Saccharomyces cerevisiae, but limited reports exist in filamentous fungi, though they are frequently the subject of nanoparticle biosynthesis and targets of antifungal metals. In most cases, microarray analyses uncovered resistance mechanisms as a response to metal exposure. In yeast, metal resistance is mainly due to the down-regulation of metal ion importers, utilization of metallothionein and metallothionein-like structures, and ion sequestration to the vacuole. In contrast, metal resistance in filamentous fungi heavily relies upon cellular ion export. However, there are instances of resistance that utilized vacuole sequestration, ion metallothionein, and chelator binding, deleting a metal ion importer, and ion storage in hyphal cell walls. In general, resistance to zinc, copper, iron, and manganese is extensively reported in yeast and partially known in filamentous fungi; and silver resistance lacks comprehensive understanding in both.Metal nanoparticles used as antifungals have increased the occurrence of fungal–metal interactions. However, there is a lack of knowledge about how these interactions cause genomic and physiological changes, which can produce fungal superbugs. Despite interest in these interactions, there is limited understanding of resistance mechanisms in most fungi studied until now. We highlight the current knowledge of fungal homeostasis of zinc, copper, iron, manganese, and silver to comprehensively examine associated mechanisms of resistance. Such mechanisms have been widely studied in  The increasing applications of fungal–metal interactions have led to the need for research on their contributions to fungal resistance ,2. In na4) hydrates to copper (II) sulfate pentahydrate (CuSO4 5H2O) and then dissociates into Cu2+ + SO42−. Upon dissociation, Cu2+ can then be reduced by fungal proteins for uptake. More recently, metals in the form of nanoparticles have gained interest for use as antifungals, which has fueled the escalation of nanoparticle production [Metals can exist in various forms such as salts, oxides, sulfates, and nanoparticles. Fungi are able to utilize metal ions from these compounds after dissociation, which leaves unbound ions available for uptake and transport. For example, in the presence of water, copper sulfate , the regulation of transcription (25%), transcription factor activity (19%), and response to chemical stimuli (15%) [c oxidate, a catalyst within the electron transport chain [Metals play critical roles in fungal homeostasis. They are required for various biochemical processes, usually as enzymatic cofactors. Metals most recognized for their importance in fungi are copper, iron, zinc, and manganese. Pertaining to zinc, approximately 5% of fungal proteomes correlate to zinc-binding proteins, and 8% of yeast genomes correlate to zinc-binding proteins. In the model yeast li (15%) ,107,108.rt chain ,48. Ironrt chain . Manganert chain ,98.Magnaporthe oryzae [Very few metals that are not considered essential have also been identified in some fungal–metal interactions; these include magnesium and molybdenum. Magnesium is a well-known micronutrient in other eukaryotic organisms, however, its homeostasis in fungi is undetermined. Only in recent years has magnesium been identified as a requirement for virulence in the agriculturally relevant fungus e oryzae . Molybdee oryzae ,129. Othe oryzae ,130,131.2Cys6), found only in Ascomycetes (with the singular exception of Lentinus edodes) [Zinc is a transition metal required for fungal survival and is necessary for various functions, including the structuring of nucleic acids, physical growth and, most predominately, protein folding ,133. In  edodes) ,135,136. edodes) ,134.Aspergillus niger, Fusarium oxysporum, and Botrytis cinerea, respectively [F. oxysporum [4)2 and zinc sulfate (ZnSO4) also inhibit mycelial growth that produces mycotoxins and reduces the production of mycotoxins themselves [In agriculture, fungal infections threaten food security by increasing global crop loss ,138. Traectively ,142,143.xysporum . This isxysporum ,146. Fusxysporum ,148,149.emselves ,151.S. cerevisiae, zinc transport occurs through several protein groups; the ZIP protein family , the CDF protein family , the ferrous transport protein Fet4, and others -IRT (iron-regulated transporter)-like protein (ZIP) family and the cation diffusor facilitator (CDF) protein family ,153. In d others  [105,107avorable ,156. In avorable ,28. In aavorable ,157,158.inc ions ,159,160.Apergillus fumigatus, genes in the ZIP family  also regulate zinc transport [zrfA and zrfB, orthologues of S. cerevisiae ZRT1 and ZRT2, respectively, encode zinc membrane transporters that operate in acidic, low-zinc environments and are activated by transcription factor ZafA [zrfC gene product is an alkaline zinc transporter activated in high pH, zinc-limiting conditions [zrfD/E/H are not restricted by pH and can function in either acidic or alkaline environments [F. oxysporum, zrfA and zrfB are also zinc importers regulated by transcription factor ZafA [F. oxysporum to adapt to a zinc-limiting environment, such as if the host enacts nutritional immunity to deprive it of this essential metal [Ustilago maydis UmZRT1 and UmZRT2 genes, and Cryptococcus neoformans Zip1 and Zip2 are homologous to S. cerevisiae ZRT1 and ZRT2, respectively, with similar transport function [C. neoformans Zrc1 is homologous to S. cerevisiae Zrc1 and mediates zinc transport into the vacuole to prevent toxicity and decrease zinc sensitivity [In filamentous Ascomycota, such as ransport ,27,161. tor ZafA . Conversnditions ,27. zrfDronments . In F. otor ZafA . During al metal . Basidiofunction ,24,26. Ssitivity .S. cerevisiae ZIP proteins and homologs. The next section will discuss how negative homeostatic interventions can result in toxicity.Mechanisms of zinc uptake and transport in fungi are mostly conserved through Malassezia spp. and induces toxicity by increasing cellular zinc uptake [Malassezia spp., ZPT toxicity in S. cerevisiae is not a result of increased zinc uptake, rather of increased copper uptake which overloads homeostatic systems [S. cerevisiae, ZnO NPs reduce ergosterol biosynthesis which, in turn, increases cellular leakage (up to 24%) and depolarization, reduces cell wall integrity, and increases the occurrence of ROS [Zinc-based antifungal compounds have mechanisms of toxicity that vary between species. Zinc pyrithione (ZPT), is a zinc ionophore often used to treat fungal dandruff caused by c uptake ,165. ZPTc uptake . In cont systems ,167,168.e of ROS . In filae of ROS . In the e of ROS . Zinc se2+ to the vacuole (up to 100 mM) in S. cerevisiae, or the endoplasmic reticulum in C. albicans (Zrc1) [S. cerevisiae, though it is not yet fully understood [2+ uptake, and in that role increases tolerance through interactions with Mg, where Mg competes with Zn2+ for uptake, increasing intracellular Mg and restricting Zn [2+ resistance [High-zinc environments can be detrimental to fungi; therefore they must possess resistance mechanisms to overcome toxicity. In yeast, resistance relies on the upregulation of Zrc1 and Cot1, which sequester Zns (Zrc1) ,108,169.derstood . They sucting Zn . In filasistance ,172,173.+, and copper(II), Cu2+ [Copper is also a transition metal and presents itself in oxidation states copper(I), CuI), Cu2+ ,48. It iI), Cu2+ ,175. Som+ (cuprous oxide); however, in the environment, it often exists as Cu2+ (cupric oxide) and must undergo reduction. In S. cerevisiae, cupric reductase Fre1, transcribed by Mac1, reduces Cu2+ to Cu+, making it readily available for uptake via high–affinity membrane transporters of the copper transporter (Ctr) protein family, Ctr1 and Ctr3 or low-affinity copper transporter Fet4 , uptake, and oxidation [FET3 is regulated by transcription factor Aft1 (activator of ferrous transport) in iron-deficient conditions and its gene product contains four Cu+ binding sites where copper serves as a cofactor for enzyme activation [After uptake, Cuxidation ,179,180.tivation ,178,181.tivation ,182.2•−) [2•− are ROS that cause cellular damage and toxicity and must be effectively dismutated to prevent stress; therefore, delivery of copper to Sod1 is critical [S. cerevisiae, the cytosolic copper chaperone Lys7 acquires Cu+ and delivers it to Sod1, with high specificity [2•− being successfully detoxified to hydrogen peroxide (H2O2) and molecular oxygen (O2); H2O2 is now readily available for further detoxification to water via catalyst Cct1 [+ transport to MTs Cup1  and Crs5 is also integral to cellular detoxification [CUP1 and CRS5 at elevated copper concentrations [+ binding sites, respectively, and are responsible for buffering cytosolic copper to maintain safe intracellular copper concentrations [CUP1 [Another enzyme dependent on copper is the cytoplasmic Cu/Zn superoxide dismutase (Sod1). This is an antioxidant for superoxide anions (O2•−) ,184. O2•critical ,185,186.cificity . Once Soyst Cct1 ,187,188.fication . Both MTtrations ,189. Cuptrations ,191. Thons [CUP1 ,191.S. pombe follows a pattern of copper transport similar to S. cerevisiae. Extracellular Cu2+ is reduced to Cu+ by cell surface reductases before uptake [+ can then be transported across the cell membrane, depending on the current cell cycle [+ uptake, and during meiosis, Mfc1  is responsible [+ and ctr5+ is regulated by transcription factor Cuf1, and expression of mfc1+ is regulated by transcription factor Mca1, both of which are activated or deactivated by the absence or presence of sufficient copper levels, respectively [+ to respective organelles [+ to unmetalated Sod1 in a copper-limited environment, activating Sod1 [+ to prevent toxic cytosolic levels [S. pombe plays a similar role to Atx1 in S. cerevisiae. In S. pombe, Atx1 is also located in the cytosol and responsible for carrying Cu+ to Ccc2 [S. cerevisiae [+ to an active site on the CAO, where copper  activates it [S. pombe‘s Cox17 is an orthologue to S. cerevisiae Cox17, sharing 38% identity and is located in the mitochondrial intermembrane space [+ it is delivered to Sco1, Sco2, and Cox11 for copper loading to cytochrome c oxidase subunits [e uptake ,36. Cu+ ll cycle ,35,36. Dponsible ,35,36. Eectively ,36. Onceganelles . Pccs ising Sod1 . In highc levels . Atx1 in to Ccc2 ,42. Peterevisiae ,42. Atx1vates it ,42,192. ne space . Once Cosubunits ,48.Aspergillus fumigatus, studies have shown similarities to S. cerevisiae and S. pombe in copper uptake. Cu2+ must also be reduced before uptake, however, there is some ambiguity regarding the reductases responsible [S. cerevisiae FRE or FRE3) [S. cerevisiae Ctr1) transport Cu+ into the cytosol and serve as enzymatic cofactors [Filamentous fungi are also important in assessing copper homeostasis, as these organisms depend on copper for growth and virulence in pathogenic species. In the pathogenic Ascomycete ponsible . This reor FRE3) ,193,194.ofactors ,39. CtrAofactors ,195,196.ofactors ,195,196.Fibroporia radiculosa and the edible white-rot fungus Pleurotus ostreatus, reported some details. In F. radiculosa, only the regulation of intracellular Cu+ concentration has been unveiled, by three, unnamed copper ATPases and one gene of unknown function, CutC, [P. ostreatus, membrane protein Ctr1 is involved in copper uptake and shares homology with the low-affinity copper transporter