Protein ubiquitination regulates almost every process in eukaryotic cells. The study of the many enzymes involved in the ubiquitination system and the development of ubiquitination-associated therapeutics are important areas of current research. Synthetic tools such as ubiquitin-based chemical probes have been making an increasing contribution to deciphering various biochemical components involved in ubiquitin conjugation, recruitment, signaling, and deconjugation. In the present minireview, we summarize the progress of ubiquitin-based chemical probes with an emphasis on their various structures and chemical synthesis. We discuss the utility of the ubiquitin-based chemical probes for discovering and profiling ubiquitin-dependent signaling systems, as well as the monitoring and visualization of ubiquitin-related enzymatic machinery. We also show how the probes can serve to elucidate the molecular mechanism of recognition and catalysis. Collectively, the development and application of ubiquitin-based chemical probes emphasizes the importance and utility of chemical protein synthesis in modern chemical biology. This article reviews the design, synthesis, and application of different classes of Ub-based chemical probes. Ubiquitination, the post-translational attachment of a 76-residue protein named ubiquitin (Ub), is orchestrated by the actions of four enzyme classes.1–3 The Ub activating enzyme E1 catalyses the formation of an E1-Ub thioester at the expense of ATP  modifications such as Nedd8, SUMO, and ISG15 have also been identified.8–10 Defects in components of the Ub/UbL processes influence disease pathogenesis, especially cancer and neurodegeneration. A more detailed understanding of Ub/UbL modifications in cellular processes such as DNA repair and immune response remains to be acquired 14 developed for capturing DUBs can react with the active Cys through 1,2-addition. Both of these reactions are reversible, the resulting complex usually being unstable under strongly reducing conditions. To solve this problem, the more powerful probe Ub-Prg  was developed.20,21 Ub-Prg can react with the active Cys of DUBs through 1,2-addition to produce a stable vinyl thioether product.The first probes  family DUBs.22Ub-based chemical probes that react with the active Cys through nucleophilic substitution include Ub-Cl, Ub-Br2, Ub-Br3, and Ub-AOMK.2.2The recruitment and binding of different Ubs by the Ub interactors is usually non-covalent in nature. The development of Ub-based chemical probes to capture or enrich Ub interactors can inform the study of these recognition events.2.2.133–40 Ub units can be either natural or mimetics that do not impact the interaction with linkage-specific interactors (Ub interactors containing one or more Ub binding domains (UBD) can bind to Ubs through non-covalent interactions. This is the basis of a simple but effective strategy, wherein Ub pull-down probes are used to capture or enrich Ub interactors from the cell lysates. Such probes usually contain one or more Ub units and an enrichment/purification tag.eractors .2.2.241–43 Common photoactive groups include aryl azide, diaziridine, and benzophenone. The aryl azide group can generate a nitrene intermediate, though may be reduced under the physiological conditions, resulting in low crosslinking efficiency.44 In contrast, diaziridine can generate a more active carbene intermediate able to insert into various C–H bonds with high labelling efficacy. Finally, the wavelength to activate benzophenone is relatively long (350–365 nm), which is less damaging to protein44 (For the Ub interactors that are difficult to capture using pull-down probes (because of weak interactions), a photocrosslinking group can be installed on the Ub chain . UV lighrotein44 .3.Synthesis of the Ub-based chemical probes usually involves two steps: first, synthesis of Ub conjugate module; second, incorporation of the active group. Ub conjugates are usually difficult to obtain through direct recombinant expression, and therefore need to be synthesized through chemical means.3.145 -thioester and Ub(A46C-G76) with a masked thioester. Native chemical ligation (NCL) between the two segments led to the formation of full-length Ub. After the conversion of Cys46 to Ala through desulfurization and activation of the masked thioester, the target Ub-thioester was successfully obtained. In addition, a Ub-hydrazide can be synthesized in a similar two-segment strategy, and used as a thioester equivalent.51–61Fmoc solid-phase peptide synthesis (SPPS) can be used to synthesize peptide thioesters, but this method is usually limited to peptides of up to 50 amino acids46–50 . Since U62 both of which disrupt the aggregation of the nascent peptide on the resin surface. Using this strategy, Ovaa et al. successfully synthesized Ub thioesters and even SUMO2 (92 amino acids).63Another approach for the synthesis of long peptides on the solid support is to incorporate “aggregation breakers” such as pseudoproline and dimethoxybenzyl dipeptides,3.1.2Several methods have been developed to introduce the “Cys-like” auxiliary group at or close to a specific Lys residue in the substrate protein .3.1.2.164 The auxiliary reacts with the Ub-thioester through NCL, and then the auxiliary can be selectively removed via photolysis to generate the desired ubiquitinated substrate. Two alternative auxiliaries are 1--2-mercaptoethyl, which can be removed by trifluoroacetic acid after ligation;63,65 and a 2-aminooxy-ethanethiol auxiliary, which exhibits faster ligation.66A photo-cleavable thiol-containing glycyl auxiliary can be introduced to the Lys of the substrate protein through SPPS.3.1.2.267,68 Both δ- and γ-mercaptolysine can be ligated with a Ub-thioester, leading to the formation of an isopeptide bond after desulfurization. Compared to the glycyl-auxiliary strategy, the mercaptolysine uses a primary amino group to participate in the ligation and has a faster ligation speed.Brik and Liu groups designed δ- and γ-mercaptolysine and incorporated them into substrate proteins.3.1.2.3This strategy employs the 76-residue isoUb unit, which is made from two adjacent Ub segments, each of which contains an N-terminal Cys and a C-terminal hydrazide to facilitate ligation. Multiple isoUb units can be assembled through sequential hydrazide-based NCL. Unlike native Ub, the isoUb unit does not aggregate, and can therefore be readily synthesized. Furthermore, the ligation of isoUb takes place at Cys with less hydrolysis by-products. Using the isoUb strategy, a K11/K48-branched hexa-Ub (456 amino acids) was synthesized.3.270–74The total chemical synthesis may present technical challenges to some biochemistry-oriented laboratories. Protein semi-synthesis methods may be a useful alternate.3.2.1N-to-S acyl transfer to produce a protein thioester with the expulsion of the intein75,76 .Ub thioesters can be produced through intein splicing, wherein an expressed protein undergoes an intramolecular ein75,76 . To obtaet al. discovered that peptidyl C-terminal Cys can undergo N-to-S acyl transfer to generate a transient thioester.77 After adding hydrazine to Ub(1-76C), N-to-S acyl transfer generates Ub(1-75)-hydrazide with a yield of ca. 50 mg L−1 of LB medium. Second, Liu et al. discovered that the Ub hydrolase YUH1 can hydrolyse Ub analogues to form thioester intermediates.78 By appending Asp to the Ub C-terminal and treating Ub(1-77D) with YUH1 and hydrazine, Ub(1-76)-hydrazide was obtained in a high yield (ca. 30–40 mg L−1 of LB medium). Third, the transesterification of E1 and Ub to form an active E1-Ub thioester can be intercepted by the addition of thiol, leading to the formation of Ub (1-76)-thioester in good yields .36,79,80 Finally, Liu et al. found that a small molecule cyanylating reagent (2-nitro-5-thiocyanatobenzoic acid) can modify a recombinant protein at its C-terminus via nucleophilic acyl substitution, generating a protein hydrazide if the nucleophile is hydrazine.81Other methods can produce recombinant Ub-hydrazide . First, Macmillan 3.2.2E. coli expression . After Boc deprotection, Ub-thioester was reacted with the substrate protein forming an isopeptide bond through Ag-catalysed thioester-amine condensation. Finally, the removal of Alloc led to the formation of the target Ub conjugate.An unnatural amino acid strategy has also been used to introduce a side-chain Boc-protected Lys into the substrate protein.3.370–73,84,85  was added to Ub-SH generating a high-energy disulfide. This intermediate was then reacted with the substrate protein bearing a Lys-to-Cys mutation at the desired position, generating a Ub-substrate disulfide adduct.3.3.288,89 The advantage of the strategy is that the protein precursors are easy to express, and the isopeptide mimetics are stable in reducing conditions. Nonetheless, the crosslinking process may produce two self-crosslinking by-products. Matthew et al. proposed the use of 1,3-dibromoacetone in a stepwise crosslinking, which generates a single crosslinked product with a high yield.90The Lys (to be ubiquitinated) on the substrate protein is mutated to Cys, and the Gly76 of the donor Ub is mutated to Cys. When 1,3-dichloroacetone is added, the substrate forms a crosslinked adduct with Ub.3.3.338,39,91 Introduction of the azide into the Ub C-terminal was accomplished through unnatural amino acid insertion, and then the alkyne was introduced into the substrate Lys through bio-orthogonal reactions. The Ub and substrate were ligated in the form of triazole via CuAAC reaction. The reaction has good substrate adaptability, and the triazole isopeptide bond mimetic is resistant to DUB hydrolysis.Cu-catalysed azide–alkyne cycloaddition (CuAAC) reactions can be used to construct isopeptide bond mimics.3.3.436,78 Ligation of the substrate with the Ub-thioester using NCL followed by auxiliary removal led to the formation of the isopeptide bond mimetics. Two different bifunctional handles were developed: one that results in mimetics bearing only one atom different from the native isopeptide bonds, which may provide ideal tools for biochemical and structural studies;78 and the other producing an N-ethyl isopeptide bond that is stable to DUB and therefore, useful for screening Ub chain binding proteins from cell lysates.36To obtain a Ub conjugate incorporating a closer mimic of the native isopeptide bond, a bifunctional handle was appended onto a substrate protein through aminoethylation reaction with Cys.3.3.5S-Ub intermediate. Cys is used as the lysine surrogate providing linkage specificity. Free-radical thiol-ene polymerization will lead to the formation of non-native isopeptide bonds between Ubs.92 Thiol-ene coupling strategy can be applied to the synthesis of site-specific isopeptide, Ub oligomers, and also branched Ub trimers.93Thiol-ene coupling can be used for nonenzymatic synthesis of Ub chains. This strategy requires the use of a Ub variant bearing a C-terminal allyl amine appendage, which can act as E2-3.3.6et al. developed a coupling strategy based on maleimide. First, Ub(1-75)-NHNH2 is converted to Ub(1-75)-maleimide with the addition of NaNO2 and N-(aminoethyl) maleimide. Then, the olefin group on the maleimide can then be linked