An irreversible step concludes the reaction sequenceto render an integrated solution for precise proteinbioconjugation. In this perspective, we highlight the key advancements,unsolved challenges, and potential opportunities.The chemical toolbox for the selective modification ofproteinshas witnessed immense interest in the past few years. The rapid growthof biologics and the need for precision therapeutics have fuelledthis growth further. However, the broad spectrum of selectivity parameterscreates a roadblock to the field’s growth. Additionally, bondformation and dissociation are significantly redefined during thetranslation from small molecules to proteins. Understanding theseprinciples and developing theories to deconvolute the multidimensionalattributes could accelerate the area. This outlook presents a  disintegrate the selectivity challenges in proteinbioconjugation,followed by the integration of their solutions usinga combination of chemical reactions.The DIN theory provides a systematictool to  The screening-driven drug discovery processes remainthe preferred routes for the latter.3 Further,proteome-wide chemoproteomics has accelerated the segment by establishingselective probes and new targets.5 On the otherhand, the data also provide insight into considerable drug promiscuityand its implications.6 These developmentshighlight the need for principles to regulate selectivity in proteinmodification.The synchronized coordination of eventswithin biomolecules regulateshuman health. In this pool, proteins have served as valuable candidatesas both therapeutics9 Besides, controlling selectivity with isolated proteins,enzymes, and antibodies would empower biologics.14 The overlap of knowledge between segments at thetwo ends of the complexity scale, from small molecules to humans,must evolve with time to meet the technological demands.In this direction, we first need the tools andmethods that canallow us to control selectivity with isolated proteins. Next, we cantranslate them sequentially from one system to another 1a. In thn-butyl amine, a Lys-bearing peptide, and a Lys-bearing protein couldbe substantially different. The trend continues with additional layersadded to the functional group ecosystem (x-axis).Next, the reagent must distinguish one functional group from others. Finally, such a chemoselectivereagent should be able to distinguish one copy of a residue from itsmultiple copies to display site selectivity (z-axis).As we move forward, the modularity beyond protein-defined reactivityorder, site or residue specificity, and protein specificity can offeradditional dimensions.Revisiting this problem from the end of small-moleculesubstratesprovides another perspective. It initiates with chemical bond formationbetween two reactive partners, such as nucleophiles and electrophiles1a. With cosystem 1a. Can wcosystem 1b. Initi16 Subsequently, In this outlook, we argue that the selectivityattributes can bedisintegrated through a multistep chemical transformation  motif creates an opportunityfor irreversible benzylic rearrangement after the nucleophilic additionof Arg.20 The interplay of reversible andirreversible pathways adds another dimension to chemoselectivity.For example, oxoaldehyde reacts with Lys in a kinetically preferredtransformation. However, Arg delivers a thermodynamically stable product,while the Lys adduct reverts through transoximization with hydroxylamine.21The nucleophilic functionalities in a protein are also basicinnature. Their pones 1a, 3, e.g., 1c) can lead tothe labeling of multiple carboxylates without distinguishing Asp,Glu, and C-terminus CO2H (2c).23 On the other hand, the oxidation potential differences between C-terminaland side-chain carboxylates enabled site-selective C-terminal radicalgeneration (2d).24 An α,β-unsaturatedester traps the relatively high-energy C-radical to render the bioconjugate.Even though the oxidative conditions or electrophilic system can compromisethe extent of conjugation, it is a positive step toward the selectivetargeting of a residue with low reactivity.Unlike Arg, carboxylic acid remains in deprotonatedcarboxylateform under physiological conditions. However, its low nucleophilicitymakes it very difficult to target with chemoselectivity. Besides,its high abundance creates a roadblock to site selectivity. For example,esterification with diazo compounds (1e).27 However, the inherentreversibility of C–S bonds results in thiol exchange and couldcompromise the chemoselectivity and site selectivity. These challengeshave been addressed by regulating the reactivity through subtle changesin the electrophile. For example, incorporating bromine,25 an exocyclic double bond,28 and the hydrolysis of the product29 has offered bioconjugates with higher stability. If an exocyclicdouble bond replaces the maleimide carbonyl, the 1,4-addition andsubsequent reduction yield the bioconjugate over pH 6.0–8.5(1f).31 However, such methylene pyrrolonesare susceptible to retro-Michael addition and thiol exchange underbasic conditions. In another set of electrophiles, the carbonyl acrylates32 offer better stability than azidoacrylates.33 Additionally, the vinylpyridinium salts offerirreversible Cys modification with negligible competition from Lys.34 Besides, the isoxazolinium ring uses intramolecularrearrangement and fragment release to render a stable and site-selectiveCys adduct.35 The polarized double bondsequipped with electron-withdrawing groups also offer pKa regulation to avoid retro-Michael addition, such asvinyl sulphone derivatives (1g).36 Polarized triple bonds, such as alkynoic amides, esters, and alkynones,offer another possibility (1h).37 In particular, they are preferred when the application needs cleavablereagents. An unsaturated vinyl sulfide linkage is created in suchcases that undergoes an addition–elimination sequence in thepresence of an external thiol. The positions of the alkyne, terminalor internal, and the electron-withdrawing functionality regulate theirreactivity. For example, an internal alkyne polarized between aryland cyano groups offers hydrolytically stable reagents.38 The alkylation reactions offer another opportunityfor chemoselective Cys bioconjugation (1i). The nucleophilicsubstitution of halogens such as bromine (bromooxetane)39 or chlorine (chlorofluoroacetamides)40 occurs under mild conditions. However, a fewbromooxetane derivatives require higher pH (8–11) and an organicsolvent. The nucleophilic aromatic substitution with benzothiazole41 or chlorotetrazine42 also renders Cys selectivity, with some challenges from Lys. Ina mechanistically different route, a free radical-mediated pathwaycan render thio-ene-43 and thio-yne-based44 Cys modifications. The former involves the photoinducedcoupling of alkenyl glycosides with the thiol group. On the otherhand, thio-yne coupling proceeds by the addition of two thiol-basedfree radicals to a terminal alkyne. The vinyl sulfide intermediateformed by the addition of the first thiol is captured by a secondthiol via a thio-ene mechanism. However, both methods also engageother residues, including disulfide bonds, and impact the structuralintegrity of the protein.Cysteine offersthe other end with respect to carboxylates, bothin terms of occurrence and reactivity. Its low frequency often meansthat the site selectivity is not applicable for achieving single-siteprotein bioconjugation. On the other hand, chemoselectivity can beachieved with a range of soft electrophiles. Multiple polarized doublebonds have been identified from this perspective. For example, theconjugate addition using maleimide derivatives has been explored extensivelyfor Cys modification 47 and thiophosphorodichloridates (2k)48 offer noteworthy control over chemoselectivityand site selectivity for single-site His modification.The reversibility becomes even moreprominent with the His sidechain due to the inherently labile imidazole-based N–C bond.This moderate frequency residue with low reactivity poses a sternchallenge for single-site modification. Substantial competition fromCys and Lys is unavoidable in most cases. Despite this, the prominentrole of His in biological pathways makes it a valuable target.Nα-NH2) and the Lys ε-amine(Nε-NH2). Cys is a prominentcompetitor for amines. However, the differences in hardness, pKa, and occurrence frequency enable their exclusivebioconjugation. The pKa difference alsomakes Nα-NH2 a preferredtarget over Nε-NH2 underphysiological conditions. The charged state ensures enhanced solventaccessibility for this high-frequency residue, making site selectivitya daunting task. Amine modification is often dominated by additionand substitution reactions. Amine acylation with NHS ester derivativesis one of the most frequently employed methods.49 The stability of an amide bond compared to that of a Cys-basedthioester enables the formation of chemoselective product. However,such methods cannot distinguish between Nα-NH2 and Nε-NH2. pH fine-tuning or the controlled addition of an NHS ester coulddeliver a site-selective Nα-NH2 modification.50 The other C-centeredamine-selective electrophiles include dichlorotriazine,51 sulfonyl acrylate .54 Inanother case, the generated imine was designed for an irreversible[3 + 3] cycloaddition (3c).55 The o-ester-substituted arenediazonium reacts withthe amine to generate an unstable triazine adduct. Next, an intramolecularreaction generates a stable benzotriazinone derivative.56 The site selectivity presents a substantialchallenge in most of these cases. However, blocking Nε-NH2 through acidic reaction conditionscould create an opportunity for N-terminus labeling. For example,this approach enabled a ketene to render single-site Nα-NH2 labeling (3d).57 In a distinct approach, a hemiaminal formedbetween Nα-NH2 and arylaldehyde with ortho-selenoester enables the N-terminusmodification (3e).58 Further,we demonstrated that a proximally placed electrophile could site-selectivelycapture the Nα-NH2 imine.59 In another example, N-hydroxypthalimideachieves the site-selective Nα-NH2 modification by shifting the rate-determining step from anintermolecular to intramolecular process while negating the requirementof slow addition (3f).60 Thesite-selective Lys modification is not accessible through single-stepprocesses, especially due to the presence of Nα-NH2.UnlikeHis, multiple electrophiles have been established to displayhigh chemoselectivity toward the N-terminus α-amine .62 Hypervalent iodine reagents also offer promisefrom this perspective (3h).63 However, selectively modifying Met under mild conditions withoutperturbing other residues is still problematic.Met presents an entirely different scenario withrespect to Lys.It is among the most hydrophobic residues and hence is often buriedin domains with minimal solvent accessibility. Moreover, it holdsthe second position from the bottom in the amino acid frequency scale.These attributes make surface-accessible Met residues rare. It isless likely to face site selectivity challenges when available, makingit a promising target for single-site modification. As it is proneto oxidation, redox-activated chemical tagging (ReACT) enables selectiveMet bioconjugation using oxaziridine-based reagents (3i).64 In another case, a heterogeneousPdNP biohybrid catalyst allowed indole C(2)–H activation inTrp under physiological conditions (3j).65 The protein  offers two Trp residues on the