Caenorhabditis elegans provide a profound advantage as a reductionist, economical model for PD research, with a short lifecycle, straightforward genome engineering and high conservation of PD relevant neural, cellular and molecular pathways. Functional models of PD genes utilising C. elegans show many phenotypes recapitulating pathologies observed in PD. When contrasted with mammalian in vivo and in vitro models, these are frequently validated, suggesting relevance of C. elegans in the development of novel PD functional models. This review will discuss how the nematode C. elegans PD models have contributed to the uncovering of molecular and cellular mechanisms of disease, with a focus on the genes most commonly found as causative in familial PD and risk factors in idiopathic PD. Specifically, we will examine the current knowledge on a central player in both familial and idiopathic PD, Leucine-rich repeat kinase 2 (LRRK2) and how it connects to multiple PD associated GWAS candidates and Mendelian disease-causing genes.For decades, Parkinson’s disease (PD) cases have been genetically categorised into familial, when caused by mutations in single genes with a clear inheritance pattern in affected families, or idiopathic, in the absence of an evident monogenic determinant. Recently, genome-wide association studies (GWAS) have revealed how common genetic variability can explain up to 36% of PD heritability and that PD manifestation is often determined by multiple variants at different genetic loci. Thus, one of the current challenges in PD research stands in modelling the complex genetic architecture of this condition and translating this into functional studies.  Affecting 2% of the population over 65 , PD is cynuclein . At the ynuclein , accountsymptoms . Hence, in vitro, in vivo and in silico models [The complex aetiology of idiopathic PD is a rapidly expanding area of research, with a plethora of genetic and environmental factors associated with the lifetime risk of PD development ,10–12. Ao models ,21, subsCommon genetic variants identified through genome-wide association studies (GWAS) targeting increased risk of developing idiopathic PD  have impThe burden of multiple, common variants within risk loci, which may be present in a single individual, contribute to a polygenic risk score of lifetime PD development . ImportaCaenorhabditis elegans (C. elegans), provides an excellent model system for understanding underlying disease biology in neurodegenerative conditions [C. elegans [in vivo gene function models. C. elegans have been widely used as a model for ageing [C.elegans are highly amenable to neurobiology, as they have a fully mapped neural connectome [C. elegans neuronal system [C. elegans , known as ROC, with an adjacent C-terminal of ROC, known as COR domain. These are the defining feature of the ROCO protein family, of which LRRK2 is the most studied member. Adjacent to the ROC-COR is a kinase domain , as showLRRK2 PD , which hLRRK2 PD , NCT0405LRRK2 PD ). SeveraLRRK2 PD , therebyLRRK2 PD ,83–88. Tin vitro ,90, althin vitro ,92. LRRKin vitro ,94, suggin vitro ,86. Thisin vitro . In addiin vitro . Intriguin vitro ,92; howein vitro ,86.in vitro [in vivo [G2019S autophosphorylation activity 4-fold [R1441C mutation, located in the ROC domain, disrupts GTP hydrolysis, enhancing both substrate phosphorylation and autophosphorylation activity by approximately 3 to 4-fold [LRRK2 mutation carriers, with higher penetrance observed for LRRK2R1441C carriers [G2019S carriers [G2019S substitution is restricted to the kinase pocket of LRRK2, any alteration in the GTPase domain is likely to exert broader biochemical and cellular effects on protein function [The most common LRRK2 mutation associated with PD, G2019S, is located in the kinase domain and causes a modest 2-fold increase in kinase activity and phosphorylation of substrates in vitro  and in v[in vivo , while iy 4-fold . The LRRo 4-fold ,97,99. Tcarriers , althougfunction .LRRK2 PD present with classical PD symptoms and exhibit variable Lewy body pathology upon post mortem examination [LRRK2 PD cases exhibit significant tau accumulation [MAPT is within a consistent idiopathic PD GWAS locus [The majority of individuals with mination ,57. Intemulation ,100–102,mulation , particumulation ; howevermulation ,9. MoreoAS locus ,103, illAS locus ,106. TheAS locus , with α-AS locus  and are AS locus . MeanwhiAS locus , progresAS locus  and fronAS locus ; hence, LRRK2 is notable for exhibiting pleiomorphic pathology in neurodegeneration and in the peripheral immune system [LRRK2 locus have recently been identified through GWAS as candidates for increased survival in the tauopathy PSP [LRRK2 have been observed to increase susceptibility to Mycobacterium leprae infection [LRRK2 have been shown to increase the risk of Crohn’s disease [LRRK2 has remained a consistent candidate emerging from idiopathic PD GWAS [LRRK2 functional modelling will provide insights into idiopathic disease, a current challenge in PD research.e system . In termathy PSP . Additioathy PSP  and its athy PSP . This sunfection , the mic disease , an infl PD GWAS ,119–121 LRRK2 is a promising, druggable target for developing a disease modifying therapeutic for PD, with the potential to be applicable in some cases of idiopathic disease presented with LRRK2 hyperactivation . Howeverin vitro and in vivo models [LRRK2 have demonstrated that in human patient-derived cells and murine humanised models, LRRK2 levels can be stably reduced, leading to reduced LRRK2-mediated phosphorylation activity [LRRK2-ASO treated mice exposed to α-synuclein, while peripheral, off target effects of loss of LRRK2 expression were avoided through direct CNS injection of the ASO [LRRK2 are also entering early stage clinical trials . Furtheactivity . Reduced the ASO . ASOs ta03976349 ), with p03976349 , illustrC. elegans as a simple in vivo model for PD genes and as a platform for high-throughput screening of chemical and genetic modifiers [The development of novel, disease modifying therapeutics for PD hinges upon the thorough understanding of perturbed pathways and the identification of druggable targets that arise, often elucidated through gene functional studies. Substantial progress has been made in utilising odifiers ; howeverDictoyostelium discoideum [Drosophila melanogaster and C. elegans have only one, dLRRK2 and LRK-1 respectively. It has been hypothesised that LRRK2 arose as a gene duplication event, following the protostome-deuterostome split [C. elegans LRK-1 and LRRK2 is not yet established. Here, we will discuss current models of LRK-1 function, in contrast with established models of LRRK2 function in order to evaluate its suitability as a model for LRRK2 in PD.The ROCO proteins are conserved even in simple eukaryotes, such as the slime mould amoeba scoideum , in whicme split ,130. Stume split . In the LRRK2 PD, or synthetically mutated to ablate catalytic activity, are conserved in C. elegans LRK-1 [C. elegans LRK-1  has demonstrated that kinase activity of LRK-1 is crucial to its function in determining polarised vesicle localisation in axons of C. elegans sensory neurons, suggesting shared conservation in enzymatic activity and functionality between LRK-1 and LRRK2 [in vitro and in vivo in PD LRRK2G2019S mutant models [C. elegans protein UNC-101, an orthologue of the clathrin adaptor protein AP-1 [C. elegans model.Data obtained upon transgenic expression of t manner , while dt manner . Accordi in vivo . Expressnd LRRK2 ,160. LRRt models . Interesein AP-1 . Alteratein AP-1  and otheein AP-1 ,167, impC. elegans the WNT signalling pathways modulate neuronal development [C. elegans LRK-1G1876S  in wild-type, or lrk-1 deletion mutant background, revealed significant defects in canal-associated neuron development [C. elegans lines overexpressing human LRRK2G2019S, endogenous nematode WNT signalling pathway components and axon guidance genes had the largest effect sizes in determining dopaminergic neuron survival [C. elegans LRK-1 and vertebrate LRRK2 models further strengthens the conserved roles of LRK-1 and LRRK2.WNT/β-catenin signalling is an essential pathway in dopaminergic neurogenesis during development and also contributes to synapse formation and neuroprotection during ageing . As PD ielopment . Intereselopment . Furthersurvival . Thus, tC. elegans, UNC-23 and HSP-1, the orthologues of BAG2 and HSC70 respectively, interacts with LRK-1, highlighting existing conservation of protein interactions [unc-23 deletion mutants phenocopy the defects in synaptic vesicle localisation [lrk-1 deletion mutants [Comparative study of LRK-1 and LRRK2 interactomes further supports functional conservation and it might provide another strategy to identify candidate pathways as novel therapeutic targets . LRRK2 hractions . Furtherlisation , as seen mutants ,133,176.C. elegans [C. elegans could provide an excellent tool to drive interactome-based functional studies. These could be investigated in the context of the ageing organism, including environmental and genetic PD risk factors, utilising the high-throughput nature of this model to assess the efficacy of genetic and chemical disease modifiers.LRRK2 has an extensively characterised interactome; a stringent search through PINOT, a resource for obtaining quality controlled PPIs in humans and  elegans , indicatRAB29, also known as RAB7L1, has been a consistent PD GWAS hit [RAB29 has been stratified through unbiased LRRK2 PPI arrays as a candidate risk gene, which may bridge the gap between familial LRRK2 and idiopathic PD [LRRK2 and idiopathic PD may be furthered by functional studies of RAB29.RAB proteins are a superfamily of small GTPases, with diverse regulatory roles in vesicle formation, trafficking and endosomal transport. A subset of these RABs are key effector substrates of LRRK2 phosphorylation . RAB29, GWAS hit  and the GWAS hit , while RGWAS hit . RAB29 hathic PD ,179,181.RAB29 knockout had no impact on basal LRRK2 phosphorylation activity [R1441C, LRRK2Y1699C and LRRK2G2019S present a 4-fold increase of LRRK2 mediated phosphorylation of RAB29 [RAB29 has been demonstrated to be a selective master regulator of LRRK2, acting upstream and leading to LRRK2 kinase recruitment, localisation and activation on the Golgi membranes, via binding of RAB29 to LRRK2s ankyrin repeat domain ,182,183,activity , suggestof RAB29 , suggestC. elegans has an orthologue for RAB29, glo-1, which shows conservation in its function in the endolysosomal transport pathway [K157R has been detected in an idiopathic PD patient [C. elegans GLO-1 and is located within the G5 loop, involved in binding of small G-proteins to nucleotides [C. elegans GLO-1 has been shown to act upstream of C. elegans LRK-1 [C. elegans orthologue for the closely related human RAB32 and RAB38, and it is most similar to these proteins in terms of protein sequence identity [In vivo model organisms do not always show exact orthologue conservation with the human gene of interest. For example, mouse models