PaCtr2 of the Ascomycete Podospora anserine (20%) and the high-affinity S. cerevisiae copper transporter, Ctr1 (20%) [S. cerevisiae and S. pombe; however, more research is needed in other Ascomycetes and Basidiomycetes.Limited knowledge exists on copper homeostasis in Basidiomycetes. Studies in two Basidiomycetes, the brow-rot fungus n, CutC, . In P. or1 (20%) . This reS. cerevisiae, cupric sulfate (CuSO4) and copper oxide nanoparticles (CuO NPs) significantly reduce growth in a dose dependent manner, with the toxicity of both potentially related to Cup2 [CUP2 increases copper sensitivity, suggesting that a mechanism of toxicity could be reducing or inactivating its regulation, resulting in decreased Cu+/MT binding and increased cytosolic Cu+ [Candida spp., CuO NPs have also shown toxic capabilities by inducing porous cell membranes [Rhizoctonia solani, a copper (II)–lignin hybrid had high efficacy and significantly reduced the number of plants attacked by R. solani [Copper contains antifungals that have been investigated against various fungi. In  to Cup2 ,198,199.olic Cu+ ,201. Giaolic Cu+ . In Candembranes . Copper responsible for cellular detoxification and Cu+ uptake [CUP1  and CRS5 (~8-fold) and the downregulation of FRE1, FRE7, and CTR1  [4 sensitivity in cup2Δ mutants indicates MTs, coupled with decreased Cu2+ reduction and decreased Cu+ uptake, are likely to be employed as mechanisms of copper resistance [Since copper is implicated as an antifungal agent, its ability to evade copper toxicity must be continuously evaluated. In + uptake . Exposurctively) . This fosistance ,200.Aspergillus spp., P-type ATPase CrpA has Cu+ exporting activity that aids in cellular detoxification, increasing Cu+ resistance [Fusarium graminearum, copper exposure upregulates FgCrpA (ATPase exporter) and the MT FgCrdA as a means to prevent over accumulation, with the predominate method being Cu+ export activity [F. oxysporum, upregulation of oxidoreductase activity may decrease susceptibility to oxidative stress that can be induced by excessive copper exposure [F. radiculosa, where increased production of copper oxalate increases resistance [Less is known about copper resistance in filamentous fungi. In sistance ,193,209.sistance ,49. In Factivity . In F. oexposure . In Basisistance . However2+) or ferric (Fe3+) iron [A. fumigatus and F. oxysporum, survival depends on the ability to sequester iron from the host and a well-functioning homeostatic system to maintain this delicate balance [Iron (Fe) is a transition metal belonging to group eight of the periodic table and can exist as ferrous (Fe3+) iron ,212. As  balance ,214. Inc balance ,216,217.S. cerevisiae, two iron uptake systems are described, the reductive and nonreductive systems. The reductive system recognizes Fe2+ salts and chelates for uptake through importers, while the nonreductive systems utilizes iron siderophores [FRE1 expression induces the reduction of Cu2+ when transcription factor Mac1 is bound, and Fe3+ reduction occurs via binding of transcription factor Aft1 [3+ reduction, Fe2+ is then ready for uptake by a six domain, transmembrane, metal transporter, Fet4 [2+ uptake in iron-restricted cells [FET4 is repressed by Rox1, which has two binding sites in the FET4 promoter region [rox1Δ mutants have increased sensitivity to Cd under aerobic conditions [2+/H complex [SMF1 also results in significant iron uptake [3+ substrates such as ferric chelates, salts, and siderophores [2+ then transitions through the Fet3/Ftr1 complex [+ binding domains that must be metalated for activation [2+ to Fe3+ for passage to the cytosol via iron permease Ftr1 [3+ is not fully elucidated. Generally, in rophores ,220,221.rophores ,223. Forrophores . Since ttor Aft1 ,181,225.er, Fet4 ,55. Fet4nditions . A secon complex ,52. Thisn uptake ,227. Higrophores ,219. Fe2 complex . Fet3 istivation ,178,181.ase Ftr1 ,178. TheS. cerevisiae is incapable of producing siderophores, but can sequester siderophores produced by other microorganisms via siderophore iron plasma membrane transporters Arn1—Arn4 [3+ storage reservoir until the cell needs iron; in this event, Fe3+ is reduced via metallo-reductases, or released via ferrichrome degradation [ARN family, responsible for transporting tri-acetyl-fusarinine to the cytosol; it is unclear if Arn2 is located anywhere else aside from the plasma membrane when tri-acetyl-fusarinine is unavailable [S. cerevisiae, but based on the function of other siderophores it may be reasonable to conclude that, upon cellular entry, ferric entero-bactin is also used as an Fe3+ storage system.The nonreductive system utilizes siderophores. rn1—Arn4 ,224. Arnrn1—Arn4 ,220,228.rn1—Arn4 ,220,228.radation . Arn2  show that heme degradation is catalyzed upon cellular entry via heme oxygenase [S. pombe also biosynthesizes heme and is encoded by hem1+, hem2+, hem3+, hem12+, hem13+, hem14+, hem15+, and ups1+ [Heme is an iron-containing compound and its acquisition and biosynthesis are the finally discussed mechanisms of iron uptake in ire iron ,241. S. and Shu1 ,57. Shu1and Shu1 ,57. The and Shu1 ,63. Ironxygenase ,57,242. nd ups1+ ,57. In ind ups1+ ,57,243.S. pombe, regulation mechanisms must be in place to prevent over-accumulation. Mercier, Pelletier, and Labbé identified the gene pcl1+ to play a role in vacuolar iron storage [pcl1+ shares homology to S. cerevisiae Ccc1, an iron vacuolar transporter, and it has been shown that pcl1Δ mutants have increased sensitivity to iron; this together with the study of Mercier, Pelletier, and Labbé suggests that Pcl1 might play a similar role in iron storage in S. pombe [S. pombe; however, there are apparent gaps in knowledge of specific processes.In addition to iron acquisition in  storage . pcl1+ sS. pombe ,239. As S. pombe ,57,245. U. maydis, a pathogenic fungus that causes corn smut disease and whose virulence is associated with iron acquisition [S. pombe [Sdi1 and Sid2, and both are negatively regulated by transcription factor Urbs1 [In filamentous fungi, iron homeostasis is less documented. It has been investigated in uisition ,121. TheS. pombe ,246,247.S. pombe ,121. In S. cerevisiae and fungal pathogens, but they have also demonstrated that targeting and interfering in iron acquisition mechanisms can also be detrimental. Reports indicate that iron or iron compounds are fungistatic against F. oxysporum and its mycotoxins in a dose dependent manner [A. fumigatus and F. oxysporum in mice [S. cerevisiae, iron toxicity is related to the ability of the cell to transport cytosolic iron to the vacuole via Ccc1 [ccc1Δ mutants could not transfer cytosolic iron to the vacuole under anaerobic conditions, even with the overexpression of iron mitochondrial transporter Mrs3, effectively inducing toxicity [Iron is involved in many biological processes, but can be toxic in excess. Studies have shown its toxicity in t manner ,217. In  in mice ,93. Resuvia Ccc1 ,248. LinS. cerevisiae achieves iron resistance through the downregulation of iron import systems via Aft1, or activation of vacuolar transporter Ccc1 [YAP5 increases iron sensitivity, while its overexpression dramatically reduces cytosolic iron [CCC1 affects iron resistance and the vacuolar ability to store excess iron in order to prevent toxicity. Another vacuolar gene, VMA13, might also play a potentially novel role in iron tolerance [+-ATPase subunit that plays a role in vacuolar acidification; however, a study involving vma13Δ mutants showed that they experienced increased sensitivity to iron deprivation, suggesting Vma13 plays a role in iron import [VMA13 in iron homeostasis combined with its role in vacuolar acidification should be studied to determine if mutants can also help increase iron resistance. Another method of iron resistance in S. cerevisiae is the expression of ferritin related genes. Ferritin is an iron storage protein found in many other eukaryotes, but is not native to fungi [HuFH, SFerH1/SFerH2, and TFH, respectively) resulted in the increased ability of yeast to store and carry higher concentration of iron [ccc1Δ mutants [ter Ccc1 ,249. Ccclic iron . It may olerance . Vma13 in import . The funto fungi ,123,124. of iron ,123,124. mutants . This isS. pombe, ferrichome production, excretion, and subsequent uptake are used to acquire extracellular Fe3+ in iron-deficient conditions [U. maydis also biosynthesizes siderophores (hydroxamate) via sid1 for iron uptake which could also be investigated for increased production for storage of excess iron [Fewer studies report on iron resistance in other fungi, but several inferences can be made based on knowledge of iron homeostasis. In nditions ,237,251.nditions ,237,251.ess iron ,121.2+ is required for virulence [2+, such as manganese-dependent peroxidase, which white-rot fungi express during lignocellulose degradation, integral to nutrient uptake [2+ and homeostatic mechanisms must exist to ensure proliferation.Manganese (Mn) is a transition metal and also an essential micronutrient in fungi. In agriculture, Mn compounds reduce mycelial growth of fungal pathogens ,253. In irulence . Some lit uptake ,256. ManS. cerevisiae, Mn2+ transporters Smf1 and Smf2  and phosphate transporter Pho84, have a diverging consensus on their roles in Mn2+ homeostasis. In the case of Smf1, it was initially determined to be a high-affinity plasma membrane transporter, which acquired extracellular Mn2+ in Mn2+ deficient environments [2+ transporter [2+ is as a cofactor for proteins such as Sod2 [2+ via Mtm1 for activation [smf2Δ mutants, the Sod2 primary protein structure accumulates in the mitochondria; however, they were mostly inactive due to inadequate Mn2+ transfer to the mitochondria, indicating that Smf2 is a requirement for S. cerevisiae Sod2 activity [2+ availability [2+ concentrations are stable or in excess (~100 nmol/(1 × 109 cells)), Smf1 and Smf2 are ubiquitinated via Rsp5 (a NEDD4 family E3 ubiquitin ligase) with the aid of Bsd2 and transferrin receptor-like proteins (Tre1 and Tre2) [tre1Δ, tre2Δ, and bsd2Δ mutants resulted in the accumulation of Smf1 and Smf2 [2+ starvation occurs, Bsd2 is depleted, Smf1 is localized to the cell surface, Smf2 is localized to intracellular vesicles, and Smf1 and Smf2 resume their Mn2+ uptake functions [Within ronments ,66. Smf2nsporter ,257. Onc as Sod2 ,258. Sodtivation ,258,259.activity ,258. Smflability . When Mnnd Tre2) ,261,262.nd Tre2) ,263,264.and Smf2 ,261,262.unctions ,261.S. cerevisiae as a high-affinity, six-domain, transmembrane, inorganic phosphate transporter [2+ transporter, along with other metals such as cobalt, zinc, and copper [pho84Δ mutants, it was shown that Mn2+ uptake was the most commonly affected  when PHO84 was removed, further proving its Mn2+ transporter role [PHO84 transcription is regulated by transcription factor Pho4, which inhibits Pho84 activity when it is phosphorylated in the presence of excess phosphate; Pho4 resumes transcription when phosphate levels are low [The final transport system discussed here is the phosphate transporter Pho84. It was initially characterized in nsporter . Howeverd copper . Throughter role . PHO84 t are low ,266.2+ is inside the cell, there are an array of destinations. Pmr1 (high-affinity Ca2+/Mn2+ P-type ATPase) and Gdt1  both transport cytosolic Mn2+ to the Golgi lumen, where Mn2+ serves as a cofactor for