The present concept demonstrates that the highly efficient COF-based ECL luminophores can be precisely designed, providing a promising direction toward COF-based ECL phosphors.Electrochemiluminescence (ECL) plays a key role in analysis and sensing because of its high sensitivity and low background. Its wide applications are however limited by a lack of highly tunable ECL luminophores. Here we develop a scalable method to design ECL emitters of covalent organic frameworks (COFs) in aqueous medium by simultaneously restricting the donor and acceptor to the COFs’ tight electron configurations and constructing high-speed charge transport networks through olefin linkages. This design allows efficient intramolecular charge transfer for strong ECL, and no exogenous poisonous co-reactants are needed. Olefin-linked donor-acceptor conjugated COFs, systematically synthesized by combining non-ECL active monomers with C Electrochemiluminescence (ECL) plays a key role in analysis and sensing but its application is limited by a lack of highly tunable luminophores. Here, the authors demonstrate the design of high efficient ECL luminophores of covalent organic frameworks (COFs) in aqueous media by simultaneously restricting the donor and acceptor to the COFs’ electron configurations and constructing charge transport networks through olefin linkages. ECL is excited by electrochemical reactions rather than by photons, endowing this technique with unique advantages of low background and high sensitivity, high selectivity, fast response, and spatial and temporal control of luminescence5. Thus, ECL has been extensively explored in analytical fields of environment, clinical, medicine, and biology10.As the first discovery of electrochemiluminescence (ECL), ~100 years ago, this discipline has been developed from an academic curiosity to a powerful electroanalytical toolECL) of the phosphors in aqueous media. The quantum efficiency of widely used inorganic phosphors such as [Ru(bpy)3]2+ and Ir(ppy)3 is difficult to be improved since their structures can hardly be modified. Although the organic phosphors  are modifiable, they function only in aprotic solvents, as their ECL is significantly quenched in aqueous media by water and oxygen via either disabling reductive/oxidative ECL precursors or quenching the formed excited states1. In addition, practical application of these phosphors in the construction of ECL-based sensors requires the utilization of noxious co-reactants ) to amplify the signals owing to the low quantum efficiency of the existed phosphors18. Thus, the development of water-compatible ECL phosphors with high quantum efficiency is urgent for wider applications. However, the reasonable design and synthesis are limited owing to the lack of an integrated tailor-made material platform.However, the wide application of ECL is limited by the low quantum efficiency , showing relatively weak π-electronic connectivity on the skeleton33. Besides, the intramolecular rotation induced by π-π stacking and the non-radiative relaxation caused by self-quenching become the main obstacles for luminescent COFs35. Recently, Luo’s group constructed the ECL-COFs with aggregation-induced (AI)-ECL monomers, where ECL was observed using H2O2 as a co-reactant and Co3O4 as an amplification element2. Similarly, Yuan’s group observed ECL of COF nanosheet with co-reactant S2O82− and co-reaction accelerator Bu4NPF6, which topologically linked the aggregation-caused quenching ECL luminophores (pyrene) and AIE groups8. However, their COFs only had very low ECL signals. To date, the highly efficient ECL of COFs constructed by non-ECL monomers has not yet been reported. In addition, systematic induction and methodology research on COF-based ECL is lacking.Covalent organic frameworks (COFs), an emerging class of bottom–up crystalline porous polymers, are connected by covalent bonds of molecular building units with pre-designed geometric structures2v symmetrical monomer 2,4,6-trimethylpyridine-3,5-dicarbonitrile (DCTP) and C3v symmetrical monomer 2,4,6-trimethyl-1,3,5-triazine (TMT) as two key acceptors subunits. Different linear and trigonal aldehyde monomers were used to ensure the generalizability of this concept. On the basis of ECL performance of the synthesized COFs, we summarized a scalable topological design protocol for efficient ECL-COFs in aqueous media with internal dissolved oxygen acting as a co-reactant , 4,4”-p-terphenyldicarboxaldehyde (TDA), and 4-[2-(4-formylphenyl)ethynyl]benzaldehyde (EDA) can be regarded as linear monomers. 4-phenyl]phenyl]benzoic acid (DAFB), 4,4,4-)tribenzaldehyde (BTTA), 1,3,5-tris(4-formylphenyl)benzene (TFPB), tris(4-formylphenyl)amine (TPA), and 2,4,6-tris(4-formylphenyl)-1,3,5-triazine (TFPT) are representative trigonal monomers.To activate the ECL responses, COFs are constructed with various combinations of building blocks Fig. . For a hPM space group, the BDA-DCTP, TDA-DCTP, and EDA-DCTP series COFs are in P2/M space group, the TPA-TMT, TFPT-TMT, TFPB-TMT, and BTTA-TMT series COFs are in P6 space group, and the BDA-TMT, TDA-TMT, and EDA-TMT are in P6/M space group  and trifluoroacetic acid (for TMT-COFs) as catalysts, respectively. The characterizations of all COFs and model compounds are described in the 39. However, the donor and acceptor molecules always tend to pile up with each other rather than to separately form the bicontinuous arrays, in which charge carriers are captured and easily annihilated by rapid binding39. The periodically stacked 2D-COFs can solve this predicament of D-A-disordered arrangement and provide an independent route to allow the conduction of electrons and holes38. However, it is challenging to arrange the D and A molecules to co-crystallize and stack against each other to form periodic, D-on-D, and A-on-A columnar COF arrays37. To address these issues, we synthesized D-A conjugated olefin-linked COFs with spatially separated D-A structures and extended π-conjugated skeletons to demonstrate the tunability of the ECL of the resulted COFs.As the charge dynamics in devices can be easily controlled, the D-A heterojunction is currently a key structure including transistors, light-emitting diodes, and photovoltaic technologies1 molecular orbitals is reduced simultaneously , with stronger electron-supplying abilities, possess smaller bandgaps of 1.82 eV, 2.08 eV and emerge ~1.4 times stronger ECL intensities than those of TFPB-COFs   Fig. . For furcur Fig. , and thucur Fig. . The the44. In this context, it is necessary to choose one beneficial linkage for preventing linkage-originated ECL quenching. Under the same experimental conditions, the two olefin-linked COFs  reveal much stronger ECL signals than the imine-linked COF (DAFB-Pa), which is also true for the three COFs of the BTTA-COFs series , the ECL strength is also significantly enhanced by 2.1 (BDA-DCTP/TDA-DCTP) and 5.2 times (BDA-TMT/TDA-TMT), respectively, when the linear monomer BDA is extended into longer TDA .47. Therefore, the migration of holes and electrons through the skeletons is crucial in luminescent molecules44. The benzene rings in building blocks connected by C–C single bond would rotate freely, which partially undermines the charge dislocation in COFs. Alkyne bonds have been proved to be fine connectors of photoactive molecular conductors owing to their highly conjugated structures and inherent abilities to modulate electronic structures and suppressing charge recombinations50, although the influence of alkynes on ECL properties of alkynes is rarely reported. Thus, we studied the ECL performance of acetylene-induced D-A COFs.The ECL generated by conjugated organic molecules essentially originates from the charge recombination of the electron excitation systems that emit light upon relaxation2, alkynyl shortens the distance between dissolved oxygen and COFs, which is favorable to promote the oxidation reaction on the electrode surface52.Using the improved reaction conditions, a series of COFs were synthesized with BTTA/EDA as donors and DCTP/TMT as receptors. The calculated HOMO-LUMO distribution profiles show that the acetylene moieties have a significant contribution to the decreased LUMO levels by ~0.1 eV Fig. . Compare2. Hence, the acetylene functionalized COFs show significant improvement in ECL strength.Our results prove that introducing acetylene is a practical approach to enhance the ECL performance of the D-A COFs, based on the roles of acetylene in regulating the electronic structures and promoting the charge separation in the conjugated structure, as well as the special ability of adsorbing O3, the prepared 10 μL 1 mM DAFB-DCTP/DMF solution was covered on the surface of a cleaned glassy carbon electrode (GCE) and dried at room temperature to complete the assembly of the COF on the electrode surface.To clarify the mechanism of electrochemistry and ECL of the COFs, DAFB-DCTP was selected as a representative of ECL-COFs in this experiment, and a series of control experiments were carried out. Considering the advantages of solid-state ECL in reducing the non-radiative relaxation and enhancing the electrocatalytic effect+), and the electrochemical and ECL behaviors of COFs were studied  and the aqueous solution (PBS with 0.1 M KCl) as shown in Supplementary Fig. 53. Coupled with these advantages on flexibility and diversity, COFs are endowed with great potentials for application in life-related ECL systems.After cathodic and anodic potential optimization, the strongest ECL signal was obtained by cyclic voltammetry (CV) within a potential window from −3 V to 0 V  of only 1.5%) after third scans Fig. . The inc54. The relative band position of DAFB-DCTP was estimated by Mott-Schottky (M-S) analysis to further investigate the electron structure. The positive slope of the M-S plots indicates that DAFB-DCTP is an n-type semiconductor  of DAFB-DCTP can be fitted to be −1.16 V vs RHE from the x-intercept of the linear region of the M-S diagram. Considering that the conduction band minimum of n-type semiconductors is slightly negative than its flat-band potential (~0.2 eV)57 and combined with the optical bandgap measured by the Kubelka–Munk function on the basis of UV-vis spectrum, the valence band potential (EVB) and conduction band potential (ECB) were summarized in the inset of Supplementary Fig. As shown in Fig. 2-saturated PBS. The ECL is enhanced by 1.2 times in O2-saturated solution, suggesting that the dissolved oxygen plays a synergistic role. We then used 0.1 mM ascorbic acid (AA) and isopropanol (IS) as the scavengers for superoxide anion radical (O2•−)58 and hydroxyl radical (OH•)4 respectively, and found that AA significantly inhibits the ECL, while IS almost has no effect. These control experiments unravel that the ECL reaction proceeds via a radical pathway, which is also confirmed by electron paramagnetic resonance measurement showing the formation of a huge number of O2•− within COFs in the ECL process, but no OH• ..2 is fir2O2, S2O82− or TPrA is needed. The research on the ECL mechanism of COF is still blank to date, as a supplement to the ECL basic research, it may provide a feasible method and reference mechanism for activating the ECL response of non-ECL molecules.Hereby, the ECL from fully conjugated D-A COFs, generating ECL based on the collision between the electron-donors and the acceptors show the much stronger ECL, slightly dissolved oxygen are borrowed to act as a co-reactant and no highly oxidizing co-reactants like H33. Only a handful of COFs has been reported the ECL phenomenon to date8. For them, even if the typical ECL groups (pyrene) were linked to form the main part of the COF framework, the observable ECL signal requires not only the highly oxidizing co-reactants (H2O2/S2O82−), but the assistance of nano-enzymes Co3O4 or accelerator Bu4NPF68. The tedious steps would greatly limit the versatility of practical applications. As for COFs per se, no distinct ECL signal was obtained. Although the design of these COFs is still limited to the improvement of the existing luminophores, these works did prove that COFs are the most anticipated luminescent material.Although COFs possess attractive designability, the inherent properties of dynamic covalent bonds generally lead to weak π-electron communication, which limits the electron transfer between componentst index and interlayer separation, and introducing the alkynyl to COF skeleton can obtain a stronger ECL signal by enhancing the conjugation degree and shortening the distance to dissolved oxygen. (5) The ECL intensity of DAFB-DCTP is superior among all the organic phosphors reported to date  In contrast to the olefin-linked COFs, other imine-linked COFs , only showed dim signals, which highlights the contribution from olefin linkages. (2) The two model compounds (MC-1 and MC-2) showed negligible ECL intensity, which verifies the importance of the extended π-conjugated framework. (3) Compared with fully conjugated D-A COFs, another olefin-linked COFs (TFPT-DCTP and TFPT-TMT) with no D-A relationship, showed no ECL signal, demonstrating that ECL indeed originates from the ICTs between the D-A building blocks. (4) As two promising enhancing approaches, increasing the chain length of the donor subunits can get narrower optical bandgaps and stronger ECL signals due to the increased Last but not least, the generality of the standpoints raised in this research is emphasized by changing the receptor and symmetry. The previous work relied on existing ECL research and only considered how to weave the phosphors into the COF skeleton. In contrast, this reference is absent for our general approach to ECL-COFs here: model compounds and monomers hardly showed ECL performance, while there is a significant difference when D-A building blocks extending in the olefin-linked crystal framework. This approach is more controllable and more abundant than AIE because the crystal structure can be designed precisely with a stable stroke. Accordingly, this contribution will provide insight for the exploration of organic emitters from small molecules or oligomers to crystalline extended frameworks.A general topology design strategy of COFs was reported as a family of ECL luminophores. We achieved the ICT to initiate ECL via restriction of the donors and acceptors to the COFs’ tight electron configurations and construction of the high-speed charge transport network through the olefin linkages. Thus, the assembled COFs exhibit the cathodic ECL in an aqueous medium using dissolved oxygen as an amplifier without adding external toxic co-reactants. Our results show that increasing the chain length and using the alkyne bonds can effectively decline the non-radiative damping of the excited states further enhance the molecular ECL strength, which may inspire researchers to customize the COFs suitable for designed applications. With the help of density functional theory (DFT) calculations and control experiments, a mechanism of COFs structure-related ECL has been suggested that the ECL of COFs is attributed to the bandgap mechanism through the free radical pathway. Providing a powerful platform for tailor-made sensor analysis, the COF-ECL system here is very suitable for dealing with various types of environmental pollution, drug analysis, and life monitoring. Furthermore, the present concept could be promising to design more efficient COFs based luminophores by combining machine learning to run high-throughput virtual screening, hopefully opening up broader applications of COF materials and ECL technology.p-terphenyldicarboxaldehyde (TDA), 4-[2-(4-formylphenyl)ethynyl]benzaldehyde (EDA), tris(4-formylphenyl)amine (TPA), and p-phenylenediamine (Pa) were purchased from Jilin Chinese Academy of Sciences-Yanshen Technology Co., Ltd. Tetrahydrofuran (THF), 1,4-dioxane, mesitylene, benzaldehyde, NaOH, KOH, EtONa, N,N-dimethylformamide (DMF), 2,2,6,6-tetramethylpiperidine (TEMP), ethanol, methanol, CH2Cl2, acetone, and n-BuOH were purchased from Sinopharm Chemical Reagent Co., Ltd. Ultrapure water was prepared from the Millipore system (18.25 MΩ-cm). All the electrochemical measurement used phosphate buffered  containing mixture solutions of 0.1 M KCl, 0.1 M K2HPO4, and 0.1 M KH2PO4 as the electrolyte.Piperidine, DBU, trifluoroacetic acid and acetonitrile (ACN) were purchased from Energy Chemical Technology (Shanghai) Co., Ltd. 2,4,6-Trimethyl-1,3,5-triazine (TMT), 2,4,6-trimethylpyridine-3,5-dicarbonitrile (DCTP), 2,4,6-tris(4-formylphenyl)-1,3,5-triazine (TFPT), 1,3,5-tris(4-formylphenyl)benzene (TFPB), 4-phenyl]phenyl]benzoic acid (DAFB), 4,4,4-)tribenzaldehyde (BTTA), 4,4’-biphenyldicarboxaldehyde (BDA), 4,4”-2Cl2, and THF, respectively. It was then Soxhlet extracted in CH2Cl2 and THF for 24 h and dried under vacuum at 60 °C for 12 h to afford DAFB-DCTP , BTTA-DCTP , TDA-DCTP , EDA-DCTP , BDA-DCTP , TPA-DCTP , TFPB-DCTP , and TFPT-DCTP , respectively.DCTP , anhydrous DMF (4.0 mL), and piperidine  were added separately with 8 different aldehydes in a 20 mL Pyrex tube. The amount of aldehydes were taken as DAFB , BTTA , TDA , EDA , BDA , TPA , TFPB , and TFPT , respectively. The mixture was degassed by three freeze–pump–thaw cycles, sealed under vacuum, stirred for 10 min, and heated in a 180 °C oil bath for 3 days. The reaction mixture was cooled to room temperature, and a yellow precipitate was collected by centrifugation, washed several times with methanol, CH−1 NH4OH solution in aqueous methanol (50 wt%), and then washed with methanol in a Soxhlet extractor for 12 h and dried under vacuum at 60 °C for 12 h to afford DAFB-TMT , BTTA-TMT , TDA-TMT , EDA-TMT , BDA-TMT , TPA-TMT , TFPB-TMT , and TFPT-TMT , respectively.TMT , 0.9 mL mesitylene, 0.9 mL 1,4-dioxane, 0.4 mL trifluoroacetic acid, and 0.05 mL ACN were added separately with eight different aldehydes in a 20 mL Pyrex tube. The amount of aldehydes were taken as DAFB , BTTA , TDA , EDA , BDA , TPA , TFPB , and TFPT , respectively. The mixture was degassed by three freeze–pump–thaw cycles, sealed under vacuum, stirred for 10 min, and heated in a 150 °C oil bath for 3 days. The reaction mixture was cooled to room temperature, and the solid was collected by centrifugation, washed with acetone and methanol, neutralized by 0.1 mol L3:H2O, anhydrous ethanol, and ultrapure water at 40% power for 1 min, respectively, and dried in the nitrogen flow. The prepared COFs/DMF solution  was covered on the surface of GCE and dried at room temperature for characterization and further use.The GCE scrubbed by soaked filter paper was polished with 1.0 μm, 0.3 μm, and 0.05 μm alumina powder slurry on chamois leather until showing a mirror surface. Subsequently, the electrodes were ultrasonic in 1:1 HNO−1, as well as the potential scan was from 0 to −3.0 V in the detection process. The cyclic voltammograms were measured at a sweep rate at 100 mV s−1 and with voltage sweeps between 0 and −3.0 V. The ECL and CV were conducted at room temperature in a classical three-electrode configuration consisting of a modified carbon glassy electrode working electrode, a platinum counter electrode, and an Ag/AgCl reference electrode.The ECL measurements were carried out on an MPI-E multifunctional electrochemical analytical system  with the voltage of the photomultiplier tube at 800 V and the scan rate at 100 mV sECL) of the COFs was obtained by the following equation, and [Ru(bpy)3]2+ was used as a reference59.° is the ECL efficiency of the [Ru(bpy)3]2+ system, I and I° represent the ECL intensities of the COFs and [Ru(bpy)3]2+, respectively. Qf and Q°f correspond to the respective faradaic charge passed for the COFs and [Ru(bpy)3]2+. The I° and Q°f for 1 mM [Ru(bpy)3]2+ was measured in ACN containing 0.1 M TBAP at 23 °C. While I and Qf for COFs, the procedures were the same as the ECL experimental described above.ECL efficiency . In order to calculate accurately, the DFT-D3 dispersion correction was used, and the excited-state calculation was calculated by TD-B3LYP method. The properties of excited states were analyzed by Multiwfn.Supplementary Information