surface,and careful control of catalyst loading allows the predominant labelingof a single site.Principally,Trp falls under a similar category as Met, as it isthe least abundant and exhibits minimal surface exposure. Hence, itprovides an equally good opportunity for single-site protein bioconjugation.A sterically unhindered and stable nitroxyl radical (ABNO) reactedselectively with Trp by forming a C(3)–O bond with indole (66 and cyclic imines (3k).67 Besides, the cyclic diazodicarboxamide derivatives havebeen successfully employed for selective Tyr bioconjugation throughthe ene-reaction under physiological conditions (3l).69 Additionally, a diazonium salt offers an appropriate handle to reactwith Tyr chemoselectively.70 In anotherapproach, N-methyl luminol derivatives under HRP-catalyzedsingle-electron transfer (SET) or electrochemically activated SETdeliver selective Tyr modification (3m).72Tyr is the third residue in this category,with a low frequencyand limited surface exposure. The C=N and N=N bondsin diverse structural motifs have served as capable electrophilicsystems for targeting the phenolic residue of Tyr. For example, Tyrreacts conveniently with an in situ formed imine5a, 73 The other initial findings established thatvarying the linker alters the reactivity and site selectivity of theepoxide in hCAII.75 This method offered the additionalflexibility of removing the ligand post-bioconjugation. A benzenesulfonamide-linkedtosyl group (5e) also offered similar control over selectivity,enabling His alkylation in hCAII.76 Theligand-enabled localization of acyl imidazole (5b) createdan opportunity for the selective acylation of a single Lys residuein a protein.77 The same ligand was alsoutilized to localize a Ru complex ([Ru(bpy)3]2+, 5f), allowing SET photocatalysis for selective Tyrmodification.78 The concept has been furtherextended to the biotin–avidin combination for the selectivelabeling of Lys using O-nitrobenzoxadiazole 79 andthe diazotransfer reaction with imidazole-1-sulfonyl azide (5c).80 The specificity of the ligand–proteininteraction limits the competitors considerably and empowers routea for selective protein modification in a biological milieu such aslive cells.The challenges of addressingreactivity, chemoselectivity, and site selectivity are amplified withlarge proteins and complex biological mixtures. The ligand–proteininteraction driven by reversible covalent or noncovalent binding offersan excellent solution by limiting the number of competitors. Subsequently,the electrophile has a much better opportunity to render a chemoselectiveand site-selective modification. In this perspective, a benzenesulfonamideligand tethered to an epoxide 5a, 5 throughintermolecular reaction will only needto regulate the site selectivity. Besides, the intermediate generatedafter the first step could redefine the reactivity order. In turn,it opens the opportunity for single-site labeling of reactivity hotspotsdistinct from route a targets. Such methods could also offer moreflexibility with the reaction parameters, at least until the firststep.The DIN theory deconvolutes the chemoselectivityand site selectivityinto two steps. In turn, it could provide enhanced control while addressingthese selectivity attributes separately route b, 2. For ex8a, 9a). This step engages multiple solvent-accessibleamines. The external nucleophiles can site-selectively capture a singlecopy of these intermediates. We demonstrated that diethyl phosphite,triethyl phosphite, and t-butyl isocyanide (Nu) coulddeliver the single-site labeling of Lys residues with a structurallydiverse set of proteins (11a).81 The reactivity order of primary amines is mostly redefined whenthey are converted from their nucleophilic form to imine-based intermediates.Often, this leads to a distinct bioconjugation site. The selectionof o-phthalaldehyde can also render stable adductswith external amines.82 However, it isaccompanied by competing pathways that create roadblocks for chemoselectivityand site selectivity. On the other hand, the latent electrophilicimine-based intermediates (9b) can be captured site-selectivelyby the Cu–acetylide complex (11b).83 Interestingly, the Nα-NH2-based imine with selected aldehydes reacts rapidlywith the proximal amide bond to produce imidazolidinone (9b). This reversible in situ protection keeps Nα-NH2 out of the competition. Besides, thisapproach could create conjugation sites distinct from those in routea, confirming the alteration of reactivity hotspots. These methodstranslate well to large proteins such as monoclonal antibodies (mAbs).The approach can also facilitate targeting a proteinogenic secondaryamine with high selectivity. For example, an N-Pro-derivediminium intermediate (9c) reacts with an external nucleophileconveniently in a borono-Mannich reaction (11c).84 The Nα-NH2-imine (9d) with certain aldehydes (8e) can also be captured with NaBH3CN (10c).85 However, N-Cys-containing proteinsrender a mixture of reductive alkylation and thiazolidine-based proteinconjugates.In one of the established examples, the reaction of analdehyde8a, 6 derivat10d) capture thethiolate for antibody modification.86 Further,the reagents with two electrophilic centers add value by rebridging.From this perspective, the reduction (9e) followed bythe thiol-selective reaction with divinylpyridine (10e) works well to render the bioconjugate (11f).87 In a principally similar manner, s-tetrazines (10f),88 allylor aryl sulfones (10g),91 and pyridazinedinones(10h)92 render the selectivetargeting of disulfide bonds. This approach requires caution, as thefunctionality often contributes to structural stability. For example,disulfide scrambling and heterogeneity are commonly observed withthe antibodies.In the absence of an accessible free Cys residue,a chemoselectivereversible reduction of the disulfide bridge provides a notable alternative.For example, ethynylphosphonamidates  through a linker gives a single-site His modification.95 The method offers simultaneous control overreactivity, chemoselectivity, site selectivity, and modularity. Atfirst, FK1 forms an imine  with all the accessible Lys residues in a reversible reaction.It allows the second electrophile (FH) to react irreversiblywith a proximal His residue to deliver site-selective labeling (15a). The site selectivity and modularity can be regulatedby the spacer design. The aldehyde (FK1) iscaptured with hydroxylamine (14) for the subsequent installationof probes. The approach also translates well to the lysine-directedsingle-site modification of a lysine residue (LDMK–K) by replacing the epoxide (FH) with an acylating reagent.96 The strategy was further extended to selectively labelLys residues in various therapeutically relevant monoclonal antibodiesusing p-phenol ester (12d) equippedwith a linchpin fragment as the leaving group.98 The o-hydroxyl group of the linchpin imine makesit highly inert toward the external nucleophiles. It creates an opportunityto use another aromatic aldehyde (FK2) to createthe second imine,99 which can be capturedin the presence of an external nucleophile to deliver a single-sitemodification. The LDM platform also extends the site-selective modificationof His or Asp using an alkylating reagent such as sulfonate esters.100 Additionally,we demonstrated that nitroolefins could shift the linchpin sites froma high-frequency residue (Lys) to Cys with low occurrence.101 This considerably reduces the number of competitorsand creates the opportunity to translate the method for protein selectivityin a complex biological milieu. The nitroolefin (FC) meetsall the criteria necessary to create an appropriate linchpin and useFK2 (12e) for the Cys-directedLys modification (LDMC–K). Here, the chemoselectivethio-Michael addition (13b) enables the site-selectiveLys acylation (15b). The linchpin’s release fromthe thio-Michael adduct is coupled with a retro-Henry reaction torender an aldehyde under mild conditions for downstream utility. Weanticipated that the geometry and conformation of LDMC–K reagents would regulate the site selectivity. Hence, the proteinmodification must happen if the distance between the linchpin andthe target site matches the FC–FK effectivelength. Further, the contribution from the dynamics of the proteinand the reagent needs to be assessed. In this perspective, the MDsimulations offer microscopic insights into the structures and dynamicsof these reaction partners. The simulation results validate that theconformational flexibility of the reagent coupled with protein-inducedrigidity regulates the effective spacer length and the bioconjugationsite. In another validation, the semioxamide vinylogous thioester-basedSTEF probes offer reversible Cys conjugation to regulate an irreversiblesite-selective Lys modification.102 A classof cleavable aryl thioethers linked with UV-activatable o-nitrobenzyl alcohol delivers the selective labeling of the Lys residue.103 The modified Cys residue is recovered by thiolexchange in this case. In a recent development, we established thatthis approach could be extended from a pair of residues to the molecularsignatures composed of three residues  and the proelectrophile (12g), bothof which were equipped with a linchpin handle for proximity control(LDC). In a principally similar approach, the reversible complexationof boronic acids (BA) with an antibody’s FC-N-glycan directs the 4-(dimethylamino)pyridine (DMAP).105 The latter forms N-acylpyridiniumintermediate with thioester-based acyl donors to yield the acylationof a proximal Lys residue. Encouraged by the linchpin-guided proteinmodification platform, 10H-phenoxazine-3,7-dicarboxaldehydewas designed for Tyr modification via photoredox catalysis.106 It would be interesting to see if integratingspacers or linkers in these reagents can add modularity to this chemo-and site-selective method.The linchpin-directedmodification (LDM) platform provides theproof of concept for this segment. Like route b, it initiates withchemoselective imine formation with all the accessible amines. However,this is where the similarity ends, as the imine does not participatein the subsequent irreversible reaction. It serves as a linchpin anddirects another functional group to the proximal residue of interest.The chemoselectivity attributes of the latter and the spacer’sdesign connecting the two functionalities determine the conjugationsite. For example, the yde FK1, 7a tetherresidues 7b.104 ItNα-NH2 coupled with another functionalgroup, such as the side chain residue of the N-terminal amino acid,offers such an opportunity. For example, the N-Cysprotein reacts reversibly with thioesters (16a to 17a) to create an opportunity for an irreversible intramolecularSCys to Nα-NH2 acyl transfer .109 In another case, the N-Cys thiol could capturethe Nα-NH2-imine (17b) to render thiazolidine (18b).110 The challenges outlined for C–S bondstability in route a are associated with these adducts. However, theycan be addressed to an extent through the use of aldehydes equippedwith boronic acids.111 Boronic acid enhancesthe reaction rate and stabilizes the thiazolidine through the B–Ndative bond along with other B-mediated coordination.112 The product stability can also be enhancedby engaging the lone pair on the thiazolidine nitrogen through intramolecularacylation.113 Additionally, the Nα-NH2 can pair with the proximalamide to render selectivity. For example, 