display significant differences in biology and biochemistry of LRRK2 when compared with human [in vivo, in vitro and in silico models, along with findings reported in studies of PD patients. Thus, C. elegans GLO-1, when taken in conjunction with findings in other models, will be a useful model for further understanding RAB29 function, along with shedding insight into its close counterparts RAB32 and RAB38. pathway . A codin patient , howeverleotides ,188. Impns LRK-1 , congrueidentity ,136. Funth human . Despiteth human ,151,152,RAB29, RAB32 and RAB38 have been highlighted as genes of interest in various neurodegenerative conditions. Functional studies of RAB29 in the presence of C9orf72 hexanucleotide repeat [RAB38 has been identified as a significant GWAS hit in a behavioural variant subtype of FTD [RAB32 has been associated with ER stress and mitochondrial dysfunction in multiple sclerosis [in vivo reductionist model of their functions focusing on GLO-1 may be applicable to further research areas.GLO-1 may not just be a potential functional model with relevance to PD, as e repeat , causatie repeat . Additioe of FTD , while Rclerosis . This ilC. elegans both endogenous LRK-1 and neuronally expressed human LRRK2 on a lrk-1 deletion background have been shown to act downstream from GLO-1 in axon termination [in vitro [C. elegans GLO-1. On the other hand, key LRRK2 phosphorylation target sites T71 and S72 of RAB29 [G2019S or LRRK2R1441C in lrk-1 deletion mutants efficiently supresses the axonal abnormality phenotype exhibited in lrk-1 or glo-1 deletion mutants [glo-1 deletion mutant background was not investigated, nor was the Golgi localisation of both proteins [C. elegans. This highlights the need for additional studies to further assess the LRK-1/GLO-1 mechanistic conservation with the comparatively better understood LRRK2/RAB29 interplay.Congruent with the human RAB29/LRRK2 axis, in mination . Notablyin vitro , are conof RAB29  are not of RAB29 . In motoof RAB29 . Transge mutants , suggestproteins . Furtherproteins , as robuRAB32 and RAB38 implicate some shared pathways with RAB29, augmenting the relevance of glo-1 for further study. RAB32 and RAB38 have been demonstrated to act co-operatively to regulate lysosomal biogenesis and modulation of lysosome-related organelles (LROs) [C. elegans, GLO-1 modulates the biogenesis of LROs, which are intracellular compartments for storage, sharing some characteristics but coexistent with conventional lysosomes [C. elegans orthologue of AP-3, via C. elegans LRK-1 [glo-1 or apb-3, or double deletion mutants of these genes, show decreased LRO biogenesis [Functional studies of human s (LROs) . In C. eysosomes ,196. Furysosomes , which iysosomes . Likewisns LRK-1 , mirrorins LRK-1 . Additioogenesis , suggestMycobacterium leprae, the causative agent of leprosy [LRRK2 have been identified through GWAS as predisposing towards leprosy [RAB32 [LRRK2, prevalent in multiple human populations [LRRK2 PD mutations, resulting in antagonistic pleiotropy, with potential advantageous roles of mutation in immunity, but increased risk of PD development in later life [Similarly to RAB29, RAB32 and RAB38 also share convergent biology with LRRK2, although their mechanisms of interaction may differ. RAB32 and RAB38 have been shown to mediate phagosome restriction of intracellular pathogens, notably  leprosy . Interes leprosy , and a py [RAB32  has alsoy [RAB32 . Howeverulations , may conter life ,202.C. elegans and RNAi knockdown of GARP subunits in C. elegans expressing human LRRK2G2019S in the dopaminergic neurons [LRRK2 physically interacts with RAB32 and RAB38 through its armadillo domain , converg neurons  induces  neurons . This prD620N, was identified in 2011 in a small number of families with late onset, autosomal dominant PD, showing similar symptomatic presentation to idiopathic PD [Saccharomyces cerevisiae [C. elegans has a well conserved, direct VPS35 orthologue, vps-35 [D620N has been shown to enhance LRRK2 kinase activity 6-fold in vitro, in CRISPR engineered murine fibroblasts and neutrophils isolated from PD patients carrying VPS35D620N [D620NVPS35 PD, further highlighting the roles of LRRK2 and the endosomal network in neurodegeneration.A mutation in Vacuolar Protein Sorting 35, VPS35athic PD . VPS35 iathic PD . VPS35 arevisiae . C. eleg, vps-35 . InteresS35D620N . This suC. elegans thus far have demonstrated that VPS-35 and the retromer have a role in modulating WNT signalling, determining neuronal migration during development [VPS35 in WNT signalling is conserved in other invertebrate models, such as, but also in mammals as observed in cell culture studies [D620N mouse models have been reported to display WNT/ß-catenin signalling dysfunction [LRRK2 PD models [C. elegans, VPS-35 has been implicated in trafficking of the nematode orthologue of the excitatory AMPA glutamate receptor, GLR-1. Deletion of vps-35 leads to a significant reduction in the number of GLR-1 puncta on the post-synaptic surface of ventral nervous cord dendrites and suggests a role for the retromer in GLR-1 recycling [D620NVPS35 PD, APMA receptor trafficking was dysregulated and receptors were mislocalised [C. elegans VPS-35 and its human orthologue suggest retained functional conservation through evolution.Studies conducted in elopment ,213–215. studies , suggestfunction . The potfunction , convergD models , implicaecycling . Coherenocalised . This shLRRK2, VPS35 has been associated with tauopathies in in vivo and in vitro modelling [D620NVPS35 PD exhibit typical α-synuclein pathology, as due to the rarity of mutations in this gene autopsy studies to date have been very limited [D620N carrier [In vivo murine models with VPS35D620N endogenously knocked in exhibit progressive neurodegeneration driven by tauopathy [in vitro [D620N resulting in enhanced LRRK2 kinase activity [D620NVPS35 PD, as tauopathy is a hallmark of LRRK2 PD [G2019S has been demonstrated to enhance neuronal transmission of tau in mouse models [Similarly to odelling ,221. It  limited . In 2008auopathy , while ractivity  further LRRK2 PD . Additioe models . Tau, a C. elegans model may shed further insight into multiple pathologies.Like tauopathies, retromer dysfunction has been implicated in multiple neurodegenerative conditions, suggesting potential common perturbed pathways and an essential role in neuronal function. In AD autopsy studies, microarrays have illustrated that retromer is depleted in the hippocampus, within the dentate gyrus and entorhinal cortex, a region in which AD pathology is initiated . FurtherC. elegans have a tau-like orthologue, ptl-1 [ptl-1 is not rescued by expression of human tau isoforms [ptl-1 and human tau. Multiple studies have used transgenic expression of human tau isoforms, investigating the impact of overexpression of wildtype or pathogenic mutant tau in C. elegans. Overexpression of wild-type tau in C. elegans, or neuronal expression of A152T mutant tau [A152T in C. elegans leads to impaired associative memory, compared with animals expressing wildtype tau [LRRK2 and VPS35 linked PD, the emerging aetiological overlap with the tauopathies and the existence of ready to use tauopathy models in C. elegans, provides further opportunities for functional studies and modifier screens of tau pathology, with impacts on multiple neurodegenerative conditions.e, ptl-1 ,227 and e, ptl-1 . Loss ofisoforms , suggesttant tau , a risk tant tau ,230 havetant tau . Expresstype tau , and thitype tau , of whicSNCA gene on chromosome 4 [SNCA (A53T) was found to segregate with PD and dementia in a large family of Greek and Italian origin, the Contursi kindred [SNCA, was identified as the major constituent of Lewy bodies [SNCA missense mutations, as well as gene duplications and triplications have been linked to dominantly inherited PD and dementia, suggesting that an increase not only in the aggregation tendency but also in the expression level of α-synuclein can induce toxicity [SNCA locus is among the major determinants of PD predisposition in the general population [SNCA, as many others, is a pleiotropic PD gene and that PD follows an oligogenic pattern of inheritance [The first gene to be directly implicated in inherited PD was the mosome 4 . In 1997 kindred . Soon afy bodies . The aggy bodies , who frey bodies ,235. Sintoxicity . More repulation , with vapulation ,238,239.eritance ,240. A keritance .C. elegans do not possess an orthologue of SNCA; however, multiple C. elegans models have been generated expressing wild-type or mutant human SNCA as a transgene, in muscles, neurons or dopaminergic neurons [C. elegans models, have demonstrated that multiple PD relevant genes, such as orthologues of PINK-1, PARKIN, ATP13A2 and DJ-1, identified in Mendelian familial studies are modifiers of α-synuclein pathology [C. elegans. neurons ,247. The neurons ,246,247  neurons , therefoathology . This susubstantia nigra of rodents and primates, where they lead to Lewy body formation and neurodegeneration. Conversely, overexpression of the wild-type or mutant human α-synuclein in rodents does not correlate with pathological aggregate formation and degeneration of dopaminergic neurons in the substantia nigra [D. melanogaster and C. elegans recapitulates the major PD hallmarks such as impaired dopaminergic behaviour and neurodegeneration despite the lack of an orthologue [Many pathways linking α-synuclein and LRRK2 have been suggested, including cytoskeletal dynamics, ER/Golgi transport, mitochondrial homeostasis and functionality of the degradative systems, leading to the hypothesis that the use of LRRK2 inhibitors might be beneficial in the treatment of synuclein pathology ,251. Lacia nigra . Howeverthologue ,244.in vivo in rodent models and in vitro human derived SH-SY5Y cultures, and contrasted with C. elegans LRK-1 [lrk-1 deletion mutant C. elegans shows reduced aggregation of α-synuclein, expressed in the muscle [LRRK2 knockout rats, injected with AAV vectors of recombinant human α-synuclein. These animals showed significantly reduced number of axons immunoreactive for α-synuclein at 12 weeks [in vitro human SH-SY5Y culture study, this phenotype was determined as kinase activity dependent, with LRRK2G2019S enhancing α-synuclein propagation, through RAB35 phosphorylation, a mechanism demonstrated to be conserved in C. elegans [lrk-1 deletion mutant reversed the phenotype of reduced α-synuclein aggregation [C. elegans, despite the simplicity and evolutionary differences of the system.Similar studies of α-synuclein propagation with regard to LRRK2 have been undertaken ns LRK-1 . Interese muscle . In the 12 weeks , consist12 weeks ,126. In  elegans . Expressregation , furtherC. elegans model for neuron-neuron propagation of α-synuclein in vivo demonstrated that RNAi silencing of PD orthologue genes, including lrk-1, pdr-1, pink-1, vps-35, resulted in increased α-synuclein propagation between neurons [lrk-1 expression at the mRNA level has been shown to significantly increase in nematodes expressing α-synuclein