Cdk5 is a proline-directed serine/threonine protein kinase that governs a variety of cellular processes in neurons, the dysregulation of which compromises normal brain function. The mechanisms underlying the modulation of Cdk5, its modes of action, and its effects on the nervous system have been a great focus in the field for nearly three decades. In this review, we provide an overview of the discovery and regulation of Cdk5, highlighting recent findings revealing its role in neuronal/synaptic functions, circadian clocks, DNA damage, cell cycle reentry, mitochondrial dysfunction, as well as its non-neuronal functions under physiological and pathological conditions. Moreover, we discuss evidence underscoring aberrant Cdk5 activity as a common theme observed in many neurodegenerative diseases. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase. Thirty years ago, Cdk5 was first discovered on the basis of its close sequence homology to the human cell division cycle protein 2 , a regulator of cell cycle progression . The kinUnlike other Cdks, Cdk5 activity is most apparent in adult mouse brain [Cdk5r1, the gene encoding p35, shows marked expression predominately in the neuronal linage [The Cdk family comprises 21 members, most of which depend on the association with specific cyclin partners to become constitutively active protein kinases . Surprisl linage  Another Cdk5 regulatory subunit, p39, was later discovered in part by its high degree of sequence identity to p35 . While pSubcellular fractionation assays reveal the enrichment of p35 and p39 proteins in membrane-bound fractions of cultured neurons , 19. TheWhile binding to a regulatory subunit is obligatory for Cdk5 activation, its activity can be further modulated by a variety of post-translational modifications. The phosphorylation of Cdk5 at two different residues within the ATP-binding sites leads to opposing effects. Phosphorylation at Tyr 15 is stimulatory but inhibitory at Thr 14 , 23. MorThere has been rapid progress in our understanding of Cdk5 function in the nervous system.. In this review, we focus on the recent findings of Cdk5, particularly its roles in neuronal/synaptic functions and circadian clock regulation under physiological conditions, as well as its pathological links to DNA damage, cell cycle reentry, mitochondrial dysfunction, and oxidative stress. The Cdk5 substrates discussed in this review and their functional categories are listed in Table Prior review articles on Cdk5 have provided a comprehensive coverage of its roles in neuronal migration, neurite outgrowth, axonal guidance, and synaptic plasticityGene-targeting studies demonstrate a role for Cdk5 in brain development, particularly in neuronal migration –40. Cdk5Drosophila model [On the basis of its unique expression and activity pattern in the developing mouse brain, Cdk5 is believed to have roles in neurogenesis. Indeed, Cdk5 is essential for neurite growth and axonal formation. Cdk5 and p35 show co-localization with actin filaments in axonal growth cones, and inactivation of Cdk5 in cultured neurons inhibits neurite outgrowth . Consistla model , and p35la model . In contla model . Cdk5 cala model , whereasla model . Cdk5 alla model . Recent la model . Cdk5 cala model , as wellla model , a microla model , a scaff2+ influx. Phosphorylation of Munc-18 by Cdk5 results in the dissociation of Munc-18 from Syntaxin 1A, facilitating synaptic fusion and release [2+ influx into the pre-synaptic cytoplasm via voltage-dependent Ca2+ channels (VDCCs), which increases the probability of channel opening and facilitates the release of neurotransmitters [Impaired long-term depression and depotentiation of long-term potentiation have been shown in p35-deficient mice , implyin release . Cdk5 al release , 62, twosmitters , 64.Change in the numbers or properties of post-synaptic receptors is one of the mechanisms for modulating synaptic plasticity at the post-synapses. Cdk5 phosphorylates the NMDA receptor subunit NR2A at Ser1232, thereby enhancing NMDA receptor function , 66. PSDSynaptic homeostasis is a compensatory process that allows neurons to adapt to altered levels of network activity. Neurons potentiate synaptic efficacy when inputs are dampened and downmodulate their firings when inputs are heightened . Thus, hAt the postsynaptic levels, Cdk5 has been implicated in depressing synaptic strength following heightened neuronal activity . Upon inPer1/2 and Cry1/2. Newly synthesized PER and CRY proteins heterodimerize, translocate into the nucleus, and inhibit CLOCK/BMAL1 activity through direct binding, resulting in the subsequent repression of downstream target genes [Circadian clocks are oscillators that synchronize daily cycles of behavior and physiology. The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian pacemaker in mammals and entrains the peripheral clocks across the body . Circadiet genes . Thus, cet genes .Per1 and Per2, is disturbed in p35 heterozygous knockout mice [Cdk5 has been implicated in the regulation of circadian clocks , 80. Theout mice . Collect98/Ala99 sequence and generates p25 in a calcium-dependent manner. Accordingly, increasing intracellular calcium level stimulates p25 generation, whereas removing calcium prevents p25 accumulation [Aberrant Cdk5 activity caused by p25 accumulation contributes to the pathogenesis of various neurodegenerative diseases . p25 is mulation .p25 causes constitutive activation and mislocalization of Cdk5. p25 activates Cdk5 through direct binding, and p25 has an approximately 5- to 10-fold longer protein half-life compared to p35 , therebyDysregulation of Cdk5 activity leads to neurotoxicity and neurodegeneration . CultureCK-p25 mice are one such p25 overexpression model. CK-p25 mice overexpress an inducible human p25 under the control of a forebrain-specific CamKII promoter, which is expressed predominantly in excitatory pyramidal neurons. The expression of p25 is induced upon the removal of the tetracycline derivative doxycycline from the animal diet . FollowiDrosophila, dysregulated Cdk5 disrupts autophagy and augments the expression of anti-microbial peptides. Anti-microbial peptides cause the hyperactivation of innate immune response and are linked to dopaminergic neuronal death in Drosophila [Aberrant Cdk5 activity is also found to trigger neuroinflammation. Neurons overexpressing p25 markedly produce and secrete a soluble lipid known as lysophosphatidylcholine, which activates glia and induces the expression of cytokines and chemokines . Prolongosophila . In summ42 peptide to cultured neurons leads to p25 generation and neuronal death [AD is the leading cause of senile dementia, which is pathologically characterized by the accumulation of amyloid plaques and neurofibrillary tangles (NFTs) . Severalal death , and accal death , 95. A pal death , 97. HerDysregulation of Cdk5 activity promotes amyloid plaque deposition by promoting amyloidogenic APP processing. The amyloidogenic APP pathway includes sequential cleavage by β- and γ-secretases, which ultimately produce a secreted form of APP (sAPPβ), C-terminal fragments (CTF99 and CTF89) and Aβ peptides . Aβ peptp35KI mice [p35KI mice, a mutant p35 resistant to calpain cleavage was designed by substituting an alaine [93NLSTF98) adjacent to the calpain-cleavage site [p35KI mice rescues AD pathology in 5XFAD, Δp35KI mice. Compared to 5XFAD mice, the 5XFAD, Δp35KI mice display a reduction in soluble Aβ peptide levels, plaque deposition, and inflammatory cytokine expression. Furthermore, the 5XFAD, Δp35KI mice show an improvement in synaptic plasticity and memory function relative to the 5XFAD mice [A previous report established a knock-in mouse model deficient in p25 generation named Δ5KI mice . In Δp35n alaine  residue age site . 5XFAD mFAD mice . CollectTau is a microtubule-associated protein mainly distributed in axons. Tau stabilizes neuronal microtubules and regulates microtubule dynamics involved in axonal outgrowth and transport . TauopatCdk5 is a tau protein kinase: Cdk5 co-purifies with tau and phosphorylates tau in vitro . OverexpMAPT gene (the gene encoding tau) are associated with FTD and Parkinsonism linked to chromosome 17 (FTDP-17) [p35KI mice reduces tau phosphorylation at residues T181 and S202 [p35KI compound mice display a reduction in tau seeding activity [Mutations in the FTDP-17) . ImportaFTDP-17) . IncreasFTDP-17) , 110. Cractivity .p35 in these lines using CRISPR/Cas9 genome-editing [p35KI) attenuates tau phosphorylation and increases the expression of synaptophysin [A previous report utilized FTD patient-derived iPSCs that harbor the tau P301L mutation, and generated isogenic lines in which the tau leucine mutation was reverted to proline, as well as targeted knock-in Δ-editing . Comparetophysin . TogetheIncreased DNA damage and cell cycle reentry have been observed in AD human brains . Most stDNA DSBs in CK-p25 mice may be critically linked to the inhibition of histone deacetylase 1 (HDAC1) , a membee.g., etoposide, methotrexate, and homocysteine) to cultured neurons increases DNA damage and Cdc25a levels, a marker of S-phase entry and DNA replication [42 peptides to cultured neurons also induces DNA damage and cell cycle reentry [Post-mitotic neurons normally do not exhibit cell cycle activity. However, re-expression of cell cycle-related proteins including Cyclin B and proliferating cell nuclear antigen (PCNA) has been documented in hippocampal pyramidal neurons of post-mortem AD brains . Fluoreslication . Importa reentry , suggestCdk5 dysfunction has been linked to cell cycle reentry. Several cell-cycle markers are aberrantly upregulated in p25-expressing neurons in the CK-p25 mouse model . MoreoveMitochondria utilize oxidative phosphorylation to produce ATP and adapt the metabolic needs of cells by fusion and fission. Mitochondrial fusion joins mitochondria together, while fission separates one mitochondrion into two or more , 119. MiDynamin-related protein 1 (Drp1) is a GTPase that regulates mitochondrial fission. Drp1 is recruited from the cytosol to the mitochondrial outer membrane (MOM), where it assembles into ring-like structures that wrap around the MOM and incise the membrane following GTP hydrolysis . In pathC. elegans after exposure to paraquat [Parkinson’s’ disease (PD) is a chronic movement disorder characterized by Lewy body formation, mitochondrial dysfunction, and loss of dopaminergic neurons in the substantia nigra . Compareparaquat .Several reports link Cdk5 hyperactivity to oxidative stress in PD. Reactive oxygen species (ROS) including hydrogen peroxide are byproducts of normal mitochondrial metabolism, and elevated levels of ROS damage lipids, proteins, and DNA, impeding a wide array of cellular processes . A largeOther pathways have been suggested for Cdk5 hyperactivity in PD pathogenesis involving mitochondrial defects and Parkin dysfunction. In a non-human primate model of PD, aberrant Cdk5 is proposed to phosphorylate Drp1 at S616, which accelerates mitochondrial dysfunction and neurotoxicity . Cdk5 alsuperoxide dismutase type 1 (SOD1) gene [G93A mouse model of ALS, along with the hyperphosphorylation of tau and neurofilament (NF) [G37R mice that exhibit different disease severity, Cdk5 activity correlates with lethality [G93A mice [Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results in selective loss of motor neurons in the spinal cord, brainstem and cerebral cortex. Patients with ALS suffer from severe motor deficits and paralysis, which lead to death within years after disease onset . Marked D1) gene . Moreoveent (NF) , 141. Inethality . ConversIn Huntington’s disease (HD), expansion of a polyglutamine domain of the