques have led to development of 2+)–calmodulin complex could be considered as the key regulator of CBF linked with both nucleotides, cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate), in the process of ciliary stimulation, although cAMP can also play a role in Ca2+-independent manner (2+ to be effective since disruption of nitric oxide (NO)–cGMP–protein kinase (PK) G pathway at any of the steps in the presence of high Ca2+ concentration eliminates its action  following stimulation of certain membrane receptors  or is transported from extracellular space via ion channels that mediate influx of Ca2+ to the ciliary cells  with a sustained moderately elevated CBF, requiring PKG activation. However, this phase is controlled predominantly by axonemal PKA in a Ca2+-independent manner  may have clinical significance when considering clearance of secretions over hours. Furthermore, despite the normal CBF, the efficacy of mucociliary clearance is dependent also on the proper ciliary beat pattern. This is well documented in patients with primary ciliary dyskinesia (PCD) .Mucociliary clearance belongs to the group of defense mechanisms in the airways. In pathological conditions associated with CBF slowing , the cough and the other antibacterial defense mechanisms can temporarily substitute it . TherefoThe overall effect of mucociliary clearance is dependent on the proper ciliary function determined by the ciliary beat frequency, on the ciliary pattern, and on the optimal airway surface hydration state involving the presence of an airway surface layer (ASL). ASL is responsible for mechanical trapping of inhaled particles and pathogens by mucous component and for facilitating ciliary beating in presence of the periciliary layer. These facts have been confirmed in primary and secondary ciliary dyskinesia or cystic fibrosis. The former is related to the ciliary dysfunction as a consequence of defects in the ciliary structure based on the genetic mutations or inflammatory mediators and pH of the cilia environment, respectively . The latSecondary ciliary dyskinesia includes acquired defects of ciliary movement, which can be caused by viral or bacterial infection or by air pollutants. This may be evident in lung diseases such as bronchial asthma, and COPD. However, a direct relationship between ciliary dysfunction with ultrastructural abnormalities and disease severity was observed only in asthma subjects . In COPDChronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable disease associated with chronic inflammation of the lung parenchyma and peripheral airways. This leads to persistent respiratory symptoms and progressive airflow limitation usually caused by significant exposure to tobacco smoke and airborne pollutants .The pharmacotherapy of COPD is strictly individualized based on the disease severity, drug efficacy, drug tolerance, patient preferences, comorbidities, and potential drug interactions. Bronchodilators and anti-inflammatory medications are one of the cornerstones of COPD management. They reduce clinical symptoms, prevent the development of airflow limitation, lower the risk and severity of exacerbations, and improve the patients’ quality of life and exercise tolerance. Despite the relative effectivity of these agents, severe or very severe stages of the disease (FEV1 post-bronchodilator less than 50% predicted) can sometimes developed probably due to progression of inflammatory process.2 agonists (LABA) reduce but do not eliminate acute exacerbations of COPD. Methylxanthines have been accepted as being effective drugs for the treatment of COPD possessing both anti-inflammatory and bronchodilator activity in the same molecule. But findings about their non-selective mechanisms responsible for their relatively unfavourable safety profile have led to the development of novel therapeutic drugs with more selective properties.Long-term inhaled treatment with corticosteroids (CS) in association with long-acting betaThe discovery of phosphodiesterase isoenzymes (PDEs) along with increased understanding of their different function based on the tissue/cell specificity was a driving force for the development of PDE selective inhibitors for the treatment of various diseases including COPD. Up-to-date, 11 isoenzymes families of PDEs have been recognized (PDE1 - PDE11), of which PDE3 and PDE4 are the major cAMP-hydrolyzing enzymes identified in the smooth muscle. They are involved in the airway smooth muscle tone regulation and inflammatory cell activity. PDE1 and PDE5 are responsible for the cGMP-hydrolytic activity in airway and vascular smooth muscle .Roflumilast, the first selective PDE4 inhibitor approved for the therapy of COPD, is an anti-inflammatory agent that improves lung function in patients with moderate to severe COPD and reduces the risk of moderate to severe exacerbations when add on to bronchodilators , or inhaled CS. Although it is generally well tolerated, systemic effect of the drug is associated with the risk of diarrhea, nausea, weight loss, headaches, and suicide limiting its use and inhaled form if this drug with more favorable safety profile would be of benefit.It is well known that most recommended drugs for COPD are given by inhalational route . It provThus, mucus hypersecretion and chronic productive cough as the two characteristic features of COPD suggest the airway ciliary dysfunction and indicate the need for improvement of ciliary motility in the severe stages of this disease. At present, there are no specific therapies available to correct the ciliary dysfunction and the empiric treatment helps only to manage the consequences of dysfunctional cilia. Therefore, inhalational route of administration and focusing on mechanisms ameliorating the ciliary beating frequency  might be of huge benefit .The activity of PDE proteins is dependent on cell type. In the human airway epithelial cells PDE4 activity is predominantly expressed in addition to lesser PDE1, PDE3, and PDE5 activity . The airChronic treatment with PDE inhibitors seems to have stronger effect on the ciliary beating since they do not directly increase the generation of cAMP, but decrease its breakdown. Therefore, their stronger action is expected in more progressed disease stages.in vivo studies with significantly increased sperm motility in men with oligoasthenoteratozoospermia (OAT) or in vitro studies during a vitrification program without adverse effects on sperm DNA or chromatin integrity , while both ASM cells and human non-polarized bronchial epithelial (16HBE 14o) cells are responding to PDE4 inhibition by roflumilast. Moreover, selective PDE4 inhibition was found to induce an effective reversion in the CBF down-regulation induced by cigarette exposure in ex vivo conditions under which expression of both PDE3 and PDE4 enzymes is dominant  human cells as well as epithelial cells express PDE4 enzymes . This isdominant . Furtherdominant . Workmaneceptors . Furthereceptors .2 agonists , whilst Ca2+ released from intracellular stores plays only little or no role  increase the ciliary beat frequency in human upper airway mucosa in vitro ;better epithelium hydration lowering the mucus viscosity ;anti-inflammatory effect (PDE4 and PDE3/4 inhibitors);bronchodilator effects ;via the inhaled route.relatively few systemic adverse effects when delivered 2 agonist mediated by cAMP accumulation, whilst PDE4 inhibitors become effective in the presence of LABA to stimulate ciliary beating. This makes the mucociliary clearance in pathological airway conditions more effective.Furthermore, PDE1A inhibitors potentiate the cilia-stimulatory effects of beta2 agonist; however, their cilia-stimulatory effect is probably mediated predominantly by PDE4 inhibition.Dual PDE3/4 inhibitors could provide additional benefits and show more rapid response when taken with betavia direct cilia-stimulatory response, when cilia dysfunction in lung diseases has developed. Further research is required to provide evidence of the effectiveness and feasibility of the inhaled selective or bifunctional PDE inhibitors with respect to their cilia-modulating properties.In summary, PDE inhibitors besides their anti-inflammatory and bronchodilator properties can prevent recurrent infection, persistent inflammation, and decline in lung functions MJ wrote the first draft of the manuscript. JM wrote sections of the manuscript. MJ, JM, and SF contributed to the manuscript revision, read, and approved the submitted version.Supported by grants VEGA 1/0160/17, VEGA 1/0356/18, VEGA 1/0253/19, and APVV-18-0084, and by the project “Measurement of Respiratory Epithelium Cilium Kinematics” (ITMS 2622022019).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DARS1 and DARS2—which encode cytosolic and mitochondrial aspartyl-tRNA synthetases, and the clinical conditions associated with mutations of these genes. We also describe attempts made at modeling these conditions in mice, which have both yielded important mechanistic insights. Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a disease caused by a range of mutations in the DARS2 gene, initially identified in 2003. Ten years later, hypomyelination with brainstem and spinal cord involvement and leg spasticity (HBSL), caused by mutations of cytosolic DARS1, was discovered. Multiple parallels have been drawn between the two conditions. The Magnetic Resonance Imaging (MRI) patterns are strikingly similar, but still set these two conditions apart from other leukodystrophies. Clinically, both conditions are characterized by lower limb spasticity, often associated with other pyramidal signs. However, perhaps due to earlier detection, a wider range of symptoms, including peripheral neuropathy, as well as visual and hearing changes have been described in LBSL patients. Both HBSL and LBSL are spectrum disorders lacking genotype to phenotype correlation. While the fatal phenotype of Dars1 or Dars2 single gene deletion mouse mutants revealed that the two enzymes lack functional redundancy, further pursuit of disease modeling are required to shed light onto the underlying disease mechanism, and enable examination of experimental treatments, including gene therapies.Aminoacyl-tRNA synthetases (ARSs) accurately charge tRNAs with their respective amino acids. As such, they are vital for the initiation of cytosolic and mitochondrial protein translation. These enzymes have become increasingly scrutinized in recent years for their role in neurodegenerative disorders caused by the mutations of ARS-encoding genes. This review focuses on two such genes— DARS1 and DARS2 genes, respectively—two genes that encode aspartyl-tRNA synthetase enzymes responsible for the accurate charging of aspartate-specific transfer ribonucleic acid (tRNA) with aspartate  and Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation (LBSL) both belong to a group of white matter disorders termed leukodystrophies. These conditions are caused by mutations of the Gln to glutamine takes place through an indirect pathway, where “mischarging” with glutamic acid by mitochondrial glutamyl-tRNA synthetase (GluRS) is the first step , DARS2 encodes its mitochondrial counterpart (mt-AspRS). AspRS catalyzes the aminoacylation reaction exclusively in the cytosol, whereas mt-AspRS is transported from the cytosol to the mitochondrial matrix where it is involved in mitochondrial protein synthesis.ARSs can be divided into two subgroups based on their location of action—cytosolic and mitochondrial ARSs. In general, each ARS functions exclusively in one compartment and is responsible for the pairing of one of the 20 amino acids with its respective tRNA. Exceptions include glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS), which are capable of functioning in both compartments, and glutamyl-prolyl-tRNA synthetase (EPRS) which is able to pair both proline and glutamic acid with their respective tRNAs in the cytosolic compartment  pattern . While the majority of LBSL patients are compound heterozygous, homozygosity has only been described in five LBSL patients from three unrelated families  have experienced infantile-onset disease with severe disease course and subsequent death  signs have also been described . This patient experienced infantile onset of symptoms and lived to 9 years of age. The most common presentation in infants is regression in motor development or developmental delay  diseases. Most leukodystrophies can be accurately diagnosed with this modality, as they present with a unique MRI signature , superior and inferior cerebellar peduncles, intraparenchymal trajectories of the trigeminal nerve, mesencephalic tracts of the trigeminal nerve and anterior spinocerebellar tract in the medulla  is often thought to be a defining feature of LBSL. However, elevated lactate levels were only detected in 68% of LBSL patients. Furthermore, even in patients where lactate was elevated, this elevation fluctuated over time  has also been performed in LBSL patients, revealing diffusion restriction in parts of the affected WM  gene was initially diagnosed with LBSL based on MRI and clinical features, prior to genetic testing . In comparison, 37.5% of reported HBSL cases are homozygous. All DARS2 gene  of mt-AspRS  at the age of 4 weeks, using tamoxifen inducible Cre expression under control of the proteolipid 1 (Plp1) promoter. Similar to the neuronal Dars2 cKO model produced by the same group, strong MRC deficiencies were observed in these mice, yet no other sequelae involving demyelination, apoptosis or inflammation followed  Cre-driver line. Maximal recombination of the Dars2 gene occurred at 2 weeks of age in cardiac and skeletal muscle (SkM) cells —a known lysosomal cysteine protease, which was also upregulated in neuronal Dars2 cKO mice  in cardiomyocytes was evident by 6 weeks of age  within the first 2 