in the muscle in the presence of the apoptosis inducer wedelolactone [C. elegans muscles. Dysregulation of LRRK2 mRNA expression has been described in post-mortem studies of individuals with idiopathic and LRRK2G2019S PD [C. elegans, with regards to the α-synuclein/LRRK2 interplay, further highlights the need to develop more precise genetic models of LRRK2 pathology in C. elegans, rather than depending solely on deletion and RNAi silencing studies.However, deletion models of PD orthologues, when used in conjunction with α-synuclein expression still need to be approached with caution. A study developing a  neurons . Furtherolactone  and the olactone . Both of2019S PD . HoweverC. elegans orthologues [C. elegans, detailed in C. elegans possess the optimal feasibility in terms of genetic manipulation, for the generation of oligogenic models, along with RNAi and chemical modulator screening to investigate the impact of genetic and environmental factors. Genome-wide RNAi screens are powerful, but could miss many potential modifiers, with subtle impairments that occur only in specific conditions. Thus, combined risk screens might constitute a novel era in PD research, for which a simple functional model is needed.The past two decades have witnessed a seismic change in our understanding of the genetic contribution to the aetiology of PD, culminating most recently in the 2019 meta-analysis of PD GWAS, identifying 90 risk loci for PD, that account for up to 36% of PD heritability . In Orthhologues , 64 of tC. elegans orthologues of Mendelian PD genes such as lrk-1 and vps-35 show promising functional conservation with their human counterparts LRRK2 and VPS35, when contrasted with a diverse range of in vitro and in vivo models. It is clear that in many model systems, LRRK2 is a key player in PD pathway disruption and may further bridge the gap in our understanding of Mendelian to idiopathic PD. GWAS risk genes, such as RAB29 (glo-1 in C. elegans), also show great promise for further functional modelling in C. elegans. This opens the possibility of developing new nematode models to further understand the functionality of novel candidate genes, rapidly emerging through the availability of vast genetic data. The development of CRISPR/Cas9 technologies has enabled the rapid generation of knockin point mutations in C. elegans orthologues of human genes [C. elegans is one of the easiest and cheapest multicellular eukaryotic organism to apply precise genome editing to, the choice of this simple nematode for studying PD biology provides fast, cheap in vivo modelling with great translational potential [C. elegans functional studies of other Mendelian PD genes and their orthologues, including PINK1, PARKIN, ATP13A2 and DJ-1, or PD risk genes, such as GBA1, have proven the strength of this invertebrate model, highlighting highly conserved functions of these genes and proteins in cellular pathways disrupted in PD, including mitophagy, lysosomal degradation and α-synuclein pathology [C. elegans models, replicating the human genetic changes and evaluating cross-talk of various cellular pathways in PD, will help in enabling the future development of novel, disease modifying therapeutics.As extensively detailed in this review, an genes ,258, altan genes ,259,260.an genes . As C. eotential ,29,51, aathology ,262–270.athology . Develop


According to the WHO, cancer was responsible for an estimated 9.6 million deaths in 2018, making it the second global leading cause of death. The main risk factors that lead to the development of this disease include poor behavioral and dietary habits, such as tobacco use, alcohol use and lack of fruit and vegetable intake, or physical inactivity. In turn, it is well known that polyphenols are deeply implicated with the lower rates of cancer in populations that consume high levels of plant derived foods. In this field, phlorotannins have been under the spotlight in recent years since they have shown exceptional bioactive properties, with great interest for application in food and pharmaceutical industries. Among their multiple bioactive properties, phlorotannins have revealed the capacity to interfere with several biochemical mechanisms that regulate oxidative stress, inflammation and tumorigenesis, which are central aspects in the pathogenesis of cancer. This versatility and ability to act either directly or indirectly at different stages and mechanisms of cancer growth make these compounds highly appealing for the development of new therapeutical strategies to address this world scourge. The present manuscript revises relevant studies focusing the effects of phlorotannins to counteract the oxidative stress–inflammation network, emphasizing their potential for application in cancer prevention and/or treatment. Seaweeds have been part of the human nutrition for centuries, particularly among the Far East populations such as China, Japan and Korea, to which seaweed cultivation and processing represent a great source of economic income. In contrast, Western countries have mainly explored seaweeds as a source of phycocolloids and certain fine biochemicals with industrial purposes, although the interest in their direct consumption emerged recently . Marine Among them, phlorotannins represent an important group of phenolic compounds, exclusively occurring in brown algae that can form simple structures of 126 Da to very large and complex polymers. Although the biosynthetic pathway of these compounds is still not consensual, it is known that they are formed via C–C and/or C–O–C oxidative coupling of several monomeric units of phloroglucinol, which in turn is known to be biosynthesized through the acetate–malonate pathway . AccordiOver the recent years, phlorotannins have drawn much attention from the scientific community due to the numerous biological activities and potential industrial applications they have demonstrated, including antioxidant, anti-inflammatory, anti-obesity and several others . Of thesIn this context, this review presents an overview of the resourcefulness of phlorotannins to modulate numerous mechanisms underlying oxidative stress and inflammation mechanisms and highlights their contribution for preventing and tackling cancer.Oxidative stress can be defined as an imbalance between the production of free radicals and reactive metabolites, commonly known as reactive oxygen and nitrogen species (ROS and RNS), and their elimination by protective mechanisms, referred to as antioxidants .In a normal situation, ROS and RNS have important signaling and physiological functions in human metabolism, which is equipped with several antioxidant mechanisms to maintain their concentration in balanced levels . HoweverThe activated immune cells respond with the generation of more reactive species in an event known as oxidative burst that promote a massive release and accumulation of ROS and RNS in the damaged area and contribute to produce more pro-inflammatory mediators . Until tCancer is a multistep process defined in three main stages: initiation, promotion and progression . OxidatiOne of the key characteristics of tumor cells is their increased survivability and capacity to resist apoptosis, which is a major barrier against tumorigenesis. Once triggered, the apoptotic signaling cascade unfolds in an orchestrated series of steps that culminate in the disruption of cellular membranes, the breakdown of nuclear and cytoplasmic skeleton, extrusion of cytosol, degradation of the chromosomes and fragmentation of the nucleus. In this process, there are essentially two types of components: the regulators, which are responsible for monitoring the extracellular and intracellular environment for conditions of normality or abnormality that influence whether a cell should live or die, and the effectors, which are responsible for the execution phase in which the cell progressively disassembles and gets consumed either by its neighbors or by phagocytic cells .c, a potent catalyst of apoptosis. These signals are however controlled by counterbalancing pro- and antiapoptotic members of the B-cell lymphoma 2 (Bcl-2) family of regulatory proteins. The antiapoptotic proteins  act by binding and suppressing the proapoptotic proteins (Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak)) in the mitochondrial outer membrane. When this suppression is lifted, Bax and Bak induce the permeabilization of the outer mitochondrial membrane causing the release of the cytochrome c, which in turn will activate the caspase signaling cascade, culminating in the activation of caspase-3, considered the most important of the executioner caspases, and other effector proteins that execute the selective obliteration of subcellular structures, organelles and genome [Many of the stimuli that trigger apoptosis converge on the mitochondria, which responds with the release of cytochrome d genome .L. Indeed, the overexpression of Bcl-2 has been linked to cancer resistance to chemo- and immunotherapy [In order to survive, tumor cells develop a variety of strategies that allow them to escape or limit apoptosis. One of the most common is the loss of function of tumor suppressor protein p53, which has been described as “the guardian of the genome” since it is responsible for controlling the stability of the DNA and upregulate the expression of Bax upon sensing DNA damage, thus inducing apoptosis . In factotherapy . Other totherapy .