huntingtin (Htt) protein leads to Htt aggregation and selective loss of medium spiny neurons in the striatum . Brain eInfection with the human immunodeficiency virus (HIV) compromises the immune system and causes a number of physiological disruptions, including cognitive impairment . ElevateRecent reports strengthen the link between Cdk5 hyperactivation and diabetes-associated neurodegeneration. Diabetes mellitus is a prevalent metabolic disorder characterized by hyperglycemia, which results in insulin resistance and insufficient insulin secretion due to the failure of β-pancreatic cells . High glInterestingly, there is an emerging role for non-neuronal Cdk5 in insulin secretion and insulin sensitivity. Upon glucose stimulation, salt inducible kinase 2 (SIK2) phosphorylates p35 at Ser 91 residue, which promotes p35 protein degradation via the ubiquitin-proteosome pathway  and thusOn the other hand, Cdk5 modulates insulin sensitivity through peroxisome proliferator-activated receptor γ (PPARγ) in adipocytes. PPARγ is a ligand-activated transcription factor that belongs to nuclear receptor superfamily . PPARγs 50 of roscovitine for Cdk5/p25 complex is 0.16 μM, whereas a higher concentration is needed to reach the same inhibition on other Cdks [Modulating the aberrant activity of Cdk5 has attracted attention as a therapeutic target for neurodegenerative diseases. Multiple synthetic inhibitors of Cdk5 have been discovered, and most of them  are purine derivates that share the basic ring structure of ATP . Mechaniher Cdks . Despiteher Cdks , 150. IdPeptide inhibitors are a promising approach for targeting Cdk5. Inhibitory peptides are typically derived from sequences of native proteins mediating protein–protein interactions, which contains a small number of key residues. Inhibitory peptides act as dominant-negative forms of the endogenous proteins, and have a greater efficacy and specificity than synthetic inhibitors .G37R mouse model of ALS improves motor deficits and delays neurological pathology [245–Ala277, inhibits Cdk5 comparably to CIP [42-treated cultured neurons, overexpression of P5 inhibits Cdk5 aberrant activity, phosphorylation of tau, and neuronal death [CIP is the first identified peptide inhibitor of Cdk5. CIP is a 126 residue-long peptide that originally derived from the C-terminal of p35 (amino residues 154 to 279), within the region that is necessary for p35 to activate Cdk5 , 166. CIathology . A 24-rey to CIP . In Aβ42al death . Importaal death –171.172. Cdk5i is a 12-amino acid peptide derived from the T-loop of Cdk5 (A148RAFGIPVRCYS159) [A recent study revealed an inhibitory peptide targeting Cdk5 named Cdk5iDespite the major role of Cdk5 in neurons, growing evidence suggests non-neuronal functions of Cdk5. In addition to insulin secretion and insulin sensitivity highlighted above, Cdk5’s role in other non-neuronal functions, especially its regulation in astrocyte activation, oligodendrocyte maturation/myelination, T cell activation, and cancer biology will be discussed below.Astrocytes regulate brain homeostasis and provide metabolic/structural support to neurons and synapses. Both p35 expression and Cdk5 activity increase in astrocytes injured by in vitro scratch assays, a condition that strongly activates astrocytes . ImpairmOligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes to form myelin, which ensures the rapid propagation of action potentials. Cdk5 activity increases during OPC differentiation . OverexpCdk5 regulates T cell activation. T cell receptor (TCR) stimulation induces T lymphocyte activation, which is accompanied by a rapid induction of Cdk5/p35 expression and Cdk5 activity . DisruptCdk5 and p35/p39 are expressed in clinical tumor specimens . PatientThree decades ago, Cdk5 was discovered based on its high similarity to Cdc2 and was predicted to function in cell cycle regulation. With the remarkable progress through the years, significant insights have unveiled the role of Cdk5 in normal brain function and neurodegeneration. Hyperactivation of Cdk5 appears to be a common theme among different neurodegenerative diseases, and blocking Cdk5 hyperactivity attenuates disease progression. Single-cell transcriptomic analyses reveal the noticeable expression of Cdk5 and p35 in non-neuronal cell types, suggesting an exciting area of Cdk5 biology remains to be explored. Future studies will define more precisely how Cdk5 hyperactivation impairs brain function, will advance our understanding of Cdk5 in non-neuronal cell types, and will generate fundamentally novel therapeutic opportunities aimed at reining in Cdk5 aberrant activity implicated in many neurological disorders.


In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg Caliciviridae family of which seven infect animals including noroviruses. Noroviruses are further divided into 10 genogroups (GI-GX) that are further subdivided into 49 genotypes: 9 GI, 27 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI and 1 genotype each for GVII, GVIII, GIX (formerly GII.15) and GX [There are 11 genera in the Efforts to make effective norovirus vaccines have been thwarted by our lack of understanding of the structural mechanisms of viral escape from antibody neutralization. In addition, noroviruses are constantly evolving and frequently generating new strains ,5,6 thatCaliciviruses are T = 3 icosahedral particles  with 1802+ and Ca2+ concentrations cause the P domains to rotate ~90° and contract onto the viral shell. Within the P domain, these conditions make the loops at the tip of the P domain convert to the ‘closed’ conformation, which blocks antibody binding while enhancing receptor binding. The results reviewed here strongly suggest that MNV escapes the immune system by shapeshifting from a contracted structure in the gut that is primed for infection to an open structure in the serum that elicits the immune response.The purpose of this review is to examine recent results demonstrating that the capsid of murine norovirus, and likely all noroviruses, is a dynamic structure that responds to several environmental/physiological cues. Under conditions found in the serum, the P domain rises more than 15 Å off the surface of the shell and is highly mobile. At the tip of the P domain are loops splayed apart in an ‘open’ conformation to which neutralizing antibodies bind. This all changes in the gut where high bile concentrations, low pH, and high MgCd300lf [Using CRISPR-Cas9 technology, a gene was identified that encodes a cell-surface protein, Cd300lf ,22. CD30Cd300lf . This isCd300lf . Unlike Cd300lf  and therUsing these soluble forms of CD300lf and the P domains, Nelson et al. also directly measured the interaction affinity using surface plasmon resonance . The monMNV is a versatile system for which all the virological tools have been developed. MNV can be propagated in a cell culture system, pathogenesis and the host immune response can be examined in the murine model, large amounts of virus can be readily produced, neutralizing monoclonal antibodies have been isolated, and an infectious clone has been developed . TherefoCaliciviridae is like MNV with highly flexible P domains that do not contact the shell surface.Since this unusual ‘floating P domain’ conformation was so different from the crystal structure of NV, the cryo-EM structure of another calicivirus, rabbit hemorrhagic disease virus (RHDV), VLP was also determined . The strSince neither MNV nor RHDV infect humans, it was important to revisit the cryo-EM structure of a human norovirus. The human norovirus Vietnam026 (GII.10) at ~10 Å resolution appeared very similar to MNV and RHDV . As withAt the time of the earlier MNV-1 studies, it was not at all clear whether the P domains of the noroviruses could transition between this extended, flexible state ,30 and tIt is also possible that the flexibility of the P domain plays a different role apart from affecting cell attachment. The flexible tether between the shell and the P domains could be sensitive to proteases that would release soluble P domains. This might act as an immune ‘smoke screen’ by presenting antigenic sites not well exposed on the viral surface. Indeed, P domains have been found in stool from NV infected volunteers , suggestIn our initial studies to understand how noroviruses might use P domain flexibility to escape antibody neutralization, we determined the cryo-EM structure of MNV complexed with the Fab fragment from a neutralizing A6.2 ,26 and tNearly identical results were observed with a second neutralizing antibody, 2D3 . CompareThe relatively low-resolution cryo-EM structures of the MNV virion complexed with Fab fragments  left sevAs was predicted from the previous pseudo-atomic structure of the MNV/A6.2 complex ,26, the The structure of the MNV virion/CD300lf complex showed the general location of the receptor but was of insufficient resolution to determine the contact details or the possibility of conformational changes . Those dInterestingly, the conformations of the A’/B’ and E’/F’ loops in the receptor complex  are nearWhat is clear from these high-resolution structures is that antibodies and CD300lf bind to opposing P domain conformations C,D. In FThese structural results suggest that conditions that shift the P domain structural equilibrium towards the closed conformation would enhance receptor binding while blocking antibody binding. Indeed, this has been shown to be the case. As reviewed above, bile salts and metal ions enhance the CD300lf binding to the P domain . Bile 11, low pH2+ binds to the three acidic groups on the G’H’ loop and brings them into proximity, thereby distorting the G’H’ loop. This structure is also nearly identical to the pH 5.0 structure without bile or metals being present [What was not immediately clear from these results was how the addition of bile, low pH, and metals could all result in the same contracted, closed structure.  