mannosyl-transferases, such as Mnn1, Mnn2, Mnn5, and Mnn9, which glycosylate proteins in the secretory pathway [2+ to the ER lumen; this is supported by a study showing that spf1Δ mutants had decreased luminal Mn2+; its overexpression had the opposite effect [2+ depletion observed in spf1Δ mutants negatively impacted luminal Mn2+ dependent processes. On the contrary, it positively impacted Mn2+ associated cytosolic processes, indicating that Spf1 is integral to S. cerevisiae manganese ER and cytosolic homeostasis [Once Mn pathway ,270,271. pathway . Concerne effect . This saeostasis .2+ accumulation can have severe consequences on cellular health, and systems must be in place to prevent subsequent events. We will discuss two defense mechanisms in S. cerevisiae, Mn2+ trafficking to vacuoles for storage and degradation and Mn2+ export. Pmr1, previously characterized as an Mn2+ Golgi lumen transporter, also serves as a detoxifier. Presented with toxic Mn2+ levels, Mn2+ is still transported to the Golgi lumen from the cytosol, but excess ions are delivered to secretory pathway vesicles, which ultimately exit the cell, completely removing toxic Mn2+  is localized in the vacuolar membrane and is responsible for trafficking cytosolic Mn2+ to vacuoles; CCC1 overexpression results in reduced Mn2+ toxicity, lower concentrations of cytosolic Mn2+, and increased vacuolar concentrations , has yet to be identified, but predicted to have a similar antioxidant activity [2+ storage and export in P. chrysosporium, respectively. In Phanerochaete sordida, PsMnt was found to be a homolog of yeast Smf2 and plays a role in Mn2+ uptake, suggesting that it could have dual functionality, but this is still unknown [2+ homeostasis in other fungi; however, due to the impact of Mn2+ on lignin-degrading enzymes in wood-rotting fungi, more studies should be conducted. Overall, Mn2+ homeostasis is critical to cellular functioning to prevent toxic Mn2+ accumulation, detoxify cells of free radicals, and provide white-rot fungi with their capacity to degrade lignin. In the absence of such mechanisms, toxicity can impede proper functioning and cause cellular damage.Manganese homeostasis has not been well characterized in higher fungi, but llutants ,276,277.llutants ,279. It llutants ,282,283. homolog . Smf1/2 homologs . Intrace mutants . When gr as Sod1 . In the activity . PcMnt a unknown . LimitedS. cerevisiae, excessive Mn2+ can overrun homeostatic systems and create a toxic ionic imbalance that negatively impacts survival rate [2+ down-regulate genes associated to histidine proteins  that are compulsory in chromatin assembly chromosome functioning, and interface in this functioning can end in cell cycle arrest [2+ impacts manganese peroxidase activity, but there is a lack of knowledge on how excess Mn2+ can be toxic towards this activity [2+ over accumulation should be further investigated [In the model yeast val rate ,285,286.e arrest ,114,287.activity ,97,98. Dstigated ,97,98. I2+ resistance is usually contingent upon homeostatic systems. In S. cerevisiae, several genes involved in resistance emanate from mutations. MNR1 , encodes a vacuolar H+/Ca2+ antiporter, but has been implicated in Mn2+ resistance [2+ sequestration to the vacuole [PHO84 is also implicated in Mn2+ resistance, where pho84Δ mutants have increased resistance, likely due to the acquired inability to import and accumulate excess Mn2+ [P. chrysosporium, where it was demonstrated that PcPho84Δ mutants increase Mn2+ resistance, as well as expression of PcMNT, which is likely to engage in Mn2+export activity [As with other metals, Mnsistance ,288,289. vacuole ,289,290.ess Mn2+ . In filaactivity . Up to this point, metals that serve as essential nutrients have been reviewed. In recent years, there has been an increase in studies on the usage of metals with no nutritional purpose, but which serve as antimicrobial agents, such as silver. This increase gives cause for further investigation into how these metals are metabolized and their intracellular functioning.+) have demonstrated their ability to control plant pathogens [Silver (Ag) is a transition metal that shares similar properties to other transition metals in groups three through twelve, and closely resembles the properties of Cu and gold (Au) ,292. In athogens ,294,295.athogens ,296,297.Silver is a non-essential metal that has no designated cellular receptors or membrane channels for ion uptake. Much of the literature has focused on silver as an antimicrobial agent, but some studies have begun to clarify homeostatic mechanisms ,116,298.S. cerevisiae, Ctr1, high-affinity Cu+ transporter, has been identified as a Ag+ importer. This is based on observed reduced Ag+ uptake in ctr1Δ mutants exposed to low silver concentrations, and transcriptional analysis that shows exposure to Ag NPs upregulates CTR1 throughout the entire transcriptome [+ homeostasis was also investigated by Hosiner et al. and Niazi et al.; both found that short-term exposure to silver resulted in increased expression of copper MTs Cup1-1 and Cup1-2, suggesting these MTs sequester Ag+ in response to silver stress [+ and Ag+ for Cup1-1 and Cup1-2 should be further investigated to determine which ion the MTs have a higher affinity for. Other metal ion transporters  have been investigated for their involvement in Ag+ uptake, but results indicate they are not [In criptome ,81. The r stress ,115. The+ destinations. AgNO3 exposure results in Ag+ accumulation in the mitochondria, which, in return, reduces Cu+ accumulation in the mitochondrial matrix [c oxidase activity, suggesting that cytosolic Ag+ is trafficked to the mitochondria via Cu+ mitochondrial transporter Pic2, potentially with a higher affinity, which can be toxic to cells by reducing the rate of cellular respiration [Once inside the cell, there are not many known Agl matrix . The dirpiration . No otheS. cerevisiae, Ag+ uptake can affect these systems, resulting in toxicity. Exposure to Ag+ can increase the efflux rate of potassium ions (K+) from S. cerevisiae, resulting in almost complete K+ efflux from the cell. S. cerevisiae requires a minimum 30mM K+, suggesting those events can be toxic if the ion concentration is not restored [+ toxicity is its ability to alter cellular structure [Yarrowia lipolytica, reducing the amount of ergosterol, which fluidizes the membrane, and increases internal lateral pressures [+ exposure can also deform the cell wall, which is a likely a response to the down-regulation of genes involved in ergosterol synthesis  in S. cerevisiae [Articulospora tetracladia, transcriptome analysis via RNAseq revealed toxicity of Ag+ and Ag NPs may result from interrupted functioning of plasma/organelle membranes and downregulation of genes associated with cellular redox [3 and Ag NPs can be useful in pathogen control of plant diseases [Efflux systems are integral to cellular homeostasis, preventing the accumulation of toxic compounds within a cell. In restored ,300. Anotructure ,103. Ionressures . Ag+ exprevisiae ,99. In tar redox . Silver diseases ,293,295.CTR3 is implicated in Ag+ resistance after an observed fold increase in its expression in a silver evolved strain of S. cerevisiae [MAC1 in the presence of Ag+ may clarify its role in resistance. It is possible that MTs Cup1-1 and Cup1-2 are also involved in resistance. It was previously described that exposure to AgNO3 and Ag NPs resulted in the increased expression of CUP1-1 and CUP1-2, proposing that the encoded MTs may also bind Ag+ and decrease sensitivity [3 exposure, where yeast had increased expression of CUP1-1 and CUP1-2  in an extended study that resulted in an evolved yeast strain, confirming the potential role of copper MTs in silver resistance [+ transporters, Pho84, Fet3, and Smf1, were not implicated in Ag+ uptake; however, significant down regulation (68.56-fold) of PHO84 in silver evolved yeast has been observed, which may indicate that Pho84 plays a role in Ag+ uptake, and may serve as a mechanism of Ag+ resistance [+ on genes involved in ergosterol biosynthesis was also investigated in a silver evolved yeast [+ toxicity could be the ability to inhibit their down regulation [A. nidulans, silver induced expression of copper exporter crpA, indicating that it may play a role in silver export and resistance [A. tetracladia, resistance may be due to increased vacuolar function [S. cerevisiae. Due to the increasing silver and Ag NP usage in many aspects of human life, silver–fungal interactions should be further investigated at the molecular level to decipher precise homeostatic and resistance mechanisms.The worldwide increase of silver usage makes studies on mechanisms of silver resistance important; presently, few studies have reported on this. revisiae . Insightsitivity ,114,115.sistance . Other Asistance ,116. Thesistance . In A. tfunction . OverallAs the potential for commercial use of antifungal metals increases, so does the need to further investigate fungal homeostasis of essential and non-essential metals. Currently, research in this area is heavily reliant on assay based methods, which can be subjective and ambiguous. In this review, many of the discoveries of homeostatic mechanisms stemmed from the use of deletion libraries, microarrays, and PCR-based methods. This can restrict the scope of the research by only analyzing known genomic or transcriptiomic signatures.The incorporation of an omics based approach is a resolution to this issue. The most popular omics utilizes bioinformatics to analyze fungal–metal interactions at a nucleotide and protein level, which can reveal novel genes and mutations. In genomics, the entirety of a genome is assessed and compared to others for similarities and differences that can contribute to an organism’s characteristics ,303. TraBioinformatics analysis is used to translate omics results via computer programming methods. In nucleotide based omics, DNA or RNA is fragmented into segments or reads prior to sequencing. After sequencing, base calling assigns a nucleotide base to an intensity signal linked to a chromatogram peak and quality control measures are taken to trim reads of adapters used in the sequencing process and trim low quality bases . Next, sSteps within these bioinformatics pipelines require the use of computational tools written into the command line. Multiple tools with varying parameters exist to complete the same function; however, the user must decide which tools fit their scientific needs. This can result in variation between datasets and across scientific disciplines, based on accepted standards and norms. However, this limitation does not deduct from the vast amounts of data received.With the increasing affordability of high-throughput omics, organisms can be analyzed at multiple omics levels. This is leading to a more comprehensive understanding of characteristics, especially in fungi where there is limited knowledge of their complexity. This type of research will also illuminate unique features of fungal metal homeostasis, toxicity, and resistance, especially of non-essential metals that are becoming conventional antimicrobial agents.S. cerevisiae. However, there was a significant lack of fundamental knowledge of such mechanisms in filamentous fungi, which play critical roles in nanoparticle biosynthesis and are targets of metal antifungals, further accentuating the need to investigate molecular systems involved in metal homeostasis. Fungal homeostasis of the non-essential metal silver was also highlighted. It showed that homeostatic mechanisms were reliant on existing copper transport systems, but were largely unclear regarding overall cellular processing. There is a need to further investigate other non-essential metals’ cellular homeostasis as their commercial usage increases, due to the current lack of knowledge of future implications. Fungal–metal interactions such as the synthesis of nanoparticles and metal used as antifungal agents are on the rise. Studies on metal toxicity and resistance have uncovered preserved homeostatic mechanisms. This review discussed metal homeostasis in various fungi types and has shown that essential metals have designated uptake and transport systems that regulate metal ion balance, mostly through the model organism