+), peroxyl (RCOO●), alkyl, HO●, and O2●− assays and a decrease of ROS in the presence of phlorotannins has been consensually observed [A. nodosum were shown to exert markedly higher DPPH● scavenging activity (IC50 = 6.3–7.7 µg/mL) than BHT (IC50 = 51 µg/mL), and those from F. vesiculosus (IC50 = 3.8–4.7 µg/mL) were even stronger than ascorbic acid (IC50 = 6.3 µg/mL) [F. vesiculosus, displayed a DPPH● scavenging activity better than α-tocopherol (IC50 = 3.76 against 5.93 µg/mL) and comparable to that of BHT (IC50 = 3.28 µg/mL) [Sargassum ringgoldianum showed approximately five times stronger O2●− scavenging activity than catechin (IC50 = 1.0 against 4.6 µg/mL) [As phenolic compounds, the most characteristic biological property of phlorotannins is their antioxidant activity, which has been extensively explored and is encompassed by a large number of studies in literature contemplating numerous phlorotannin-rich extracts or isolated phlorotannins from multiple algal species and geographic origins, obtained with different extraction procedures and evaluated by a multitude of antioxidant assays . Not onlobserved ,36,37,383 µg/mL) . Likewis8 µg/mL) . In a di6 µg/mL) .Ecklonia stolonifera, namely eckstolonol, dieckol, phlorofucofuroeckol A also revealed lower IC50 values  than ascorbic acid (10.3 µM) on DPPH● scavenging assay, thus evidencing remarkable antioxidant activity [Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, were twice more effective scavengers of DPPH●  than catechin, ascorbic acid and α-tocopherol . The same tendency was observed for the O2●− scavenging in which these compounds exhibited considerably lower IC50 values  than resveratrol, ascorbic acid or α-tocopherol  [Some isolated compounds from activity . In turnctively) .F. vesiculosus, F. serratus or A. nodosum [E. cava [2O2 and Cl−, were also observed in HL-60 cells treated with phlorofucofuroeckol A, diphlororethohydroxycarmalol, 7-phloroeckol and 6,6′-bieckol prior to a pro-oxidant stimulus [Lessonia vadosa and Macrocystis pyrifera were able to prevent UV-B-induced ROS and malformations in zebrafish [E. cava, were reported as effective inhibitors of intracellular ROS, lipid peroxidation and DNA damage either in ethanol, 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH), high glucose or UV-B-exposed zebrafish [Promising results have been observed in different cellular systems of oxidative stress as well, with several authors reporting a decrease of the intracellular ROS on cells treated either with phlorotannin-rich extracts or pure compounds ,47,48,49 nodosum ,51,52,53[E. cava ,55. Signstimulus ,56. In aebrafish . Likewisebrafish ,35,58,59Sargassum kjellmanianum by Kunming mice (5.0 g/kg body weight/day over 30 days) was shown to significantly protect the liver against the lipid peroxidation induced either by CCl4 or FeSO4-VC [A. nodosum phenolic-rich extract [In a different model, the prolonged consumption of high molecular weight phlorotannins from FeSO4-VC . Notably extract .F. vesiculosus, F. serratus, Pelvetia canaliculata and A. nodosum) [A. nodosum, H. elongata, F. serratus, F. vesiculosus, Pelvetia canaliculata, E. cava and Eisenia bicyclis phlorotannin extracts [2O2-induced HepG2 cells [2O2-induced HepG2 cells [Apart from the direct antioxidant properties, the capacity of phlorotannins to boost the auto-antioxidant defenses is a subject that has also shown promising results. For instance, superoxide dismutase (SOD) and catalase (CAT), which are two important detoxifying enzymes that are usually downregulated in cells under oxidative stress, were shown to be increased on different cell lines treated either with phlorotannin-rich extracts (from nodosum) ,51,52 ornodosum) ,68,69,70extracts ,50,51,71extracts ,65,70,72extracts . Indeed,G2 cells . SimilarG2 cells  and H2O2G2 cells , both thH. elongata in restructured meat given to hypercholesterolemic rats over 35 days significantly contributed for the restoration of the levels of GR and Cu,Zn-SOD in their livers. In a different approach, the administration of 55 mg/kg/day of L. japonica extract to streptozotocin-induced diabetes in Sprague-Dawley rats over five days not only significantly reduced the expression of XO, but also increased the levels of GSH, GPx and GR in the liver of the treated animals [Sargassum pallidum to Wistar rats with CCl4-induced hepatic injury was shown to remarkably increase both GSH and SOD levels in the livers of the treated animals [The phlorotannins capacity to upregulate the auto-antioxidant defenses has even been confirmed in vivo. According to Moreira et al. , the inc animals . Similar animals . Upregul animals  or dieck animals ,66. Phlo animals .In addition to their antioxidant properties, phlorotannins are also closely related to numerous inflammatory events, being capable of inhibiting the expression of pro-inflammatory cytokines, regulate the expression and/or activity of important enzymes and even interfere with the transcriptional regulation , making Eisenia and Ecklonia are perhaps the most studied in this field. The majority of the phlorotannins obtained from these seaweeds were proven of being able to inhibit the expression of cellular adhesion molecules, like ICAM-1 and VCAM-1 and cytokines, namely TNF-α, IL-1β and IL-6, in several cells of the immune system [● and PG2, as well as the expression of enzymes responsible for their synthesis, i.e., iNOS and COX-2, respectively, were found reduced in cells treated with E. cava, E. stolonifera and E. arborea extracts [Fucus, namely F. vesiculosus, F. guiryi, F. spiralis and F. serratus, dose-dependently inhibited the LPS-induced NO● secretion in Raw 264.7 macrophages, which is in line with previous studies from Zaragozá et al. [● production on PMA-stimulated macrophages after being treated with a phlorotannin-rich F. vesiculosus ethanol extract. Likewise, phlorotannin extracts of several species of Sargassum including S. horneri, S. sagamianum and S. patens were shown to affect the expression of NO●, PG2, IL-1β, IL-6 and TNF-α in LPS-stimulated Raw 264.7 macrophages [S. fulvellum ethanolic extract prevented the expression of NO●, PG2, TNF-α, COX-2 and iNOS [Macrocystis pyrifera not only exerted inhibitory effects towards NO●, TNF-α and iNOS but also stimulated the expression of IL-10 which is an important anti-inflammatory cytokine responsible for the suppression of cytokines secretion [Due to their wide distribution and ecological importance, seaweeds from the genus e system ,80,81,82extracts ,85,86,87extracts ,89. Otheextracts  recentlyá et al. , who alsrophages ,92,93, wand iNOS . In turnE. cava ethanolic extract to LPS-stimulated Raw 264.7 macrophages dose-dependently diminished the levels of TNF-α, IL-1β, IL-6, iNOS, COX-2, NO● and PG2 via inhibition of the nuclear NF-κB translocation [E. cava ethanolic extract ability to inhibit NF-κB nuclear translocation and binding to DNA in LPS-stimulated BV2 microglia cells was caused by the inhibition of both IκBα degradation and mitogen-activated protein kinase (MAPK) pathway, which also contributes for the regulation of pro-inflammatory cytokines biosynthesis [Evidence have shown that the anti-inflammatory capacity of these compounds is much likely to be related with their modulatory effects on important transcription factors that mediate the inflammatory signaling cascade. In fact, it has been proven that the addition of location . Notablyynthesis . Yayeh eynthesis  reportedynthesis  and 6,6′ynthesis . In phloynthesis .25–35-stimulated PC12 pheochromocytoma cells, PMA-induced MG-63 human osteosarcoma cells, high glucose-induced HUVEC endothelial cells, hypoxia-induced primary mouse hepatocytes and gamma-irradiated primary rat hepatocytes [In a different study, Lee et al.  reportedatocytes ,111,112,Myagropsis myagroides ethanolic extract in ICR mice ears (90 μg/ear) as well as 6,6′-bieckol (30 μg/ear) isolated from the same species prior to the induction of ear edema with PMA significantly suppressed the ear swelling in approximately 67% and 64%, respectively, which was very close to the effects observed for indomethacin, used as positive control (at 1 mg/ear) [Cystoseira sedoides, Cladostephus spongiosis and Padina pavonica as well as pure compounds such as eckol, 6,6′-bieckol, 6,8′-bieckol, 8,8′-bieckol, phlorofucofuroeckol A and phlorofucofuroeckol B in mice ear edemas induced with several different sensitizers including arachidonic acid (AA), 12-O-tetradecanoylphorbol-13-acetate (TPA), oxazolone (OXA) and xylene (XYL) [C. sedoides [Sargassum fulvellum phlorotannin purified fraction prior to UV-B irradiation effectively suppressed the protein expression of TNF-α, COX-2 and iNOS, and their downstream products, PGE2 and NO●, respectively [● [● levels as well as iNOS and COX-2 expression were described on zebrafish exposed to high glucose levels and treated with dieckol [●, PGE2 and high-mobility group box-1 (HMGB-1), improving their survival rates in a dose-dependent manner [4-injured livers [S. horneri ethanol extract by oral ingestion over 15 days. Notably, in an intervention study carried out on human volunteers, the oral administration of a capsule containing A. nodosum polyphenol extract seems to attenuate the ex vivo production of IL-6 in blood samples cultured with LPS, although no statistical significance was achieved [A common method to evaluate the anti-inflammatory properties of a potential therapeutic compound in vivo is through the induction of ear or paw edema in mice, usually with PMA or carrageenan. Indeed, using this method, Joung et al.  found th mg/ear) . Similarne (XYL) ,104,105.sedoides . In BALBectively , while iively [● . Likewis dieckol . Oral adt manner , whereasd livers  reportedachieved .A. nodosum and Alaria esculenta exhibited dose-dependent anti-proliferative effects on Caco-2 growth showing IC50 values of 33 μg/mL and 7 μg/mL, respectively. Similarly, the phenolic-rich extracts of three different Cystoseira species  demonstrated promising growth inhibitory effects against three different tumor cell lines, namely A549 lung cell carcinoma cells, HCT15 colon cell carcinoma cells and MCF-7 breast adenocarcinoma cells, although their IC50 values (17.9–90.3 µg/mL) were much higher when compared with those of cisplatin (1.5–1.9 µg/mL), an important chemotherapeutic drug used for the treatment of several neoplasms. However, when tested on normal cells, i.e., non-tumor cells (Mardin–Darby canine kidney cells and rat fibroblasts), it was found that all the three Cystoseira extracts presented much lower toxicity than cisplatin, suggesting that, although their antitumor capacity may be weaker compared to the commercial drug, their specificity for tumor cells is much higher, and thus administration of higher doses could be possible for achieving the desired antitumor effect without compromising the viability of normal cells [The fact that phlorotannins can counteract oxidative and inflammatory states make them able to indirectly display some potential to prevent and/or mitigate oxidative stress- or inflammation-based tumor initiation and progression. Notwithstanding, the role of phlorotannins as anti-neoplastic agents has been suggested to go much further, with several authors reporting that these compounds may act as anti-proliferative, antimetastatic and anti-angiogenic agents at different types of cancer . Accordial cells . In factal cells  carried Fucus evanescens, Laminaria cichorioides and Costaria costata, the 60% ethanolic extract from the former, which was the highest in polyphenols (10.1% dry matter), displayed the strongest inhibitory effects on DLD-1 and HT-29 human colorectal adenocarcinoma cells growth , while the extract from L. cichorioides harvested in May, which had the lowest polyphenol content (1.4% dry matter), exhibited the lowest inhibitory capacity (9% and 23% respectively). The authors reported identical observations for C. costata, since extracts from samples collected in May (phenolic content of 2.0% dry matter) exhibited slightly lower inhibitory effects than the extract from samples collected in July (phenolic content of 2.7% dry matter). These results are in agreement with a previous study carried out on HeLa cervical adenocarcinoma cells in which Macrocystis integrifolia and Nereocystis leutkeana methanolic extracts, both containing a phenolic content of 3.9 µg gallic acid equivalents/g extract, showed stronger anti-proliferative effect than the extract of Laminaria setchellii, which contained only 1.8 µg gallic acid equivalents/g extract [S. muticum, collected along the North Atlantic coast during the same harvesting season, exhibited different