to another protein.94Brik 3.3.7et al. also reported a strategy based on a set of well-defined conjugates bearing an oxime bond. Ub-methyl 3-mercaptopropionate thioester is incubated with 1,2-bisaminoxy ethane and the Ub C-terminus is modified with an oxyimino group. Through isopeptide ligation via an oxime bond, polyUb chains can be synthesized.95Brik 3.4Ub-based chemical probes incorporate two types of active groups: one to capture the active Cys residue, and the other to capture protein-interacting interface amino acids.3.4.1Three strategies have been used to incorporate the chemical groups to capture the Cys residue .3.4.1.1et al. used the protein semi-synthesis method to prepare an HA-labelled Ub-thioester and then introduced the active group to the C-terminus of Ub through direct aminolysis.22 This approach was used to make a series of monoUb active probes including Ub-Michael acceptor and Ub-halides. Zhuang et al. attached a bifunctional linker containing a masked Michael acceptor at the C-terminus of the remote Ub thioester.96 The ketal group in the linker was then deprotected to generate an α-bromo ketone. After a thiol substitution reaction was carried out with the α-bromo ketone intermediate, a diUb probe was obtained.Kessler 3.4.1.2et al. mutated the remote Ub's Gly76 to Cys and loaded it onto the Lys side chain amino group of the receptor Ub through SPPS.24 After ligation with the remote Ub thioester through NCL, 2,5-dibromohexanediamide was used to convert the Cys76 residue to Dha. In this manner, a diUb probe was obtained. Ovaa et al. designed an NCL ligation handle, which allows the ligation of Ub onto a protein. This building block was loaded on the proximal Ub and then reacted with Ub(1-75)-thioester to construct via NCL. Through in situ selective thiol elimination, a diUb probe was constructed.Brik 3.4.2Three methods have been used to introduce the photocrosslinking groups into the Ub conjugates .3.4.2.1et al. introduced photoleucine containing a photocrosslinking group into the sequence of Ub through SPPS, and then enzymatically assembled it into a Ub chain to generate a photocrosslinking polyUb probe.97Ovaa 3.4.2.2et al. used an evolved engineered pair of tRNA and aminoacyl-tRNA synthetase to embed p-benzoyl-l-phenylalanine (Bpa) into the sequence of Ub.41 This Ub mutant was covalently put onto the active Cys residue-to-Lys mutated E2 to construct an E2-Ub photocrosslinking probe.Virdee 3.4.3.3et al. mutated Ub's Ile44 to Cys and obtained a diUb conjugate through hydrazide based NCL.98 The two Cys residues of the synthetic conjugate were then modified with photocrosslinking groups (diazirine or aryl azide) through selective substitution reaction, leading to the generation of a diUb photocrosslinking probe.Tian 4.4.14.1.199 In 1997, Cohen et al. captured a 37 KD subunit with previously unknown DUB activity from proteasome 19S regulatory complex using an I125-isotope labelling Ub-CN probe.99 Subsequently, it was identified as UBCH37.100,101As early as 1987, Ubal was shown to be an effective probe for capturing DUBs.et al. developed Ub-VS that can form irreversible adducts.15 This probe reacted efficiently with UCH-L3, and also showed high activity with an array of Ub carboxyl terminal hydrolases such as Ubp1, Ubp2, Ubp6, Ubp15, and Yuh1. Using this probe, a unique DUB, i.e. USP14, was identified in the mammalian 26S proteasome. In 2002, more monoUb probes were developed, including HA-Ub-Cl, HA-Ub-Br2, HA-Ub-Br3, HA-Ub-VS, HA-Ub-VME, HA-Ub-VSPh, and HA-Ub-VCN.22 These probes exhibited distinct DUB profiling, enabling the identification of DUBs by affinity-tag based mass proteomics (AP-MS). A total of 23 active DUBs were identified in EL4 cells. HSPC263, a protein labelled by HA-Ub-Br2, had no sequence homology with known DUB and was determined to belong to a new OUT family of DUB.The covalent product of Ub-CN and DUB was not compatible with the reducing conditions of SDS/PAGE. In 2001, Ploegh et al. developed a ‘reactive-site-centric chemoproteomics’ method for the detection of probe-labelled residues by enhanced monoUb-probes. A C-terminal alkyne was added and attached with a cleavable biotin-azide tag via click reaction.102 Using the new probe, a previously unannotated DUB, ZUFSP with high Lys63-linked specificity was identified.103To overcome false positives caused by non-covalent interactions and non-selective capturing, Wertz 104 For instance, HA-Ub-VS was used to study the function of USP7 during lipogenesis. Transcriptional coregulator Tip60, whose expression is regulated by polyubiquitination on multiple sites, plays a key role in adipocyte differentiation. Combining activity monitoring using Ub-VS and further in vitro and in vivo experiments, it was found that early adipogenesis is regulated through USP7-mediated de-ubiquitination of the Tip60.105Finally, mono-Ub probes can be used for the identification of DUBs in defined cellular compartments.4.1.2106–108 Additionally, fluorescently labelled mono-Ub probes for visualizing activity of Ub-related enzymatic can be used to validate drug targets and design novel inhibitors. Ub aminomethyl coumarin (Ub-Amc) was the first fluorescent probe to report DUB activity.109 Ub-Rho110, bearing a substituted fluorescent molecule rhodamine-110, was also developed for high-throughput screening.110Mono-Ub probes can be used for screening of small-molecule inhibitors of DUB. For example, the inhibition of USP7 enables activation of the tumour suppressor p53 – a typical ‘undruggable’ target in various cancer cells. In the effort to find and optimize potent, specific inhibitors of USP7, several probes such as Ub-VS, Ub-Br2, and Ub-PA were used to measure their potency by the activity competitive assay.4.1.3et al. solved the crystal structure of UCH-L3 and Ub-VME complex (1.45 Å) and found that a loop region (residue 147–167) of complexed UCHL3 exhibited a significant conformational rearrangement.17 Comparison with UCHL3 apo state showed that the loop is invisible and may traverse the active site cleft; but once bound it changed to a well-ordered structure, exposing the interior active site. This has been proposed as the key to Ub binding in the whole family members of UCH.17 Further studies showed that changes of the length and flexibility of the active-site crossover loop also contribute to the substrate specificity of UCH family.111In 2005, Gaudet 18 However, the consequences of binding are different: RNP13 exerts activation activity, whereas INO80 inhibits activity. To explain regulatory mechanisms, Ub-Prg was used to capture the catalytic conformation by which RPN13DEUBAD activates UCH-L5; and the catalytic conformation that truncated INO80DEUBAD lacks to inhibit UCH-L5. On the basis of multiple intermediate structures, it was found that RPN13DEUBAD activates UCH-L5 by positioning its domains while INO80DEUBAD inhibits UCH-L5 by blocking Ub binding.18Another study elucidated the extent to which activation and inhibition of UCH-L5 was tuned by its two adaptor RPN13 and INO80. Both adaptors bind to UCH-L5 as demonstrated by the crystal structures.31 including Ub-AMSN probe to mimic the adenylate intermediate, and Ub-AVSN to mimic the tetrahedral intermediate. These two structures have shown significant conformational changes, from an open conformation before release of pyrophosphate to a closed conformation required for thioester bond formation.19 Similar probes have also been applied to capture Sumo E1's active intermediates and study structural mechanism.19Finally, monoUb probes can also be employed to study the mechanism for activation and thioester bond formation in E1s. Two mono-Ub probes were used to capture E1 intermediates,4.1.4et al. introduced a vinyl sulfone moiety to the C-terminus of Nedd8, ISG15, and SUMO-1, and proved that these UbL-VS probes enable the exploration of the Ubl enzymatic system in complex cellular environments16 , 5 OTUs, and 1 Machado–Josephin domain (MJD). MS-based quantitation showed that most USPs have little selectivity towards diUbs while OTU family DUBs exhibited highly chain selectivity, e.g. OTU7B shows a strong preference for K11 diUb probes and OTUB1 favoured K48 diUb.The earliest attempt at profiling DUB's Ub linkage selectivity was carried out by Franke -diUbs33 . Pull-doet al. used K11 and K48 diUb probes to label Cezanne  and found that Cezanne can be exclusively labelled by K11 diUb active probes.62 Brik et al. found that IsoT, USP2, OTUB1, and OTUB2 hydrolyse K48 diUb based on dehyrdoalanine (Dha) probe.24To circumvent the steric hindrance of the diUb probes, other diUb probes incorporating a Michael acceptor that more closely resemble native diUbs were developed. For example, Ovaa et al. reported a photocrosslinking K27 diUb probe bearing an aryl azide group,115 which probe captured K27-selective DUBs from cell lysates (OTUD2 and USP13). A similar photocrosslinking K29-diUb probe also captured K29 specific DUBs (ZRANB1 and OTUD2).Nonetheless, activity-based diUb probes cannot readily capture K27-active DUBs due to the steric hindrance of the K27 isopeptide bond. Li 4.2.2et al. used Lys11 diUb probes to illustrate a non-canonical kcat-driven catalytic cycle of Cezanne. A K11 diUb probe was used to react with Cezanne to form a Cezanne-K11 diUb complex for crystallization. By comparing the structures of Cezanne alone (2.2 Å) and in complex with K11 diUb probe (2.8 Å),34 as well as the results of dynamic H/D exchange mass spectrometry experiments, a three-step hydrolytic cycle was postulated accounting for Cezanne's Lys11 specificity. First, the priming of distal Ub at the S1 site of Cezanne opens the autoinhibition of apo-state by releasing Cys-loop. Next, a transient conformation is remodelled to form S1′ site in situ only if Lys11-linked diUb is bound, relying on the key interaction Lys33  and Glu157 (in Cezanne). Finally, upon hydrolysis, the S1′ site is destroyed the proximal Ub expelled, leading to a rearrangement back to the apo-Cezanne.Komander via at least two binding sites of S2–S1, rather than S1–S1′.116 To elucidate the structural basis of this selectivity, a distal diUbK48 probe was developed to crosslink SARS PLpro, and the covalent complex crystalized and solved at 2.85 Å.117,118 It was found that at least three Ub-binding sites cooperated to cleave K48 Ub. The S1 and S2 sites of PLpro remodelled K48 diUb to an extended conformation, resulting in K48 specificity.Severe acute respiratory syndrome coronavirus papain-like protease (SARS PLpro) is a DUB which recognizes K48 polyUb chains 35 For example, USP9X, a DUB regulating multiple important cellular processes including apoptosis and stem cell self-renewal, displays multiple Ub-binding sites and selectivity for K11, K48, and K63 Ubs. In order to investigate its cleavage mode, Zhuang et al. developed K11, K48, and K63 tri-Ub probes, in which the active group is located at the isopeptide bond between the distal and middle Ubs. Experiments with these probes revealed that USP9X cleaved the K11, K48, and K63 Ubs in different modes .Finally, a triUb probe with well-defined cross-linking site can be used to investigate whether a DUB performs endo- and exo-Ub chain cleavage.4.2.3et al. to identify