2-pyridinecarboxaldehyde114 or 1H-1,2,3-triazole-4-carbaldehyde (TA4C)115 forms an imine that prefers to react with the penultimateamide to yield imidazolidinone (18c). The Lys Nε-NH2-based imine lacks suchsupport to facilitate the irreversible step. Under similar conditions,pyridoxal-5-phosphate  generates the Nα-NH2-imine that tautomerizesto render a glyoxyl imine.116 Its hydrolysisyields an aldehyde or ketone (18d) for chemically orthogonaltransformations.Additionally, targeting a pair of functionalitiesfrom the sameresidue can offer a unique signature to drive selectivity 8. Nα-NH2Nα-NH2-imine  could generate a nucleophilic intermediate(21).117 A proximal H-bondstabilizer in the aldehyde plays a critical role in the process. Itallows the N-terminus Cα-H functionalityto be paired with Nα-NH2 and creates a unique signature for bioconjugation. Hence, the Nε-NH2-imine would be excludedfrom any irreversible transformation. Additionally, the method candistinguish the Cα-H of an unsubstitutedamino acid from those of all the substituted analogs. Hence, it offersexclusive N-Gly selectivity and renders precisionlabeling of proteins without affecting any internal residue. It alsoenables protein selectivity by distinguishing N-Glyfrom all the other N-terminal amino acids in a complex mixture ofproteins. In another report, the Gly tag technology inspired azidopyridoxal derivatives that delivered residue-specific azidolation.118 Engaging a unique combination of functionalitiesfor an irreversible transformation can offer new gateways for preciselyengineered bioconjugates. It would be interesting to see if chemicaltransformations can harness a Cα-H pKa of another unique residue. Besides,the side chain residues can generate a proximal electrophilic systemto engage the Cα-H-enabled nucleophilicintermediate. One can also imagine a side-chain residue to generatean intermediate that can serve as a leaving group in cooperation with Cα-H abstraction. Such a case could leadto the formation of chemically orthogonal dehydroalanine. If extendedto an internal residue, these approaches will likely encounter multiplesolvent-accessible copies. Hence, the question of N-residue specificitywill be redefined to site selectivity.As we noticed in route c, treating the proteinwith an aldehydechemoselectively converts a nucleophilic landscape to its electrophiliccounterpart. If an additional step can follow this to generate a distinctreactive intermediate, it could disintegrate additional selectivitychallenges. For example, we demonstrated that the mine 20, 9 with anProtein bioconjugates have established theirimmense value at thechemistry–biology–medicine interface. However, chemicalmethods enabling control over precision are vital to meet the technologicaldemands. This makes it essential to understand the bottlenecks inthe development of new routes for selectively modifying proteins.The functional group ecosystem of a protein is complex, and the interplayof multiple operating parameters adds to the challenge. That is whyhit-and-trial or directed screening has been a preferred option fordeveloping a new method.The DIN theory, in this Outlook, arguesthat the selectivity attributescan be disintegrated through a multistep chemical transformation.In turn, it could enable the exploration of new reactivity dimensions,better prediction of potential products, and hypothesis-driven research.We have outlined three representative routes (b–d) to exemplifyhow such deconvolutions led to additional selectivity or specificityattributes. It would be interesting to see how new dimensions addto this repertoire with time. The methods addressing all the selectivityattributes in a single step often fall short of site selectivity.Besides, their flexibility and translation for protein selectivityin complex systems are limited. Additionally, the precision in suchexamples displays a very high sensitivity toward the reaction parameters.The disintegration of chemoselectivity and site selectivity in twointermolecular steps renders a unique reactivity landscape and hotspotsfor single-site modification. Shifting the latter to an intramolecularstep also opens the platform to regulating modularity. Further, themultistep deconvolution could deliver the simultaneous regulationof chemoselectivity, residue specificity, and protein selectivity.Such processes also offer access to a broader reaction parameter windowwithout compromising the overall selectivity.In the comingyears, the DIN theory could encourage the examinationof multiple unique reactive intermediates through reversible transformations.In turn, we will have access to distinct reactivity orders leadingto unique bioconjugation sites. These findings will likely extendthe reach of single-site bioconjugation to low-reactivity residues.It will also be interesting to see if such deconvolutions can empowerhigh-energy intermediates for site-selective modification, contraryto their behavior. We can expect it to accelerate the field’sgrowth while contributing to the rapidly growing segment of biologicssuch as ADCs, AFCs, and conjugate vaccines. Besides, such regulationscan expedite the discovery of molecules, such as covalent inhibitors,for the selective targeting of a protein.


Neoplastic cells displayed altered metabolism with accelerated glycolysis. Therefore, these cells need a mammoth supply of glucose for which they display an upregulated expression of various glucose transporters (GLUT). Thus, novel antineoplastic strategies focus on inhibiting GLUT to intersect the glycolytic lifeline of cancer cells. This review focuses on the current status of various GLUT inhibition scenarios. The GLUT inhibitors belong to both natural and synthetic small inhibitory molecules category. As neoplastic cells express multiple GLUT isoforms, it is necessary to use pan-GLUT inhibitors. Nevertheless, it is also necessary that such pan-GLUT inhibitors exert their action at a low concentration so that normal healthy cells are left unharmed and minimal injury is caused to the other vital organs and systems of the body. Moreover, approaches are also emerging from combining GLUT inhibitors with other chemotherapeutic agents to potentiate the antineoplastic action. A new pan-GLUT inhibitor named glutor, a piperazine-one derivative, has shown a potent antineoplastic action owing to its inhibitory action exerted at nanomolar concentrations. The review discusses the merits and limitations of the existing GLUT inhibitory approach with possible future outcomes. The most prominent GLUTs are highly overexpressed in cancer cells and encoded by the SLC2A gene family . In addi glucose .The credit for discovering glucose transporters goes to The GLUT1 and GLUT3 display about 64% resemblance of amino acid composition. in silico structure and docking prediction tools have greatly added to deciphering the binding sites of glucose and inhibitors on GLUT and predicting the ‘Modus Operandi’ of the transport process , and Hox antisense intergenic RNA (HOTAIR) . Given tSmall molecule GLUT inhibitors mainly belong to natural, seminatural and synthetic chemical origins . have elin silico docking tools to decipher the mechanism of GLUT1 inhibition and identified the amino acids and binding sites through which cytochalasin B exerted its inhibitory action. However, being a blocker of microfilament polymerization, its utility for antineoplastic application in vivo needs to be verified vis à vis validation of safety for other organs and tissues. Moreover, the cytoskeleton plays a crucial role in the functioning of many tumor-infiltrating cells like macrophages and dendritic cells, where microfilament poisoning by cytochalasin B needs to be controlled while utilizing it for anticancer therapeutic approaches.A cell-permeable mycotoxin named cytochalasin B, which is also a microfilament poison and used to regulate cytoskeletal elements is yet another GLUT inhibitor of natural origin that is extensively investigated for its mechanism of GLUT inhibition and utility for antineoplastic applications . Kapoor streptomyces of the actinomycetes family. Glucopiericidin A has been remarkable for its low LD50 values and is predicted to have less toxicity   . Thus, tIn vitro experiments on Caco-2 cells revealed that exposure to anthocyanin-rich plant extract inhibited the expression of SGLT1 and GLUT2 phenyl)-3-oxobutanamide  is a sma and TNF  later it and TNF . However and TNF . Moreove and TNF  which isin vitro and in vivo to explore its dual inhibitory action on GLUT1 and NAMPT. However, the utility of the same will depend on the expression levels of GLUT1 and NAMPT, which may vary in a tumor-to-tumor manner. Moreover, it is also reported that if cancer cells express GLUT2, they can overcome the inhibitory action of STF-31 and that it does not harm normal cells  triple-negative breast cancer (TNBC) cell lines  to GLUT1 has shown a promising inhibitory effect on M.D. Anderson - Metastatic Breast 231 (MDA-MB-231) and ll lines . Similarll lines . Wang etll lines . Thus, tApproaches to transfecting neoplastic cells with GLUT1 antisense cDNA have been demonstrated to usher in a decline in the gene expression of GLUT1 and an increase in radiosensitivity of neoplastic cells . The comin vivo animal models is essential before exploring the clinical potential. As stated above, selecting those GLUT inhibitors with a pan-GLUT inhibitory potential at low concentrations will be necessary to avoid toxicity on normal healthy cells and organs. Assessing safety on crucial physiological parameters, including blood profile, renal and hepatic function, will also be essential. The half-life of the GLUT inhibitors and their renal clearance needs to be determined for dose optimization. As GLUT plays a critical role in neurological functions, the ability of GLUT inhibitors to cross the blood-brain barrier must be critically evaluated.The review above shows that inhibition of GLUT can be a promising antineoplastic therapeutic approach, which needs to be vigorously approached at preclinical and clinical levels. It will be essential to consider the safety of the combinatorial approach of GLUT inhibitors. As a massive fraction of the human population is coming under various metabolic disorders, including diabetes, GLUT inhibitors’ impact must be carefully evaluated in such patients. However, overcoming these lacunas and unaddressed issues indicates a bright future for using GLUT inhibition approaches in anticancer regimens.1) It is worked out to be a Pan-GLUT inhibitor and hence is supposed to exert a more potent inhibitory action on glucose uptake2) It has been screened against 94 cancer cell lines and is found to be effective in more than half of cell lines for its cytostatic action, and no other small molecule inhibitor has been tested on such a broad range of target cancer cells of diverse etiology.3) Its antineoplastic action is exerted in the nanomolar concentration range and has been shown to have the least toxic effect on normal cells.4) Its easy accessibility at the commercial level for further investigation.Based on the criteria for selection of GLUT inhibitors, it emerges that glutor is one of the best to choose for further investigations owing to the following points in its favor:Therefore, based on our evaluation glutor has an edge over other pan-GLUT inhibitors for further investigations at preclinical and clinical level.