anti-proliferative activities towards HT-29 cells according to their total phlorotannin content, i.e., the extracts from samples collected in Norway which had the highest phlorotannin contents (approximately 5 mg/g extract) displayed growth inhibitions almost two times stronger than those observed for samples from Portugal, which contained approximately 4 mg/g extract [Many authors have reported a causality link between the phlorotannin content of the extracts and the extracts’ anti-proliferative effects. This is the case of the work reported by Imbs et al. , who fou extract . More re extract .F. vesiculosus acetone extract was shown to induce apoptosis in different pancreatic cell lines through cell cycle arrest, which is comparable to the effects of common chemotherapeutic drugs clinically used, such as gemcitabine [c from the mitochondria or the expression of several caspases including caspase-3, -7, -8 and -9 in different tumor cell lines [L was described in HT-29 cells treated with increasing concentrations of phloroglucinol (12.5–50 µg/mL) [One of the most common mechanisms responsible for phlorotannins anti-proliferative activity is their capacity to interact with multiple mediators of the apoptotic signaling cascade and thus trigger cell death by inducing apoptosis ,131,133.citabine . Compounll lines ,133. Addll lines  and to dll lines , while ill lines . Likewis0 µg/mL)  reported0 µg/mL) .c release and caspases expression), the administration of dieckol (40 mg/kg bw over 15 weeks) to Wistar rats with N-nitrosodiethylamine-induced hepatocarcinogenesis significantly inhibited the expression of vascular endothelial growth factor (VEGF), involved in the tumor angiogenesis, and metalloproteinases-2 and -9, both participating in the extracellular matrix processing and intimately related with tumor invasion and migration [In vivo antitumor effects of eckol in a sarcoma xenograft-bearing mice model, were demonstrated to be not only resultant from its capacity to interfere with the expression of caspase-3, caspase-9, Bcl-2 and Bax, but also from its capacity to inhibit the expression of epidermal growth factor receptor (EGFR), a transmembrane protein that is usually overexpressed in many cancer types, and to stimulate the mononuclear phagocytic system, recruiting and activating dendritic cells, promoting the tumor-specific Th1 responses, increasing the CD4+/CD8+ T lymphocyte ratio, and enhancing cytotoxic T lymphocyte responses. Therefore, in addition to its anti-proliferative properties, eckol also exerts antitumor effects via stimulation of the host immune response . Using aigration .E. cava phlorotannin-rich extracts on human fibroblasts, HT1080 fibrosarcoma cells [Ishige okamurae were shown to not only inhibit MMP-2 and MMP-9 expressions, but also to enhance the levels of tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 in HT1080 cells, at both gene and protein levels [Indeed, antimetastatic effect is another characteristic of phlorotannins that greatly contributes for their antitumor properties. One of the mechanisms by which phlorotannins exert antimetastatic effects is through inhibition of the expression of matrix metalloproteinases (MMPs), which are important enzymes involved in degradation of extracellular matrix proteins and tissue remodeling and are associated with various physiological or pathological processes such as morphogenesis, angiogenesis and metastasis. Among these, MMP-2 and MMP-9 are of particular relevance since they are usually overexpressed in tumor cells and are capable of degrading type IV collagen of basement membrane, the first barrier for cancer invasion . Inhibitma cells  and A549ma cells , while dn levels . In TPA-n levels , while in levels , evidencn levels . In a din levels  demonstrAn alternative antimetastatic mechanism of dieckol was demonstrated in A549 lung cancer cells, in which migratory and invasive phenotype were remarkable abrogated via activation of tumor suppressor protein E-cadherin, an adhesion molecule that plays a key role in the epithelial-mesenchymal transition process, i.e., the mechanism by which epithelial cells lose their cell-cell adhesion characteristics and gain migratory/invasive properties .The recovery of phlorotannins from brown macroalgae is a subject that is triggering a great economic interest, thus a domain open to technological innovations. These innovations are often translated in patents, i.e., legal documents that ensure the right of innovative inventions, in which the new product or process developed to answer a particular problem is described, with very meticulous technical information. Patent review is therefore a useful approach to have an idea of the progress made in a certain technological field. In Ecklonia cava extract with high antioxidant activity describing a process for producing an extract from E. cava containing 10 to 15% of phlorotannins using supercritical extraction with CO2. The extracts produced this manner are claimed to exert high antioxidant activity and are intended for application as raw ingredients in the formulation of cosmetics and functional foods [A. nodosum containing 44.4 mg of phloroglucinol equivalents/g extract and B. bifurcata containing 62.4 mg of phloroglucinol equivalents/g extract. This invention was developed aiming the application of these extracts as active ingredients in cosmetic and food formulations [In 2006, Cosmax Co. (South Korea) filed the patent al foods . Later, ulations .Ecklonia stolonifera Okamura having anti-oxidative activity”. This composition contains eckstolonol, eckstolonol pentaacetate, eckol, phlorofucofuroeckol A and dieckol isolated from E. cava and is aimed to be used either as a food preservative, to prevent oxidative spoilage, or as a pharmaceutical ingredient for the preparation of powders, granules, tablets, capsules, suspensions, emulsions, syrups and/or other formulations with anti-aging properties via inhibition of cell membrane lipid peroxidation [Ishige okamurae”, describing a procedure to retrieve pure phlorotannin compounds from I. okamurae, namely phloroglucinol, 6,6’- bieckol and diphlorethohydroxycarmalol, through a conventional extraction method using methanol followed by solvent partitioning with different solvents and chromatography separation on Sephadex LH-20. The inventors showed that the compounds obtained with this method were highly effective at scavenging free radicals and reducing oxidative stress in different cells. As such, the inventors further describe an antioxidant composition using these compounds which is intended to be used as an active ingredient for the development of novel foods, cosmetics or pharmaceuticals targeting ROS-related diseases [Ecklonia cava extract as an effective component” consists of an anti-inflammatory composition composed of phlorofucofuroeckol-A isolated from E. cava and E. stolonifera via conventional extraction with ethanol and further purification using solvent partitioning and silica gel column chromatography. The inventors claim this composition has suppressive effects against both iNOS and COX-2 expression, addressing it as an active ingredient to be applied in the formulation of pharmaceutical products including injectables, tablets, capsules or syrups for treating inflammatory diseases. An additional application as a functional ingredient for developing health promoting beverages is also included in this document [Two applications for phlorotannin-based antioxidant compositions were filed by the Pukyong National University (South Korea). The first was filed in 2003 for a “Composition comprising phlorotannins isolated from the extract of xidation . In 2008diseases . A thirddocument .Ascophyllum sp. and Fucus sp. via conventional extraction with water, following additional purifications and diafiltrations for removing alginates, iodine and low-molecular weight compounds. This extract, claimed to contain between 11 to 15% of polyphenols, was employed in an anti-inflammatory composition to be used with medicinal purposes for the prevention/treatment of inflammatory events, mainly targeting arthritis and allergy [In 2007 a patent entitled “Algae extract containing polyphenols”, was filed by Diana Naturals (France), describing a method for producing an anti-inflammatory extract from  allergy .An innovative composition with anti-matrix metalloproteinase effects was registered in 2006 by Pukyong National University Industry-University Cooperation Foundation (South Korea) under the title “Composition containing phlorotannin for inhibition of matrix metalloproteinase activities”. This innovative product contains phlorotannins from the eckol type, particularly dieckol and bieckol, which are claimed to inhibit the activity of the matrix metalloproteinase-1, -2, -8 and -9 more effectively than the tetracycline type pharmacological agents. Matrix metalloproteinases are collagenolytic enzymes involved in numerous diseases, including inflammation, hyperparathyroidism, diabetes, corneal ulcer, osteoporosis, gastric ulcer, cancer and metastasis. Therefore, this composition is aimed to be applied as an active ingredient in the formulation of powders, tablets, capsules, ointment compositions, solutions, gels, pastes, patches or granules for the multiple therapeutic approaches such as treating wound, cancer metastasis, rheumatoid arthritis, inflammation, hyperparathyroidism, diabetes, corneal ulcer, osteoporosis, stomach ulcer, acne, burn, periodontal diseases, arteriosclerosis and fracture . The samOverall, the research and innovation in this field of phlorotannins applied as antioxidants, anti-inflammatory or anti-tumor are slowly but steadily moving forward in a positive direction, with the Asian countries, particularly Korea, clearly leading the way since most of the patents filled are registered in this country.In conclusion, the data herein gathered make it clear that brown algae phlorotannins and phlorotannin-rich extracts are unanimously acknowledged by their remarkable effects on critical steps involved in the pathogenesis of cancer, either directly acting as pro-apoptotic, anti-proliferative, anti-metastatic or antiangiogenic agents or indirectly through inhibition of the multiple mechanisms of the oxidative stress-inflammation network which are intimately linked to tumorigenesis. These compounds are, therefore, endowed with a high versatility and capacity to promote and improve health status by acting on different fronts, opening a path for possible innovations on different fields. In fact, some important steps have already been taken in this direction, which can be measured by the number of phlorotannin-related patents that have been slowly but steadily emerging over the last years, meaning that these compounds are indeed object of great economic interest. This strengthens the hypothesis that phlorotannins could be used as valuable naturally occurring pharmacological alternatives for the development of novel therapeutic strategies for treating not only cancer but also other diseases.