Ub linkage interactors from cell lysates.37 The triazole bond is resistant to endogenous DUBs. Eight different diUb probes were examined to obtain a global Ub interactome by Ub interactor affinity enrichment-mass spectrometry (UbIA-MS). TAB2 ZNF and TAB3 ZNF domains were identified as new interactors of K6 diUb, while UCHL3  was found to be a new interactor for K27 Ubs. By comparing the differences of Ub interactors in HeLa cells, mouse embryonic stem cells (ESCs), and neuronal precursor cells (NPCs), the authors found that K27, K29, K33, and K6 Ub interactors showed cell-type dependence.DiUb probes bearing a triazole structure were used by Ovaa et al. developed polyUb probes bearing up to ten Ub units.38 These probes were applied to proteomic detection, from which 70, 44, and 37 proteins were found to be interactors of K27, K29, and K33 Ub, respectively. The authors also used gel eluted liquid fraction entrapment electrophoresis to separate the Ubs into Ub2, Ub4, Ub6+ to examine their interactors separately.39 Significant differences were observed in the interactors of Ubs with different lengths. For example, Ub-associated domain-containing protein 1 (Ubac1), RING finger 123 (RNF123), and USP15 only interact with long Ub chains .In another study of Ub interactors, Stengel N-ethyl isopeptide bonds.36 These probes were used to profile the interactome of K29 Ubs, which captured some new interactors not identified by the triazole probes. The main limitation of the Ub pull-down probes is that weak interactors cannot be captured. Tian et al. reported diazirine-based photoaffinity probes that can capture K48- and K63-Ub interactors in cell lysates.98 Meanwhile, Glickman et al. developed photo-crosslinking polyUb probes for detecting proteasome subunits that interact with Ubs.97In addition, triazole-free DUB-resistant Ub probes were developed containing 4.2.4et al. designed M1, K63 and K48 diUb probes containing a FRET dye pair.119 These probes were used to measure the conformational change in the presence of recognition protein. It was found that Ub chains are present in a multi-conformational equilibrium in solution. K63 and M1 diUb exhibited an extended “open” and “semi-compact” conformation, while K48 diUb barely exhibited any “open” conformation in isolation. Some DUB such as AMSH-LP (for K63 diUb), OTUB1 (for K48 diUb) and USP21 (for M1 and K63 diUb) chose the pre-existing conformation to achieve the cleavage, but USP21 must remodel K48 diUb into an “open” conformation in order to undertake enzymatic activity.Komander 4.34.3.1et al. reported a α-globin-Ub-Dha probe for the identification of α-globin specific DUBs30  reacted with the probes only in the presence of Ser65-phosphorylated ubiquitin (p-Ub), indicating that p-Ub is a prerequisite for p-parkin sustaining its transthiolation activity. A panel of parkin mutations associated with Parkinson's disease was profiled with the probes and revealed that nearly all contributed to defects in transthiolation activity. The probes were further applied to cell extracts or patient tissues to investigate parkin activation. Licchesi et al. later showed these probes react with HECT E3, such as NEDD4, UBE3C, and HECTD1.120Virdee  RBR E3s .28 The pIn vitro and in vivo experiments verified NMNAT2 (nicotinamide mononucleotide adenyltransferase) was the substrate of MYCBP2.29More recently, a variant of the probe E2-Ub-AVS was developed, which showed improved labelling efficiency and was applied to profiling HECT or RBR E3s in neuroblastoma SH-SY5Y cell extracts. About 40 HECT/RBR E3s were profiled, consisting of 80% known HECT/RBR ligase. Notably, 33 RING ligases were also enriched, of which a novel RING ligase MYCBP2 was discovered to utilizes a unique RING-Cys relay (RCR) mechanism mediating the transfer of Ub onto the substrate threonine and serine residues. et al. reported an E2-Ub-Dha probe  and several RING E3s were also enriched, similar to E2-Ub-AVS probe.Shi ha probe , containet al. developed a photo-reactive E2-SUMO probe to trap RING type SUMO E3 ligase.42 C93 of SUMO E2 Ubc9 was mutated to 2,3-diaminopropionic acid to form a stable amide-linked E2-SUMO conjugate  revealed that H2B-Ub altered the binding orientation of hDot1L on nucleosome, which may place the active site of the Dot1L proximal to H3K79. This finding is consistent with the early proposed “corralling” mechanism, in which Ub locates on the surface of the nucleosome to block the unproductive hDot1L binding direction.Dot1L-mediated methylation of histone 3 K79 was reported to be stimulated by ubiquitination of histone 2B at K120.me probe .43 The p4.44.4.1et al. developed an Ub-Dha probe to capture E1, E2, and E3  of either the TAT sequence or cyclic polyarginine cR10 ]2 ]2. Also, the probe was demonstrated to inhibit USP2a in DU45 cells when treated with the Pd complex. This work provides the first case of delivery and activation of a synthetic protein in cells and opens new opportunities for studying dynamic regulation of ubiquitination in cells.Brik allyl)]2  ]2 . A cR10 4.4.4et al. reported a reversible monoUb probe with a warhead containing a disulfide group  can release the probe and regain their activities under mild reducing conditions. The probe was utilized to capture DUBs in HeLa cell lysate, and most of the captured DUBs can be re-captured by the Ub-PA after being reduced by DTT. The reversibility of the probe made it useful for isolating active DUBs or DUB complexes from natural environment and study their activity in vitro.Ovaa de group , which c4.4.52+, where no covalent intermediate is generated. Therefore, the aforementioned probes that target Cys are not applicable for capturing this family of DUBs. Ovaa et al. reported a mono-Ub probe (129 Inhibition assays showed that the probe can inhibit Rpn11/Rpn8 with an IC50 value about 2 μM. When incubated with HeLa cell lysates, the probe captured metalloDUBs POH1, AMSH, and AMSH-LP.The active site of the JAMM/MPN + family DUBs contains ZnUb probe  with a z5.in vivo profiling are also increasingly needed. New Ub-based chemical probes will continue to emerge, inspired both by the innovation and progress of chemical protein synthesis, and the fascinating biology and clinical importance of ubiquitination.This article reviews the development and application of the state-of-the-art Ub-based chemical probes, highlighting the need for innovative technologies and novel concepts to study the ever-increasingly complex facets of Ub biology. Special attention has been given to the use of chemical probes to capture and monitor the enzymes or Ub interactors previously not targeted by conventional probe designs. Progress in this regard will enable a more in-depth dissection of the many enigmatic aspects of ubiquitination in the cells, and elucidation of the mechanisms behind the complex ubiquitination machinery. Furthermore, it should be pointed out that proteomic identification of trapped proteins by the synthetic and semisynthetic probes is still not easy and has become the bottleneck in the field. Also, chemical probes capable of There are no conflicts to declare.


However, the biological mechanism underlying this phenomenon is yet to be elucidated. The elicitation of a systemic response by cryotherapy with documented abscopal effect was rarely reported, although the immune response induction is presumably similar to a radiotherapy-induced one. The report is a combination case study and review of the abscopal effect in melanoma treated with checkpoint inhibitors.Therapy targeting immune checkpoints represents an integral part of the treatment for patients suffering from advanced melanoma. However, the mechanisms of resistance are responsible for a lower therapeutic outcome than expected. Concerning melanoma, insufficient stimulation of the immune system by tumour neoantigens is a likely explanation. As shown previously, radiotherapy is a known option for increasing the production of tumour neoantigens and their release into the microenvironment. Consequently, neoantigens could be recognized by antigen presenting cells (APCs) and subjected to effector T lymphocytes. Enhancing the immune reaction can trigger the therapeutic response also at distant metastases, a phenomenon known as an abscopal effect . To illustrate this, we present the case of a 78-year old male treated by anti-CTLA-4/ipilimumab for metastatic melanoma. The patient received the standard four doses of ipilimumab administered every three weeks. However, the control CT scans detected disease progression in the form of axillary lymph nodes metastasis and liver metastasis two months after ipilimumab. At this stage, palliative cryotherapy of the skin metastases was initiated to alleviate the tumour burden. Surprisingly, the effect of cryotherapy was also observed in untreated metastases and deep subcutaneous metastases on the back. Moreover, we observed the disease remission of axillary lymph nodes and liver metastasis two months after the cryotherapy. The rarity of the abscopal effect suggests that even primed anti-tumour CD8 Immune checkpoint inhibitors represent a milestone in the immunotherapy of cancer, including melanoma. The monoclonal antibody targeting CTLA-4 (ipilimumab) was the pioneer checkpoint inhibitor on the market, achieving a significant improvement in overall survival (OS) in metastatic melanoma patients. Despite this success, the therapeutic response observed in patients treated by ipilimumab is only about 10% . Despiteinsufficient production and release of neoantigens by the tumour (presumably due to a low mutation load) with consequent insufficient stimulation of immune cells by these neoantigens ,4;insufficient tumour infiltration by tumour infiltrating lymphocytes (TILs) resulting in immunologically “cold” or “deserted” tumours ;absence of PD-1-expressing T cells and only transient infiltration of PD-L1-expressing tumour-associated macrophages (TAMs) in metastasis, which might be documented in biopsies at the beginning of therapy ;presence of an innate transcriptional “signature” of anti-PD-1 resistance  ;insufficient signalling of interferon γ ,9,10,11.The mechanisms of resistance to immunotherapy in melanoma are numerous and reflect the complexity of tumour cell interactions with the immune system. Not all of these interactions are fully documented and understood. The principal mechanisms of immunotherapy resistance include:Low stimulation of the immune system by tumour-specific neoantigens is indeed one of the critical factors responsible for immunotherapy resistance . The entThe abscopal effect is based on these principles of immune system surveillance . The absThe abscopal effect has also been observed after various other types of local treatments such as electroporation or intra-tumoral injection of therapeutics ,21. HoweMechanistically, the disintegration of tumour cells due to local treatment can release tumour neoantigens. This prerequisite could consequently result in the stimulation of the immune system. Recognition of the tumour-specific neoantigens, their intake and presentation are mediated by dendritic cells (DCs).