Deposition ice nucleation of ferrihydrite occurred at a relative humidity with respect to ice, RHi, between ∼120–138% and temperatures, T ∼ 232 K. However, we also observed water uptake on ferrihydrite at the same T when deposition ice nucleation did not occur. Although, immersion freezing of ferrihydrite both in pure water droplets and in aqueous citric acid occurred at or slightly below conditions for homogeneous freezing, i.e. the effect of ferrihydrite particles acting as a heterogeneous ice nucleus for immersion freezing was small. Microcline K-rich feldspar mixed with xanthan gum was also used in INXCell experiments. Deposition ice nucleation occurred at conditions when xanthan gum was expected to be highly viscous (glassy). At less viscous conditions, immersion freezing was observed. We extended a model for heterogeneous and homogeneous ice nucleation, named the stochastic freezing model (SFM). It was used to quantify heterogeneous ice nucleation rate coefficients, mimic the competition between homogeneous ice nucleation; water uptake; deposition ice nucleation and immersion freezing, and predict the T and RHi at which ice was observed. The importance of ferrihydrite to act as a heterogeneous ice nucleating particle in the atmosphere using the SFM is discussed.Ice nucleation is one of the most uncertain microphysical processes, as it occurs in various ways and on many types of particles. To overcome this challenge, we present a heterogeneous ice nucleation study on deposition ice nucleation and immersion freezing in a novel cryogenic X-ray experiment with the capability to spectroscopically probe individual ice nucleating and non-ice nucleating particles. Mineral dust type particles composed of either ferrihydrite or feldspar were used and mixed with organic matter of either citric acid or xanthan gum. We observed  in situ using X-ray spectro-microscopy in a new experiment, which is applied to mineral aerosol particles composed of ferrihydrite or feldspar and associated organic matter.Ice nucleation can now be imaged  Snow, hail, and about two-thirds of rain begins as ice crystals. Therefore, the initial formation of ice crystals, or ice nucleation, in the upper atmosphere is a critical step in the Earth's water cycle. Particles present in the air or within cloud droplets can catalyze ice nucleation in what is called heterogeneous ice nucleation. We studied this with a range of particle types in a newly developed cryogenic environmental cell that allows nano-focused X-ray experiments to be performed while ice nucleation is occurring. We have found that particles of an important mineral in airborne dust, ferrihydrite, aids ice nucleation and so is important for cloud formation. When the concentrations of such particle types are known, our work can be used to accurately predict the amount of ice formed for better representation of clouds in weather and climate models.11–3 of stratospheric water vapor,4,5 of multi-phase chemical reactions on ice,6–9 and thus for climate projection overall.10–12 Furthermore, ice plays a large role in precipitation, not only for snow, sleet and hail, but also for rain – about two-thirds of rain begins as ice crystals.13 In mixed-phase clouds where ice particles and liquid droplets co-exist, ice crystals grow and remove water vapor by sedimentation, which can affect cloud lifetime and reflectivity of solar radiation.5 Cirrus clouds are composed entirely of ice crystals and on average, they have a warming effect (positive radiative forcing) due to them being fairly transparent to solar radiation and strongly absorbing terrestrial radiation.14,15 Aerosol particles are playing a key role in the formation of ice. They have varied composition and properties, including organic and inorganic matter, as well as water soluble and insoluble components.16–18 This has led to a wide range of thermodynamic conditions over which they can nucleate ice. Aerosol-cloud interactions can influence Earth's radiation balance, atmospheric temperature, and cloud evolution.5,11,19–21 However, accurate representation of aerosol–ice cloud interactions and any resulting climatic effects requires a significant reduction in the uncertainties of quantifying how aerosol particles influence ice cloud abundance, albedo, height, and especially overall ice particle numbers in clouds.5,21Ice formation in the atmosphere is important for accurate representation of the hydrological cycle,20,21 Homogeneous ice nucleation can occur from micrometer sized liquid cloud droplets and aqueous aerosol particles below about 235 K.22–25 Heterogeneous ice nucleation in the atmosphere can occur from the liquid or the vapor phase of water on aerosol particles, termed ice nucleating particles (INPs). Furthermore, the ability to form ice can be highly variable between different substrates including insoluble particles, crystalline particles, biogenic particles or macromolecules, compressed surfactants on droplets and highly viscous (or glass-like) solid aerosol particles.20,23,26–28 Atmospheric particles experience continuously changing relative humidity with respect to water, RHw, and temperature, T, for a wide range of water vapor pressures. Therefore, knowing the precise RHw and T that define which phase transitions to ice are dominant is of great value. Immersion freezing (liquid-to-ice) is a heterogeneous ice nucleation mode that describes liquid water in an aqueous aerosol or cloud droplet initiating freezing on a particle surface immersed within the liquid. Another heterogeneous ice nucleation mode is deposition ice nucleation (vapor-to-ice), whereby water molecules from the supersaturated vapor phase nucleate ice and subsequently form an ice crystal on a particle surface without the involvement of a bulk liquid aqueous phase. Other freezing modes include contact freezing, condensation freezing, pore-condensation freezing or inside-out freezing,29–32 however these are not considered here. Surfactant substrates can nucleate ice heterogeneously,33–37 although, the impact of ambient organic surfactants on ice formation in atmospheric clouds is fairly understudied compared with laboratory based investigations.38 In particular, the kinetics of ice nucleation for immersion freezing have been quantified using a universal approach based on aqueous solution water activity in laboratory and field studies applicable for a variety of substrates previously mentioned including mineral, organic, biological, surfactant, and soot.39–42 Use of water activity in this study to predict freezing is detailed in a later section. It is important to note that an ice nucleating substrate can be solid-like (glassy or highly viscous), but not necessarily water insoluble.20,43–45 In this study, we have aimed to investigate whether deposition ice nucleation, immersion freezing or homogeneous ice nucleation is favored over the other depending on the particle types present.Predicting atmospheric ice cloud formation from aerosol particles is difficult, partly because ice can form in different ways depending on the aerosol composition and phase.46 and found in the residuals of ice crystals.47–50 Laboratory and model studies have revealed that common minerals and clay such as feldspar, a highly important atmospheric ice nucleating particle, kaolinite and other iron containing minerals are ice forming particles.40,51–59 Iron is frequently incorporated into airborne clay mineral particles and can be at about 5 weight percent as inclusions of iron oxy-hydroxides.60 Additionally, iron mass abundance is typically equal to that of calcium and aluminum in fine dust.46 Anthropogenic iron from urban and combustion sources can be present in about 1 in 20 particles61 and make up a high fraction of soluble iron globally, suspected to be due to an abundance of ferrihydrite.62 Ferrihydrite, or colloidal iron oxy-hydroxides, is an amorphous mineral phase responsible for the transition between soluble iron (i.e. iron ions) and hematite or goethite.63 Iron minerals such as hematite and goethite are heterogeneous ice nucleators and their freezing efficiency increases (i.e. freezing can occur at warmer temperatures) when milled or when particle surface area is increased by other chemical or physical treatments.64,65 The authors of these studies explain their results with milled and unmilled particles by correlating the freezing temperatures with differences in OH group surface concentration and arrangement, in addition to the degree of ice crystal lattice-match.64,65Iron containing aerosol particles are a subset of mineral dust particles that are abundant66,67 and to a quarter of iron containing particles from oil combustion.66 Although it is thermodynamically unstable,68,69 ferrihydrite is ubiquitous in nature70 and can remain untransformed for a few hundred days in the atmosphere.71 This long lifetime has been reportedly due to silica, clay minerals, and a range of organic substances  that contribute to its stabilization.72 Importantly, ferrihydrite is also considered to be a product from the weathering process of iron containing clays such as illite or smectite69 and indeed present at the surface of such clay minerals.69,73 Unlike goethite and hematite, ferrihydrite has no clear structure and previously observed to be in the form of nanoaggregates.63,74,75 It exists in two types, exhibits 2 or 6 lines in X-ray diffractograms and has no fixed composition.63 Due to its porous nanostructure, ferrihydrite has a high specific surface area and offers a high number of accessible sites for adsorption and reaction. It is also considered to be the main source of bio-available iron, i.e. leached from the mineral and absorbed by organisms and thus is critically important to marine life.63,74,75 Thus, ferrihydrite can significantly impact atmospheric chemistry, iron fertilization in oceans, and in particular, ice cloud formation. Although, its capability to nucleate ice has not previously been identified, likely due to the difficulty in isolating it from ambient air and from other minerals, as well as being difficult to synthesize in the laboratory. Being quite unstable, ferrihydrite is also difficult to accurately identify and characterize in laboratory samples.Ferrihydrite is an amorphous iron oxy-hydroxide. In general, iron oxy-hydroxides amount to more