Glioblastoma (GBM) is one of the most commonly pivotal malignant caners. Numerous reports have revealed the crucial roles of immune infiltration in the initiation and progression of GBM. In this study, we first identified differentially expressed genes (DEGs) in the progression of GBM using CGGA databases. Totally, 156 upregulated DEGs and 251 downregulated DEGs were revealed. By constructing a protein-protein interaction network, KIF2C was identified as a hub gene in GBM. Further analysis revealed an evidently positive association existing in KIF2C expression and the advanced stages of gliomas. Higher expression of KIF2C was in WHO grade IV samples relative to that in grade III and grade II samples. In addition, our results showed that KIF2C was higher in IDH1 wild-type samples than IDH1 mutant glioma samples, in 1p/19q noncodel samples than 1p/19q code glioma samples, and in recurrent samples than primary glioma samples. Moreover, our results showed that higher expression of KIF2C correlated with shorter survival time in both primary and recurrent gliomas and could act as a potential biomarker for the prognosis of GBM. Further analysis demonstrated that higher expression of KIF2C was related to higher levels of endothelial cell, T cell CD8+ naïve, common lymphoid progenitor, T cell CD4+ Th2, T cell CD4+ Th2, macrophage, macrophage M1, T cell CD4+ memory, and T cell CD4+ effector memory, but was related to lower levels of NK cell, B cell plasma, T cell CD4+ Th1, T cell regulatory (Tregs), neutrophil, and T cell NK. We thought this study could provide potential biomarkers for the prediction of prognosis and immune infiltration of gliomas. Glioblastoma (GBM) derived from neuroepithelium is the widely occurring main malignant neoplasm in the central nervous system (CNS) of adults . More th6A demethylase ALKBH5 preserves the tumorigenesis of GBM stem-like cells via maintaining the expression of FOXM1 and the program of cell proliferation  > 1 were thought to be DEGs. Finally, 593 DEGs with upregulation and 1021 DEGs with downregulation were found in mRNA-seq-693 database  , and hubThe dysregulation of hub DEGs was correlated to the survival time in patients with glioma.Then, a forest map was produced to analyze the correlation between hub DEGs and OS analysis. After calculating the hazard ratio and confidence interval, we observed that 8 hub genes in hub network 1 were related to the poor prognosis of gliomas, including PBK, KIF2C, CENPE, KIF14, MND1, FAM83D, NEIL3, and CDKN3 . In addiOn the basis of CGGA data, we have drawn Kaplan-Meier plots and the data demonstrated the impacts of these top hub genes on survival probability. Among these hub genes, KIF2C showed the most significant correlation to prognosis, which had been reported to be a key role in cell cycle regulation. We chose KIF2C here to validate the links existing in the expression and its clinicopathological values in CGGA cohort. The data showed different degrees of KIF2C expression in multiple histology of gliomas . We founSurvival curve of the gene was also generated to evaluate the correlation between KIF2C expression and survival time in primary and recurrent gliomas using CGGA data . Our resIn order to validate the assumption that KIF2C was an independent factor for prognosis of gliomas patients, we conducted univariate and multivariate Cox regression analysis. The result showed that KIF2C expression, age, grade, and radiation therapy were evident factors that were related to glioma prognosis . In addiWe in-depth established a monograph containing the prognostic factors, such as KIF2C expression, age, and radiation therapy, to supply a quantitative base to forecast the possibility of one-year, three-year, and five-year OS in glioma patients for clinicians . What isIn the present study, we analyzed the correlation between KIF2C expression and immune infiltration in gliomas using the Xcell dataset. As presented in GBM is a widespread and precarious malignant brain neoplasm, of which symptoms include abdominal distension, pelvic pain, difficulty in eating, and frequent urination . As a tyThe present study analyzed three independent datasets in CGGA databases to define the DEGs between low-grade and high-grade glioma samples. Finally, 407 DEGs associated with glioma development were uncovered. Among them, 156 genes were increased and 251 genes were reduced. Bioinformatics analysis showed that genes with upregulation primarily participated in synaptic transmission and cAMP signaling pathway. Of note, our analysis revealed that upregulated genes were related to multiple key signaling in carcinoma progression, such as angiogenesis, mitotic cell cycle, and PI3K-Akt, TNF, and p53 signaling pathways.The PI3K (PI3 kinase) family displays an intricate role in biological process, such as metabolism , 20. In In order to identify the hub genes involved in glioma progression, we constructed PPI network analysis, and two hub PPI networks were identified. Hub network 1 included 52 genes, and hub network 2 included 18 genes. Also, we observed 13 hub genes were related to the poor prognosis of gliomas, including PBK, KIF2C, CENPE, KIF14, MND1, FAM83D, NEIL3, CDKN3, F5, IGFBP5, TNC, SCG3, and IGFBP3. Of note, the functional importance of several DEGs had been demonstrated in gliomas in previous reports. For example, PBK, a serine/threonine kinase, belonged to the family of mitogen-activated protein kinase kinase (MAPKK), which participated in modulating cell proliferation, metastasis, and autophagy . In glioKIF2C was chosen for subsequent studies amid the selected hub genes. A previous study showed KIF2C is a marker for prognosis in human gliomas . In thisEmerging studies had demonstrated tumor-infiltrating lymphocytes (TILs) could affect the immune response and tumor initiation and progression. TILs had also been found to be enriched in GBM. Very interestingly, several genes had been reported to modulate the immune infiltrating. For example, LRRK2 correlates with macrophage infiltration in GBM . MeanwhiSeveral limitations should be noted. First, the functional roles of KIF2C were not confirmed using experimental methods. Gain- or loss-of-function assays should be performed in the future study. Second, despite the fact that two independent datasets were used to explore the clinical significance of KIF2C in GBM, further validation using clinical samples is still needed. Finally, the association between KIF2C expression and immune infiltration was determined using bioinformatics analysis, thus lacking more confirmation.Collectively, our current study identified 407 progression-related DEGs in gliomas. Bioinformatics analysis showed that these DEGs were related to cAMP signaling, mitotic cell cycle, and PI3K-Akt and p53 signaling pathways. Among these DEGs, 13 hub genes were demonstrated to be associated with the poor prognosis of gliomas, including PBK, KIF2C, CENPE, KIF14, MND1, FAM83D, NEIL3, CDKN3, F5, IGFBP5, TNC, SCG3, and IGFBP3. It is found that the DEGs, especially KIF2C, may provide diagnostic and prognostic value for GBM. In the future study, further research is required to unclose the underlying mechanisms and develop novel therapeutic strategies for GBM.


Pediococcus acidilactici (pA1c) in a murine model of high-fat diet (HFD)-induced T2D. For that purpose, C57BL/6 mice were given HFD enriched with either probiotic (1 × 1010 CFU/day) or placebo for 12 weeks. We determined body weight, fasting blood glucose, glucose tolerance, HOMA-IR and HOMA-β index, C-peptide, GLP-1, leptin, and lipid profile. We also measured hepatic gene expression  and examined pancreatic and intestinal histology . We found that pA1c supplementation significantly attenuated body weight gain, mitigated glucose dysregulation by reducing fasting blood glucose levels, glucose tolerance test, leptin levels, and insulin resistance, increased C-peptide and GLP-1 levels, enhanced pancreatic function, and improved intestinal histology. These findings indicate that pA1c improved HFD-induced T2D derived insulin resistance and intestinal histology, as well as protected from body weight increase. Together, our study proposes that pA1c may be a promising new dietary management strategy to improve metabolic disorders in PreD and T2D.Prediabetes (PreD), which is associated with impaired glucose tolerance and fasting blood glucose, is a potential risk factor for type 2 diabetes mellitus (T2D). Growing evidence suggests the role of the gastrointestinal microbiota in both PreD and T2D, which opens the possibility for a novel nutritional approach, based on probiotics, for improving glucose regulation and delaying disease progression of PreD to T2D. In this light, the present study aimed to assess the antidiabetic properties of  Prediabetes (PreD) refers to an intermediary phase between healthy state and diabetes disease where glycaemia is altered but still below the threshold for the diagnosis of diabetes. PreD is characterized by an impaired glucose tolerance (IGT) and fasting blood glucose (FBG) , and it At this juncture, mounting evidence has suggested that the gastrointestinal microbiota (GM) is a relevant environmental factor in PreD and T2D development. The proposed mechanistic links include changes in intestinal permeability, energy homeostasis, metabolism, and especially inflammation, which is closely related to IR ,7,8,9. TLactobacillus or Bifidobacterium spp. .Part of the results that serve as the basis for this manuscript have been included in the patent entitled:


Laminin-332 pemphigoid is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone. Laminin-332 pemphigoid is characterized by variable inflammatory infiltrate and the predominance of non-complement-fixing antibodies. Given these findings, we hypothesized that IgG autoantibodies to laminin-332 directly resulted in keratinocyte expression of inflammatory factors. We performed RNA-seq on primary human keratinocytes treated with IgG from patients with laminin-332 pemphigoid. Genes for numerous cytokines and chemokines were upregulated, including CSF2, CSF3, CXCL1, CXCL5, CXCL3, CXCL8, CXCL10, CXCL1, IL6, IL7, IL15, IL23, IL32, IL37, TGFB2 as well as metalloproteases. Considering the pro-inflammatory and proteolytic effect of autoantibodies from patients with laminin-332 pemphigoid identified in our initial experiment, we next questioned whether the reactivity against specific laminin subunits dictates the inflammatory and proteolytic keratinocyte response. Then, we treated keratinocytes with IgG from a separate cohort of patients with reactivity against individual subunits of laminin-332. We identified upregulation of IL-1α, IL-6, IL-8, CXCL1, MMP9, TSLP, and GM-CSF at the protein level, most notably in keratinocytes treated with IgG from laminin β3-reactive patients. We for the first time demonstrated a pro-inflammatory response, similar to that described in keratinocytes treated with IgG autoantibodies from patients with bullous pemphigoid, providing novel insight into the pathogenesis of laminin-332 pemphigoid and laminin-332 biology. Laminin-332 pemphigoid, a subtype of mucous membrane pemphigoid (MMP), is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone (BMZ) –3. LaminSeveral animal models have supported the pathogenicity of anti-laminin-332 IgG –14. The Aside from their structural role, hemidesmosomal proteins may additionally regulate the local inflammatory response. For example, IgG autoantibodies to collagen XVII in bullous pemphigoid (BP) lead to internalization of collagen XVII in keratinocytes , with upGiven the heterogeneity of cutaneous inflammation and the predominance of non-complement-fixing antibodies in laminin-332 pemphigoid, we hypothesized that IgG autoantibodies to laminin-332 directly resulted in keratinocyte expression of inflammatory factors.All analyses of human materials were performed in accordance with the principles in the Declaration of Helsinki and approved by the ethical committee at the University of Illinois at Chicago, Rush University Medical Center, Philipps University, and Kurume University School of Medicine. Diagnosis of laminin-332 mucous membrane pemphigoid in the first cohort of patients was based on clinical presentation of MMP, direct immunofluorescence (DIF) demonstrating linear IgG deposition at the cutaneous BMZ, and with indirect immunofluorescence (IIF) against the dermal side of salt-split skin. All patients were screened and negative for BP180/BP230 antibodies . ELISA wIgG extraction was performed using Pearl IgG Purification Kits  according to manufacturer’s instructions. Isolated IgG was subsequently washed and concentrated using Amicon Ultra-15 centrifugal filter units with a 100kDa filter in PBS .2 at 37°. Fresh culture media were replaced every 48 h. When confluency reached 80-90%, cells were treated with the patients’ IgG (3.5 ug/ul) or normal human IgG for 24 h. Each sample represents a distinct culture. Finally, cells were washed twice with PBS and were stored at −80°C until RNA extraction. Culture supernatants were stored at −80°C until Luminex and ELISA assays were performed.Primary adult human keratinocytes (PHKs)  were cultured in EpiLife Medium with Human Keratinocyte Growth Supplement  in a humidified atmosphere of 5% CO-ΔΔCT values were compared using student’s T-test with significance defined as P < 0.05. Primers used in this study were synthesized by Integrated DNA Technologies Inc. . Primers used are provided in Total RNA from PHKs was isolated using miRNeasy Mini Kit following the manufacturer’s instructions . On-Column DNase I Digestion was performed to prevent genomic DNA contamination. The RT and real-time PCR were performed as previously described . BrieflyLibrary preparation was performed as previously described . RNA quahttp://geneontology.org/). Heat maps were generated using FPKM values using the Morpheus online server (https://software.broadinstitute.org/morpheus).Filtered clear reads were mapped to the reference genome by HISAT2 . Mapped Luminex multiplex kits for IL-1a, IL-6, IL-8, CXCL1, matrix metalloproteinase 9 (MMP9), and TSLP were purchased from R&D systems . Supernatants were diluted per manufacturer recommendations using a Flexmap 3D system , performed at the Rush University Medical Center Biomarker Development Core. GM-CSF and HSPA5 ELISA were purchased from Thermo Fisher Scientific  and MyBioSource , respectively. Samples in Luminex and ELISA assays were run in duplicate. ELISAs were performed per the manufacturer’s instructions and read on a Tecan Spark plate imager . Standard curves were calculated for all markers. To compare concentrations between controls and each laminin-subunit reactive, we used a one-way ANOVA with Tukey’s test (GraphPad Prism 6). Significance was set at P < 0.05. ClustVis was used to perform principal component analysis . Express. Whole transcriptome sequencing was used to profile expression of primary keratinocytes treated with IgG from patients with untyped laminin-332 pemphigoid (n=4) or control serum (n=3). We obtained an average of 40.1 million clean reads per sample. An average of 96% genes mapped to the human genome. Principal component analysis demonstrates two distinct clusters between keratinocytes treated with laminin-332 IgG or control IgG Figure 1As hypothesized, genes for numerous cytokines and chemokines were significantly upregulated, including CSF2, CSF3, CXCL1, CXCL5, CXCL3, CXCL8, CXCL10, CXCL1, IL6, IL7, IL15, IL23, IL32, IL37, TGFB2. Only TGFB3 and IL34 genes were downregulated. Interestingly, numerous heat shock genes were differentially expressed with HSP90B1, HSP90B2P, HSP90B3P, and HSPA5 highly upregulated, while HSPA2, HSPA4L, HSPA14, HSPB8, HSPE1P2, and HSPA6 were down regulated. Numerous proteases were additionally upregulated including MMP1, MMP2, MMP9, MMP10, MMP12, MMP14, MMP15, MMP19, MMP24, ADAM8, ADAM12, ADAM19, and ADAM21. Of these, MMP9 and ADAM19 were most highly upregulated. Lastly, hemidesmosomal genes COL17A1, COL7A1, LAMA3, LAMB3, and LAMC2 were upregulated. Heat map of select proteases, heat-shock proteins, cytokines, chemokines, and structural proteins are shown in Figure 1To confirm next generation sequencing findings, RT-qPCR was performed on a subset of genes from the same samples. These genes covered several functional clusters such as proteases, structural proteins, inflammatory molecules, and heat shock response. RT-qPCR confirmed significant upregulation (P < 0.05) in all assessed genes: ADAM19, MMP9, COL17A1, LAMA3, VIM, ICAM-1, CSF2, CSF3, IL23a, IL-32, HSPA5, and HSP90B1 Figure 2.Gene ontology assessment for biologic processes demonstrated significant enrichment of extracellular structures, epidermal development, and inflammasome activation (response to lipopolysaccharide), as well as various aspects of DNA replication. Functional enrichment for cellular components demonstrated enhancement in notable pathways including extracellular matrix genes and basement membranes genes, while enrichment for molecular function demonstrated notable effects on glycosaminoglycan binding, cytokine binding, integrin binding, and transmembrane receptor protein kinase activity pathways Considering the pro-inflammatory and proteolytic effect of IgG autoantibodies from patients with laminin-332 pemphigoid on keratinocytes identified in our initial experiment, we next looked to validate several of the key findings in a larger cohort of patients with laminin-332 pemphigoid. Additionally, we questioned whether the reactivity against specific laminin subunits affects the inflammatory and proteolytic keratinocyte response. We treated PHK with IgG from a separate cohort of patients with reactivity against only either laminin α3 (n=5), laminin β3 (n=4), or laminin γ2 (n=5). Patient demographics are shown in We next evaluated IL-1α, IL-6, IL-8, CXCL1, MMP9, GM-CSF protein expression using Luminex and ELISA assays, confirming overall upregulation. We additionally evaluated TSLP as a pro-inflammatory marker which has been described in BP; this too was elevated at the protein level, though not significantly increased at the mRNA level by RNA-seq. Inflammatory markers were consistently most elevated in patients with reactivity against the laminin β3 subunit Figure 3Local inflammatory responses in laminin-332 pemphigoid vary significantly in human disease and in different in vivo models –15, 28. A few limitations must be noted. We selected patients with IgG autoantibodies to each subunit specifically. However, patients often harbor IgG autoantibodies to several domains of laminin-332 concurrently. As such, synergistic effects of autoantibodies targeting different subunits were not identified in this approach. Additionally, the presence of concomitant autoantibodies may contribute a small degree to these findings. For example, BP180 antibodies are known to induce a pro-inflammatory response. As these antibodies were detected by ELISA, but no epidermal staining was appreciated on IIF, two samples were included.We did not exclude patients who had positive ELISA for BP180/BP230 when looking at laminin subtype. It should be noted that IIF was only positive on dermal side for all of these, so BP180/BP230 autoantibodies are likely at a far lower concentration relative to laminin sub-unit specific antibodies or are not against pathogenic epitopes. Patient LAMA3-IgG3 had antibodies to BP180/BP230 but did not appear to significantly differ from other LAMA3 sera on PCA analysis. When evaluating the protein expression heatmap, the presence of BP180 antibodies do not appear to affect the inflammatory response. In fact, patient LAMB3-IgG1, and LAMA3-IgG3 do not demonstrate particularly high expression relative to the other LAMB3. Thus, the presence of these antibodies appears insufficient to explain our pro-inflammatory findings. While patient LAMC2-IgG3 additionally had autoantibodies against the p200 antigen detected by immunoblot, the patient’s clinical presentation was consistent with laminin-332 mucous membrane pemphigoid. Nevertheless, we cannot rule out a contributory role of p200 antibodies. Analysis of LAMC2 treated patient does not reveal comparable clustering to other subunits. Yet, the most notable ‘outlier’ is patient LAMC2-IgG2, who only expressed autoantibodies to LAMC2. Lastly, we were unable to correlate histopathological findings with keratinocyte cytokine expression levels due to a limited number of specimens with histology.In addition to identifying potential complement-independent inflammatory mechanisms in laminin-332 pemphigoid, these findings shed light on laminin-332 as an active regulator of cell signalling and function. Extracellular matrix proteins not only serve as structural proteins but affect cell signalling through protein-protein interactions . Blockadin vivo models of epidermolysis bullosa acquisita, though its mechanism of action appears to be tied towards neutrophil function  can be found below: The studies involving human participants were reviewed and approved by the ethical committee at the University of Illinois at Chicago, Rush University Medical Center, Philipps University, and Kurume University School of Medicine. The patients/participants provided their written informed consent to participate in this study.KA and LB designed the research study. LB, FS, JL, DD, and PP performed the experiments. LB, XL, HQ, and KA analyzed the data. KA and PP drafted the manuscript. LB, JL, FS, DD, XL, HQ, NI, TH, and MH provided critical revisions. All authors contributed to the article and approved the submitted version.The work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) FOR 2497/TP02  and the Albert H. and Mary Jane Slepyan Endowed Fellowship (LB).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.