+ T cells by MHC molecules class I and to CD4+ T cells by MHC molecules class II. Antigen presentation efficiently performed by mature DCs also contributes to the cytotoxic immune response by activating NK cells /Lymphocytes abs.: 1.35 [0.80–4.00]) before initiating ipilimumab therapy, and NRL was 1.74 (Neutrophils abs.: 3.66/Lymphocytes abs.: 2.10) , and the median fraction size was 4 Gy (range: 1.8–25 Gy). The median time between radiotherapy and ipilimumab was one month . The effSome tumours lack the appropriate inflammatory cytokines and chemokines to attract immune cells, such as DCs, macrophages and cytotoxic T cells, to the tumour site. Indeed, we have also observed this phenomenon in the initial biopsy. On the other hand, the expression of immunosuppressive ligands and death ligands inhibits T cells’ function and activation of the anti-tumour effect . The dowRadiotherapy can promote the release of tumour neoantigens and potentially lead to the stimulation of immune responses with an anti-tumour effect. Reits and co-workers observed that radiotherapy at doses 10–26 Gy could enhance MHC-I expression in vitro and in vivo studies. This fact increased the presentation of tumour antigens and stimulated T cell attack to the tumour site . The nexThe change in the tumour microenvironment by radiotherapy is relatively fundamental. Localized radiotherapy induces a burst release of cytokines and chemokines, giving rise to an inflammatory tumour microenvironment. The main factor is the strengthening of interferons’ expression (IFNs), crucial for anti-tumour immune response. Type I IFN is essential for the activation and function of DCs and T cells, which, in turn, is responsible for the release of IFN-γ and tumour control. Inhibition of expression IFN-γ is one of many factors responsible for resistance to immunotherapy by checkpoint inhibitors ,45.+ T cells, which are crucial effectors in anti-tumour immunity. However, the MHC pathway is a complex of interactions that, alone, are insufficient to lead to T cells’ activation. Subsequent activation is necessary for co-stimulatory signals, such as the expression of CD80, CD40 L, and CD28 [Radiotherapy induces cell apoptosis called immunogenic cell death (ICD). This type of apoptosis responds to the release of tumour neoantigens for stimulation of antigen-presenting cells (APCs). It is the stimulation of these cells that is responsible for the possible systemic immune response and the induction of the abscopal effect . Calretiand CD28 . O ne doand CD28 . Therefoand CD28 ,19,33. Iand CD28 ,53. PD-Land CD28 . In thisand CD28 . Tsui anand CD28 .Cryotherapy with liquid nitrogen is a possible option to treat non-melanoma skin cancers such as basal cell carcinoma . In melaThe direct cellular injury in cryotherapy is due to the freezing of water, leading to disruptive necrosis and water transmembrane movement. Of note, thawing is also associated with cellular damage and can be equally destructive. The mechanisms by which cryoablation kills cells are therefore multiple and complex. Mechanistically, the cooling and warming rate during cryosurgery has a significant impact on extracellular and intracellular ice formation and also osmotic changes. In our case, we have indeed observed massive necrosis and marked infiltration of immune cells in the third presented biopsy I–L.+, CD45+ and CD68+ cells in tumours and their vicinity after cryotherapy  such as DNA and heat shock proteins (HSPs) from damaged or dying cells can stimulate Toll-like receptors of macrophages and dendritic cells, leading to the release of various cytokines, namely IL-6 as a key secreted cytokine.It was observed that the number of local infiltrated macrophages and serum IL-6 levels peaked at day 7 and decreased at day 14 after cryoablation . Gu and + and CD8+ T cells in the abscopal tumour was also significantly higher [Takahashi and co-workers used cryosurgery in the mouse melanoma and Lewis lung carcinoma model. This study suggested that cryosurgery generates the most favourable immune-regulatory response for abscopal tumours and activation of anti-tumour immune cells, and increased secretion of pro-inflammatory cytokines such as IL-1β, IL-2, IL-6, IL-12β, IFN-γ and TNF-α. The infiltration of CD4y higher .+ T-cell response with a potential systemic effect [Mechanistically, cryosurgery results in an acute response of the tissue similarly to normal wounding. Following the lysis of tumour cells, tumour-derived antigens are released. Highlighting this boosting effect of cryosurgery, some authors have referred to this ability of cryotherapy to load dendritic cells and consequently present to effectors as producing an “in-vivo dendritic cell vaccine” . After cc effect .However, the tumour microenvironment, including malignant melanoma, is very similar to that of a chronic wound ,68. ThisWe can hypothesize that the wound healing-dependent activation of immune response in cryo-ablated tumour tissue can synergize with immune checkpoint inhibition such as anti-CTLA-4/ipilimumab therapy of melanoma.s to −189 °C with tissue warming up to 37 °C in between. The photodocumentation was performed during the first cryotherapy  to destroy metastatic tissues was performed using contact Cryocauter . First, we made a shave excision of five bleeding exophytic metastases to achieve a more reliable cryodestruction. Every metastasis base was cryocauterised by the method of partially overlapping fields; every field was exposed twice for 30 otherapy C. The seotherapy D. The biTissue samples were fixed for 24–48 h in 4% neutral buffered formalin at room temperature and routinely processed to paraffin blocks. Sections (3 µm thickness) were deparaffinized and rehydrated through xylene and ethanol, and PBS. Afterwards, heat-induced epitope retrieval was performed using citrate buffer (pH 6.0) in an autoclave at 120 °C for 3 min with consequent gradual cooling to room temperature over 60 min. Unspecific binding of antibodies was inhibited using Protein Block system  followed by treatment with 3% hydrogen peroxide  for 20 min. Sections were incubated overnight at 4 °C with biotinylated primary antibodies . The laboratory complies with national accreditation standards , which guarantee the accuracy of verified results. Normal value range and pathological limits were determined by the producer. S100B  was performed by Electro-chemiluminescence immunoassay (ECLIA) for the in vitro quantitative determination of S100 in human serum. LDH  was performed by International Federation of Clinical Chemistry (IFCC) methods, and CRP (Roche) was performed by nephelometry using a Dade Behring BNII instrument. Blood count, neutrophils abs. and lymphocytes abs. were performed by automatic analyzer. The serological markers used, and the examination of the blood count, physiological limits and critical values are summarized in The abscopal effect is mediated by a systemic anti-tumour immune response elicited by focal treatment and followed by regression at distant sites . The absThe biological rationale behind the abscopal effect triggering remains unclear. However, the great success of immunotherapy achieved in melanoma recently also reignited interest in the abscopal effect. The discussed studies with the achieved abscopal effect are summarized in Our case report presents the successes achieved with immunotherapy and cryotherapy in combination. The induction of a systemic response with an abscopal effect by cryotherapy is rare even though the immune system’s stimulation is similar to radiotherapy or oncolytic viruses ,72. The


Report a rare case of retinal capillary macroaneurysm with associated subretinal fluid.A 71-year-old male underwent full ophthalmic examination including Optical Coherence Tomography (OCT), Fluorescein Angiography (FA). Fundus examination showed moderate non-proliferative diabetic retinopathy of both eyes with scattered microaneurysms. On initial visit, FA displayed a hyperfluorescent lesion with leakage on late frames in the left eye. OCT revealed the lesion to be spheroid with a hyperreflective wall and hyporeflective lumen in the inner retina, corresponding to a capillary macroaneurysm. Intraretinal cystic fluid surrounded the lesion. On subsequent visit 7 months later, subretinal fluid in the location of the capillary macroaneurysm was noted on OCT. Vision was maintained at 20/30–2 OD, 20/40 OS throughout. No treatment was necessary.Subretinal fluid from the capillary macroaneurysm likely developed from its juxtafoveal location and discontinuity of the external limiting membrane (ELM); a barrier preventing flow of intraretinal fluid to the outer retina. Aneurysms are defined as fusiform or round dilatation, otherwise known as outpouching, of vasculature. Aneurysms of retinal vessels can involve all aspects of the circulation and may develop from arteries, veins, collateral circulation or capillaries . When thA 71-year-old male, followed for 5 years for diabetes mellitus presented to the University Eye Clinic for yearly examination. Patient had no complaints including blurry vision, metamorphopsias or scotomas. He had a history of stable, well-controlled diabetes mellitus 2 with a hemoglobin A1c of 6.5 the month before. On record, patient never had a hemoglobin A1c above 7.0. He also had a past medical history of hypertension, hyperlipidemia and prostate cancer 19 years prior which was cured after a complete resection. His medications included atorvastatin, metformin, glimepiride and losartan. On examination his best corrected visual acuity was 20/30–2, 20/40–2 and intraocular pressure 11, 14 mmHg in the right and left eyes respectively. Pupillary examination, extraocular muscles and confrontation visual fields were all within normal limits. Slit lamp examination showed a right upper eyelid nevus and bilateral nasal pterygium stable for many years.On fundus examination of the left eye, a large juxta-foveal aneurysm with surrounding intraretinal fluid was present inferior to the fovea. Both eyes had scattered microaneurysms and mild peripheral retinal degeneration nasally. Initially, Optical Coherence Tomography (OCT) of the right eye was normal Fig. a. The leOn subsequent in-person visit, 7 months after initial visit, patient reported no visual changes and maintained a visual acuity of 20/30–2 and 20/40–2 of the right and left eyes respectively. OCT of the right eye was normal  with associated subretinal fluid. Blood-brain barrier disruption in exudative macroaneurysms results in a slowly prolonged leakage of plasma components through the weakened walls into the retina. Usually, this fluid spreads to the outer plexiform layer (OPL) and ONL where it concentrates due to the external limiting membrane (ELM) acting as a relative barrier to protein and osmotic forces . The chaAn important differential diagnosis is Perifoveal Exudative Vascular Anomalous Complex (PEVAC) defined by the presence of a unilateral, isolated perifoveal aneurysm in otherwise healthy individuals . Our patCapillary Macroaneurysms should be added to a differential diagnosis in cases of fusiform dilatation of vasculature in association with subretinal fluid. In our case, we hypothesize that the juxtafoveal location and discontinuity of the ELM played a part in the formation of subretinal fluid.