than half of all iron in Asian and African dust aerosol particles,in situ with stability and control of RHw and RHi, which was not previously achievable in a STXM experiment. Environmental scanning electron microscopy (ESEM) has previously been used for imaging ice nucleation in situ.76–78 Although spatial resolution of SEM is superior to STXM, energy-dispersive X-ray spectroscopy used with SEM has inferior chemical selectivity compared with STXM/NEXAFS.79,80 Our method also does not require sample transport or vacuum exposure in between ice nucleation observations and chemical investigation as in offline studies,48,50 eliminating sudden exposure of particles to vast swings of T and RHw. STXM/NEXAFS requires thin (∼100 nm) and delicate silicon nitride windows for an X-ray transparent support for the particles, however this results in very poor survivability of samples for offline studies due to transport, handling and preparation. Our INXCell also facilitates easier identification of single ice nucleating particles. This avoids the difficulty for offline studies due to the differences in orientation, contrast generation and spatial resolution between various microscopes, e.g., optical and X-ray, that can hinder re-identification of particles. These difficulties are evident due the fact that only one particle observed to nucleate ice was ever observed using STXM/NEXAFS offline,50 and ice was only tentatively observed in another X-ray environmental cell without knowledge of the nucleating T or RHi conditions.81 When considering ice nucleation capability, spatial resolution and chemical selectivity, the INXCell is superior to other techniques, e.g. Raman spectro-microscopy, typically having a 1 × 1 μm2 pixel size.82 This is important when investigating submicrometer sized particles or ambient particles that can be highly diverse.Here, we have used a unique pairing between scanning transmission X-ray microscopy coupled to near-edge X-ray absorption fine structure spectroscopy (STXM/NEXAFS) and a new ice nucleation experiment, termed the INXCell, to simultaneously observe the formation of ice in the vicinity of specific particles under tightly controlled temperature and humidity conditions. The composition and chemical state of particles (those that did and did not nucleate ice) were imaged 83,84 Xanthan gum is a proxy for polymeric or oligomeric compounds common in atmospheric organic particles85,86 and primary marine organic matter.87,88 The glass transition of xanthan gum was previously documented,89 and beneficial to explain the impact on ice nucleation mode when particles are more solid or liquid-like. The INXCell encloses a volume between two X-ray transparent membranes where the local gaseous environment is provided from a controlled external flow and the temperature is controlled by blowing cold nitrogen gas against the membrane supporting the test particles. A platinum wire sensor printed onto the membrane was used to measure the temperature with high precision and accuracy. The pair of membranes provide a window through which the sample volume of INXCell can be observed in situ with STXM/NEXAFS spectroscopy.79,80,90 This allows the composition and chemical state of particles to be mapped in situ, as well as having the stability and control to image particles inside ice crystals and water droplets at saturation with respect to liquid or ice . Additionally, we have advanced a previous model for homogeneous ice nucleation and heterogeneous ice nucleation from the vapor phase to complement our experimental results that builds on our previous work.40 This model is designed to derive heterogeneous ice nucleation rates with carefully estimated uncertainties. Together, our new method and model makes it possible to investigate single ice nucleating particles in situ as a function of T, RHw, and RHi.We quantify the ice nucleation ability of bare ferrihydrite, ferrihydrite mixed with citric acid, and K-rich microcline feldspar mixed with xanthan gum. Citric acid was used as a proxy for oxygenated organic material common in atmospheric aerosol particles and has a similar viscosity behavior.279,80,90 traditional cryogenic cold-stage optical microscopy ice nucleation techniques,91,92 and in general, water uptake and ice formation are imaged and spectroscopically probed.81,93–100 Controlling the environmental conditions with enough precision to navigate the phase changes while also keeping the sample area thin enough for transparency of the soft X-ray beam is very challenging, as illustrated by the fact that only one study has ever tentatively detected ice under controlled conditions,81 and that pure water droplets formed from the vapor phase have never before been observed using STXM/NEXAFS. A key feature in the design of the INXCell was the use of lithographic patterning to fabricate high precision temperature sensors directly onto sample substrates, placing the sensor directly in the measurement region and allowing precise determination of the thermodynamic conditions when ice nucleates on particles within the INXCell. Previous in situ ice nucleation studies with higher spatial resolution than optical microscopy have utilized environmental-scanning electron microscopy (E-SEM), in which a sample is imaged while resting on a thick, cold, and thermally conductive block in a humidified atmosphere.51,76–78,101 These studies have advanced our understanding of how morphological features of individual particles or surfaces can impact freezing and crystallization. However, E-SEM does not have chemical sensitivity, compared to STXM/NEXAFS, which can identify organic coatings and provide information on chemical bonding and metal speciation within individual particles. This is important due to the great chemical complexity that ambient INPs can have.50,78 Both INPs50 and the residuals of ice crystals48 have been probed using X-ray spectro-microscopy in offline analysis. In terms of in situ analysis, previous studies have successfully produced X-ray transparent, layered structures for the purpose of investigating time resolved changes in magnetic domains,102–105 electrochemical investigation106 and calorimetric studies.107,108In situ microreactors have been previously developed and used in the field of atmospheric chemistry and physics for investigating water uptake and phase transformations,81,93–97 and chemical98,99 and photochemical reaction cycling.100,109The INXCell is based on STXM/NEXAFS,2.1102,105 and inspired by a previous study.108 Briefly, a 40 nm thick film of platinum was deposited with a serpentine pattern in a PMMA/MMA (poly methyl methacrylate/methyl methacrylate) bilayer resist prepared by electron beam lithography with a direct current magnetron sputter coater (AJA) at a base pressure <2 × 10−8 torr and deposition pressure of 3 mTorr. The platinum and resist were on a 1 mm square silicon nitride window having a 100 nm membrane thickness supported by a 0.2 mm thick silicon frame that was 5 mm square. The exposed resist was developed by immersion in a 1 : 3 by volume mixture of methyl isobutyl ketone and isopropyl alcohol and rinsed in pure isopropyl alcohol. A lift-off step was performed by immersing the patterned membrane in pure acetone to remove the resist and extraneous platinum over layer, but retaining the platinum wire pattern. Contacts extended from the membrane to the supporting silicon frame and were wire bonded (TPT HB05) to a custom printed circuit board102,105 connected to a high precision electrometer (Keithley Series 4200). The sensor resistance was around 600 Ω at room temperature and a sample specific temperature calibration, described in a later section, was performed.108 and so the sample particles near the center of the membrane cannot be efficiently cooled by the cell body. Therefore, an additional form of cooling must be applied from a direction out of the plane of the membrane. This is further hindered by the space restriction of the STXM optics; the Fresnel zone plate (X-ray diffraction focusing optic) requires an order selecting aperture (OSA) positioned within a few hundred micrometers of the focus in order to block the unfocused, zero-order light, while allowing the first-order focus to pass through, as illustrated in 2 to the front of the cell, removing heat from the center of the membrane and then being pumped from the experiment chamber by a turbomolecular pump. In some instances, a 40 nm layer of Al was deposited on the reverse side of the sample membrane to marginally increase thermal conductivity across its surface. The total pressure inside the cell was maintained at 150 mbar and the vacuum chamber pressure was ∼10−3 mbar. Finally, the body of the INXCell was also cooled and temperature controlled at about 1–2 K warmer than the OSA. It is this two-step cooling design, i.e. cooling of the INXCell body and further cooling of the sample surface utilizing the OSA, resulted in the sample position having the lowest temperature in the gas flow system and being the only area for ice nucleation to occur.The second important design consideration was to ensure that the sample of particles on the silicon nitride membrane was the coldest surface to prevent icing in undesirable locations of the INXCell construction. The requirement for X-ray transparency limits the thickness of the membranes enclosing the cell and supporting the sample particles to about 0.1 μm. This means that the membrane transports heat easily through the thickness of the membrane, but very poorly across its much larger width,93,98,99 with minor improvements such as thermally insulating the mounting plate attached from below. Dry helium was used as the carrier gas inside the cell at 20 cm3 min−1 . First, all residual water vapor was removed from the He flow using a liquid nitrogen trap. The flow was split and directed to either a humidifier or a bypass. The temperature controlled humidifier was half-filled with ice held at −16 °C and separated from the air above using a layer of Nafion film. The humidified flow was mixed with the dry bypass flow to achieve a controlled frost point temperature down to about 215 K. Pressure was measured just before the inlet gas flow line of the INXCell and controlled at 150 mbar using a vacuum pump and a proportional-integral-derivative (PID) controlled solenoid