weeks of life in the heart of Ckmm Dars2 cKO mice. This was evident through the upregulation of Atf4, a key component of the ISR, at 16 weeks of age  pathway was also activated in neuronal Taken together, an imbalance in protein homeostasis, which is evident through a rise in ubiquitinated proteins, appears to be the initial trigger for the disease . While homozygous Dars1D367Y/D367Y mice failed to model HBSL, mice compound heterozygous for the Dars1D367Y and the Dars1null mutation captured some aspects of the disease.For HBSL, our lab is currently working on a Dars2 was rendered functionally inactive postnatally. Since CNS development is accelerated in mice compared to humans, these knockouts occur relatively late during development. In contrast, DARS2 mutations are evident from birth in LBSL patients. These mutations greatly reduce, but do not completely abolish mt-AspRS function, throughout the developmental period.In summary, attempts at modeling HBSL and LBSL have contributed significantly to our understanding of both conditions. Nevertheless, these studies have their limitations. In all studies, Dars1 or Dars2 were both embryonically lethal. This indicates a lack of functional redundancy between the two enzymes and puts to rest questions on possible compensatory mechanisms between the two. However, other findings from these studies continue to offer support for a possible shared disease mechanism between LBSL and HBSL. Firstly, the finding of inflammatory processes and immune responses likely underlying neuronal dysfunction and cell-death in LBSL is reminiscent of the study by Wolf et al., who postulated that HBSL might possess a neuroinflammatory component based on clinical findings and the responsiveness of HBSL patients to steroids (Wolf et al., In mice, complete knockout of Dars2 cKO in myelinating cells. Moreover, one study reported findings of axonal degeneration of myelinated fibers in histopathological analysis of tissues from LBSL patients. This was accompanied by detection of myelin ovoids in the peripheral sural nerve, and the absence of segmental demyelination, hence the pattern of peripheral neuropathy in these patients was suggested to be mainly axonal (Yamashita et al., Secondly, studies in LBSL models suggest the primary pathology to lie in neuronal or axonal dysfunction rather than in myelin-producing cells or the myelin itself. These findings are supported by the absence of noticeable phenotypic changes in mice following in vitro for LBSL (Synofzik et al., Management of HBSL and LBSL are currently solely supportive and symptomatic. While these methods can improve the quality of life, the benefits are neither sustained nor equally effective for all patients. Hence, there is a need for the development of novel treatment strategies for patients affected by these conditions. Some medications have been shown to be effective in patients and when tested For the symptomatic management of LBSL, the use of allied health specialties such as speech, nutrition and physiotherapy has been reported, to support patient disability (Navarro Vázquez et al., In both conditions, steroid medications have been shown to reduce the severity of symptoms experienced by patients. Particularly in HBSL, 5 out of 16 patients reported in the literature showed improvements following administration of steroids, three of whom were infantile-onset patients (Taft et al., DARS2 intron 2 splice site in vitro (Van Berge et al., DARS2 following cantharidin application. While cantharidin is toxic and unsuitable for clinical use, the use of other protein phosphatase 1 and 2A inhibitors could be explored for therapeutic use. Alternatively, antisense oligonucleotides have also been shown to increase the efficiency of splicing at this site (van Berge et al., Lately, some drugs have gained special attention for the treatment of LBSL. Firstly, the carbonic anhydrase inhibitor acetazolamide was reported to improve ataxia in one patient (Synofzik et al., Finally, gene therapy is often considered for inherited conditions. This is a particularly viable option for HBSL and LBSL, as they are both monogenic disorders and therefore highly amenable to such a treatment approach. With the recent advances in creating animal models for HBSL and LBSL, attempts at establishing such a treatment modality will follow and hopefully provide a cure for these devastating conditions.AM and DF led the project and the manuscript production. MK and GH contributed to the manuscript preparation. All authors read and approved the final manuscript.MK is an employee of Boehringer Ingelheim Pharma GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Living beings spend their lives and carry out their daily activities interacting with environmental situations that present space-time variations and that involve contact with other life forms, which may behave as commensals or as invaders and/or parasites. The characteristics of the environment, as well as the processes that support the maintenance of life and that characterize the execution of activities of daily life generally present periodic variations, which are mostly synchronized with the light–dark cycle determined by Earth’s rotation on its axis. These rhythms with 24-h periodicity, defined as circadian, influence events linked to the interaction between hosts and hosted microorganisms and can dramatically determine the outcome of this interplay. As for the various pathological conditions resulting from host–microorganism interactions, a particularly interesting scenario concerns infections by viruses. When a viral agent enters the body, it alters the biological processes of the infected cells in order to favour its replication and to spread to various tissues. Though our knowledge concerning the mutual influence between the biological clock and viruses is still limited, recent studies start to unravel interesting aspects of the clock–virus molecular interplay. Three different aspects of this interplay are addressed in this mini-review and include the circadian regulation of both innate and adaptive immune systems, the impact of the biological clock on viral infection itself, and finally the putative perturbations that the virus may confer to the clock leading to its deregulation. Circadian rhythms, generated by the endogenous circadian clock, allow living beings to anticipate environmental daily changes and provide an evolutionary advantage that favours survival despite strong selective pressure. The circadian clock is evolutionary conserved and thought to be roughly 2.5 billion years old, tracing back to cyanobacteria, which started releasing vast amounts of oxygen during the Great Oxidation Event, prior (700 million years) to the divergence of plants, animals, and fungal lineages [Rotation of the planet Earth around its axis generates rhythmic day/night alternations with a period of approximately 24 h (circadian). The term circadian originates from the Latin lineages ,2,3.The biological clock is a molecular network which generates oscillations in gene and protein expression that control cellular functioning and allows for the timely separation of biological processes that cannot or should not coexist. These include redox reactions and DNA replication , switcheIn mammals, the system driving the generation of internal timing and ultimately the regulation of physiology and behaviour is formed by a hierarchical network of biological oscillators with two hubs in the hypothalamic suprachiasmatic nuclei (SCN) . The SCNThe molecular clockworks generate rhythmic oscillations through “transcription-translation feedback loops” (TTFL), i.e., core-clock transcription factors positively turn on the expression of genes encoding circadian proteins that subsequently inhibit transcription via negative-feedback loops, with a time delay indispensable for the proper functioning of the biological clock . The TTFBMAL1 and fine-tune its transcription [In addition, the nuclear receptors REV-ERBα and REV-ERBβ compete with RORα, RORβ, and RORγ at ROR-binding elements (ROREs) in the promoter region of cription . The CLOcription .Synchronization with the external environment and anticipation of predictable environmental changes confer competitive advantages to living beings, especially in the wild. Accordingly, in humans experiencing dysregulation of circadian timing, for example, due to shift work or social jet lag, there is increased incidence of various degenerative, inflammatory, metabolic, and neoplastic diseases ,36. BesiMounting evidence shows that time-of-day-related treatment schedules and chrono-modulated drug delivery significantly affect both effectiveness and side effects of pharmacological therapy ,38,39. DThe circadian regulation of the immune system response allows for its time-dependent success against different pathogens in a rhythmic fashion ,42,43,44BMAL1 silencing lead to more pronounced asthma-typical airway changes, which suggests a role for BMAL1 in the regulation of lung-specific antiviral responses and the subsequent development of asthmatic symptoms. This study reinforces the previous findings from Majumdar et al., who reported a role for BMAL1 as a regulator of innate immunity and showed that