The efficient transport of solid particles using polymeric fluids is an important step in many industrial operations. Different viscoelastic fluids have been designed for this purpose, however, the effects of elasticity have not been fully integrated in examining the particle-carrying capacity of the fluids. In this work, two elastic fluid formulations were employed to experimentally clarify the effect of elasticity on the particle drag coefficient as a proxy model for measuring carrying capacity. Fluids were designed to have a constant shear viscosity within a specific range of shear rates,  The dynamics of solid particles flowing through polymeric fluids is strongly affected by viscoelasticity of the fluid ,2. Elastc [Due to the lack of physics and theoretical models, or computational power, state-of-the-art fracturing simulators ignore the relevance of the flow properties  of polymeric fluids ,11,12,13ation, c ,23. Diluntration ,24.Despite the complexity of such systems, the particle-carrying capacity of a fluid is estimated by mapping the translation of a single sphere in inertia-less steady-state conditions ,26. In hIn many practical cases, because of the strong interactions between elasticity and viscosity of the polymeric fluids, the hindrance due to elasticity was intermingled with the inherent shear-thinning properties, i.e., the fact that the viscosity of the polymeric fluid surrounding the falling sphere decreases during the sphere motion . Blyton The current study is undertaken to fulfill two main goals: (i) outline an appropriate scheme to infer the particle-carrying capacity of polymeric fluids, and (ii) generate unique static settling, or drag coefficient, data in moderate levels of elasticity. To this end, first, the theoretical background is briefly reviewed for the particle dynamics and rheology measurements  to accurately determine the parameters required to infer the drag coefficient. Then, two fluid formulations are designed with distinct longest relaxation times to carry out the inertia-less particle settling tests at low to moderate level of elasticity. Next, the experimental setup and velocity measurement procedures are elaborated, and the experimental results are presented and weighted against the approximate drag model developed by Faroughi et al. . Finallya settling through the fluid. Here, U, of the particle. For a Newtonian fluid, the Weissenberg number is The interplay among different mechanisms controlling particle transport can be studied by decoupling different relevant forces acting on particles. The most important ones induced by the viscoelastic fluids are the drag, inertial, and transversal forces for which the theoretical developments are very limited . DimensiThe presence of coiled or stretched polymers also impacts the effective shear viscosity of the polymeric fluid through hydrodynamic and physical interactions similar to the presence of a cloud of solid particles ,36. Thisr fluids , the relequation  that besequation . Severalequation ,40,41. Aequation  is gener and 0.5 ) and accAnother important dimensionless number is the Reynolds number representing the ratio between inertial and viscous forces, which is defined as,w regime ,23.p,For a single particle settling in a viscoelastic fluid, one may carry out the drag coefficient on the surface of the particle using a surface integration of the total stress comprising the polymeric and solvent stress contributions, U, measured for a sphere settling under the action of gravity, g, through a fluid. A relationship between the drag coefficient and terminal velocity can be deduced using the drag and gravitational force balance, leading to,In experimental studies, the drag coefficient can be calculated using the terminal velocity, quations  and 5)  U, measu CD=24Re . At highnetworks ).Due to strong interactions of the fluid viscoelasticity and the complex kinematics of the mixed shearing and extensional flow around the particles, an exact solution to Equation  that peri et al.  at Wi≤1 duces toχ=CD(24/RTo determine the drag coefficient using Equations  and 7),,7), one nd Colby , Kulickend Colby . This meEquation .The determination of the relaxation time,  methods . For an  methods . The rel methods . This su methods ,51. Othe methods . Sometim methods ,52,53,54 methods ,55. In t methods .T = 20 Two different elastic fluids were used to investigate the influence of elasticity on the drag coefficient for slow flow around a sphere. The first fluid was composed of 0.1 wt.% polyacrylamide (5–6 MDa) dissolved in a solvent made of 90 wt.% glycerol and 10 wt.% DI water (this fluid is tagged as PAM/GLY for the rest of this paper). Polyacrylamide is known to adopt a relatively extended conformation in low salinity, and a random coil conformation in solutions containing high concentrations of ions. Deionized water is then used as a solvent to remove the reduction in extensibility of this polyacrylamide-based Boger fluid prepared for different measurements. The second fluid was composed of dissolving 16 wt.% high molecular weight polystyrene (20 MDa) in a solvent made of 70 wt.% low molecular weight polystyrene 500 Da) and 30 wt.% tricresyl phosphate (this fluid is tagged as PS/TCP for the rest of this paper). The mixture of tricresyl phosphate with low molecular weight polystyrene is known to be a good solvent for high molecular weight polystyrene, and results in a high extensibility for the solution ,57. For au model ,59. The au model ,61 and μ0 Da and To probe a broad range of Weissenberg numbers, different Boger fluids possessing different relaxation times are needed. Due to the difficulty to formulate Boger fluids , various sizes and types of spherical particles were used with R = 15.25 cm) is captured and measured using two techniques, (i) digital image processing (DIP) following Kim et al. [The transient motion of a sphere along the center line of a glass cylindrical tube . All rheological measurements were performed at carefully controlled room temperature, L, from the inlet position. This step is performed by dividing the cross-section of the microchannel arbitrarily into six bands for which the average velocities and cross-sectional area are calculated, see Del Giudice et al. [k, and H, and the confinement ratio defined as The longest relaxation time for the PAM/GLYC system could not be measured using conventional rheometry due to technical limitations of the instruments. Instead, the ion time ,54. The H, the following equation,Q represents the imposed volumetric flow rate. Here, specifically, polystyrene particles having a 10 It is important to note that the master curve plotted in e in IDL .Results of the bulk shear rheology measurements are shown in fined as ,(11)c*=issolved . The ovenditions ,65. The  present . Therefon is the flow index accounting for the shear-thinning of the solution. For Boger fluids, the flow index should approach unity, Both polymer solutions maintain a constant viscosity, as one of the most important characteristics of the Boger fluid, within the full range of shear rates present in the settling experiments at au model ,12)η=ηη=ηγ˙<50 extendeAt higher Weissenberg numbers, no data was produced for the PAM/GLY solution due to its very low relaxation time and the limitation on the particle size satisfying terature ,23 and EEquation  very welEquation ) extendeThe experimental data plotted in quations  and 7),,Wi≤1), ad Sharma  and Hu ed Sharma . TherefoWhen polymeric solutions are strongly shear-thinning, the effect of elasticity on the drag coefficient reduction or enhancement can be masked ,23. SheaIn this work, we performed an experimental campaign using different polymer solutions and rheological techniques to validate a theoretical model introduced previously  to descr


Replicative immortality is a hallmark of cancer, and can be achieved through telomere lengthening and maintenance. Although the role of telomere length in cancer has been well studied, its association to genomic features is less well known. Here, we report the telomere lengths of 392 localized prostate cancer tumours and characterize their relationship to genomic, transcriptomic and proteomic features. Shorter tumour telomere lengths are associated with elevated genomic instability, including single-nucleotide variants, indels and structural variants. Genes involved in cell proliferation and signaling are correlated with tumour telomere length at all levels of the central dogma. Telomere length is also associated with multiple clinical features of a tumour. Longer telomere lengths in non-tumour samples are associated with a lower rate of biochemical relapse. In summary, we describe the multi-level integration of telomere length, genomics, transcriptomics and proteomics in localized prostate cancer. Despite the known role of telomere length in cancer, its association with genomic features remains unclear. Here, the authors integrate telomere length, genomics, transcriptomics and proteomics in localized prostate cancer and reveal links between telomere maintenance, disease drivers and clinical outcomes. Telomeres protect chromosomal ends from degradation by the DNA double-strand break (DSB) response pathway. Due to the linearity of chromosomes and chromosomal replication, telomeres are shortened by approximately 50 bp during mitosis3. When telomeres become substantially shortened, cell cycle progression halts and cells enter replicative senescence; further replication leads to cellular crisis and eventually cell death4. Telomere maintenance and lengthening is essential for cancer cell proliferation and enables replicative immortality: a fundamental hallmark of cancer5. Telomere regulation occurs through two known mechanisms: activation of telomerase or alternative lengthening of telomeres (ALT) which relies on homology-directed DNA replication6.Telomeres, which make up the ends of chromosomes, consist of a repeat TTAGGG sequence8, the role of telomere maintenance in individual tumour types is poorly understood. Moreover, the relationship between telomere length and biologically relevant genomic indices, such as percentage of the genome altered , and other measures of mutational density has not been assessed, nor has the association between telomere length and clinical outcome in prostate cancer.Despite the pan-cancer studies analysing telomere length from various tumour types18 the most frequently diagnosed non-skin malignancy in North American men . Localized prostate cancer is a C-class tumour19, characterized by a paucity of driver single-nucleotide variants (SNVs) and a relatively large number of structural variants (SVs), including copy number aberrations (CNAs) and genomic rearrangements (GRs). Several of these aberrations, including mutations in ATM and amplifications of MYC – which drive DSB repair and cell proliferation, respectively—are associated with significantly reduced time to biochemical and metastatic relapse after local therapy20. Intriguingly, both of these mutations have also been associated with telomere maintenance22 and telomere shortening–relative to adjacent epithelium23. Similarly, an interaction between hypoxia, dysregulated PTEN, TERT abundance and telomere shortening were recently illustrated15. Despite this, no well-powered study exists evaluating the association between telomere length, somatic features and clinical outcome in prostate cancer.We and others have described the genomic, transcriptomic and proteomic landscape of localized, non-indolent prostate cancer:To fill this gap, we quantify the telomere length and somatic mutational landscapes of 392 localized prostate tumours. We explore associations between telomere length and the tumour methylome, transcriptome and proteome. Using rich clinical annotation, we further assess the relationship between telomere length and outcome. Taken together, these data establish the role and regulation of telomere length in localized prostate cancer, and establish clear links between telomere maintenance and drivers of prostate cancer development and clinical aggression.24. We estimated both tumour and non-tumour  TLs for each sample using TelSeq v0.0.125 and TelomereHunter v1.0.426. After quality control, 381 samples were retained for further analysis (see Methods). All tumours were treatment-naive, and detailed clinical information was collected. The cohort consisted of 11.2% ISUP Grade Group (GG) 1, 52.8% GG2, 24.6 % GG3, 7.8% GG4 and 3.4% GG5. For the majority of samples, the tumour was confined to the prostate . The mean tumour coverage was 73.1x ± 20.6x (median ± standard deviation); the mean non-tumour coverage was 44.1x ± 13.4x. Median clinical follow-up time was 7.46 years. TLs for each sample, along with clinical and genomic summary data are in Supplementary Data n = 40) were longer than those in blood tissue  and tumour tissue  on the clinico-genomics of prostate tumours, we exploited whole genome sequencing (WGS) of 392 published tumour–reference pairs14. The relationship of each of these features with tumour TL is shown in Fig. CHD1, RB1 or NKX3-1 deletions had shorter tumour TL . Similar to tumour TL, the TL ratio did not significantly differ between samples with any of the recurrent prostate cancer-related GRs or CNAs but samples with a somatic SNV in the gene 27 scores from a subset of samples in this cohort (n = 170)14. There was no correlation between scores representing chromothripsis events in either tumour TL  or TL ratio .We also assessed the association of telomere