valve. Outside of the INXCell in the STXM/NEXAFS vacuum chamber, a 100 cm3 min−1 flow of N2 was directed through the cooled OSA onto the reverse side of the sample substrate. This resulted in a vacuum chamber pressure of ∼10−3 mbar, sufficiently low to minimize the X-ray absorption outside of the gas cell. The N2 flow was pre-dried in the same water vapor trap as for the He flow with an additional pressure regulator.The INXCell chamber dimensions and airflow are identical to previous designs,2.2Tcell, and of the OSA supporting arm, TOSA, was measured with calibrated Pt-100 sensors and controlled using PID modules. Prior to starting any ice nucleation experiments, a temperature calibration of each lithographically patterned Pt sample sensor was performed. To do this, the sample resistance was measured while dry gas was flowing through the INXCell and OSA. The position of the INXCell and OSA was the same as when obtaining a focused STXM image. Then, the OSA and INXCell were cooled to the same T, i.e. TOSA = Tcell and the resistance of the Pt sample sensor, Rp, was measured. Therefore, the temperature of the particles on the membrane, Tp, was equal to TOSA and Tcell, and corresponded to Rp. This was repeated every 10 K over the range 210–290 K to derive a temperature-resistance calibration curve.The temperature of the INXCell, TOSA and Tcell were initially set to the same temperature and RHi was set to just under <90%. When a cooling ramp began, Tcell was fixed and TOSA was cooled at a rate of 0.3 K min−1, which led to a change in RHi of about 2.8% min−1 and similar to other methods.92,110 During cooling, we continuously imaged the sample area at a single X-ray energy until ice formed. Ice crystals grew to hundreds of micrometers in size over a few minutes and were clearly identifiable in the STXM images as areas of dark contrast. As soon as ice was detected, TOSA was immediately increased to ∼ 1–3 K warmer than the temperature at which ice nucleated in attempt to stop crystal growth. Water uptake was identified when imaged particles instantaneously and clearly increased in size and X-ray absorption while being cooled. It is notable that these effects were much less pronounced than ice crystal appearance and growth92,110  or subsaturated (<100%), respectively. We determined Tp at which an ice crystal did not change size over multiple images, i.e. for a time of ∼10 min. The measured temperature at which ice crystals did not change size is the so-called frost point temperature, Tfst. Then, we calculated  where  is the saturation vapor pressure over ice as a function of temperature. In some instances, ice nucleation did not occur and instead, only water droplets formed. Then,  where Tdew, is the so-called dew point temperature. The same calibration procedure was implemented except determining the temperature at which water droplets did not grow or shrink, equivalent to Tdew. After calibration, ice crystals were sublimated while continuously being imaged. The residual particles remaining beneath the sublimated crystals were then investigated using the highest spatial and X-ray energy resolution possible. The error on resistance was <±0.3 Ω, which translated to <±0.2 K in T. The uncertainty in both Tdew and Tfst was then chosen as ±0.2 K. This was propagated to  or  and then to the error in RHi found in RHi of about 2%, which is far less than the error given in N times given in 92,111Calibration of 2.3112 where 100 ml of Fe(NO3)3 at 0.1 M was prepared in a beaker and titrated by 0.1 M of NaOH under strong stirring until the pH of the solution reached 7.5–8. The product in the form of precipitate was then washed six times with twice its volume of distilled water through a centrifugation procedure. Finally, the product was dried in an oven for 48 h at 40 °C.112 When desired, aqueous citric acid droplets were deposited on top first by nebulizing a 3 weight percent solution, and impacting them on top of the ferrihydrite particles. The solution droplets were generated using an ultrasonic nebulizer.98,99 K-rich feldspar particles mixed with xanthan gum were also dry deposited from a powder. Feldspar was purchased from the Bureau of Analysed Sample Ltd (BCS-CRM no. 376/1 SGT FELDSPAR 1) and sieved through a 250 μm mesh. This was milled again with a ball-milling machine for 5 min and sieved through a 64 μm mesh. A bulk sample was prepared by mixing a small volume of xanthan gum, feldspar and water in equal weight ratio. After drying for about two weeks, the mixture turned to a glass state, which was then crushed into a powder using a mortar and pestle. The nebulizer was cleaned before each use, first by scrubbing using laboratory detergent, then rinsing in distilled and deionized water (resistivity of 18.2 MΩ) and third, soaking in 10% weight percent HCl solution for one hour followed by another rinse prior to use. Tubing and pumps that supplied liquid to the nebulizer were cleaned by circulating the HCl solution for one hour, followed by a rinsing with distilled and deionized water. The mortar and pestle were cleaned following the same procedure. This ensured that undesired compounds were below detection, which was confirmed by generating sodium chloride particles after a cleaning procedure without detecting iron or carbon using STXM/NEXAFS. Confirmation of coatings on mineral particles was performed using STXM/NEXAFS where particles were imaged at the carbon pre-edge at 280.0 eV and either the peak absorption for citric acid at 288.5 eV or xanthan gum at 289.4 eV. X-ray absorption increased uniformly over the particles and appeared slightly enlarged indicating a complete coating. However, STXM images are 2-D and thus we cannot exclude the possibility that some bare feldspar was exposed at the top of the particles. However, we find this unlikely and maintain the presumption that feldspar was completely coated. Future studies should take care to quantify coating amount and heterogeneity. Multiple images of particles were used to derive 2-D projected surface area, which were then translated to 3-D particle surface area estimates assuming spherical geometry. The particle surface area per field of view from multiple images was scaled up to the total sample area estimated to be about 0.16 mm2. Due to time constraints using the synchrotron X-ray beamline, our experiments were limited to the investigation of ferrihydrite particles, ferrihydrite particles coated with citric acid and feldspar coated with xanthan gum. We suggest further experiments on ferrihydrite particles coated with xanthan gum and feldspar particles without any coatings as an addition to the current dataset.Ferrihydrite particles were dry deposited on the silicon nitride membrane. Briefly, ferrihydrite synthesis followed an adapted version of Schwertmann's method,2.479 X-rays were first focused using a Fresnel zone plate to a minimum pixel size of either 50 or 35 nm at the position of the sample, depending on the specific zone plate used. The custom designed OSA was positioned between the zone plate and sample to block unfocused and unwanted light. The transmission of X-ray photons through the particle was measured and converted to optical density, OD = − ln(I/I0), where I0 and I are the incident and transmitted photon count, respectively, and with an uncertainty of  and  The sample was scanned over a desired field of view (FOV), while the focused X-rays remained at a fixed position. An image was acquired from hundreds to thousands of individual pixels, where OD was calculated for each pixel. Multiple OD images over the same FOV taken over a range of X-ray energies, referred to here as a “stack”, were aligned and processed using publicly available software.113 We primarily investigated the X-ray energy ranges between 278–320 eV, 525–550 eV and 700–735 eV, which correspond to the carbon K-edge, oxygen K-edge and iron L2,3-edges, respectively.The INXCell was developed and operated at the PolLux beamline of the Swiss Light Source using STXM/NEXAFS.33.1T and RHi and shown in RHi = 132% seen as the filled blue diamond, which is the average of multiple freezing cycles. Individual RHi and T values for each cycle are shown as small blue open diamonds. Water uptake (open circles) for ferrihydrite particles was observed for only one cycle at T = 231 K, while it was always observed when T = 236 K. Note that water condensation was observed at subsaturated conditions for these temperatures, i.e. RHw < 100%. After water uptake, ice nucleation occurred (filled circles) at or slightly below conditions expected for homogeneous ice nucleation. This result implies that there was a competition between deposition ice nucleation and water uptake. At T = 240 K, ice nucleation was not observed, and water uptake occurred close to RHw = 100%. When ferrihydrite was present with citric acid, ice nucleation was observed at or slightly below conditions for homogeneous freezing when T = 230 K. Water uptake due to citric acid already occurred when RHi < 90% and was not quantified in this study. Additional information about freezing results and particle sample properties are found in 64 having a similar sample surface area shown as small filled diamonds. Note, that these data points are onset conditions, i.e. the RHi and T at which ice and water uptake was first observed. Ice nucleation on hematite occurred at considerably less RHi with roughly the same scatter in the data. Clearly, ice nucleation for ferrihydrite is not so efficient compared to hematite, although its importance to atmospheric ice formation will be evaluated in a later section. Highly concentrated aqueous solutions of citric acid can be highly viscous and kinetically limit water uptake.83,114 The glass transition temperature at RHi = 100% has previously been estimated at 211.8 K . Ice formation did not occur at this low temperature while the liquid-vapor equilibrium was maintained for hours, demonstrating our ability to well-control thermodynamic conditions in the INXCell. If ice formed heterogeneously elsewhere on the sample, e.g. on the silicon nitride substrate or the Pt temperature sensor, the vapor pressure decrease would result in rapid evaporation of the droplet. This did not occur and implies that the substrate and Pt wire did not nucleate ice heterogeneously. In the presence of citric acid, droplets