BMAL1-deficent cells are more susceptible to infection of the RNA viruses RSV  and PIV3 (parainfluenza virus type 3) [The rhythmic function of the immune system influences the appearance of pathological conditions, for instance, inflammatory lung disease and asthma, which show circadian fluctuations in symptoms and occurrence. In a recent study using both acute and chronic models of virus-induced airway disease (based on the Sendai virus (SeV)), disturbance of the 24-h periodicity negatively affected development, progression, and exacerbation of asthma . In this type 3) .PER2-deficient mice did not show the observed time variation of immune response. Likewise, the antibody response to viral challenge in humans was also reported to be time dependent: immunization with hepatitis A and influenza virus vaccines in the morning led to higher antibody response as compared to afternoon vaccination [The efficacy of vaccination is also thought to be under circadian control as suggested by results obtained in mice and human studies. Silver et al. have shown in immunized mice that vaccination at the time of higher TLR9 (Toll-like receptor 9) expression led to enhanced immune response, likely due to the circadian expression pattern of TLR9 . Interescination ,54,55.BMAL1 expression enhances viral disease [BMAL1, the levels of virus replication were independent of the time of day at which the viral infection was performed [Cellular and tissue functions exhibit rhythmic fluctuations over the course of 24 h that impact the susceptibility to or progression of the viral infection. Several core-clock elements have been reported to modulate viral infection. BMAL1 and PER2 are regulators of antiviral immunity, as described in the previous section, and additional roles for PER1 and REV-ERBα, as well as melatonin have been highlighted, as described in the following. The seasonal variation of BMAL1 expression, reaching its lowest values in winter, seems to contribute to the high occurrence of respiratory viral diseases in this season since low  disease ,57. An e disease . In miceerformed . The efferformed . The timerformed .PER1 was identified as a negative regulator of HIV-1 transcription and its expression inversely correlated with viral loads, likely in relation to different latencies of the virus in resting CD4+ T-cells [Moreover, the core-clock gene  T-cells .Liver infection caused by hepatitis B and C viruses was also reported to be under circadian control. REV-ERBα plays an indirect role in Hepatitis C Virus (HCV) infection by negatively regulating miR122 , a microAnother important interaction involves BMAL1 and the HSV transcriptional machinery. BMAL1 is able to associate with the regulatory protein ICP0 ; it formCLOCK, PER1, and PER2 and downregulation of BMAL1, PER3, CRY1, CRY2, and CKIɛ [Viral infection may also alter circadian gene expression as reported in different studies. Simian immunodeficiency virus (SIV) infection in monkeys caused changes in amplitude and values of body temperature and locomotor activity circadian rhythmicity after the acute retroviral syndrome stage. These changes were not linked to modifications detected in the acute febrile response triggered by virus inoculation. On the other hand, hypothalamic microglia infiltration and macrophage accumulation were shown to occur in animals sacrificed upon appearance of strong circadian anomalies, hinting at analogously significant physiological and psycho-cognitive consequences in human subjects with HIV infection . Moreoveand CKIɛ .A worldwide problem is represented by seasonal epidemics of influenza. Influenza is a disease with global impact that causes enormous morbidity and mortality on an annual basis. The severity of the infection depends on both the virus strain and a number of host factors, primarily age and the presence of comorbid conditions. The mortality and usage of healthcare resources associated with influenza is focused in the elderly and in those with a coexisting disease such as chronic obstructive pulmonary disease (COPD). In patients with COPD, the morbidity and mortality caused by an influenza virus infection are considerably greater.Bmal1 knockout mice infected with IAV showed amplified lung inflammatory and pro-fibrotic responses.In a COPD/emphysema mouse model, influenza A virus (IAV) infection reworked lung clock gene expression and decreased the amplitude in the rhythms of locomotor activity. This effect was more prominent in C57BL/6J mice that were chronically exposed to cigarette smoke and was accompanied by decreased body weight and augmented mortality; likewise, fl/fl