present . In thisThe above discussion is a model for how bile, low pH, and metals push the P domain towards the closed conformation to which the receptor binds but antibodies cannot. However, the next question is how these conditions can also cause the contraction of the P domain onto the surface of the shell ,20,37,402 of the total ~1800 Å2 dimer interface surface area [From the analysis of all structural information collected to date, this rotation/contraction seems likely governed by the movement in the C’D’ loop that, in turn, is linked to the G’H’ loop. This correlation is made evident when all the conditions and structures are compared . In the ace area , so chanViruses exist on the edge of disaster; they need to be stable enough to survive movement between cells and hosts while being sufficiently unstable to respond to environmental stimuli required for infection and genome release. For example, a number of plant viruses just need the relatively slight push of neutral pH conditions and low metal concentrations to start uncoating . WitMNV differs from these viruses in that it uses at least three cues to alter its structure in response to the changing environment: pH, bile, cationic metals. Since MNV is an enteric virus, it needs to adapt to wildly changing conditions in the gastrointestinal tract. . In thisOnce the viral infection spreads from the gut lumen to the serum, the pH is neutral, and the bile and metal concentrations drop to low levels. Under these conditions, the P domain rises off the shell and the apical loops change to the open conformation. In this way, MNV uses all the environmental cues in the gut to simultaneously protect itself from digestion and denaturation, block antibody binding, and enhance cell binding. Once the infection spreads outside the gut, the virus adopts the expanded (floating) P domain conformation with the loops at the tip of the P domain splayed out in the open conformation.It is interesting to note that all of the MNV monoclonal antibodies raised from infected mice that we have studied to date ,34,36,37These studies have shown that the calicivirus capsid is not merely a static container moving viral genome from host to host. What we have seen is that MNV has two modes of flexibility, each with different biological purposes. In the first mode, the entire P domain changes from being loosely tethered to the shell to being contracted to the shell surface. While the reversible contraction of the P domain onto the shell remains to be shown in other noroviruses, the tethered, loose association with the shell is a common feature ,27. The The most unexpected result from these studies is that the conformational changes occurring within the P domain appear to drive the contraction of the capsid and most biological functions. We have shown that bile, low pH, and metals rotate the P domains within the spike dimers to create a complementary surface between the P domain and the shell that leads to contraction onto the shell. This rotation, in turn, appears to be related to the C’D’ loop movement  that canIt is important to note that these results show that conversion of the P domain to the closed conformation and the resulting rotation of the P domains about each other drives the contraction of the P domain onto the shell rather than the other way around. As summarized in In summary, MNV has evolved a remarkable strategy to infect the host and evade the immune system. MNV enters the alimentary canal with its target being the Peyer’s patches in the gut. It uses the extreme conditions in the gut to contract into a tightly packed shell, protecting itself against protease digestion and low pH denaturation. These same conditions alter the P domain structure itself to bind better to the receptor and the target tissue. Once it replicates and escapes into the lamina propria, the neutral pH, low metal, and low bile concentration cause the P domains to rise off the shell and the apical loops spread open. There, the B cells are trained to what they see, the open conformation. Antibodies raised against this serum form of the virus are of little use in protecting the gut against infection.Perhaps similar processes are involved in human noroviruses, and these may be the reason for the relatively short-lived protection against subsequent infections . There MNV uses metabolites and environmental cues to create one shape in the gut and a completely different one to the immune system in the serum. Recent studies on SARS-CoV-2 suggest that other viruses might also use metabolites to thwart antibody binding . Specifi


P16-induced cell cycle arrest, antiapoptotic activity, and immune evasion caused by malfunction of tumor antigen processing. The arrested cell cycle in lgCMN is consistent with its phenotype and rare malignant transformation. Antiapoptotic activity and immune evasion might explain how such heterogeneous cells have avoided elimination. Major histocompatibility complex (MHC) class I-mediated tumor antigen processing was the hub pathway that was significantly downregulated in lgCMN, and ITCH, FBXW7, HECW2, and WWP1 were identified as candidate hub genes. In conclusion, our research provides new perspectives for immunotherapy and targeted therapy.Large to giant congenital melanocytic nevus (lgCMN) is a benign cutaneous tumor that develops during embryogenesis. A large number of lgCMN patients are ineligible for surgical treatment; hence, there is an urgent need to develop pharmacological treatments. Clinically, tumorigenesis and progression essentially halt after birth, resulting in the homeostasis of growth arrest and survival. Numerous studies have employed whole-genome or whole-exome sequencing to clarify the etiology of lgCMN; however, transcriptome sequencing of lgCMN is still lacking. Through comprehensive transcriptome analysis, this study elucidated the ongoing regulation and homeostasis of lgCMN and identified potential targets for treatment. Transcriptome sequencing, identification of differentially expressed genes and hub genes, protein–protein network construction, functional enrichment, pathway analysis, and gene annotations were performed in this study. Immunohistochemistry, real-time quantitative PCR, immunocytofluorescence, and cell cycle assays were employed for further validation. The results revealed several intriguing phenomena in lgCMN, including  Congenital melanocytic nevus (CMN) is a neurocutaneous benign tumor that results from abnormal proliferation and migration of melanocytes during embryogenesis. Based on the measurement of the largest projected diameter of nevus at adulthood, CMN is classified into small <1.5 cm), medium 1.5–20 cm), large 20–40 cm), and giant (>40 cm) types [.5 cm, me0 cm, lar0 cm, andSurgical resection and tissue expansion remain the mainstay of lgCMN treatment , 8. HoweNRAS and BRAF in CMN. NRAS mutations are identified in 50–80% of patients with lgCMN [BRAF mutations are frequently detected in small CMN [BRAF and NRAS somatic mutations [in vivo and in vitro, lgCMN cells are nontumorigenic with a finite lifespan [Previous studies have reported common somatic mutations in th lgCMN , 11, andmall CMN . Similarutations , 13. Howutations . Both inlifespan  and are lifespan . These fIn this study, based on the transcriptome analysis results, we propose intriguing phenomena that are seen with lgCMN: growth arrest and immune evasion. Functional enrichment was performed to determine the regulation between different pathways or processes. Finally, we unveiled the hub pathways and candidate genes that provide a framework for further research in targeted therapy and immunotherapy.We enrolled patients diagnosed with lgCMN at the Shanghai Ninth People's Hospital between 2019 and 2020. The projected adult size of the nevus was estimated to obtain an accurate diagnosis. All patients underwent surgical resection as treatment. We obtained 12 lgCMN samples and 4 lgCMN-adjacent normal skin samples for transcriptome analysis. Expression data of transcriptome is provided in Supplementary Materials For all samples in For cDNA construction, total RNA was extracted from the samples and processed using a TruSeq RNA Sample Preparation Kit , and cDNA libraries were used to generate paired-end reads. Sequencing was performed on each library to generate 100 bp paired-end reads for transcriptome sequencing on an Illumina High-Seq 2000 platform  by a commercial service provider (Sangon).P value  and Benjamini–Hochberg false discovery rate were applied to balance the discovery of significant genes and to limit of false positives. Log | FC | >2 and Q value < 0.05 were considered significant in this study.The DEGs in lgCMN tissue samples were screened using the limma package in R . The adjk‐score = 2.The PPI network was constructed using STRING . CytoscaQ value < 0.05 was considered significant.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) , 23. The2. For cell cycle assays, A375 cells, P2 NHM, and P2 lgCMN cells were prepared in phosphate-buffered saline (PBS) at a density of 5 × 105 cells/mL. The cell suspensions were pipetted into 70% ethanol on ice and kept for two hours. After washing the cells with PBS, they were stained using Cell Cycle and Apoptosis Analysis Kit  for two hours. A flow cytometer, Beckman CytoflexS , was used to evaluate the cell cycle.Normal human melanocytes (NHM) were isolated from healthy skin tissues, and lgCMN cells were isolated from patients' tumor lesions, following previously described procedures , 26. BotImmunohistochemical staining of formalin-fixed, paraffin-embedded normal skin tissues, lgCMN tissues, and lgCMN-developed melanoma tissues was performed on a Leica BOND MAX Immunostainer . The sections were pretreated using heat-mediated antigen retrieval with citrate buffer (pH 6.0) for 30 min. The sections were then incubated with Ki67 , P16 , SOX10 , CD3 , CD4 , CD8 , BCL2 , or caspase-3  for 1 h at 20–30°C and detected using an HRP-conjugated compact polymer system. AEC was used as the chromogen. The sections were then counterstained with hematoxylin.The cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% PBS-TritonX-100 for 5 min, and then blocked with 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h. The cells were then incubated overnight at 4°C with anti-Ki67 antibody /anti-SOX10 antibody  or anti-P16 antibody /anti-SOX10 antibody, followed by further incubation at 20–30°C for 1 h with goat anti-rabbit IgG H&L  and goat anti-mouse IgG H&L (Cy3 ®) preadsorbed  secondary antibodies. Nuclear DNA was labeled blue with DAPI.