always formed followed by ice nucleation.2,3-edges shown in Our ability to detect droplet formation, ice nucleation and identify residual particles from sublimating ice crystals is shown in 3.3116 The homogeneous ice nucleation rate coefficient, Jhom, with units of cm−3 s−1 predicts water and aqueous solution droplet freezing in the atmosphere.22,24,116,117 The homogeneous ice nucleation rate is ωhom = JhomVd, where Vd is the volume of a droplet. A heterogeneous ice nucleating substrate reduces the energy barrier for ice nucleation, and the heterogeneous ice nucleation rate coefficient is Jhet, with units of cm−2 s−1. From this, the heterogeneous ice nucleation rate, ωhet, scales with the particle surface area, Ap, where ωhet = JhetAp. Immersion freezing from laboratory generated particles, from ambient particles and in atmospheric cloud models has been successfully described using the water activity, aw, of the bulk solution around the immersed particle,39–41,118,119 being either pure water or aqueous solution. The aw-based immersion freezing model (ABIFM) quantifies Jhet over a range of atmospherically relevant T and RHw using aw and shown to be valid thus far for every investigated organic, biogenic and mineral type of particles and for every organic or inorganic solute.39,40,119 Due to its vast applicability for heterogeneous freezing both at and below water saturation, we use the ABIFM to derive Jhet for immersion freezing due to ferrihydrite with and without coatings of citric acid, as well as immersion freezing due to feldspar coated with xanthan gum. Jhet for immersion freezing of feldspar in aqueous solution was previously derived,40 although this was not tested with feldspar in xanthan gum solutions. A aw-based deposition ice nucleation model (ABDINM) was not previously considered, because aqueous solution is thought not to be involved in deposition ice nucleation. Despite this, we use the ABIFM and ABDINM as convenient and reliable functional forms of Jhet for model implementation described below.Ice formation is well-established in nucleation theory, which is used to derive a freezing rate that is crucial to the nucleation process. Water clusters form with increasing numbers of molecules coinciding with increasing free energy until a potential energy barrier is overcome triggering energy release and thus nucleation followed by bulk crystallization.Jhet,im, as a function of the water activity criterion, Δaw,39,40 is parameterized asmim and cim are parameters specific to either ferrihydrite or feldspar. The term, Δaw, is the difference between solution water activity and the water activity along the ice melting point line at constant T, oraw is assumed equal to RHw the particle is exposed to in the INXCell, and aiw(T) is the ice–liquid equilibrium curve.22,120 As previously mentioned, a major advantage of aw defines a unique point in the T versus aw phase diagram where Jhet,im is constant and independent of any solute. 39,40For immersion freezing, mdep and cdep are parameters different from those for immersion freezing. We have expanded the stochastic freezing model (SFM) presented in Alpert and Knopf40 to predict the freezing probability, Pfrz, of a particle due to immersion freezing, deposition ice nucleation and homogeneous freezing asJhom is taken from literature22,120 and used in t, is included in i.e. T(t), of the sample material. The updated SFM presented here simulates freezing by single particles,40 each having a unique Ap. The uncertainty in quantifying Ap was previously shown to significantly impact freezing results.40 Therefore, we derived Ap and the distribution of particles sizes and total surface area on a sample from measurements of particle sizes on the sample estimated from STXM images. These were used to produce a surface area probability distribution from which the SFM randomly sampled Ap for one simulation. The temperature in the SFM decreases continuously and after a small temperature step, ΔT = 0.2 K, freezing of a particle is sampled from a binomial distribution as a Bernoulli trial using a probability parameter from 5 times to determine the average conditions of T, RHw and RHi at which ice occurs and their uncertainty, which is derived from the multiple simulations and from Poisson statistics at 99.9% confidence following Alpert and Knopf.40 It is important to note that each simulation run will sample a new particle population from the measured size distribution to account for the uncertainty in surface area. Finally, parameters mim, mdep, cim and cdep were varied to best fit the average thermodynamic conditions at which ice was observed to form, while also matching the standard deviation from multiple cooling cycles.To represent deposition ice nucleation, we have used the same functional form as RHi and T at which freezing was observed. In addition, we evaluated the variability in RHi and T that could be attributed to random freezing. As previously stated, only the first ice nucleation event on a sample with many other particles was observed and due to time constraints, we were limited on the number of repeated cooling cycles  reach 10−2 L−1 of air before immersion or homogeneous freezing occurred at a rate, Pi < 10−2 min−1 L−1 near RHi = 100%. These initial freezing events are due to the largest coarse mode particles that have more surface area and thus a higher probability to freeze. At a maximum, Nnuc = 21 L−1 and Pi = 8 min−1 L−1 from deposition ice nucleation alone. Water uptake was modelled at RHw = 94.5%, which was similar to observations for ferrihydrite particles. After water uptake, deposition ice nucleation was not possible and immersion and homogeneous freezing was allowed. Homogeneous freezing contributed far more to the total Nnuc toward the end of the model run than immersion freezing shown in −1 of air with a 10 s resident time and using a 10 min averaging interval. These CFDC instruments are routinely deployed in aircraft studies for measuring INP concentrations.42,123,124 Again, using Poisson statistics at 99.9% confidence, we derive upper and lower fiducial limits125 of Nnuc and Pi seen as the shaded areas. Ambient INPs and ice crystal concentrations are observed from 10−1 to 103 L−1 in atmospheric clouds, however these numbers are highly uncertain in general and not well-known for cirrus clouds.28 We find that low numbers of ice crystals in clouds could be due to deposition ice nucleation from ferrihydrite in dust, but also we claim that these low numbers, and thus low sampling statistics, are the leading cause of the great uncertainty associated with predictions. Reducing the uncertainties would require high volume sampling and long particle residence times in ice nucleation instrumentation to detect high numbers of INPs, which poses a significant experimental challenge. However, despite low numbers of ice nucleation events, careful evaluation of uncertainties, as done here, can still yield increased understanding of ice nucleating particle chemistry and physics. We have demonstrated that ferrihydrite particles have the ability to nucleate ice and should be considered in future deposition ice nucleation studies and derived quantitative values of Jhet and corresponding uncertainties following water activity to predict ice particle production.in situ identification and composition mapping of organic coatings and metal oxidation states of atmospheric INPs will give valuable insights on the nature of ice nucleation. In doing so, future studies will be able to discriminate in situ INP types and their composition with discrimination of immersion freezing and deposition ice nucleation. We recommend further investigation on ambient particles using our new INXCell and newly developed SFM. Furthermore, experiments on complex laboratory generated particles using the INXCell would greatly benefit from spectroscopic and composition mapping data. This could be applied in future ice nucleation studies, e.g. on both fully or partially coated particles.Our INXCell and SFM are also well-suited for measuring and modelling the ice nucleation ability of field collected particles in combination with their chemical morphology. Due to the wide variability of particle types, 4in situ environmental spectroscopy. In this way, the detection of specific chemical components on and inside single particles could be distinguished such as inorganic material, organic material, iron, and corresponding carbon bonding functionalities and oxidation state. In particular, single ice nucleating ferrihydrite particles were identified through X-ray images and NEXAFS spectroscopy. Ferrihydrite nucleated ice at significantly lower values of RHi than expected for homogeneous ice nucleation. This occurred only when water uptake was completely avoided. When water uptake did occur, ice nucleation occurred at conditions close to homogeneous ice nucleation. One surprising finding was that even at the same humidity conditions, repeat cycles were performed that either did and did not result in observed water uptake. Therefore, heterogeneous ice nucleation via deposition ice nucleation from the water vapor phase was in competition with water uptake onto ferrihydrite particles. This was possibly due to the stochastic nature of heterogeneous ice nucleation, i.e. if ice formation did not occur by chance, then water uptake would eliminate any further chance of deposition ice nucleation. We also observed single ferrihydrite particles immersed inside of supercooled water droplets without any indication of ice nucleation and rapid crystallization. Feldspar deposition ice nucleation in the presence of xanthan gum was observed and was in agreement with previous ice nucleation results at water subsaturated conditions for feldspar ice nucleation studies without xanthan gum. Although xanthan gum is hygroscopic, no water uptake was observed on the mixed particles, likely due to the xanthan gum being a glass at the humidity and temperature at which ice nucleation was observed.Ice nucleation due to ferrihydrite particles with and without citric acid, as well as feldspar with xanthan gum, was observed in a new instrument, referred to as the INXCell, that combines X-ray spectro-microscopy, a humidified environmental chamber and a cryogenic cold stage. This work showed that ice nucleation on