Mantle cell lymphoma (MCL) is considered one of the most aggressive lymphoid tumors. However, it sometimes displays indolent behavior in patients and might not necessitate treatment at diagnosis; this has been described as “smoldering MCL” (SMCL). There are significant differences in the diagnosis, prognosis, molecular mechanisms and treatments of indolent MCL and classical MCL. In this review, we discuss the progress in understanding the molecular mechanism of indolent MCL to provide insights into the genomic nature of this entity. Reported findings of molecular features of indolent MCL include a low Ki-67 index, CD200 positivity, a low frequency of mutations in TP53, a lack of SOX11, normal arrangement and expression of MYC, IGHV mutations, differences from classical MCL by L-MCL16 assays and MCL35 assays, an unmutated P16 status, few defects in ATM, no NOTCH1/2 mutation, Amp 11q gene mutation, no chr9 deletion, microRNA upregulation/downregulation, and low expression of several genes that have been valued in recent years . Based on the above molecular characteristics, we may distinguish indolent MCL from classical MCL. If so, indolent MCL will not be overtreated, whereas the treatment of classical MCL will not be delayed. Mantle cell lymphoma (MCL) is a mature B-cell neoplasm that accounts for 5% to 10% of all lymphomas . MCL repA low Ki-67 index may imply a more indolent form of MCL . Kimura CD200 expression in MCL indicates a unique subgroup. This subgroup is frequently accompanied by IGHV mutations and SOX11 negativity and portends an indolent clinical course , 10. CD2Mutations and deletions of the tumor protein p53 (TP53) gene are the most frequent genetic alterations detected in human tumors, although they are rather less common in lymphomas . HoweverOverall, indolent MCL carries almost no TP53 mutation/deletion. However, a small portion of indolent MCL may harbor subclonal TP53 mutations. TP53 mutation and TP53 deletion are both associated with a poor prognosis independent of the MIPI, and TP53 mutations are associated with significantly worse outcomes than TP53 deletions. Moreover, TP53 mutations are correlated with CD38 negativity and hypermutated IGHV.The clinical features of MCL are closely related to the expression level of SOX11. Leukemic mantle cell lymphoma limited to the blood and bone marrow is characterized by a lack of SOX11, mild-moderate lymphocytosis, and interstitial low-level bone marrow involvement . SOX11, Overall, the expression of the SOX11 gene is almost absent in indolent MCL. SOX11 negativity indicates a better prognosis. However, the significance of SOX11 negativity in indolent MCL is still inconclusive.Velden and colleagues suggested that B-cell prolymphocytic leukemia is a specific subtype of MCL . InteresThus, the role of MYC amplification is currently unclear, with this aberrancy reported in studies on both indolent and aggressive MCLs.As early as 2003, Orchard et al. suggested that mutated immunoglobulin heavy chain variable region (IGHV) genes might help identify indolent MCL . In receThus, most patients with indolent MCL carry IGHV mutations. However, the significance of hypermutated IGHV in patient survival is still conflicting.In a recent study, Guillem et al. developed a novel gene expression assay that could identify classical MCL and leukemic non-nodal MCL— the L-MCL16 assay . The L-MTherefore, the L-MCL16 assay and MCL35 assay may be used to screen out indolent MCL from all variants of MCL. However, their prognostic value remains unclear.Deletions of the P16 gene are closely associated with aggressive MCL subtypes, such as the blastoid variant of MCL, with a poor prognosis , 78. P16The frequency of defects in ataxia telangiectasia mutated (ATM) gene has been reflected to be much lower in the indolent variants of MCL . The ATMThe frequency of defects in ATM is much lower in indolent MCL. Furthermore, indolent MCLs with aberrations in ATM are more aggressive. However, some recent studies have suggested that ATM has little prognostic value.The frequency of mutations in the NOTCH1 gene has been reported to be much lower in the indolent variants of MCL . Other sCompared to those with a low Ki-67 level, AH-MCL patients with Ki-67 ≥ 50% have exclusive mutations in TP53, RANBP2, SMARCA4, KMT2C, SPEN, and NSD2 WHSC1) , 88. The , 88. ThIn conclusion, the frequency of mutations in SPEN, SMARCA4 (SWI/SNF), RANBP2, KMT2C, NSD2 (WHSC1), Del 9p and/or Del 9q, CARD11, FBXW7, SP140, BIRC3, KMT2D (MLL2), PCDH10, CELSR3 and HNRNPH1 is much lower in indolent MCL. Conversely, Amp 11q gene mutations without chr9 deletion have a better prognosis.Indolent MCL is characterized by kappa light chain restriction. In contrast, a more typical lambda light chain restriction is presented in cMCL , 96. TheIndolent MCL is characterized by kappa light chain restriction, a low incidence of frameshift Bax mutations, low expression of CD34, low or no expression of HMG, low PARP1 protein content, upregulation of BACH2, inhibition of the Akt pathway and low expression of Mcl-1.Javeed et al. suggested that a set of stroma-associated microRNAs might define a more indolent group of MCL . MicroRNIn conclusion, the expression level of miR-20b in indolent MCL is lower than that in classical MCL. MiR-127-3p and miR-615-3p are related to the OS of patients with indolent MCL. Moreover, miR-29, miR-708, miR-455-5p/3p, miR-223, miR-132-3P and miR-18b are highly expressed in indolent MCL.MCL is one of the most aggressive lymphoid tumors, and the toxicity of chemotherapeutic drugs is unfavorable to every patient. Indolent MCL is a subtype of mantle cell lymphoma. Indolent MCL has a better prognosis than classical MCL. However, the process of “wait and watch” puts these patients at the potential risk of evolution to classical and aggressive MCL . TherefoIn this review, we summarize and analyze the common molecular characteristics of indolent MCL described in previous studies Fig. . Further


Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL). Despite the genetic heterogeneity of the disease, most patients are initially treated with a combination of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), but relapse occurs in ~50% of patients. One of the hallmarks of DLBCL is the occurrence of genetic events that inhibit apoptosis, which contributes to disease development and resistance to therapy. These events can affect the intrinsic or extrinsic apoptotic pathways, or their modulators. Understanding the factors that contribute to inhibition of apoptosis in DLBCL is crucial in order to be able to develop targeted therapies and improve outcomes, particularly in relapsed and refractory DLBCL (rrDLBCL). This review provides a description of the genetic events inhibiting apoptosis in DLBCL, their contribution to lymphomagenesis and chemoresistance, and their implication for the future of DLBCL therapy.Diffuse large B cell lymphoma (DLBCL) is curable with chemoimmunotherapy in ~65% of patients. One of the hallmarks of the pathogenesis and resistance to therapy in DLBCL is inhibition of apoptosis, which allows malignant cells to survive and acquire further alterations. Inhibition of apoptosis can be the result of genetic events inhibiting the intrinsic or extrinsic apoptotic pathways, as well as their modulators, such as the inhibitor of apoptosis proteins, P53, and components of the NF-kB pathway. Mechanisms of dysregulation include upregulation of anti-apoptotic proteins and downregulation of pro-apoptotic proteins via point mutations, amplifications, deletions, translocations, and influences of other proteins. Understanding the factors contributing to resistance to apoptosis in DLBCL is crucial in order to be able to develop targeted therapies that could improve outcomes by restoring apoptosis in malignant cells. This review describes the genetic events inhibiting apoptosis in DLBCL, provides a perspective of their interactions in lymphomagenesis, and discusses their implication for the future of DLBCL therapy. Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma and comprises about 25–40% of all non-Hodgkin lymphomas (NHL) in the Western world . PatientBCL2 translocations are almost exclusive to GCB DLBCL. Other genetic events, such as TP53 mutations, are observed in both types of DLBCL [DLBCL is classified into molecular subtypes that share common survival pathways and mechanisms that inhibit apoptosis. The most widely used classification stratifies DLBCL according to cell-of-origin (COO) molecular signatures, determined by gene expression profiling (GEP). These include the germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes, with 10–20% of cases having an intermediate or “unclassifiable” profile. GCB and ABC subtypes have distinct genomic alterations and clinical outcomes, with the latter being associated with an inferior overall survival ,10,11,12of DLBCL . An alteof DLBCL . The claof DLBCL ), and a of DLBCL ,16,17.Apoptosis in the normal germinal center reaction is regulated by several pro- and anti-apoptotic proteins that have been previously reviewed . In the A hallmark of cancer, including DLBCL, is the occurrence and accumulation of genetic alterations that promote malignant cell survival via inhibition of apoptosis . This suThe intrinsic apoptotic pathway, also known as the mitochondrial or BCL2-regulated apoptotic pathway, is predominantly under the control of the BCL2 protein family ,26. ThesBCL2 is normally silenced in germinal center (GC) B cells to allow low affinity B cells generated through somatic hypermutation (SHM) to undergo apoptosis. Thus, its expression in DLBCL is pathogenic [BCL2 is located on chromosome 18q21 [BCL2 translocations to immunoglobulin genes (IG), t  , t   and t  , involving the IgH, IgK and IgL loci, respectively, are present in 20–25% of DLBCL, almost exclusively of the GCB subtype [BCL2 translocations are also a dominant feature in the new DLBCL classification systems, occurring in 71% of C3 DLBCL, 89% of BCL2 DLBCL, and 78% of EZB DLBCL [BCL2 by the enhancer elements within the active IG loci [BCL2 overexpression, the t  translocation is associated with a significantly higher rate of SHM-associated BCL2 mutations, which is the most commonly mutated gene in DLBCL [BCL2 promoter mutations might increase BCL2 protein expression by preventing binding and transcriptional repression by BCL6 [BCL2 decrease calcium-mediated apoptosis by preventing binding of BCL2 to the inositol 1,4,5-triphosphate receptor (IP3R), a channel that promotes apoptosis by facilitating calcium conductance from the endoplasmic reticulum to the mitochondria [BCL2 mutations do not seem to affect prognosis [BCL2 gains and amplifications occur in up to 25% of DLBCL, mostly of the ABC subtype. They typically are the result of copy number aberration of chromosome 18q21.33 and are associated with increased BCL2 expression and worse prognosis [BCL2 is the most common and important anti-apoptotic protein inhibiting apoptosis in DLBCL. thogenic . BCL2 isme 18q21 . In addime 18q21 . BCL2 prme 18q21 . BCL2 tr14;18) q; q21, t ;18 q; q2ZB DLBCL ,17. They IG loci . In addiin DLBCL . The rolin DLBCL . Some of by BCL6 . Mutatio by BCL6 . Mutatiochondria ,36. Howerognosis . BCL2 garognosis .MCL1 were observed in 26% of ABC DLBCL. However, MCL1 mutations were identified in less than 1% of cases [MCL1 is an anti-apoptotic protein from the BCL2 family that is essential in B cell development and germinal center formation. It is therefore