length with chromothripsis using published ShatterProofvia the TP53 pathway28. Failure to block cell growth can lead to telomere crisis and subsequent translocations, chromothripsis or chromosome fusions29. We explored the association of TL and the number of gene fusions present in a tumour. There was a negative correlation between the number of gene fusions and tumour TL  but no correlation with TL ratio  or TL ratio  and MIK67 RNA abundance  or TL ratio , and in a metastatic dataset 3% , likely reflecting the early-stage of our cohort. Mutations in ATRX and DAXX, which have been correlated with longer telomeres32, were rare in our cohort: only two samples harboured a CNA in DAXX, and only four samples had an alteration in ATRX.A pan-cancer study reported that TERT RNA abundance was not correlated with tumour TL or TL ratio . These data suggest that TERT signalling is not significantly abrogated in localized prostate cancer either by somatic aberrations or through gene expression changes.Tumour tio Fig. . SamplesTERT RNA abundance and tumour TL further, we considered known activating transcription factors. Transcription of TERT can be activated by MYC and SP1 and repressed by AR33. MYC amplifications occurred in 14.5% of our samples . TERT and MYC mRNA abundance was positively correlated  but MYC abundance was unrelated to tumour TL  but no significant correlation between SP1 and TERT abundance  and related that to tumour TL (n = 241). Methylation of almost half of all genes  was significantly correlated with tumour TL . No proteins were significantly associated with tumour TL after FDR adjustment although 9.3% proteins showed correlation to tumour TL before adjustment . There were 112 genes with methylation, transcription and proteome correlations to tumour TL. Remarkably, these showed no functional enrichment. Several genes showed methylation positively correlated with tumour TL but negatively correlated with RNA and protein abundance  of genes with overlapping data also had a significant correlation between RNA abundance and TL ratio and none between protein abundance and TL ratio . These results suggest that tumour TL, not TL ratio, is associated with tumour gene expression.We similarly investigated whether TL ratio was associated with methylation and found that the methylation levels of 33.7% 5,218/15,492) of genes were significantly correlated with TL ratio  and the membrane metallo-endopeptidase gene, MMEL1 , shelterin interacting proteins (PINX1 and RTEL1), and the components of telomerase (TERT and TERC). There were no differences in the tumour TL  or TL ratio  in the four CNA subtypes identified from aCGH arrays and associated with prognosis9  identified from 284 OncoScan SNP arrays14 but not with tumour TL , higher ISUP Grading and tumour size and extent are all associated with worse outcome. Therefore, we considered whether there was interplay between TL and the clinical features of a tumour. Tumour TL was not significantly correlated to age at diagnosis,  and TL ratio , an early surrogate endpoint in intermediate-risk prostate cancer. Cox proportional hazards (Cox PH) models were fit, splitting patients (n = 290) into two groups based on their TL with increasing cutoff thresholds using the group with the longer TL as the reference group. . There was no association between tumour TL and BCR  and TL ratio  while specific CNAs, GRs and SNVs are not Fig. . This suOne limitation in the estimation of TL using short-read whole genome sequencing is the difficulty in estimating chromosome specific telomere lengths. Junction spanning reads from paired-end experiments, in which one read maps within the first or last band of the chromosome and the other read maps within the telomere region, are scarce. Further studies should be performed using-long read sequences, in which these regions may have more coverage and can be used to determine chromosome specific shortening and its association to specific genomic events or biochemical relapse.These data highlight the complicated relationship between telomere length in both tumour and non-tumour cells, and molecular and clinical tumour phenotypes. They highlight the need for increased study of telomere length across cancer types, and for long-read sequencing to introduce chromosome-specific analyses.11, phs000330.v1.p112, EGAS0000100090014, phs000178.v11.p813, EGAS0000100040024, phs001648.v2.p145). Cellularity was also determined in silico from OncoScan SNP arrays via qpure (v1.1)46. Detailed clinical characteristics of patients are provided in Supplementary Data Published whole genome sequences of tumour and matched non-tumour samples, as well as, available clinical information were downloaded from public repositories (phs000447.v1.p147) at the lane level. Picard  was used to merge the lane-level BAMs from the same library and mark duplicates. Library level BAMs from each sample were also merged without marking duplicates using Picard. Local realignment and base quality recalibration was carried out on tumour/non-tumour pairs together using the Genome Analysis Toolkit . Tumour and non-tumour sample level BAMs were extracted, headers were corrected using SAMtools , and files were indexed with Picard.Raw sequencing reads were aligned to the human reference genome, GRCh37, using BWA-mem  and TelomereHunter (v1.0.4)26 on BAM files generated using BWA-mem  and GATK . TelSeq estimates telomere length with the following formula l = tksc, where tk is the abundance of telomeric reads , c is a constant for the genome length divided by the number of chromosome ends and s is the fraction of all reads with GC composition between 48–52%. TelomereHunter also identifies telomeric reads based on the number to repeat sequences with a read and normalizes by the number of reads with GC content between 48–52%. This value is multiplied by 106 to calculate TRPM (telomeric reads per GC content-matched million reads) values. As a quality measure, TelSeq estimates for each sample were generated per sequencing lane. Reads from lanes that contained too few reads to calculate an estimate (marked as UNKNOWN), and outlier lanes as identified by grub’s test, were removed from input BAMs using BAMQL (v1.6)50. After outliers were removed, TelSeq was run again ignoring read groups with the −u parameter. Samples with telomere estimates <0.25 were removed from further analysis. To account for differences in TL due to sequencing centre, a linear model was fit with TL as the response variable and sequencing centre as the predictor variable. A separate model was fit for tumour and non-tumour length.Tumour and non-tumour telomere lengths were estimated using TelSeq  was used to call SNVs on bases with at least 17x coverage in tumours and 10x in non-tumours. Coding versus non-coding SNVs were determined using Annovar52. Genomic rearrangements were identified using Delly . Gene fusion events involving ERG or ETV were collectively referred to as ETS events. Genomic rearrangement calls were examined to determine if breakpoints led to a TMPRSS2:ERG fusion or if breakpoints were found in both 1 Mbp bins surrounding the following gene pairs: ERG:SLC45A3, ERG:NDRG1, ETV1:TMPRSS2, ETV4:TMPRSS2, ETV1:SLC45A3, ETV4:SLC45A3, ETV1:NDRG1 and ETV4:NDRG1. ERG immunohistochemistry and deletion calls between TMPRSS2 and ERG loci in OncoScan SNP array data provided further support for these fusions.Single-nucleotide variants (SNVs) and genomic rearrangements (GRs) were called using standardized pipelines across all datasets. SomaticSniper  to GRCh37 with GENCODE v24lift3755. RSEM (v1.2.29) was used to quantify gene abundance56.Total RNA was extracted with mirVana miRNA Isolation Kit (Life Technologies) according to manufacturer’s instructions. RNA samples were sent to BGI Americas where it underwent QC and DNase treatment. Two hundred nanogram of total RNA was used to construct a TruSeq strand specific library with the Ribo-Zero protocol (Illumina), and all samples were sequenced on a HiSeq2000v3 to a minimal target of 180 million paired-end reads. Reads were mapped using the STAR aligner . Quality control was conducted using the minfi package . Raw methylation intensity levels were then pre-processed using Dasen. For each probe, a detection P-value was computed to indicate whether the signal for the corresponding genomic position was distinguishable from the background noise. Probes having 1% of samples with a detection P < 0.05 were removed. We also filtered probes based on SNPs and non-CpG methylation probes. Next, we used the DMRcate package (v1.4.2) to further filter out 27,309 probes that are known to cross-hybridize to multiple locations in the genome and 17,168 probes that contain an SNP with an annotated minor allele frequency of >5% with a maximum distance of two nucleotides to the nearest CpG site. Annotation to chromosome location, probe position, and gene symbol was conducted using the IlluminaHumanMethylation450kanno.ilmn12.hg19 package (v0.6.0).Illumina Infinium HumanMethylation 450k BeadChip kits were used to assess global methylation, using 500 ng of input genomic DNA, at McGill University and the Genome Quebec Innovation Centre . All samples used in this study were processed from fresh-frozen prostate cancer tissue. The IDAT files were loaded and converted to raw intensity values with the use of wateRmelon package  was tested using a two-sided Mann–Whitney U test in 47 previously identified18 recurrent gene fusions.The association between gene fusion status and tumour TL and TL ratio  with default settings. Two-sided Spearman’s correlation between the maximum ShatterProof score per sample and telomere length was calculated using samples with both available metrics (n = 170).Chromothripsis scores were previously generated using ShatterProof  were calculated using the AS-89 algorithm59 as implemented in the base R stats package.The correlation matrix of methylation and mRNA abundance levels from TCGA was downloaded from n = 138;14) and protein abundance  and TL were calculated using the AS-89 algorithm59 as implemented in the base R stats package.Two-sided Spearman’s correlations between TL and RNA (35 (v0.2.1) R package using genes in which there was a significant association between TL and methylation or RNA separately using KEGG collection of pathways36.Pathway analysis was performed with the gprofiler238. Briefly, for genes that are overlap among pathways, each gene is only allowed membership in one of the pathways. This membership is determined by the highest correlation between the gene and the PC1 of the other genes in the pathway. An one-sided Fisher’s Exact test was then used to determine enrichment of TL correlated genes in the reduced pathway membership.To account for crosstalk effects caused by gene overlap in pathway analysis, we implemented the principle component analysis method proposed byTM for OncoScan 3 Software was used to call copy number aberrations using the SNP-FASST2 algorithm. Gene level copy number aberrations for each patient were identified by overlapping copy number segments from OncoScan SNP 3.0 data, with RefGene (2014-07-15) annotation using BEDTools . Genes with the same copy number profile across patients were then collapsed into contiguous regions. Contiguous gene segments with aberrations in <5% of patients were removed from the analysis. To find associations between TL and copy number segments, a two-sided Mann–Whitney U test was used to compare the mean TL between samples with a copy number aberration and those without (n = 381). The copy number aberration state (either amplified or deleted) was determined as the status with the largest proportion of samples. Samples with aberrations in the other class was merged into the without group. For example, three samples have an amplification in CHD1, while 49 samples have a deletion. The three samples would be grouped with copy number neutral samples and the Mann–Whitney test performed comparing the two groups. P-values were FDR adjusted to account for multiple testing.SNP microarrays were performed with 200 ng of DNA on Affymetrix OncoScan FFPE Express 2.0 and 3.0 arrays. Analysis of the probe assays was performed using.OSCHP files generated by OncoScan Console (v1.1) using a custom reference. BioDiscovery’s Nexus ExpressR package survival (v3.2–7) using TL as a continuous or discrete variable. Age at diagnosis was controlled for in the model. Kaplan Meier plots were generated by dichotomizing samples based on the optimal cut point analysis, in which samples were dichotomized using increasing thresholds of 50 bp.Cox proportional hazards models were fit with the R statistical environment (v4.1.1). Visualization in R was performed through the BoutrosLab Plotting General package . We chose to use Spearman’s rank correlation because it is a nonparametric test and does not assume that values are sampled from a population that follows a Gaussian distribution or that there is a linear relationship between the variables. P-values from two-sided Spearman’s correlations were calculated using the AS-89 algorithm59 as implemented in the base R stats package. Box plots depict the upper and lower quartiles, with the median shown as a solid line; whiskers indicate 1.5 times the interquartile range (IQR).All statistical analyses were performed within the Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


In this article , the wroThe in-text reference citations has been arranged in numerical ascending order and the corresponding list has been arranged accordingly.