Ercre+Bmal1 mice, and this time-of-day observed variation was abolished in −/−Bmal1 mice. The rhythmic fluctuation of IAV-dependent effects was driven by inflammatory changes in the lung favouring better survival and related to a time point with high proportion of natural killer cells and of natural killer T lymphocytes and small percentages of inflammatory monocytes [Bmal1 deletion in pulmonary airway epithelial cells (AECs), cellular elements that vitally shape the host/environment boundary, induced alterations in neutrophil infiltration, biomechanical function, and responses to influenza infection in the lungs. Analysis of the 24-h time-resolved RNA sequencing performed on laser-captured AECs indicates that extensive transcriptomic changes in circadian enriched pathways are linked to lipid metabolism, xenobiotic detoxification, extracellular matrix, and chemokine signalling [Furthermore, the time of day of virus infection crucially impacts the outcome of IAV and this effect is observed under different types of light–dark cycles (12-h light/12-h darkness versus constant darkness), as well as in genetic circadian disruption models. The time of infection impacted late-viral clearance and IAV survival in onocytes . Accordignalling .Bmal1 knockout findings suggest that proper functioning of the molecular clockwork is necessary for the immune response towards viral infections and the subsequent disease [The  disease .Epigenetic regulatory mechanism, which includes DNA methylation and histone posttranslational modifications (PTM), leads to chromatin remodelling and regulated gene expression. Chromatin remodelling represents a critical process through which inputs, such as light or food, are transduced by the cell to generate permissive and silencing histone modifications to influence gene transcription and ultimately signalling pathway activity. CLOCK-BMAL1-dependent activation of clock-controlled genes is connected to circadian changes in histone PTM at their own promoters. Numerous chromatin remodelers, such as the deacetylases Sirtuin 1  and histone deacetylase 3 (HDAC3), are recruited to the promoter region of the clock-controlled genes in a circadian manner ,73. ImpoPrevious studies report the existence of an interplay between the molecular clockwork, epigenetic modifications, and hepatitis C virus (HCV) in the liver, which affects ~3% of the population worldwide. Infection with HCV is considered one of the main risk factors for hepatocellular carcinoma (HCC) . HCV is Bmal1 or overexpression/pharmacological activation of REV-ERB was able to counteract HCV replication by modulating the circadian expression of the liver-specific microRNA miR-122 with consequent derangements of signalling pathways involved in lipid metabolism [In the in vitro model of Huh-7 cells transfected with HCV core protein of different genotypes  , cellulatabolism . A betteInfectious agents exploit infected organisms in order to survive, multiply, settle, and eventually spoil. While some infections may remain asymptomatic or scarcely manifest, others are treatment resistant and have a grim prognosis. The immune system efficiently shields the body against infectious agents but eventually may fail, with harsh outcomes for the infected organism. Viruses are intracellular parasites hinging on the host cellular structures and cellular processes to replicate, survive, and disseminate. The biological clock drives circadian rhythmicity of cellular processes and immune functions, thus shaping the host response to viral infections and affecting the patterns of host–pathogen interaction . SimilarEven though much has been investigated in the circadian clock/immune system field in recent years, several open and outstanding questions remain to be addressed. Are different branches of the immune system differentially regulated by circadian rhythms? If autonomous ticking is dampened in peripheral tissues, do peripheral immune cells alter their rhythms/responses relative to immune cells in the thymus and lymph nodes? Do responses to vaccines that need boosters show different time-dependent patterns? Would different adjuvants boost vaccine responses in a time-of-day-dependent manner? Does deregulation of circadian rhythms correlate with reactivation of viruses known to cause latent infections ? Do hibernating animal species mount different antiviral responses during hibernation? These are extremely interesting topics for future research, and the answers to the questions above will help pave new directions in the study of circadian–immune network interactions in the context of viral infections.