Mitochondria are the vital organelles in heart tissue. Mitochondrial Ca2+ homeostasis is particularly important for maintaining the physiological and pathological mechanisms of the heart. Mitochondrial Ca2+ homeostasis plays a key role in the regulation of cardiac energy metabolism, mechanisms of death, oxygen free radical production, and autophagy. The imbalance of mitochondrial Ca2+ balance is closely associated with cardiac remodeling. The mitochondrial Ca2+ uniporter (mtCU) protein complex is responsible for the uptake and release of mitochondrial Ca2+ and regulation of Ca2+ homeostasis in mitochondria and consequently, in cells. This review summarizes the mechanisms of mitochondrial Ca2+ homeostasis in physiological and pathological cardiac remodeling and the regulatory effects of the mitochondrial calcium regulatory complex on cardiac energy metabolism, cell death, and autophagy, and also provides the theoretical basis for mitochondrial Ca2+ as a novel target for the treatment of cardiovascular diseases.Mitochondria are the sites of oxidative metabolism in eukaryotes where the metabolites of sugars, fats, and amino acids are oxidized to harvest energy. Notably, mitochondria store Ca However, the importance of mitochondrial Ca2+ buffering capacity, the kinetics of mitochondrial Ca2+ uptake/release, and the molecular mechanism of [Ca2+]m-mediated ATP and ROS production remain controversial.High cytoplasmic Ca+ uptake , and somntration . Althougeostasis ,133. VDAchondria ,41,134.  limited , and VDAthe cell . Furtherate Ca2+ ,137. Vasscovered . A year  (MICU1) , which r and NCX , which mnium red ,41. In tscovered ,143,144.nium red . In mammnium red . Recentlreported ,147. Theting MCU . The Ca2xchanger . The uptr sodium . When inmyocytes . Additioocardium  and flow via NCX . Increas2+ influx through mitochondrial Ca2+ channels and transporters m and plays an important role in regulating mitochondrial Ca2+ homeostasis c) changes to the MCU complex and activates mitochondrial in situ Ca2+ uptake [2+] sensor on the matrix side m uptake rate, increased mtROS, and enhanced cell death induced by [Ca2+]m overload c has been shown to be increased in failing guinea pig hearts relative to [Na+]c in normal hearts [2+ outflow and reduces [Ca2+]m in failing hearts ), suggesting that mitochondrial Ca2+ overload is a key determinant of heart failure  or substantially affect cardiac function under basic conditions [The increased incidence of malignant arrhythmias can contribute to heart failure, diabetic cardiomyopathy, senile cardiac insufficiency, and hereditary diseases . Abnormadiseases . Garcia diseases . Thus, tnditions . The incnditions .2+ transport and Ca2+-induced membrane permeability transition, inhibit the activation of the MAPK/JNK pathway, inhibit foam-cell formation, and reduce the progression of atherosclerosis [Studies have shown that drugs targeting mitochondria interfere with mitochondrial Caclerosis . CsA is clerosis . In a Phclerosis ,279.2+ regulation. These peptides primarily include Ca2+ channels and pumps that regulate ER localization and small peptides or proteins that regulate mitochondrial Ca2+ channels, affecting Ca2+ transport from ER to mitochondria  as a polypeptide has been shown to inhibit IP3R-mediated Ca2+ release and protect cells from pro-apoptotic Ca2+ transfer to mitochondria [2+-induced Ca2+ release via Ca2+ influx. Further studies have shown that a short fragment of the cytosolic II–III loop of the DHPR, called peptide A, induces RyR1-mediated Ca2+ release [2+ uptake by directly or proximally acting on VDAC and MCU. Short peptides from the N-terminal of VDAC1 and LP4  may significantly inhibit mitochondrial Ca2+ uptake and lung cancer cell migration by blocking the interaction between VDAC1 and Bcl-XL, McL-1 [2+ levels, and this event was associated with VDAC1 oligomerization, cytochrome c release, and apoptotic cell death. Peptides derived from accessory protein sequences of VDAC1 show potential therapeutic applications [2+ uptake into mitochondria, making cells more resistant to the pro-apoptotic release of Ca2+ from the MAM-targeted ER [2+ signaling, such as ischemia-reperfusion injury [2+ flux or homeostasis. The fungus-derived cyclic peptide cyclosporine A (CsA) desensitizes mPTP to Ca2+ and inhibits pore opening. In recent years, an increasing number of CypD-selective and non-immunosuppressive derivatives of CsA  have been developed as promising cardioprotective agents, as their ability to reduce the harmful effects of acute myocardial infarction has been observed in different models [2+ retention in diabetic animals, reduce oxidative damage of heart tissue, and improve the glucose utilization rate [2+ permeable hemichannels, contributing to a mitochondrial Ca2+ overload and loss of energy and ion gradients, leading to cell death [2+ overload and infarct size during cardiac ischemia-reperfusion [2+ crosstalk that deserve further study. In conclusion, preclinical data using decoy or regulatory peptides acting on major Ca2+ channels in the ER-mitochondria will be needed to facilitate the rapid development of these tools into practical therapies.It is promising to develop therapeutic peptides targeting mitochondrial Cachondria . Anti-apchondria . An IP3Rchondria . The mec release . The lip release . Some pe release . CalcinsL, McL-1 , or HKI.ications . Furthergeted ER . Owing tn injury , Alzheimn injury , and Parn injury . Studiesn injury . The rect models . Anothert models . Studiesion rate . More reion rate . Furtherll death ,296. RNYerfusion . In addi


Prostate cancer (PC) is the second most common cancer worldwide and steroid hormone plays an important role in prostate carcinogenesis. Most patients with PC are initially sensitive to androgen deprivation therapy (ADT) but eventually become hormone refractory and reflect disease progression. The aim of the study was to investigate the genes which regulate the steroid hormone functional pathways and associate with the disease progression of PC. We identified a panel of eight-gene signatures that modulated steroid-hormone pathways and predicted the prognosis of PC using integrative bioinformatics analysis of multiple datasets validated from external cohorts. This panel could be used for predicting the prognosis of PC patients and might be associated with the drug response of hormonal therapies. Moreover, these genes in the signature could be potential targets to develop a novel treatment for castration-resistant PC therapy.CA2, CYP2E1, HSD17B, SSTR3, SULT1E1, TUBB3, UCN, and UGT2B7 was established. Patients with higher expression of this gene signature had worse progression-free interval in both univariate and multivariate cox models adjusted for clinical variables. The expression of the gene signatures also showed the aggressiveness consistently in two external cohorts, PCS and PAM50. Our findings demonstrated a validated eight-gene signature could successfully predict PC prognosis and regulate the steroid hormone pathway.The importance of anti-androgen therapy for prostate cancer (PC) has been well recognized. However, the mechanisms underlying prostate cancer resistance to anti-androgens are not completely understood. Therefore, identifying pharmacological targets in driving the development of castration-resistant PC is necessary. In the present study, we sought to identify core genes in regulating steroid hormone pathways and associating them with the disease progression of PC. The selection of steroid hormone-associated genes was identified from functional databases, including gene ontology, KEGG, and Reactome. The gene expression profiles and relevant clinical information of patients with PC were obtained from TCGA and used to examine the genes associated with steroid hormone. The machine-learning algorithm was performed for key feature selection and signature construction. With the integrative bioinformatics analysis, an eight-gene signature, including  In 2020, prostate cancer (PC) was the second most frequent cancer and the fifth leading cause of cancer death among males worldwide, with an estimated 1.4 million newly diagnosed cases, causing approximately 374,000 deaths [The strategy of treating prostate cancer is based on the risk group. For lower-risk patients, localized therapy, including surgery or radiotherapy is recommended. For higher-risk or metastatic group, androgen deprivation therapy (ADT) plays a major key role in the treatment . Many paBRCA1, BRCA2, CDK12, ATM, FANCD2, and RAD51C were identified in 19% of tissue samples from 333 PC patients collected in a TCGA dataset [Prostate cancer is often associated with somatic mutations that occur in the tumor, with an estimated 89% of metastatic CRPC tumors containing a potentially actionable mutation . These m dataset . Olapari dataset .To identify the targeted genes related to cancer progression, Cox proportional hazard regression is traditionally well-accepted as the standard algorithm for survival analysis. However, in these high-throughput datasets, the number of variables is substantially higher than the number of samples. Compared to traditional regression statistics, machine learning (ML) algorithm has shown the ability to detect the key features predicting cancer prognosis from these complex datasets , and althttps://portal.gdc.cancer.gov/) and genes were annotated with ENSEMBLE v91 [https://www.cell.com/cms/10.1016/j.cell.2018.02.052/attachment/bbf46a06-1fb0-417a-a259-fd47591180e4/mmc1.xlsx in 1 January 2018). The RNA-seq profiles, clinical data and survival data were matched according to the TCGA barcode. Only patients with prostate adenocarcinoma (PRAD) were retrieved for further analysis.The processed RNA sequencing (RNA-seq) profile and the corresponding clinical data of patients were curated from the DriverDB database ,18,19. BMBLE v91   , Kyoto E2 fold-change value greater than 1 and adjusted p-values, calculated by DEseq2, smaller than 0.05.DEseq2  was usedBoth univariate and multivariate survival analyses were conducted under the R environment with Survival library package . Two surLasso (Least Absolute Shrinkage and Selection Operator), was a combination of ridge regression and was used to select gene feature ,26. A pohttp://www.thepcta.org/; accessed on 1 December 2021) platform.The expression of the construct gene signature was examined with external cohorts. Two PC classifiers, Prostate Cancer Classification System (PCS) and PAM 50, were used from the public Prostate Cancer Transcriptome Atlas , pathway names in KEGG, and reaction names in REACTOME, which contained more than or equal to 3 of 8 candidate genes  were dow2 fold change was greater than 1 with the p-value less than 0.05, between TP and