TM) is a novel technology that noninvasively quantifies lung fluid levels. Trends in ReDS values following hemodialysis remain uninvestigated. In a 64-year-old man with clinically stable hemodynamics, 2.7 L of fluid was drained during hemodialysis whereas the ReDS value remained almost unchanged (from 32 to 30%). In a 60-year-old woman with unstable hemodynamics, only 1.8 L of fluid was drained during hemodialysis, whereas ReDS value decreased considerably from 37 to 27%. Given our initial experience measuring ReDS values during hemodialysis, the ratio of fluid removal by hemodialysis between systemic plasma and lung fluid might vary in each patient. ReDS value might be a promising marker to determine the degree of fluid removal in addition to the conventional multidisciplinary index, particularly for those with unstable hemodynamics. The implications of ReDS-guided hemodialysis remain a future concern.Remote dielectric sensing (ReDS A dry weight for hemodialysis is determined by multidisciplinary modalities including congestive symptoms, physical examination, echocardiography, and chest X-ray . Of noteTM) system, which is a non-invasive wearable device to quickly quantify the lung fluid level [Recently, a remote dielectric sensing . Blood pressure trended from 128/80 to 113/72 mmHg . EchocarMedical history: The second patient was a 60-year-old woman with a history of type-2 diabetes mellitus, hypertension, and dyslipidemia. She received coronary artery bypass grafting five years ago. She initiated hemodialysis one year ago due to diabetic end-stage renal dysfunction. She was hospitalized due to worsening heart failure five months ago. She received a percutaneous coronary intervention due to unstable angina pectoris three months ago. Her medications were as follows: olmesartan 20 mg/day, aspirin 100 mg/day, clopidogrel 75 mg/day, atorvastatin 10 mg/day, and ezetimibe 10 mg/day, as well as bisoprolol transdermal patch 4 mg/day and insulin degludec 20 units per hemodialysis.2 polysulfone dialyzer with heparinization 3 times per week. Her systolic blood pressure decreased from 200 mmHg down to 100 mmHg during the hemodialysis despite etilefrine hydrochloride and amedinium methyl sulfate supports. Her laboratory data are summarized in Hemodialysis history: She received 4 h hemodialysis using 1.5 mIndex hemodialysis and ReDS measurement: During the index hemodialysis, her body weight decreased from 57.3 kg to 55.8 kg by 1.8 kg of fluid removal. We could not achieve the targeted fluid removal given a decrease in systolic blood pressure down to 92 mmHg during the hemodialysis. ReDS value decreased from 37% to 27%. Blood pressure before and after the hemodialysis was 176/71 and 138/68 mmHg , respectThere have been no established methodologies to accurately assess the lung fluid levels, which should be one of the essential components for the fluid volume management in hemodialysis patients. Chest X-ray and computed tomography are conventional tools, but these require expert technique to assess. Human atrial natriuretic peptide is referenced to consider fluid valance during hemodialysis, but the optimal range remains uncertain . Plasma ReDS is a novel device that can estimate lung fluid levels noninvasively, quickly, and easily . ReDS vaBoth patients had relatively high ReDS values at baseline (32% and 37%) considering the manufacturer-suggested normal range (between 20% and 35%), although their chest X-ray seemed to have almost no pulmonary congestion. ReDS might be a useful tool to clarify the existence of sub-clinical pulmonary congestion that other modalities cannot find.In the case 1 patient, systemic plasma volume reduction was high, whereas ReDS reduction was low. On the contrary, in the case 2 patient, systemic plasma volume reduction was low, whereas ReDS reduction was considerable.Given these findings, the ratio of fluid reduction during hemodialysis between systemic plasma and lung fluid might not necessarily be similar in all patients. Consistently, in case 2, despite the intradialytic plasma decrease being small, indicating an overhydrated status , the ReDWhen the achievement of fluid removal to the level of dry weight is challenging due to unstable hemodynamics, as with the case 2 patient, ReDS measurement might become one of the practical indicators to set the degree of fluid removal. For example, in the case 2 patient, we attempted fluid removal aggressively to achieve the dry weight despite unstable hemodynamics. However, given the considerable reduction in lung fluid level, which was quantified by ReDS measurement, it might be permitted to weaken the degree of fluid removal for the prevention of worsening pulmonary congestion as well as intradialytic hypotension.Although this is just a proof-of-concept, ReDS measurement might be a promising supportive tool to determine optimal dry weight. Optimal ReDS values that associate with favorable clinical outcomes remain the next concern. Clinical implications of ReDS-guided hemodialysis also remain a future concern.


Twitter is one of the largest online platforms where people exchange information. In the first few years since its emergence, researchers have been exploring ways to use Twitter data in various decision making scenarios, and have shown promising results. In this review, we examine 28 newer papers published in last five years (since 2016) that continued to advance Twitter-aided decision making. The application scenarios we cover include product sales prediction, stock selection, crime prevention, epidemic tracking, and traffic monitoring. We first discuss the findings presented in these papers, that is how much decision making performance has been improved with the help of Twitter data. Then we offer a methodological analysis that considers four aspects of methods used in these papers, including problem formulation, solution, Twitter feature, and information transformation. This methodological analysis aims to enable researchers and decision makers to see the applicability of Twitter-aided methods in different application domains or platforms. Some consider it as a microblogging platform , 51, 60.hey read . WhateveOne may intuitively think that anything that enters people’s conversation could leave a trace on Twitter. Indeed, as we will show soon, researches have been linking real-world phenomena, such as stock price movement, disease spread, and traffic jam, to some Twitter data. Twitter has been supporting research efforts by providing free-of-charge and easy-to-use application programming interfaces (APIs) that allows researchers to obtain Twitter data. With some restrictions, the Streaming APIcalm, had significant causal relationship with stock market movements. In a 2013 work, Culotta proposed to use tweets to help influenza monitoring [2010-2015 is a period of initial exploration when several important works showed the potential of using tweets in various decision making scenarios. Here are some examples. In a 2010 work, Sakaki et al. proposed to use tweets to detect earthquakes in Japan . By clasnitoring . ExtendiHow useful is Twitter in various decision making scenarios?How to use Twitter in these scenarios?These initial explorations showed that Twitter can be useful as a reference data source in various decision making scenarios, even though Twitter is not designed to provide such aids. We can say that researchers have invented ways to used subsets of Twitter data and information generated from them in their own problems. After initial successes, many works have followed the same direction and developed the idea further. When reviewing these later works, we focus on two research questions: The first question is about findings. Given an established decision making problem where traditional solutions exist, we would like to know by using Twitter, how much of the results can be improved. The second question is about methodologies. We would like to know the techniques used for analyzing tweets, processing text, time series, locations, and network data, and their applicability in general. Accordingly, when writing this survey paper, we divide our discussion into two parts, first we discuss the findings, then we make a summarizing effort of methodologies and techniques involved in these works.Several previous attempts had been made to summarize works that use Twitter in prediction problems. For example, an early attempt made by Kalampokis et al. in 2013 provided a summary of techniques used in a number of works that use Twitter and other social media in prediction problems . They idOur review focuses on decision making scenarios and have clear business implication. While existing reviews generally discussed data analytics performed on Twitter, we narrow the selected works to those that have clear business impact.We offer in-depth examination of the methodologies. We recognize and discuss four aspects of methodologies used in this topic, including problem formulation, solution, Twitter features, and information transformation. We examine the methodology this way so that the applicability in different scenarios and platforms can be easily understood.We cover newer papers that came out in the last five years, in or after 2016. These papers reflect latest trends in data analytics, such as deep learning, which have not been thoroughly examined in existing reviews.The main purpose of this review is to provide an overview of recent developments in Twitter-aided decision making, which may encourage and help future researches in this topic. In summary, we make the following contribution with this review: The remainder of this paper is organized as the following. In Section There are many kinds of data analysis that involve Twitter data. In this review, we will cover three modes that use Twitter data to help real-world decision making. They are shown as (A), (B) and (C) in Fig. Given above data usage modes, we decide on some application scenarios where Twitter-aided decision making can be performed. Previous surveys have covered scenario categories such as health, politics, natural disaster and crime, finance, stock, sales, sports events, and transportation , 53, 67.Twitter and tweets, and the name of the application scenario. Google Scholar ranks papers by relevance to the keywords and their impact, i.e., the number of citations. Therefore we only look at first 100 search results, and consider the remaining search results as either irrelevant or have little importance. After obtaining some relevant papers, we also look at the papers these papers cited, and the papers citing these papers. In this step we consider the rank of the journal or conference and citation numbers. We only pick papers from reputable journals and conferences. We recognize the reputation of journals and conference through ranking sources, including Scientific Journal RankingsThe solution proposed in the work addressed a problem that is independent to the existence of Twitter. Moreover, Twitter data was used as one of the three modes  shown in Fig. The work was published on a reputable venue, and preferably have attracted relatively high number of citations, taking into account their publication year.The work was published in or after 2016.After deciding on these scenarios, we set out to search for relevant works. Basically, we search Google ScholarFinally, we decide on 28 papers, 5 to 7 for each of the application scenarios, which are the main papers to be analyzed in this review. Although this is not an exhaustive search, we consider that the selected papers are representative for the on-going research in the field, and cover most impactful works.In this section, we will discuss the findings in the examined papers. The focus is on answering our first research question: how useful is Twitter in various decision making scenarios? More specifically, we discuss the improvements of solution performance, such as increases of prediction accuracy or decreases of error rates, when Twitter data is considered.Traditionally, predicting product sales considers many factors including competition, perceived quality of the product, state of the economy, and marketing efforts . Social Product sales prediction with Twitter has been generally treated as a correlation discovery problem, and the goal is to find meaningful correlation between sales numbers and relevant tweet volumes. For example, Pai and Liu proposed to use tweets and stock market values to predict vehicle sales . Their tIn some other works, event is studied instead of sales numbers. Events are usually considered as anomalies in a time series, such as peaks that deviate largely from the mean. Some temporal activities, such as flash sales, can be also considered as events. Kolchyna et al. made an attempt to predict sales spikes using Twitter . The maiStock market prediction works have in many years assumed the efficient market hypothesis, which states that the current market price fully reflects all available information . AlthougR2 when stock prices up to 20-days into the future