ΔΔCt− method. GAPDH is applied to normalize the data. The primers are listed in Supplementary Materials Real-time quantitative PCR was performed according to the MIQE guidelines . After Rt-test was used for the analysis of differences between two groups, and the ANOVA was conducted for comparisons among the groups. Data are presented as the mean ± SD. A P value < 0.05 was considered to be statistically significant.Statistical analyses were performed using GraphPad Prism 8. Student's P value < 0.05, 165 upregulated and 1397 downregulated genes were detected. This result is shown as a volcano plot  of the pathways enriched by all DEGs were also found to be enriched by the downregulated genes Figures . That imP = 3.39e − 04), RAS (P = 0.0012), MAPK (P = 0.0015), melanoma (P = 0.0023), and WNT signaling pathways (P = 0.0073), were significantly downregulated , proteoglycans in cancer (P = 5.00e − 06), and regulation of the actin cytoskeleton (P = 1.49e − 05). Several pathways that induce tumor proliferation, differentiation, and migration were significantly downregulated in lgCMN, which may explain how lgCMN retains its benign characteristics and growth arrest.Pathways in tumorigenesis, including PI3K-AKT  and its downstream NF-κB pathway (P = 0.0128) was also inhibited, causing a reduction in inflammatory factor levels and affecting the process of apoptosis and immune response. Moreover, caspase-3 (Log  | FC | = −2.80) and caspase-7 (Log | FC | = −1.56), the key effectors inducing apoptosis, levels were also downregulated.Expression of TNF signaling pathway  which plays crucial roles in growth homeostasis was significantly upregulated in lgCMN samples . Among the three components  of the pathway, MHC I-mediated antigen processing was responsible for enrichment, and it was the initiator of tumor antigen recognition and CD8+ T cell activation [Based on the functional enrichment of key modules , primarytivation . The rooITCH, FBXW7, HECW2, and WWP1 as candidate hub genes among the E3 ubiquitin ligases that participate in antigen processing based on gene annotations and reported studies. ITCH is involved in the regulation of many aspects of immune responses, including lymphoid cell differentiation, T cell activation, and the strength of the T cell receptor signal [ITCH greatly influences the antigen recognition and activation of the adaptive immune system, thus leading to the inhibition of tumor immune response [FBXW7. Dysfunctional FBXW7 is responsible for defects in antigen peptide formation and presentation of tumor development and malignancies [FBXW7 loss promotes resistance to anti-PD-1 therapy in melanoma, and the reactivation of FBXW7 could improve the response to PD-1 blockade [HECW2 and WWP1 were also considered hub genes because of their participation in neural development during the embryonic period. Melanocytes and nevus cells are derived from neural crest cells after a series of differentiation and migration processes. Remarkably, HECW2 plays a vital role in the proliferation, migration, and differentiation of neural crest cells, which may be involved in lgCMN pathogenesis [WWP1 is indispensable for the proper polarization of developing neurons, and its deregulation may cause abnormal neuronal morphology and dysfunction [ITCH, FBXW7, HECW2, and WWP1 as candidate hub genes because of their function in immune responses and melanocyte development.Next, gene annotations of the downregulated E3 coding genes were conducted to probe their potential pathogenesis. We considered r signal . The dowresponse . Anothergnancies . Additioblockade . HECW2 aogenesis . WWP1 isfunction . In summITCH, FBXW7, HECW2, and WWP1 were also downregulated , the key cell cycle inhibitor, was upregulated in both our samples and the GEO samples, and inhibitory downstream CDK4 and CDK6 activity was downregulated.Gene expression data of the 12 lgCMN samples were downloaded from the GSE8525 dataset . The rested Fig. . As the Clinically, we observed that lgCMN generally demonstrates a state of growth arrest and survival after birth. Therefore, a series of regulatory mechanisms may flourish after birth to prevent the malignant transformation of lgCMN. Although lgCMN does not progress, the abnormal nevus cells are not cleared by the immune system and survive in the tissues, and the reasons for this remain unknown.According to the results, arrested proliferation and immune evasion are the two main aspects that should be intensively explored. The arrested proliferation or migration of lgCMN cells is consistent with its phenotype and rare malignant transformation, and immune evasion explains how such heterogeneous cells have avoided elimination.P16 expression was considerably upregulated in lgCMN, whereas its inhibitory downstream CDK6, RB1, and E2F transcription factor levels were downregulated (Supplementary Materials P16-induced senescence-like cell cycle arrest of acquired nevus that prevented it from developing into melanoma [P16 deletion or its downregulation, inducing tumor malignancy and metastasis [P16-induced homeostasis of growth arrest in lgCMN, which plays a crucial role in preventing malignant transformation. In the case of the extensive expression of P16 in lgCMN, further research on targeting P16 and P16-induced senescence-like homeostasis in lgCMN cells will be enlightening.Attention should be paid to cell cycle arrest as it was discovered in both the DEGs and key module analyses Among the cyclin-dependent kinase inhibitors, melanoma . By conttastasis . Stefanatastasis  investigAntiapoptotic activity and immune suppression were the other interesting biological processes occurring in lgCMN, mainly through the downregulated TNF signaling and antigen processing pathways. TNF kills tumors directly and promotes the differentiation of T and B cells , 40. Fur+ T cells. By recognizing the antigen peptides, CD8+ T cells eliminate tumor cells and subsequently mobilize the adaptive immune system [+ T cells is blocked, resulting in immune evasion of lgCMN [ITCH, FBXW7, HECW2, and WWP1 were considered candidate hub genes in lgCMN according to gene annotations. To the best of our knowledge, immune-related studies in lgCMN have not yet been reported, and our results may inspire research on immunotherapy for lgCMN.Finally, the role of the most significantly enriched pathway in our study, MHC class I-mediated antigen processing, should be highlighted . Tumor ae system . As the e system . Howeverof lgCMN –43. AmonITCH-, FBXW7-, HECW2-, and WWP1-induced immune evasion in lgCMN. These discoveries may provide new perspectives for targeted therapy and immunotherapy.In this study, we provide the first comprehensive transcriptome analysis of lgCMN. We revealed intriguing phenomena, including P16-induced growth arrest, antiapoptotic activity, and


Mycoplasma pneumoniae has been identified as a preceding factor of Guillain–Barré–Stohl syndrome. The Guillain–Barré–Stohl syndrome is triggered by an immune reaction against the major glycolipids and it has been postulated that M. pneumoniae infection triggers this syndrome due to bacterial production of galactocerebroside. Here, we present an extensive comparison of 224 genome sequences from 104 Mycoplasma species to characterize the genetic determinants of galactocerebroside biosynthesis. Hidden Markov models were used to analyse glycosil transferases, leading to identification of a functional protein domain, termed M2000535 that appears in about a third of the studied genomes. This domain appears to be associated with a potential UDP‐glucose epimerase, which converts UDP‐glucose into UDP‐galactose, a main substrate for the biosynthesis of galactocerebroside. These findings clarify the pathogenic mechanisms underlining the triggering of Guillain–Barré–Stohl syndrome by M. pneumoniae infections.Infection by  Mycoplasma pneumoniae is a preceding factor of Guillain‐Barré‐Stohl syndrome, triggered by an autoimmune reaction against a glycolipid named galactocerebroside, produced by the bacterium. In this work, we reconstruct the pathway and identify the functional protein domain responsible for the synthesis of galactocerebroside in Mycoplasmas. Mycoplasma species are bacteria representing the smallest free‐living organisms on earth. They include several pathogens infecting human, animals and plants. Mycoplasma is characterized by the lack of a cell wall, reason for which glycolipids of the membrane are exposed to the host’s immune system upon an infection. Mycoplasma pneumoniae is a human respiratory pathogen causing atypical  pneumonia, accounting for approximately 20–30% of all types of pneumonia worldwide   and ceramide glucosyltransferase (or glucosylceramide synthase – reaction EC 2.4.1.80) of the glycosyltransferase family. The enzyme ceramide galactosyltransferase appears in viruses and cellular organisms; it synthetizes the biosynthesis of galactocerebroside by binding UDP‐galactose to a ceramide molecule, releasing UDP.M. pneumoniae, encoded by gene mpn483, has been shown to synthesize galactosylceramide (most likely the beta‐variant) using ceramide and UDP‐glucose as substrates, both with phosphatidylglycerol or cardiolipin as activators. M. pneumoniae has access to ceramide and galactose. It imports ceramide both from in vitro growth in axenic medium and from the host in vivo during infection  performed with Clustal Omega 1.2.4  were built and searched for with HMMER v3.3 Eddy,  – througSequence logos have been generated with WebLogo version 3  codes for a thymidine phosphorylase (EC 2.4.2.2) that contains a ‘glycosyl transferase family 3’ domain with InterPro identifier IPR000312, mpn028, mpn483 and mpn075 contain a ‘glycosyltransferase 2‐like’ domain, ( IPR001173), associated with proteins that have been linked to glycosphingolipids biosynthesis pathways and, specifically, to proteins that have been proven to own glycosyltransferase activity  to a phospholipid (ceramide) and building the glycosyl bond. Our functional analysis identified 4 genes containing a glycosyltransferase domain in C. jejuni, with E‐values of 10‐29  and 10‐27 . Focusing on the Mycoplasma species, we therefore assumed the protein sequences containing the Pfam domain PF00535 to be the ones associated with galactocerebroside synthesis. Thus, we continued our analysis focusing on PF00535 and discarded further analysis of PF13632. The distribution of the E‐values of this signature, presented in Fig. M. pneumoniae contain only the second motif M200535. The HMM for M200535 motif is provided in File C. jejuni match M200535 with much higher significance than PF00535: the beta‐1,3‐galactosyltransferase with E‐value 10‐43 and putative galactosyltransferase with E‐value 10‐42. An additional glycosyltransferase family 2 protein matching M200535 with E‐value 10‐42 was identified in C. jejuni, and full results are given in File The InterPro IPR001173 domain comprises two Pfam domains: ‘Glyco_trans_2_3’ (PF13632) and ‘Glyco_transf_2’ (PF00535); this last is found in two glycosyltransferases of The two new M100535 and M200535 motifs show some substantial differences: the sequence alignments of the proteins carrying the domains reveal M200535 to be almost a twice as long as M100535. Most differences between the two motifs are present in the first part of the domain, where