NT. After differential expression analysis, 92 significantly differentially expressed (DE) genes were identified , with these oncogenes showing positive association with the disease progression. As a consequence, these genes may serve as targets to improve outcome of PC [p-value and hazard ratio (HR) of PFI, are shown in To identify the genes associated with PC progression, the univariate CoxPH model was conducted to analyze each gene in the aspect of 5-year PFI among 497 patients from TCGA-PRAD cohort, respectively. PFI is an endpoint including cancer progression or cancer death. Two of 497 patients were excluded given incompatible gene expression levels, while 495 patients were stratified according to the median RNA expression levels. Genes with a me of PC . The corCA2, CYP2E1, HSD17B, SSTR3, SULT1E1, TUBB3, UCN, and UGT2B7 was selected, and the corresponding Lasso coefficients were estimated. The linear combination of eight gene expression values weighted by the corresponding Lasso coefficients was used to construct the eight-gene signature score. Patients were stratified into two groups, a high-risk group (n = 248) and a low-risk group (n = 247), according to the median gene-signature score. The eight-gene signature was evaluated by the univariate CoxPH model, and the prognostic ability of the 8-gene signature was evaluated by the log-rank test. The correspondent Kaplan–Meier plot of the patients from TCGA-PRAD database demonstrated that the patients with higher expression of eight-gene signature had significantly worse 5-year PFI compared to those with lower expression . The higher Gleason scores (>7) also had significantly worse PFI compared to lower Gleason score . Other parameters, i.e., age, PSA level, and TNM staging, did not reach statistical significance in this model.A multivariate CoxPH model was conducted to adjust possible clinical confounding variables, including age, baseline prostate-specific antigen (PSA) level, Gleason score, and TNM staging. As shown in p < 0.001; Ranksums test fold change = 0.385, p < 0.001, p < 0.001; Rank-sums test Fold change = 0.22, p = 0.023, In this study, we examined the expression of identified eight-gene panel based on these two well-known PC classifiers, PCS and PAM50, using data from an independent cohort . ProstatCA2, CYP2E1, HSD17B, SSTR3, SULT1E1, TUBB3, UCN, and UGT2B7 were involved in regulating “steroid hormone biosynthesis” in the KEGG database; “metabolism of steroid hormones” and “HSP 90 chaperone cycle for steroid hormone receptors” in Reactome; and “cellular response to steroid hormone stimulus”, “steroid hormone biosynthetic process” and response to steroid hormone in GO. In order to elucidate the crucial functions regulated by the identified eight genes, functional annotation was performed using GO, KEGG, and Reactome pathway databases. Only the pathways regulated simultaneously by at least 3 genes among the 8-gene signature were regarded as key functions. The cross interaction of these 8 genes among KEGG, Reactome, and GO databases is shown in As shown in CA2, CYP2E1, HSD17B, SSTR3, SULT1E1, TUBB3, UCN, and UGT2B7, respectively. Among these eight genes, five genes, including HSD17B, SSTR3, SULT1E1, TUBB3, and UGT2B7, were identified to be associated with both PC prognosis and hormone refractoriness previously. HSD17B3, which is the key enzyme for the metabolism of progestins to adrenal androgens and subsequent conversion to testosterone, is significantly upregulated in castration-resistant metastases compared to untreated PC [SULT1E1 were identified to be associated with time to treatment failure (TTF) in 68 patients with CRPC under the treatment of abiraterone [Tubulin-β3 (TUBB3) gene is one of the seven β-microtubule proteins normally expressed in neuronal cells and testis. TUBB3 has been reported to be associated with phosphatase and tensin homolog (PTEN) and neuroendocrine differentiation, which might induce an aggressive type of PC [UGT2B7 gene, belonging to UDP-glucuronosyltransferase (UGT) enzymes, is known particularly for its wide spectrum of specificity for all classes of steroids, such as conjugating (1) 5a-reduced metabolites of mineralocorticoids, glucocorticoids, progestins and androgens, and (2) 5b-reduced C21 and C19 steroids [CYP2E1 and UCN, have shown the association with prostate carcinogenesis only but revealed no clear role of the prognosis of PC receiving ADT. The differential expression of urocortin (UCN) between prostate malignancy and normal tissue has been indicated [CYP2E1 gene might be associated with a two-fold increased risk for the development of PC [Steroid hormone receptors, especially AR, provide a critical pathway for PC progression, and ADT remains the backbone for PC treatment. However, it has been revealed that genetic aberrations result in the resistance of ADT, through either androgen-dependent or androgen-independent mechanisms . The fireated PC ,33. Amoneated PC . The oveeated PC . Estrogeeated PC . Six sinraterone . Tubulinpe of PC . The exppe of PC  but is ape of PC . The UGTsteroids . The rolsteroids . In the ndicated ,44. The nt of PC . Lastly,nt of PC  and breant of PC . To sum Although the univariate cox analyses revealed statistical significance of our eight-gene signature in terms of PFI, there may be clinical confounding variables that led to bias in the analyses. To overcome this issue, a multivariate Cox regression model adjusting for commonly seen clinical variables in PC was conducted. These clinical variables, including age, TNM staging, PSA at diagnosis, and Gleason score, were selected given clinical practice guidelines using these four factors together to stratify the subgroup of PC and further different treatments . The Glen = 1321) of human PC transcriptome profiles from 38 distinct cohorts [External cohort validation is needed for bioinformatics analysis. Two well-validated classifiers (PCS and PAM50) were used in our study. PCS is a classification system derived from a large cohort ( cohorts . Analysi cohorts ,51. Zhao cohorts . The expUGT2B7 expression is associated with the poor prognosis of PC, suggesting that UGT2B7 might play a critical role in driving castration resistance.Functional annotation has been widely applied on analyzing the biological processes of collecting genes based on molecular function, biological role, subcellular location, and the regulatory pathways ,53. The CA2, CYP2E1, HSD17B, SSTR3, SULT1E1, TUBB3, UCN, and UGT2B7, predicting the prognosis of PC. These signature clinical and biological associations are consistent among multiple datasets, including TCGA, DriverDBv3, PCS, PAM50 GO, KEGG, and Reactome. These genes not only regulate the pathways related to steroid hormone but modulate the function of metabolism and signal transduction as well. This eight-gene signature could become a promising panel of biomarkers to screen the prognosis of PC patients and might be pharmacological targets to develop therapies for CRPC. Further large prospective cohort studies are required for further validation, and additional experimental studies in vivo are also needed to provide robust evidence of the role of anti-cancer therapy.In conclusion, the mechanisms regarding PC resistance to anti-androgens are incompletely understood, thus the novel and precisely targeted therapy for CRPC is still in demand. Using the ML-based bioinformatics analysis, we successfully identified an eight-gene signature consisting of


Under severe Fe2+ toxicity, reactive oxygen species (ROS) are crucial, although molecular mechanisms and associated ROS homeostasis genes are still unknown. In this study, a comparative RNA-Seq based transcriptome analysis was conducted to understand the Fe2+ toxicity tolerance mechanism in aromatic Keteki Joha. About 69 Fe homeostasis related genes and their homologs were identified, where most of the genes were downregulated. Under severe Fe2+ toxicity, the biosynthesis of amino acids, RNA degradation, and glutathione metabolism were induced, whereas phenylpropanoid biosynthesis, photosynthesis, and fatty acid elongation were inhibited. The mitochondrial iron transporter (OsMIT), vacuolar iron transporter 2 (OsVIT2), ferritin (OsFER), vacuolar mugineic acid transporter (OsVMT), phenolic efflux zero1 (OsPEZ1), root meander curling (OsRMC), and nicotianamine synthase (OsNAS3) were upregulated in different tissues, suggesting the importance of Fe retention and sequestration for detoxification. However, several antioxidants, ROS scavenging genes and abiotic stress-responsive transcription factors indicate ROS homeostasis as one of the most important defense mechanisms under severe Fe2+ toxicity. Catalase (CAT), glutathione (GSH), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were upregulated. Moreover, abiotic stress-responsive transcription factors, no apical meristem (NAC), myeloblastosis (MYB), auxin response factor (ARF), basic helix-loop-helix (bZIP), WRKY, and C2H2-zinc finger protein (C2H2-ZFP) were also upregulated. Accordingly, ROS homeostasis has been proposed as an essential defense mechanism under such conditions. Thus, the current study may enrich the understanding of Fe-homeostasis in rice.Lowland acidic soils with water-logged regions are often affected by ferrous iron (Fe Fe functions as an electron acceptor or donor actively participating in photosynthesis and respiration  is an essential micronutrient, participating in various vital processes in cell metabolism due to its redox status between ferrous  across the root plasma membrane. Strategy II is based on the uptake of Fe3+ phytosiderophore complexes. Although being a Strategy II plant, rice (Oryza sativa L.) possesses Fe2+ transporter genes (OsIRT1 and OsIRT2) that can directly uptake Fe2+ from the soil  on the root surface acts as a barrier to the uptake of Fe2+ into root tissues. Fe plaque is formed due to the rhizospheric oxidation of Fe2+ to Fe3+ by oxygen transport from shoots to roots  plays an important role by retaining excess Fe in root and avoiding translocation into the shoot. Defense 3  includes compartmentalization, disposal, or storage of Fe inside shoots by vacuolar iron transporter (OsVIT1/OsVIT2), and OsFER1/2. Defense 4 [reactive oxygen species (ROS) detoxification] includes enzymatic detoxification, scavenging of ROS by antioxidants. Defense 1–3 was highlighted to work on mild to moderate Fe excess conditions and do not seriously affect rice growth. In contrast, defense 4 was hypothesized to work at a molecular level under Fe severe conditions that causes bronzing and inhibit plant growth and development  related to the ROS scavenging system. The results provided a comprehensive transcriptome resource that may enrich the understanding of