single particles could be paired with T and RHi at which ice formed using our newly developed model, the SFM, and carefully determined corresponding uncertainties. This was an extension of our previously developed immersion freezing model using a Monte Carlo technique.40 This model uses a water activity based description for quantifying freezing kinetics (Jhet) and thus, independent of any solute type present and applicable for both water saturated and water subsaturated conditions. The SFM could reproduce our observations of ice nucleation due to ferrihydrite and feldspar with and without citric acid or xanthan gum aqueous solution and within our experimental error. The range of RHi at which particle froze predicted by the SFM was almost identical to the scatter in our observations. Since the SFM variability is entirely based on a stochastic freezing process, we found that stochastic freezing was the major source of error for deposition ice nucleation and immersion freezing due to ferrihydrite particles and deposition ice nucleation due to feldspar particles. On the other hand, the variability of RHi predicted by the SFM for immersion freezing caused by feldspar was much smaller and thus, experimental temperature error was likely the major source of variability. The fact that stochastic freezing has the ability to explain our data scatter and error gives evidence that heterogeneous ice nucleation due to ferrihydrite and feldspar is dominated by stochastic freezing and validated the use of a nucleation rate coefficient following nucleation theory.We quantified ice nucleation rate coefficients using observation of 1 L−1 when RHi increased up to 145%. These crystal numbers are typical for cirrus cloud formation, and thus, ferrihydrite could potentially be important for atmospheric ice production if water uptake is avoided. Immersion freezing insignificantly contributed to the total ice particle concentration compared to homogeneous freezing, and therefore, immersion freezing of ferrihydrite is concluded to not be important for atmospheric ice production.We have also investigated the potential importance of ferrihydrite as an atmospheric ice nucleating particle using our SFM, considering the competition between immersion freezing, deposition ice nucleation and homogeneous ice nucleation. Concentrations of ice particles from deposition ice nucleation were predicted on the order of 10i.e. at 100% relative humidity, with and without particle immersed using STXM/NEXAFS has never been done until now. The INXCell is advantageous for future studies of, e.g. phase transitions, chemical reactions, or multiphase chemistry, but potentially in other disciplines such as electrochemistry106 for fuel cell performance,126,127 involving the aqueous phase of liquid water where water saturation is maintained for long time scales.This work could only be possible using our new INXCell, which features a platinum resistive temperature sensor that was lithographically patterned onto the sample surface and a novel cryogenic cooling solution. This ensured a high precision measurement of the sample temperature as low as 230 K, demonstrated in this study. The major challenge to overcome was to ensure that the coldest area of the gas flow path throughout the inside of the entire INXCell construction was located at the X-ray transparent silicon nitride membrane where particles sit, and co-located where the temperature of the particles was measured. This yielded unprecedented accuracy and control of temperature and thus humidity. Inside the cell, this means that maximum relative humidity conditions are central, localized, predictable and uniform across a defined sample area. To our knowledge, measurement and equilibration of pure water droplets, https://doi.org/10.5281/zenodo.6034243. This includes schematic drawings of the order selecting aperture.All data are publicly available online at https://doi.org/10.5281/zenodo.6034243.All codes are publicly available online at P. A. A. wrote the manuscript. P. A. A., B. W., C. P. and M. A. conceptualized and planned the study. P. A. A. planned and conducted STXM/NEXAFS experiments supervised by B. W., C. P. and M. A., A. B., S. Y., H. Y. and K. K. also conducted STXM/NEXAFS experiments. P. A. A. and S. F. designed the INXCell supervised by B. W., S. F. performed lithography. Z. L. performed sputter coating and characterized the temperature sensors. P. A. A. conducted STXM/NEXAFS data analysis and interpretation of results. P. A. A. wrote and developed the SFM. All co-authors discussed the results and commented on the manuscript.We declare no conflict of interest.EA-002-D1EA00077B-s001


SECISBP2) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2, we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNASec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3′ of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2-mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific R543QSecisbp2-mutant brains. Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 ( Several cis- and trans-acting factors are needed to promote the efficient co-translational insertion of selenocysteine (Sec) into selenoproteins directed by UGA/Sec codons . SelenopSec to the ribosome O3) overnight. After rinsing with 1× PBS, cells were lysed in RIPA buffer. Fifty micrograms of protein lysate were electrophoresed in a 12% SDS-polyacrylamide gel. Staining of the gel with Coomassie brilliant blue was carried out to show equal loading. The gel was dried using a gel dryer (Bio-Rad) and exposed for one day to a Phosphorimager screen, which was developed by a BAS-1800 II Phosphoimager (Fujifilm), as described in .,27.SECISSECISBP2 . These snt serum . These o in vivo . In addiSec, several selenoproteins show ribosomal pausing at or upstream of the UGA/Sec codon [Secisbp2 was inactivated [SECISBP2 can delay for a significant time premature termination when bound to a ribosome with a UGA/Sec codon in the A-site. In mouse models of low selenium availability or mutation of tRNAec codon ,28. Pausctivated . This obSec positioned in the A-site. Such RPF are characterized by their length of 28/29 nucleotides (bRPF), and the codon adjacent to the A-site is 15 nucleotides from their 5′ terminus (see Methods). Clearly, selenoprotein transcripts with reduced UGA/Sec read-through are expected to have fewer of such RPFs. Likewise, if recruitment of Sec-tRNASec was impaired, one would expect an increased number of RPFs with UGA/Sec adjacent to an empty A-site. Such RPF have a size of 21/22 nucleotides. We believe this type of RPF is analyzed for the first time in the present study. SELENOH and SELENOO are the transcripts with the most abundant RPF coverage on the UGA/Sec codon in our HAP1 cell model and are both resistant to NMD. Thus, RPF density is not confounded by changing levels of mRNA. In both cases, the increase in sRPF  is clearly seen in SECISBP2-mutant cells. We conclude from this data that such an observation is compatible with a function of SECISBP2 in recruitment of EEFSEC:Sec-tRNASec to the ribosomal A-site—possibly by stabilizing a conformation of the SECIS that can interact with EEFSEC [All the measures to estimate read-through of UGA/Sec in individual transcripts via Ribo-Seq are based on the density of RPF 3′ of the UGA/Sec and, depending on the method, are influenced by RPF density 5′ of the UGA or by mRNA instability. We wanted to harness the full power of Ribo-Seq and analyzed our Ribo-Seq data in this study at codon resolution around the UGA/Sec codon. Ribosomes reading the UGA/Sec codons have the tRNAh EEFSEC .Next, the most direct way to look at Sec incorporation using Ribo-Seq is to assess the density of RPFs with UGA/Sec positioned in the P-site (pUGA). Such RPF directly reflect successful elongation through UGA. This measure can be calculated irrespective of the occupancy of the A-site 3′ from the UGA/Sec. Thus, by pooling sRPF and bRPF, the data base is improved on the level of individual transcripts. pUGA nicely correlates with 3′RPM but can be calculated also for selenoproteins carrying the UGA/Sec codon close to the termination codon e. Hence,RF1-mutant E. coli was skipped in a +3 frameshifting event [SECISBP2D623−630, Δ631N. The mRNA with the biggest effect is SELENOW but also GPX1 and SELENOF showed a reduced fraction of RPF in frame 0 3′ from the UGA/Sec codon. We failed to pinpoint a specific “slippery” sequence in these selenoproteins, but it is obvious that UGA/Sec would qualify as a “hungry codon”, since Sec is by far the rarest proteinogenic amino acid. In the case of the Ty3 retrotransposon in yeast, +1 frameshifting in the P-site has been shown at a GCG/Ala codon followed by the “hungry” AGU/Ser codon without the need of a “slippery site” [SELENOW, the P-site codon GCU also encodes Ala. It is interesting that even in the presence of functional SECISBP2, there seems to be increased +1 frameshifting in SELENOW. We found the same increased frameshifting 3′ from the UGA/Sec in Selenow and Selenof in mice expressing the SECISBP2R543Q mutant [Translational frameshifting has, to our knowledge, not been explored in the context of canonical selenoproteins. There is, however, a known instant where a synthetic UAG/Sec codon in GPX4 recombinantly expressed in an ng event . This mang event . Often, ng event ,57,58. IQ mutant . The seqConsidering that our present experiment assigns more than 85% of all RPF to reading frame 0 in the control HAP1 cells, we consider this Ribo-Seq experiment to be of high quality. The RPF assigned to other reading frames are the result of incomplete nuclease digestion at the 5′ end of the RPF and merely reflects a technical limitation. If a clear increase in RPF outside frame 0 is observed in only specific selenoproteins and only 3′ of the UGA/Sec, we take this as evidence for frameshifting. Clearly, future studies should investigate in detail the codon context and conditions in favor of frameshifting.When we consider pUGA, we assume that the tRNA occupying the P-site is Sec. However, our Ribo-Seq experiment cannot inform us about the identity of this tRNA. In addition to elongation with Sec, there is the possibility of misreading with a near-cognate tRNA. Mis-incorporation of Trp, Arg, and Cys at UGA/Sec have been demonstrated upon aminoglycoside treatment . Thus, t