normally expressed in GC B cells ,39. It dof cases . Other pof cases , and decof cases .BCLX produces two proteins via alternative splicing: BCL-XL, an anti-apoptotic protein, and BCL-XS, a BCL2 inhibitor [BCLW is a relatively understudied anti-apoptotic gene, as it was initially shown to be only essential in spermatogenesis [BCLX and BCLW in DLBCL, and the role of the proteins encoded by these two genes is minor in comparison to BCL2 and MCL1 [nhibitor . BCL-XL nhibitor ,48,49,50nhibitor ,49. A manhibitor . BCLW isogenesis ,53. Overogenesis ,55. Howeogenesis . Overalland MCL1 .Although genetic events amplifying the effect of anti-apoptotic proteins are well characterized, pro-apoptotic defects in DLBCL remain a relatively understudied phenomenon. Functional assessment of the intrinsic apoptotic pathway through BH3 profiling in DLBCL cell lines and primary samples has revealed defects in BH3-only pro-apoptotic proteins (class A apoptotic block) and in apoptotic effectors (class B block). Such pro-apoptotic defects are a factor contributing to resistance to the BCL2 inhibitor venetoclax ,57. HoweBCL2 are characteristic of GCB DLBCL, while BCL2 and MCL1 gains and amplifications are more common in the ABC subtype. These proteins can also be upregulated through other mechanisms that do not require direct alteration of the gene loci. The therapeutic relevance of these alterations is discussed at the end of this review.In summary, deregulation of the intrinsic apoptotic pathway is a clear contributor to the pathogenesis of DLBCL. Translocations and mutations affecting The extrinsic apoptotic pathway is important in the regulation of the germinal center reaction and prevention of lymphomagenesis . It is aFAS, located on chromosome 10q23 and containing 9 exons, encodes the FAS cell surface death receptor [FAS is highly expressed in germinal center B cells and is critical for the negative selection of suboptimal or self-reactive B cells during the germinal center reaction [FAS are associated with autoimmune lymphoproliferative syndrome, a rare disorder characterized by lymphadenopathy, splenomegaly, autoimmune cytopenias, and a significantly increased risk of B cell lymphoma [FAS mutations are consistently identified in GCB and ABC DLBCL, with reported frequencies of 5–15% [FAS deletions have been observed in 7% of DLBCL [FAS [FAS mutations in DLBCL is limited, but decreased FAS or FasL expression has been associated with decreased survival [receptor ,63. The receptor . FAS is reaction ,66. The reaction ,68,69,70reaction . Deregulreaction ,73,74. Hlymphoma . Somaticof 5–15% ,17,24. Tof 5–15% , or are of 5–15% . These mof 5–15% . Mutatioof 5–15% . In addiof DLBCL . AnotherBCL [FAS . Prognossurvival . Preclinsurvival . Howeversurvival ,83.TRAIL-R1 (DR4) or TRAIL-R2 (DR5) [TRAIL-R1 and TRAIL-R2, are both located on chromosome 8p21 [TRAIL-R1 or TRAIL-R2 mutations in 5 of them (10.9%), all of which were inside or in close proximity to the region encoding the death domain [TRAIL-R1 and TRAIL-R2 are also common in DLBCL [TRAIL (APO2-L) is a ligand from the tumor necrosis factor (TNF) family that can trigger extrinsic apoptosis by binding to either R2 (DR5) ,85. The ome 8p21 ,87. Threome 8p21 . TRAIL-mome 8p21 ,89,90. Home 8p21 ,92,93. Iome 8p21 . Resistaome 8p21 . A studyh domain . 8p21 dein DLBCL . TRAIL iin DLBCL . TRAIL ain DLBCL ,98. In aThe inhibitor of apoptosis proteins (IAPs) are a family of antiapoptotic proteins that inhibit the intrinsic and extrinsic apoptotic pathways, mainly via inhibition of caspases 3,7, and 9. Eight IAPs have been identified including XIAP, cIAP1, cIAP2, and survivin . XIAP isTP53, located on chromosome 17p13.1 and containing 11 exons, encodes the tumor suppressor P53 and is the most frequently mutated gene in human cancers [TP53 cause Li-Fraumeni syndrome, a cancer predisposition syndrome associated with breast cancer, brain tumors, adrenocortical carcinoma, leukemias, and many other malignancies [ cancers ,109,110.gnancies . P53 is gnancies . Its strgnancies . Under ngnancies ,114,115.gnancies . Activatgnancies .TP53 and its associated proteins are common in DLBCL. TP53 mutations are seen in more than 20% of GCB and ABC DLBCL and are associated with poor prognosis in the GCB subtype [MDM4 and RFWD2 encode two other P53 inhibitors and can both be amplified with gains of chromosome 1q23.3, reported in 15% of DLBCL [BCL2L12, an atypical BCL2 protein that inhibits P53 and caspases 3/7, have been observed in 10% of cases [CDKN2A encodes the ARF (p14) and INK4a (p16) tumor suppressors [CDKN2A deletions are present in 19–35% of DLBCL. They are associated with an ABC subtype and a decreased survival [PERP (caspase 8 activator) and SCOTIN (pro-caspase 3/7 activator), which are deleted in 27% and 8% of DLBCL, respectively [Genomic alterations in  subtype ,119,120. subtype . Chromos subtype . MDM4 anof DLBCL . Amplifiof cases . CDKN2A pressors . ARF propressors . CDKN2A the ARF p and INK4survival ,126. Altectively .TP53 is the most commonly mutated gene in rrDLBCL, with mutations observed in up to half of cases [TP53 mutations in the plasma ctDNA in patients undergoing chemotherapy has the potential to detect and monitor early resistant clones [of cases ,24. Clonof cases ,127,128.t clones ,130.In addition to alterations in the apoptotic pathways themselves, resistance to apoptosis in DLBCL is driven by the constitutive activation of transcriptional regulators that decrease apoptotic signals and potentiate anti-apoptotic proteins. Inhibition of apoptosis by the nuclear factor kappa beta (NF-kB) pathway is the most notable example of such transcriptional regulation in DLBCL . The rolCD79B occur in 10–25% of ABC DLBCL and 3% of GCB DLBCL, and lead to increased BCR signaling [CD79A mutations can also be observed but are rare [LYN, LAPTM5, PTPN6, GRB2, PRKCD, DGKZ, SLA, and MAP4K1 have collectively been identified in almost 40% of all DLBCL [In ABC DLBCL, the NF-kB pathway is constitutively activated by chronic active BCR signaling . This inignaling ,144,146.CARD11 occur in approximately 10% of ABC DLBCL and in a smaller proportion of GCB DLBCL. These mutations occur almost exclusively in the coiled-coil domain of CARD11 and result in a gain of function by preventing functioning of the autoinhibitory domain [BCL10 are identified in up to 20% of DLBCL and are more common in the GCB subtype [BCL10 amplifications can also occur but are rare [BCL10 also has pro-apoptotic functions [BCL10 mutations occur in about 5% of DLBCL [BCL2, MALT1 is located on chromosome 18q21 and can therefore be gained in the same copy number alteration events [As described above, the CBM complex is essential for NF-kB activation in lymphocytes. In resting cells, CARD11, a scaffold protein from the membrane-associated guanylate kinase family, is inactivated by its autoinhibitory domain. B cell activation leads to phosphorylation and activation of CARD11, recruitment of BCL10 and MALT1, formation of the CBM complex, and NF-kB activation ,148. Misy domain . Chromos subtype ,150. BCLunctions . BCL10 mof DLBCL  and resuof DLBCL . As for n events .NFKB2 (encoding p100) and constitutive protein activation [REL (encoding c-Rel), which are mostly seen in GCB DLBCL [REL amplifications is unclear, as they do not correlate with NF-kB target gene expression [A20 is located on chromosome 6q23.3 and encodes a ubiquitin-modifying enzyme that can downregulate the NF-kB response [A20 have been identified in 24% of ABC DLBCL [A20 can also be the result of 6q23 deletions [TNIP1, encoding an A20-binding inhibitor of NF-kB, can also be seen [MYD88 encodes an adaptor protein that upregulates the NF-kB pathway by mediating toll and interleukin-1 signaling. The L265PMYD88 mutation, a gain-of-function mutation that leads to constitutive NF-kB activation, is observed in 18–30% of ABC DLBCL and is associated with poor prognosis [NF-kB is a family of five dimeric transcription factors  that are involved in numerous processes such as cellular development and proliferation, immune cell activation, and regulation of apoptosis. Two other proteins, p100 and p105, are precursors of p52 and p50, respectively . These 7tivation , and ampCB DLBCL . Howeverpression . Mutatiopression . A20 is response . Loss-ofBC DLBCL . Inactiveletions ,157. Mut be seen . MYD88 erognosis ,16,158. rognosis ,16,17.Inhibition of apoptosis is the main mechanism by which NF-kB contributes to the pathogenesis of DLBCL, particularly of the ABC subtype . This anThe central role of inhibition of apoptosis in DLBCL and other malignancies has led to increasing investigation of apoptotic pathways and proteins as potential therapeutic targets . VenetocExtrinsic apoptosis is of particular importance in the use of therapies that involve cytotoxic T cells, namely immune checkpoint inhibitors, chimeric antigen receptor T (CAR T) cells, and bispecific T cell engagers (BiTEs) ,169,170.TP53 mutations [An important consideration in the choice of therapy for rrDLBCL is a high rate of utations . Loss ofutations . This imutations . BH3 mimutations . Howeverde novo ABC DLBCL has shown promising results in phase II trials [de novo ABC DLBCL [Another potential therapeutic strategy in DLBCL is to modulate transcriptional regulators of apoptosis. The predominant effect of NF-kB on inhibition of apoptosis in DLBCL, particularly of the ABC subtype, makes it a therapeutic target of interest. Inhibition of BCR signaling with the BTK tyrosine kinase inhibitor ibrutinib has initially shown good tolerability but modest response rates in ABC DLBCL ,182. PhaI trials ,188, butI trials . BortezoI trials , has shoBC DLBCL ,192,193.TP53 mutations [DLBCL genetically reprograms itself to inhibit apoptosis. This inhibition of apoptosis is a consequence of genetic events leading to dysregulation of several interacting pathways that modulate intrinsic and extrinsic apoptosis . Overallutations ,194,195.BCL2 translocations [In addition to improving the understanding of the pathogenesis of DLBCL, characterizing the genetic events inhibiting apoptosis have several important clinical implications. Indeed, newer DLBCL classification systems, which are based on genetic defects, recapitulate the different mechanisms of inhibition of apoptosis more accurately than the COO classification. Once a consensus classification system is established, it would likely replace the current COO classification in genetic studies and clinical trials. Sequencing lymphomas in clinical practice may reveal mutation patterns that would help in establishing the diagnosis of DLBCL. Monitoring these mutations in the plasma ctDNA over time would be a non-invasive approach to identify the emergence of resistant subclones. This could then lead to the timely implementation of targeted therapies. For instance, ibrutinib could be beneficial in MCD and BN2 DLBCL, which are characterized by predominant B cell receptor-dependent NF-kB activation, while BH3 mimetics might be particularly beneficial in the EZB subtype, which shows frequent ocations ,16,17.A better understanding of the genetic events contributing to the inhibition of apoptosis in DLBCL is essential. It gives insight into the mechanisms involved in disease development and progression. The identification of potential therapeutic targets in the plasma could help guide physicians to select effective therapies based on DLBCL subtype or apoptotic profile. This precision medicine may ultimately improve the survival of patients with rrDLBCL.