Fe-homeostasis under FeThe Keteki Joha rice variety seeds were collected from Regional Agricultural Research Station, Assam Agricultural University, Titabor, Assam, India . Seeds were surface sterilized with 0.01% sodium hypochlorite (NaOCI) and germinated in Petri dishes by incubation at 28°C for 3-5 days. Germinated seedlings were grown in a plastic pots, containing 400 ml of Hoagland nutrient solutions, prepared in distilled water  value of more than 6.5 was used to prepare the sequencing library prepared by Truseq Stranded RNA Library Prep Kit-Plant . Finally, 100–150 bp RNA Seq was performed by Illumina HiSeq 2000 platform .Rice Genome Annotation Project was used as a reference sequence database for assembly . GO terms were ranked by employing the Fishers' exact statistical test and classic algorithm of topGO. The ggplot2 package of the R program was used to represent the graphical representation , KEGG entry no T01015, and GHOSTX score ≥100 were considered for pathway mapping. Finally, the KEGG pathway was analyzed using the KEGG mapper. Expression value (log2FoldChange) of the DEGs were integrated into their corresponding KEGG gene of each sample, and multiple states pathway maps were rendered by the Pathview web tool  was used to manipulate the featureCounts data to create the DEXSeq object.Differential exon usage of the DEGs was inferred by Bioconductor package DEXSeq  was compared with control (70 μM Fe). Briefly, about 100 mg of fresh tissues with 3 biological replicates were grounded to powder with Liquid Nitrogen (LN2). Total RNA was isolated using the RNAqueous Phenol-free total RNA isolation kit of Invitrogen, Thermo Fisher Scientific. The cDNA synthesis was performed using the iScript Reverse Transcription Supermix for RT-qPCR kit, Bio-Rad. Gene-specific primers were designed with t-test performed in the R program  compared with control.The statistical analysis of gene expression by qRT-PCR was analyzed with a minimum of 3 biological replicates. The significant mean difference between the treated (2.5 mM Fe) and control (70 μM Fe) was estimated by the student's 2+ toxicity , vacuolar mugineic acid transporter (OsVMT), and acireductone dioxygenase 1/submergence-induced protein 2 (OsARD1/OsSIP2) were highly upregulated. Other upregulated genes include nicotianamine synthase 3 (OsNAS3), transporter of mugineic acid 3 (OsTOM3), mitochondrial iron transporter (OsMIT), root meander curling (OsRMC), OsFER1/OsFER2, ABC transporter of mitochondrion 3 (OsATM3), yellow stripe-like 13 (OsYSL13), FRD3-Like Protein 1 (OsFRDL1), natural resistance-associated macrophage protein 2 (OsNRAMP2), and positive regulator of iron homeostasis 2 (OsbHLH58/OsPRI2). The Fe2+ transporter OsIRT1, OsIRT2, OsNRAMP1, and Fe/Mn/Cd transporter OsNRAMP5 were downregulated. In addition, Fe-phytosiderophore transporters like OsYSL15 and OsYSL2 were also downregulated. Other downregulated genes include OsTOM1, OsNAS1, OsNAS2, mitochondrial iron-regulated gene (OsMIR), iron man (OsIMA1), phosphoribosyl pyrophosphate synthetase (OsPRPPS), dehydratase-enolase-phosphatase (OsDEP), Fe-deficiency-induced protein 4 (OsIDI4), basic helix-loop-helix protein 133 (OsbHLH133), deoxymugineic acid synthase 1 (OsDMAS1), ribose 5-phosphate isomerase (OsRPI), oligopeptide transporter 7 (OsOPT7), formate dehydrogenase (OsFDH), efflux transporter of nicotianamine 1 (OsENA1), iron-related transcription factor 2 (OsIRO2), OsNRAMP1, OsIDI2, FER-like Fe deficiency-induced transcription factor (OsbHLH156/OsFIT) etc. , OsYSL9, OsTOM2, IDE-binding factor 2 (OsIDEF2), OsYSL5, OsPEZ2, ferric reductase oxidase 1 (OsFRO1), and OsFRO2 were not expressed in roots  etc. . Besidesin roots .OsVIT2 and OsFER1/OsFER2 were highly upregulated in the seedling stage, whereas they were not differentially expressed in the mature stage , OsENA1 were highly upregulated, followed by OsYSL13, OsMIT, OsRab6a, OsYSL10, OsTOM3, OsIDI1L/OsARD1, OsVMT, OsbHLH58/OsPRI2, etc. , etc. , etc. . A total1), etc. .Comparative analysis of the top 500 up/down regulated genes indicates that genes are distinctly expressed in both roots and leaves. Only one gene was found to be commonly upregulated in the roots and leaves of both seedlings as well as of the mature stage. Similarly, only 21 genes were found to be downregulated in all of the tissue samples . On comp2+ toxicity, stress-responsive, and source of H2O2 generation genes such as respiratory burst oxidase homolog (Rboh) were differentially expressed in both roots and leaves , receptor-like cytoplasmic kinase 253 (OsRLCK253), phosphate dikinase (OsPPDKA), and cyclin-like F-box domain-containing protein (OsMsr9) were differentially expressed. In addition, submergence tolerance genes pyruvate decarboxylase (OsPDC4), phosphoenolpyruvate carboxykinase (OsPEPCK), OsARD1/OsSIP2, and B12D protein were differentially expressed. Low nutrient responses genes haemoglobin 1 (OsHB1), OsHB2, and low phosphate root 2 (OsLPR2) were mostly upregulated , response to stimulus (GO:0050896), secondary metabolic process (GO:0019748), response to abiotic stimulus (GO:0009628), lipid metabolic process (GO:0006629), etc. . Similar2+ toxicity. Among the several pathways, significantly and differentially expressed pathways were evaluated by the pathview-web tool. Log2FoldChange value of each DEGs was integrated into their respective KEGG gene ID, and their active or passive state was evaluated, and accordingly, multiple states pathways were presented , OsPEZ2, OsSAM1, OsIDEF2, OsTOM3, and OsVMT used different exons between the control and treated groups  to investigate how it responds under stress conditions. Fe homeostasis genes were well characterized in Fe deficient condition; however, the molecular mechanism of Fe toxicity in rice plants is still not well empathized  was upregulated in all the tissues, thereby involved in metal transport. The characterization of the OsTOM3 gene needs to be further studied for better clarification about its role in Fe2+ toxicity. The OsPEZ1 induced in root tissue plays a significant role in the efficient translocation of protocatechuic acid (PCA) and caffeic acid from roots to shoots in rice -nicotianamine and iron (III)-deoxymugineic acid into the cell. The expression of OsYSL10 is like OsYSL9 and may also participate in a similar mechanism. OsYSL13 was expressed in roots, and mature leaves participate in the distribution of Fe from older leaves to new leaves . In the current study, OsARD1 showed high upregulation in all the tissue types that might enhance rice tolerance under Fe2+ toxicity. Overexpression of OsARD1 also leads to the tolerance of abiotic stresses like submergence, drought, and salinity , i.e., ROS detoxification has been hypothesized as a possible mechanism of tolerance against severe Fe2+ toxicity. Excess Fe2+ is lethal to plants under acidic conditions as they catalyze H2O2 and produce a highly toxic hydroxyl free radical, termed as Fenton reaction  as the ultimate reductant . However, Mn-OsSOD1 was not found to be differentially expressed in roots, whereas it was fractionally upregulated in leaves of both stages. The remaining H2O2 is detoxified by the PRX-TRX system or by the enzymes of the ascorbate-glutathione cycle  group and as a precursor of phytochelatins (PCs)  and drought stress  and NAD(P)H dehydrogenase (NDs), play an essential role in minimizing ROS production , OsIDEF2 (NAC TF family), and OsARF16 (ARF family) reported to modulate the expression of Fe-related genes and integrate auxin signals, respectively, thus play a critical role in Fe homeostasis  and WRKY TFs were hypothesized to regulate severe Fe2+ toxicity tolerance in rice  was reported to promote root tolerance to Fe toxicity via a nitric oxide pathway  transcription factors were downregulated in roots, whereas upregulated in leaves. Overall, differentially expressed TFs under Fe2+ toxicity are very intense, which need to be further studied to elucidate their specific role in understanding Fe2+ toxicity in rice. However, identification of their downstream target genes will remain important to uncover the possible signaling pathways in rice. Recent computational analysis has identified several cis-regulatory elements (CREs) and conserved motifs among the Fe homeostasis related genes which are important for their regulation by transcription factors  was also upregulated in all tissue types, which plays an essential role in the plant metabolism in response to biotic and abiotic stresses  signaling pathway are highly upregulated in leaves, under Fe2+ toxicity. Apart from the ROS homeostasis genes, differential expression of various abiotic stress-related genes might indicate the existence of multiple stress tolerance strategies in rice. Besides all the above, L-lactate dehydrogenase B (LDH-B) was the topmost upregulated gene in roots, which catalyzes the reversible NAD-dependent interconversion of pyruvate to L-lactate. KEGG pathway analysis revealed that it is involved in glycolysis/gluconeogenesis, cysteine and methionine metabolism, pyruvate metabolism, propanoate metabolism, and biosynthesis of secondary metabolites. Its stereoisomer D-2-hydroxyglutarate dehydrogenase/D-lactate dehydrogenase was recently characterized to be conferred multiple abiotic stresses by maintaining cellular homeostasis in rice (Mitsuhara et al., 2+ toxicity in rice.In addition, upregulation of low nutrient responsive genes indicates nutrient deficiency upon Fe2+ detoxification under severe Fe2+ toxicity, regulated by transcription factors. Future molecular research work targeting the characterization of the strongly induced genes will be important to understand the overall Fe2+ detoxification mechanism in rice. In addition, the genes identified in this study may serve as a valuable insight for further research and development of rice genotypes for Fe2+ toxicity tolerance.In this study, ROS homeostasis was identified as the key mechanism for FeThe original contributions presented in the study are publicly available. This data can be found here: Bioproject: PRJNA680441. BioSamples: SAMN16879912, SAMN16879913, SAMN16879914, SAMN16879915, SAMN16879916, and SAMN168799.PR: conceptualization, methodology, writing–original draft preparation, data curation, and analysis. SD: manuscript writing and analysis. MR: methodology. AP: manuscript writing. UC: editing. BT: investigation, writing–reviewing, and editing. AT: reviewing and supervision. SP: reviewing, editing, and supervision. All authors contributed to the article and approved the submitted version.The RNA Sequencing was funded by the Department of Biotechnology, Government of India with a grant no. DBT-NER/AGRI/29/2015.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.