M200535‐containing sequences show a predominance of aspartic acid in positions 344, 348, 428, 430, 436 and 455  comprised in the InterPro domain IPR016040 , found in sequences annotated as UDP‐6‐glucose 4‐epimerases that interconvert UDP‐glucose into UDP‐galactose. Therefore, we assume M. pneumoniae has the ability of converting UDP‐glucose into UDP‐galactose.Mycoplasma pneumoniae in vivo is most likely as represented in Fig. We can conclude the pathway for galactocerebroside synthesis in Mycoplasma species shows that almost only the species containing at least one domain, M200535 matched the UDP‐glucose‐epimerase domains Pfam PF16363 and/or PF01370  and Mycoplasma iowae (containing a glycosyltransferase domain different from M200535).Domain Pfam PF01370 is associated with a functionally equivalent epimerase. Interestingly, analysis of all 370 Fig. . ExceptiM. pneumoniae analysed have at least three glycosyltransferases encoded in the genome  that can potentially perform the synthesis of galactocerebroside. The highly significant match of M200535 with C. jejuni galactosyltransferases indicates that the sequences responsible of galactocerebroside synthesis contain the functional domain M200535, which, in this study, was found to be a distinctive motif with specific feature, while previously included in the Pfam domain PF00535. The Mycoplasma species and strains containing at least a protein with the functional domain M200535 are the only Mycoplasma  showing concomitant presence of a UDP‐glucose epimerase domain, which converts UDP‐glucose into UDP‐galactose. UDP‐galactose is used in the galactocerebroside synthesis and favoured, over UDP‐glucose, as a substrate by the glycosyltransferase operating the linkage. In fact, M200535 contains conserved aspartic acid residues, which in other microorganisms such as E. coli have been found to be catalytically essential for glycosyltransferase to exploit their β‐transferase activity on UDP‐sugars  and respiratory tract‐related live biotherapeutics (www.pulmobio.com). Therefore, an experimental application of our computational results would represent a suitable approach to render this bacterium safe in its numerous applications. In fact, other attempts to avoid biosynthesis of galactocerebroside seem to be not trivial: it would not be possible to knock‐out genes involved in sphingolipid transporter since this transporter would not only import ceramide but also very important lipids such as sphingomyelin that were shown to be essential to the survival of M. pneumoniae  that can synthetize glycosphingolipids such as galactocerebroside. Most likely, MPN_028 and MPN_075 perform the same synthesis, as they show high significant match for motif M200535. This motif was found as well in C. jejuni, the first microorganism linked to GBS through galactocerebroside biosynthesis. While the access of M. pneumoniae to galactose in vivo, when the import of glucose is at higher rate, remains unclear, the presence of a UDP‐glucose epimerase MPN_257, converting UDP‐glucose into UDP‐galactose, could make the synthesis of galactocerebroside possible. We can conclude that all wild‐type strains of M. pneumoniae are potentially capable of synthetizing galactocerebroside and will most likely be able to do so even with substitution or knock‐out of the galactosyltransferase MPN483 or the epimerase MPN_257, which will be problematic for certain medical applications, that is human vaccines and live biotherapeutics. Similarly, this will be the case for Mycoplasma species with the functional domain M200535, presenting also a UDP‐glucose epimerase domain. Our data show a set of Mycoplasma species that could serve as alternatives for such biomedical applications or that could provide pathway genes to modify M. pneumoniae accordingly.All Patent application n. EP20174842.3 by the Wageningen University & Research. Author Vitor A.P. Martins dos Santos has interests in the company LifeGlimmer GmbH. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.JF suggested the study. EG, JJK, JF, MS‐D and VAPMdS conceived the project. JJK performed the functional domain analysis. EG developed the motif model. EG wrote the manuscript with input of MS‐D, JF, VAPMdS and JJK. JF, MS‐D and VAPMdS supervised the project. All authors read and approved the manuscript.File S1. List of Mycoplasma genome sequences used in the study, obtained from NCBI repository.Click here for additional data file.File S2. Motif M200535, suggested to be responsible for galactocerebroside biosynthesis, as Hidden Markov Model.Click here for additional data file.File S3. Result of HMM search of motif M200535 in Campilobacter jejuni.Click here for additional data file.File S4. Consensus sequences for motifs M100535 and M200535.Click here for additional data file. Click here for additional data file.


Wheat bran incorporation into biscuits may increase their nutritional value, however, it may affect dough rheology and baking performance, due to the effect of bran particles on dough structure and an increase in water absorption. This study analyzed the enrichment effect of wheat bran and arabinoxylans, the most important non-starch polysaccharides found in whole wheat flour, on dough rheology and thermal behaviour during processing of rotary-moulded biscuits. The objective was to understand the contribution of arabinoxylans during biscuit-making and their impact when incorporated as wheat bran. Refined flour was replaced at 25, 50, 75, or 100% by whole flour with different bran particle sizes . The isolated effect of arabinoxylans was examined by preparing model flours, where refined flour was enriched with water-extractable and water-unextractable arabinoxylans. Wheat bran had the greatest impact on dough firmness and arabinoxylans had the greatest impact on the elastic response. The degree of starch gelatinization increased from 24 to 36% in biscuits enriched with arabinoxylans or whole flour and coarse bran. The microstructural analysis  suggested that fibre micropores may retain water inside their capillaries which can be released in a controlled manner during baking. Wheat bran constitutes about 16% of the weight of the wheat grain. It is the main by-product of wheat milling and is mostly used for animal feed . AccordiWhole wheat flour has been used to enrich baked products with bran fibre, including bread ,7, crackSugary biscuit doughs are quite different from bread or saltine-cracker doughs. Sweet biscuits are formulated with higher amounts of sugar  and fat , and with less water  than bread or saltine-cracker doughs . During As mentioned above, arabinoxylans (AX) are the most important non-starch polysaccharides found in whole wheat flour and refined flour, and they have the highest water holding capacity among the flour polymers. Native starch, damaged starch and gluten proteins can hold 0.30–0.45, 1.0–10.0 and ~2.8 g of water per gram of dry matter, respectively, while arabinoxylans can hold 4–10 g of water per gram of dry matter . Most ofThe incorporation of wheat bran to biscuits would increase their nutritional value, however, its addition may affect dough rheology and baking performance, impacting final biscuits attributes due to the effect of bran particles on dough structure as well as to an increase in water absorption, where arabinoxylans may play a pivotal role. Accordingly, the aim of this study was to analyze the effect of water-unextractable and water-extractable wheat arabinoxylans addition and the effect of wheat bran flour enrichment on the dough rheology and starch gelatinization and on the resultant microstructure of rotary-moulded biscuits.Soft wheat was supplied by Molinera San Cristobal . Soluble wheat arabinoxylan  and insoluble wheat arabinoxylan  were purchased from Megazyme Ltd. . Palm oil  was kindly provided by Team Foods Spa . Granulated sucrose was obtained from IANSA , soy lecithin from Cargill , ammonium bicarbonate from Pistor AG , sodium bicarbonate from Andimex S.A. , monocalcium phosphate from Blumos , and salt from K + S Chile S.A. .Soft wheat was subjected to a milling process using a RT-1 pin mill  to obtain refined flour (RF) and the wheat coarse bran fraction . The flour extraction rate was 75.7%. Part of the wheat bran was subjected to additional milling using a Mikro-Pulverizer MP1  to reduce its particle size and obtain the fine wheat bran fraction .Whole flours with different bran particle sizes were reconstituted based on the flour extraction rate (75.7%). The coarse or fine bran fraction was used to obtain whole flour with coarse bran (WFL) and whole flour with fine bran (WFS), respectively  and it was expressed as mg xylose equivalent. The water-unextractable arabinoxylan (WUAX) was obtained as the difference between TAX and WEAX.The arabinoxylan content of flours was determined using the phloroglucinol colorimetric method, as described by Ramseyer et al. . To deter 10 min . A standg for 10 min. The supernatant was carefully discarded, and the pellet was drained for 15 min. Finally, the pellet in the tube was weighed, and the WRC was quantified by subtracting the initial mass of the sample and tube. The results were expressed as g water retained per g dry sample.Water retention capacity (WRC) was measured in triplicate using the procedure described by Hemdane et al. . Flour  as part of the creaming phase. Thereafter, the flour and monocalcium phosphate were added, and the mix was blended for 2 min at low speed (60 rpm) to obtain the biscuit dough. The dough was sheeted to a thickness of 3 mm, moulded in rectangular moulds (53 mm × 34 mm), and then baked at 170 °C for 13 min until a final moisture content of 2.3 ± 0.3% (w.b.) was reached.Dynamic oscillatory rheological measurements of biscuit doughs were conducted on a Physica MCR 501 rheometer  following Ahmed et al.  with somThe firmness of the biscuit doughs was determined using a penetration test in a TA.HDplusC Texture Analyzer , following the procedure described by Colakoglu et al. . The douThe hardness and fracturability of the biscuits were measured two days after they were made using a TA.XT Texture Analyzer . Twenty biscuits per batch (3 batches per condition) were analysed using a three-point bending test (HDP/3PB probe) with a support span of 36 mm and test speed of 1 mm/s over a distance of 5 mm. The maximum breaking force (N) upon compression was used as a texture descriptor of hardness in biscuits, and the distance (mm) to the breaking point was used to represent fracturability .DSC analysis was performed in triplicate using a Mettler Toledo DSC823e  in order to