Lung metastasis of malignant tumor signifies worse prognosis and immensely deteriorates patients' life quality. Spatholobi Caulis (SC) has been reported to reduce lung metastasis, but the mechanism remains elusive. P value <0.05. Network of SC-active components-intersection targets-KEGG pathway was accomplished with Cytoscape. Molecular docking between hub targets and active components was performed, analyzed, and visualized by AutoDockTools, AutoDock Vina, PLIP Web tool, and PYMOL.  The active components and corresponding targets of SC were obtained from the Traditional Chinese Medicine Database and Analysis Platform (TCMSP) database and the SwissTargetPrediction database. The disease targets were acquired from DisGeNET and GeneCards databases. Venn map was composed to figure out intersection targets by using R. The PPI network was constructed through STRING and Cytoscape, and MCODE plug-in was used to sift hub targets. Gene Ontology (GO)-Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out by utilizing clusterProfiler package (R3.6.1) with adjusted  24 active components and 123 corresponding targets were screened, and the number of disease targets and intersection targets was 1074 and 47, respectively. RELA, JUN, MAPK1, MAPK14, STAT3, IL-4, ESR1, and TP53 were the 8 hub targets. GO analysis and KEGG analysis elucidated that SC could ameliorate lung metastasis mainly by intervening oxidative stress, AGE-RAGE signaling pathway, and microRNAs in cancer. All 8 hub targets were proven to combine successfully with active components of SC.  Inflammation is the core factor that integrates all these targets, biological process, and signaling pathways, which indicates that SC prevents or reduces lung metastasis mainly by dispelling inflammation. It is estimated that the number of worldwide new cancer cases and cancer deaths has exceeded 19 million and 10 million, respectively, in 2020 . Since tThe question of why metastatic cancer cells manifest the feature of organotropism has brought about extensive discussions. Paget put forward the “seed and soil” hypothesis that prometastatic tumor cells (the “seed”) colonize in specific organ sites (the “soil”) where the microenvironment is favorable for metastasis , 8. In 2Spatholobus suberectus Dunn., is a typical kind of traditional Chinese medicine that possesses functions of invigorating blood circulation and eliminating stasis. It has been proven that 80% ethanol extracts of SC significantly reduced the pulmonary metastasis and prolonged the life span of Balb/c mice, which had been injected with 4T1-luc cells [Spatholobi Caulis (SC), the vine stem of uc cells . The undhttps://tcmsp-e.com/) [http://new.swisstargetprediction.ch/) [https://pubchem.ncbi.nlm.nih.gov/) [http://www.uniprot.org/uniprot/) [With the thresholds of oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18, the active components of SC and its corresponding targets were obtained from the Traditional Chinese Medicine Database and Analysis Platform (TCMSP) (-e.com/) . To thosion.ch/)  was utilih.gov/) . To ensuniprot/) .http://www.disgenet.org) [https://www.genecards.org/) [Homo sapiens,” only those targets that appear in both databases simultaneously were chosen as targets relevant to lung metastasis of malignant tumor.The academic name of lung metastasis “secondary malignant neoplasm of lung” was searched in the DisGeNET database (net.org)  and Geneds.org/) . With thR (v3.6.1) software , we created two files in “csv” format to store the targets of the active components of SC and targets relevant to lung metastasis of malignant tumor, respectively. The two files were read by R (v3.6.1) software, and the VennDiagram package was employed to get the intersection targets between SC and lung metastasis.Before using Setting minimum required interaction score as 0.9, the intersection targets were imported into STRING 11.0  to gain P value <0.05 [Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of intersection targets were conducted to show the top 15 items by using clusterProfiler package (R3.6.1) with adjusted ue <0.05 .This network was constructed by Cytoscape 3.7.2 to visualize mechanisms of SC treating lung metastasis.Homo sapiens”; (ii) the original 3D structures should possess at least one eutectic ligands; and (iii) only the 3D structures with the least “resolution” value would be chosen. The selected 3D structures of the hub targets were downloaded from the PDB database (http://www.rcsb.org/) [https://pubchem.ncbi.nlm.nih.gov/) [https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) [We followed three key principles to select the most suitable 3D structures of the hub targets: (i) the source organism should be “sb.org/) , and remih.gov/)  and werep/index)  to analyAccording to the criterion, 24 active components were recorded, and 123 targets of the active components were identified. The basic information of 24 active components is listed in R software  for further process, shown in p. adjust ＜0.05, 1846 items of biological process (BP) were acquired, and the top 15 items are illustrated in With Taking the advantage of Cytoscape, we created the network of SC, 24 active components, 47 intersection targets, and top 5 signaling pathways, containing 77 nodes and 248 edges shown in . The sizα-L-arabinosylluteolin, MAPK1-Calycosin, MAPK14-Catechin, STAT3-Medicago, IL-4-Vestitol, ESR1-Catechin, and TP53-Hederagenin. After analyzing noncovalent interactions, details including affinity, hydrogen bonds, hydrophobic interactions, π-stacking, π-cation interactions, and relative amino acid residues are exhibited in Supplementary π-stacking and π-cation interactions were present by red and purple dotted line, respectively.In consideration of affinity and hydrogen bonds, we ultimately chose the most practical conformation for each hub target, which were RELA-Aloe-emodin, JUN-8-C-α-L-arabinosylluteolin was supported by 7 hydrogen bonds, which is the most among all 8 conformations. Hydrophobic interaction is the force that reduces the free energy and stabilizes the contact between two nonpolar regions. Except for STAT3-Medicago, at least 1 hydrophobic interaction formed in the remaining 7 conformations, and there were 7 hydrophobic interactions formed between ESR1 and catechin, ranking 1st in our research. MAPK14-Catechin was the only conformation that contains π-stacking and π-cation interactions, which made a great contribution for the stable combination.Affinity is the concept that measure binding force between ligand and macromolecular protein, and it is widely acknowledged that the spontaneous combination is likely to occur when the affinity is smaller than −5 kcal/mol. In our research, affinity of all 8 conformations was smaller than −5 kcal/mol and MAPK14-Catechin exhibited the strongest binding force with affinity reaching −8.9 kcal/mol. The formation of hydrogen bonds mainly results from electrostatic force, which stems from charge transfer between donor and acceptor. In our research, at least 2 hydrogen bonds took shape, which offered powerful electrostatic force to form stable conformation, and JUN-8-C-Compared with primary tumor, the colonies of lung metastasis often mean worse prognosis and less effectiveness of therapeutic plans. Therefore, preventing or reducing lung metastasis of malignant tumor will extend life expectancy and mitigate the distress of patients with advanced stages of tumor.β-Sitosterol is a bioactive phytosterol compound that is naturally present in the plant cell membrane, and it was proven to decrease lung metastasis in PC-3 and 4T1 cells in vivo and in vitro experiments [β-sitosterol for 7 days, the number of metastatic colonies in the lungs of B16BL6 melanoma cells was prominently less than that of the control group, which might be the result of enhanced immune surveillance activity evidenced by the increase of NK cells and immune response cytokines such as IL-12 and IL-18. Aloe-emodin is a natural anthraquinone derivative of many Chinese herbs, and it is reported that the administration of aloe-emodin dampened the burden of lung metastasis through the inhibitory effect on capabilities of invasion and migration of MDA-MB-231 cells [16H12O4) has a promising anticancer effect through influencing a variety of mechanisms, which pertains to a class of 7-hydroisoflavones [β-catenin expression [μM) of luteolin remarkably inhibited VEGF secretion in MDA-MB-231 (4175) LM2 cells, thereby suppressing the occurrence of pulmonary metastasis [In this study, 24 active components were screened in the aggregate, among which many components have been confirmed to suppress lung metastasis with convincing proof. eriments . Imanakaeriments  discover31 cells , 29. Witflavones . Zhou etflavones . Luteoliflavones . A previpression . The restastasis .In all, there were 47 intersection targets obtained between SC and secondary malignant neoplasm of lung and 8 of them were regarded as hub targets after utilizing MCODE plug-in unit of Cytoscape, namely RELA, JUN, MAPK1, MAPK14, STAT3, IL-4, ESR1, and TP53.κB), participates in cancer development and metastasis by forming chronic inflammation. Upon the phosphorylation of S276 amino acid residue mediated by TNF (tumor necrosis factor), RELA leads to the promoter-specific methylation and transcriptional repression of tumor metastasis suppressor gene BRMS1 (breast cancer metastasis suppressor 1) via direct recruitment of DNMT-1 (DNA (cytosine-5)-methyltransferase 1) to chromatin [P < 0.05) and decrease the ability of MDA-MB-231 cells to adhere in vivo and in vitro, demonstrating that RELA can induce lung metastasis by suppressing BRMS1 [κB p65 protein [RELA (transcription factor p65), which is the p65 subunit of nuclear factor NF-kappa-B  and MAPK14 both belong to MAPK, which plays a vital role in the switch from extracellular signals to intracellular responses . Gagliartastases . The ext [α MAPK .JUN (transcription factor AP-1), a downstream factor of ERK pathway, is involved in gene reprogramming in many tumorigenic processes . The bloUpregulated in approximately 50% of all human tumors, STAT3  is a key signaling protein engaged by a multitude of growth factors and cytokines to elicit diverse biological outcomes including cellular growth, differentiation, and survival . AccordiSimilar to STAT3, IL-4 (interleukin-4) assists lung metastasis mainly through M2 polarization of macrophages , which rα (hypoxia-inducible factor 2α). It has been revealed that pretreatment of luteolin reversed oxidative stress induced by H2O2 in a dose-dependent manner [The GO.BP enrichment analysis spotlights the fundamental position of response to oxidative stress in SC treating lung metastasis of malignant tumor. Lung colonies of B16F10 cells increased apparently caused by oxidative stress, assessed by p22phox and SOD mRNA levels and the NRF2 protein level . In addit manner .Among the top 15 results of KEGG pathway enrichment analysis, the AGE-RAGE signaling pathway and microRNAs in cancer are the most relevant to lung metastasis of malignant tumor.Synthesized during chronic hyperglycemic conditions or aging, AGEs (advanced glycation end products) are the products of a nonenzymatic reaction between the free reducing sugars and proteins, lipids, or nucleic acids . The comMicroRNAs (miRNAs) are small noncoding regulatory RNAs with sizes of 17–25 nucleotides and can be transported to distant organs by cancer-secreted exosomes to regulate local microenvironment for engraftment and colonization of circulating tumor cells . miR-25-According to the results above, we found that the majority of hub targets and biological process including RELA, MAPK1, MAPK14, STAT3, IL-4, and oxidative stress are directly associated with inflammation, which is a key feature of premetastatic niche . As the Last but not least, limitations are also clear that further experiments are necessary to verify the results, and the studies cited in this paper are mainly based on animal or cell experiments, indicating deficiency of clinical research as proof. In addition, data of SC and lung metastasis could be more comprehensive by retrieving more literature and databases.Integrating network pharmacology with molecular docking, our research elaborated that SC could prevent or reduce lung metastasis of malignant tumor to some extent by its active components through targeting RELA, JUN, MAPK1, MAPK14, STAT3, IL-4, ESR1, and TP53. Oxidative stress, AGE-RAGE signaling pathway, and microRNAs in cancer also play important roles in lung metastasis of malignant tumor, which are likely to be reversed by SC, laying the foundation for further experiments. Moreover, inflammation is the core factor that integrates all these targets, biological process, and signaling pathways together, which indicates that SC prevents or reduces lung metastasis mainly by dispelling inflammation.