were considered, much higher than that given by sentiment of tweets from many followers. Chen et al. made an attempt to classify numbers in financial tweets using various tweet text features [R2. However, the number of tweets for the stock seemed to be an important factor. With the stock of another firm that ranked 9th in number of tweets, treating users equally turned out to be better. Duz Tan and Tas extended previous works by investigating tweet sentiment correlation with international stocks, including those in U.S., European and emerging markets [Similar to product sales prediction, stock selection using Twitter is also often treated as a correlation discovery problem. Some works have been exploring additional information on Twitter other than tweet volumes. Sul et al. made an investigation on the effect of Twitter user influence on stock price movements . Their nfeatures . After efeatures . Differe markets . They peSome researches also considered Twitter activities as events. Ranco et al. made an investigation on the effect of tweet sentiments on individual stocks . They coDeep learning has also appeared as a solution in this problem. Xu and Cohen followed a deep learning approach to predict stock price movements from tweets . They coA recent development in the mission to fight crime is so-called predictive policing . StudiesSome works aims to extend Gerber’s model with more advanced methods. For example, Vomfell et al. added taxi flow data to the model . They foSome researches also proposed original models to solve the problem. Aghababaei et al. modeled crime prediction as a trend classification problem, with two classes of trends, namely increase and decrease in crime number . They coTraditionally, tracking epidemics such as influenza are based on clinical data or self-reporting effortszika and mosquito and compared them with weekly ZIKV cases reports provided by the government. Testing several lag values, they found that tweets with one week lag reached the highest correlation with government ZIKV reports, with a 0.67 Pearson correlation coefficient, 2 to 17 percent higher than other lag values. Consequently, building an auto-regressive (AR) prediction model, they found that adding one-week-lag tweet data substantially improved future case count prediction, comparing to the model without using tweets, increasing R2 from 0.61 to 0.74. They also found that tweets with the keyword mosquito did not add more information to the model since they strongly correlated to tweets with the keyword zika. Missier et al. made an attempt to classify tweets in order to track the development of dengue epidemic [In some recent works, disease monitoring using Twitter has been treated as a correlation discovery problem, similar to the product sales prediction. Masri et al. studied using tweets to improve the prediction of ZIKV epidemic . They coepidemic . The unsepidemic . After cepidemic . An impoepidemic . They teSome recent works also consider the spatial information of the epidemic, similar to crime prediction. Broniatowski et al. made an attempt to track influenza with tweets . Where asocial sensing, Twitter was also taken as a sensing platform [Traditionally, road traffic monitoring is done through sensors . After tplatform . It is fplatform . Early wplatform . Recent discovery, a big data tool for traffic-related event detection from Twitter data in Saudi Arabia [Event-based traffic monitoring in recent works still focus on tweet classification. Dabiri and Heaslip developed a Twitter-based traffic event detection model using deep learning architectures . They coi Arabia . The tooi Arabia . They teIn addition to events, some researches also performed spatial analysis in traffic monitoring. Wang et al. proposed a Twitter-based railway delay detection method based on topic propagation analysis of geo-tagged tweets between railway stations . They coWe have seen by now that Twitter data can be useful in various decision making scenarios. Now we are to analyze the methods used to achieve these results. Sometimes a method is very general and can be applied outside Twitter platform. For example, we have seen papers performed sentiment extraction on filtered tweets and related them to a real-world phenomenon such as product sales or stock price changes. This method can work as long as there are some related texts on which text sentiment analysis can be performed. However, in some papers, exclusive Twitter features such as follower and mention are taken into account, and thus cannot be easily applied in another platform. To understand the methods and their applicability, we analyze four aspects in each method, including problem formulation, solution, Twitter feature used, and information acquisition and transformation. A summary of these four aspects in each of the 28 examined paper is showed in Table In different application area where Twitter data is used, it is required to formulate a problem which can be solved with computational means. This problem is an abstract from the surface application. It means that different application may have a same problem formulation. For example, sales prediction and epidemic tracking can be both formulated as a correlation analysis problem , 55. On One of the fundamental problems in computational intelligence is to predict a value. This value can be the stock price next day, the number of infections in an area where clinical data is absent, or the number of crimes of a neighborhood in the future. This is done usually using regression techniques in combination with a number of predictors. The regression techniques used in the examined papers include linear regression , random In computational intelligence settings, classification outputs a prediction that is one in many categories. If the total number of categories is two, then it is a binary classification problem. This is a very common problem especially in decision making. It gives a distinct suggestion on, for example, whether stock market will go up or down tomorrow, or whether there is a traffic jam in a specific street now. Similar to value prediction, the typical solution is to use a supervised classifier with a number of predictors. The classifiers used in the examined paper include logistic regression , random Sometimes instead of running value prediction, the mere correlation between tweets and a target variable can reveal the usefulness of tweets. Correlation analysis focuses more on explaining the predictiveness of tweets, instead of improving prediction accuracy. Such analysis usually relies on techniques such as Pearson’s R  and GranWhile value prediction and classification try to provide a likely outcome, event detection tries to capture abnormalities. Sometimes also considered as anomaly detection, event detection has been applied to find unusual behaviors in data, such as sudden rises in sales number, stock price, or tweet frequency in a geographical area. By detecting events, one can correlate anomaly in tweets with that in, for example, stock price  or traffThe most used Twitter feature is tweet text, which is the main body of Twitter data. Discussions of product, stock, crime, infectious disease, and traffic, are all contained in tweet text. Thus methods proposed by most examined papers involved extracting information from tweet text. In the 28 examined papers, only two papers did not consider tweet text. In literature , crime nApart from tweet text, other features offered by Twitter have also been used. These features include time series (built after quantifying tweets over time units), location (tagged by mobile devices or provided by the user) , 49, folTwitter has support research community greatly by making acquisition of Twitter data easy and free-of-charge. The Filter APIWith Twitter APIs, one can easily collect a large number of tweets. However, unstructured data such as tweet text are difficult to be used in a computational solution. Therefore most of the examined paper made efforts to filter relevant data during acquisition, and transform Twitter data into a form convenient for computational solutions.Given a chunk of tweets, in many applications, one needs to first extract a subset of relevant tweets. This is called tweet filtering. For example, to relate tweets to stock market, it is necessary to filter tweets that discuss stocks, which usually contain stock symbols . SimilarAnother popular transformation is sentiment extraction. This is an established task in natural language processing (NLP) that assigns a sentiment score to a piece of text. The sentiment score can be bi-polar , or involving multiple sentiment dimensions. Similar to tweet filtering, the common methods in sentiment extraction include keyword-based method and supervised machine learning. The former makes use of a sentiment word dictionary to assign score to texts , while tSometimes tweet filtering is not performed because it is difficult to know beforehand which tweets are related to the problem. For example, in some Twitter-based crime analysis, all tweets are considered at first, and some words that are particularly relevant to crime can be extracted later . Also inMore advanced transformation techniques include part-of-speech (POS) tagging, bag-of-words (BOW), and word embedding. POS tagging is mainly used to select certain words in the text, such as nouns or adjectives . BOW andIn the above we discussed problems, solving techniques, Twitter features used, and transformation. We found that some methods have more strengths in certain setting combinations than others. For example, when solving classification problem with BOW text representation, many works used random forest classifier, which in some reports achieved best accuracy , compariWe also see that when doing correlation analysis of tweets and a real-world phenomenon , the common choice is Pearson’s correlation coefficient, although linear regression is also used. However, when the number of data samples is large, the significance value in such analysis may not reflect the real correlation in the data. A special case of this problem is spurious correlation, which means a correlation was found between two variables but this is due to mere co-incident, and there is actually no relation between these two variables. The number of variables is usually large when Twitter data is considered, and avoiding spurious correlation can actually be quite challenging.For the problem of event detection, we see two types of solving techniques. The first is supervised learning, with techniques such as SVM and naive Bayes. The second is unsupervised learning such as LDA and k-means. The advantages of unsupervised learning include removing the necessity of manual annotations, and the ability to discover unexpected new events. The disadvantage, on the other hand, is that the results from unsupervised methods are harder to interpret for human reader. We acknowledge that the unsupervised method may be a better future direction for solving event detection problem, as it has higher generality and applicability, although the interpretation issue remains a challenging problem.In this review, we analyzed 28 papers that proposed methods for using Twitter data in decision making scenarios. We first discussed the findings, which showed that decision making performances with the help of Twitter data have further improved in the past five or six years. For instances, the prediction of stock price movement has become more accurate, and the predictive policing has become more effective. Then we offered a methodology analysis, which showed whether methods proposed in these works could be applied to different problems and new platforms. And it became clear that, a set of techniques, such as tweet filtering, sentiment extraction, and correlation analysis, could be easily applied to different problems and platforms to establish links between public conversations and real-world phenomena.However, we also see that current explorations still remain in a shallow level. For instances, the majority of works use Twitter as the sole data, with only five works out of 28 added Twitter data to existing data, and four works use Twitter data together with other data sources. There is much space to explore in multi-source data integration. With regard to techniques, only four works out of the 28 made use of the deep neural network, the latest and rapidly growing trend in computational intelligence. Moreover, only three works out of the 28 considered user and interaction features, which should contain rich information but often overlooked. New techniques such as graph mining can potentially leverage such information, but we have yet to find works that use such techniques. We can see, after analyzing the methodologies, that there is still very large space to advance Twitter-aided decision making, both in features considered, and in techniques used. We hope, by offering this review, we have made a stimulation, and provided a ground on which future researches in this topic can be built.