quantify the degree of starch gelatinization (DG) in biscuits after baking following Molina et al. . Rotary-Rotary-moulded biscuits were defatted prior to SEM analysis in order to reduce the fat interference during the visualization as suggested by Pareyt et al. . The defThe microstructure of rotary-moulded biscuits was characterized on a Skyscan 1272 X-ray computed microtomography system . The image acquisition was carried out with an X-ray source operated at a voltage of 40 kV and a current of 250 µA using an exposure time of 800 s per frame over an interval of 0–180° with a rotation step of 0.2° and two frames averaging on each rotation step. Three biscuits were scanned for each condition.Around 962 projection images were obtained from the image acquisition, and they were reconstructed using an adapted cone-beam filtered back projection algorithm , and recThe experimental data were acquired in triplicate (3 batches per formulation) and analysed with R, version 3.6.1. For the statistical analysis, the bootstrap method introduced by Efron  was usedThe WEAX and TAX fractions were determined in refined and whole flours . The TAX and WEAX contents were 2.20 ± 0.21% and 0.49 ± 0.05% in refined flour, 5.84 ± 0.44% and 0.51 ± 0.07% in whole flour with coarse bran, and 5.09 ± 0.25% and 0.55 ± 0.07% in whole flour with fine bran, respectively. The WUAX fraction, an important structural component in the cell wall of the endosperm and bran , constitp-value < 0.05) was observed in the water retained by flour blends enriched with whole flour, in particular at a replacement equal or over 50%. Bran particle size marginally affected this value; blends with fine bran particles retained ~2% more water than blends with coarse bran, but differences were not always significant. The water retention capacity in model flours prepared with arabinoxylans was significantly higher  than in blends enriched with whole flour . This shows that isolated fibre can retain more water than when it is part of the bran matrix. The WUAX/WEAX ratio in model flours increased from 3.5 (RF) to 7.5 and 6.3 in 50MFL and 50MFS, and to 10.9 and 8.7 in 100MFL and 100MFS, respectively. This suggests that both WUAX and WEAX, but especially WUAX, may influence the increase in flours’ water-retention capacity due to their strong tendency to absorb water, as discussed by Courtin et al. [n et al. . Also, dn et al. . WEAX arn et al. , so thatn et al. . Overalln et al. . This prn et al. .p-value < 0.05) dough firmness compared to dough made with refined flour, which aligns with results reported by Nandeesh et al. [p-value < 0.05) this value. The firmness was ~8% higher in dough with fine bran particles compared to the dough with coarse bran at a replacement of 25%, and it rose ~17% at 50 and 75% substitution levels. The highest values of firmness were obtained in whole flour , while dough containing finer bran had the greatest firmness. These results suggest that bran particles, especially smaller ones, produce a physical hindrance along the dough structure, as previously Sozer et al. [The viscoelastic properties of dough may influence the moulding stage and baking performance, impacting the final attributes of rotary-moulded biscuits. As such, firmness and rheological parameters were measured in fibre-enriched biscuit doughs. h et al.  for rotah et al.  for sugar et al. .control) and loss tangent (G’’/G’) curves of doughs enriched with whole flours (control) increased much more in doughs with fine bran particles. The different particle size of bran particles may explain these results, as finer bran particles can be better distributed along the dough structure compared to coarse bran particles, creating a more discontinuous structure , which can increase the storage modulus [p-value < 0.05) in doughs containing finer bran particles  of rotary-moulded biscuits was quantified. Results are presented in The difference in starch gelatinization due to bran particle size A suggestp-value > 0.05) in biscuits prepared with 50MFS and 50MFL (mean DG of 23.6 or 23.7%), this value increased up to ~33.5 and ~36.3% when biscuits were formulated with 100MFS and 100MFL, respectively. These results show that the amount of arabinoxylans (WUAX and WEAX) affected the degree of starch gelatinization as well as the increase in the WUAX/WEAX ratio, as discussed in previous sections. Additionally, the DG of these biscuits (100MFL and 100MFS) was similar to the DG obtained in biscuits enriched with 100% of whole flour with coarse bran, despite the slightly amount of additional water in the dough. With regard to the microstructure, Arabinoxylans’ direct inclusion also modified the degree of starch gelatinization B. AlthouSEM and X-ray micro-CT images showed that bran fractions were distributed along the biscuit matrix. This aspect may influence the final texture. Additionally, differences in dough rheology and thermal transition due to fibre enrichment were mainly observed at 50% whole flour replacement and above. To better understand this aspect on the texture of rotary-moulded biscuits, the hardness and the fracturability of biscuits prepared with 50 or 100% whole flour replacement were measured, as shown in p-value < 0.05) the fracturability of biscuits, as shown in Enrichment with whole flour and bran particle size significantly affected (This research focused on the enrichment effect of wheat AX and wheat bran with different particle sizes during processing of rotary-moulded biscuits. The results showed that refined flour enriched with wheat bran retained less water than when AXs were incorporated. Regarding the rheological properties, the firmness of the dough was affected by the wheat bran particle size. Smaller fractions produced a discontinuous and compact structure that increased the overall strength of dough compared to coarse bran particles. Additionally, the elastic response increased in dough prepared with bran or arabinoxylans, but it was even greater with the latter. This suggests that arabinoxylans contribute to the elasticity of biscuit dough while wheat bran contributes to its stiffness.A partial starch gelatinization occurred in rotary-moulded biscuits, and a higher degree of gelatinized starch was obtained in biscuits enriched with AX or coarse bran particles. From a microstructural perspective, it was proposed that the micropores of coarse wheat bran or WUAX may retain water inside of their capillaries which could be released in a controlled manner during cooking, thus promoting this phenomenon.Overall, the processing of rotary-moulded biscuit doughs was not affected by whole flour incorporation. The dough rheology was slightly modified with fine bran particles but did not alter the moulding stage, which is a critical part in this type of biscuits. Fine bran particles are a feasible option to elaborate rotary-moulded biscuits because their incorporation allows to control the degree of starch gelatinization and the fracturability of biscuits is practically unaltered compared to biscuits elaborated with refined flour. Accordingly, these biscuits may constitute a convenient and inexpensive source of dietary fibre.


PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.The ancient paralogs premelanosome protein ( GPNMB (Glycoprotein nonmetastatic melanoma protein B)  and PMEL (premelanosome protein)  have generated increased interest from disparate biomedical fields. GPNMB and PMEL are related genes that encode for proteins with a common functional domain architecture: a signal peptide, core amyloid fragment, Kringle-like domain and a single transmembrane domain XXXL[LI] sequence D 130]. . 130]. AThe strong conservation between the PKAT family members may be considered surprising, if one interprets the phenotypic effects as relatively minor in these loss of function mutations across a variety of species (The repeat domain (RPT) is a low complexity region unique to PMEL, and in mammals, it contains a variable number of proline/serine/threonine-rich imperfect repeats . The RPTTo investigate the evolutionary relationship between orthologous PMEL RPT domains, we used RADAR predictions ,133 and The primary sequences of individual repeats are also drastically different between species. The human RPT contains two motifs that we noted are highly conserved amongst the repeats: GTT and PTXE; in contrast, the chicken RPT domain has a much more divergent TXXXTXX[DE] motif, despite also being a similar 12-mer-repeating unit. These striking differences in sequences are especially remarkable considering that the chicken RPT domain is sufficient to rescue the fibrilogenesis defects of human PMEL lacking a RPT domain, while the zebrafish RPT domain can provide a partial rescue . These dd-galactopyranoside causes a collapsed PMEL amyloid  ladder” , blunt-snouted clingfish (Gouania willdenowi) and Atlantic herring (Clupea harengus) each possess many repeat units that are almost perfectly identical in amino acid sequence within the species. However, comparing the near-perfect regions between species reveals that these closely related species underwent considerable sequence divergences (An interesting observation made during the analysis of the RPT domain sequences was that some species had near-perfect repeating protein sequences. For example, the RPT domain of zebrafish (ergences A. We theergences B. For thOne might speculate that these near-perfect repeat units confer an evolutionary advantage to the species examined or that imperfect repeats confer an evolutionary disadvantage. However, a far greater number of sequences from individual animals than are currently available would be required to test this hypothesis. Despite this divergence, these three species did have regularly spaced D/E ladders within all of the repeats, separated by speciation events. The PKAT family of genes are linked by a common ancestor and share a common domain architecture. However, the functional roles of PMEL and GPNMB have few commonalities, whereas practically nothing is known about the most ancient member, TMEM130. In this manuscript, we investigated the commonalities in the mutant phenotypes, evolutionary origin and functional domains between GPNMB and PMEL and analyzed the major difference: the RPT domain. Furthermore, we established that TMEM130 is well-suited to serve as an outgroup for future analyses comparing PMEL and GPNMB functions. We hope that this analysis assists with improving the understanding of the PKAT family and encourages further investigations into their evolution and diversification to perform such disparate functions.